BioMed Research International

New Biomaterials and Regenerative

Medicine Strategies in
Periodontology, Oral Surgery,
and Esthetic and Implant Dentistry
Guest Editors: David M. Dohan Ehrenfest, Hom-Lay Wang,
Jean-Pierre Bernard, and Gilberto Sammartino
New Biomaterials and Regenerative Medicine
Strategies in Periodontology, Oral Surgery,
Esthetic and Implant Dentistry
BioMed Research International

New Biomaterials and Regenerative Medicine

Strategies in Periodontology, Oral Surgery,
Esthetic and Implant Dentistry

Guest Editors: David M. Dohan Ehrenfest, Hom-Lay Wang,

Jean-Pierre Bernard, and Gilberto Sammartino
Copyright © 2015 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “BioMed Research International.” All articles are open access articles distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
Contents
New Biomaterials and Regenerative Medicine Strategies in Periodontology, Oral Surgery, Esthetic and
Implant Dentistry, David M. Dohan Ehrenfest, Hom-Lay Wang, Jean-Pierre Bernard, and Gilberto
Sammartino
Volume 2015, Article ID 210792, 3 pages

Histological and Histomorphometric Human Results of HA-Beta-TCP 30/70 Compared to Three

Different Biomaterials in Maxillary Sinus Augmentation at 6 Months: A Preliminary Report,
Susanna Annibali, Giovanna Iezzi, Gian Luca Sfasciotti, Maria Paola Cristalli, Iole Vozza, Carlo Mangano,
Gerardo La Monaca, and Antonella Polimeni
Volume 2015, Article ID 156850, 7 pages

Oral Streptococci Biofilm Formation on Different Implant Surface Topographies,

Pedro Paulo Cardoso Pita, José Augusto Rodrigues, Claudia Ota-Tsuzuki, Tatiane Ferreira Miato,
Elton G. Zenobio, Gabriela Giro, Luciene C. Figueiredo, Cristiane Gonçalves, Sergio A. Gehrke,
Alessandra Cassoni, and Jamil Awad Shibli
Volume 2015, Article ID 159625, 6 pages

Influence of Leukocyte- and Platelet-Rich Fibrin (L-PRF) in the Healing of Simple Postextraction
Sockets: A Split-Mouth Study, Gaetano Marenzi, Francesco Riccitiello, Mariano Tia, Alessandro di Lauro,
and Gilberto Sammartino
Volume 2015, Article ID 369273, 6 pages

Clinical Evaluation of the Regenerative Potential of EMD and NanoHA in Periodontal Infrabony
Defects: A 2-Year Follow-Up, Andrea Pilloni, Matteo Saccucci, Gabriele Di Carlo, Blerina Zeza,
Marco Ambrosca, Michele Paolantonio, Gilberto Sammartino, Claudio Mongardini, and Antonella Polimeni
Volume 2014, Article ID 492725, 9 pages

Adhesion and Proliferation of Human Periodontal Ligament Cells on Poly(2-methoxyethyl acrylate),

Erika Kitakami, Makiko Aoki, Chikako Sato, Hiroshi Ishihata, and Masaru Tanaka
Volume 2014, Article ID 102648, 14 pages

Physicochemical Characteristics of Bone Substitutes Used in Oral Surgery in Comparison to

Autogenous Bone, Antoine Berberi, Antoine Samarani, Nabih Nader, Ziad Noujeim, Maroun Dagher,
Wasfi Kanj, Rita Mearawi, Ziad Salemeh, and Bassam Badran
Volume 2014, Article ID 320790, 9 pages

Biological Width around One- and Two-Piece Implants Retrieved from Human Jaws, Ricardo Judgar,
Gabriela Giro, Elton Zenobio, Paulo G. Coelho, Magda Feres, Jose A. Rodrigues, Carlo Mangano,
Giovanna Iezzi, Adriano Piattelli, and Jamil Awad Shibli
Volume 2014, Article ID 850120, 5 pages

Effect of Low-Level Laser on Bone Defects Treated with Bovine or Autogenous Bone Grafts: In Vivo
Study in Rat Calvaria, Mércia J. S. Cunha, Luis A. Esper, Michyele C. Sbrana, Paula G. F. P. de Oliveira,
Accácio L. do Valle, and Ana Lúcia P. F. de Almeida
Volume 2014, Article ID 104230, 9 pages

Osteoconductive Potential of Barrier NanoSiO2 PLGA Membranes Functionalized by Plasma Enhanced

Chemical Vapour Deposition, Antonia Terriza, Jose I. Vilches-Pérez, Emilio de la Orden, Francisco Yubero,
Juan L. Gonzalez-Caballero, Agustin R. González-Elipe, José Vilches, and Mercedes Salido
Volume 2014, Article ID 253590, 10 pages
Vertical Ridge Augmentation of the Atrophic Posterior Mandible with Sandwich Technique: Bone Block
from the Chin Area versus Corticocancellous Bone Block Allograft—Clinical and Histological
Prospective Randomized Controlled Study, Luigi Laino, Giovanna Iezzi, Adriano Piattelli,
Lorenzo Lo Muzio, and Marco Cicciù
Volume 2014, Article ID 982104, 7 pages

Surface Characteristics and Bioactivity of a Novel Natural HA/Zircon Nanocomposite Coated on Dental
Implants, Ebrahim Karamian, Amirsalar Khandan, Mahmood Reza Kalantar Motamedi,
and Hesam Mirmohammadi
Volume 2014, Article ID 410627, 10 pages

Solubility of Two Resin Composites in Different Mouthrinses, Sezin Ozer, Emine Sen Tunc,
Nuray Tuloglu, and Sule Bayrak
Volume 2014, Article ID 580675, 4 pages
Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 210792, 3 pages
http://dx.doi.org/10.1155/2015/210792

Editorial
New Biomaterials and Regenerative Medicine Strategies in
Periodontology, Oral Surgery, Esthetic and Implant Dentistry

David M. Dohan Ehrenfest,1,2 Hom-Lay Wang,1

Jean-Pierre Bernard,3 and Gilberto Sammartino4
1
Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA
2
LoB5 Research Unit, School of Dentistry & Research Center for Biomineralization Disorders,
Chonnam National University, Gwangju 500-757, Republic of Korea
3
Department of Stomatology, Implantology and Dental and Maxillofacial Radiology, School of Dental Medicine,
University of Geneva, 1205 Geneva, Switzerland
4
Department of Oral Surgery, Faculty of Medicine, University of Naples Federico II, 80131 Naples, Italy

Correspondence should be addressed to David M. Dohan Ehrenfest; [email protected]

Received 1 March 2015; Accepted 1 March 2015

Copyright © 2015 David M. Dohan Ehrenfest et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Periodontology, Oral Surgery, Esthetic and Implant Dentistry found in dentistry nowadays. Implantable biomaterials were
(the POSEID disciplines) are strongly interconnected clinical particularly investigated and have significantly evolved in
and research fields, even if they are taught as separate the recent years: new dental implant design and surfaces
disciplines in most academic environments. Depending on [1], new bone biomaterials [2], new surgical adjuvants such
the professional organization of each country, dental implants as platelet concentrates [3], and so forth. The objectives
are often placed by periodontists or oral surgeons (and of all these biomaterials and technologies are not only to
even sometimes prosthodontists), even if implant dentistry is replace missing or damaged tissues but also now to promote
today one of the most active independent fields of education tissue regeneration [4]. This regenerative medicine approach
and research by itself. Esthetic dentistry is also a general term and the many recent technological evolutions are associated
that concerns in fact most of the Dental Arts: per definition, with the development of new therapeutic strategies that still
all dental treatments are expected to lead to an esthetic result require to be duly evaluated and validated. We can cite,
nowadays. The separation between these dental disciplines among the main current research biomaterial themes in the
appears sometimes quite artificial, even if each discipline POSEID disciplines, the development of new biomaterials
has clearly its own themes and specificities. Behind this and techniques of bone grafting or bone regeneration and
question of terminology and limits between the disciplines for periodontal and peri-implant soft tissue management
the question of transdisciplinarity in dental therapeutics and [4], the development of new bone substitutes and healing
the need for a global conception of the dental rehabilitations membranes for periodontal and implant reconstructions [5],
rises. However, when it is related to the development of new the development of new technologies and strategies in oral
technologies, the borders between these disciplines vanish in regenerative medicine and bioengineering (e.g., the use of
front of the paradigm of translational transversal research. growth factors or fibrin-based healing surgical adjuvants)
These POSEID dental disciplines are currently very [3], the development of new pharmacological technologies
active and are the source of development of many new and strategies during periodontal treatments and oral surgery
techniques and technologies. It is in their domains that (e.g., mouthwash and oral gel), or new dental implant surfaces
the highest number of innovation and publications can be and design for the improvement of osseointegration [1].
2 BioMed Research International

All these themes are very transdisciplinary/transversal, be described in all transversal fields, such as regenerative
both in their clinical impact (it concerns almost all aspects medicine [9], platelet concentrates for surgical use [3], or
of periodontology, oral surgery, implantology, and even den- bone materials [2]. The specialty-centered culture is often
tistry in general) and in their research methods (it concerns a big limitation for the development of these sciences and
physics/biophysics, pharmacology, biology, and material sci- a better integration of this transdisciplinarity has to be
ences). promoted.
These themes are also very transversal, as they are con- Finally, it is also important to notice that many review
cerning many clinical disciplines outside of dentistry, such as articles can be found in these fields about various aspects
orthopedics/sports medicine or plastic surgery. For example, of these biomaterials and their clinical impact, but we are
regenerative medicine strategies with platelets (Platelet-Rich lacking specific review articles making the synthesis of
Plasma (PRP) and Platelet-Rich Fibrin (PRF)) or advanced new approaches in these fields and leading to international
cell therapies (with various forms of stem cells) are a frequent consensus. In many review articles, the conclusions are too
theme in the POSEID field [4], but they are also widely often that data are missing. Concrete perspectives with new
developed in medical research [6]. If Leukocyte- and Platelet- approaches are often lacking, while many data from other
Rich Fibrin (L-PRF) is sometimes perceived as a dental fields could allow drawing more perspectives and feeding
biomaterial (due to historical reasons and its frequent use in the debates and discussions. This need for multidisciplinary
oral surgery and implant dentistry) [4], it is also very promis- debates and consensus is very strong [1, 3], but works
ing in orthopedics, sports medicine [7, 8], and regenerative promoting this transcollaborative culture are still scarce, even
strategies of the chronic ulcer wounds [9]. Transdisciplinarity if officially promoted by most academic institutions.
is very strong in most POSEID research themes. As a conclusion, it is a real need nowadays to consider
These research fields are also the most active transla- the research about biomaterials and biotechnologies in the
tional research topics in orofacial sciences, as any research interconnected fields of periodontology, oral surgery, and
about new biomaterials or techniques requires basic sciences esthetic and implant dentistry as a whole. It is the essence
research, in vitro and in vivo. For example, the develop- of the POSEIDO Academic Consortium (Periodontology,
ment of new healing growth factors based materials (such Oral Surgery, Esthetic & Implant Dentistry Organization) to
as Leukocyte- and Platelet-Rich Fibrin (L-PRF)) requires promote this transdisciplinary philosophy [15]. First, these
pharmacologic, biological, and tissue engineering concepts disciplines are so tightly interconnected that research in
to be tested, validated, optimized, and finally redeveloped this domain deserves often to be regrouped under such an
for extended applications in other fields [6, 9, 10]. On the acronym (POSEID disciplines). Second, this transdisciplinar-
other hand the development of new mouthwash solutions or ity has to be extended to all related medical, biological, and
healing gels implies biological and biophysical research (e.g., engineering disciplines able to collaborate on these topics.
with microencapsulated bioactive molecules) for the produc- Finally, the culture of translational sciences is really needed to
tion of new technologies and their final validation. Finally, promote development in this domain. It is expected that this
the implantable materials are ideal examples of translational POSEIDO philosophy will continue to develop and become
research (e.g., titanium implants or bone substitute materials) a standard in this field.
[1, 2] as they require very accurate engineering of the chemi-
cal and morphological characteristics of the materials (using
physical instruments from surface and material science) [1,
Acknowledgments
11], its correlation and validation with biological behaviors
and concepts [1, 12], its validation in vivo and in humans, and This work and special issue about new biomaterials and
finally the understanding of its long-term clinical outcomes regenerative medicine strategies in the POSEID disciplines
and eventual pathologies (such as associated peri-implantitis were supported by the POSEIDO Academic Consortium
mechanisms). In this case, many parameters are integrated (Periodontology, Oral Surgery, Esthetic & Implant Dentistry
(surface, macrodesign, and biomechanics) [13] and have a Organization), by a Grant from the National Research Foun-
strong impact in several domains (orthopedics particularly). dation of Korea (NRF) funded by the Korean Government
It is a good example of translational transversal research. (MEST) (no. 2011-0030121) and by the LoB5 Foundation for
The quantity of new biomaterials and biotechnolo- Research, France. The authors also want to thank Ms. Lidia M.
gies with direct clinical applications in the interconnected Wisniewska, from the Department of Didactics and School
POSEID fields is considerable and reflects very strongly Organization, Faculty of Education Sciences, University of
this need for transdisciplinarity and translational culture Granada, Granada, Spain, and Department of International
[4]. However, the borders between these disciplines are Relations, Paris Sorbonne University, Paris, France, for her
often artificially maintained and the conception of these help and contribution in the management of this special issue.
themes is still frequently lacking this global approach. For
example, it can be often observed in the dental literature
that teams working on implant materials (e.g., surfaces) are
neglecting the material science expertise of other specialties David M. Dohan Ehrenfest
(e.g., surface engineers) [1, 12, 14], leading sometimes to Hom-Lay Wang
a very dental simplification of some topics and to serious Jean-Pierre Bernard
approximations and flaws in the literature [1]. The same could Gilberto Sammartino
BioMed Research International 3

References [14] B. S. Kang, Y. T. Sul, S. J. Oh, H. J. Lee, and T. Albrektsson,

“XPS, AES and SEM analysis of recent dental implants,” Acta
[1] D. M. Dohan Ehrenfest, P. G. Coelho, B. S. Kang, Y. T. Biomaterialia, vol. 5, no. 6, pp. 2222–2229, 2009.
Sul, and T. Albrektsson, “Classification of osseointegrated [15] D. M. Dohan Ehrenfest, G. Sammartino, and J. P. Bernard, “The
implant surfaces: materials, chemistry and topography,” Trends periodontology, oral surgery, esthetic and implant dentistry
in Biotechnology, vol. 28, no. 4, pp. 198–206, 2010. organization (POSEIDO) and open journal: an international
[2] H. Browaeys, P. Bouvry, and H. de Bruyn, “A literature review on academic and scientific community for a new approach of open-
biomaterials in sinus augmentation procedures,” Clinical access publishing,” POSEIDO, vol. 1, no. 1, pp. 1–5, 2013.
Implant Dentistry and Related Research, vol. 9, no. 3, pp.
166–177, 2007.
[3] D. M. Dohan Ehrenfest, L. Rasmusson, and T. Albrektsson,
“Classification of platelet concentrates: from pure platelet-rich
plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF),”
Trends in Biotechnology, vol. 27, no. 3, pp. 158–167, 2009.
[4] M. Del Corso, A. Vervelle, A. Simonpieri et al., “Current
knowledge and perspectives for the use of platelet-rich plasma
(PRP) and platelet-rich fibrin (PRF) in oral and maxillofacial
surgery part 1: periodontal and dentoalveolar surgery,” Current
Pharmaceutical Biotechnology, vol. 13, no. 7, pp. 1207–1230, 2012.
[5] A. Simonpieri, M. Del Corso, A. Vervelle et al., “Current
knowledge and perspectives for the use of platelet-rich plasma
(PRP) and platelet-rich fibrin (PRF) in oral and maxillofacial
surgery part 2: bone graft, implant and reconstructive surgery,”
Current Pharmaceutical Biotechnology, vol. 13, no. 7, pp. 1231–
1256, 2012.
[6] P. A. Everts, M. M. Hoogbergen, T. A. Weber, R. J. Devilee, G.
van Monftort, and I. H. de Hingh, “Is the use of autologous
platelet-rich plasma gels in gynecologic, cardiac, and general,
reconstructive surgery beneficial?” Current Pharmaceutical
Biotechnology, vol. 13, no. 7, pp. 1163–1172, 2012.
[7] A. Mishra, K. Harmon, J. Woodall, and A. Vieira, “Sports
medicine applications of platelet rich plasma,” Current Pharma-
ceutical Biotechnology, vol. 13, no. 7, pp. 1185–1195, 2012.
[8] M. A. Zumstein, S. Berger, M. Schober et al., “Leukocyte- and
platelet-rich fibrin (L-PRF) for long-term delivery of growth
factor in rotator cuff repair: review, preliminary results and
future directions,” Current Pharmaceutical Biotechnology, vol.
13, no. 7, pp. 1196–1206, 2012.
[9] A. Cieslik-Bielecka, J. Choukroun, G. Odin, and D. M. Dohan
Ehrenfest, “L-PRP/L-PRF in esthetic plastic surgery, regen-
erative medicine of the skin and chronic wounds,” Current
Pharmaceutical Biotechnology, vol. 13, no. 7, pp. 1266–1277, 2012.
[10] T. M. Bielecki, T. S. Gazdzik, J. Arendt, T. Szczepanski, W. Krol,
and T. Wielkoszynski, “Antibacterial effect of autologous
platelet gel enriched with growth factors and other active
substances: an in vitro study,” The Journal of Bone and Joint
Surgery: British Volume, vol. 89, no. 3, pp. 417–420, 2007.
[11] D. M. Dohan Ehrenfest, M. Del Corso, and B.-S. Kang, “Identifi-
cation card and codification of the chemical and morphological
characteristics of 62 dental implant surfaces. Part 1: description
of the Implant Surface Identification Standard (ISIS) codifica-
tion system,” POSEIDO, vol. 2, no. 1, pp. 7–22, 2014.
[12] Y. T. Sul, B. S. Kang, C. Johansson, H. S. Um, C. J. Park, and T.
Albrektsson, “The roles of surface chemistry and topography in
the strength and rate of osseointegration of titanium implants in
bone,” Journal of Biomedical Materials Research: Part A, vol. 89,
no. 4, pp. 942–950, 2009.
[13] M. M. Al-Nsour, H. L. Chan, and H. L. Wang, “Effect of the
platform-switching technique on preservation of peri-implant
marginal bone: a systematic review,” The International Journal of
Oral & Maxillofacial Implants, vol. 27, no. 1, pp. 138–145, 2012.
Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 156850, 7 pages
http://dx.doi.org/10.1155/2015/156850

Clinical Study
Histological and Histomorphometric Human
Results of HA-Beta-TCP 30/70 Compared to Three Different
Biomaterials in Maxillary Sinus Augmentation at 6 Months:
A Preliminary Report

Susanna Annibali,1 Giovanna Iezzi,2 Gian Luca Sfasciotti,1 Maria Paola Cristalli,3
Iole Vozza,1 Carlo Mangano,4 Gerardo La Monaca,1 and Antonella Polimeni1
1
Department of Oral and Maxillofacial Sciences, Oral Surgery Unit, School of Dentistry, Sapienza University of Rome,
Via Caserta 6, 00161 Rome, Italy
2
Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara, Chieti, Italy
3
Department of Biotechnologies and Medical Surgical Sciences, Sapienza University of Rome, Rome, Italy
4
Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy

Correspondence should be addressed to Susanna Annibali; [email protected]

Received 19 September 2014; Accepted 13 December 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2015 Susanna Annibali et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Objective. The aim of this investigation was to examine the bone regenerative potential of newly biphasic calcium phosphate
ceramics (HA-𝛽-TCP 30/70), by assessing histological and histomorphometric results of human specimens retrieved from sinuses
augmented with HA-𝛽-TCP 30/70, and comparing them to anorganic bovine bone (ABB), mineralized solvent-dehydrated bone
allograft (MSDBA), and equine bone (EB), after a healing period of 6 months. Materials and Methods. Four consecutive patients with
edentulous atrophic posterior maxilla were included in this report. A two-stage procedure was carried out for sinus augmentation
with HA-𝛽-TCP 30/70, ABB, MSDBA, and EB. After 6 months, specimens were retrieved at the time of implant placement and
processed for histological and histomorphometric analyses. Results. At histological examination, all biomaterials were in close
contact with the newly formed bone and showed the same pattern of bone formation; the grafted granules were surrounded by
a bridge-like network of newly formed bone. A limited number of ABB particles were partially covered by connective tissue. The
histomorphometric analysis revealed 30.2% newly formed bone for Ha-𝛽-TCP 30/70, 20.1% for ABB, 16.4% for MSDBA, and 21.9%
for EB. Conclusions. Within the limitations of the present investigation, these results support the successful use of HA-𝛽-TCP 30/70
for sinus augmentation.

1. Introduction potential intraoperative and postoperative complications [5–

Deficiencies in the volume and quality of available bone can 7]. After implant placement, a resorption with up to 49.5%
complicate implant placement in the resorbed maxillary jaw. marked volume loss has been reported in the literature after 6
Sinus grafting in the maxillary posterior region is a highly months from sinus grafting [8, 9]. A number of graft materials
predictable procedure to increase the vertical volume of sinus with different origin and mechanism of bone regeneration
floor bone to accommodate dental implants [1, 2]. have been used alone or in combination with autografts
Autologous bone is considered the gold standard for bone in sinus floor augmentations [10–20]. Therefore the current
regeneration [3, 4], even if different bone substitutes have issue is the definition of the best filling material for the sinus
been proposed to overcome the limits related to its use, cavity. The presence or absence of autogenous bone in a graft
specifically, donor site morbidity, increased operating time, did not affect implant survival [21, 22] and different studies
need of a second surgical site to obtain the transplant, and have suggested that some alternative augmentation materials
2 BioMed Research International

may not adversely influence clinical outcomes and implant 2. Materials and Methods
survival [23, 24].
Among the graft materials used in maxillary sinus aug- 2.1. Patient Selection. Four healthy patients (3 males, 1
mentation procedures, biphasic calcium phosphate ceramics, females, all nonsmokers, mean age 52 years, range 36–70
reached by mixing hydroxyapatite (HA) and tricalcium phos- years), scheduled for sinus augmentation in the atrophic
phate (TCP), are considered biocompatible, osteoconductive, posterior maxilla to receive fixed restorations, were recruited
and suitable to obtain bone formation [24]-[25]. The HA, for this study. Inclusion criteria were maxillary partial eden-
undergoing slow resorption, works as a scaffold to maintain tulism in the premolar/molar areas and subsinus residual
space, while the TCP that underwent quick dissolution bone height, measured on computerized tomography (CT)
leads to more interparticle space and releases calcium and scan, ranging from 2 to 4 mm and bone thickness 3–5 mm.
phosphorus ions that could stimulate new bone formation, Exclusion criteria were smoking, patients with systemic
promoting osteogenic activity [26]. Currently several bioce- diseases and maxillary sinus pathology. The clinical proce-
ramic materials are used and differences in HA/TCP ratio, dures were performed in accordance with the Declaration
phase composition, formulation, sizes, and shapes affect their of Helsinki and the Good Clinical Practice Guidelines. All
biological and mechanical properties [10, 18], [24], [27–29]. patients signed a written informed consent form. After
reviewing medical history and making a preliminary clinical
The newly biphasic calcium phosphate ceramics (HA-𝛽- examination, digital panoramic radiography and CT scan
TCP 30/70) with reticular structure, tested in the present were performed. A two-stage procedure was carried out
investigation, seem to have a better resorption and an for sinus floor augmentation with HA-𝛽-TCP 30/70, ABB,
increased bone formation [18]. MCBA, and EB. These materials were allocated to the par-
Anorganic bovine bone (ABB) is a deproteinized ster- ticipant’s sinus under randomized conditions. After a healing
ilized bovine bone, constituted by a calcium-deficient car- period of 6 months, specimens were retrieved at the time
bonate apatite, with 75% porosity and a crystal size of about of implant placement and processed for histological and
10 nm in the form of granules. Due to its natural structure, histomorphometric analyses.
it is chemically and physically comparable to human bone
and its porous nature, greatly increasing the surface area
of the material, promotes angiogenesis and immigration of 2.2. Surgical Procedure. Surgery was performed under sterile
osteogenic cells [19]. The usage of ABB is widely documented conditions and local anaesthesia with mepivacaine 2% with
and shows that it can well integrate in host bone tissue in epinephrine 1 : 100.000 (Carbocaine, AstraZeneca, Italy). On
different clinical and histological results [30–32]. the day of surgery, each patient was administered 2 g
amoxicillin + clavulanic acid (Augmentin, GlaxoSmithKline,
MSDBA is a solvent-dehydrated, limited-dose, gamma-
London, UK) 1 hour prior to surgery and rinsed with a
irradiated portions of human iliac crest bone wedge. The
chlorhexidine digluconate solution 0.2% (Corsodyl, Glaxo-
allograft mixture contains 50% cortical and 50% cancellous
SmithKline, Belgium) for 2 min immediately prior to the
microchips with a particle size of 1-2 mm. MSDBA showed
intervention. Sinus floor elevation was performed utilizing a
significant histological results in terms of new bone formation
lateral window technique. A slightly palatal crestal incision
after sinus augmentation procedures [10, 11, 33].
was made and supplemented by buccal releasing incisions
Equine-derived bone (EB) is an equine-derived bone mesially and distally. Full thickness flaps were elevated to
tissue deantigenated by a proteolytic low-temperature pro- expose the alveolar crest and the lateral sinus wall. An
cess that eliminates the organic components but leaves the oval-shaped osteotomy, with the inferior border about 5 mm
mineral structure of the hydroxyapatite unaltered, saving superior to the alveolar bone margin, was made on the
the resorption potential [12]. Recent investigations showed lateral aspect of the sinus wall using a round bur under cold
that also the equine-derived bone (EB) is able to induce sterile saline irrigation. The center of the bony window was
bone formation in maxillary sinus augmentations [12, 13, 34, gently in-fractured with care to ensure that its most superior
35]. portion was left intact. The sinus membrane was carefully
Although the number of newly formed bones in the elevated and rotated, together with the osteotomy window,
augmented sinus is not directly related to the survival rate of inward and upward. The exposed sinus cavity was augmented
the implants, histological and histomorphometric analysis of with HA-𝛽-TCP 30/70 ABB, MCBA, or EB. The graft was
specimens, retrieved at the time of implant placement (after placed and carefully packed into the area between the elevated
a healing period of 6 months), represents a reliable indicator Schneiderian membrane and sinus floor without excessive
to assess and compare the performance of the graft materials pressure. A resorbable membrane was placed over the lateral
[19]. wall of the sinus and the mucoperiosteal flap was replaced
The aim of this investigation was to examine the bone and stabilized with monofilament, nonresorbable, expanded
regenerative potential of a newly biphasic calcium phosphate polytetrafluoroethylene (e-PTFE) (Gore-Tex Suture, W. L.
ceramics (HA-𝛽-TCP 30/70), by assessing histological and Gore, Flagstaff, AZ, USA) sutures. No Schneiderian mem-
histomorphometric results of human specimens retrieved branes were perforated during any of the sinus augmentation
from sinuses augmented with HA-𝛽-TCP 30/70, and com- procedures. For the postoperative phase, the patients were
paring them to anorganic bovine bone (ABB), mineralized protected from infection by administration of prophylactic
solvent-dehydrated bone allograft (MSDBA), and equine antibiotics (Augmentin, GlaxoSmithKline, London, UK) 1 g,
bone (EB), after a healing period of 6 months. 2 times daily for 1 week; an analgesic and antiphlogistic
BioMed Research International 3

medication (Ibifen, 200 mg -IBI-Lorenzini, Aprilia, Italy)

was prescribed 3 times daily. The patient was instructed NB
to avoid use of a removable prosthesis, not to blow the
HA
nose, and to rinse twice daily for a period of 2 weeks with
0.2% chlorhexidine gluconate. Following a healing period
of 2 weeks, sutures were removed. Six months after the
augmentation procedure, clinical and radiographic exami-
nation were undertaken and each patient was reappointed Figure 1: Grafted HA-𝛽-TCP 30/70 (HA) particles, almost com-
pletely surrounded by newly formed bone (NB) (acid fuchsin-
for biopsy at the time of implant placement in the same
toluidine blue; original magnification 12x).
location. Under local anesthesia, a vertical incision was made
buccally and continued horizontally and distally at the palatal
side of the alveolar crest. A full thickness flap was raised
and mobilized for tension-free closure. During this surgery,
a total of 4 bone samples were retrieved with a 3.5 trephine
bur under sterile saline solution irrigation, one from each HA
NB
augmented site. From 1 to 6 implants were placed into each
patient’s augmented sinus according to the manufacturer’s
directions.
MS
2.3. Histological Procedure. The bone cores were retrieved
and were immediately stored in 10% buffered formalin and
processed to obtain thin ground sections. The specimens were
processed using the Precise 1 Automated System (Assing,
Rome, Italy) [36]. The specimens were dehydrated in a graded Figure 2: Marrow stromal cells (MS) and blood vessels were found
series of ethanol rinses and embedded in a glycolmethacrylate in the vicinity of newly formed bone (NB) surrounding HA-𝛽-
resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). TCP 30/70 particles (HA) (acid fuchsin-toluidine blue; original
After polymerization, the specimens were sectioned, along magnification 100x).
their longitudinal axis, with a high precision diamond disk
at about 150 𝜇m and ground down to about 30 𝜇m with a
specially designed grinding machine Precise 1 Automated
System (Assing, Rome, Italy). Three slides were obtained 3.2. Histological and Histomorphometric Results
from each specimen. These slides were stained with acid
fuchsin and toluidine blue and examined with transmitted 3.2.1. Biphasic Calcium Phosphate Ceramics (HA-𝛽-TCP
light Leitz Laborlux microscope (Leitz, Wetzlar, Germany). 30/70). At low magnification, a large number of grafted
Histomorphometry of the percentages of newly formed bone, biomaterials, completely surrounded by newly formed bone,
residual grafted material, and marrow spaces was carried were observed (Figure 1). In some fields osteoblasts were
out using a light microscope (Laborlux S, Leitz, Wetzlar, detected in the process of apposing bone directly on the
Germany) connected to a high-resolution video camera particle surface. No gaps were present at the bone-particles
(3CCD, JVC KY-F55B, JVC, Yokohama, Japan) and interfaced interface, and the bone was always in close contact with
with a monitor and PC (Intel Pentium III 1200 MMX, Intel, the particles. Marrow stromal cells and blood vessels were
Santa Clara, CA, USA). This optical system was associated found inside the marrow spaces. A vascular growth was
with a digitizing pad (Matrix Vision GmbH, Oppenweiler, also observed next to the newly formed bone (Figure 2).
Germany) and a histometry software package with image cap- No inflammatory cells or foreign body reaction was noted
turing capabilities (Image-Pro Plus 4.5, Media Cybernetics around the grafted particles. Histomorphometry showed that
Inc., Immagini & Computer Snc, Milano, Italy). newly formed bone represented 30.2%, marrow spaces 40.7%,
and the residual graft material 29.1%.

3. Results
3.2.2. Anorganic Bovine Bone (ABB). At low magnification,
3.1. Clinical Results. In all cases, no perforations of the trabecular bone with large marrow spaces was observed
Schneiderian membrane were present, primary stability of (Figure 3). Some of the particles appeared to be cemented
the implants was achieved, regardless of the bone graft by this newly formed bone. At higher magnification, the
substitute used, and postoperative complications were absent. bone presented wide osteocyte lacunae (Figure 4). A limited
The healing process after sinus augmentation procedure was number of anorganic bovine bone particles were partially
uneventful and no clinical signs of sinus pathology were covered by connective tissue. No inflammatory cell infiltrate
observed. Six months after augmentation, the radiographic was present around the particles or at the interface with
examination showed in all patients the presence of dense bone. Histomorphometry showed that newly formed bone
bone in the maxillary sinuses where the biomaterials were represented 20.1%, marrow spaces 60.8%, and the residual
inserted. graft material 19.1%.
4 BioMed Research International

ABB

B B

B G
MS
ABB

Figure 3: Trabecular bone (B) with large marrow spaces and resid-
ual ABB (ABB) particles can be observed (acid fuchsin-toluidine
blue; original magnification 30x).
Figure 5: Trabecular bone (B) with large marrow spaces (MS)
and residual MSDBA (G) particles can be observed (acid fuchsin-
toluidine blue; original magnification 10x).

ABB

Os

NB

Os

Figure 4: Newly formed trabecular bone (NB) is in close contact

with ABB (ABB) particle, with no gaps at the bone-particle interface
(acid fuchsin-toluidine blue; original magnification 400x). Figure 6: In some fields, osteoblasts (Os) were observed in the
process of apposing bone directly on the MSDBA (G) particles’
surface (acid fuchsin-toluidine blue; original magnification 200x).

3.2.3. Mineralized Solvent-Dehydrated Bone Allograft

(MSDBA). At low magnification, trabecular bone with
large marrow spaces was observed (Figure 5). Few particles 4. Discussion
presented irregularly shaped margins, probably due to a
resorption process. There were no gaps at the bone-particle In sinus augmentation procedures, the choice of bone substi-
interface and the new bone was in strict contact with tutes plays a key role, because their different properties affect
the granules. In some fields, osteoblasts were observed medium and long-term success of implant rehabilitation.
in the process of apposing bone directly on the particle However, clinical and histological outcomes about bone
surface (Figure 6). Newly formed bone was characterized substitute materials and management and timing of implant
by large osteocyte lacunae and bridged up most part of the placement and their follow-up still remain open questions,
biomaterial particles. Histomorphometry showed that newly because there are no clear guidelines for the use of autogenous
formed bone represented 16.4%, marrow spaces 65.1%, and bone or bone substitutes [24]. Numerous studies have com-
the residual graft material 18.5%. pared grafting materials after sinus augmentation, reporting
small numbers of histology samples as our investigation
[11, 19, 21, 22, 26, 27, 30]. Therefore, it is very important to
3.2.4. Equine Bone (EB). At low magnification, trabecular histologically evaluate the healing process of bone substitute
bone with large marrow spaces was observed. In a portion materials, after two-stage sinus augmentation, in order to
of the specimen, preexisting bone with areas of remodeling increase the knowledge about their biological behavior in
was present (Figure 7). Some of the particles appeared to humans [37].
be cemented by the newly formed bone. At higher magnifi- In the present preliminary investigation, to examine the
cation, the bone showed wide osteocyte lacunae (Figure 8). bone regenerative potential of a newly biphasic calcium
Some trabeculae of the grafted material were bridged by phosphate ceramic, the histologic process of healing and
newly formed bone. No inflammatory cells or multinucleated bone formation of HA-𝛽-TCP (30/70) was compared to
cells were observed around the particles or at newly formed those of the most frequently used and well documented
bone-biomaterial interface in the marrow spaces. Histomor- biomaterials after six months from sinus augmentation [10–
phometry showed that newly formed bone represented 21.9%, 13, 17–20]. The present scaffold was developed by the direct
marrow spaces 54.9%, and the residual graft material 23.2%. rapid prototyping technique dispense-plotting, as previously
BioMed Research International 5

completely surrounded by newly formed bone, that was

always in close contact, without gaps, with the particles.
Furthermore, osteoblasts, in some fields, were detected in the
B apposing bone directly on the particle surface, the marrow
MS stromal cells and blood vessels were found inside the marrow
EB spaces and the vascular growth was observed next to the
B
newly formed bone. These histological data, in agreement
with those reported in a similar study about evaluation
of HA-𝛽-TCP (40/60) in sinus elevation after a healing
period of 6 months [42], demonstrated that the HA-𝛽-TCP
is an osteoconductive and resorbable material. The absence
of inflammatory cells or foreign body reaction around the
Figure 7: Trabecular bone (B) with large marrow spaces (MS) and grafted particles testifies safety and biocompatibility.
residual EB (EB) particles were present (acid fuchsin-toluidine blue; For the ABB sample a limited number of grafted particles
original magnification 12x). were found partially covered by connective tissue while some
of the particles appeared to be cemented by this newly formed
bone. The ABB sample was smaller than the others and
this was due to the fracture of the bone biopsy retrieved
from the ABB augmented site during the removal from the
trephine bur. ABB is a nonresorbable bone substitute and
has osteoconductive properties. Its structure could represent
EB a type of protection against bone resorption, guaranteeing
long-term stability of the augmented maxillary sinus [10]. The
MSDBA sample confirmed the biocompatibility, ease of use,
and ability to form and maintain new bone in the maxillary
NB
sinus as reported by previous studies [11, 26]. No signs of
acute inflammation were present, and the percentage of newly
formed bone and residual graft material were comparable to
those reported for other biomaterials [43]. The EB sample
Figure 8: Newly formed bone (NB) with wide osteocyte lacunae showed some trabeculae bridged by newly formed bone. Our
(black arrow) in tight contact with EB (EB) particles (acid fuchsin- results are comparable to other investigations [12, 13]. It is
toluidine blue; original magnification 200x). evident resorption phenomena, and its ability to achieve a
more rapid and intense vascularization is very important
also in influencing long-term integration and predictability
of implant-prosthetic rehabilitation in regenerated sites. Its
described by Deisinger et al. [38] and Mangano et al. [18]. higher resorption ability could probably be due to a deanti-
This method enables tuning the HA/𝛽-TCP ratio, surface, genation process this grafting material undergoes [13]. The
structure, and micro- and macroporosity which in turn results of the present investigation revealed that HA-𝛽-TCP
influence the regenerative potential. Indeed, the pattern of (30/70) could provide a better result in sinus augmentation
resorption and the healing process would be influenced by procedures compared to the other biomaterials. No histo-
the variation of the ratio between the component at slow logical signs of adverse reactions were observed. Results
resorption (HA) and those at more fast resorption (𝛽-TCP), from this preliminary investigation have shown that HA-𝛽-
even if no statistical significant difference was found by TCP (30/70) has good biocompatibility, with no histological
Schopper et al. [39], comparing two different ratios (30/70 signs of adverse reactions, and osteoconductivity, with bone
versus 50/50) of HA/TCP in a sheep model, although a better formation directly on the biomaterial surface. Nevertheless
integration into the host bone was observed in the 30/70 the presence of a high quantity of the biomaterial particles
composition. On the contrary in a comparative study of after 6 months will require long-term histological studies
biphasic calcium phosphates with different HA/TCP ratios to better understand the times and the modalities of the
in mandibular bone defects of minipig, the BCP (20/80), resorption of this graft.
unlike BCP (60/40) and BCP (80/20), showed results similar In conclusion, within the limitations of the present
to those of autogenous bone grafts, indicating that the ratio of report the histological and histomorphometric observations
HA/TCP (60/40) might not be optimal for bone healing [40]. support the fact that Ha-TCP (30/70) can be suitable for
The three-dimensional network with an interconnecting pore successful augmentation procedures of the maxillary sinus
structure promotes the vascularization that is essential for the and represents a very interesting option.
proliferation and differentiation of osteoblasts and therefore
for the ingrowth of new bone into the graft [41].
At the histologic examination, the HA-𝛽-TCP (30/70) Conflict of Interests
scaffold showed a pattern of bone formation similar to the
other biomaterials tested, with a large number of grafts, The authors declare that they have no conflict of interests.
6 BioMed Research International

Acknowledgment calvaria autologous bone: immunohistochemical evaluation of

OPG/RANKL in humans,” European Journal of Histochemistry,
The work was partially supported by the Ministry of Edu- vol. 57, no. 1, p. e10, 2013.
cation, University and Research (MIUR), Rome, Italy (PRIN [14] J. Bassil, N. Naaman, R. Lattouf et al., “Clinical, histological, and
2010ZLNJ5), without any influence or interference. histomorphometrical analysis of maxillary sinus augmentation
using inorganic bovine in humans: preliminary results,” Journal
of Oral Implantology, vol. 39, no. 1, pp. 73–80, 2013.
References
[15] C. Mangano, B. Barboni, L. Valbonetti et al., “In vivo behavior
[1] M. Chiapasco, P. Casentini, and M. Zaniboni, “Bone augmenta- of a custom-made 3D synthetic bone substitute in sinus aug-
tion procedures in implant dentistry,” The International Journal mentation procedure in sheep,” Journal of Oral Implantology. In
of Oral & Maxillofacial Implants, vol. 24, supplement, pp. 237– press.
259, 2009. [16] B. Barboni, C. Mangano, L. Valbonetti et al., “Synthetic bone
[2] T. L. Aghaloo and P. K. Moy, “Which hard tissue augmentation substitute engineered with amniotic epithelial cells enhances
techniques are the most successful in furnishing bony support bone regeneration after maxillary sinus augmentation,” PLoS
for implant placement?” International Journal of Oral and ONE, vol. 8, no. 5, Article ID e63256, 2013.
Maxillofacial Implants, vol. 22, pp. 49–70, 2007. [17] A. Mordenfeld, M. Hallman, C. B. Johansson, and T. Albrekts-
[3] M. Hallman and A. Thor, “Bone substitutes and growth factors son, “Histological and histomorphometrical analyses of biop-
as an alternative/complement to autogenous bone for grafting in sies harvested 11 years after maxillary sinus floor augmentation
implant dentistry,” Periodontology 2000, vol. 47, no. 1, pp. 172– with deproteinized bovine and autogenous bone,” Clinical Oral
192, 2008. Implants Research, vol. 21, no. 9, pp. 961–970, 2010.
[4] R. J. Klijn, G. J. Meijer, E. M. Bronkhorst, and J. A. Jansen, “A [18] C. Mangano, B. Sinjari, J. A. Shibli et al., “A human clinical,
meta-analysis of histomorphometric results and graft healing histological, histomorphometrical, and radiographical study on
time of various biomaterials compared to autologous bone biphasic ha-beta-tcp 30/70 in maxillary sinus augmentation,”
used as sinus floor augmentation material in humans,” Tissue Clinical Implant Dentistry and Related Research, 2013.
Engineering—Part B: Reviews, vol. 16, no. 5, pp. 493–507, 2010. [19] G. Iezzi, M. Degidi, A. Piattelli et al., “Comparative histological
[5] C. M. Misch, “Autogenous bone: is it still the gold standard?” results of different biomaterials used in sinus augmentation
Implant Dentistry, vol. 19, no. 5, p. 361, 2010. procedures: a human study at 6 months,” Clinical Oral Implants
[6] D. Rickert, J. J. R. H. Slater, H. J. A. Meijer, A. Vissink, and Research, vol. 23, no. 12, pp. 1369–1376, 2012.
G. M. Raghoebar, “Maxillary sinus lift with solely autogenous [20] Y.-K. Kim, J. Lee, J.-Y. Yun, P.-Y. Yun, and I.-W. Um, “Com-
bone compared to a combination of autogenous bone and parison of autogenous tooth bone graft and synthetic bone
growth factors or (solely) bone substitutes. A systematic review,” graft materials used for bone resorption around implants after
International Journal of Oral & Maxillofacial Surgery, vol. 41, no. crestal approach sinus lifting: a retrospective study,” Journal of
2, pp. 160–167, 2012. Periodontal & Implant Science, vol. 44, no. 5, pp. 216–221, 2014.
[7] G. D. Rabelo, P. M. de Paula, F. S. Rocha, C. Jordão Silva, [21] S. S. Wallace and S. J. Froum, “Effect of maxillary sinus
and D. Zanetta-Barbosa, “Retrospective study of bone grafting augmentation on the survival of endosseous dental implants. A
procedures before implant placement,” Implant Dentistry, vol. systematic review,” Annals of periodontology, vol. 8, no. 1, pp.
19, no. 4, pp. 342–350, 2010. 328–343, 2003.
[8] P. Galindo-Moreno, I. Moreno-Riestra, G. Ávila et al., “Histo- [22] M. Chiapasco, P. Casentini, and M. Zaniboni, “Bone augmenta-
morphometric comparison of maxillary pristine bone and com- tion procedures in implant dentistry,” The International Journal
posite bone graft biopsies obtained after sinus augmentation,” of oral & Maxillofacial implants, vol. 24, pp. 237–259, 2009.
Clinical Oral Implants Research, vol. 21, no. 1, pp. 122–128, 2010. [23] E. Nkenke and F. Stelzle, “Clinical outcomes of sinus floor
[9] R. J. Klijn, J. J. J. P. van den Beucken, E. M. Bronkhorst, S. augmentation for implant placement using autogenous bone
J. Berge, G. J. Meijer, and J. A. Jansen, “Predictive value of or bone substitutes: a systematic review,” Clinical Oral Implants
ridge dimensions on autologous bone graft resorption in staged Research, vol. 20, no. 4, pp. 124–133, 2009.
maxillary sinus augmentation surgery using Cone-Beam CT,” [24] S. S. Jensen, N. Broggini, E. Hjørting-Hansen, R. Schenk, and
Clinical Oral Implants Research, vol. 23, no. 4, pp. 409–415, 2012. D. Buser, “Bone healing and graft resorption of autograft, anor-
[10] C. M. Schmitt, H. Doering, T. Schmidt, R. Lutz, F. W. Neukam, ganic bovine bone and 𝛽-tricalcium phosphate. A histologic
and K. A. Schlegel, “Histological results after maxillary sinus and histomorphometric study in the mandibles of minipigs,”
augmentation with Straumann BoneCeramic, Bio-Oss, Puros, Clinical Oral Implants Research, vol. 17, no. 3, pp. 237–243, 2006.
and autologous bone. A randomized controlled clinical trial,” [25] S. V. Dorozhkin, “Self-setting calcium orthophosphate formula-
Clinical Oral Implants Research, vol. 24, no. 5, pp. 576–585, 2013. tions,” Journal of Functional Biomaterials, vol. 4, no. 4, pp. 209–
[11] S. Annibali, M. P. Cristalli, G. La Monaca et al., “Human max- 311, 2013.
illary sinuses augmented with mineralized, solvent-dehydrated [26] S. V. Dorozhkin, “Calcium orthophosphates in dentistry,” Jour-
bone allograft: a longitudinal case series,” Implant Dentistry, vol. nal of Materials Science: Materials in Medicine, vol. 24, no. 6, pp.
20, no. 6, pp. 445–454, 2011. 1335–1363, 2013.
[12] M. Nevins, F. Heinemann, U. W. Janke et al., “Equine-derived [27] S. V. Dorozhkin, “Biocomposites and hybrid biomaterials based
bone mineral matrix for maxillary sinus floor augmentation: a on calcium orthophosphates,” Biomatter, vol. 1, no. 1, pp. 3–56,
clinical, radiographic, histologic, and histomorphometric case 2011.
series,” The International Journal of Periodontics and Restorative [28] C. Mangano, V. Perrotti, J. A. Shibli et al., “Maxillary sinus
Dentistry, vol. 33, no. 4, pp. 483–489, 2013. grafting with biphasic calcium phosphate ceramics: clinical and
[13] S. Tetè, R. Vinci, V. L. Zizzari et al., “Maxillary sinus aug- histologic evaluation in man,” The International Journal of Oral
mentation procedures through equine-derived biomaterial or & Maxillofacial Implants, vol. 28, no. 1, pp. 51–56, 2013.
BioMed Research International 7

[29] C. Lindgren, A. Mordenfeld, and M. Hallman, “A prospective Biomedical Materials Research. Part B Applied Biomaterials, vol.
1-year clinical and radiographic study of implants placed after 97, no. 2, pp. 245–254, 2011.
maxillary sinus floor augmentation with synthetic biphasic [42] J. W. F. H. Frenken, W. F. Bouwman, N. Bravenboer, S. A.
calcium phosphate or deproteinized bovine bone,” Clinical Zijderveld, E. A. J. M. Schulten, and C. M. ten Bruggenkate,
Implant Dentistry and Related Research, vol. 14, no. 1, pp. 41–50, “The use of Straumann Bone Ceramic in a maxillary sinus floor
2012. elevation procedure: a clinical, radiological, histological and
[30] S. J. Froum, S. S. Wallace, S.-O. Cho, N. Elian, and D. P. Tarnow, histomorphometric evaluation with a 6-month healing period,”
“Histomorphometric comparison of a biphasic bone ceramic to Clinical Oral Implants Research, vol. 21, no. 2, pp. 201–208, 2010.
anorganic bovine bone for sinus augmentation: 6- to 8-month [43] A. Scarano, M. Degidi, G. Iezzi et al., “Maxillary sinus augmen-
postsurgical assessment of vital bone formation. A Pilot Study,” tation with different biomaterials: a comparative histologic and
International Journal of Periodontics and Restorative Dentistry, histomorphometric study in man,” Implant Dentistry, vol. 15, no.
vol. 28, no. 3, pp. 273–281, 2008. 2, pp. 197–207, 2006.
[31] C. M. Schmitt, T. Moest, R. Lutz, F. W. Neukam, and K. A.
Schlegel, “Anorganic bovine bone (ABB) vs. autologous bone
(AB) plus ABB in maxillary sinus grafting. A prospective non-
randomized clinical and histomorphometrical trial,” Clinical
Oral Implants Research, 2014.
[32] G. Iezzi, A. Scarano, C. Mangano, B. Cirotti, and A. Piattelli,
“Histologic results from a human implant retrieved due to
fracture 5 years after insertion in a sinus augmented with
anorganic bovine bone,” Journal of Periodontology, vol. 79, no.
1, pp. 192–198, 2008.
[33] S. J. Froum, S. S. Wallace, N. Elian, S. C. Cho, and D. P. Tarnow,
“Comparison of mineralized cancellous bone allograft (Puros)
and anorganic bovine bone matrix (Bio-Oss) for sinus aug-
mentation: histomorphometry at 26 to 32 weeks after grafting,”
International Journal of Periodontics and Restorative Dentistry,
vol. 26, no. 6, pp. 543–551, 2006.
[34] S. Tetè, V. L. Zizzari, R. Vinci et al., “Equine and porcine bone
substitutes in maxillary sinus augmentation: a histological and
immunohistochemical analysis of VEGF expression,” Journal of
Craniofacial Surgery, vol. 25, no. 3, pp. 835–839, 2014.
[35] L. Artese, A. Piattelli, D. A. Di Stefano et al., “Sinus lift with
autologous bone alone or in addition to equine bone: an
immunohistochemical study in man,” Implant Dentistry, vol. 20,
no. 5, pp. 383–388, 2011.
[36] A. Piattelli, A. Scarano, and M. Quaranta, “High-precision, cost-
effective cutting system for producing thin sections of oral
tissues containing dental implants,” Biomaterials, vol. 18, no. 7,
pp. 577–579, 1997.
[37] Y. K. Kim, P. Y. Yun, S. G. Kim, and S. C. Lim, “Analysis of the
healing process in sinus bone grafting using various grafting
materials,” Oral Surgery, Oral Medicine, Oral Pathology, Oral
Radiology and Endodontology, vol. 107, no. 2, pp. 204–211, 2009.
[38] U. Deisinger, S. Hamisch, M. Schumacher, F. Uhl, R. Detsch,
and G. Ziegler, “Fabrication of tailored hydroxyapatite scaffolds:
comparison between a direct and an indirect rapid prototyping
technique,” Key Engineering Materials, vol. 361–363, pp. 915–918,
2008.
[39] C. Schopper, F. Ziya-Ghazvini, W. Goriwoda et al., “HA/TCP
compounding of a porous CaP biomaterial improves bone
formation and scaffold degradation. A long-term histological
study,” Journal of Biomedical Materials Research Part: B Applied
Biomaterials, vol. 74, no. 1, pp. 458–467, 2005.
[40] S. S. Jensen, M. M. Bornstein, M. Dard, D. D. Bosshardt, and
D. Buser, “Comparative study of biphasic calcium phosphates
with different HA/TCP ratios in mandibular bone defects. A
long term histomorphometric study in minipigs,” Journal of
Biomedical Materials Research B, vol. 90, pp. 171–181, 2009.
[41] C. R. Campion, C. Chander, T. Buckland, and K. Hing, “Increas-
ing strut porosity in silicate-substituted calcium-phosphate
bone graft substitutes enhances osteogenesis,” Journal of
Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 159625, 6 pages
http://dx.doi.org/10.1155/2015/159625

Research Article
Oral Streptococci Biofilm Formation on Different Implant
Surface Topographies

Pedro Paulo Cardoso Pita,1 José Augusto Rodrigues,1,2 Claudia Ota-Tsuzuki,1

Tatiane Ferreira Miato,1 Elton G. Zenobio,3 Gabriela Giro,1
Luciene C. Figueiredo,1 Cristiane Gonçalves,1 Sergio A. Gehrke,4
Alessandra Cassoni,1 and Jamil Awad Shibli1
1
Department of Periodontology and Oral Implantology, Dental Research Division, Guarulhos University,
Praça Tereza Cristina 229, 07023-070 Guarulhos, SP, Brazil
2
Department of Operative Dentistry, Dental Research Division, Guarulhos University, Praça Tereza Cristina 229,
07023-070 Guarulhos, SP, Brazil
3
Department of Oral Implantology, PUC Minas, Avenida Dom Cabral 500, Prédio 46, Coração Eucarı́stico,
30535-901 Belo Horizonte, MG, Brazil
4
Biotecnos-Tecnologia e Ciência Ltda, Rua Dr. Bozano, 571 Centro, 97015-001 Santa Maria, RS, Brazil

Correspondence should be addressed to José Augusto Rodrigues; [email protected]

Received 7 September 2014; Revised 4 November 2014; Accepted 4 November 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2015 Pedro Paulo Cardoso Pita et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The establishment of the subgingival microbiota is dependent on successive colonization of the implant surface by bacterial species.
Different implant surface topographies could influence the bacterial adsorption and therefore jeopardize the implant survival. This
study evaluated the biofilm formation capacity of five oral streptococci species on two titanium surface topographies. In vitro biofilm
formation was induced on 30 titanium discs divided in two groups: sandblasted acid-etched (SAE- 𝑛 = 15) and as-machined (M-
𝑛 = 15) surface. The specimens were immersed in sterilized whole human unstimulated saliva and then in fresh bacterial culture
with five oral streptococci species: Streptococcus sanguinis, Streptococcus salivarius, Streptococcus mutans, Streptococcus sobrinus,
and Streptococcus cricetus. The specimens were fixed and stained and the adsorbed dye was measured. Surface characterization was
performed by atomic force and scanning electron microscopy. Surface and microbiologic data were analyzed by Student’s 𝑡-test
and two-way ANOVA, respectively (𝑃 < 0.05). S. cricetus, S. mutans, and S. sobrinus exhibited higher biofilm formation and no
differences were observed between surfaces analyzed within each species (𝑃 > 0.05). S. sanguinis exhibited similar behavior to form
biofilm on both implant surface topographies, while S. salivarius showed the lowest ability to form biofilm. It was concluded that
biofilm formation on titanium surfaces depends on surface topography and species involved.

1. Introduction However, the different implant surface topographies

could influence the bacterial adsorption [6–8]. Physical
Establishment of the dental subgingival microbiota is depen- and chemical factors may affect the attachment of biofilms
dent on successive colonization of the tooth surface by several to hard surfaces. The surface roughness at micrometer
bacterial species [1]. Each of these bacterial species appears level can increase the surface area and hence increase the
to facilitate enamel surface colonization by the next wave bacterial colonization. Roughness also provides protection
of bacterial settlers, resulting in the establishment of an from shear forces and increases the difficulty of cleaning
anaerobic Gram-negative microbiota [2]. So far, it is believed methods. Furthermore, Kolenbrander et al. [2] have shown
that a similar pattern of the same colonization process may that supragingival plaque formation, after initial bacterial
occur on the titanium implant surfaces [3–5]. colonization, was faster on a rough surface. The roughness
2 BioMed Research International

of different dental implant surfaces can work like grooves for Streptococcus cricetus (ATCC 19642) were used in this study
initial periodontal pathogen adhesion [6, 9, 10]. in biofilm formation.
The oral streptococci are members of the indigenous mi-
crobiota mainly in the supragingival environment [11] and 2.4. Saliva Coating of the Specimens. Unstimulated saliva
species of mutans group such as Streptococcus mutans, Strep- from 6 healthy nonsmoker and systemic healthy donors was
tococcus sobrinus, and Streptococcus cricetus were related collected for one hour per day, for seven days. Then the
to individuals with teeth because they are able to adhere saliva samples were sterilized and frozen at −20∘ C until a
to nonshedding surfaces, with S. mutans being the most total of 500 mL was collected per donor. All donors signed
prevalent species in humans [12]. Other species such as Strep- the informed consent. Subsequently, the saliva samples
tococcus sanguinis and Streptococcus salivarius are commonly were pooled and centrifuged (30 min; 4∘ C; 27,000 ×g). The
found in healthy periodontal individuals and the latter is supernatant was pasteurized (60∘ C, 30 min) to inactivate
related to mucosal surfaces; besides it can contribute to the endogenous enzymes, recentrifuged (30 min, 4∘ C; 27,000 ×g)
coaggregation of pathogenic bacteria, such as Porphyromonas in sterile bottles, and stored at −20∘ C. The pasteurization
gingivalis. Thus the oral streptococci are considered the efficacy was evaluated by plating 100 𝜇L of saliva onto brain
pioneer colonizers and might participate in the process, heart infusion (BHI) agar and by observing the absence
which can lead to implant failure on the long term [6]. of bacterial growth after 72 hours. The sterile disks were
Therefore, the aim of this in vitro study was to verify the placed in a sterile 24-well polystyrene cell culture plate
ability of five oral streptococci species to form biofilm on two containing 500 𝜇L of saliva for 4 hours to allow salivary
different titanium surface topographies. pellicle formation.

2.5. Biofilm Formation Assay. After coating period, saliva

2. Material and Methods was aspirated from each well and replaced with 500 𝜇L
2.1. Implant Surface Topography. Thirty discs (5 mm diameter of BHI broth (double concentrated) and 500 𝜇L of saliva.
and 3 mm thickness) made of grade-4 titanium (Implacil De Inocula were prepared by harvesting each standard reference
Bortoli, Sao Paulo, SP, Brazil) were prepared with 2 surface strain cell from BHI agar plates previously inoculated and
topographies: as-machined (M) and sandblasted acid-etched incubated under microaerophilic conditions for 24 hours
(SAE) surfaces. The titanium discs with sandblasted acid- (candle jar, 37∘ C). The bacterial cells were suspended in
etched surface were blasted with 50–100 𝜇m TiO2 particles. sterile saline solution, adjusting the turbidity to OD630 0.15
After sandblasting, the specimens were ultrasonically cleaned (∼106 UFC/mL). Each well was inoculated with 100 𝜇L of this
with an alkaline solution, washed in distilled water, and inoculum suspension. Plates were then incubated for 16 hours
pickled with maleic acid. under microaerophilic conditions. Afterwards, the specimens
were gently washed in sterile saline solution three times in
order to remove unattached cells.
2.2. Implant Surface Characteristics. The samples were first
The specimens with remaining attached bacteria were
checked for chemical composition with XPS/ESCA (X-ray
fixed using 0.25 mL of 2.5% glutaraldehyde per well for 15 min
photoelectron spectroscopy/electron spectroscopy for chem-
and, subsequently, air-dried. The specimens were transferred
ical analysis), and no significant pollution was detected [13].
to clean well plates and were stained with 0.25 mL of crystal
The topographies at the microscale were then visualized using
violet for 5 min. Excess stain was rinsed off by placing the
routine scanning electron microscopy (SEM) control. At the
microplate under running tap water, and after this it was
nanoscale, the SEM confirmed that both surface types were
air-dried. The specimens were transferred to clean tubes,
nanosmooth, following the current definition [13, 14]. The
and, in order to resolubilize the dye bound to the adherent
sole difference between these 2 tested implant types was
cells on specimen surfaces, 0.3 mL of ethanol was added
therefore the specific surface microtopography.
per well. The supernatant was transferred to a clean 96-
Atomic force microscopy (AFM, PicoSPM I plus 2100 well microplate, and the absorbance was measured at 570 nm
PicoScan Controller, in contact mode) was used for the using an automated 96-well microplate reader.
surface topography analysis, in contact mode. The AFM
scanned areas of 60 𝜇m × 60 𝜇m of each specimen. The
2.6. Statistical Analysis. The surface characterization was
measured parameters, such as the arithmetic average of all
tested using Student’s 𝑡-test. Two-way analysis of variance
profile point absolute values (Ra), the root-mean-square of
(ANOVA) was used in order to compare the groups of species
all point values (Rq), and the average absolute height values within the same group of implant surface topography and to
of the five highest peaks and the depths of the five deepest verify possible differences among specimen surfaces within
valleys (Rz), were measured for each group. Representative the same species (𝛼 = 0.05).
images of the surfaces of each group of specimens were also
taken by scanning electronic microscopy.
3. Results
2.3. Strains. Streptococcus sanguinis (ATCC 10556), Strepto- 3.1. Surface Characterization of Implants Surfaces Substrata.
coccus salivarius (ATCC 7073), Streptococcus mutans (ATCC Scanning electronic microscopy showed that M group exhib-
25175) and Streptococcus sobrinus (ATCC 33478), and ited only the grid of machining (Figure 1(a)). On the other
BioMed Research International 3

(a) (b)

Figure 1: Scanning electron microphotograph of the implant surface topography: (a) as-machined implant surface and (b) sandblasted acid-
etched surface.

3.5

(𝜇m)
(𝜇m)

1.3
0.0 0.0

y: 60 𝜇m x: 60 𝜇m y: 60 𝜇m x: 60 𝜇m

(a) (b)

Figure 2: Atomic force microscopy (AFM) of the implant surface topography: (a) as-machined implant surface and (b) sandblasted acid-
etched surface.

hand, the SAE exhibited peaks and valleys with diverse Table 1: Mean ± standard deviation of the as-machined (MS) and
irregularities (Figure 1(b)). titanium discs blasted with titanium oxide particles and washed with
The surfaces were characterized by atomic force micro- maleic acid solution (SAE) profilometry.
scopy, which revealed differences between the surfaces (𝑃 <
Implant surface
0.0001). M showed only the machining grids with peaks of topography∗
Ra (𝜇m) Rq (𝜇m) Rz (𝜇m)
1.3 𝜇m and some regions that were almost flat (Figure 2(a)).
As-machined (M) 0.14 ± 0.02 0.16 ± 0.01 1.61 ± 0.10
The SAE exhibited irregular surfaces with peaks of about
6.5 𝜇m (Figure 2(b)). The roughness values are shown in Sandblasted
acid-etched 0.87 ± 0.14 1.12 ± 0.18 5.14 ± 0.69
Table 1.
surface (SAE)
∗
Statistically significant between the implant surface topographies (Student’s
3.2. In Vitro Determination of Microbial Adhesion. The bio- 𝑡-test 𝑃 = 0.0001), M < SAE; Ra: arithmetic average of the absolute values of
film forming ability was evaluated and the means of readings all profile points; Rq: the root-mean-square of the values of all points; Rz: the
are shown in Figure 3. The group mutans streptococci (S. average value of the absolute heights of the five highest peaks and the depths
cricetus, S. mutans, and S. sobrinus) exhibited higher levels of of the five deepest valleys.
biofilm formation and no differences were observed between
surfaces analyzed within each species (𝑃 > 0.05). It was 4. Discussion
observed that although S. cricetus exhibited the highest ability
to form biofilm on SAE, among all species, within this species Titanium has been widely used as a component of dental
this difference was not significant (𝑃 > 0.05) between the implants since the 1970s. More than the improvements in
surfaces analyzed. biomechanical performance, these modifications on implant
S. sanguinis exhibited a similar behavior to form biofilm surfaces lead to other biological responses, such as differences
on both implant surface topographies (Figure 4), and their in the protein adsorption profiles [15, 16], attachment, cell
ability to do so was lower than that of the group mutans proliferation and differentiation, and fibrin adhesion [17]. The
streptococci species. The lowest ability was observed for S. present study presented the biofilm forming ability of 5 oral
salivarius. streptococci species on two different types of surfaces.
4 BioMed Research International

0.40 S. cricetus, S. mutans, S. sobrinus, and S. sanguinis. Among

AB
them, S. salivarius and S. sanguinis exhibited the lowest
0.35
a ab capacity to form biofilm. Two aspects of biofilm forming
0.30 A ability must be pointed out: the specific traits of each species
and surface topographies.
0.25 B b Differences on adhesion to glass surface among mutans
C streptococci group were already observed [27]. The authors
0.20
found that S. rattus adhered less than the other species (S.
C c
0.15 c sobrinus, S. mutans, and S. cricettus) and attributed these
results to different properties of the S. rattus surface like
0.10 negative zeta-potentials. In the present study, three species
0.05 of mutans streptococci group (S. mutans, S. cricetus, and
S. sobrinus) were evaluated and although the raw values
0.0 showed a high capacity of S. mutans to accumulate biofilm
S. sobrinus
S. cricetus

S. sanguinis
S. mutans

S. salivarius
on titanium surface followed by S. cricetus and S. sobrinus,
statistical differences were observed only between S. mutans
and S. sobrinus (𝑃 < 0.05).
Although roughness seems to promote an increase in the
SAE amount of plaque, the biofilm composition did not show
M substantial changes and the establishment of irreversible
Figure 3: Mean ± standard deviation of the amount of adsorbed attachment in the surface irregularities, where microorgan-
dye released after the assay (𝑃 > 0.05; two-way ANOVA). Letters: isms are protected against mechanical shear [10]. Despite this,
differences among biofilm accumulated by each species (𝑃 < 0.05; the results of our study demonstrated that biofilm formation
two-way ANOVA/Tukey test). Different letters indicate groups with does not increase markedly on rougher surfaces.
distinct characteristics. Capital letters compare SAE surfaces, while Oral strains, most of them having high surface free
lower case letters compare M surfaces. energy, might adhere better to hydrophilic substrata [28].
Differences with regard to surface hydrophobicity could be
The blasting process with titanium oxide particle (50– attributed to the acid etching, which could introduce − OH
100 𝜇m) and maleic acid solution etching modified sub- groups on the surface, thus modifying its chemical properties
stantially the surface, which was indeed confirmed by SEM [29]. According to this hypothesis, these treatments can
and AFM. AFM revealed higher density of irregularities originate different surfaces, and, consequently, new patterns
on R surface as well higher peaks. An earlier study [17] of adsorbed substances will be originated, which may offer
detected different profiles of plasma adsorption depending on different profiles of receptors for bacterial colonization.
surface treatment (acid etching only and blasting plus acid Another issue concerns virulence traits of each species
etching processes) and attributed this difference mainly to like tooth colonization mechanisms; S. mutans apparently
the changes in physical properties, since minor alterations in attach by adhesin and glucan mediated mechanisms, whereas
chemical composition were detected. On the other hand, Li S. sobrinus utilize primarily the latter process [30].
et al. [18] found differences on titanium surfaces after applica-
tion of different treatments, including chemical changes such 5. Conclusions
as an oxide layer and surface contamination, and these might
exert some influence on biocompatibility issues. Recently, In conclusion, within the limitations of the study, the present
it has been shown that nanosurfaces could impair bacterial findings showed the following: (a) biofilm formation by
adsorption, suggesting that further studies must be done to oral streptococci might vary according to the species; (b) S.
evaluate the role of implant surface topography on bacterial salivarius and S. sanguinis showed the lowest ability to accu-
colonization [19]. mulate biofilm; (c) group mutans streptococci accumulated
However, there is an unclear debate about the link higher amounts of biofilm; (d) the substratum roughness is
between bacterial contamination and peri-implantitis [20, not the only issue to be considered with regard to bacterial
21]. These papers suggested that peri-implantitis is pathology biofilm formation.
of bone-to-implant interface and that bacterial contamina-
tion is only the associated consequence, not the triggering Conflict of Interests
factor. However, we must point out that, until now, there are
now clear and consistent evidences to follow this idea. The authors declare that there is no conflict of interests
In addition, these surface changes might also influence regarding the publication of this paper.
biofilm formation, since the earlier steps of this process
are related to contact surface extension, surface free energy, Authors’ Contribution
topography, wettability, hydrophobicity, and other surface
traits [17, 18, 22–26]. Jamil Awad Shibli, Claudia Ota-Tsuzuki, and José Augusto
The results of the present study revealed differences Rodrigues were in charge of the elaboration of the study
as regards the biofilm forming ability among S. salivarius, proposal and the financial support of the study, and they
BioMed Research International 5

(a) (b)

Figure 4: Representative scanning electron microscopy (×10,000) in a back scattering mode (BSE) of the Streptococcus sanguinis in (a) as-
machined (M) and (b) sandblasted acid-etched (SAE) surface. Note proliferation of the S. sanguinis in the pitches and notches of the SAE
surface.

participated in the elaboration of the paper. Luciene C. Journal of Oral and Maxillofacial Implants, vol. 4, no. 4, pp. 321–
Figueiredo and Elton G. Zenobio were in charge of the 326, 1989.
statistical analysis, the implant surface characterization, and [7] S. Sardin, J.-J. Morrier, G. Benay, and O. Barsotti, “In vitro
the financial support for the study. Pedro Paulo Cardoso Pita, streptococcal adherence on prosthetic and implant materials.
Tatiane Ferreira Miato, Sergio A. Gehrke, Gabriela Giro, and Interactions with physicochemical surface properties,” Journal
Cristiane Gonçalves were in charge of the saliva collection, of Oral Rehabilitation, vol. 31, no. 2, pp. 140–148, 2004.
laboratory processing, and oral biofilm maintenance and [8] J. A. Shibli, M. C. Martins, L. H. Theodoro, R. F. M. Lotufo, V.
they participated in the elaboration of the paper. Claudia G. Garcia, and E. J. Marcantonio, “Lethal photosensitization in
Ota-Tsuzuki and Alessandra Cassoni were in charge of the microbiological treatment of ligature-induced peri-implantitis:
laboratory processing of the samples and participated in the a preliminary study in dogs,” Journal of Oral Science, vol. 45, no.
data analyses and elaboration of the paper. 1, pp. 17–23, 2003.
[9] B. Größner-Schreiber, M. Griepentrog, I. Haustein et al.,
“Plaque formation on surface modified dental implants—an in
Acknowledgments vitro study,” Clinical Oral Implants Research, vol. 12, no. 6, pp.
543–551, 2001.
Dr. Miato received grant from the University of Guarulhos [10] M. Quirynen, H. C. van der Mei, C. M. Bollen et al., “An in vivo
(fellowship PIBIC-UnG). Implacil De Bortoli, Sao Paulo, study of the influence of the surface roughness of implants on
Brazil, provided the titanium discs. the microbiology of supra- and subgingival plaque,” Journal of
Dental Research, vol. 72, no. 9, pp. 1304–1309, 1993.
References [11] M. Quirynen, H. C. van der Mei, C. M. Bollen et al., “The
influence of surface-free energy on supra- and subgingival
[1] S. S. Socransky and A. D. Haffajee, “Periodontal microbial plaque microbiology. An in vivo study on implants,” Journal of
ecology,” Periodontology 2000, vol. 38, pp. 135–187, 2005. Periodontology, vol. 65, no. 2, pp. 162–167, 1994.
[2] P. E. Kolenbrander, R. J. Palmer Jr., A. H. Rickard, N. S. [12] H. K. Kuramitsu, X. He, R. Lux, M. H. Anderson, and W. Shi,
Jakubovics, N. I. Chalmers, and P. I. Diaz, “Bacterial interactions “Interspecies interactions within oral microbial communities,”
and successions during plaque development,” Periodontology Microbiology and Molecular Biology Reviews, vol. 71, no. 4, pp.
2000, vol. 42, no. 1, pp. 47–79, 2006. 653–670, 2007.
[3] J. A. Shibli, L. Melo, D. S. Ferrari, L. C. Figueiredo, M. Faveri, [13] A. L. Coykendall, “Classification and identification of the
and M. Feres, “Composition of supra- and subgingival biofilm viridans streptococci,” Clinical Microbiology Reviews, vol. 2, no.
of subjects with healthy and diseased implants,” Clinical Oral 3, pp. 315–328, 1989.
Implants Research, vol. 19, no. 10, pp. 975–982, 2008. [14] D. M. D. Ehrenfest, B. S. Kang, G. Sammartino et al., “Guidelines
[4] J. A. Shibli, M. C. Martins, R. F. M. Lotufo, and E. Marcan- for the publication of articles related to implant surfaces and
tonio Jr., “Microbiologic and radiographic analysis of ligature- design from the POSEIDO: a standard for surface characteriza-
induced peri-implantitis with different dental implant surfaces,” tion,” POSEIDO, vol. 1, no. 1, pp. 7–15, 2013.
The International Journal of Oral & Maxillofacial Implants, vol. [15] A. Shibli and D. M. Dohan Ehrenfest, “In dental implant
18, no. 3, pp. 383–390, 2003. surfaces, NanoWar has begun... but NanoQuest is still at stake!,”
[5] J. A. Shibli, T. R. C. Vitussi, R. V. Garcia et al., “Implant POSEIDO, vol. 1, no. 3, pp. 131–140, 2013.
surface analysis and microbiologic evaluation of failed implants [16] J. P. Davidas, “Looking for a new international standard for
retrieved from smokers,” The Journal of Oral Implantology, vol. characterization, classification and identification of surfaces in
33, no. 4, pp. 232–238, 2007. implantable materials: the long march for the evaluation of
[6] G. Nakazato, H. Tsuchiya, M. Sato, and M. Yamauchi, “In dental implant surfaces has just begun,” POSEIDO, vol. 2, no.
vivo plaque formation on implant materials,” The International 1, pp. 1–5, 2014.
6 BioMed Research International

[17] M. N. Sela, L. Badihi, G. Rosen, D. Steinberg, and D. Kohavi,

“Adsorption of human plasma proteins to modified titanium
surfaces,” Clinical Oral Implants Research, vol. 18, no. 5, pp. 630–
638, 2007.
[18] D. Li, S. J. Ferguson, T. Beutler et al., “Biomechanical com-
parison of the sandblasted and acid-etched and the machined
and acid-etched titanium surface for dental implants,” Journal of
Biomedical Materials Research, vol. 60, no. 2, pp. 325–332, 2002.
[19] E. Elizabeth, G. Baranwal, A. G. Krishnan, D. Menon, and M.
Nair, “ZnO nanoparticle incorporated nanostructured metallic
titanium for increased mesenchymal stem cell response and
antibacterial activity,” Nanotechnology, vol. 25, no. 11, Article ID
115101, 2014.
[20] R. Trindade, T. Albrektsson, P. Tengvall, and A. Wennerberg,
“Foreign body reaction to biomaterials: on mechanisms for
buildup and breakdown of osseointegration,” Clinical Implant
Dentistry and Related Research, 2014.
[21] T. Albrektsson, C. Dahlin, T. Jemt, L. Sennerby, A. Turri, and A.
Wennerberg, “Is marginal bone loss around oral implants the
result of a provoked foreign body reaction?” Clinical Implant
Dentistry and Related Research, vol. 16, no. 2, pp. 155–165, 2014.
[22] E. M. C. X. Lima, H. Koo, A. M. Vacca Smith, P. L. Rosalen,
and A. A. Del Bel Cury, “Adsorption of salivary and serum
proteins, and bacterial adherence on titanium and zirconia
ceramic surfaces,” Clinical Oral Implants Research, vol. 19, no.
8, pp. 780–785, 2008.
[23] K. Mustafa, A. Wennerberg, J. Wroblewski, K. Hultenby, B. S.
Lopez, and K. Arvidson, “Determining optimal surface rough-
ness of TiO2 blasted titanium implant material for attachment,
proliferation and differentiation of cells derived from human
mandibular alveolar bone,” Clinical Oral Implants Research, vol.
12, no. 5, pp. 515–525, 2001.
[24] L. le Guéhennec, A. Soueidan, P. Layrolle, and Y. Amouriq,
“Surface treatments of titanium dental implants for rapid
osseointegration,” Dental Materials, vol. 23, no. 7, pp. 844–854,
2007.
[25] W. Teughels, N. Van Assche, I. Sliepen, and M. Quirynen,
“Effect of material characteristics and/or surface topography on
biofilm development,” Clinical Oral Implants Research, vol. 17,
no. 2, pp. 68–81, 2006.
[26] A. Leonhardt, J. Olsson, and G. Dahlén, “Bacterial colonization
on titanium, hydroxyapatite, and amalgam surfaces in vivo,”
Journal of Dental Research, vol. 74, no. 9, pp. 1607–1612, 1995.
[27] H. J. Busscher and H. C. van der Mei, “Physico-chemical
interactions in initial microbial adhesion and relevance for
biofilm formation,” Advances in Dental Research, vol. 11, no. 1,
pp. 24–32, 1997.
[28] M. M. Fürst, G. E. Salvi, N. P. Lang, and G. R. Persson, “Bacterial
colonization immediately after installation on oral titanium
implants,” Clinical Oral Implants Research, vol. 18, no. 4, pp. 501–
508, 2007.
[29] A. H. Weerkamp, H. M. Uyen, and H. J. Busscher, “Effect of zeta
potential and surface energy on bacterial adhesion to uncoated
and saliva-coated human enamel and dentin,” Journal of Dental
Research, vol. 67, no. 12, pp. 1483–1487, 1988.
[30] H. M. W. Uyen, J. M. Schakenraad, J. Sjollema et al., “Amount
and surface structure of albumin adsorbed to solid substrata
with different wettabilities in a parallel plate flow cell,” Journal
of Biomedical Materials Research, vol. 24, no. 12, pp. 1599–1614,
1990.
Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 369273, 6 pages
http://dx.doi.org/10.1155/2015/369273

Clinical Study
Influence of Leukocyte- and Platelet-Rich Fibrin (L-PRF) in the
Healing of Simple Postextraction Sockets: A Split-Mouth Study

Gaetano Marenzi,1 Francesco Riccitiello,2 Mariano Tia,3

Alessandro di Lauro,1 and Gilberto Sammartino1
1
Division of Oral Surgery and Implantology, Department of Neurosciences, Reproductive and Odontostomatological Sciences,
University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy
2
Department of Neurosciences, Reproductive and Odontostomatological Sciences, University of Naples “Federico II”,
Via Pansini 5, 80131 Naples, Italy
3
Private Practice, Via Austria 6, Sant’Antimo, Naples, Italy

Correspondence should be addressed to Gaetano Marenzi; [email protected]

Received 12 August 2014; Revised 11 November 2014; Accepted 11 November 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2015 Gaetano Marenzi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

The aim of this study was to evaluate the effects of leukocyte- and platelet-rich fibrin (L-PRF) on the pain and soft tissue healing
after tooth extractions. Twenty-six patients (9 males and 17 females) were treated with multiple extractions (2 to 8), with a total
of 108 extractions. This was an exploratory single blinded randomized clinical trial with a split-mouth design. The pain after the
surgery was assessed in each patient by the VAS scale (1 to 10) at intervals of 24-48-72-96 hours. The soft tissue healing was clinically
evaluated at 3, 7, 14, and 21 days after surgery by the same examiner surgeon, using the modified Healing Index (4 to 12). The mean
value of postextraction pain was 3.2 ± 0.3 in the experimental sides and 4.1 ± 0.1 in the control sides. After 7 days from the extractions,
the values of modified Healing Index in the experimental and control groups were, respectively, 4.8 ± 0.6 and 5.1 ± 0.9. The use of
L-PRF in postextraction sockets filling can be proposed as a useful procedure in order to manage the postoperative pain and to
promote the soft tissue healing process, reducing the early adverse effects of the inflammation.

1. Introduction platelet-rich plasma (L-PRP), pure platelet-rich fibrin (P-

PRF), and leukocyte- and platelet-rich fibrin (L-PRF) [11].
Many studies revealed that platelet concentrates for surgical Each family of products has different aspect, biological con-
use can be used as efficient adjuvants for tissue repair [1– tent, and potential application [12].
5]. The growth factors (particularly platelet-derived growth The PRPs were already tested in many oral surgery
factors (PDGF), transforming growth factors (TGF-𝛽), and applications, with mixed results depending on the kind of
vascular endothelial growth factors (VEGF)) and the other preparations [13–16]. Numerous protocols have attempted to
molecules (fibrinogen, fibronectin, and vitronectin) con- optimize the preparation of the autologous factors, using var-
tained in platelets (𝛼-granules) give to these products the ious performances standards and centrifugation parameters
ability to modulate many phases of the healing process like [17, 18]. Several authors demonstrated the effectiveness of
the hemostasis and the neoangiogenesis [6]. The clinical some PRP types during tooth extractions to stimulate soft
results of these products are interesting but remain quite tissue healing and wound control [19, 20] and in prevention of
mixed and controversial in the literature, depending on the postoperative bleeding in anticoagulated patients undergoing
kind of preparation [7–10]. Platelet concentrates are classified oral surgery. However, these PRP techniques remain quite
into 4 main families depending on their leukocyte and fibrin complex and expensive on a daily use basis, and their use may
content: pure platelet-rich plasma (P-PRP), leukocyte- and not be justified for daily oral surgery applications [13, 14].
2 BioMed Research International

On the other hand, L-PRF represents a more recent gen-

eration of platelet concentrates. The development of L-PRF
is very significant in oral and maxillofacial surgery, with many
validated applications in periodontal surgery [13, 21] and
implant dentistry [14–22]. L-PRF is easy and inexpensive to
prepare for frequent use in private practice, and it exists in
the form of L-PRF clots or membranes (after compression).
The membrane releases a significant quantity of autologous
growth factors (particularly PDGF-AB, TGF𝛽, and VEGF)
[23], cytokines, and healing proteins (fibronectin, etc.) during
more than 7 days in vitro [24], while other platelet gels
dissolve in vitro in 3 days [12]. In another study, when com- Figure 1: Presurgery clinical occlusal view. For orthodontic motive,
this patient needed the bilateral extraction of the upper first
pared with a procedure for platelet-rich plasma (PRP), L-PRF
premolars.
released more than 15-fold VEGF and more than 2-fold
TGF𝛽1 [25]. According to the literature, L-PRF was a useful
tool in postextraction hemostasis control [26] and in preven-
tion of hemorrhagic complications in cardiopathic patients
[27].
The aim of this study is to evaluate the effectiveness of L-
PRF to improve the soft tissues healing and to reduce pain
after tooth extractions.

2. Materials and Methods

2.1. Study Population. From January 2012 to July 2013 at the
Unit of Oral Surgery and Implantology of the University
of Naples “Federico II,” 26 healthy patients were selected, Figure 2: The postextraction sockets.
including 9 males and 17 females with a mean age of 53 ± 4
years. The selected patients were nonsmokers or light smok-
ers (<5/day); they did not have systemic diseases that could and to give adequate support to the L-PRF filling (Figures
interfere with the healing process (such as diabetes, liver dis- 1 and 2). All extraction sites were simple with alveolar
ease, heart disease, or immune-disorders) or diseases of the walls preserved. All alveolar sockets were sutured with a
oral mucosa. The study was designed as a prospective split- 3/0 Vicryl (Ethicon/Johnson & Johnson, Somerville, NJ,
mouth trial on patients who needed bilateral paired dental USA). Each patient also served as the control (split-mouth
extractions; on the side chosen to be the study side, the sock- design): a socket was treated with L-PRF application (study
ets were filled with L-PRF, whereas on the other side (control), socket) whereas the other (control socket) had to undergo
they were allowed to undergo natural healing. Test and natural healing by clot formation without socket filling
control sites were chosen with coin toss randomization. This (Figure 3). The number of paired extractions per patient
cross-sectional split-mouth research study was conducted in ranged from 2 to 4 for a total of 108 extractions. Indica-
accordance with the requirements of Helsinki Declaration of tion for tooth extraction included root or crow fractures,
1975 as revised in 2008. Patients were verbally informed about residual roots, no restorable caries, periapical granuloma,
the sample to be taken and gave their written consent. The and orthodontic reasons. Patients showing anatomic and
patients who did not sign the agreement and also the patients pathologic conditions not comparable between the study and
with poor oral hygiene, patients with local infections of the control sites were excluded as study subject. Antibiotic pro-
soft tissues, patients undergoing bisphosphonates therapy, phylaxis was undertaken (amoxicillin 875 mg and clavulanic
patients irradiated to the jaws in the past, and patients with acid 125 mg) starting 2 days before surgery up to 3 days
psychiatric illness or pregnant patients were excluded from after it.
the study.
2.3. L-PRF Preparation Protocol. The L-PRF was prepared
2.2. Surgical Procedure. An alveolar nerve block infiltration through a single centrifugation of blood according to the pro-
was administrated with local or regional anesthesia, depend- tocol of Dohan Ehrenfest et al. (now marketed as Intra-Spin
ing on the dental arch, using 2% mepivacaine. Mepivacaine L-PRF kit, Intra-Lock, Boca-Raton, FL, USA) for a period
does not contain epinephrine, so it was used to prevent of 12 minutes at 2700 rpm. Blood was taken in 9 mL tubes,
restriction of the blood supply. To prevent interference 30 minutes before the surgery, immediately centrifuged, and
with the healing process, no intraligamentous or intrapap- used for the filing of the experimental sites. The total amount
illary infiltration was made. The teeth were extracted in of blood collected (from 18 mL to 54 mL) was related to the
a nontraumatic manner without elevation of full-thickness number of tooth extractions, in order to obtain the complete
flaps and preserving the buccal and lingual walls of the filling of the sockets with the L-PRF. After centrifugation,
alveolar sockets in order to minimize the possible trauma each L-PRF clot was separated from the portion of red blood
BioMed Research International 3

Table 1 Table 2
Number of Number of paired Tooth extracted Healing Index 3 days 7 days 14 days 21 days
patients extractions per patient L-PRF 4.8 ± 0.6 4.5 ± 0.5 4.2 ± 0.2 4.1 ± 0.1
10 1 Premolar CTRL 5.1 ± 0.9 4.9 ± 0.3 4.3 ± 0.3 4.2 ± 0.2
7 2 Canine/premolar/molar 𝑃 0.197 0.05 0.01 0.0002
6 3 Canine/premolar/molar L-PRF = study site; CTRL = control site.
3 4 Canine/premolar/molar

Figure 4: Clinical occlusal view 3 days after surgery. In the study site,
the epithelialization process was more advanced than in the control
Figure 3: The upper right socket (study site) was filled with L- site. In the study site, the inflammatory reaction was reduced.
PRF, while the upper left socket (control site) had to follow natural
healing. Both sites were sutured.
severely impaired healing. The Wilcoxon signed-rank test for
comparison of 2 correlated samples matched pairs with the
cells (red thrombus), obtaining a fibrin clot with a red small level of significance predetermined at 0.05 was used.
portion in order to include the “buffy” coat richer in large
leucocytes [24]. The L-PRF clot was condensed and modeled 3. Results
on a sterile surgical plate before the application in the sockets
[24]. All patients completed the study. No cases of bleeding,
L-PRF was used within 60 minutes after the preparation. infection, alveolar osteitis, or other surgical complications
It was accurately positioned in the extraction sites and were reported.
stabilized with a resorbable suture. In the control sites, the Regarding the postextraction pain, patients enrolled in
same suture was used (Figure 3). All patients were advised to the study reported a mean value of the study sites of 3.2 ± 0.3,
follow soft and liquid diet, avoiding hot food in the following which is lower (𝑃 < 0.0001) than the mean value of the
hours. In all cases, the sutures were removed after one week. control sites (4.5 ± 0.7), with a statistical difference average of
Table 1 reports the number and the type of paired extractions 0.9 ± 0.3. The VAS score was nearly equal for the 2 sides after
per patient. 4 days (decreasing to 0).
Results concerning the healing of the socket are reported
2.4. Study Variables and Statistical Methods. The predictor in Table 2. Comparisons between values relative to the study
variable was the treatment group status: L-PRF versus control and control sides showed better healing and faster socket
socket. The outcome variables of interest were as follows: closure for the side treated with L-PRF, with differences
pain, postsurgical complications to soft and hard tissues, and statistically significant at days 3 and 7 (Figures 4 and 5).
the Healing Index modified.
A 10-point visual analog scale (VAS) with a score of 0 that 4. Discussion
equals “no pain” and a score of 10 that equals “very severe
pain” was used by the same patient to assess the postoperative This study was designed to test the efficacy of L-PRF in foster-
pain at 24, 48, and 72 hours. Between groups comparisons for ing socket healing after tooth extractions. The 26 split-mouth
VAS outcomes were carried out by means of univariate anal- case control extractions that constituted our study were
ysis of variance, considering the group (i.e., PRF versus CTR) statistically enough to prove the ability of L-PRF to improve
and the recording time point as factors and VAS score as the early healing phases (hemostasis and epithelial closure),
dependent variable. reducing the inflammatory process and the risk of infection.
The quality of the socket soft tissue healing was clin- The reported results of the experimental sites showed, in the
ically evaluated at 3, 7, 14, and 21 days after surgery by first 7 days after the tooth extractions, a fast evolution of the
an examiner surgeon, using the Healing Index modified healing and a positive effect on pain. After a week, minor
[28] which involved 3 scoring levels for each of the four differences between the two groups are reported (Figures
parameters considered: bleeding, suppuration, tissue color, 6 and 7). These effects could be related to the biochemical
and consistency of the healing tissue. The scoring scale ranged and structural features of the L-PRF [29], which collects a
from 4, corresponding to excellent healing, to 12, indicating large quantity of leukocytes (about 60% of the initial blood
4 BioMed Research International

Figure 5: Clinical occlusal views 7 days after surgery. The sutures Figure 7: At 21 days after surgery, both postextraction healing sites
were removed. Both postextraction socket cavities presented a were completely closed with the soft tissues.
decreased volume and appeared epithelialized.

reach a very clean result, but it does not reflect the real
strength and advantages of L-PRF. This material is particu-
larly useful and efficient in complex situations, when some
walls are destroyed and the bone regeneration is difficult, but
an accurate split-mouth study with this kind of cases is virtu-
ally impossible to standardize. It is however the needed next
step of evaluation and validation of the use of L-PRF during
tooth extractions.

5. Conclusion
Even if the selected samples are limited, the reported results
Figure 6: Clinical follow-up at 14 days after surgery. Both postex- suggested that the use of L-PRF in postextraction sockets
traction sockets were completely closed with the soft tissues. filling is an efficient and useful procedure in order to manage
the postoperative pain and to enhance the alveolar soft tissue
healing process, especially in the first days after the extrac-
tions, reducing the early adverse effects of the inflammation.
harvest) and platelets embedded in a fibrin matrix [30, 31]. This study represents a preliminary clinical trial, which could
The fibrin architecture of L-PRF, constituted by connected be used as baseline for further histologic studies.
trimolecular junctions (or equatorial), due to a slow poly-
merization of the platelet concentrate and due to the absence
of heterologous thrombin, induces a flexible fibrin network, Conflict of Interests
able to promote the gradual release of growth factors and
The authors have no conflict of interests to declare regarding
leukocytes migration. The fibrin membrane promotes the
the devices used for this study. The current research was not
mechanical protection of the surgical site and, biologically,
influenced by any secondary interests, such as financial gain.
it interacts with the physiological mechanisms of healing
favoring the angiogenesis [13, 14]. The fibrin induces the
expression of 𝛼v-𝛽3 integrin by endothelial cells, allowing References
the links with structural proteins, such as fibronectin and
[1] T. Bielecki and D. M. Dohan Ehrenfest, “Leukocyte- and
vitronectin, supporting the process of formation of capillaries platelet-rich Plasma (L-PRP)/fibrin (L-PRF) in medicine—past,
[12]. In relation to the previous properties, the fibrin also present, future,” Current Pharmaceutical Biotechnology, vol. 13,
allows the association of some growth factors, such as FGFb no. 7, pp. 1–2, 2012.
(fibroblast growth factor basic) and PDGF (platelet-derived
growth factor) involved in the angiogenic process and useful [2] R. Gruber, F. Varga, M. B. Fisher, and G. Watzek, “Platelet stim-
as chemotactic factors, favoring diapedesis of white blood ulate proliferation of bone cells:involvement of platelet-derived
growth factor, microparticles and membranes,” Clinical Oral
cells [12]. The immunological properties of the L-PRF, result-
Implants Research, vol. 13, pp. 529–535, 2002.
ing from its content in leukocytes, could be useful to prevent
the surgical site infections, such as postextraction alveolitis, [3] N. E. Carlson and J. R. B. Roach, “Platelet-rich plasma: clinical
with a consequent reduction of the inflammation symptoms. application in dentistry,” The Journal of the American Dental
The presence of leukocytes is a very important parameter to Association, vol. 133, pp. 1383–1386, 2002.
stimulate healing and wound control [32]. [4] A. R. Sanchez, P. J. Sheridan, and L. I. Kupp, “Is platelet-rich
The main limitation of this exploratory study was that the plasma the perfect enhancement factor? A current review,” The
extraction sites were voluntarily very simple, with all alveolar International Journal of Oral & Maxillofacial Implants, vol. 18,
walls preserved. It allowed standardizing the study easily to pp. 93–103, 2003.
BioMed Research International 5

[5] E. Anitua, I. Andia, B. Ardanza et al., “Autologous platelets as a [18] P. Borzini, V. Balbo, and L. Mazzucco, “Platelet concentrates for
sourge of proteins for healing and tissue regeneration,” Throm- topical use: bedside device and blood transfusion technology.
bosis and Haemostasis, vol. 91, pp. 4–15, 2004. Quality and versatility,” Current Pharmaceutical Biotechnology,
[6] R. E. Marx, E. R. Carlson, R. M. Eichstaedt, S. R. Schimmele, J. E. vol. 13, no. 7, pp. 1138–1144, 2012.
Strauss, and K. R. Georgeff, “Platelet-rich plasma: growth factor [19] C. Rivera, F. Monsalve, J. Salas, A. Morán, and I. Suazo, “Platelet-
enhancement for bone grafts,” Oral Surgery, Oral Medicine, Oral rich plasma, plasma rich in growth factors and simvastatin in
Pathology, Oral Radiology, and Endodontics, vol. 85, no. 6, pp. the regeneration and repair of alveolar bone,” Experimental and
638–646, 1998. Therapeutic Medicine, vol. 6, no. 6, pp. 1543–1549, 2013.
[7] P. A. M. Everts, M. M. Hoogbergen, T. A. Weber, R. J. J. Devilee, [20] G. Sammartino, M. Tia, T. Bucci, and H. L. Wang, “Prevention
G. van Monftort, and I. H. J. T. de Hingh, “Is the use of autol- of mandibular third molar extraction-associated periodontal
ogous platelet-rich plasma gels in gynecologic, cardiac, and defects: a comparative study,” Journal of Periodontology, vol. 80,
general, reconstructive surgery beneficial?” Current Pharma- no. 3, pp. 389–396, 2009.
ceutical Biotechnology, vol. 13, no. 7, pp. 1163–1172, 2012. [21] Y. C. Chang and J. H. Zhao, “Effect of platelet-rich fibrin on
[8] T. Yuan, S.-C. Guo, P. Han, C. Q. Zhang, and B. F. Zeng, human periodontal ligament fibroblasts and application forpe-
“Applications of leukocyte- and platelet-rich plasma (L-PRP) in riodontal infrabony defects,” Australian Dental Journal, vol. 56,
trauma surgery,” Current Pharmaceutical Biotechnology, vol. 13, pp. 365–371, 2011.
no. 7, pp. 1173–1184, 2012. [22] R. Toeroek and D. M. Dohan Ehrenfest, “The concept of
[9] M. del Fabbro, M. Bortolin, and S. Tascheri, “Is autologous Screw-Guided Bone Regeneration (S-GBR). Part 3: fast screw-
platelet concentrate beneficial for post-extraction socket heal- guided bone regeneration (FS-GBR) in the severely resorbed
ing? A systematic review,” International Journal of Oral and preimplant posterior mandible using allograft and leukocyte-
Maxillofacial Surgery, vol. 40, pp. 891–900, 2011. and platelet-rich fibrin (L-PRF): a 4-year follow-up,” POSEIDO,
vol. 1, no. 2, pp. 93–100, 2013.
[10] M. Esposito, M. G. Grusovin, J. Rees et al., “Effectiveness
of sinus lift procedures for dental implant rehabilitation: a [23] M. A. Zumstein, S. Berger, M. Schober et al., “Leukocyte- and
Cochrane systematic review,” European Journal of Oral Implan- platelet-rich fibrin (L-PRF) for long-term delivery of growth
tology, vol. 3, pp. 7–26, 2010. factor in rotator cuff repair: review, preliminary results and
future directions,” Current Pharmaceutical Biotechnology, vol.
[11] D. M. Dohan Ehrenfest, G. Sammartino, J. A. Shibli, H. L. Wang,
13, no. 7, pp. 1196–1206, 2012.
D. R. Zou, and J. P. Bernard, “Guidelines for the publication of
articles related to platelet concentrates (Platelet-Rich Plasma— [24] D. M. Dohan Ehrenfest, G. M. de Peppo, P. Doglioli, and
PRP, or Platelet-Rich Fibrin—PRF): the international classifica- G. Sammartino, “Slow release of growth factors and throm-
tion of the POSEIDO,” POSEIDO, vol. 1, no. 1, pp. 17–27, 2013. bospondin-1 in Choukroun’s platelet-rich fibrin (PRF): a gold
standard to achieve for all surgical platelet concentrates tech-
[12] D. M. Dohan Ehrenfest, T. Bielecki, R. Jimbo et al., “Do the
nologies,” Growth Factors, vol. 27, no. 1, pp. 63–69, 2009.
fibrin architecture and leukocyte content influence the growth
factor release of platelet concentrates? An evidence-based [25] F. Passaretti, M. Tia, V. D’esposito et al., “Growth-promoting
answer comparing a pure platelet-rich plasm (P-PRP) gel and action and growth factor release by different platelet deriva-
a leukocyte- and platelet-rich fibrin (L-PRF),” Current Pharma- tives,” Platelets, vol. 25, no. 4, pp. 252–256, 2014.
ceutical Biotechnology, vol. 13, no. 7, pp. 1145–1152, 2012. [26] D. M. Dohan Ehrenfest and L. Vazquez, “Pulling out, extraction
[13] M. del Corso, A. Vervelle, A. Simonpieri et al., “Current knowl- or avulsion?” Implant Dentistry, vol. 17, no. 1, p. 4, 2008.
edge and perspectives for the use of platelet-rich plasma (PRP) [27] G. Sammartino, D. M. Dohan Ehrenfest, F. Carile, M. Tia, and
and platelet-rich fibrin (PRF) in oral and maxillofacial surgery P. Bucci, “Prevention of hemorrhagic complications after dental
part 1: periodontal and dentoalveolar surgery,” Current Pharma- extractions into open heart surgery patients under anticoagu-
ceutical Biotechnology, vol. 13, no. 7, pp. 1207–1230, 2012. lant therapy: the use of leukocyte- and platelet-rich fibrin,” The
[14] A. Simonpieri, M. del Corso, A. Vervelle et al., “Current knowl- Journal of Oral Implantology, vol. 37, no. 6, pp. 681–690, 2011.
edge and perspectives for the use of platelet-rich plasma (PRP) [28] M. Mozzati, G. Gallesio, S. di Romana, L. Bergamasco, and R.
and platelet-rich fibrin (PRF) in oral and maxillofacial surgery. Pol, “Efficacy of plasma-rich growth factor in the healing of
Part 2. Bone graft, implant and reconstructive surgery,” Current postextraction sockets in patients affected by insulin-dependent
Pharmaceutical Biotechnology, vol. 13, no. 7, pp. 1231–1256, 2012. diabetes mellitus,” Journal of Oral and Maxillofacial Surgery, vol.
[15] N. Geurs, A. Ntounis, P. Vassilopoulos, U. van der Velden, B. G. 72, no. 3, pp. 456–462, 2014.
Loos, and M. Reddy, “Using growth factors in human extrac- [29] D. M. Dohan Ehrenfest, M. del Corso, F. Inchingolo, and J. B.
tion sockets: a histologic and histomorphometric evaluation Charrier, “Selecting a relevant in vitro cell model for testing
of short-term healing,” The International Journal of Oral & and comparing the effects of a Choukroun’s platelet-rich fibrin
Maxillofacial Implants, vol. 29, no. 2, pp. 485–496, 2014. (PRF) membrane and a platelet-rich plasma (PRP) gel: tricks
[16] P. Kaur and A. Maria, “Efficacy of platelet rich plasma and and traps,” Oral Surgery, Oral Medicine, Oral Pathology, Oral
hydroxyapatite crystals in bone regeneration after surgical Radiology and Endodontology, vol. 110, no. 4, pp. 409–413, 2010.
removal of mandibular third molars,” Journal of Maxillofacial [30] J. Choukroun, A. Diss, A. Simonpieri et al., “Platelet-rich fibrin
and Oral Surgery, vol. 12, no. 1, pp. 51–59, 2013. (PRF): a second-generation platelet concentrate. Part IV: clini-
[17] G. Weibrich, W. K. G. Kleis, and G. Hafner, “Growth factor levels cal effects on tissue healing,” Oral Surgery, Oral Medicine, Oral
in the platelet-rich plasma produced by 2 different methods: Pathology, Oral Radiology, and Endodontics, vol. 101, pp. 56–60,
curasan-type PRP kit versus PCCS PRP system,” International 2006.
Journal of Oral & Maxillofacial Implants, vol. 17, no. 2, pp. 184– [31] Y.-H. Kang, S. H. Jeon, and J.-Y. Park, “Platelet-rich fibrin
190, 2002. is a bioscaffold and reservoir of growth factors for tissue
6 BioMed Research International

regeneration,” Tissue Engineering Part A, vol. 17, pp. 349–359,

2011.
[32] T. Bielecki, D. M. Dohan Ehrenfest, P. A. Everts, and A.
Wiczkowski, “The role of leukocytes from L-PRP/L-PRF in
wound healing and immune defense: new perspectives,” Cur-
rent Pharmaceutical Biotechnology, vol. 13, no. 7, pp. 1153–1162,
2012.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 492725, 9 pages
http://dx.doi.org/10.1155/2014/492725

Clinical Study
Clinical Evaluation of the Regenerative Potential of EMD and
NanoHA in Periodontal Infrabony Defects: A 2-Year Follow-Up

Andrea Pilloni,1 Matteo Saccucci,1 Gabriele Di Carlo,1

Blerina Zeza,1 Marco Ambrosca,1 Michele Paolantonio,2 Gilberto Sammartino,3
Claudio Mongardini,1 and Antonella Polimeni1
1
Department of Oral and Maxillofacial Science, Sapienza University of Rome, Via Caserta 6, 00161 Rome, Italy
2
Department of Medical, Oral and Biotechnological Sciences, University “G. d’Annunzio”, Chieti, Italy
3
Department of Oral Surgery, Faculty of Medicine, University Federico II, Naples, Italy

Correspondence should be addressed to Matteo Saccucci; [email protected]

Received 4 July 2014; Revised 24 July 2014; Accepted 14 August 2014; Published 8 September 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Andrea Pilloni et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction. The aim of this retrospective study was to compare the clinical efficacy of four different surgical techniques in
promoting periodontal regeneration in patients with infrabony defects: open flap debridement, application of enamel matrix
derivatives (EMD), nanohydroxyapatite (nanoHA) application, and combined nanoHA and EMD application. Probing attachment
level (PAL), pocket depth (PD), and position of gingival margin at completion of therapy (REC) were measured. Materials and
Methods. Data were collected from 64 healthy patients (34 women and 30 men, mean age 37,7 years). Clinical indices were measured
by a calibrated examiner at baseline and at 12, 18, and 24 months. The values obtained for each treatment were compared using
nonparametric tests. Results. All treatments resulted in a tendency toward PD reduction over time, with improvements in REC and
PAL. The differences in PD, REC, and PAL values at baseline compared with values after 12, 18, and 24 months were statistically
significant for all treatments. Statistically significant differences in PAL and PD were detected between nanoHA and nanoHA +
EMD at 12, 18, and 24 months. Conclusion. In this study, EMD and nanoHA used together in patients with infrabony periodontal
lesions had better clinical efficacy than nanoHA alone, EMD alone, or open flap debridement.

1. Introduction sources [3, 4]. The ideal material for periodontal wound
healing and regeneration would combine biologics with
In the past three decades periodontal regenerative treatment an easy-to-use, moldable, space-providing, biocompatible,
has received increasing attention as an alternative to tooth bioadhesive, porous, and biodegradable matrix for local
extraction in patients with periodontal disease. Implant inser- applications [2]. The simultaneous use of enamel matrix
tion, although highly predictable, has been shown to be less derivative (EMD) and nanohydroxyapatite (nanoHA) seems
predictable than saving a periodontally compromised tooth to fulfill the aforementioned criteria for an ideal combination.
with regenerative therapy [1]. Given the proper conditions The in vitro combination has shown promising results, with
for optimal wound healing (wound stability, adequate space, each material stimulating periodontal fibroblasts differently,
and healing by primary intention), the periodontal tissues enhancing the potential of each [5]. Amelogenins, present
are capable of significant regeneration [2]. Unfortunately, in EMD, are extracellular matrix proteins that induce the
systemic and local factors can interfere with these conditions, formation of acellular cementum when absorbed on the
making periodontal regeneration difficult without the use of root surface [6] and that stimulate the proliferation and
grafting biomaterials, biologics, and devices for periodontal differentiation of periodontal fibroblasts and osteoblasts [7,
regeneration [2]. Biomaterials in periodontal regeneration, 8]. The role of amelogenins in periodontal regeneration is
as for bone reconstruction around implants, are of different primarily related to regeneration of the periodontal ligament
2 BioMed Research International

and cementum. In contrast, nanoHA is an alloplastic material PD values at baseline by age

8
chemically similar to the inorganic component of bone
matrix and is known for its osteoinductive and osteocon-

Number of patients
ductive properties in alveolar bone regeneration [9]. The 6
clinical relevance of EMD is supported by its long-term
outcome stability in studies with 10–15 years of follow-up [10]. 4
In contrast, the literature on nanoHA remains scarce and
contradictory. In 2008, Kasaj et al. [11] reported significant 2
clinical improvement in patients treated with nanoHA com-
pared with open debridement. However, in 2013, Horváth et
0
al. [12] could not confirm those findings. In 2014, Al Machot 5 6 7 8 9 10 11 12
et al. [13] reported that EMD and nanoHA powder performed PD values
similarly and resulted in significant bone filling and clinical
improvement. The aim of the present study was to compare 29−34 39−42
the clinical efficacy of EMD and nanoHA applied individually 35−38 43−50
and in combination, using open flap debridement (OFD) as a
Figure 1
control.
PD values at baseline by gender
2. Materials and Methods
10
2.1. Study Design and Population. This retrospective study
was conducted in the Department of Oral and Maxillofacial Number of patients 8
Science, Sapienza University of Rome, Italy. Data were col-
lected between 2009 and 2012. Sixty-four generally healthy
6
patients (34 women and 30 men; mean age 37,7 years)
were screened for inclusion. Study inclusion criteria were as
4
follows:
(i) age over 18 years; 2
(ii) systemic health: lack of acute or chronic condition
that would contraindicate oral surgery; 0
5 6 7 8 9 10 11 12
(iii) bone defect characteristics: a single 1-, 2-, or 3-wall PD values
infrabony defect more than 3 mm deep on radio-
graphs, with pocket depth (PD) ≥5 mm; F
(iv) tooth condition: periodontally, endodontically, and M
prosthetically healthy tooth. Figure 2
Patients were excluded for the following reasons:
(i) pregnancy or lactation;
2.2. Treatments. Patients were randomly allocated to one of
(ii) taking medications that could interfere with the the following surgical treatment groups:
healing of periodontal tissues;
(i) open flap debridement;
(iii) furcation involvement;
(ii) EMD application (Emdogain Straumann, Basel,
(iv) overhanging restorations;
Switzerland);
(v) teeth with grade 2 or higher mobility;
(iii) nanoHA (NeoActive Ghimas, Casalecchio di Reno,
(vi) teeth with endodontic lesions. Italy);
Baseline measurements of probing attachment level (PAL), (iv) Sandwich technique combining nanoHA and EMD.
position of gingival margin at completion of therapy (REC),
and PD, according to gender and age, are shown in Figures 1, All patients underwent the same presurgical and surgical
2, 3, 4, 5, and 6. Chronic periodontitis was diagnosed in all procedure, differing only in the material used for bone
patients, because the location of defects and number of teeth regeneration.
affected did not meet the criteria of aggressive periodontitis,
even in those younger than 30 years of age. Each patient 2.3. Presurgical Treatment. All patients underwent phase I
participating in this study signed a consent form approved therapy with revaluation within 3 months. If the full-mouth
by the Ethical Committee of the Faculty of Medicine, G. plaque score and full-mouth bleeding score were under 15% at
D’Annunzio University, Chieti, Italy. The study protocol was recheck, the patient entered the surgical protocol. Otherwise,
performed in accordance with the Declaration of Helsinki of an additional motivation and professional cleaning phase was
1975, revised in Tokyo in 2004. performed.
BioMed Research International 3

REC values at baseline by age PAL values at baseline by gender

14
14
12 12
Number of patients

10 10

Number of patients
8
8
6
4 6

2 4
0
−1 0 1 2
REC values
0
29−34 39−42 5 6 7 8 9 10 11 12
35−38 43−50 PAL values

Figure 3 F
M

REC values at baseline by gender Figure 6

15 2.4. Periodontal Surgery. After local anesthesia, intrasulcular

incisions were made, in conjunction with vertical releasing
Number of patients

10
incisions if needed for bone defect exposure. A full-thickness
flap was then prepared, the bone defect exposed, and granula-
tion tissue removed with manual and ultrasonic root planing
5 instruments. The area of the infrabony defect was rinsed
with saline, dried, and treated according to treatment group
allocation:
0
−1 0 1
(i) chemical root conditioning (EDTA, 24%) before
REC values application of EMD to the root surface;
F (ii) application of adequate nanoHA powder to fill the
M defect and maintain root surface area sufficient for its
biological width to be reestablished;
Figure 4
(iii) chemical root conditioning (EDTA, 24%), EMD
application, bone defect filling with nanoHA, and
PAL values at baseline by age layer of EMD over of the bone substitute, using the
sandwich technique;
10
(iv) open flap debridement.
8 Gingival flaps were closed with 5–0 nylon suture in a simple
Number of patients

interrupted pattern (Figures 7, 8, 9(a), 9(b), 10(a), 10(b), 11(a),

6 and 10(b)).

4 2.5. Postsurgical Treatment. Patients received a prescription

for amoxicillin, 1 g every 12 h for 5 days, and for ibuprofen,
600 mg every 12 h for 3 days. The use of synthetic ice in
2 5-minute intervals for the first 30 minutes was prescribed
immediately after completion of surgery. All patients used
0 0.12% chlorhexidine rinse for 1 min twice daily, not within 1
5 6 7 8 9 10 11 12
hour of tooth brushing. Patients were examined each week
PAL values for the first month and each month during the first year.
Frequency of rechecks after the first year was customized
29−34 39−42
35−38 43−50 based on the patient’s plaque control and level of compliance.
During recheck appointments patients underwent profes-
Figure 5 sional oral hygiene treatment.
4 BioMed Research International

efficacy of each treatment after 12, 18, and 24 months, the

null hypothesis was that the mean of PD, REC, and PAL in
each treatment group was the same at baseline and after 12,
18, and 24 months. The normal distribution of data within
each group was confirmed with the Shapiro-Wilks test (SW
test) [14]. If the hypothesis of normal distribution of the data
was confirmed, comparisons among groups were made with
a 𝑡-test or analysis of variance. If the hypothesis of normal
distribution was rejected, then comparisons among groups
were performed with Wilcoxon’s test for paired data (W test)
[15]. Values of 𝑃 < 0.05 were considered significant. Median
and mean differences in the location parameters for PD,
Figure 7: Probing at baseline showing PAL = 7 mm on the mesial
aspect of tooth number 9.
REC, and PAL among the four treatment groups at 12, 18,
and 24 months were tested for statistical significance. If the
hypothesis of normality in the data was rejected according
to the SW test, the nonparametric Kruskal-Wallis test (KW
test) was performed in place of analysis of variance. If
the null hypothesis where the median and mean location
parameters were the same in each group was rejected, Siegel
and Castellan’s post hoc multiple comparison was performed
at 5% significance level.

3. Results
The group that received EMD+HA showed greater mean
reduction in PD (5.75 mm) and improvement in gingival
recession compared with the EMD-only group. The group
Figure 8: Periapical X-ray at baseline. that received nanoHA alone had the poorest clinical results,
although they did improve from baseline. All treatments
resulted in PD reduction over time, with improvements in
2.6. Examiner and Surgeon. One individual was selected to REC and PAL.
make clinical measurements and a second to perform surgical
treatments. Surgical treatments were performed by a highly 3.1. Differences between Baseline and Follow-Ups
experienced surgeon (AP).
3.1.1. PD Index. According to the SW test, the hypothesis of
2.7. Clinical Parameters. Patients were evaluated at baseline normality could be confirmed only for baseline data in the
and at 12, 18, and 24 months after regenerative therapy by nanoHA + EMD group (𝑃 = 0.26). In the other groups the
the above-mentioned calibrated dental hygienist. Clinical hypothesis was rejected, because 𝑃 values were near 5% or
parameters measured at each time point included PD, REC, much lower. The differences between PD levels at baseline and
and PAL. All measurements were recorded using a standard after 12, 18, and 24 months were statistically significant (W
periodontal probe (PCP-15, UNC) at six sites per tooth: test, 𝑃 < 0.01) for all treatments. The reductions in median
mesiobuccal, midbuccal, distobuccal, mesiolingual, midlin- and mean PD levels shown in Figures 12, 13, 14, and 15 are
gual, and distolingual. The highest value obtained for PD and significant.
PAL and the lowest for REC were considered for statistical
analysis. As suggested by Kasaj et al. [11], the cementoenamel 3.1.2. REC Index. According to the SW test, the hypothesis
junction or restoration margin was used as the fixed reference of normality could be accepted at 5% significance level only
point. for nanoHA at 12 months and for EMD at 18 and 24 months.
In the other groups the hypothesis was rejected, because 𝑃
2.8. Statistical Analysis. Statistical analyses were performed values were lower or much lower than 5%. The differences in
with R software, version 3.1.0. Clinical parameters were REC levels at baseline compared with levels after 12, 18, and
described as minimum, 1st quartile, median, 3rd quartile, 24 months were statistically significant (W test, 𝑃 < 0.01)
maximum, mean, and standard deviation and were calculated for all treatments. The reductions in median and mean REC
by treatment (OFD, nanoHA, EMD, and nanoHA + EMD), levels shown in Figures 16, 17, 18, and 19 can be considered
time (baseline, 12 months, 18 months, and 24 months), significant.
site (mesiovestibular, distovestibular), and position (anterior,
posterior). First, we evaluated differences in PAL, REC, and 3.1.3. PAL Index. According to the SW test, the hypothesis
PD among patients assigned to each treatment. We accepted of normality could be accepted at 5% significance level only
the hypothesis of equal location parameters at 5%. To test the in the EMD treatment group, so both the 𝑡-test and W test
BioMed Research International 5

(a) (b)

Figure 9: (a) Root surface appearance after flap reflection and degranulation. (b) EMD applied on root surface.

(a) (b)

Figure 10: (a) NanoHA + EMD application on root surface using sandwich technique. (b) Sutured flap.

(a) (b)

Figure 11: (a) X-ray at 24 months. (b) Clinical aspect showing attachment gain.

were performed. In the other treatment groups the hypothesis significant differences between nanoHA and nanoHA + EMD
of normality was rejected (𝑃 values lower than 5%). The (observed difference (obs diff) = 33.01, critical difference
differences in REC levels at baseline compared with levels (crit diff) = 21.12) and between nanoHA + EMD and OFD
after 12, 18, and 24 months were statistically significant at (obs diff = 24.29, crit diff = 21.12). For REC, post hoc
10% for defects treated with nanoHA after 18 and 24 months multiple comparison at 5% showed statistically significant
and at 5% for the other treatments. The median and mean differences between EMD and nanoHA (obs diff = 22.05, crit
reductions in PAL levels shown in Figures 20, 21, 22, and 23 diff = 21.12). Post hoc multiple comparison for PAL at 5%
were significant. showed statistically significant differences between nanoHA
and nanoHA + EMD (obs diff = 24.33, crit diff = 21.12) in favor
of nanoHA + EMD.
3.2. Differences among Treatments after 12 Months. The null
hypothesis where location parameters were the same among
treatments was rejected for PD, REC, and PAL (𝑃 < 0.03). For 3.3. Differences among Treatments after 18 Months. The null
PD, post hoc multiple comparison at 5% showed statistically hypothesis where location parameters were the same among
6 BioMed Research International

PD versus time in NANO + EMD treatment PD versus time in OFD treatment

12 9

8
10
7
8
6

6 5

4
4
3
2 2
0 12 18 24 0 12 18 24
(Months) (Months)

Figure 12 Figure 15

REC versus time in NANO + EMD treatment

PD versus time in NANO treatment
3
9

8
2
7

6 1
5

4 0

3
−1
2
0 12 18 24
0 12 18 24
(Months)
(Months)
Figure 16
Figure 13

REC versus time in NANO treatment

PD versus time in EMD treatment
3
9

8 2
7
1
6

5 0

4
−1
3
−2
2
0 12 18 24
0 12 18 24
(Months)
(Months)
Figure 17
Figure 14

For PD, post hoc multiple comparison at 5% showed

treatments was accepted for REC (𝑃 = 0.53) and rejected statistically significant differences between nanoHA and
for PD and PAL (𝑃 < 0.01). Median and mean location nanoHA + EMD (obs diff = 31.38, crit diff = 21.12) and
parameters for REC were not statistically different among between nanoHA + EMD and OFD (obs diff = 24.13, crit
treatments; parameters were significantly different for PD and diff = 21.12). These were the same results as at the 12-month
PAL (𝑃 < 0.03). evaluation. For PAL, post hoc multiple comparison at 5%
BioMed Research International 7

REC versus time in EMD treatment PAL versus time in NANO treatment
4 9

8
3
7
2
6
1
5
0 4

−1 3
0 12 18 24 0 12 18 24
(Months) (Months)

Figure 18 Figure 21

REC versus time in OFD treatment

3 PAL versus time in EMD treatment
10
2
8
1
6
0
4
−1

2
−2
0 12 18 24
0 12 18 24
(Months)
(Months)
Figure 19
Figure 22
PAL versus time in NANO + EMD treatment
12 PAL versus time in OFD treatment
9

10 8

8 7

6
6
5

4 4

3
0 12 18 24
(Months) 0 12 18 24
(Months)
Figure 20
Figure 23

showed statistically significant differences between nanoHA

and nanoHA + EMD (obs diff = 26.75, crit diff = 21.12) and treatments was accepted for REC (𝑃 = 0.28) and rejected
between nanoHA + EMD and OFD (obs diff = 26.25, crit diff for PD and PAL (𝑃 < 0.02). Median and mean location
= 21.12). parameters were not statistically different among treatments
for REC, but they were different for PD and PAL. The same
3.4. Differences among Treatments after 24 Months. The null results were obtained as for the 18-month evaluation (𝑃 <
hypothesis that location parameters were the same among 0.03).
8 BioMed Research International

For PD, post hoc multiple comparison at 5% showed sta- EMD acts as a chemoattractant, while nanoHA paste is a
tistically significant differences between EMD and nanoHA synthetic extracellular matrix component in its coated form.
(obs diff = 22.27, crit diff = 21.12), between nanoHA and Al Machot et al. [13] hypothesized a potential synergic effect
nanoHA + EMD (obs diff = 33.58, crit diff = 21.12), and of combining the materials with resulting possible beneficial
between nanoHA + EMD and OFD (obs diff = 25.26, crit effects on wound healing.
diff = 21.12). For PAL, post hoc multiple comparison at 5% Avoiding the Hawthorne effect is supposed to provide a
showed statistically significant differences between nanoHA more realistic picture of how a treatment functions in every-
and nanoHA + EMD (obs diff = 21.55, crit diff = 21.12). day practice. However, retrospective treatment evaluations
have limitations that could bias the results. However, this
bias can be reduced by using multiple control groups as in
4. Discussion
the present study, which compared four treatment groups,
All four methods employed in this study fulfilled the main and by the triple blinding of the operator, the patient, and
purposes of periodontal surgery: controlling periodontal the analyst. Moreover, restricting patient selection criteria
infection and providing periodontal maintainable sites. Thus, reduced confounding factors. One important tool in ana-
reduction of PD was found in all four treatment groups. How- lyzing clinical outcome from the patient’s perspective is the
ever, periodontal regeneration requires more than control of visual analog scale, which can confirm or cast doubt on the
infection and gingival recession. Calculation of the clinical effects of biomaterials and techniques as regenerative strate-
attachment level reflects the degree of regeneration obtained. gies [18]. Such evaluation could have been useful in our study;
In this study, gingival recession showed an improving trend postoperative data, including patient perception, could have
over time in all treatment groups. Regeneration without been matched to clinical outcome, in both short- and long-
significant gingival recession was statistically important for term evaluation. One limit of the present study is that clinical
OFD, EMD, and nanoHA + EMD but not for nanoHA parameters alone were used to evaluate the regeneration
alone. NanoHA alone showed less beneficial clinical effect process. Previous studies have associated these parameters
than removal of granulation tissue without use of defect- with radiographic measurements to reduce possible biases.
filling material. This was an unexpected result, because However, clinical and radiographic parameters cannot fully
biomaterials, especially osteoconductive materials such as replace histological analysis to confirm the true regeneration
nanoHA, are supposed to provide support for soft tissues and of all periodontal tissues. Therefore, even using both sources
to prevent their prolapse. However, the clinical observations of information could be considered insufficient. Because the
in the present study are in accordance with the histological purpose of treatment is controlling infection by eliminating
results of Horváth et al., showing the limited potential of periodontal pockets and at the same time preventing soft
nanoHA in promoting periodontal regeneration over a 7- tissue collapse, clinical data can be considered a primary
month period [12]. This finding could be explained by the outcome in everyday clinical practice. The combined use of
rationale of Susin and Wikesjö [2], who reported that most nanoHA and EMD seems to fulfill such clinical criteria.
biomaterials interfere with rather than support periodontal
regeneration because of their sequestration within connec- 5. Conclusions
tive tissues. Susin and Wikesjö [2] and Trombelli et al.
[16] evaluated a minimally invasive surgical protocol as a In summary, within the limitations of this study, we found
stand-alone approach or in combination with membranes that EMD and nanoHA played a synergic role in promoting
and hydroxyapatite-based biomaterials. No significant dif- restoration of the tooth-supporting apparatus.
ferences were observed between experimental groups; sites
treated with a minimally invasive technique exhibited clinical
attachment gain averaging 4.7 ± 2.5 mm, probing depth Conflict of Interests
reduction of 5.3 ± 2.4 mm, and gingival recession of 0.4 ± The authors declare that there is no conflict of interests
1.4 mm. Similarly, Cortellini and Tonetti [17] evaluated a regarding the publication of this paper.
minimally invasive surgical technique, alone and in conjunc-
tion with an EMD and a bovine bone-based biomaterial.
No significant differences between treatments were observed; References
the minimally invasive surgical approach without additions
[1] N. Donos, L. Laurell, and N. Mardas, “Hierarchical decisions on
achieved substantial clinical attachment gain of 4.1 ± 1.2 mm teeth vs. implants in the periodontitis-susceptible patient: the
and radiographic bone fill of 77 ± 19%. In contrast, our modern dilemma,” Periodontology 2000, vol. 59, no. 1, pp. 89–
results suggest a significant difference between nanoHA and 110, 2012.
nanoHA + EMD in terms of changes in PD and PAL indices, [2] C. Susin and U. M. E. Wikesjö, “Regenerative periodontal ther-
measured at each follow-up interval. Our results suggest that apy: 30 years of lessons learned and unlearned,” Periodontology
EMD plays a prominent role in promoting better results 2000, vol. 62, no. 1, pp. 232–242, 2013.
when used in conjunction with nanoHA, compared with [3] Y.-K. Tu, A. Woolston, and C. M. Faggion Jr., “Do bone grafts
nanoHA alone. This clinical outcome is supported by a 12- or barrier membranes provide additional treatment effects for
month follow-up study conducted by Kasaj et al. [11], who infrabony lesions treated with enamel matrix derivatives? A net-
reported that EMD had advantages over nanoHA in patient work meta-analysis of randomized-controlled trials,” Journal of
comfort and adverse effects. Kasaj et al. [5] reported that Clinical Periodontology, vol. 37, no. 1, pp. 59–79, 2010.
BioMed Research International 9

[4] I. Bouchlariotou, J. P. Bernard, J. P. Carrel, and L. Vazquez, [17] P. Cortellini and M. S. Tonetti, “Clinical and radiographic
“Long-term stability of osseointegrated implants in bone regen- outcomes of the modified minimally invasive surgical tech-
erated with a collagen membrane in combination with a depro- nique with and without regenerative materials: a randomized-
teinized bovine bone graft: 5-year follow-up of 20 implants,” controlled trial in intra-bony defects,” Journal of Clinical Peri-
POSEIDO Journal, vol. 1, no. 1, pp. 1–64, 2013. odontology, vol. 38, no. 4, pp. 365–373, 2011.
[5] A. Kasaj, B. Willershausen, R. Junker, S.-I. Stratul, and M. [18] J. L. Wennström and J. Lindhe, “Some effects of enamel
Schmidt, “Human periodontal ligament fibroblasts stimulated matrix proteins on wound healing in the dento-gingival region,”
by nanocrystalline hydroxyapatite paste or enamel matrix Journal of Clinical Periodontology, vol. 29, no. 1, pp. 9–14, 2002.
derivative. An in vitro assessment of PDL attachment, migra-
tion, and proliferation,” Clinical Oral Investigations, vol. 16, no.
3, pp. 745–754, 2012.
[6] S. Gestrelius, C. Andersson, A. C. Johansson et al., “Formulation
of enamel matrix derivative for surface coating kinetics and cell
colonization,” Journal of Clinical Periodontology, vol. 24, no. 9,
pp. 678–684, 1997.
[7] D. B. Palioto, R. D. Coletta, E. Graner, J. C. Joly, and A. F.
Martorelli de Lima, “The influence of enamel matrix derivative
associated with insulin-like growth factor-I on periodontal
ligament fibroblasts,” Journal of Periodontology, vol. 75, no. 4,
pp. 498–504, 2004.
[8] J. He, J. Jiang, K. E. Safavi, L. S. W. Spångberg, and Q. Zhu,
“Emdogain promotes osteoblast proliferation and differentia-
tion and stimulates osteoprotegerin expression,” Oral Surgery,
Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics,
vol. 97, no. 2, pp. 239–245, 2004.
[9] A. Pilloni, G. Pompa, M. Saccucci et al., “Analysis of human
alveolar osteoblast behavior on a nano-hydroxyapatite sub-
strate: an in vitro study,” BMC Oral Health, vol. 20, no. 1, pp.
14–22, 2014.
[10] M. Silvestri, G. Rasperini, and S. Milani, “120 Infrabony defects
treated with regenerative therapy: long-term results,” Journal of
Periodontology, vol. 82, no. 5, pp. 668–675, 2011.
[11] A. Kasaj, B. Röhrig, G.-G. Zafiropoulos, and B. Willershausen,
“Clinical evaluation of nanocrystalline hydroxyapatite paste in
the treatment of human periodontal bony defects: a randomized
controlled clinical trial: 6-month results,” Journal of Periodon-
tology, vol. 79, no. 3, pp. 394–400, 2008.
[12] A. Horváth, A. Stavropoulos, P. Windisch, L. Lukács, I. Gera,
and A. Sculean, “Histological evaluation of human intrabony
periodontal defects treated with an unsintered nanocrystalline
hydroxyapatite paste,” Clinical Oral Investigations, vol. 17, no. 2,
pp. 423–430, 2013.
[13] E. Al Machot, T. Hoffmann, K. Lorenz, I. Khalili, and B. Noack,
“Clinical outcomes after treatment of periodontal intrabony
defects with nanocrystalline hydroxyapatite (Ostim) or enamel
matrix derivatives (Emdogain): a randomized controlled clin-
ical trial,” BioMed Research International, vol. 2014, Article ID
786353, 9 pages, 2014.
[14] S. S. Shapiro and M. B. Wilk, “An analysis of variance test for
normality: complete samples,” Biometrika, vol. 52, pp. 591–611,
1965.
[15] M. Hollander and D. A. Wolfe, “Nonparametric statistical
methods,” Wiley Series in Probability and Statistics, vol. 17, no.
8, pp. 526–1975, 2007.
[16] L. Trombelli, A. Simonelli, M. Pramstraller, U. M. E. Wikesjö,
and R. Farina, “Single flap approach with and without guided
tissue regeneration and a hydroxyapatite biomaterial in the
management of intraosseous periodontal defects,” Journal of
Periodontology, vol. 81, no. 9, pp. 1256–1263, 2010.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 102648, 14 pages
http://dx.doi.org/10.1155/2014/102648

Research Article
Adhesion and Proliferation of Human Periodontal Ligament
Cells on Poly(2-methoxyethyl acrylate)

Erika Kitakami,1 Makiko Aoki,1 Chikako Sato,1 Hiroshi Ishihata,2 and Masaru Tanaka1
1
Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa, Yamagata 992-8510, Japan
2
Graduate School of Dentistry, Division of Periodontology and Endodontology, Tohoku University, Seiryo-machi 4-1,
Sendai, Miyagi 980-8575, Japan

Correspondence should be addressed to Masaru Tanaka; [email protected]

Received 15 May 2014; Accepted 29 June 2014; Published 6 August 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Erika Kitakami et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Human periodontal ligament (PDL) cells obtained from extracted teeth are a potential cell source for tissue engineering. We
previously reported that poly(2-methoxyethyl acrylate) (PMEA) is highly biocompatible with human blood cells. In this study,
we investigated the adhesion, morphology, and proliferation of PDL cells on PMEA and other types of polymers to design an
appropriate scaffold for tissue engineering. PDL cells adhered and proliferated on all investigated polymer surfaces except for
poly(2-hydroxyethyl methacrylate) and poly[(2-methacryloyloxyethyl phosphorylcholine)-co-(n-butyl methacrylate)]. The initial
adhesion of the PDL cells on PMEA was comparable with that on polyethylene terephthalate (PET). In addition, the PDL cells on
PMEA spread well and exhibited proliferation behavior similar to that observed on PET. In contrast, platelets hardly adhered to
PMEA. PMEA is therefore expected to be an excellent scaffold for tissue engineering and for culturing tissue-derived cells in a
blood-rich environment.

1. Introduction have the ability to form cementum/periodontal ligament-

like tissue in vivo [3, 4]. Human PDL stem cells, similar to
Periodontal diseases, caused by the bacterial biofilm, can bone marrow mesenchymal stem cells, are able to suppress
affect up to 90% of adults worldwide [1]. Severe periodontitis immune responses and inflammatory reactions, which sug-
leads to losing connective tissue and bone support and finally gests that PDL stem cells may be used in allogeneic stem cell-
losing teeth [1]. Therefore, much research is being conducted based therapies [5]. In the first clinical application of human
on periodontal tissue regeneration for restoring the alveolar autologous PDL-derived cells, including PDL stem cells,
support of the teeth. The periodontal ligament (PDL) is an the transplanted cells were used to reconstruct periodontal
important structure that is composed of periodontal tissue, intrabony defects in 3 patients, and a significant improvement
in which PDL cells generate connective tissue fibers that span of periodontal disease was achieved, which suggests that
the gap between the cementum and the alveolar bone [2] PDL stem cell transplantation may be an efficacious and safe
to suspend the tooth. This complex structure of PDL tissue alternative for the treatment of human periodontitis [6].
comprises several different cell populations [3], including Furthermore, human induced pluripotent stem cells (iPS
PDL cells, which are predominantly fibroblasts and play cells) have been generated from adult human periodontal
crucial roles in maintaining and regenerating periodontal ligament fibroblasts [7]. Nomura et al. reported that the
tissue [2, 3]. reprogramming efficiency of human PDL fibroblasts was
Human PDL cells obtained from extracted teeth are a 0.025%, which is not lower than the reprogramming effi-
promising cell source for periodontal tissue regeneration, ciency of dental stem cells, even though the stem cells already
regenerative medicine, and tissue engineering [3–6], because express a number of ES cell-associated genes; therefore,
they include stem cells that have a high capacity for prolifera- human PDL fibroblasts may be an optimal cell source for
tion, self-renewal, and multilineage differentiation and also generating iPS cells [7]. Additionally, the use of PDL cells
2 BioMed Research International

is advantageous because it efficiently utilizes extracted teeth. 300,000, and 600,000, respectively. The chemical structure
However, for periodontal tissue regeneration, it is necessary of each polymer PMEA, PHEMA, and PMPC is shown
to culture PDL cells on a biocompatible scaffold to deliver in Figure 1 in Supplementary Material available online
the cells to the wound site and also to provide space for at http://dx.doi.org/10.1155/2014/102648, and the polymers
the formation of the new periodontal tissue. Biomaterial were prepared on polyethylene terephthalate (PET film:
scaffolds designed for tissue-engineered constructs must T100E125; Mitsubishi Plastics, Tokyo, Japan; 14 mm diameter
accommodate cell viability, growth, and function [8]. There and 125 𝜇m thickness) using a spin coater (MS-A100; Mikasa,
is increasing interest in designing new biomaterials for scaf- Tokyo, Japan). Exactly 40 𝜇L of a 0.2 wt% solution of each
folds with minimal or no immune response that encourage polymer was cast twice onto the PET films. Analytical grade
stable implant/tissue interaction [9]. In addition, the surface methanol (Kanto Chemical Co., Inc) was used as the solvent
characterization of biomaterials is important to design new for each solution. The surfaces of the polymer films were
implantable materials [10–14]. analyzed by X-ray photoelectron spectroscopy (XPS) (ESCA-
We previously reported that poly(2-methoxyethyl acry- 1000; Shimadzu, Kyoto, Japan) to confirm the coverage of the
late) (PMEA) shows excellent biocompatibility with human coated polymer. The take-off angle was 45∘ .
blood coagulation and complement systems and does not For cell culture, the films were sterilized by UV exposure
activate leukocytes, erythrocytes, and platelets in vitro and for 2 h. Subsequently, the films were soaked in medium
ex vivo relative to other polymer surfaces during the early composed of Minimum Essential Medium (MEM-Alpha; Life
stages of immune reactions [15–19]. On the basis of our Technologies) containing 10% heat-inactivated fetal bovine
results, superior biocompatible catheters and oxygenators serum (Equitech-Bio, Inc., Kerrville, TX) and antibiotic solu-
coated with PMEA were approved by the Food and Drug tion (100 U/mL penicillin G sodium, 100 𝜇g/mL streptomycin
Administration (FDA) and made available to the global mar- sulfate, and 0.25 𝜇g/mL amphotericin B; Life Technologies)
ket [16–19]. We further determined why PMEA has excellent (culture medium) for one hour (preconditioning).
biocompatibility [15, 20–22]. In particular, the low extent of
platelet adhesion and spreading observed was closely related 2.2. Characterization of Polymer Films. We analyzed each
to a low degree of denaturation and high dissociation rate for polymer film by atomic force microcopy (AFM; Agilent Tech-
proteins adsorbed onto PMEA [15]. nologies 5550 Scanning Probe Microscope, Agilent Tech-
The objective of this study was to examine the hypothesis nologies, Inc., Santa Clara, CA). The maximum scan range
that PMEA is a biocompatible polymer for tissue engineering was approximately 10 𝜇m × 10 𝜇m using a cantilever with a
that can facilitate adhesion and proliferation of PDL cells with force constant of 21–78 N/m, resonance frequency of 250–
low platelet adhesion. Cell-material interactions determine 390 kHz, and tip height of 10–15 𝜇m (NCH-10, Nano World,
many cellular processes such as adhesion, spreading, and Zurich, Switzerland). AFM was performed in air acoustic AC
proliferation and are thus essential for tissue engineering [23– mode. AFM image analysis was performed using Pico Image
27]. However, the influence of the chemical components of Software (Agilent Technologies).
synthesized polymers on the biology of PDL cells remains The wettability of the polymer surfaces was characterized
unclear. To investigate PDL cell-material interactions, we by contact angle measurement [15]. The static contact angle
characterized the localization of focal adhesions, which are on each polymer surface was measured using the sessile drop
multifunctional organelles that mediate cell-material adhe- method at room temperature. For the sessile drop method,
sion, force transmission, and cytoskeletal regulation and sig- 2 𝜇L of deionized water was dropped on a dried polymer film
naling [28]. To analyze the formation of focal adhesions, we using a microsyringe. The static contact angle was observed
evaluated the localization of vinculin, which is a membrane- 30 s later under a microscope (G-1-1000; ERMA Inc., Tokyo,
cytoskeletal protein present in focal adhesions that is involved Japan). After at least five readings which were obtained
in the linkage of integrin adhesion molecules to the actin for different areas of the polymer, the measurements were
cytoskeleton [29]. averaged to arrive at a final contact angle (𝑛 = 6).

2. Material and Methods 2.3. Cell Preparation and Culture. Primary PDL cells were
obtained as previously reported [30]. Fibroblast-like PDL
2.1. Preparation of Polymer Surfaces. PMEA was prepared by cells were derived from the periodontal ligament of human
free-radical polymerization using 2,2󸀠 -azobisisobutyronitrile third molars extracted from healthy individuals aged 17–21
(Kanto Chemical Co., Inc., Japan) as the initiator and 2- years who had no clinical signs of chronic periodontal dis-
methoxyethyl acrylate (MEA) as the monomer. The MEA was ease. Informed consent was obtained prior to each extraction.
obtained from Wako Pure Chemical Industries, Ltd. (Osaka, The cells were obtained from the Dental Faculty of Tohoku
Japan), poly(2-hydroxyethyl methacrylate) (PHEMA) was University. Periodontal ligament tissues were dissected into
obtained from Scientific Polymer Products, Inc. (Ontario, small pieces from the midportion of the root with a sharp
NY), and poly[(2-methacryloyloxyethyl phosphorylcholine)- blade. The pieces were then cultivated in tissue culture dishes
co-(n-butyl methacrylate)] (PMPC) was obtained from (Asahi Glass Co., LTD, Tokyo, Japan) until the formation of
NOF corporation (Tokyo, Japan). The molecular weight a confluent cell monolayer using culture medium. After con-
of each polymer was estimated by gel permeation chro- fluence was achieved, the cells were washed with phosphate-
matography using polystyrene standards. The molecular buffered saline (PBS; Takara Bio Inc., Shiga, Japan) and
weight (Mw) of PMEA, PHEMA, and PMPC was 85,000, resuspended with 0.075 g/L protease and 0.1 g/L EDTA to
BioMed Research International 3

enable passage. These experiments were approved by the overnight at 4∘ C. They were next washed three times with
Ethics Committee of the Dental Faculty of Tohoku University PBS and then with pure water and subsequently air-dried. The
and the Graduate School of Science and Engineering of dried samples were coated with carbon using an ion sputter
Yamagata University, Japan. coater (HPC-1SW; Vacuum Device Inc., Ibaraki, Japan).
We used PDL cells at passages six and eight for adhesion
and proliferation assays. PET, PMEA, PHEMA and PMPC 2.6. Platelet Adhesion Test. To investigate the number of
films were put in 24-well polystyrene plates (Asahi Glass Co., platelets adhering to the polymers, blood was drawn from 3
LTD). After preconditioning of these films, PDL cells were healthy volunteers (nonsmokers; age 22 male, age 33 female,
seeded at 1 x 104 cells/cm2 onto the tested films, and grown and age 42 male) and mixed with a 1/9 volume of 3.2% sodium
for up to 1 hour (1 h), 1 day, 3 days, and 7 days using culture citrate. Platelet-rich plasma (PRP) and platelet-poor plasma
medium. During culturing, the cells were maintained at 37∘ C (PPP) were obtained by centrifugation of citrated blood at
in 5% CO2 and 95% air, and the medium was changed every 1,500 rpm for 5 min and 4,000 rpm for 10 min, respectively.
three days. The progression of the cultures was examined by Plasma containing 3-4 × 107 cells/cm2 was prepared by
using phase contrast microscopy (CKX41; Olympus, Tokyo, mixing PRP with PPP. Then, 200 𝜇L of the platelet suspension
Japan). was placed on each polymer surface and incubated for 1 h at
37∘ C. After the films were washed three times with PBS, they
2.4. Immunofluorescence Staining. The adhesion, prolifera- were immersed in 1% glutaraldehyde in PBS for 120 min at
tion, and focal adhesion formation of the cultured PDL 4∘ C to fix the adhered platelets. The samples were dried and
cells were observed by confocal laser scanning microscopy sputter-coated in platinum-palladium using an ion sputter
(CLSM; FV-1000; Olympus). To visualize cell adhesion, coater prior to SEM (JSM-7600FA, JEOL Ltd., Tokyo, Japan).
spreading, proliferation, and focal adhesion formation on the The number of adherent platelets on the polymer films was
polymers, staining of vinculin, actin fibers, and cell nuclei was counted in five randomly selected SEM images (𝑛 = 6).
performed. After culture for the indicated period, the cells
were washed with PBS twice. After washing, the cells were 2.7. Data Analyses. The results were analyzed using Student’s
fixed with PBS containing 4% paraformaldehyde obtained 𝑡-test. 𝑃 < 0.05 was used as the threshold for statistical
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) significance between groups.
for 10 min at 37∘ C and washed again three times with PBS.
Subsequently, the cells were permeated three times with 1% 3. Results and Discussion
Triton-X-100 (MP Biomedicals, LLC, Solon, OH) in PBS for
10 min at room temperature and then immersed in 0.02% 3.1. Characterization of Polymer Films. The surface rough-
Tween-PBS (MP Biomedicals, LLC) three times for 10 min ness of each polymer film was analyzed by AFM (Table 1).
each. To assess PDL cell-material interactions, PDL cells The AFM topographical values (root mean squared; RMS)
on polymers were stained for vinculin, which is localized for PET, PMEA, PHEMA, and PMPC were 10, 6.7, 5.8,
at focal adhesions, using a mouse antivinculin monoclonal and 6.5 nm, respectively. The polymer-coated films were
antibody (Millipore, Temecula, CA) as a primary antibody smoother than the PET film.
for 1 h, followed by treatment with Alexa Fluor 546 goat Figure 1(a) shows the XPS spectrum of the PET film
anti-mouse IgG (Life Technologies as a secondary antibody coated nitrogen-modified PET film (Figures 1(a)–1(c), line
for 1 h. For actin staining, the samples were incubated with A). C 1s and O 1s peaks derived from PMEA were observed,
Alexa Fluor 488 phalloidin (Life Technologies) for 1 h. For whereas an N 1s peak for the nitrogen-modified PET film was
detection of Ki-67 antigen as a cell proliferation marker, not observed (Figure 1(a), line B). The XPS spectrum of the
the samples were treated with rabbit anti-Ki-67 antigen coated PHEMA also showed the same result (Figure 1(b)).
monoclonal antibody (Life Technologies) for 2 h. The samples The XPS spectra of coated PMPC also did not show an N 1s
were then incubated with Alexa Fluor 488 goat anti-rabbit peak but showed a P 2s peak (Figure 1(c), line B). These results
antibody (Life Technologies) for 1 h (𝑛 = 9). The stained cells indicate that the PET film surface was completely covered
were rinsed three times with PBS and subsequently immersed with each polymer.
in PBS for 10 min. All specimens were placed on glass slides, The static contact angle (𝜃) on each polymer surface was
mounted by using ProLong Gold antifade regent with DAPI measured by the sessile drop method as shown in Table 2. The
(Life Technologies), and covered with glass cover slips. 𝜃 values determined by the sessile drop method for deionized
The specimens were imaged by CLSM, and cell mor- water were 69∘ ± 2.6∘ , 45∘ ± 2.2∘ , 36∘ ± 2.7∘ , and 105∘ ± 3.3∘ on
phology parameters were quantified by Olympus Fluoview PET, PMEA, PHEMA, and PMPC, respectively. These data
software. The total number of adherent cells on polymer films indicate that the hydrophilicity of PMEA is between that of
was counted in five randomly selected CLSM images (𝑛 = 3). PET and PHEMA. The static contact angle of each polymer
was consistent with values in the literature [31, 32].
2.5. Scanning Electron Microscopy. To assess the morphol-
ogy of adherent PDL cells cultured on each polymer for 3.2. Morphology of PDL Cells on Polymer Surfaces. Cell
1 h, the cells were observed by field emission scanning morphology and proliferation behavior observed by CLSM
electron microscopy (SEM; SU-8000, Hitachi, Ltd., Tokyo, demonstrated that PDL cells adhered to PMEA and other
Japan). Cultured cells were fixed with 2.5% glutaraldehyde polymer surfaces, except PMPC, with a round shape within
(Polysciences, Inc., Warrington, PA) in PBS and incubated 1 h (Figure 2). After 1 day, PDL cells had spread across the
4 BioMed Research International

O 1s C 1s
N 1s
O 1s
(A) N 1s C 1s

Intensity (CPS) ×102

Intensity (CPS) ×102

(A)

(B)

(B)

1000 900 800 700 600 500 400 300 200 100 0 1000 900 800 700 600 500 400 300 200 100 0
Binding energy (eV) Binding energy (eV)
(a) (b)
O 1s C 1s
Intensity (CPS) ×102

N 1s
(A)

P 2s
(B)

1000 900 800 700 600 500 400 300 200 100 0
Binding energy (eV)
(c)

Figure 1: XPS spectrum of the PET film surface coated with PMEA (a), PHEMA (b), and PMPC (c). (A) indicates the XPS spectrum of the
PET film surface. (B) indicates the XPS spectrum of the coated film. The atomic compositions determined from the XPS spectra match the
expected composition based on the structure of each polymer.

Table 1: AFM topographical data. RMS: root mean squared rough- Table 2: Static contact angles of polymer surfaces.
ness. Scan size 10 × 10 𝜇m2 .
Polymer Sessile drop (degrees), (±SD)
Polymer RMS (nm) PET 69.2 (±2.6)
PET 10 PMEA 45.0 (±2.2)
PMEA 6.7 PHEMA 36.0 (±2.7)
PHEMA 5.8 PMPC 105.2 (±3.3)
PMPC 6.5

on PET contained some pseudopodia (white circle) and

polymers. After 3 days, PDL cells had spread further and spikes (white arrow). PDL cells on PMEA contained some
showed proliferation behavior on all polymers. After 7 days, pseudopodia (white circle) and many spikes (white arrows).
PDL cells on PET and PMEA were confluent, and developed PDL cells on PHEMA contained lamellipodia. The surfaces of
actin fibers were observed. PDL cells on PHEMA were not adherent PDL cells adherent to PET and PMEA were rougher
confluent but had aggregated. PDL cells did not adhere and than those on PHEMA.
proliferate on PMPC throughout the experiment.
3.4. Quantification of Projected Area, Perimeter, and Long
3.3. Initial Adhesion of PDL Cells. The nuclei of adherent PDL Axis of PDL Cells. The morphology of the adherent PDL
cells on each polymer surface were counted under CLSM. cells was quantified using CLSM images (Figure 4(a)).
PDL cells adhered to PMEA and the other polymer surfaces, Figure 4(b) shows the projected cell area on each polymer.
except for PMPC, upon incubation for 1 h (Figure 3(a)). The After 1 h, the projected cell areas were 270 ± 100 𝜇m2 , 230 ±
number of adherent PDL cells on PMEA was almost identical 90 𝜇m2 , and 270 ± 110 𝜇m2 on PET, PMEA, and PHEMA,
to that on PET and was 5 times higher than that on PHEMA. respectively. After 1 day, the projected cell areas were 1390 ±
The cell morphology observed by SEM showed differ- 660 𝜇m2 , 1770 ± 610 𝜇m2 , and 690 ± 360 𝜇m2 on PET, PMEA,
ences for each polymer after 1 h (Figure 3(b)). PDL cells and PHEMA, respectively. The projected cell areas increased
BioMed Research International 5

PET 1h Day 1 Day 3 Day 7

100 𝜇m
PMEA

100 𝜇m
PHEMA

100 𝜇m
PMPC

Figure 2: CLSM images of PDL cells cultured on polymer surfaces. Scale bars: 300 𝜇m. Blue: nucleus, green: actin, and red: vinculin. Time
points are 1 h, 1 day, 3 days, and 7 days. Polymers: PET, PMEA, PHEMA, and PMPC.

by 5.1-, 7.7-, and 2.6-fold on PET, PMEA, and PHEMA, PHEMA, respectively. After 1 day, the lengths were 85 ±
respectively, from 1 h up to 1 day. 37 𝜇m, 110 ± 44 𝜇m, and 50 ± 33 𝜇m on PET, PMEA, and
Figure 4(c) shows the perimeter of adherent PDL cells PHEMA, respectively. The length of adherent cells increased
on each polymer. After 1 h, the perimeters were 75 ± 15 𝜇m, by 3.9-, 5.0-, and 2.3-fold on PET, PMEA, and PHEMA,
80 ± 17 𝜇m, and 82 ± 27 𝜇m on PET, PMEA, and PHEMA, respectively, from 1 h up to 1 day. After 1 h, the short axes (i.e.,
respectively. After 1 day, the perimeters were 300 ± 85 𝜇m, width) were 14 ± 5 𝜇m, 9 ± 2 𝜇m, and 13 ± 2 𝜇m on PET,
380 ± 130 𝜇m, and 180 ± 80 𝜇m on PET, PMEA, and PHEMA, PMEA, and PHEMA, respectively. After 1 day, the widths
respectively. The perimeter of the adherent cells increased were 21 ± 10 𝜇m, 24 ± 9 𝜇m, and 18 ± 6 𝜇m on PET, PMEA, and
by 4.0-, 4.8-, and 2.2-fold on PET, PMEA, and PHEMA, PHEMA, respectively. The width of adherent cells increased
respectively, from 1 h up to 1 day. by 1.5-, 2.7-, and 1.4-fold on PET, PMEA, and PHEMA,
Figure 4(d) shows each axis of the adherent PDL cells respectively, from 1 h up to 1 day. Figures 4(a)–4(d) show that
on each polymer. After 1 h, the long axes (i.e., length) were PDL cells on PMEA were more spread than cells on any other
22 ± 5 𝜇m, 22 ± 7 𝜇m, and 22 ± 8 𝜇m on PET, PMEA, and polymer surface.
6 BioMed Research International

3500
∗∗
3000 NS

Cell number (cells/cm2 )

2500 ∗∗

2000

1500

1000

500

PET

PMEA

PHEMA

PMPC
(a)
PET PMEA PHEMA

(b)

Figure 3: Initial adhesion of PDL cells on polymer surfaces after 1 h. (a) The number of adherent PDL cells on polymer surfaces. Polymers:
PET, PMEA, PHEMA, and PMPC. ∗∗ 𝑃 < 0.01 versus PMEA, mean ± standard deviation, 𝑛 = 3. (b) SEM images of PDL cells on polymer
surfaces. Top scale bars: 10 𝜇m, bottom scale bars: 1.0 𝜇m. Polymers: PET, PMEA, and PHEMA. White circles indicate pseudopodia formation.
White arrows indicate spike formation.

3.5. Proliferation of PDL Cells. PDL cells adhered and pro- 3 days, only type II adherent PDL cells on PMEA showed a
liferated on all polymer surfaces, except for PMPC, during statistically significant difference relative to cells on PHEMA
the culture period (Figure 5). After 1 day and 3 days, the (Figure 6(b)), which indicates that the number of proliferat-
number of adherent PDL cells was almost identical on all of ing PDL cells on PMEA was higher than that on PHEMA.
the polymer surfaces. After 7 days, the number of PDL cells
on PMEA was almost the same as that on PET. The number of 3.6. Localization of Vinculin in Two- and Three-Dimensional
PDL cells on PHEMA was lower than that on PET and PMEA. Observation. Figures 7(a)–7(c) show a top view and cross
Figures 6(a)–6(c) show the percentage of Ki-67-positive sections of adherent cells. We classified the localization of
cells during the culture period. Ki-67-positive cells (prolif- vinculin into 4 types: (i) focal adhesions (FAs) and (ii)
erating cells; Figures 6(d)-6(e)) were categorized into types nonfocal adhesions (non-FAs), where FAs were localized at
I and II. Type I showed stronger staining, while type II basal cell surfaces, and non-FAs were localized at apical cell
showed weaker staining. Ki-67-negative cells (quiescent or surfaces; (iii) vinculin rods that were composed of a complex
resting cells) were categorized as type III (Figures 6(d)-6(e)). of FAs and non-FAs and that were connected vertically and
PDL cells did not show a statistically significant difference penetrated the cells; and (iv) vinculin fibers that were mainly
between 1 day (Figure 6(a)) and 7 days (Figure 6(c)). After oriented along the long axis of the cells.
BioMed Research International 7

PET PMEA PHEMA

1h
Day 1

(a)
3000 700
NS ∗∗ NS ∗∗
600
Projected cell area (𝜇m2 )

2500
500
Perimeter (𝜇m)

2000
400
1500
300
1000
NS NS 200 NS NS
500 100
0 0
PET

PMEA

PHEMA

PET

PMEA

PHEMA

PET

PMEA

PHEMA

PET

PMEA

PHEMA

1h Day 1 1h Day1
(b) (c)
200
180 ∗∗
160
Length axis (𝜇m)

140
120
100
80
60
40 ∗ ∗∗
20
0
PET
PMEA
PHEMA

PET
PMEA
PHEMA

PET
PMEA
PHEMA

PET
PMEA
PHEMA

1h Day 1 1h Day 1
Long axis Short axis
(d)

Figure 4: Quantification of adherent PDL cell morphologies for 1 h and 1 day. (a) CLSM images for the quantification of adherent PDL cell
morphology on polymer surfaces. Scale bars: 100 𝜇m. Blue: nucleus, green: actin, and red: vinculin. Polymers: PET, PMEA, and PHEMA. (b)
Projected cell area. (c) Perimeter of adherent PDL cells. (d) Long and short axes of adherent PDL cells. Polymers: PET, PMEA, and PHEMA.
∗∗
𝑃 < 0.01 and ∗ 𝑃 < 0.05 versus PMEA, mean ± standard deviation, 𝑛 = 10.
8 BioMed Research International

10.0 to the shape of cells on the other polymers (Figures 7(a)

9.0 ∗∗ and 7(b)). Large vinculin spots were localized at apical
∗ cell surfaces in non-FAs. FAs and vinculin rods were also
Cell number (×104 cells/cm2 )

8.0
observed. After 3 days, the PDL cells had spread out. Vinculin
7.0
rods were also observed. FAs and non-FAs were observed
6.0 in addition to vinculin fibers. The vinculin fiber-formation
5.0 process is summarized in Figure 7(d). PDL cells on PMEA
4.0 contained few vinculin rods, whereas PDL cells on PET and
PHEMA contained many vinculin rods.
3.0
2.0 ∗∗
∗∗
3.6.1. Relationship between Cell Proliferation and Ki-67 Protein
1.0 Production. As shown in Figures 2, 3(a)-3(b), 4(a)–4(d),
0 and 5, PDL cells on PMEA showed similar adhesion and
PET
PMEA
PHEMA
PMPC

PET
PMEA
PHEMA
PMPC

PET
PMEA
PHEMA
PMPC
proliferation behavior to cells on PET at all-time points.
The low proliferation on PHEMA was consistent with the
results of Peluso et al., who found that human embryonic
Day 1 Day 3 Day 7
lung fibroblasts did not proliferate on PHEMA [33]. Based
Figure 5: The number of PDL cells on polymer surfaces. Time on this finding, we focused on differences in the cell cycle
points are 1 day, 3 days, and 7 days. Polymers: PET, PMEA, PHEMA, and quantified the percentage of Ki-67-positive cells on the
and PMPC. ∗∗ 𝑃 < 0.01 and ∗ 𝑃 < 0.05 versus PMEA, mean ± polymers to identify differences in cell proliferation. As
standard deviation, 𝑛 = 3. shown in Figure 6(b), the percentage of Ki-67-positive cells
on PMEA at 3 days was higher than that on PHEMA. These
data suggest that the higher proliferation of PDL cells on
PMEA was related to the higher percentage of Ki-67-positive
Figure 7(a) shows PDL cells cultured on PET. After cells at 3 days (Figures 5 and 6(b)).
1 h, adherent PDL cells were shaped similarly to gourds
(Figure 7(a)). The cells were spherical with a thickness of 3.6.2. Relationship between Vinculin Localization and Cell
approximately 15 𝜇m, and their actin and vinculin were Proliferation. Next, we analyzed the localization of vinculin
undeveloped. After 4 h, the cells had spread to form disc- in 2 and 3 dimensions to investigate the cause of difference
like shapes and FAs. Large spots of actin and vinculin were in the proportion of Ki-67-positive cells and to analyze cell-
localized at the apical cell surfaces. The thickness of the material interactions. Our results suggest that PDL cells on
adherent PDL cells was approximately 10 𝜇m. Vinculin was PMEA have stronger PDL cell-material interactions than
mainly localized at basal cell surfaces in FAs and apical cells on PHEMA because cells on PMEA exhibited high
cell surfaces in non-FAs. After 1 day, the cells were spread vinculin localization and Ki-67 protein production. In our
out thinly. Large vinculin spots were localized at apical cell next study, we will attempt to elucidate the influence of the
surfaces in non-FAs, and FAs were also observed in addition chemical structure of the synthetic polymer on cell behavior
to vinculin rods. After 3 days, the cells were spread out more by altering the composition of the main chain and/or the
thinly. Many vinculin rods were observed. FAs, non-FAs, and terminal functional group of the side chain.
vinculin fibers were also observed. As shown in Figures 7(a)–7(d), we observed some unique
Figure 7(b) shows PDL cells cultured on PMEA. After vinculin localization in non-FAs, vinculin rods, and vinculin
1 h, the adherent PDL cells were shaped similarly to gourds. fibers. Kanchanawong et al. reported that focal adhesions
The cells were spherical with a thickness of approximately link the extracellular matrix to the actin cytoskeleton, and
11 𝜇m. Their actin and vinculin were undeveloped. After 4 h, vinculin localized in focal adhesions probably links integrin
PDL cells had spread to form disc-like shapes and FAs. Large to actin directly, as the distribution of vinculin is consistent
actin spots and small vinculin spots were localized at the with its binding to sites along the talin rod domain and actin,
apical cell surfaces in non-FA. The thickness of the cells was which may serve to buttress the integrin-talin-actin linkage
approximately 10 𝜇m. Vinculin was mainly localized at basal [29]. Therefore, vinculin localization was expected to occur
cell surfaces in FAs and apical cell surfaces in non-FAs. After at basal cell surfaces contacting the materials; however, we
1 day, the cells were spread out and spindle shaped. Large observed that vinculin localized in non-FAs at apical cell sur-
vinculin spots were localized at apical cell surfaces in non- faces and in vinculin rods that were composed of a complex
FAs, and FAs were also observed in addition to vinculin of FAs and non-FAs that were connected vertically and pene-
fibers. After 3 days, the cells were spread out more thinly. trated the cells (Figures 7(a)–7(d)). In addition, we observed
FAs and non-FAs were observed, and vinculin fibers were also that vinculin fibers appeared to be similar to the supermat-
observed. uration of focal adhesions reported by Dugina et al., who
Figure 7(c) shows PDL cells cultured on PHEMA. After showed that increased extradomain A fibronectin expres-
1 h, the adherent cells were spherical with a thickness of sion induced by transforming growth factor 𝛽 (TGF𝛽) was
approximately 15 𝜇m. Their actin and vinculin were undevel- accompanied by 𝛼-smooth muscle actin expression and focal
oped. After 4 h, the cells were slightly spread out, and they adhesion supermaturation in fibroblasts [34]. The non-FAs
possessed FAs. After 1 day, the cells had a round shape relative probably link extracellular matrix proteins such as fibronectin
BioMed Research International 9

100 100

Ki-67 positive cells (%)

Ki-67 positive cells (%)
80 80 ∗∗

60 60

40 40

20 20

0 0
PET
PMEA
PHEMA

PET
PMEA
PHEMA

PET
PMEA
PHEMA

PET
PMEA
PHEMA

PET
PMEA
PHEMA

PET
PMEA
PHEMA
I II III I II III
(a) (b)
100
II II
Ki-67 positive cells (%)

80 III

II II
60

40 III

20 II II
0 III
III
PET
PMEA
PHEMA

PET
PMEA
PHEMA

PET
PMEA
PHEMA

I II I II I
I
I II III
(c) (d) (e)
Figure 6: Percentage of Ki-67-positive cells on polymer surfaces: (a) 1 day, (b) 3 days, and (c) 7 days. Polymers: PET, PMEA, and PHEMA.
∗∗
𝑃 < 0.01 and ∗ 𝑃 < 0.05 versus PMEA, mean ± standard deviation, 𝑛 = 9. (d)-(e) Classification of Ki-67 staining. The images show Ki-67
staining of PDL cells on PHEMA after 3 days. Scale bars: 100 𝜇m. Blue: nucleus, green: Ki-67-positive cells. (d) CLSM images of PDL cells
showing the nucleus and Ki-67 staining. (e) CLSM images of PDL cells showing Ki-67 staining. Types I and II: the Ki-67 antigen is present
in the nucleus during the G1, S, and G2 phases of cell division and during mitosis. Type III: quiescent or resting cells in the G0 phase do not
express the Ki-67 antigen.

with the apical cell surface. In future studies, we will evaluate platelet adhesion [35–39]. PDL cells hardly adhered and pro-
the role of the unique vinculin localization in non-FAs, liferated on PMPC, which was consistent with the findings of
vinculin rods, and vinculin fibers with regard to cell behavior. a previous study by Iwasaki et al., who reported that adhesion
of human promyelocytic leukemia cells and human uterine
3.7. Comparison of Platelet and PDL Cell Adhesion on PMEA cervical cancer cells was completely suppressed on PMPC
and PMPC. Figure 8(a) shows the adherent platelets on [36]. We previously reported that, we previously reported that
each polymer as determined by SEM. Figure 8(b) shows PMEA has excellent biocompatibility with human blood cells
the number of adherent platelets and PDL cells on each [15]. As shown in Figures 8(a)-8(b), the number of platelets
polymer relative to that on PET. Platelet adhesion on PMEA on PMEA was not significantly different from the number of
and PMPC was low, and the number of platelets on PMEA platelets onPMPC. In contrast, PDL cells adhered to PMEA,
was not significantly different from that on PMPC. Many and the number of PDL cells on PMEA was higher than that
adherent PDL cells were observed on PMEA and PET, on PHEMA and PMPC (Figure 8(b)). These findings raise the
whereas relatively few adherent PDL cells were observed on question of why PDL cells adhere to biocompatible PMEA but
PHEMA and PMPC. PDL cells adhered to PMEA, whereas platelets do not, especially when both platelets and PDL cells
platelets hardly adhered to PMEA. In contrast, both platelets do not adhere to PMPC in a limited manner. Although we
and PDL cells did not adhere to PMPC. have no clear evidence to answer this question or to explain
why PDL cells demonstrate higher growth rates on PMEA,
3.7.1. Relationship between Biocompatibility and Cell Adhe- we can offer the following speculations in terms of the 3 steps
sions. Conventional synthetic biocompatible polymers such required for cell adhesion on polymer surfaces.
as PMPC, poly(sulfobetaine methacrylate), poly(carboxybe-
taine methacrylate), and poly(ethylene glycol) (PEG) are 3.7.2. Relationship between Adsorbed Proteins and Cell Adhe-
known to demonstrate low protein adsorption and/or no sion. Initially, when a polymer surface comes in contact with
10 BioMed Research International

Top view Cross section Top view Cross section Top view Cross section
1h

1h
1h
4h

4h
4h

Day 1
Day 1

Day 1
Day 3

Day 3

Day 3
(a) (b) (c)
PET/PHEMA PMEA
(i) Vinculin spot (i) Vinculin spot
FA and/or non-FA FA and/or non-FA

(ii) Vinculin rod

FA + non-FA

(iii) Vinculin fiber (iii) Vinculin fiber

FA + FA or FA + FA or
FA + non-FA FA + non-FA
(d)

Figure 7: Localization of nucleus, actin, and vinculin in adherent PDL cells on polymer surfaces. Scale bars: 10 𝜇m. In the cross-section
images, the top panel shows the nucleus (blue), the second panel shows the nucleus and actin (green), the third panel shows the nucleus and
vinculin (red), and the bottom panel shows a merged image. The time points are 1 h, 4 h, 1 day, and 3 days. (a) PDL cells on PET. (b) PDL cells
on PMEA. (c) PDL cells on PHEMA. White arrows indicate focal adhesions that were localized at the basal cell surface. Yellow arrows indicate
nonfocal adhesions (non-FA) localized at the apical cell surface. White arrowheads indicate vinculin rods that were connected vertically and
penetrated the adherent cell. White circles indicate vinculin fibers that were mainly oriented along the long axis of the adherent cells. (d)
Schematic representation of vinculin fiber formation.

cell culture medium, it absorbs water, and a specific water conformational change of the adsorbed proteins occurs, and
structure is formed on the polymer surface [21]. On PET many cell-binding sites are exposed. Intermediate water in
or PHEMA, the absorbed water creates a nonfreezing water PMEA does not induce a conformational change in the
layer and a free water layer. We have also reported that PMEA adsorbed proteins (bovine serum albumin and fibrinogen),
and PMPC form another layer called the intermediate water and, thus, potential platelet-binding sites are minimally
layer [20, 23, 40]. exposed [15, 20].
Proteins in the cell culture medium then adsorb to the Finally, platelets and PDL cells adhere to the cell-
water layer. When proteins adsorb to the nonfreezing water binding sites of the adsorbed proteins. We recently found
layer on polymer surfaces such as PET and PHEMA, a strong that adsorption-induced deformation of fibrinogen (platelet
BioMed Research International 11

PET PMEA PHEMA PMPC

(a)
1.8
NS
1.6
Initial cell adhesion (relative to PET)

1.4 NS
1.2 ∗∗

1.0
0.8
0.6 ∗
0.4
∗∗
0.2
0
PET PMEA PHEMA PMPC
(b)

Figure 8: Comparison of platelet and PDL cell adhesion after 1 h. (a) SEM images of adherent platelets on polymer surfaces. Top scale bars:
10 𝜇m, bottom scale bars: 1.0 𝜇m. Polymers: PET, PMEA, PHEMA, and PMPC. (b) Comparison of platelet and PDL cell adhesion after 1 h.
Gray bar indicates platelet adhesion and white bar indicates PDL cell adhesion, respectively, relative to PET. Polymers: PET, PMEA, PHEMA,
and PMPC. ∗∗ 𝑃 < 0.01 and ∗ 𝑃 < 0.05 versus PET, mean ± standard deviation, platelets: 𝑛 = 6, PDL cells: 𝑛 = 9.

adhesion ligand), which is required for the adhesion of

platelets, does not occur on PMEA [41]. In contrast,
Biocompatibility

PMPC PMEA
fibronectin (PDL cell adhesion ligand) was deformed on
PHEMA PMEA [41]. Therefore, we concluded that PDL cells and not
platelets are capable of adhering to PMEA based on this
protein deformation difference between polymer films. We
suppose that the existence of an intermediate water layer
alters the amount of exposed cell-binding sites, which results
in differing cell adhesion on each polymer.
PET
3.7.3. Relationship between Biocompatibility and Intermediate
PDL cell adhesion
Water. In addition, we have reported that hydrated PMPC
and PEG, as well as various proteins and polysaccharides that
Figure 9: Relationship between PDL cell adhesion and biocompat- are well-known biocompatible polymers, contain intermedi-
ibility on PET, PMEA, PHEMA, and PMPC. ate water [20, 23, 42, 43]. In contrast, poorly biocompatible
12 BioMed Research International

polymers do not contain intermediate water [31]. Free water challenge the widely accepted notion that biocompatible
has high mobility and is unable to shield the polymer surface (blood-compatible) polymers (such as PMPC) do not permit
or the nonfreezing water layer on the polymer surface [23]. cell adhesion as shown in Figure 9. We observed PDL cell
Because intermediate water is weakly bound to the polymer adhesion on the biocompatible (blood-compatible) polymer
molecules or to nonfreezing water, it forms a more stable PMEA in the absence of incorporated, substrate-bound, cell-
structure than free water [23]. Based on these findings, adhesive ligands and antibodies. We therefore consider that
we hypothesized that intermediate water, which prevents PMEA could be used in smart biomaterials. Different cell
proteins and platelets from directly contacting the polymer types may thus be selected by PMEA based on differences
surface or nonfreezing water on the polymer surface, plays an in cell adhesion strength. It should be noted that PMEA has
important role in biocompatibility and cell adhesion [23], and been approved by the FDA and can be used in a blood-rich
the amount of intermediate water affects protein adsorption environment. Therefore, biocompatible PMEA may provide
and cell adhesion [20, 44, 45]. an excellent scaffold for tissue engineering using PDL cells in
humans as well as for culturing tissue-derived cells in a blood-
3.7.4. Relationship between Intermediate Water Content and rich environment.
Cell Adhesion. Intermediate water content in hydrated
PMEA (4.5 wt%) [31] prevented platelet adhesion but did not
prevent PDL cell adhesion. In contrast, higher intermediate 4. Conclusion
water content in hydrated PMPC (28.5 wt%) [40] prevented We found that PDL cells, but not platelets, adhered to
the adhesion of both platelets and PDL cells. The value of biocompatible PMEA. We also observed unique vinculin
the intermediate water content in hydrated PMPC reflects localization in non-FAs, vinculin rods, and vinculin fibers. In
the value for the MPC homopolymer because Kitano et al. addition, PDL cells on PMEA proliferated better than those
confirmed that the MPC-rich domain is directed toward the on PHEMA. Therefore, PMEA may provide an excellent
surface in water [32]. We assume that the higher intermediate scaffold material for tissue engineering using PDL cells in
water content in hydrated PMPC relative to PMEA prevented humans and also for culturing tissue-derived cells in a blood-
cell adhesion because the thick intermediate water layer may rich environment.
have shielded the polymer surface or nonfreezing water layer.
In contrast, the thin intermediate water layer in hydrated
PMEA likely prevented platelet adhesion but did not prevent Conflict of Interests
PDL cell adhesion.
The authors declare that there is no conflict of interests
3.7.5. Relationship between Cell Properties and Cell Adhesion. regarding the publication of this paper.
We also consider the possibility that cell characteristics
affected cell adhesion. Platelets are floating cells and mainly Acknowledgments
adhere to surfaces via glycoprotein IIb/IIIa [46]. PDL cells
are anchorage-dependent and mainly adhere to surfaces The authors thank Ayano Sasaki for introduction to AFM
via integrin 𝛼5 𝛽1 [47]. Furthermore, differences of cell size measurements. This work is supported by Grants-in-Aid
and weight are present, as PDL cells are larger (10–15 𝜇m) and Special Coordination Funds for Promoting Science and
and heavier than platelets (2–4 𝜇m); therefore, PDL cells Technology of the Ministry of Education, Culture, Sports,
may demonstrate increased adhesion simply because of their Science and Technology of Japan. This work is also part
weight. In our next study, we will attempt to clarify the of the financial support from Funding Program for Next
molecular mechanisms underlying cell adhesion on PMEA Generation World-Leading Researchers (NEXT Program,
with regard to the intermediate water content and cell Japan). Erika Kitakami is also supported in part by a Grant-
adhesion. in-Aid for Japan Society of the Promotion of Science (JSPS)
Fellows, Grant no. 248746, Japan.
3.8. PMEA: Applications for Tissue Engineering Scaffolds in a
Blood-Rich Environment. As shown in Figures 2, 3(a), 5, and References
8(b), PDL cells adhered to and proliferated on PET, whereas
PET was not biocompatible for human platelets. PDL cells [1] B. L. Pihlstrom, B. S. Michalowicz, and N. W. Johnson, “Peri-
on PHEMA proliferated but had aggregated (Figures 2 and odontal diseases,” The Lancet, vol. 366, no. 9499, pp. 1809–1820,
5), and PHEMA was thus also found to be nonbiocompatible 2005.
for tissue engineering using PDL cells. As shown in Figures [2] H. Choi, W. Noh, J. Park, J. Lee, and J. Suh, “Analysis of
2, 3(a), 5, and 8(b), PDL cells adhered and proliferated on gene expression during mineralization of cultured human
biocompatible PMEA without platelet adhesion; however, periodontal ligament cells,” Journal of Periodontal and Implant
PDL cells did not adhere and proliferate on biocompatible Science, vol. 41, no. 1, pp. 30–43, 2011.
PMPC. PMEA and PMPC have been previously identi- [3] B. M. Seo, M. Miura, S. Gronthos et al., “Investigation of multi-
fied as blood-compatible (nonplatelet-adhesive) polymers. potent postnatal stem cells from human periodontal ligament,”
However, recent advances in medicine require the use of The Lancet, vol. 364, no. 9429, pp. 149–155, 2004.
blood-compatible polymers that do not exhibit blood cell [4] I. C. Gay, S. Chen, and M. MacDougall, “Isolation and char-
attachment to isolate stem cells from blood. Our results acterization of multipotent human periodontal ligament stem
BioMed Research International 13

cells,” Orthodontics & Craniofacial Research, vol. 10, no. 3, pp. in a porcine model,” Annals of Thoracic Surgery, vol. 71, no. 5,
149–160, 2007. pp. 1603–1608, 2001.
[5] N. Wada, D. Menicanin, S. Shi, P. M. Bartold, and S. Gron- [20] M. Tanaka, T. Hayashi, and S. Morita, “The roles of water
thos, “Immunomodulatory properties of human periodontal molecules at the biointerface of medical polymers,” Polymer
ligament stem cells,” Journal of Cellular Physiology, vol. 219, no. Journal, vol. 45, no. 7, pp. 701–710, 2013.
3, pp. 667–676, 2009.
[21] M. Tanaka, A. Mochizuki, T. Motomura, K. Shimura, M. Onishi,
[6] F. Feng, K. Akiyama, Y. Liu et al., “Utility of PDL progenitors for and Y. Okahata, “In situ studies on protein adsorption onto
in vivo tissue regeneration: a report of 3 cases,” Oral Diseases, a poly(2-methoxyethylacrylate) surface by a quartz crystal
vol. 16, no. 1, pp. 20–28, 2010. microbalance,” Colloids and Surfaces A: Physicochemical and
[7] Y. Nomura, M. Ishikawa, Y. Yashiro et al., “Human periodontal Engineering Aspects, vol. 193, no. 1–3, pp. 145–152, 2001.
ligament fibroblasts are the optimal cell source for induced
[22] M. Tanaka, A. Mochizuki, T. Shiroya et al., “Study on kinetics of
pluripotent stem cells,” Histochemistry and Cell Biology, vol. 137,
early stage protein adsorption on poly(2-methoxyethylacrylate)
no. 6, pp. 719–732, 2012.
(PMEA) surface,” Colloids and Surfaces A: Physicochemical and
[8] L. Petrovic, A. K. Schlegel, S. Schultze-Mosgau, and J. Wiltfang, Engineering Aspects, vol. 203, no. 1–3, pp. 195–204, 2002.
“Different substitute biomaterials as potential scaffolds in tissue
engineering,” International Journal of Oral and Maxillofacial [23] M. Tanaka, “Design of novel 2D and 3D biointerfaces using self-
Implants, vol. 21, no. 2, pp. 225–231, 2006. organization to control cell behavior,” Biochimica et Biophysica
Acta, vol. 1810, no. 3, pp. 251–258, 2011.
[9] R. Schmelzeisen, R. Shimming, and M. Sittinger, “Making bone:
implant insertion into tissue-engineered bone for maxillary [24] M. J. Dalby, N. Gadegaard, R. Tare et al., “The control of human
sinus floor augmentation-a preliminary report,” Journal of mesenchymal cell differentiation using nanoscale symmetry
Cranio-Maxillofacial Surgery, vol. 31, no. 1, pp. 34–39, 2003. and disorder,” Nature Materials, vol. 6, no. 12, pp. 997–1003,
[10] J. P. Davidas, “Looking for a new international standard for 2007.
characterization, classification and identification of surfaces in [25] M. Tanaka, K. Nishikawa, H. Okubo et al., “Control of hep-
implantable materials: the long march for the evaluation of atocyte adhesion and function on self-organized honeycomb-
dental implant surfaces has just begun,” Poseido, vol. 2, no. 1, patterned polymer film,” Colloids and Surfaces A: Physicochem-
pp. 1–5, 2014. ical and Engineering Aspects, vol. 284-285, pp. 464–469, 2006.
[11] D. M. Dohan Ehrenfest, B. S. Kang, G. Sammartino et al., [26] A. Saminathan, K. J. Vinoth, H. H. Low, T. Cao, and M.
“Guidelines for the publication of articles related to implant C. Meikle, “Engineering three-dimensional constructs of the
surfaces and design from the POSEIDO: a standard for surface periodontal ligament in hyaluronan-gelatin hydrogel films and
characterization,” Poseı́do, vol. 1, no. 1, pp. 7–15, 2013. a mechanically active environment,” Journal of Periodontal
[12] N. Metoki, L. Liu, E. Beilis, N. Eliaz, and D. Mandler, Research, vol. 48, no. 6, pp. 790–801, 2013.
“Preparation and characterization of alkylphosphonic acid self- [27] M. Nune, P. Kumaraswamy, U. M. Krishnan, and S. Sethuraman,
assembled monolayers on titanium alloy by chemisorptions and “Self-assembling peptide nanofibrous scaffolds for tissue engi-
electrochemical deposition,” Langmuir, vol. 30, no. 23, pp. 6791– neering: novel approaches and strategies for effective functional
6799, 2014. regeneration,” Current Protein and Peptide Science, vol. 14, no. 1,
[13] R. França, T. D. Samani, G. Bayade, L. Yahia, and E. Sacher, pp. 70–84, 2013.
“Nanoscale surface characterization of biphasic calcium phos- [28] B. Geiger, A. Bershadsky, R. Pankov, and K. M. Yamada,
phate, with comparisons to calcium hydroxyapatite and 𝛽- “Transmembrane extracellular matrix-cytoskeleton crosstalk,”
tricalcium phosphate bioceramics,” Journal of Colloid and Inter- Nature Reviews Molecular Cell Biology, vol. 2, no. 11, pp. 793–
face Science, vol. 420, pp. 182–188, 2014. 805, 2001.
[14] M. Catauro, F. Bollino, and F. Papale, “Preparation, char-
[29] P. Kanchanawong, G. Shtengel, A. M. Pasapera et al., “Nanoscale
acterization, and biological properties of organic-inorganic
architecture of integrin-based cell adhesions,” Nature, vol. 468,
nanocomposite coatings on titanium substrates prepared by sol-
no. 7323, pp. 580–584, 2010.
gel,” Journal of Biomedical Materials Research A, vol. 102, no. 2,
pp. 392–399, 2014. [30] H. Ishihata, M. Tanaka, N. Iwama et al., “Proliferation of
[15] M. Tanaka, T. Motomura, M. Kawada et al., “Blood compatible periodontal ligament cells on biodegradable honeycomb film
aspects of poly(2-methoxyethylacrylate) (PMEA)-relationship scaffold with unified micropore organization,” Journal of Biome-
between protein adsorption and platelet adhesion on PMEA chanical Science and Engineering, vol. 5, no. 3, pp. 252–261, 2010.
surface,” Biomaterials, vol. 21, no. 14, pp. 1471–1481, 2000. [31] M. Tanaka and A. Mochizuki, “Effect of water structure
[16] X. M. Mueller, D. Jegger, M. Augstburger, J. Horisberger, and L. on blood compatibility-thermal analysis of water in
K. von Segesser, “Poly2-methoxyethylacrylate (PMEA) coated poly(meth)acrylate,” Journal of Biomedical Materials Research
oxygenator: An ex vivo study,” The International Journal of A, vol. 68, no. 4, pp. 684–695, 2004.
Artificial Organs, vol. 25, no. 3, pp. 223–229, 2002. [32] H. Kitano, M. Imai, T. Mori, M. Gemmei-Ide, Y. Yokoyama, and
[17] B. L. Greenfield, K. R. Brinkman, K. L. Koziol et al., “The K. Ishihara, “Structure of water in the vicinity of phospholipid
effect of surface modification and aprotinin on cellular injury analogue copolymers as studied by vibrational spectroscopy,”
during simulated cardiopulmonary bypass,” The Journal of Langmuir, vol. 19, no. 24, pp. 10260–10266, 2003.
Extra-Corporeal Technology, vol. 34, no. 4, pp. 267–270, 2002. [33] G. Peluso, O. Petillo, J. M. Anderson et al., “The differen-
[18] F. D. Rubens, “Cardiopulmonary bypass technology transfer: tial effects of poly(2-hydroxyethyl methacrylate) and poly(2-
musings of a cardiac surgeon,” Journal of Biomaterials Science, hydroxyethyl methacrylate)/poly(caprolactone) polymers on
Polymer Edition, vol. 13, no. 4, pp. 485–499, 2002. cell proliferation and collagen synthesis by human lung fibrob-
[19] H. Suhara, Y. Sawa, M. Nishimura et al., “Efficacy of a new lasts,” Journal of Biomedical Materials Research, vol. 34, no. 3,
coating material, PMEA, for cardiopulmonary bypass circuits pp. 327–336, 1997.
14 BioMed Research International

[34] V. Dugina, L. Fontao, C. Chaponnier, J. Vasiliev, and G.

Gabbiani, “Focal adhesion features during myofibroblastic dif-
ferentiation are controlled by intracellular and extracellular
factors,” Journal of Cell Science, vol. 114, no. 18, pp. 3285–3296,
2001.
[35] K. Ishihara, H. Nomura, T. Mihara, K. Kurita, Y. Iwasaki, and N.
Nakabayashi, “Why do phospholipid polymers reduce protein
adsorption?” Journal of Biomedical Materials, vol. 39, no. 2, pp.
323–330, 1998.
[36] Y. Iwasaki, E. Tabata, K. Kurita, and K. Akiyoshi, “Selective
cell attachment to a biomimetic polymer surface through the
recognition of cell-surface tags,” Bioconjugate Chemistry, vol. 16,
no. 3, pp. 567–575, 2005.
[37] F. Zhang, G. Li, P. Yang, W. Qin, C. Li, and N. Huang, “Fabri-
cation of biomolecule-PEG micropattern on titanium surface
and its effects on platelet adhesion,” Colloids and Surfaces B:
Biointerfaces, vol. 102, pp. 457–465, 2013.
[38] Y. Inoue and K. Ishihara, “Reduction of protein adsorption on
well-characterized polymer brush layers with varying chemical
structures,” Colloids and Surfaces B: Biointerfaces, vol. 81, no. 1,
pp. 350–357, 2010.
[39] T. Kondo, K. Nomura, M. Gemmei-Ide et al., “Structure of
water at zwitterionic copolymer film-liquid water interfaces as
examined by the sum frequency generation method,” Colloids
and Surfaces B: Biointerfaces, vol. 113, pp. 361–367, 2014.
[40] T. Hatakeyama, M. Tanaka, and H. Hatakeyama, “Studies on
bound water restrained by poly(2-methacryloyloxyethyl phos-
phorylcholine): comparison with polysaccharide-water sys-
tems,” Acta Biomaterialia, vol. 6, no. 6, pp. 2077–2082, 2010.
[41] T. Hoshiba, M. Nikaido, and M. Tanaka, “Characterization
of the attachment mechanisms of tissue-derived cell lines to
blood-compatible polymers,” Advanced Healthcare Materials,
vol. 3, no. 5, pp. 775–784, 2014.
[42] T. Hatakeyma, H. Kasuga, M. Tanaka, and H. Hatakeyama,
“Cold crystallization of poly(ethylene glycol)-water systems,”
Thermochimica Acta, vol. 465, no. 1-2, pp. 59–66, 2007.
[43] M. Tanaka and A. Mochizuki, “Clarification of blood compat-
ibility mechanism by controlling water structure,” Journal of
Biomaterials Science Polymer Edition, vol. 21, no. 14, pp. 1849–
1863, 2010.
[44] M. Tanaka, E. Kitakami, S. Yagi et al., “Biocompatible polymers
with intermediate water,” Patent 256467, Japan, 2010.
[45] T. Hayashi, Y. Tanaka, Y. Koide, M. Tanaka, and M. Hara,
“Mechanism underlying bioinertness of self-assembled mono-
layers of oligo(ethyleneglycol)-terminated alkanethiols on gold:
protein adsorption, platelet adhesion, and surface forces,” Phys-
ical Chemistry Chemical Physics, vol. 14, no. 29, pp. 10196–10206,
2012.
[46] S. J. Shattil, H. Kashiwagi, and N. Pampori, “Integrin signaling:
the platelet paradigm,” Blood, vol. 91, no. 8, pp. 2645–2657, 1998.
[47] S. Ivanovski, M. Komaki, P. M. Bartold, and A. S. Narayanan,
“Periodontal-derived cells attach to cementum attachment
protein via 𝛼5𝛽1 integrin,” Journal of Periodontal Research, vol.
34, no. 3, pp. 154–159, 1999.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 320790, 9 pages
http://dx.doi.org/10.1155/2014/320790

Research Article
Physicochemical Characteristics of Bone Substitutes Used in
Oral Surgery in Comparison to Autogenous Bone

Antoine Berberi,1 Antoine Samarani,2 Nabih Nader,1 Ziad Noujeim,1

Maroun Dagher,3 Wasfi Kanj,1 Rita Mearawi,1 Ziad Salemeh,1 and Bassam Badran2
1
School of Dentistry, Lebanese University, P.O. Box 4, Hadath, Lebanon
2
Ecole Doctorale, PRASE, Lebanese University, P.O. Box 4, Hadath, Lebanon
3
Faculty of Dental Medicine, Saint Joseph University, P.O. Box 17-5208, Beirut, Lebanon

Correspondence should be addressed to Antoine Berberi; [email protected]

Received 11 May 2014; Accepted 8 June 2014; Published 8 July 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Antoine Berberi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Bone substitutes used in oral surgery include allografts, xenografts, and synthetic materials that are frequently used to compensate
bone loss or to reinforce repaired bone, but little is currently known about their physicochemical characteristics. The aim of
this study was to evaluate a number of physical and chemical properties in a variety of granulated mineral-based biomaterials
used in dentistry and to compare them with those of autogenous bone. Autogenous bone and eight commercial biomaterials of
human, bovine, and synthetic origins were studied by high-resolution X-ray diffraction, atomic absorption spectrometry, and
laser diffraction to determine their chemical composition, calcium release concentration, crystallinity, and granulation size. The
highest calcium release concentration was 24. 94 mg/g for Puros and the lowest one was 2.83 mg/g for Ingenios 𝛽-TCP compared
to 20.15 mg/g for natural bone. The range of particles sizes, in terms of median size D50, varied between 1.32 𝜇m for BioOss and
902.41 𝜇m for OsteoSponge, compared to 282.1 𝜇m for natural bone. All samples displayed a similar hexagonal shape as bone,
except Ingenios 𝛽-TCP, Macrobone, and OsteoSponge, which showed rhomboid and triclinic shapes, respectively. Commercial
bone substitutes significantly differ in terms of calcium concentration, particle size, and crystallinity, which may affect their in vivo
performance.

1. Introduction Autogenous bone is osteogenic (cells within a donor graft

synthesize new bone at implantation sites), osteoinductive
Bone is a living tissue that serves for structural support and (new bone is formed by active recruitment of host mesenchy-
calcium metabolism. Bone matrix is organic and consists of a mal stem cells from surrounding tissue, which differentiate
network of collagen protein fibers impregnated with mineral into bone-forming osteoblasts), osteoconductive (vascular-
salts (85% of calcium phosphate, 10% of calcium carbonate,
ization and new bone formation into the transplant), and
and 5% of calcium fluoride and magnesium fluoride). The
highly biocompatible [3, 4]. These characteristics should be
mineral compartment of bone is predominantly present in
present in an ideal substitute and all bone grafting materials
the form of calcium hydroxyapatites (Ca10 [PO4 ]6 [OH]2 ).
Bone tissue also contains negligible quantities of noncollagen can be classified according to these characteristics [5].
proteins, including the family of bone morphogenetic pro- Bone substitutes (BS) are frequently used in oral and
teins (BMPs) [1]. maxillofacial surgery, periodontics, and orthopedics. They
Calcium (Ca) plays an important role in osteoconductiv- include inorganic or organic, natural or synthetic materials to
ity and may enhance bone tissue integration by entrapping compensate for bone loss or to reinforce new bone ingrowth
and concentrating circulating bone growth factors (BMPs) into defect sites [6–17]. This second option is, in fact, the role
and osteoprogenitor cells, thus imparting osteoinductive played by calcium phosphate and constitutes materials that
properties to calcium-based bone graft materials [2, 3]. show the closet similarity to the mineral component of bone
2 BioMed Research International

[14]. The greatest success in bone grafting has been achieved used for dental applications as bone substitutes and to
with autogenous bone (gold standard), which fulfills all compare them with autogenous bone.
essential physicochemical and biological properties needed
in a bone graft material, despite its inherent limitations in 2. Materials and Methods
availability and postoperative pain at donor sites [6, 8, 10, 11,
13, 18–20]. This study evaluated the physicochemical characteristics of
Numerous BS biomaterials have been successfully used, the eight commercially available bone substitutes of human,
such as allografts (human), xenografts (porcine, equine, bovine, and synthetic origins. Each material was used in
or bovine, and synthetic calcium-based materials (cal- its lowest available particle size range, and all samples were
cium phosphates [𝛽-tricalcium phosphate/𝛽-TCP, hydrox- obtained directly from their manufacturers in sealed vials and
yapatite/HA], bioactive glasses), calcium sulfate, calcium evaluated without alteration.
hydroxide), and a combination of these with or without the
use of membrane and screws [6–23]. (i) DynaBlast (Keystone Dental, Inc., Burlington, MA) is
a combination of mineralized and demineralized allo-
Allografts do not have the drawbacks of autografts but are
genic bone that is mixed with a proprietary poloxamer
less successful in clinical practice. They also display several
reverse-phase resorbable medium and processed into
other disadvantages: risk of disease transmission or infec-
a paste or puttylike form [28, 36].
tion, difficulties in obtaining and processing, possible rapid
resorption, [8–10, 24], and partial loss of mechanical strength (ii) Puros bone allograft (Zimmer Dental, Inc., Carls-
after sterilization [25, 26]. Xenogenic bone substitutes of bad, CA) is an allogenic graft material treated by a
porcine, bovine, or, more recently, equine origin are used proprietary process (Tutoplast, RTI Biologics, Inc.,
because of their chemical and structural composition simi- Alachua, FL) designed to inactivate pathogens and
larity when compared to human bone [27]. They represent remove fat, cells, and antigens, while preserving the
an unlimited supply of available material and may reduce minerals and collagen matrix of the native bone
morbidity by eliminating the donor site [5, 10, 22, 23]. Heat or tissue. After processing, the material is preserved by
other treatments are used to deproteinate bone particles and solvent dehydration, which can also help to ensure
eliminate immunogenicity risks [25, 28]. Synthetic calcium pathogen inactivation [13]. It is available in cortical,
phosphate ceramics with their excellent biocompatibility are cancellous, and a cortical-cancellous mix in particle
common alternatives to autogenous bone [15]. sizes ranging from 0.25 to 2 mm [26, 37].
Ideally, a BS should have specific biological and clinical (iii) OsteoSponge allograft (Bacterin International, Inc.,
particularities. Biologically, it should mediate recruitment of Belgrade, MT) consists of 100% of demineralized
mesenchymal cells derived from host site and have bioactive human cancellous bone, with no additional carrier
effects on ossification (osteoinduction). Furthermore, it must materials. It is prepared using undisclosed methods
be osteoconductive, providing three-dimensional scaffolds that reportedly preserve native growth factors [25].
for the ingrowth of vessels and osteoprogenitor cells. Finally, The granule size varies from 1 to 4 mm.
it should be resorbable. Clinically, a BS should be easy to (iv) BioOss (Geistlich Pharma AG, Wolhusen, Switzer-
use, cost effective, and with adequate density to allow easy land) xenogenic spongiosa granules are reported to
radiographic recognition during the entire healing process be a natural bone mineral derived from bovine bone
[27, 29]. This feature is particularly important to radiograph- which contain carbonate apatite. Granules are ren-
ically follow the rate of resorption/substitution [30, 31]. dered nonorganic through a proprietary extraction
Regarding material structure, particle size affects not only process that involves treatment with strong alkalis and
contact area but also the packing characteristics of the mate- organic solvents under high-temperature processing
rials, which ultimately determines the macroporosity of a up to 300∘ C, which allegedly renders the substrate
particulate graft [32, 33]. It is also known that pore size exerts antigenic and protein-free [37]. The material was used
a major influence over the interaction of osteogenic cells in granules of 0.25–1 mm.
with the biomaterial surface [34, 35]. Biological integration
requires pores that are greater than 100–150 mm in diameter (v) Cerabone (AAP Biomaterials GmbH, Berlin, Ger-
to provide a blood supply to the tissues [27]. A BS should many) xenogenic granulate is a bovine bone material
gradually degrade with time until it is completely replaced sintered at high temperature (>1200∘ C), which retains
with vital new bone tissue. Moreover, a material’s resorption the inorganic part of bone (hydroxyapatite) [38]. The
rate should match the formation rate of the new bone tissue material used was granulate of 0.5–1.0 mm in size.
[29]. Biomaterial degradation that occurs too rapidly can (vi) Macrobone (Euroteknika Groupe, Sallanches,
exert a negative effect on bone regeneration processes, [24] France) is a high-porosity (90%), synthetic bone sub-
and the presence of residual BS graft particles after bone stitute made of pure 𝛽-TCP that is completely and
healing may lead to composite tissue repair rather than to rapidly resorbable [39]. Particle size varies between
bone tissue regeneration [27]. 0.15 mm and 2 mm.
The aim of this study was to evaluate some of physical (vii) IngeniOs 𝛽-TCP (Zimmer Dental, Inc.) is a bioactive
and chemical properties in a variety of commercially available material made of silicated 𝛽-TCP of non-biologic ori-
granulated mineral-based biomaterials that are frequently gin. The structure is a porous biocompatible synthetic
BioMed Research International 3

scaffold of ceramic material [40]. The size of the par- orthogonal direction to the crystal plane according to the
ticles is 0.25–1 mm. following formula:
(viii) IngeniOs HA (Zimmer Dental Inc.) is a synthetic
spongious bone substitute. The structure is a porous 0.9𝜆
𝑋𝑠 = , (1)
scaffold that resembles cancellous bone. Particles (FWHM × cos 𝜃)
are biocompatible and made of 100% hydroxyapatite
ceramic with a putty phase of ≥95%, and granules where 𝑋𝑠 is the crystallite size in nanometer, 𝜆 is the
range 1-2 mm in size [41]. wavelength of X-ray beam in nanometer (𝜆 = 0.15406 nm in
our case), and FWHM is the full width at half maximum for
(ix) Autogenous bone samples were collected during
the diffraction angle at 2𝜃 = 25.9∘ that was selected according
mandibular third-molar surgery, rinsed with ethanol,
to (002) Miller’s plane family [47].
dried in vacuum at room temperature, ground in
an agate mortar, and sterilized by gamma irradiation
[42, 43]. 3. Results
Atomic Absorption Spectroscopy (AAS) (WFX-210, Ray- AAS results of calcium concentration over the observation
Leigh, BRAIC, China) was used to determine the concentra- period are summarized in Table 1. Cerabone showed less
tion of calcium ions in the bone substitutes by quantifying the calcium release than BioOss. In the synthetic xenograft
release of calcium and phosphorous from the graft material category, Macrobone displayed a high calcium release
in demineralized water. For this aim, standards for calcium concentration (17.30 mg/g), compared to IngeniOs HA
and phosphorous within the range between 0.5 and 10 𝜇g/L (2.92 mg/g) and IngeniOs 𝛽-TCP (2.83 mg/g). In the allograft
were prepared, and 0.4 mg of each biomaterial (all nine group, OsteoSponge revealed the lowest calcium release
samples) was immersed in 100 mL of 0.9% NaCl and the pH concentration (4.05 mg/g). The calcium concentration of
was adjusted at 7 by using hydrochloric acid (0.1 N). The Puros (24.94 mg/g) was comparable to autogenous bone
variation of Ca concentration was determined at D0 (day 0), (20.15 mg/g).
D2 (day two), and each week after, until the sixth week. The The particles median size D50 (in volume percentages),
concentration was calculated based on the Beer-Lambert law the particle size range expressed by the 10% and 90% per-
[44]. centiles (D10 and D90 ), and the particles size ranges reported
LASER Diffraction (LD) was used to determine particle by the manufacturers as determined by the LD measurements
size by evaluating the distribution of the granules using a laser are all presented in Table 2.
scattering particle size analyzer (Patrica LA-950 V2 Horiba Results showed that BioOss had the lowest median
Instruments, Japan). The measurement method relied on the particle size (1.32 𝜇m) followed by Ingenios 𝛽-TCP (6.72 𝜇m),
Mie scattering theory [45]. Using an ultrasonic probe with while OsteoSponge had the highest one (902.41 𝜇m).
measuring time of 20 s at a frequency of 20 kHz, the unit’s The median size of Macrobone (262.37 𝜇m) was close to
measuring range varied between 0.01 and 3.00 𝜇m. The devise autogenous bone (282.1 𝜇m). The narrowest size distribution
was equipped with an optical system of two light sources, was observed with BioOss (0.26–8.92 𝜇m), followed by Inge-
a laser diode of approximately 1.6 mW with 𝜆 = 650 nm, nios 𝛽-TCP (3.90–15.18 𝜇m). The widest size distribution was
and a 405 nm light emitting diode of approximately 0.3 mW. observed with OsteoSponge (174.62–2301.84 𝜇m) followed by
Large particles scatter light at small angles relative to the DynaBlast (39.24–1754.62 𝜇m).
laser beam and small particles scatter light at large angles. X-ray diffractograms for all bone substitutes are shown
The particle size is reported as a volume equivalent sphere in Figure 1. They represent the intensity of X-ray (cps) as a
diameter [43, 46]. Samples were well mixed and homogenized function of the diffraction angles (2 theta, 𝜃).
in their powder state prior to their analysis. Average particle Results of the XRD experiments that are indicative for
size and distribution were calculated for all nine biomaterials the chemical composition of the BS are shown in Table 3
and autogenous bone. except for DynaBlast, as the puttylike material was not
X-ray Diffraction (XRD) (D8 Advance, Bruker Corpora- granular in form. All study materials showed small amounts
tion, Billerica, MA) was used to identify phase and composi- of impurities. These materials diffract more and less the X-
tion features and qualitatively evaluate the crystallinity of all ray, which means diverse degrees of crystallinity, as indicated
study materials. Homogenized powder samples (1-2 g) were in the different peaks widths.
compressed in polyvinyl chloride lenses (diameter 2.5 cm, The common crystal phase was calcium phosphate silicate
thickness 2 mm) and measured using a diffract meter (copper hydroxide (Ca5 (PO4 )2.85 (SiO4 )0.15 (OH)) in BioOss, Ingenios
anticathode 𝜆K𝛼 = 0.154060 nm). A range of 2𝜃 between 𝑥∘ HA, Puros, OsteoSponge, and autogenous bone. Macrobone
and 𝑦∘ was chosen to obtain maximum information about was composed from calcium phosphate (Ca3 (PO4 )2 ). Cer-
crystal phases. Collected diffract grams were analyzed by abone and Ingenios 𝛽-TCP were composed, respectively,
software EVA (EVA, Bruker Corporation) based on powder of calcium gadolinium oxide phosphate (Ca8 Gd2 (PO4 )6O2 )
diffraction files provided by the International Center for and sodium calcium iron phosphate (Na2 Ca19 Fe0.667 (PO4 )14 )
Diffraction Data (Newtown Square, PA). Crystallite size as the main crystal phases. Except for Ingenios 𝛽-TCP,
analysis was calculated using the peak broadening of XRD Macrobone, and Osteosponge, all samples were crystallized
reflection that is used to estimate the crystallite size in an at different levels of crystallinity in hexagonal systems.
 4

Table 1: The calcium concentration as derived from AAS experiments by brand names and time period.
Ca (mg/g) BioOss Cerabone Macrobone Ingenios B-TCP Ingenios HA Puros OsteoSponge Dyna Blast Autogenous bone
Day 0 1.977 0.98 1.4485 0.892 0.7485 2.104 1.521 2.768 3.77
Day 2 2.643 1.061 3.0735 1.276 2.3862 3.613 1.723 3.7132 4.2
Week 1 7.849 2.605 10.9865 1.663 2.5515 6.99 1.859 4.871 6.73
Week 3 8.79 3.308 15.301 2.306 2.7427 15.1535 2.018 5.6507 16.4
Week 4 9.451 3.445 16.081 2.682 2.7502 18.879 2.18 5.8985 17.96
Week 5 10.205 4.023 16.11 2.834 2.8282 23.11 2.493 6.0485 19.64
Week 6 11.8 4.194 17.308 2.835 2.9275 24.942 4.051 6.2 20.15
BioMed Research International
BioMed Research International 5

Table 2: Particle size parameters in volume percentage of the samples.

Median size Size range Size range reported

Sample
(D50 𝜇m) (D10 –D90 𝜇m) By producers (𝜇m)
Bio-Oss 1.32 0.26–8.92 250–1000
Cerabone 663.31 174.62–1337.48 500–1000
Macrobone 262.37 22.79–517.2 150–500
Ingenios B-TCP 6.72 3.90–15.18 250–1000
Ingenios HA 592.39 8.82–1337.48 1000–2000
Puros 630.47 174.62–1167.72 250–2000
OsteoSponge 902.41 152.45–2301.84 1000–4000
Dyna Blast 777.14 39.24–1754.62 Nonindicated
Autogenous bone 282.1 90.5–465.15

Ingenious-HA Ingenious 𝛽-TCP Macrobone

700 280 260
260 240
600 240 220
220 200
500

Lin (Cps)
Lin (Cps)

Lin (Cps)

200 180
180 160
400 160 140
140 120
300 120 100
100 80
200 80 60
60
100 40 40
20 20
0 0 0
3 10 20 30 40 50 60 70 80 3 10 20 30 40 50 60 70 80 5 10 20 30 40 50 60 70 80
2-theta-scale 2-theta-scale 2-theta-scale

Cerabone BioOss
200
180
90
160 80
70
Lin (Cps)

140
60
Lin (Cps)

120
100 50
80 40
60 30
40 20
20 10
0 0
3 10 20 30 40 50 60 70 80 3 10 20 30 40 50 60 70 80
2-theta-scale 2-theta-scale
(a)
OsteoSponge Puros Autogeneous bone
400 70 110
100
60 90
300
Lin (Cps)

50 80
Lin (Cps)

Lin (Cps)

70
200 40 60
30 50
40
100 20 30
10 20
10
0 0 0
3 10 20 30 40 50 60 70 80 3 10 20 30 40 50 60 70 80 3 10 20 30 40 50 60 70 80
2-theta-scale 2-theta-scale 2-theta-scale
(b)

Figure 1: X-ray diffraction data for all investigated samples. (a) Ingenios HA, Ingenios 𝛽-TCP, Marrowbone, Cerabone, and BioOss have well
defined peaks, which reflects their well-crystallized components. (b) OsteoSponge, Puros, and autogenous bone have noisy diffractograms
revealing less crystallinity.

The 𝑎/𝑐 or 𝑏/𝑐 ratios (9.42/6.89) indicated a flat structure other bones crystallized in hexagonal system. Nevertheless,
parallel to 𝐴 6 axis. Such geometry may enhance the settle- 𝑐 length (37.3 Å) in Ingenios 𝛽-TCP and Macrobone was
ment properties of these particles. For Ingenios 𝛽-TCP and 5.4 times greater than 𝑐 (6.89 Å) length in the other bones.
Macrobone, 𝑎 and 𝑏 crystal dimensions in the rhombohedra Hence, settlement may be oriented preferably orthogonal to
system were relatively too close to 𝑎 and 𝑏 dimensions of the 𝐴 3 axis. In Ingenios 𝛽-TCP and Macrobone, crystal size was
 6

Table 3: Chemical compositions and shapes of samples, except for Dyna Blast due to its puttylike form, as derived from X-ray diffraction.
Product Compound name Formula System 𝑎 (Å) 𝑏 (Å) 𝑐 (Å) Alpha (∘ ) Beta (∘ ) Gamma (∘ )
Bio-Oss Calcium phosphate silicate hydroxide Ca5 (PO4 )2.85 (SiO4 )0.15 (OH) Hexagonal 9.42 9.42 6.89 90 90 120
Cerabone Calcium gadolinium oxide phosphate Ca8 Gd2 (PO4 )6 O2 Hexagonal 9.39 9.39 6.89 90 90 120
Macrobone Calcium phosphate Ca3 (PO4 )2 Rhomboid 10.4 10.4 37.4 90 90 120
Ingenios B-TCP Sodium calcium iron phosphate Na2 Ca19 Fe0.667 (PO4 )14 Rhomboid 10.4 10.4 37.3 90 90 120
Ingenios HA Calcium phosphate silicate hydroxide Ca5 (PO4 )2.85 (SiO4 )0.15 (OH) Hexagonal 9.42 9.42 6.89 90 90 120
Puros Calcium phosphate silicate hydroxide Ca5 (PO4 )2.85 (SiO4 )0.15 (OH) Hexagonal 9.42 9.42 6.89 90 90 120
OsteoSponge Calcium phosphate silicate hydroxide Ca5 (PO4 )2.85 (SiO4 )0.15 (OH) Triclinic 6.25 11.9 5.6 97 114 93
Dyna Blast
Autogenous Bone Calcium phosphate silicate hydroxide Ca5 (PO4 )2.85 (SiO4 )0.15 (OH) Hexagonal 9.42 9.42 6.89 90 90 120
BioMed Research International
BioMed Research International 7

30 between the sizes of particles and calcium concentration with

the time (Figure 2).
X-ray diffractograms give a clear idea about the crys-
25
tallinity of the analyzed materials and their crystal phases.
HA, Ingenios 𝛽-TCP, Macrobone, Cerabone, and BioOss
have well-defined peaks which reflects their well-crystallized
Calcium release concentration

20
components; OsteoSponge, Puros, and autogenous bone have
noisy diffractograms revealing the presence of amorphous
15 structure or at least noncrystallized faces of the materials.
All bone substitutes show a typical and most intense
diffraction at 2𝜃 of 32∘ since phosphate is the common com-
10 ponent in all used materials. Crystal phases were identified
using the powder diffraction files, provided by the Interactive
Center for Diffraction Data.
5
XRD diffractograms of various materials, including
human bone, were quite similar to common crystal phase
0 calcium phosphate silicate hydroxide (Ca5 (PO4 )2.85 ⋅
0 5 10 15 20 25 30 35 40 45 (SiO4 )0.15 (OH)) except for Cerabone, which showed the
Number of days presence of gadolinium in its composition, and Ingenios
𝛽-TCP, which showed the presence of iron. However,
BioOss Puros silicates, when they are present, are not major components
Cerabone OsteoSponge
of the crystal phases, since their stoichiometry compared to
Macrobone Dyna blast
Ingenios B-TCP Autogenous bone
phosphate (2.85) were considerably negligible (0.15). Iron and
Ingenios HA sodium are also negligible compared to calcium in Ingenios
𝛽-TCP. Along with calcium, they help to compensate the
Figure 2: AAS results from calcium concentration over the obser- negative charges of phosphate. It must be highlighted that,
vation period of all tested bone substitutes. 𝑌-axes represent calcium in bone and all bone substitutes, Ca to P ratios fluctuated
release in mg/g and 𝑋 axes represent day’s number. between 1.75 and 1.33. This could mean that calcium was the
major element that compensated phosphate charges.
It is not clear why gadolinium was present in the crystal
greater than that of the other bones. OsteoSponge was the phase of Cerabone. One possible explanation could be the
only sample crystallized in the triclinic system. iron oxidized at high temperature since the product was
subjected to high-temperature calcination ±1200∘ C [55].
Another explanation could be that it was used for its property
4. Discussion to enhance the resistance of alloys against oxidation. It
should be noted that natural gadolinium occurs in monazite
The higher the calcium concentration in a biomaterial, the mineral (rare earth phosphate) and gadolinium salt has an
more prone it will be to degradation [3, 42, 43]. The acidic exceptionally high absorption of neutrons and therefore is
buffer, to some extent, mimics the acidic environment during used for shielding in radiography as a contrast agent [56].
osteoclastic activity or bone resorption [3, 42, 43]. In our Nevertheless, we could not assert if gadolinium in those
study, different biomaterials had different calcium releasing studied samples naturally occurred or was purposely added.
characteristics. This could be explained by the fact that the XRD diffractograms showed that all samples, including
speed of BS biodegradability in vivo or in vitro depends on the natural bone, proved to have the same anisotropic crystal
material’s composition, particle size, crystallinity, porosity, size (9.42 Å in 𝑎- and 𝑏-directions and 6.89 Å in 𝑐-direction
and preparation [3, 38, 42, 43, 48]. with alpha and beta 90∘ and gamma 120∘ ); Ingenios 𝛽-TCP
From the particle size data, it can be concluded that, and Macrobone showed different anisotropic size 10.4 Å in
in general, size ranges measured for tested materials were 𝑎- and 𝑏-directions and 37,3 Å in 𝑐-direction with alpha
different from those reported by manufacturers who do and beta 90∘ and gamma 120∘ , 6.25 Å in 𝑎-direction, 11.9 Å
not specify the technique used in the crystalline material’s in 𝑏-direction, and 5.6 Å in 𝑐-direction with alpha 97 and
characterization and could explain the noticed differences beta 114∘ and gamma 93∘ , respectively. These results demon-
[49–54]. However, it should be kept in mind that the granules strated that crystal shapes of the BS and autogenous bone
under analysis differed not only in their size but also in their had a similar, hexagonal shape; only Ingenios 𝛽-TCP and
physicochemical properties. Macrobone showed a rhomboid design and OsteoSponge a
The influence of properties and characteristics of BS triclinic shape. This structure is the poorest system in the
on biological response cannot be easily predicted as the symmetric properties [57].
published studies involve different types of BS in different Eight different bone-grafting materials were herein inves-
particle size ranges. Regarding the ranges of particle size that tigated, and the results were compared to autogenous bone.
were tested in the present investigation, there was no relation Even when similar chemical characteristics were found,
8 BioMed Research International

significant differences were detected in terms of calcium con- [6] M. S. Block and J. N. Kent, “Sinus augmentation for dental
centrations, particle sizes, and crystallinity. Although these implants: the use of autogenous bone,” Journal of Oral and
morphological differences greatly influence in vivo behavior Maxillofacial Surgery, vol. 55, no. 11, pp. 1281–1286, 1997.
of the biomaterial, they are often not taken into consideration [7] S. L. Wheeler, “Sinus augmentation for dental implants: the
when the samples’ biological performance is evaluated. It is use of alloplastic materials,” Journal of Oral and Maxillofacial
believed that results provided for biomaterials investigated Surgery, vol. 55, no. 11, pp. 1287–1293, 1997.
will be most useful to fully understand their clinical behavior [8] A. S. Greenwald, S. D. Boden, V. M. Goldberg, Y. Khan, C.
and response. Since the bone substitute of choice depends T. Laurencin, and R. N. Rosier, “Bone-graft substitutes: facts,
largely on the possible clinical application and its associated fictions, and applications,” Journal of Bone and Joint Surgery A,
biological and mechanical needs, it is important not to vol. 83, no. 2, pp. 98–103, 2001.
assume that all bone substitutes will show the same pattern [9] S. N. Parikh, “Bone graft substitutes: past, present, future,”
of performance and that the validation of a bone substitute Journal of Postgraduate Medicine, vol. 48, no. 2, pp. 142–148,
2002.
in one clinical site may not necessarily predict its identical
performance in another anatomical location. Hopefully in the [10] C. G. Finkemeier, “Bone-grafting and bone-graft substitutes,”
Journal of Bone and Joint Surgery A, vol. 84, no. 3, pp. 454–464,
future, hybrid or complex combination products that include
2002.
cells, growth factors, and/or gene therapy in combination
[11] G. K. B. Sandor, T. C. Lindholm, and C. M. L. Clokie, “Bone
will be likely to provide oral surgeons more effective tools
regeneration of the cranio-maxillofacial and dento-alveolar
for bone defects reparation. In this regard, it is obvious that
skeletons in the framework of tissue engineering,” in Topics
further studies are warranted and a new international stan- in Tissue Engineering, N. Ashammakhi and P. Ferretti, Eds.,
dard for characterization, classification, and identification of chapter 7, pp. 1–46, 2003.
implantable materials is needed. [12] B. Ben-Nissan, “Natural bioceramics: from coral to bone and
beyond,” Current Opinion in Solid State and Materials Science,
5. Conclusion vol. 7, no. 4-5, pp. 283–288, 2003.
[13] D. A. di Stefano, L. Artese, G. Iezzi et al., “Alveolar ridge regen-
Commercial bone substitutes significantly differ in terms of eration with equine spongy bone: a clinical, histological, and
calcium concentration, particle size, and crystallinity from immunohistochemical case series,” Clinical Implant Dentistry
autogenous bone, which may affect their clinical applications and Related Research, vol. 11, no. 2, pp. 90–100, 2009.
and performance. [14] M. Vallet-Regı́ and J. M. González-Calbet, “Calcium phosphates
as substitution of bone tissues,” Progress in Solid State Chemistry,
vol. 32, no. 1-2, pp. 1–31, 2004.
Conflict of Interests [15] K. A. Hing, L. F. Wilson, and T. Buckland, “Comparative per-
formance of three ceramic bone graft substitutes,” The Spine
The authors do not have any financial interests in the products Journal, vol. 7, no. 4, pp. 475–490, 2007.
or information listed in this paper. The authors declare that
[16] D. M. Dohan Ehrenfest, B. S. Kang, G. Sammartino et al.,
they have no conflict of interests. “Guidelines for the publication of articles related to implant
surfaces and design from the POSEIDO: a standard for surface
Acknowledgments characterization,” POSEIDO, vol. 1, no. 1, p. 15, 2013.
[17] J. P. Davidas, “Looking for a new international standard for
The authors would like to thank Manal Houhou, Sarah characterization, classification and identification of surfaces in
Hadda, Sahar Rihan, and Hussein Bassal for their excellent implantable materilals: the long march for the evaluation of
technical assistance. This project was supported by a grant dental implant surfaces has just began,” POSEIDO, vol. 2, no.
from the Ecole Doctorale, Lebanese University. 1, pp. 1–5, 2014.
[18] J. M. Rueger, W. Linhart, and D. Sommerfeldt, “Biological
reaction to calcium-phosphate ceramic implants. Results of
References animal experiments,” Orthopade, vol. 27, no. 2, pp. 89–95, 1998.
[1] M. I. Kay, R. A. Young, and A. S. Posner, “Crystal structure of [19] M. Bohner, “Calcium orthophosphates in medicine: from
hydroxyapatite,” Nature, vol. 204, no. 4963, pp. 1050–1052, 1964. ceramics to calcium phosphate cements,” Injury, vol. 31, no. 4,
pp. D37–D47, 2000.
[2] U. Ripamonti and R. M. Klar, “Regenerative frontiers in cran-
[20] M. Bohner, “Physical and chemical aspects of calcium phos-
iofacial reconstruction: Grand challenges and opportunities
phates used in spinal surgery,” European Spine Journal, vol. 10,
for the mammalian transforming growth factor-𝛽 proteins,”
no. 2, pp. S114–S121, 2001.
Frontiers in Physiology, vol. 1, no. 1, article 143, 2010.
[21] I. Bouchlariotou, J. P. Bernard, J. P. Carrel, and L. Vazquez,
[3] R. Z. LeGeros, “Calcium phosphate-based osteoinductive mate- “Long-term stability of osseointegrated implants in bone regen-
rials,” Chemical Reviews, vol. 108, no. 11, pp. 4742–4753, 2008. erated with a collagen membrane in combination with a depro-
[4] H. Orimo, “The mechanism of mineralization and the role of teinized bovine bone graft: 5-year follow-up of 20 implants,”
alkaline phosphatase in health and disease,” Journal of Nippon POSEIDO, vol. 1, no. 1, pp. 45–53, 2013.
Medical School, vol. 77, no. 1, pp. 4–12, 2010. [22] R. Toeroek and D. M. Dohan Ehrenfest, “The concept of Screw-
[5] R. A. Kenley, K. Yim, J. Abrams et al., “Biotechnology and bone Guided Bone Regeneration (S-GBR). Part 3: Fast Screw-Guided
graft substitutes,” Pharmaceutical Research, vol. 10, no. 10, pp. Bone Regeneration (FS-GBR) in the severely resorbed preim-
1393–1401, 1993. plant posterior mandible using allograft and Leukocyte- and
BioMed Research International 9

Platelet-Rich Fibrin (L-PRF): a 4-year follow-up,” POSEIDO, [38] D. Tadic and M. Epple, “A thorough physicochemical charac-
vol. 2, no. 2, pp. 93–100, 2013. terisation of 14 calcium phosphate-based bone substitution
[23] R. Toeroek R and D. M. D. Ehrenfest, “The concept of screw- materials in comparison to natural bone,” Biomaterials, vol. 25,
guided bone regeneration (S-GBR). Part 2: S-GBR in the no. 6, pp. 987–994, 2004.
severely resorbed preimplant posterior mandible using bone [39] D. Chappard, B. Guillaume, R. Mallet, F. Pascaretti-Grizon, M.
xenograft and leukocyte- and platelet-rich fibrin (L-PRF): a 5- F. Baslé, and H. Libouban, “Sinus lift augmentation and 𝛽-TCP:
year follow-up,” POSEIDO, vol. 2, no. 2, pp. 85–92, 2013. a microCT and histologic analysis on human bone biopsies,”
Micron, vol. 41, no. 4, pp. 321–326, 2010.
[24] H. Sung, C. Meredith, C. Johnson, and Z. S. Galis, “The effect of
scaffold degradation rate on three-dimensional cell growth and [40] A. M. Pietak, J. W. Reid, M. J. Stott, and M. Sayer, “Silicon sub-
angiogenesis,” Biomaterials, vol. 25, no. 26, pp. 5735–5742, 2004. stitution in the calcium phosphate bioceramics,” Biomaterials,
vol. 28, no. 28, pp. 4023–4032, 2007.
[25] J. Glowacki, “A review of osteoinductive testing methods and
[41] M.-J. Lee, S.-K. Sohn, K.-T. Kim et al., “Effect of hydroxyapatite
sterilization processes for demineralized bone,” Cell and Tissue
on bone integration in a rabbit tibial defect model,” Clinics in
Banking, vol. 6, no. 1, pp. 3–12, 2005.
Orthopedic Surgery, vol. 2, no. 1, pp. 90–97, 2010.
[26] S. T. Moore, J. M. Katz, R. M. Zhukauskas et al., “Osteoconduc- [42] F. Peters, K. Schwarz, and M. Epple, “The structure of bone
tivity and osteoinductivity of Puros (R) DBM putty,” Journal of studied with synchrotron X-ray diffraction, X-ray absorption
Biomaterials Applications, vol. 26, no. 2, pp. 151–171, 2011. spectroscopy and thermal analysis,” Thermochimica Acta, vol.
[27] T. Traini, A. Piatelli, S. Caputi et al., “Regeneration of human 361, no. 1-2, pp. 131–138, 2000.
bone using different bone substitute biomaterials,” Clinical [43] M. Figueiredo, J. Henriques, G. Martins, F. Guerra, F. Judas,
Implant Dentistry and Related Research, 2013. and H. Figueiredo, “Physicochemical characterization of bio-
[28] C. Schöpf, W. Daiber, and D. Tadic, “Tutoplast processed materials commonly used in dentistry as bone substitutes—
allografts and xenografts,” in 3D Block Technique in from Image comparison with human bone,” Journal of Biomedical Materials
Diagnostics to Block Graft Bone Regeneration, M. Jacotti and P. Research B, vol. 92, no. 2, pp. 409–419, 2010.
Antonelli, Eds., chapter 5, pp. 54–75, RC Libri, Milano, Italy, [44] R. Garcı́a and A. P. Báez, “Atomic Absorption Spectrometry
2005. (AAS),” in Atomic Absorption Spectroscopy, M. A. Farrukh, Ed.,
[29] C. P. Klein, A. A. Driessen, K. de Groot, and A. van den Hooff, chapter 1, pp. 1–13, InTech, 2012.
“Biodegradation behavior of various calcium phosphate mate- [45] T. Wriedt, “Mie theory: a review,” in The Mie Theory, W. Hergert
rials in bone tissue,” Journal of Biomedical Materials Research, and T. Wriedt, Eds., vol. 169 of Springer Series in Optical Sciences,
vol. 17, no. 5, pp. 769–784, 1983. pp. 53–71, Springer, Berlin, Germany, 2012.
[30] S. Platzer, A. Wildburger, M. Lorenzoni et al., “Human cadaver [46] S. Marković, L. Veselinović, M. J. Lukić et al., “Synthetical
study evaluating a new measurement technique for graft vol- bone-like and biological hydroxyapatites: a comparative study
umes after sinus floor elevation,” Clinical Implant Dentistry and of crystal structure and morphology,” Biomedical Materials, vol.
Related Research, vol. 16, no. 2, pp. 212–222, 2012. 6, no. 1, pp. 45–50, 2011.
[47] P. K. Harold and E. L. Alexander, X-Ray Diffraction Procedures:
[31] C. Dellavia, S. Speroni, G. Pellegrini, A. Gatto, and C. Maiorana,
For Polycrystalline and Amorphous Materials, Wiley- Inter-
“A new method to evaluate volumetric changes in sinus aug-
science, New York, NY, USA, 2nd edition, 1974.
mentation procedure,” Clinical Implant Dentistry and Related
[48] G. Hannink and J. J. C. Arts, “Bioresorbability, porosity and
Research, vol. 19, 2013.
mechanical strength of bone substitutes: what is optimal for
[32] Z. Schwartz and B. D. Boyan, “Underlying mechanisms at the bone regeneration?” Injury, vol. 42, no. 2, pp. S22–S25, 2011.
bone-biomaterial interface,” Journal of Cellular Biochemistry, [49] Geistlich Biomaterials Inc., “Product information on BioOss,”
vol. 56, no. 3, pp. 340–347, 1994. http://www.geistlich.ch/index.cfm?dom=2&rub=42&id=100064.
[33] X. Li, C. A. van Blitterswijk, Q. Feng, F. Cui, and F. Watari, “The [50] Bacterin, product information on Osteosponge, 2014, http://
effect of calcium phosphate microstructure on bone-related bacterin.com/products/osteosponge/.
cells in vitro,” Biomaterials, vol. 29, no. 23, pp. 3306–3316, 2008. [51] Zimmer dental, products, regenerative, bone grafts, infor-
[34] A. C. C. da Cruz, M. T. Pochapski, J. B. Daher, J. C. Z. da Silva, G. mation on Puros, HA, 𝛽-TCP, 2014, http://www.zimmerden-
L. Pilatti, and F. A. Santos, “Physico-chemical characterization tal.com/products/regenerative/rg overview.aspx.
and biocompatibility evaluation of hydroxyapatites,” Journal of [52] Botiss Biomaterials, Product Information on Cerabone, 2014,
Oral Science, vol. 48, no. 4, pp. 219–226, 2006. https://www.botiss.com/en/content/cerabone%C2%AE.
[35] A. L. Carvalho, P. E. P. Faria, M. F. M. Grisi et al., “Effects of [53] KeyStone Dental Inc. product information on DynaBlast, 2014,
granule size on the osteoconductivity of bovine and synthetic http://www.keystonedental.com/products/dynablast/.
hydroxyapatite: a histologic and histometric study in dogs,” The [54] Euroteknika, “Product information on MacroBone,” 2014,
Journal of Oral Implantology, vol. 33, no. 5, pp. 267–276, 2007. http://www.euroteknika-implants.com/EN/IMG/pdf/FP Mac-
[36] T. Irinakis, “Efficacy of injectable demineralized bone matrix as robone EN.pdf.
graft material during sinus elevation surgery with simultaneous [55] A. Gschneidner Jr., V. K. Pecharsky, and A. O. Tsokol, “Recent
implant placement in the posterior maxilla: clinical evaluation developments in magnetocaloric materials,” Reports on Progress
of 49 sinuses,” Journal of Oral and Maxillofacial Surgery, vol. 69, in Physics, vol. 68, no. 6, pp. 1479–1539, 2005.
no. 1, pp. 134–141, 2011. [56] I. E. Chesnick, C. B. Fowler, J. T. Mason, and K. Potter, “Novel
[37] C. M. Schmitt, H. Doering, T. Schmidt, R. Lutz R, F. W. Neukam, mineral contrast agent for magnetic resonance studies of
and K. A. Schlegel, “Histological results after maxillary sinus bone implants grown on a chick chorioallantoic membrane,”
augmentation with Straumann, BoneCeramic, Bio-Oss, Puros, Magnetic Resonance Imaging, vol. 29, no. 9, pp. 1244–1254, 2011.
and autologous bone. A randomized controlled clinical trial,” [57] H. D. Flack, “Chiral and achiral crystal structures,” Helvetica
Clinical Oral Implants Research, vol. 24, no. 5, pp. 576–585, 2013. Chimica Acta, vol. 86, no. 4, pp. 905–921, 2003.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 850120, 5 pages
http://dx.doi.org/10.1155/2014/850120

Clinical Study
Biological Width around One- and Two-Piece Implants
Retrieved from Human Jaws

Ricardo Judgar,1 Gabriela Giro,1 Elton Zenobio,1,2 Paulo G. Coelho,3

Magda Feres,1 Jose A. Rodrigues,1 Carlo Mangano,4 Giovanna Iezzi,4
Adriano Piattelli,4 and Jamil Awad Shibli1
1
Department of Periodontology and Oral Implantology, Dental Research Division, University of Guarulhos,
Praca Tereza Cristina 229, 07023-070 Guarulhos, SP, Brazil
2
Department of Oral Implantology, Pucminas, Belo Horizonte, MG, Brazil
3
Department of Biomaterials and Biomimetics, New York University, 45 E 24th Street, New York, NY 10010, USA
4
Department of Stomatology and Biotechnologies, University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy

Correspondence should be addressed to Jamil Awad Shibli; [email protected]

Received 15 May 2014; Accepted 2 June 2014; Published 23 June 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Ricardo Judgar et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Several histologic studies regarding peri-implant soft tissues and biological width around dental implants have been done in
animals. However, these findings in human peri-implant soft tissues are very scarce. Therefore, the aim of this case series was to
compare the biological width around unloaded one- and two-piece implants retrieved from human jaws. Eight partially edentulous
patients received 2 test implants in the posterior mandible: one-piece (solid implants that comprise implant and abutment in one
piece) and two-piece (external hexagon with a healing abutment) implants. After 4 months of healing, the implants and surrounding
tissue were removed for histologic analysis. The retrieved implants showed healthy peri-implant bone and exhibited early stages of
maturation. Marginal bone loss, gaps, and fibrous tissue were not present around retrieved specimens. The biologic width dimension
ranged between 2.55 ± 0.16 and 3.26 ± 0.15 to one- and two-piece implants, respectively (𝑃 < 0.05). This difference was influenced
by the connective tissue attachment, while sulcus depth and epithelial junction presented the same dimension for both groups
(𝑃 > 0.05). Within the limits of this study, it could be shown that two-piece implants resulted in the thickening of the connective
tissue attachment, resulting in the increase of the biological width, when compared to one-piece implants.

1. Introduction ranged between 1.5 and 2 mm and connective tissue between

1 and 2 mm. The oral epithelium presents an extension with
Biologic width (BW) is a physiologically formed and stable a thin junctional epithelium facing the implant surface and
vertical dimension of the dentogingival junction that com- extending about 1.64–2.35 mm from the mucosal margin. The
prised the sulcus depth (SD), junctional epithelium (JE), dimension from the marginal portion of the peri-implant
and connective tissue attachment (CTA) [1]. In a similar mucosa to the marginal level of bone-to-implant contact has
manner to the teeth, dental implants have also a similar peri- been found to be about 3 mm.
implant soft tissue structure [2–4]. On the dental implants, Although the precise mechanism responsible for the
the biologic width is influenced by several factors, and among crestal bone remodeling in 2-piece implants is currently
them, the macrostructure, that is, one- or two-piece implants, under research, factors such as microbial colonization of the
seems to be most relevant [4]. The two-piece implants present microgap, micromovements of the abutment, or an inter-
a microgap placed at the crestal bone level, while one-piece ruption of the blood supply when implants and abutments
implants have no gap at this region [5]. are placed transmucosally and have been also hypothesized
Early studies have shown [6–8] that the mucosal barrier [9–12]. Complementary to these factors, several studies have
around dental implants is composed by sulcular epithelium demonstrated a second discrete inflammatory cell infiltrate,
2 BioMed Research International

Figure 1: Clinical and radiographic view of the implants evaluated in the study.

additionally to the connective tissue lateral to the abutment after crestal incision, mucoperiosteal flap was raised and
fixture junction [5, 12, 13]. This infiltrated connective tissue conventional implant was placed in accordance with the sur-
(abutment ICT) was consistently separated from the plaque- gical/prosthetic plan prepared for each patient. Afterwards,
associated infiltrate by a zone of normal, noninflamed tissue the experimental implants were placed in suitable areas,
[5, 12–14]. mostly in the second and third molar region, that is, posterior
As these data were obtained mainly from animal studies to the most distal conventional implant. The experimental
[5, 6, 8, 10, 12, 13] and only few studies have investigated implant recipient sites were prepared with a 2.8 mm diameter
human peri-implant soft tissues [2, 4, 14], the aim of this case twist drill. All drilling and implant placement procedures
series was to evaluate the biological width around one-and were completed under profuse irrigation with sterile saline.
two-piece implants retrieved from human jaws. If the experimental implant showed low primary stability, a
backup surgical site was prepared. The experimental implants
2. Material and Methods (one- and two-piece) were placed at the level of the alveolar
2.1. Patient Population. Eight partially edentulous subjects crest. The flaps were sutured to allow nonsubmerged healing
(5 women; 3 men) with a mean age of 56.40 ± 4.7 years, (Figure 1).
referred for oral rehabilitation of the posterior mandible Amoxicillin was administered every 8 hours for 7 days,
with dental implants at the Oral Implantology Facility at in order to avoid postsurgical infection. The sutures were
the University of Guarulhos, were included in this study. removed 10 days post-operatively. Also, 0.12% chlorhexidine
Patients presenting mandibular bone height lower than rinses were prescribed twice daily for 14 day in order to
11 mm, smoking habit, pregnancy, nursing, or any systemic enable the postoperative dental biofilm control. Following
condition that could affect bone healing were excluded from the healing period of 4 months, the test implants and the
this study. The Ethics Committee for Human Clinical Trials at surrounding tissues were retrieved with a trephine bur, and
University of Guarulhos approved the study protocol (CEP# the specimens were fixed by immediate immersion in neutral
201/03) following the World Medical Association Declaration formalin at 4%.
of Helsinki requirements. The protocol of the study was
explained to each subject that signed the informed consent. 2.3. Specimen Processing and Histometric Analyses. The biop-
sies were processed to obtain thin ground sections as previ-
2.2. Experimental Implants. Sixteen screw-shaped implants ously described (Precise 1 Automated System, Assing, Rome,
with sandblasted acid-etched surface, 3.3 mm diameter, and Italy) [18]. The specimens were dehydrated in an ascending
8 mm length were used in this study. The implants were series of alcohol rinses and embedded in glycol methacrylate
divided in 2 groups (𝑛 = 8): the one-piece implant group resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany).
(solid implants that comprise implant and abutment in one After polymerization, the specimens were sectioned length-
piece) and the two-piece implant group (external hexagon wise along the longer axis of the implant, using a high-
with a healing abutment) (Figure 1). precision diamond saw, to about 150 𝜇m, and ground down
All patients received an implant of each group (two to approximately 30 𝜇m. Two slides were obtained from each
implants were installed per patient). The implants were implant and then averaged for each group. The slides were
placed under the protocol previously reported [15–17]. Briefly, stained with basic fuchsin and toluidine blue.
BioMed Research International 3

The peri-implant tissue width was measured as follows:

PM cJE
(i) sulcus depth (SD): the distance between the mucosal
margin (MM) and the most coronal point of junc-
tional epithelium (CJE);
(ii) junctional epithelium (JE): the distance between the
most coronal point of JE and the most apical point of aJE
the JE;
(iii) connective tissue attachment (CTA): distance
CT
between the most apical point of JE and the first
bone-to-implant contact.
Therefore, the biological width (BW) was obtained after
the sum of SD + MM + CTA. CTA
Bone-to-implant contact (BIC%), defined as the amount
of mineralized bone in direct contact with the implant PB
surface, was also evaluated.
These measurements were performed using a light micro-
scope connected to a high-resolution video camera and Figure 2: Photomicrograph of the ground section of the two-piece
interfaced to a monitor and personal computer. This optical implant group (20x) and the high magnification of the biological
system was associated with a digitizing pad and a histometry width (100x).
software package with image-capture functionalities (Image-
Pro Plus 4.5, Media Cybernetics Inc., Immagini & Computer
Snc, Milan, Italy). A single trained examiner performed all 2 pieces 1 piece
histometric parameters.
The mean and standard deviation of histometric variables
were calculated for each group. Nonparametric mixed models
[19] were applied to evaluate the data clustered within the
subject. The significance test was 2-tailed and conducted at
a 0.05 level of significance.
PB
PB
3. Results
All 16 implants presented no mobility or clinical signs of
infection after healing period. Bone density on the site
of implant placement was almost D2 [20]. The retrieved
implants showed healthy peri-implant bone. Osteocytes were
present in their lacunae, although areas of woven bone
could be distinguished. The newly formed peri-implant bone
exhibited early stages of maturation. No gaps, marginal bone
loss, or fibrous tissue were found surrounding retrieved Figure 3: Photomicrographs of the one- and two-piece implant
implants. group (200x) near the first bone-to-implant contact. Note the
The sulcular epithelium was composed of about 4–6 lay- disorganization around the peri-implant bone close to the microgap
ers of parakeratinized epithelial cells. The junctional epithe- on the two-piece implant group.
lium (JE) was composed of about 5–10 layers of epithelial
cells. The middle and apical portion of the JE consisted of 3–5
layers of epithelial cells. No acute or chronic inflammatory implants group, respectively (𝑃 = 0.001). This difference
cell infiltrate was present. Epithelial downgrowth was not was influenced by the CTA, since one-piece implants showed
depicted in any ground section (Figures 2 and 3). a CTA length average of 1.24 mm while two-piece implants
In the abutment area of both groups, the connective presented 1.87 mm (𝑃 < 0.05). Sulcus depth (SD) and
tissue contained few blood vessels, and dense collagen fibers, epithelial junction (EJ) presented no statistical significant
oriented parallel to the longitudinal axis of the abutment, difference between the groups. (𝑃 > 0.05). Moreover, BIC%
were present. Collagen fibers oriented in a perpendicular way showed no statistical difference between the groups as well.
and inserting directly contacting on the abutment surface
were not observed in any of the specimens. 4. Discussion
The mean dimensions of SD, JE, and CT for the 16
implants were reported in Table 1. An increase of the bio- The current histological case series evaluated the influence
logical width’s (BW’s) dimension was observed, with mean of implant macrostructure (one- or two-piece implants) on
values of 2.55 ± 0.16 and 3.26 ± 0.15 to the one- and two-piece human peri-implant soft tissues. Specifically, the biological
4 BioMed Research International

Table 1: Mean ± standard deviation for the histometric variables of both groups. Wilcoxon rank test (∗ 𝑃 < 0.05) (𝑛 = 8 subjects).

Histometric variables 1-piece 2-piece 𝑃 value

SD (mm) 0.33 ± 0.07 0.36 ± 0.12 0.98
JE (mm) 1.03 ± 0.06 1.05 ± 0.04 0.89
CTA (mm) 1.24 ± 0.23 1.87 ± 0.20 0.02∗
BW (mm) 2.55 ± 0.16 3.26 ± 0.15 0.001∗
BIC (%) 67.56 ± 4.56 66.45 ± 5.01 0.92
SD: sulcus depth; JE: junctional epithelium; CTA: connective tissue attachment; BW: biologic width; BIC: bone-to-implant contact.

width dimension was examined in implants retrieved from have reported a tight adaptation of the connective tissue
human jaws. The biologic width dimension ranged between to the abutment presenting a thin avascular and collagen
2.5 to 3.2 mm for one- and two-piece implants, respectively fiber-rich, as a scar-like tissue characteristics [13, 23]. In the
(𝑃 < 0.001). This difference was influenced by the connective present specimens, the CT distant from the implant was
tissue attachment, while sulcus depth and epithelial junction composed by abundant collagen fibers, running in several
presented the same dimension for both groups (𝑃 > 0.05). directions and appearing to be functionally organized in a
There is a lack of information regarding the histological 3-dimensional network. Similar results have been reported
features of supracrestal peri-implant soft tissue, since the in human studies [2–4, 14]. This differentiated network of
present knowledge is basically constituted by animal studies fibers may have clinical relevance as a mechanical protection
data, using dogs and nonhuman primates [11] and some of the underlying bone [4]. These human histologic data are
patient reports [3, 14, 21]. Although these data presents such extremely valuable to validate and confirm those obtained
important role in this field, sometimes the animal studies from studies performed on animal models [8, 10, 11].
results cannot be faithfully transposed to the per-implant
tissue behavior in humans [22]. A classical report using
human teeth [1] showed that BW is a physiologically formed 5. Conclusions
and stable dimension whose level is dependent upon the Therefore, within the limits of this histologic report, it could
location of the alveolar bone crest. Around dental implants, be suggested that the two-piece implant leads to a thicker
BW determines the minimum dimensions to ensure adequate biological width. These data must be carefully analyzed,
JE and CT to obtain an optimal seal and to provide protection and further prospective longitudinal studies are required to
from mechanical and external biological agents [23]. An clarify the clinical relevance of these findings.
external agent invading the BW would induce a response
from the epithelium that migrates beyond this agent trying
to isolate it [2, 3, 23]. The resulting bone resorption produces Conflict of Interests
a reestablishment of the BW dimension. Regarding dental
implants, the dimension of the BW was reported to be The authors declare that there is no conflict of interests related
dependent on the presence/absence of a microgap and on the to this study.
location of the microgap in relation to the bone crest [5–10].
Studies have reported the dimension of JE around Authors’ Contribution
implants, in animal studies, comprised between 1.16 mm and
1.90 mm [6, 8, 10, 11, 13], while JE around retrieved implants Jamil Awad Shibli was in charge of the elaboration of the
from human jaws ranged between 1.8 and 3.4 mm [4, 14], study proposal and the financial support of the study, and
differing from the findings of this study that showed values he participated in the elaboration of the paper and the
similar to those reported in animal studies (∼1.05 mm). treatment planning of each case. Carlo Mangano and Paulo G.
Connective attachment dimension, in animal models, Coelho were in charge of the statistical analysis, the implant
ranged between 1.01 mm and 2.01 mm [10, 11, 13]. Loading surface characterization, and the financial support for the
conditions have also been reported to influence not only study. Ricardo Judgar, Gabriela Giro, Elton Zenobio, and
the dimension of JE but also the dimension of the CT. Jose A. Rodrigues were in charge of the treatment planning
Previous canine model study [10] has shown that the CT of each case and the implant placement surgery and they
dimension was significantly higher on unloaded implants participated to the elaboration of the paper. Magda Feres,
when compared to different load conditions. The results of the Elton Zenobio, and Gabriela Giro were in charge of the
present study could confirm a tendency for a larger size of the patients’ monitoring after surgery, the biopsies collection,
CT around unloaded implants. In fact, in human unloaded and the oral rehabilitation and they participated in the
specimens, CT has been comprised between 1.8 mm and elaboration of the paper. Giovanna Iezzi and Adriano Piattelli
3.4 mm [4, 14]. were in charge of the laboratory processing of the samples
The supracrestal CT was, in animal studies, characterized and the histology and participated in the data analyses and
by a 3D network of collagen fibers running in different elaboration of the paper. Adriano Piattelli also participated
directions [6, 8, 10]. In addition, several animal studies in the elaboration of the study proposal.
BioMed Research International 5

References [14] A. Piattelli, A. Scarano, M. Piattelli, R. Bertolai, and E. Panzoni,

“Histological aspects of the bone and soft tissues surround-
[1] A. W. Gargiulo, F. M. Wentz, and B. Orban, “Dimensions of the ing three titanium non-submerged plasma-sprayed implants
dentogingival junction in humans,” Journal of Periodontology, retrieved at autopsy. A case report,” Journal of Periodontology,
vol. 32, pp. 261–267, 1961. vol. 68, no. 7, pp. 694–700, 1997.
[2] M. Degidi, A. Piattelli, A. Scarano, J. A. Shibli, and G. [15] J. A. Shibli, C. Mangano, S. D'Avila et al., “Influence of
Iezzi, “Peri-implant collagen fibers around human cone Morse direct laser fabrication implant topography on type IV bone:
connection implants under polarized light: a report of three a histomorphometric study in humans,” Journal of Biomedical
cases,” The International Journal of Periodontics & Restorative Materials Research A, vol. 93, no. 2, pp. 607–614, 2010.
Dentistry, vol. 32, no. 3, pp. 323–328, 2012. [16] J. A. Shibli, S. Grassi, A. Piattelli et al., “Histomorphometric
[3] M. Degidi, V. Perrotti, J. A. Shibli, A. B. Novaes, A. Piattelli, evaluation of bioceramic molecular impregnated and dual
and G. Iezzi, “Equicrestal and subcrestal dental implants: a acid-etched implant surfaces in the human posterior maxilla,”
histologic and histomorphometric evaluation of nine retrieved Clinical Implant Dentistry and Related Research, vol. 12, no. 4,
human implants,” Journal of Periodontology, vol. 82, no. 5, pp. pp. 281–288, 2010.
708–715, 2011. [17] J. A. Shibli, C. Mangano, F. Mangano et al., “Histomorphometric
[4] R. Glauser, P. Schüpbach, J. Gottlow, and C. H. F. Hämmerle, evaluation of Direct Laser Metal Forming (DLMF) implant
“Peri-implant soft tissue barrier at experimental one-piece surface in the type IV bone: a controlled study in human jaw,”
mini-implants with different surface topography in humans: a Poseido, vol. 1, no. 3, pp. 149–156, 2013.
light-microscopic overview and histometric analysis,” Clinical [18] A. Piattelli, A. Scarano, and M. Quaranta, “High-precision, cost-
Implant Dentistry and Related Research, vol. 7, supplement 1, pp. effective cutting system for producing thin sections of oral
S44–S51, 2005. tissues containing dental implants,” Biomaterials, vol. 18, no. 7,
pp. 577–579, 1997.
[5] N. Broggini, L. M. McManus, J. S. Hermann et al., “Peri-implant
inflammation defined by the implant-abutment interface,” Jour- [19] E. Brunner and F. Langer, “Nonparametric analysis of ordered
nal of Dental Research, vol. 85, no. 5, pp. 473–478, 2006. categorical data in designs with longitudinal observations and
small sample sizes,” Biometrical Journal, vol. 42, no. 6, pp. 663–
[6] J. S. Hermann, D. Buser, R. K. Schenk, F. L. Higginbottom, 675, 2000.
and D. L. Cochran, “Biologic width around titanium implants.
[20] C. E. Misch, “Divisions of available bone in implant dentistry,”
A physiologically formed and stable dimension over time,”
The International Journal of Oral Implantology: Implantologist,
Clinical Oral Implants Research, vol. 11, no. 1, pp. 1–11, 2000.
vol. 7, no. 1, pp. 9–17, 1990.
[7] G. Schierano, G. Ramieri, M. Cortese, M. Aimetti, and G. Preti, [21] M. Degidi, A. Piattelli, A. Scarano, V. Perrotti, and G. Iezzi, “Soft
“Organization of the connective tissue barrier around long- tissues around an acid-etched healing abutment: a histological
term loaded implant abutments in man,” Clinical Oral Implants and histomorphometrical analysis,” Poseido, vol. 1, no. 3, pp.
Research, vol. 13, no. 5, pp. 460–464, 2002. 157–163, 2013.
[8] A. Quaranta, A. Piattelli, A. Scarano, M. Quaranta, G. Pompa, [22] M. Degidi, A. Piattelli, J. A. Shibli et al., “Bone formation around
and G. Iezzi, “Light-microscopic evaluation of the dimensions a dental implant with a platform switching and another with
of peri-implant mucosa around immediately loaded and sub- a TissueCare connection: a histologic and histomorphometric
merged titanium implants in monkeys,” Journal of Periodontol- evaluation in man,” Titanium, vol. 1, pp. 8–15, 2009.
ogy, vol. 79, no. 9, pp. 1697–1703, 2008.
[23] I. Abrahamsson, T. Berglundh, J. Wennström, and J. Lindhe,
[9] X. Vela-Nebot, X. Rodrı́guez-Ciurana, C. Rodado-Alonso, and “The peri-implant hard and soft tissues at different implant
M. Segalá-Torres, “Benefits of an implant platform modification systems: a comparative study in the dog,” Clinical Oral Implants
technique to reduce crestal bone resorption,” Implant Dentistry, Research, vol. 7, no. 3, pp. 212–219, 1996.
vol. 15, no. 3, pp. 313–320, 2006.
[10] J. S. Hermann, D. Buser, R. K. Schenk, and D. L. Cochran,
“Crestal bone changes around titanium implants. A histomet-
ric evaluation of unloaded non-submerged and submerged
implants in the canine mandible,” Journal of Periodontology, vol.
71, no. 9, pp. 1412–1424, 2000.
[11] C. H. Siar, C. G. Toh, G. Romanos et al., “Peri-implant soft tissue
integration of immediately loaded implants in the posterior
macaque mandible: a histomorphometric study,” Journal of
Periodontology, vol. 74, no. 5, pp. 571–578, 2003.
[12] J. S. Hermann, D. L. Cochran, P. V. Nummikoski, and D.
Buser, “Crestal bone changes around titanium implants: a radio-
graphic evaluation of unloaded nonsubmerged and submerged
implants in the canine mandible,” Journal of Periodontology, vol.
68, no. 11, pp. 1117–1130, 1997.
[13] J. S. Hermann, J. D. Schoolfied, R. K. Schenk, D. Buser, and D. L.
Cochran, “Influence of the size of the microgap on crestal bone
changes around titanium implants. A histometric evaluation
of unloaded non-submerged implants in the canine mandible,”
Journal of Periodontology, vol. 72, no. 10, pp. 1372–1383, 2001.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 104230, 9 pages
http://dx.doi.org/10.1155/2014/104230

Research Article
Effect of Low-Level Laser on Bone Defects Treated with Bovine
or Autogenous Bone Grafts: In Vivo Study in Rat Calvaria

Mércia J. S. Cunha,1,2,3 Luis A. Esper,2,3 Michyele C. Sbrana,2,3 Paula G. F. P. de Oliveira,1,3

Accácio L. do Valle,3,4 and Ana Lúcia P. F. de Almeida3,4
1
Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo, Brazil
2
Bauru School of Dentistry, USP, Brazil
3
Faculdade de Odontologia de Bauru (FOB), Universidade de São Paulo (USP), Alameda Dr. Octávio Pinheiro Brisolla 9-75,
Vila Universitária, 17012 901 Bauru, SP, Brazil
4
Department of Prosthodontics, Bauru School of Dentistry, USP, Brazil

Correspondence should be addressed to Ana Lúcia P. F. de Almeida; [email protected]

Received 28 February 2014; Revised 17 April 2014; Accepted 12 May 2014; Published 28 May 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Mércia J. S. Cunha et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Objective. The purpose of this study was to histologically evaluate the effect of low-level laser (LLL) on the healing of critical size
defects (CSD) in rat calvaria, filled with autogenous or inorganic bovine bone grafts. Methods. Sixty rats were divided into 6 groups
(𝑛 = 10): C (control—filled with blood clot), LLL (low-level laser—GaAlAs, 𝜆 780 nm, 100 mW, 210 J/cm2 , Φ 0.05 cm2 ; 6 J/point),
AB (autogenous bone), ABL (autogenous bone + low-level laser), OB (inorganic bovine bone), and OBL (inorganic bovine bone
+ LLL). Material and Methods. The animals were killed after 30 days. Histological and histometric analyses were performed by
light microscopy. Results. The groups irradiated with laser, LLL (47.67% ± 8.66%), ABL (39.15% ± 16.72%), and OBL (48.57% ±
28.22%), presented greater area of new bone formation than groups C (9.96% ± 4.50%), AB (30.98% ± 16.59%), and OB (11.36%
± 7.89%), which were not irradiated. Moreover, they were significantly better than group C (Kruskal-Wallis test followed by Dunn
test, 𝑃 < 0.05). Conclusion. The laser accelerated the healing of bone defects and the resorption of particles of the graft material.

1. Introduction due to its similarity to human bone [3]. Promising results have
been demonstrated by its use in clinical and animal studies
Currently, bone grafting has been widely used. It is estimated [9, 10]. Despite its excellent osteoconduction [8, 10], it lacks
that approximately 2.2 million bone graft procedures are osteoinductive properties, which has encouraged researchers
performed worldwide [1, 2] to repair defects in orthopedics, to find ways to further improve its behavior in vivo [9]. In
neurosurgery, and dentistry [2].
addition, the use of low-level laser (LLL) has been studied as
Among the graft materials used for bone regeneration,
an alternative to speed healing in larger bone defects [10, 11].
autogenous bone has been considered the ideal material [1, 3,
The effects related to LLL include increased vascularity,
4]. Even though it is the “gold standard” for reconstructions
[1, 3, 5], its collection is associated with 8.5 to 20% of increased osteoblastic activity [12], organization of collagen
complications, including hematoma [2], damage to anatomic fibers, and changes in mitochondrial and intracellular levels
structures [6], infections [2, 6], pain at the donor site [7, 8], of adenosine triphosphate [12, 13]. It is a noninvasive method
and unpredictable graft resorption [3, 5]. to stimulate osteogenesis [13, 14] and accelerate the healing of
For these reasons, several bone substitutes from different bone defects [12–14].
sources are available, with the advantages of unlimited supply Positive [11, 13, 15] and negative [16, 17] results have been
and no need for a donor site. The inorganic bovine bone is the reported in both in vivo and in vitro studies [11, 15] regarding
most researched graft material and is widely used in dentistry the repair of soft or mineralized tissue [18, 19], but few
2 BioMed Research International

studies have evaluated the role of LLL associated with bone performed for group ABL, which was thereafter submitted to
substitutes [20, 21]. LLL application.
Therefore, the purpose of this study was to histologically To standardize the amount of OB used in each defect, the
evaluate the effect of low-level laser on the healing of critical AB was removed from the ground calvaria using a syringe
size defects (CSD) in rat calvaria, filled with autogenous or with millimeter markings and weighed on a precision scale.
inorganic bovine bone grafts. The same weight (0.02 g) and volume (1 mm3 ) were used for
the OB.
2. Material and Methods The flap was then repositioned and sutured with 4–0 silk
suture. Each animal received an intramuscular injection of
2.1. Experimental Design. The experimental protocol was 24,000 units of penicillin G-benzathine.
approved by the Institutional Review Board on Animal All surgical procedures were performed by a single
Studies of Bauru School of Dentistry, University of São Paulo. operator, previously trained in a previous study [13].
A total of 60 male rats (Rattus norvegicus, albinus, Wistar)
weighing between 250 and 300 g were utilized. The animals 2.3. Protocol of Low-Level Laser Therapy. The laser used was
were maintained in an environment with 12-hour cycle
Theralase DMC (GaAlAs, 𝜆 = 780 nm, 100 mW, Φ 0.05 cm2 ,
of light per day and temperature between 22 and 24∘ C.
210 J/cm2 of energy density, 60 s/point, 6 J/point, continuous
Throughout the experiment, the animals received selected
mode). The applications were made at four points on the
solid diet and water ad libitum. The animals were randomly
surgical wound surface following a clockwise direction (12 h,
assigned to the following experimental groups (𝑛 = 10):
3 h, 6 h, and 9 h positions) and a central point [13]. In the LLL
(1) group C—control defect filled with blood clot; (2) group
group, application was performed after filling with blood clot,
LLL—LLL (Theralase DMC, São Carlos, Brazil); (3) group
while in the other groups application was performed after
AB—autogenous bone; (4) group ABL—autogenous bone
insertion of the respective graft material (AB and OB).
+ LLL; (5) group OB—inorganic bovine bone/0.25–1 mm
(Bio-Oss—Geistlich Pharma AG, Wolhusen, Switzerland);
(6) group OBL—inorganic bovine bone + LLL. 2.4. Tissue Processing. The animals were killed at 30 days
postoperatively with 5 mg/mL of the association of ketamine
and xylazine. The original surgical defect area and surround-
2.2. Surgical Procedure. The animals were anesthetized by
ing tissues were removed en bloc. The specimens were fixed
an intramuscular injection of xylazine (0.02 mL/kg) and
in 10% formalin solution, rinsed in water, and decalcified in
ketamine hydrochloride (0.4 mL/kg). After trichotomy and
an 18% ethylene diamine tetraacetic acid solution.
antisepsis of the dorsal part of the skull of each animal,
After decalcification, each specimen was longitudinally
a semilunar incision was made in the calvaria and a full-
divided into two blocks, exactly over the center of the original
thickness flap was raised in posterior direction. A 5 mm
surgical defect, using the main axes of each amalgam marking
diameter CSD was created with a trephine at low speed under
as reference. In addition, cross-sections were performed
thorough irrigation with sterile saline. The defect included
tangentially to the lowest axis on both “L” markings, so that
a portion of the sagittal suture. The dura mater and the
the end of each specimen measured 9 mm in length. This
brain were preserved during craniotomy. The full thickness
allowed accurate determination of the boundaries of the
of parietal bone was gently removed [13, 22].
original surgical defect during histometric analysis [13, 22].
With the aid of a previously made surgical guide, two L-
The specimens were then processed and embedded in
shaped marks were made, one being at 2 mm anteriorly and
paraffin. Longitudinal serial sections with 6 𝜇m thickness
the other one being at 2 mm posteriorly to the margins of
were obtained starting from the center of the original surgical
the surgical defect, with a FG-700 truncated carbide cone bur
defect. The sections were stained with hematoxylin and eosin
under continuous irrigation with sterile saline and then filled
(HE) for light microscopy analysis.
with amalgam [13, 22].
The major axis of each “L” was located on a craniocaudal
longitudinal imaginary line that divided the surgical defect 2.5. Histomorphometric Analysis. The histological and histo-
into half. These markings were useful to identify the middle of metric analyses were performed by a previously calibrated
the original surgical defect during laboratory processing and examiner blinded to the experimental groups. Four histologi-
also to locate the original bone margins during histometric cal sections were selected, representing the central area of the
analysis [13, 22]. original surgical defect. Images of the histological sections
After fabrication of the L-shaped mark, the defect was were captured by a digital camera SPOT RT3-2540 Color
filled according to each group. In group C, it was only filled Slider 2.0 MP connected to the Olympus BX50 microscope
with blood clot. Group LLL was filled with blood clot and at 2x magnification and saved on a computer. For each
was submitted to LLL application. In group OB, the defects animal, new bone formation (NBF) values were calculated
were filled with 0.02 g of inorganic bovine bone, and group by the arithmetic mean of three most central histological
OBL was filled with the same amount of inorganic bovine sections of the calvaria, and another section was used for
bone followed by LLL application. In group AB, the calvaria histological analysis. The histometric analysis was performed
defect was filled with autogenous bone obtained from ground on the ImageLab 2000 software (Bio Diracon Informática
calvaria bone harvested with the trephine; the same was Ltd., Vargem Grande do Sul, SP, Brazil) [13, 22].
BioMed Research International 3

(a)

(b) (c)

Figure 1: Photomicrographs of group C. (a) Panoramic view of the defect (4x); (b) defect filled with bundles of collagen fibers (40x); (c) small
amount of newly formed bone (asterisk) along the margins of the surgical defect (10x). Hematoxylin and eosin.

The following criteria, based on the methodology pro- test power was verified with a minimum power of 0.86. The
posed by Furlaneto et al. [22], were followed to standardize differences were considered statistically significant when 𝑃 <
the histometric analysis. 0.05, at a confidence level of 95%.
(1) The total area (TA) to be analyzed corresponded to the
total area of the original surgical defect. This area was 3. Results
determined by identifying the external and internal
surfaces of the original calvaria and the left and right During the laboratory processing, 1 specimen from group C,
margins of the surgical defect. These surfaces were 3 specimens from group LLL, 1 specimen from group AB, and
connected with lines drawn following their respective 2 specimens from group ABL were lost.
curvatures. Considering the total length of the histo-
logical specimen (9 mm), 2 mm was measured from 3.1. Qualitative Histological Analysis. In all groups absence of
the left and right ends of the specimen toward the inflammatory infiltrate was observed.
center in order to determine the boundaries of the In group C, virtually the entire length of the surgical
original surgical defect. wound was filled by connective tissue with collagen fibers
(2) The area of new bone formation (NBF) and the orientated parallel to the wound surface. A small amount of
remaining particle areas (RPA) of the implanted new bone formation was observed along the margins of the
materials were delineated within the boundaries of surgical defect (Figure 1). Complete regenerated bone repair
the TA. of the defect did not occur in any specimen.
New bone formation surrounded by an osteoid matrix
(3) The TA was measured in mm2 and 100% of the area was observed in some specimens in group LLL. The tissues
being analyzed was considered. The NBF and RPA presented parallel oriented bundles of collagen fibers and
were also measured in mm2 and calculated as percent- absence of inflammatory infiltrate. New bone formation
ages of TA in accordance with the following formula: extending linearly toward the center of the original defect was
NBF (mm2 )/TA (mm2 ) ×100. observed in two specimens. Areas of remodeled bone were
also observed at the region of old bone, which was preserved
2.6. Statistical Analysis. For each animal, the values of NBF (Figure 2).
and RPA were represented by the arithmetic mean of the In group AB, the connective tissue was well organized
four most central histological sections of the calvaria. The within the surgical defect, with formation of osteoid matrix,
values found did not pass the normality test (Shapiro-Wilk). presence of fibroblasts, and absence of inflammatory infil-
Thus, they were subjected to the nonparametric Kruskal- trate. The new bone formation was present in variable
Wallis test followed by the Dunn test. Analysis of the statistical extensions at the margins of the defect and around the grafted
4 BioMed Research International

(a)

(b) (c)

Figure 2: Photomicrographs of group LLL. (a) Panoramic view of the surgical defect (4x); (b) bone formation extending toward the center
of the original surgical defect (10x); (c) bone remodeling in the region of old bone that was preserved (40x). Hematoxylin and eosin.

(a)

(b) (c)

Figure 3: Photomicrographs of group AB. (a) Panoramic view of the surgical defect (4x); (b) newly formed bone tissue along the margins of
the surgical defect and bone graft particles (10x); (c) autogenous bone particle surrounded by new bone formation (40x).

bone particles (Figure 3). In only one specimen, bone graft extensions. Three specimens (37.5%) showed new bone for-
particles were not observed. mation toward the center of the surgical defect. Grafted bone
The osteoid matrix was observed in all specimens in particles were also observed, most of which had new bone
group ABL. New bone formation was present in variable tissue at the periphery (Figure 4).
BioMed Research International 5

(a)

(b) (c)

Figure 4: Photomicrographs of group ABL. (a) Panoramic view of the surgical defect (4x); (b) autogenous bone particles with new bone
formation at the periphery (10x); (c) area of new formation and remodeling of the autogenous bone particle (40x).

Table 1: Means and standard deviations of the amount of newly formed bone.

Groups 𝑁 Mean Standard deviation (sd) Q25 Median Q75

Ca 9 9.96 4.50 8.58 9.52 13.33
Lb 7 4.67 8.66 43.96 44.58 55.41
ABa,b 9 30.98 16.59 20.96 25.10 36.24
ABLb 8 39.15 16.72 26.89 40.41 53.32
BOa 10 11.36 7.89 6.30 9.49 11.57
BOLb 10 48.57 28.22 35.76 42.22 74.66
Same letters represent no statistical difference (significance level of 5%).

In OB group, parallel oriented collagen fibers were The groups irradiated with LLL had lower RPA averages.
observed. Inorganic bovine bone particles were present, There was statistically significant difference (𝑃 < 0.05)
many with osteoclasts in their periphery. In most specimens between OB × OBL and OB × ABL groups (Table 2, Figure 8).
there was a slight bone formation at the margins of the defect
(Figure 5).
Two specimens in group OB presented new bone forma-
4. Discussion
tion extending toward the center of the defect, maintaining
the original thickness of the calvaria. New bone tissue and The improvement of vascularization after LLL in this study
osteoclasts were observed at the periphery of the remaining is one of the possible mechanisms for the clinical efficacy of
inorganic bovine bone particles. Inflammatory infiltrate was that treatment [12, 13, 23–25]. It has also been reported that
not observed (Figure 6). LLL increases the osteoblast and osteoclast activity [26] and
stimulates production of the bone matrix and the formation
3.2. Histomorphometric and Statistical Analysis. The groups of bone callus [25, 27] but also accelerates the dynamics of the
irradiated with LLL showed higher NBF averages. Correla- bone matrix by modifying the expression of the extracellular
tions were statistically significant (𝑃 < 0.05) between groups matrix components and increasing the area of new bone
OB × OBL; LLL × OB; OB × ABL; OBL × C; C × LLL; C × ABL formation, which reduces the time necessary for bone healing
(Table 1, Figure 7). [28].
6 BioMed Research International

Table 2: Means and standard deviations of the remaining particle.

Groups 𝑁 Mean Standard deviation (sd) Q25 Median Q75

ABa,b 9 9.16 7.10 4.35 7.04 13.46
ABLb 8 3.66 2.79 1.08 4.04 5.84
BOa 10 38.73 6.95 35.72 37.71 42.78
BOLb 10 16.74 15.25 0.00 22.42 28.23
Same letters represent no statistical difference (significance level of 5%).

(a)

(b) (c)

Figure 5: Photomicrographs of group OB. (a) Panoramic view of the surgical defect (4x); (b) bone formation along the margins of the defect
(10x); (c) osteoclasts in the vicinity of bovine bone graft particles (40x).

Another explanation for the accelerated bone healing studies [13, 15], this work intended to establish guidelines
observed for groups irradiated with LLL is that the undiffer- for a transoperative protocol immediately involving a single
entiated mesenchymal cells can be positively biomodulated laser application in direct contact with the wound area and
to become osteoblasts and evolve to osteocytes faster. It is confirmed the beneficial effects of a single session irradiation
known that the osteogenic potential of the mesenchymal cells for bone healing of the defect, demonstrating that this type of
depends, in addition to genetic factors, on induced local and treatment may be feasible, easy, and fast.
systemic factors. LLL could act as such an inductor factor When evaluating the area of new bone formation in this
[24, 29, 30]. study, the results for groups irradiated with LLL were similar
It has been reported that the biomodulation of the LLL to the AB and ABL groups. This would suggest that only
depends on the wavelength used, since tissue components the application of laser would already be beneficial in bone
can influence the dispersion of light [12, 23, 29]. In the regeneration with this application protocol. Moreover, the
infrared spectrum the laser can provide increased osteoblast association of AB and LLL showed superior results when
proliferation, collagen deposition, and bone formation [12, compared to the treatment with AB alone. The lack of
29]. In this study, there was greater bone formation in the statistically significant difference between the AB and ABL
group irradiated with laser (group LLL) compared to the groups can be assigned to the fact that autogenous bone
nonirradiated group (group C). alone can already be considered a very good grafting material.
There are no universally accepted parameters for using Starting from a high level of excellence, the laser would not be
the LLL. Different irradiation protocols are found with able to add benefits to the point that it would be statistically
different activation materials, wavelengths, and even dose and different.
number of applications, precluding the comparison of results Although no statistically significant difference was
and choice of treatment parameters [31]. Similar to previous observed in the NBF and RPA between groups AB and
BioMed Research International 7

(a)

(b) (c)

Figure 6: Photomicrographs of group OB. (a) Panoramic view of the surgical defect (4x); (b) bone formation along the margins of the defect
(10x); (c) osteoclasts in the vicinity of bovine bone graft particles (40x).

100
Area of new bone formation (NBF) (%)

90
80
70
60
50
40
30
20
10
0
C LLL AB ABL OB OBL
Experimental groups

Figure 7: Distribution of new bone formation area for all experimental groups.

ABL, it is believed that the laser has also been able to speed The resorption of inorganic bovine bone particles is
up the process of bone remodeling when the allograft was still a conflicting issue in the literature. There are reports
used, since the histological analysis revealed that, in the that particles in the interior of bone defects fail to resorb
irradiated group, there were specimens with new bone and remain like a motionless body surrounded by the host
formation toward the center of the surgical defect, and most bone [31, 32]. Moreover, after months of healing, osteoblastic
graft particles showed new bone formation at the periphery. activity is observed in the particles and it is believed that, with
Thus, the association of LLL and AB could be suggested as time, these particles remodel themselves and meanwhile the
advantageous to accelerate cell proliferation and increase the new bone is formed; however, it appears to be a slow process
new bone volume, thus aiding the integration of the graft [33]. It is believed that the laser has accelerated the process
into the recipient area, corroborating the findings of other of bone formation and resorption of such particles, since the
studies [13, 14, 23]. This is recommended as an additional OBL group showed a statistically significant difference in the
treatment modality in the regeneration of bone defects, since NBF and RPA when compared to the OB. This may be due
it is a noninvasive method to stimulate osteogenesis [14] and to the fact that the laser improves vascularization [12, 23–25],
accelerate the healing of bone defects [12–14]. increases the osteoclastic [26] and osteoblastic activity [12],
8 BioMed Research International

100
90

Area of remaining particles (%)

80
70
60
50
40
30
20
10
0
AB ABL OB OBL
Experimental groups

Figure 8: Distribution of area of the remaining particle for groups receiving graft material.

stimulates the production of the bone matrix [34], and can and autologous bone. A randomized controlled clinical trial,”
act as an osteoinductive factor [24, 29]. Clinical Oral Implants Research, vol. 24, no. 5, pp. 576–585, 2013.
In the present study, the fact that all groups irradiated [5] G. F. Rogers and A. K. Greene, “Autogenous bone graft: basic
with LLL presented superior results to group C and groups science and clinical implications,” Journal of Craniofacial Sur-
receiving only grafts suggests that this type of therapy may gery, vol. 23, no. 1, pp. 323–327, 2012.
be effective in the healing of bone defects, especially when [6] A. S. Herford and J. S. Dean, “Complications in bone grafting,”
associated with a filling material. Oral and Maxillofacial Surgery Clinics of North America, vol. 23,
no. 3, pp. 433–442, 2011.
5. Conclusion [7] P. Amerio, G. Vianale, M. Reale, R. Muraro, A. Tulli, and A. Piat-
telli, “The effect of deproteinized bovine bone on osteoblast
The LLL accelerated the healing of bone defects and the growth factors and proinflammatory cytokine production,”
resorption of the graft material particles. Clinical Oral Implants Research, vol. 21, no. 6, pp. 650–655, 2010.
[8] L. Ohayon, “Ridge enlargement using deproteinized bovine
bone and a bioresorbable collagen membrane: a tomoden-
Conflict of Interests sitometric, histologic, and histomorphometric analysis,” The
International Journal of Periodontics & Restorative Dentistry,
The authors declare that there is no conflict of interests vol. 31, no. 3, pp. 237–245, 2011.
regarding the publication of this paper.
[9] J. Torres, F. M. Tamimi, I. F. Tresguerres et al., “Effect of solely
applied platelet-rich plasma on osseous regeneration compared
Acknowledgment to Bio-Oss: a morphometric and densitometric study on rabbit
calvaria,” Clinical Implant Dentistry and Related Research, vol.
The authors would like to thank São Paulo Research Foun- 10, no. 2, pp. 106–112, 2008.
dation (FAPESP) for the Master’s Scholarship (2010/13170-3) [10] E. Fávaro-Pı́pi, D. A. Ribeiro, J. U. Ribeiro et al., “Low-level
and for the Research Grant (2010/10538-0). laser therapy induces differential expression of osteogenic genes
during bone repair in rats,” Photomedicine and Laser Surgery,
vol. 29, no. 5, pp. 311–317, 2011.
References
[11] A. N. S. Júnior, A. L. Pinheiro, M. G. Oliveira, R. Weismann, L.
[1] K.-U. Lewandrowski, J. D. Gresser, D. L. Wise, and D. J. Tran- M. Ramalho, and R. A. Nicolau, “Computerized morphometric
tolo, “Bioresorbable bone graft substitutes of different osteo- assessment of the effect of low-level laser therapy on bone
conductivities: a histologic evaluation of osteointegration of repair: an experimental animal study,” Journal of Clinical Laser
poly(propylene glycol-co-fumaric acid)-based cement implants Medicine & Surgery, vol. 20, no. 2, pp. 83–87, 2002.
in rats,” Biomaterials, vol. 21, no. 8, pp. 757–764, 2000. [12] D. Barbosa, R. A. de Souza, M. Xavier, F. F. da Silva, E. Â.
[2] P. V. Giannoudis, H. Dinopoulos, and E. Tsiridis, “Bone substi- Arisawa, and A. G. J. B. Villaverde, “Effects of low-level laser
tutes: an update,” Injury, vol. 36, no. 3, pp. S20–S27, 2005. therapy (LLLT) on bone repair in rats: optical densitometry
[3] M. Hallman and A. Thor, “Bone substitutes and growth factors analysis,” Lasers in Medical Science, vol. 28, no. 2, pp. 651–656,
as an alternative/complement to autogenous bone for grafting in 2013.
implant dentistry,” Periodontology 2000, vol. 47, no. 1, pp. 172– [13] A. L. P. F. de Almeida, I. L. Medeiros, M. J. S. Cunha, M. C.
192, 2008. Sbrana, P. G. F. de Oliveira, and L. A. Esper, “The effect of low-
[4] C. M. Schmitt, H. Doering, T. Schmidt, R. Lutz, F. W. Neukam, level laser on bone healing in critical size defects treated with
and K. A. Schlegel, “Histological results after maxillary sinus or without autogenous bone graft: an experimental study in rat
augmentation with Straumann BoneCeramic, Bio-Oss, Puros, calvaria,” Clinical Oral Implants Research, 2013.
BioMed Research International 9

[14] R. V. da Silva and J. A. Camilli, “Repair of bone defects treated matrix,” Photochemistry and Photobiology, vol. 88, no. 5, pp.
with autogenous bone graft and low-power laser,” Journal of 1293–1301, 2012.
Craniofacial Surgery, vol. 17, no. 2, pp. 297–301, 2006. [29] A. L. B. Pinheiro and M. E. M. M. Gerbi, “Photoengineering of
[15] H. Pretel, R. F. Z. Lizarelli, and L. T. O. Ramalho, “Effect of low- bone repair processes,” Photomedicine and Laser Surgery, vol.
level laser therapy on bone repair: histological study in rats,” 24, no. 2, pp. 169–178, 2006.
Lasers in Surgery and Medicine, vol. 39, no. 10, pp. 788–796, 2007. [30] L. Abramovitch-Gottlib, T. Gross, D. Naveh et al., “Low level
[16] E. J. Luger, S. Rochkind, Y. Wollman, G. Kogan, and S. laser irradiation stimulates osteogenic phenotype of mesenchy-
Dekel, “Effect of low-power laser irradiation on the mechanical mal stem cells seeded on a three-dimensional biomatrix,” Lasers
properties of bone fracture healing in rats,” Lasers in Surgery and in Medical Science, vol. 20, no. 3-4, pp. 138–146, 2005.
Medicine, vol. 22, no. 2, pp. 97–102, 1998. [31] J. P. da Silva, M. A. da Silva, A. P. F. Almeida, I. Lombardi Junior,
[17] G. Anneroth, G. Hall, H. Rydén, and L. Zetterqvist, “The effect and A. P. Matos, “Laser therapy in the tissue repair process: a
of low-energy infra-red laser radiation on wound healing in literature review,” Photomedicine and Laser Surgery, vol. 28, no.
rats,” British Journal of Oral and Maxillofacial Surgery, vol. 26, 1, pp. 17–21, 2010.
no. 1, pp. 12–17, 1988. [32] R. Ewers, W. Goriwoda, C. Schopper, D. Moser, and E. Spassova,
[18] A. Stein, D. Benayahu, L. Maltz, and U. Oron, “Low-level laser “Histologic findings at augmented bone areas supplied with two
irradiation promotes proliferation and differentiation of human different bone substitute materials combined with sinus floor
osteoblasts in vitro,” Photomedicine and Laser Surgery, vol. 23, lifting,” Clinical Oral Implants Research, vol. 15, no. 1, pp. 96–
no. 2, pp. 161–166, 2005. 100, 2004.
[19] A. Leonida, A. Paiusco, G. Rossi, F. Carini, M. Baldoni, and G. [33] T. Berglundh and J. Lindhe, “Healing around implants placed in
Caccianiga, “Effects of low-level laser irradiation on prolifer- bone defects treated with Bio-Oss: an experimental study in the
ation and osteoblastic differentiation of human mesenchymal dog,” Clinical Oral Implants Research, vol. 8, no. 2, pp. 117–124,
stem cells seeded on a three-dimensional biomatrix: in vitro 1997.
pilot study,” Lasers in Medical Science, vol. 28, no. 1, pp. 125–132, [34] K. M. AlGhamdi, A. Kumar, and N. A. Moussa, “Low-level laser
2013. therapy: a useful technique for enhancing the proliferation of
[20] A. L. B. Pinheiro, F. D. A. L. Limeira Júnior, M. E. M. Gerbi, L. various cultured cells,” Lasers in Medical Science, vol. 27, no. 1,
M. P. Ramalho, C. Marzola, and E. A. C. Ponzi, “Effect of low pp. 237–249, 2012.
level laser therapy on the repair of bone defects grafted with
inorganic bovine bone,” Brazilian Dental Journal, vol. 14, no. 3,
pp. 177–181, 2003.
[21] A. L. B. Pinheiro, L. G. P. Soares, A. F. S. Barbosa, L. M.
P. Ramalho, and J. N. dos Santos, “Does LED phototherapy
influence the repair of bone defects grafted with MTA, bone
morphogenetic proteins, and guided bone regeneration? A
description of the repair process on rodents,” Lasers in Medical
Science, vol. 27, no. 5, pp. 1013–1024, 2012.
[22] F. A. C. Furlaneto, M. J. H. Nagata, S. E. Fucini, T. M. Delib-
erador, T. Okamoto, and M. R. Messora, “Bone healing in
critical-size defects treated with bioactive glass/calcium sulfate:
a histologic and histometric study in rat calvaria,” Clinical Oral
Implants Research, vol. 18, no. 3, pp. 311–318, 2007.
[23] J. B. B. Weber, A. L. B. Pinheiro, M. G. de Oliveira, F. A.
M. Oliveira, and L. M. P. Ramalho, “Laser therapy improves
healing of bone defects submitted to autologus bone graft,”
Photomedicine and Laser Surgery, vol. 24, no. 1, pp. 38–44, 2006.
[24] N. S. Aboelsaad, M. Soory, L. M. A. Gadalla et al., “Effect
of soft laser and bioactive glass on bone regeneration in the
treatment of infra-bony defects (a clinical study),” Lasers in
Medical Science, vol. 24, no. 3, pp. 387–395, 2009.
[25] Y. Ozawa, N. Shimizu, G. Kariya, and Y. Abiko, “Low-energy
laser irradiation stimulates bone nodule formation at early
stages of cell culture in rat calvarial cells,” Bone, vol. 22, no. 4,
pp. 347–354, 1998.
[26] R. A. Nicolau, V. Jorgetti, J. Rigau, M. T. T. Pacheco, L. M. dos
Reis, and R. A. Zangaro, “Effect of low-power GaAIAs laser
(660 nm) on bone structure and cell activity: an experimental
animal study,” Lasers in Medical Science, vol. 18, no. 2, pp. 89–
94, 2003.
[27] S. K. Shakouri, J. Soleimanpour, Y. Salekzamani, and M. R.
Oskuie, “Effect of low-level laser therapy on the fracture healing
process,” Lasers in Medical Science, vol. 25, no. 1, pp. 73–77, 2010.
[28] L. A. D. S. Merli, V. P. de Medeiros, L. Toma et al., “The low
level laser therapy effect on the remodeling of bone extracellular
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 253590, 10 pages
http://dx.doi.org/10.1155/2014/253590

Research Article
Osteoconductive Potential of Barrier NanoSiO2 PLGA
Membranes Functionalized by Plasma Enhanced Chemical
Vapour Deposition

Antonia Terriza,1 Jose I. Vilches-Pérez,2 Emilio de la Orden,2

Francisco Yubero,1 Juan L. Gonzalez-Caballero,2 Agustin R. González-Elipe,1
José Vilches,2 and Mercedes Salido2
1
Nanotechnology on Surfaces Laboratory, Instituto de Ciencia de Materiales de Sevilla (CSIC), Universidad de Sevilla,
Avenida Américo Vespucio 49, 41092 Seville, Spain
2
Facultad de Medicina, Servicios Centrales de Investigación en Ciencias de la Salud, University of Cadiz, Dr. Marañon 3,
11002 Cadiz, Spain

Correspondence should be addressed to Mercedes Salido; [email protected]

Received 26 February 2014; Accepted 1 April 2014; Published 4 May 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Antonia Terriza et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The possibility of tailoring membrane surfaces with osteoconductive potential, in particular in biodegradable devices, to create
modified biomaterials that stimulate osteoblast response should make them more suitable for clinical use, hopefully enhancing bone
regeneration. Bioactive inorganic materials, such as silica, have been suggested to improve the bioactivity of synthetic biopolymers.
An in vitro study on HOB human osteoblasts was performed to assess biocompatibility and bioactivity of SiO2 functionalized
poly(lactide-co-glycolide) (PLGA) membranes, prior to clinical use. A 15 nm SiO2 layer was deposited by plasma enhanced chemical
vapour deposition (PECVD), onto a resorbable PLGA membrane. Samples were characterized by X-ray photoelectron spectroscopy,
atomic force microscopy, scanning electron microscopy, and infrared spectroscopy (FT-IR). HOB cells were seeded on sterilized test
surfaces where cell morphology, spreading, actin cytoskeletal organization, and focal adhesion expression were assessed. As proved
by the FT-IR analysis of samples, the deposition by PECVD of the SiO2 onto the PLGA membrane did not alter the composition
and other characteristics of the organic membrane. A temporal and spatial reorganization of cytoskeleton and focal adhesions
and morphological changes in response to SiO2 nanolayer were identified in our model. The novedous SiO2 deposition method
is compatible with the standard sterilization protocols and reveals as a valuable tool to increase bioactivity of resorbable PLGA
membranes.

1. Introduction in vitro, as cells interact with native or topographically and

chemically tailored structures in many ways [1–5].
Cell-biomaterial interaction is a major challenge for tissue The GBR therapeutic approach is based on the concept
engineering, since not only the physic-mechanic-chemical that regeneration of osseous defects is attainable via the
features of the natural extracellular matrix must be mimicked, application of occlusive membranes, resorbable or nonre-
but also a successful cell to biomaterial adhesion should be sorbable, which mechanically exclude nonosteogenic cell
achieved in the shortest time. Cell adhesion is critical in populations from the surrounding soft tissues. Nowadays
the first stages of the tissue engineering methodology where GBR is subject of growing interest due to the increasing needs
adhesion is the basis and the key process that allows other for permanent, temporary, or biodegradable orthopaedic
important cell activities such as migration and growth. In devices designed for bone repair and regeneration [6–11].
the fields of tissue engineering and guided bone regeneration A variety of polymeric bioresorbable GBR membranes
(GBR), an appropriate cell culture scaffold is required to that permit single-step procedures, thus reducing patient
enhance cell attachment, proliferation, and differentiation discomfort and costs, and potential surgical complications
2 BioMed Research International

are currently available. Polymeric coatings for biomedical 2. Material and Methods
devices are especially interesting due to the diversity of their
chemical and physical properties and natural biopolymers 2.1. Cell Culture. HOB human osteoblasts were incubated in
appear promising as biomimetic coatings, as a result of Osteoblast Growing Medium with 10% foetal calf serum at
their similarity to human tissues. Although these membranes 37∘ and 5% CO2 until the experiments were started. HOB
are biocompatible, nontoxic, or immunogenic and permit cells did not exceed ten population doublings. Test surfaces
mechanical support during bone formation, they do not were sterilized under ultraviolet (u.v.) light for 20 min each
actually possess bioactive properties to induce osteogenesis side, in a laminar flow chamber prior to cell seeding. HOB
[6, 9, 12–14]. osteoblasts were seeded at a density of 5000 cells/cm2 on test
Poly(lactide-co-glycolide) (PLGA) is among the few syn- surfaces, that is, bare PLGA or SiO2 /PLGA membranes, and
thetic polymers approved for human clinical use due to immunolabelled after 24, 48, and 72 hours. HOB osteoblasts,
its biocompatibility, controllable degradability, and relatively media, and sera were obtained from Promocell (Heidelberg,
good processability and has been used in the design of Germany).
scaffolds for bone tissue engineering and for drug delivery
purposes [15, 16]. However, the polymer itself is quite bioinert 2.2. PLGA Membranes. The PLGA membranes used as sub-
and does not elicit a significant biological response in bone strates were prepared from a 1.5 wt% PLGA dichloromethane
cells [8, 16]. solution by evaporation of the solvent on a teflon plate. The
The physicochemical properties of the material at the size of the membranes was 15 × 15 mm2 . The thickness of the
cell-material interface are decisive for the cell-material inter- membranes was of the order of 50 microns.
action, due to its interfacial role between the material and
the host tissue, and are able to trigger a wide variety of 2.3. Deposition and Characterization of SiO2 Thin Film Layers.
processes, from the initial inflammatory reaction to ultimate SiO2 was deposited onto the PLGA membranes by PECVD in
tissue remodelling [13, 17]. Although the bulk properties of a plasma reactor with a remote configuration. The deposition
materials are important for the overall properties, especially system consisted of a quartz tube with a funnel shape
for mechanical strength, a topographical modification of the termination attached to a stainless-steel chamber where
cell-substrate interface is of utmost importance. Thus, the the samples were placed. The plasma source is a surfatron
possibility of tailoring membrane surfaces, in particular in (Sairem) launcher that was supplied with a microwave power
biodegradable devices with bioactive properties, to create of 60 W. These conditions provide a typical plasma electron
modified biomaterials that mimic natural bone extracellular density of about 1 ⋅ 1011 cm−3 . Distance from substrate to the
matrix (ECM) and stimulate osteoblast response should make quartz tube was 5 cm. The plasma gas and the precursor used
them more suitable for clinical use, hopefully enhancing bone were pure Ar (7.5 sccm) and hexamethyldisiloxane (HDMSO,
regeneration in the near future [1, 2, 18]. 5 sccm), respectively. Synthesis of the films was carried out
Bioactive inorganic materials, such as silica granules, at room temperature, working at a pressure of 0.3 Torr. The
have been suggested to improve the bioactivity of synthetic gas flow was controlled by mass flow controllers (MKS). Both
biopolymers in promoting the osteogenic performance of the stainless steel receptacle which contained the precursor
osteoblast-like cells. It has been strongly suggested that sili- and the dosing line were heated at 323 K to prevent any
con/silica is functionally active during bone formation with condensation in the tube walls. Before depositing SiO2 , the
a close relationship between silicate and calcium deposition. polymeric substrate was exposed to plasma of pure argon
Silica is a low cost and biocompatible inorganic material for 2 min, a pretreatment that favours the adhesion of the
having excellent chemical stability with great commercial oxide layer onto the polymer surface. A thin layer of SiO2
value and ideal for tailoring surface topographies [19–24]. of 15 nm, determined by measuring with an adjacent quartz
We have designed an in vitro model in order to assess crystal monitor, was deposited on the polymeric substrate.
normal human osteoblasts, HOB, response to a series of SiO2 These measurements were calibrated with the data obtained
functionalized PLGA membranes intended to be used as for thicker layer of SiO2 deposited on a silicon substrate that
barrier membranes for bone guided regeneration. were cleaved and examined by scanning electron microscopy.
For the synthesis of the PLGA foils we have followed clas- Before the treatments, Ar or the mixture Ar + HDMSO was
sical synthesis procedures based on the solution/evaporation kept flowing for at least 30 min to ensure the purity of the
method [25–27]. A problem encountered by the surface plasma gas in the reactor [36]. The nonfunctionalized and
functionalization of these membranes by classical sol-gel or SiO2 functionalized membranes will be designated as PLGA
similar wet chemical routes is the degradation of the PLGA and SiO2 /PLGA, respectively. The samples were characterized
when it is exposed to liquid media or treated at relatively by X-ray photoelectron spectroscopy (XPS) to determine
high temperatures [28–34]. To avoid these problems, we have their surface chemical state and composition, atomic force
followed a new approach consisting of the deposition at room microscopy (AFM) to ascertain their roughness, scanning
temperature by plasma enhanced chemical vapour deposition electron microscopy (SEM) to assess the film microstructure,
(PECVD) of a very thin layer of SiO2 on the surface of the infrared spectroscopy (FT-IR) to determine the bonding
PLGA membranes (SiO2 /PLGA) [35], in order to achieve a structure, and water contact angle measurements to check
strict control of the deposition conditions without inducing the wetting characteristics of their surfaces. XPS spectra were
any damage in the PLGA that could efficiently promote the recorded with a SPECS PHOIBOS-100 spectrometer working
development of bone regenerating cells. in the constant pass energy mode fixed at a value of 20 eV.
BioMed Research International 3

The Mg Ka radiation was used as excitation source. For PLGA SiO2 /PLGA
calibration of the binding energy scale, a value of 284.6 eV
was considered for the C1s component attributed to C–
H and C–C bonds. Surface composition was estimated by
calculating the area behind the C1s , Si2p , and O1s peaks
and by correcting the obtained values with the sensitivity
factors of these elements. AFM images were taken with a
Cervantes AFM microscope driven with a Dulcinea control
system (Nanotec, Madrid, Spain) working in tapping mode
and using high frequency cantilevers. The images were
processed with Nanotec WSxM software. SEM images were
acquired in a Hitachi S4800 microscope. FT-IR spectra were
collected in attenuated total reflectance (ATR) mode in a RMS = 0.34 nm RMS = 0.40 nm
JASCO FT/IR6200 spectrometer, performing 64 scans with Figure 1: AFM images of PLGA (left) and SiO2 /PLGA (right).
a resolution of 0.96 cm−1 and a spot size of 1 cm of diameter.
Static contact angles of water were determined by the Young
method in a SCA20 instrument (Data Physic Instruments,
later a two-way ANOVA contrast with the ranges of this
GmbH), with an estimated error of 3%.
variable [37]. Post hoc contrasts were carried out to detect the
differences among the experimental groups. For shape vari-
2.4. Cell Morphology and Spreading. HOB osteoblasts seeded ables, a factor analysis was performed in order to reduce the
on test surfaces were daily examined with a DM ILLED Leica dimension and a two-way ANOVA with the resulting factors
phase contrast microscope in order to evaluate cell morphol- to analyze the influence of the experimental conditions. All
ogy and spreading, cell alignment, and initial adhesion phase the analysis was performed with SPSS program.
to surfaces, prior to fluorescence and CLSM examination.

2.5. Immunolabelling for Actin Cytoskeletal Organization 3. Results

and Focal Adhesion Expression. At the designed experi-
3.1. PLGA Membranes: Deposition and Characterization of
mental time, cells in each group were washed with pre-
SiO2 Thin Film Layers. The SiO2 thin films prepared by
warmed phosphate buffered saline (PBS) and fixed with 3.7%
PECVD presented a homogeneous microstructure and a
paraformaldehyde at room temperature, permeabilized with
conformal growth on the PLGA membrane. The 15 nm film
0.1% Triton x-100, preincubated in 1% bovine serum albumin
surface was rather flat as shown in AFM images (Figure 1) of
in PBS, and then immunolabelled for actin cytoskeleton with
PLGA and SiO2 -PLGA samples. In the latter, the deposition
rhodamine-phalloidin and for focal adhesion identification
of the SiO2 film only increased the RMS roughness from
with monoclonal antivinculin FITC conjugate. After 20 min
0.32 to 0.43 nm, while surface topology slightly changed with
samples were twice PBS rinsed prior to mounting with
the formation of finer grains. Even if the SiO2 thickness was
Vectashield (Vector, CA). All chemicals were obtained from
rather thin (15 nm), the PLGA surface was totally covered
Sigma-Aldrich (St. Louis, Mo) unless noted.
by SiO2 , as can be directly retrieved from the XPS spectra
(Figure 2).
2.6. Immunolabelled Cells Examination. Samples were visu- The C1s spectrum of the PLGA depicts three components
alized in a Leica TCS-SL confocal microscope. At least five at 284.6, 286.4, and 288.6 eV, attributed to C–C/C–H, C–O,
samples were analysed for each experimental group to assess and >C=O/COOH species [22, 23]. This complex spectrum
surface influence on cytoskeletal organization, focal adhesion transformed when the PLGA membrane was covered by
number and development, and cell morphology. Samples SiO2 , where only a C1s component at 284.6 eV, attributed
were exposed to the lowest laser power that was able to to spurious carbon due to contamination, can be identified.
produce a fluorescent signal, with a pinhole of 1 airy. Images An equivalent modification appears in the O1s spectra, with
were acquired at a resolution of 512 × 512, mean voxel size of clearly different widths of PLGA and SiO2 spectra. The Si2p
209.20 nm. spectra confirms that the PLGA membrane was free from this
element, while the SiO2 /PLGA samples depict a well-defined
2.7. Image Analysis. To analyze the differences in focal Si 2p peak at 103.9 eV, attributed to Si4+ ions in SiO2 [38, 39].
adhesion number between different sample groups, images It must be stressed that the deposition by PECVD of the SiO2
were collected and processed. Area, perimeter, roundness, onto the PLGA membrane did not alter the composition and
circularity, solidity, and axial ratio were analysed as shape other characteristics of the organic membrane as proved by
variables. Sample images were collected as frames obtained the FT-IR analysis of samples. The spectral regions reported
at 40x magnification and processed using Leica imaging soft- (Figure 3) for the PLGA and SiO2 -PLGA samples were very
ware and Image J software (NIH, http://rsb.info.nih.gov/ij/). similar and are characterized by the typical shape of the
spectra of this polymer. The small increase in the height of
2.8. Statistical Analysis. For the variable number of contacts, the peak at 1050 cm−1 observed for the SiO2 /PLGA sample
we used first the nonparametric contrast Kruskal-Wallis and can be attributed to the contribution at this wavenumbers of
4 BioMed Research International

Intensity (a.u.)

Intensity (a.u.)

Intensity (a.u.)
295 290 285 280 540 535 530 110 105 100 95
BE (eV) BE (eV) BE (eV)
PLGA PLGA PLGA
SiO2 /PLGA SiO2 /PLGA SiO2 /PLGA

Figure 2: From left to right, C1s, O1s, and Si2p photoelectron spectra of PLGA and SiO2 /PLGA as indicated.

754

%T (a.u.)
1460 1371
%T (a.u.)

1428

2942 1753 1185

1069-984
2994
3100 3050 3000 2950 2900 2850 2800 1800 1600 1400 1200 1000 800 600
Wavenumber (cm−1 ) Wavenumber (cm−1 )
PLGA PLGA
SiO2 /PLGA SiO2 /PLGA

Figure 3: FT-IR spectra of the PLGA and SiO2 /PLGA films measured in ATR mode.

the Si–O–Si stretching vibration of the silica network in the weeks of immersion in liquid media. The exposure of PLGA
SiO2 film [36]. The other features in the spectra correspond and SiO2 /PLGA membranes to u.v. irradiation under similar
to well-documented vibrations of PLGA polymers: 2994, conditions to those used in the sterilization protocol prior to
2946, 2840 (CH bend), 1753 (C=O ester), 1460, 1428, 1371 cell seeding did not produce any detectable change.
(CH3 ), 1185, 1080–980 (C–O stretch), and 754 (CH-bend)
cm−1 [40]. No significant spectral changes are expected due to
3.2. Living Cell Examination: Cell Morphology and Spreading.
the different thickness of the PLGA membrane (50 𝜇m) and
Living HOB cells examination revealed a successful cell
the SiO2 films (15 nm) and the typical thickness proved by
spreading on silica treated membranes, with cell elongation
ATR analysis (a few tens of microns).
and marked filopodial emission, and the establishment of
Since wetting behavior and surface tension are important
clear cell contacts and attachment to the functionalized
parameters for cell development of cells on bioactive materi-
surface in the first 24 hours. Cells clustered and elongated
als [41, 42] the water contact angle was measured for both the
during the next hours, and after 48 hours in culture, cell
PLGA and SiO2 /PLGA membranes and the values obtained
spreading evolved to a clear cell packaging, with osteoblasts
were 99.3∘ and 93.8∘ , respectively, indicating no significant
well adhered to the silica treated surface and to the neigh-
differences in wetting between these two surfaces.
boring cells (Figures 4(b) and 4(d)). Cells grown on bare
During the experimental time, no clear signs of degra-
PLGA membranes spread evenly although clearly adhered to
dation were observed in the membranes, During the exper-
surfaces, showing some lamellipodia in the first 24 hours but
imental time, no clear signs of degradation were observed in
without clear cell clustering after 48 hours in culture (Figures
the membranes. FT-IR, AFM, and XPS analysis of the surface
4(a) and 4(c)).
of the PLGA and SiO2 /PLGA membranes extracted from
the PBS and gentled dried in air did not reveal significant
changes suggesting any significant degradation. membrane 3.3. Actin Cytoskeletal Organization and Vinculin Immuno-
degradation (loss of physical integrity, changes in color, etc.) labelling of Focal Adhesion Sites. Osteoblasts grown on the
could only be observed after 20 weeks of immersion in liquid SiO2 functionalized PLGA membranes showed defined stress
media, although clear signs of degradation (loss of physical fibers formation, with a clear polarization of actin cytoskele-
integrity, changes in color, etc.) could be observed after 20 ton towards strongly evident vinculin positive sites. Cell
BioMed Research International 5

(a) (b)

(c) (d)

Figure 4: HOB cells distribution and spreading after 24 (a, b) and 48 (c, d) hours in culture. Both (b) and (d) represent osteoblasts grown on
silica treated PLGA membranes and (a) and (c) represent osteoblasts grown on bare PLGA. All images were obtained in the phase contrast
microscope, magnification 40x.

(a) (b)

(c) (d)

Figure 5: HOB cells grown on bare PLGA and imunolabelled, 48 hours (a, b) and 72 hours (c, d) after seeding, with rhodamine-phalloidin
for actin cytoskeleton (red) and antivinculin antibody (green) for focal adhesion sites.

polarization was clearly determined by SiO2 functionalized more numerous and well developed in those cells grown
surfaces, with osteoblasts showing extensive lamellipodial on the SiO2 functionalized membranes HOB grown on bare
emission reinforced with vinculin positive focal adhesion PLGA randomly orientated and showed a reduced stress
in the leading edge and supported by a highly defined fibers and focal adhesion development at the same exper-
actin network. Focal adhesions appeared to be significantly imental time. Although some evenly distributed filopodia
6 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

Figure 6: HOB cells grown on SiO2 functionalized membranes, immunolabelled with rhodamine-phalloidin for actin cytoskeleton (red)
and antivinculin antibody (green) for focal adhesion sites, 48 hours after seeding. Representative images of cytoskeletal features and focal
adhesions, showing in detail (a, b) stress fibers development, (c, d) cell clustering, and (e, f) lamellipodial and (g, h) filopodial emissions.

Table 1: Variable number of contacts. Descriptive parameters.

Groups N Minimum Maximum Median Mean St. dev.

1 PLGA CLEAN 48 H 11 96 274 143 154.27 49.601
2 PLGA SILICON 48 H 11 159 311 229 230.73 46.866
3 PLGA CLEAN 72 H 9 63 198 145 128.89 51.163
4 PLGA SILICON 72 H 10 194 340 241.5 250.50 46.049

were observed, cells did not show a well-organized actin the analysis of the effects of each factor and their interaction
network or polarization, and vinculin positive sites appeared with the ranks of the variable [38], our results indicate that the
randomly distributed in the cell periphery (Figures 5, 6, model is able to explain 58.8% and that silicon is a significant
and 7). factor (𝑃 = 0.000), while time factor is not (𝑃 = 0.929).
Furthermore, silicon treated groups (post hoc contrast of
3.4. Image Analysis: Statistical Analysis of Focal Adhesion Scheffé, 𝑃 = 0.819) and untreated groups (𝑃 = 0.873) can
Behaviour. The high significance obtained after the quanti- be considered homogeneous at any time, and both groups are
tative analysis of focal adhesion number by Kruskal-Wallis different from each other (𝑃 = 0.05) (Figure 8, Table 1).
nonparametric contrast for the 4 groups obtained with the
combination of the factors silicon and time (H = 23.506, 3.5. Image Analysis: Statistical Analysis of the Shape Vari-
𝑃 = 0.000, df = 3) indicates that silicon, time, or both influ- ables. The descriptive analysis of the shape variables in
ence focal adhesion number. Using the two-way ANOVA, the osteoblasts grown in the four experimental groups did
BioMed Research International 7

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 7: HOB cells grown on SiO2 functionalized membranes, immunolabelled with rhodamine-phalloidin for actin cytoskeleton (red) and
antivinculin antibody (green) for focal adhesion sites, 72 hours after seeding. Representative images for (a, b, e, f) cell clustering, (c, d) stress
fibers distribution, and (e, f) lamellipodial emissions.

not establish a clear relationship with the factors silicon or area and perimeter and can, therefore, be interpreted as the
time. Then, an exploratory factor analysis was performed, cell size, with higher values for larger cells.
in the attempt to reduce the number of morphometric Growing cells were scored for the morphometric indices
indexes (latent factors). The correlation matrix and the according to the values obtained in each one by each
factor loadings matrix were obtained first by the method of individual cell. The two-way ANOVA contrast performed
principal components and later by varimax rotation, which for these indices showed that the experimental factor silicon
quantify the relationship between the original variables and significantly increased (𝑃 = 0.000) the mean score for SR
the indexes achieved. The first index positively related to index, estimating this increase by CI95% = (0.94, 1.71), and
circularity and solidity and negatively related to perimeter. also caused a decrease in ST index (𝑃 = 0.016), estimated
This first index represents the property of solid roundness by CI95% = (−1.03, −0.11). Moreover, the factor time caused a
(SR) showing higher values in those cells that are circular, decrease in the average score for the ST index (𝑃 = 0.012),
without voids, and with a medium perimeter. The second estimated by CI95% = (−1.06, −0.14), while a significant
index contrasts roundness and AR, thus representing stretch interaction (𝑃 = 0.029) was obtained for the index size, as cell
(ST) or lengthening, with the highest values for the more size decreases in cells grown on bare PLGA surfaces, CI95% =
elongated cells, with high AR, and negative values more (−1.28, 0.65), and increases in silicon treated ones, CI95% =
circular cells, with AR close to 1. The third index relates to (0.32, 1.30). These results indicate that silica treatment,
8 BioMed Research International

350 52 practically unaltered after the SiO2 as revealed by FT-IR

analysis (Figure 3). In comparison with wet chemical routes,
300 which may affect the integrity of this sensitivity substrate,
2 PECVD has been demonstrated to be a quite controllable
250 and friendly procedure for the surface functionalization of
resorbable polymers. The absence of a significant damage has
No contacts

200 been proved here by the AFM analysis of the SiO2 /PLGA
substrate showing that the surface roughness is not signifi-
150 cantly altered after the deposition of SiO2 . Stability of these
membranes after 72 hours of immersion in a PBS liquid and
100 after UV irradiation sustains that the in vitro cell growth
analysis basically refers to the “as prepared” samples without
50 any significant alteration during its manipulation or during
their immersion in the culture medium.
PLGA PLGA PLGA PLGA Cell adhesion onto a biomaterial surface is a major
CLEAN 48 H SILICON 48 H CLEAN 72 H SILICON 72H
contributing factor of its cytocompatibility and the host
Groups
response when implanted, ultimately determining device bio-
Figure 8: Box-Whisker graphics reporting the variable number of compatibility. Without adhesion, osteoblasts cannot spread
contacts among cells on each sample. or differentiate. A princeps issue in cell adhesion is the
development of focal adhesions which, furthermore, are
established sites for signal transduction, acting as conveyors
of mechanical forces directly to the nucleus via the cytoskele-
independently of time, favors the presence of cells that were tal network by the complex process of mechanotransduction,
circular and solid (without voids). On the other hand, and where vinculin is required for a strong network formation [1,
independently of silica treatment, the experimental factor 4, 44]. Based on the fact that the size and shape of cell spread-
time favors a lower ST, or roundness, and increases the size ing area, as well as the number and distribution of focal
in cells grown on silica treated membranes. adhesion plaques, are decisive for migratory, proliferative,
and differentiation behaviour of anchorage-dependent cells,
4. Discussion we performed animage-based quantitative feature extraction
design in which focal adhesion quantification has been used
There are several ways to modify the surface of a material in as marker to assess the osteoconductive potential of the
order to enhance its attractiveness for cell colonization and substrata, together with actin cytoskeleton arrangement [2,
osteogenic cell differentiation. For biological applications, 17, 20, 45].
there is an additional need for versatile deposition methods The relation between focal adhesion number, function-
in the case of devices with potential clinical applications, ality, cell movement, and growth of skeletal cells has been
which should also stand, without alterations, the sterilization described, by our group and others [46], and the formation
processes [13, 16, 18]. We have successfully demonstrated that of super-mature adhesions has also been related to osteo-
PECVD TiO2 functionalization methodology employed by genesis support [47]. Actin polymerization and cytoskele-
our group enhances HOB cells response to nonresorbable ton rearrangement induced by topographical and chemical
PET devices and stands for standard u.v. sterilization [43]. We cues present in the growing surface are known to serve
now herein present a novel design of barrier membrane for as driving forces for directional migration and morpho-
GBR purposes, in which HOB cells response to PECVD SiO2 logical alterations. Of particular interest is the temporal
functionalized PLGA membranes has been assessed. and spatial reorganization of cytoskeleton and the focal
Trace amounts of silicon have been described to substan- adhesion formation in response to SiO2 layer identified in our
tially contribute to strengthening of bones with promotion model, parameters that have been established as important
of bone formation especially during ageing. Well-dispersed mediators of the mechanotransductive processes, parameters
nanosized inorganic particles have been described to enhance that have been established as important mediators of the
the mechanical properties of organic materials [10]. A mechanotransductive processes [17, 47, 48].
recent study on biosilica influence on the OPG-RANKL Adherent cells require extracellular matrix anchorage to
ratio in SaOS-2 cells in vitro suggests both osteoinductive proliferate and undergo differential function. As shown by
and osteogenic potential of biosilica since it combines an our data, HOB cells grown on SiO2 functionalized PLGA
upregulation of the genes required for the differentiation with membranes actively probe the physical properties of the
the potential to increase proliferation of the osteogenic cells ECM, with their contractile machinery clearly facilitating the
[18, 24]. formation of polarized lamellipodia and evenly distributed
Our PLGA membranes were fabricated by using a con- filopodia, which gather spatial, topographical, and chemical
ventional resorbable PLGA polymer as substrate and then information from the surface. Initial cell tethering and filopo-
functionalized by depositing a very thin layer of SiO2 , as dia exploration are followed by lamellipodia ruffling mem-
active layer, by PECVD. A key feature of this method is that brane activity and cellular spreading. The results, obtained
it preserved the integrity of the substrate, which remained after a carefully designed in vitro study, are reinforced with
BioMed Research International 9

a powerful statistical treatment and reveal that in vitro [6] P. Gentile, V. Chiono, C. Tonda-Turo, A. M. Ferreira, and G. Cia-
HOB cell morphologies are significantly different and vary rdelli, “Polymeric membranes for guided bone regeneration,”
greatly depending on the substrata and are consistent with Biotechnology Journal, vol. 6, no. 10, pp. 1187–1197, 2011.
an increased cytoskeletal tension related to focal adhesion [7] T.-S. Jang, E.-J. Lee, J.-H. Jo et al., “Fibrous membrane of nano-
increase in SiO2 treated membranes. hybrid poly-L-lactic acid/silica xerogel for guided bone regen-
eration,” Journal of Biomedical Materials Research B: Applied
Biomaterials, vol. 100, no. 2, pp. 321–330, 2012.
5. Conclusions [8] E. Pamula, E. Filová, L. Bačáková, V. Lisá, and D. Adamczyk,
“Resorbable polymeric scaffolds for bone tissue engineering:
The methodology described provides a biocompatible and the influence of their microstructure on the growth of human
durable functionalization on our PLGA barrier membranes osteoblast-like MG 63 cells,” Journal of Biomedical Materials
that elicits a significant osteoblast response. Both the mor- Research A, vol. 89, no. 2, pp. 432–443, 2009.
phological changes and the focal adhesion development iden- [9] M. Retzepi and N. Donos, “Guided bone regeneration: bio-
tified in our model significantly related to SiO2 treated PLGA logical principle and therapeutic applications,” Clinical Oral
both the morphological changes and the focal adhesion Implants Research, vol. 21, no. 6, pp. 567–576, 2010.
development identified in our model are significantly related [10] J.-H. Song, H.-E. Kim, and H.-W. Kim, “Collagen-apatite
to SiO2 treated PLGA. The novedous SiO2 deposition method nanocomposite membranes for guided bone regeneration,”
reveals as a valuable tool to increase bioactivity of PLGA Journal of Biomedical Materials Research B: Applied Biomateri-
membranes by combining cell nanotopography cues with the als, vol. 83, no. 1, pp. 248–257, 2007.
incorporation of bioactive factors, that in addition, stands the [11] S.-H. Teng, E.-J. Lee, P. Wang, D.-S. Shin, and H.-E. Kim,
standard sterilization protocols. “Three-layered membranes of collagen/hydroxyapatite and chi-
tosan for guided bone regeneration,” Journal of Biomedical
Materials Research B: Applied Biomaterials, vol. 87, no. 1, pp. 132–
Conflict of Interests 138, 2008.
[12] R. Dimitriou, G. I. Mataliotakis, G. M. Calori, and P. V.
The authors declare that there is no conflict of interests Giannoudis, “The role of barrier membranes for guided bone
regarding the publication of this paper. regeneration and restoration of large bone defects: current
experimental and clinical evidence,” BMC Medicine, vol. 10,
article 81, 2012.
Acknowledgments [13] V. Gribova, R. Auzely-Velty, and C. Picart, “Polyelectrolyte
multilayer assemblies on materials surfaces: from cell adhesion
The authors thank the Junta de Andalucı́a (Projects P09-CTS-
to tissue engineering,” Chemistry of Materials, vol. 24, no. 5, pp.
5189, TEP5283, and FQM-6900) and the Spanish Ministry 854–869, 2012.
of Economy and Competitiveness (Projects CONSOLIDER
[14] T. Tirri, J. Rich, J. Wolke, J. Seppälä, A. Yli-Urpo, and T.
CSD 2008-00023, MAT2010-21228, and MAT2010-18447) O. Närhi, “Bioactive glass induced in vitro apatite formation
and Instituto de Salud Carlos III (FIS PI 09/00508) for on composite GBR membranes,” Journal of Materials Science:
financial support (patent P201232018 (Resorbable membrane Materials in Medicine, vol. 19, no. 8, pp. 2919–2923, 2008.
for guided bone regeneration) PCT pending). [15] Y. Amano, M. Ota, K. Sekiguchi, Y. Shibukawa, and S. Yamada,
“Evaluation of a poly-l-lactic acid membrane and membrane
fixing pin for guided tissue regeneration on bone defects
References in dogs,” Oral Surgery, Oral Medicine, Oral Pathology, Oral
[1] M. J. Biggs and M. J. Dalby, “Focal adhesions in osteoneogene- Radiology, and Endodontics, vol. 97, no. 2, pp. 155–163, 2004.
sis,” Proceedings of the Institution of Mechanical Engineers H, vol. [16] M. Vandrovcová and L. Bačáková, “Adhesion, growth and
224, no. 12, pp. 1441–1453, 2010. differentiation of osteoblasts on surface-modified materials
developed for bone implants,” Physiological Research, vol. 60,
[2] M. J. P. Biggs, R. G. Richards, and M. J. Dalby, “Nanotopo-
no. 3, pp. 403–417, 2011.
graphical modification: a regulator of cellular function through
focal adhesions,” Nanomedicine: Nanotechnology, Biology, and [17] C. J. Bettinger, R. Langer, and J. T. Borenstein, “Engineering
Medicine, vol. 6, no. 5, pp. 619–633, 2010. substrate topography at the Micro- and nanoscale to control cell
function,” Angewandte Chemie (International Edition), vol. 48,
[3] E.-J. Lee, S.-H. Teng, T.-S. Jang et al., “Nanostructured poly(𝜀- no. 30, pp. 5406–5415, 2009.
caprolactone)-silica xerogel fibrous membrane for guided bone [18] N. Takeuchi, M. Machigashira, D. Yamashita et al., “Cellu-
regeneration,” Acta Biomaterialia, vol. 6, no. 9, pp. 3557–3565, lar compatibility of a gamma-irradiated modified siloxane-
2010. poly(lactic acid)-calcium carbonate hybrid membrane for
[4] T. Orita, M. Tomita, and K. Kato, “Regulation of cellular guided bone regeneration,” Dental Materials Journal, vol. 30, no.
responses to macroporous inorganic films prepared by the 5, pp. 730–738, 2011.
inverse-opal method,” Colloids and Surfaces B: Biointerfaces, vol. [19] C. K. Choi, M. T. Breckenridge, and C. S. Chen, “Engineered
84, no. 1, pp. 187–197, 2011. materials and the cellular microenvironment: a strengthening
[5] J. A. Sanz-Herrera and E. Reina-Romo, “Cell-biomaterial interface between cell biology and bioengineering,” Trends in
mechanical interaction in the framework of tissue engineering: Cell Biology, vol. 20, no. 12, pp. 705–714, 2010.
insights, computational modeling and perspectives,” Interna- [20] F. Intranuovo, P. Favia, E. Sardella et al., “Osteoblast-like cell
tional Journal of Molecular Sciences, vol. 12, no. 11, pp. 8217–8244, behavior on plasma deposited micro/nanopatterned coatings,”
2011. Biomacromolecules, vol. 12, no. 2, pp. 380–387, 2011.
10 BioMed Research International

[21] V. Puddu and C. C. Perry, “Peptide adsorption on silica [37] D. A. Shah and L. V. Madden, “Nonparametric analysis of
nanoparticles: evidence of hydrophobic interactions,” ACS ordinal data in designed factorial experiments,” Phytopathology,
Nano, vol. 6, pp. 6356–6363, 2012. vol. 94, no. 1, pp. 33–43, 2004.
[22] Y. Tang, Y. Zhao, X. Wang, and T. Lin, “Layer-by-layer assembly [38] T. L. Barr, “An XPS study of Si as it occurs in adsorbents,
of silica nanoparticles on 3D fibrous scaffolds: enhancement catalysts, and thin films,” Applications of Surface Science, vol. 15,
of osteoblast cell adhesion, proliferation, and differentiation,” no. 1–4, pp. 1–35, 1983.
Journal of Biomedical Materials Research, 2013. [39] J. Finster, D. Schulze, F. Bechstedt, and A. Meisel, “Interpreta-
[23] M. Vallet-Regı́, M. Colilla, and B. González, “Medical applica- tion of XPS core level shifts and structure of thin silicon oxide
tions of organic-inorganic hybrid materials within the field of layers,” Surface Science, vol. 152-153, no. 2, pp. 1063–1070, 1985.
silica-based bioceramics,” Chemical Society Reviews, vol. 40, no. [40] M. M. Stevanovic, B. Jordovic, and D. P. Uskokovic, “Prepa-
2, pp. 596–607, 2011. ration and characterization of poly(D, L-lactide-co-glycolide)
[24] X. Wang, H. C. Schröder, M. Wiens, H. Ushijima, and W. E. nanoparticles containing ascorbic acid,” Journal of Biomedicine
G. Müller, “Bio-silica and bio-polyphosphate:applications in and Biotechnology, vol. 2007, Article ID 84965, 8 pages, 2007.
biomedicine (bone formation),” Current Opinion in Biotechnol- [41] H. Aita, N. Hori, M. Takeuchi et al., “The effect of ultraviolet
ogy, vol. 23, pp. 570–578, 2012. functionalization of titanium on integration with bone,” Bioma-
[25] H. Shen, X. Hu, J. Bei, and S. Wang, “The immobilization of terials, vol. 30, no. 6, pp. 1015–1025, 2009.
basic fibroblast growth factor on plasma-treated poly(lactide- [42] W. Att, N. Hori, F. Iwasa, M. Yamada, T. Ueno, and T. Ogawa,
co-glycolide),” Biomaterials, vol. 29, no. 15, pp. 2388–2399, 2008. “The effect of UV-photofunctionalization on the time-related
[26] Y. Wan, X. Qu, J. Lu et al., “Characterization of surface property bioactivity of titanium and chromium-cobalt alloys,” Biomate-
of poly(lactide-co-glycolide) after oxygen plasma treatment,” rials, vol. 30, no. 26, pp. 4268–4276, 2009.
Biomaterials, vol. 25, no. 19, pp. 4777–4783, 2004.
[43] A. Terriza, A. Diaz-Cuenca, F. Yubero et al., “Light induced
[27] C. Witt and T. Kissel, “Morphological characterization of hydrophilicity and osteoblast adhesion promotion on amor-
microspheres, films and implants prepared from poly(lactide- phous TiO2 ,” Journal of Biomedical Materials Research A, vol.
co-glycolide) and ABA triblock copolymers: is the erosion 101, pp. 1026–1035, 2013.
controlled by degradation, swelling or diffusion?” European
[44] M. J. P. Biggs, R. G. Richards, N. Gadegaard et al., “Interactions
Journal of Pharmaceutics and Biopharmaceutics, vol. 51, no. 3,
with nanoscale topography: adhesion quantification and signal
pp. 171–181, 2001.
transduction in cells of osteogenic and multipotent lineage,”
[28] J. Gao, L. Niklason, and R. Langer, “Surface hydrolysis of Journal of Biomedical Materials Research A, vol. 91, no. 1, pp.
poly(glycolic acid) meshes increases the seeding density of 195–208, 2009.
vascular smooth muscle cells,” Journal of Biomedical Materials
[45] L. Bacakova, E. Filova, F. Rypacek, V. Svorcik, and V. Stary,
Research, vol. 42, pp. 417–424, 1998.
“Cell adhesion on artificial materials for tissue engineering,”
[29] A. C. R. Grayson, M. J. Cima, and R. Langer, “Size and
Physiological Research, vol. 53, supplement 1, pp. S35–S45, 2004.
temperature effects on poly(lactic-co-glycolic acid) degradation
and microreservoir device performance,” Biomaterials, vol. 26, [46] M. Salido, J. I. Vilches-Perez, J. L. Gonzalez, and J. Vilches,
no. 14, pp. 2137–2145, 2005. “Mitochondrial bioenergetics and distribution in living
human osteoblasts grown on implant surfaces,” Histology and
[30] C. E. Holy, S. M. Dang, J. E. Davies, and M. S. Shoichet, “In vitro
Histopathology, vol. 24, no. 10, pp. 1275–1286, 2009.
degradation of a novel poly(lactide-co-glycolide) 75/25 foam,”
Biomaterials, vol. 20, no. 13, pp. 1177–1185, 1999. [47] L. E. McNamara, T. Sjöström, K. E. V. Burgess et al., “Skeletal
stem cell physiology on functionally distinct titania nanoto-
[31] H. Kranz, N. Ubrich, P. Maincent, and R. Bodmeier, “Physi-
pographies,” Biomaterials, vol. 32, no. 30, pp. 7403–7410, 2011.
comechanical properties of biodegradable poly(D, L-lactide)
and poly(D, L-lactide-co-glycolide) films in the dry and wet [48] M. J. Dalby, “Topographically induced direct cell mechan-
states,” Journal of Pharmaceutical Sciences, vol. 89, pp. 1558– otransduction,” Medical Engineering and Physics, vol. 27, no. 9,
1566, 2000. pp. 730–742, 2005.
[32] M. Källrot, U. Edlund, and A.-C. Albertsson, “Surface func-
tionalization of degradable polymers by covalent grafting,”
Biomaterials, vol. 27, no. 9, pp. 1788–1796, 2006.
[33] J. S. C. Loo, C. P. Ooi, and F. Y. C. Boey, “Degradation of
poly(lactide-co-glycolide) (PLGA) and poly(L-lactide) (PLLA)
by electron beam radiation,” Biomaterials, vol. 26, no. 12, pp.
1359–1367, 2005.
[34] H. Tsuji, Y. Tezuka, and K. Yamada, “Alkaline and enzymatic
degradation of L-lactide copolymers. II. crystallized films of
poly(L-lactide-co-D-lactide) and poly(L-lactide) with similar
crystallinities,” Journal of Polymer Science B: Polymer Physics,
vol. 43, no. 9, pp. 1064–1075, 2005.
[35] A. Barranco, J. Cotrino, F. Yubero et al., “Synthesis of SiO2 and
SiOxCyHz thin films by microwave plasma CVD,” Thin Solid
Films, vol. 401, no. 1-2, pp. 150–158, 2001.
[36] A. Barranco, F. Yubero, J. Cotrino et al., “Low temperature
synthesis of dense SiO2 thin films by ion beam induced chemical
vapor deposition,” Thin Solid Films, vol. 396, no. 1-2, pp. 9–15,
2001.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 982104, 7 pages
http://dx.doi.org/10.1155/2014/982104

Clinical Study
Vertical Ridge Augmentation of the Atrophic Posterior Mandible
with Sandwich Technique: Bone Block from the Chin Area
versus Corticocancellous Bone Block Allograft—Clinical and
Histological Prospective Randomized Controlled Study

Luigi Laino,1 Giovanna Iezzi,2 Adriano Piattelli,2 Lorenzo Lo Muzio,1 and Marco Cicciù3
1
Department of Clinical and Experimental Medicine, University of Foggia, FO, Italy
2
Department of Stomatology and Oral Science, University of Chieti, Italy
3
Human Pathology Department, School of Dentistry, University of Messina, Via Consolare Valeria, 98100 Messina, Italy

Correspondence should be addressed to Marco Cicciù; [email protected]

Received 29 March 2014; Accepted 15 April 2014; Published 29 April 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Luigi Laino et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aim of the present study is to compare the histological aspects of bone formation in atrophic posterior mandibles augmented
by autologous bone block from chin area with corticocancellous bone block allograft used as inlays with the sandwich technique.
Materials and Methods. Sixteen patients with bilateral partial edentulism in the posterior mandible were selected. The residual bone
height, preliminarily measured by computed tomography scans, ranged between 5 and 7 mm from the inferior alveolar nerve. All
patients required regeneration procedure with autologous bone block from chin area (control group) versus bone block allograft
Puros (Zimmer Dental, 1900 Aston Avenue, Carlsbad, CA, USA) (test group). Histological and histomorphometric samples were
collected at the time of implant positioning in order to analyze the percentage of newly formed bone, the residual graft material,
and marrow spaces/soft tissue. Results. No statistically significant differences between the two groups were found regarding the
percentage of newly formed bone. The percentage of residual grafted material was significantly higher in the test group, whilst the
percentage of marrow spaces was higher in control group. Conclusions. In conclusion, both procedures supported good results,
although the use of bone blocks allograft was less invasive and preferable than harvesting bone from the mental symphysis.

1. Introduction mandible. The use of short implants represents a simpler

and faster alternative to the augmentation procedure, even
The rehabilitation of posterior mandible with dental implants if in some “critical cases” the residual bone crest above the
represents today a hard challenge for clinicians due to the lack inferior alveolar nerve is only 5–7 mm in height, and therefore
of supporting bone. The alveolar nerve presence and lifting the surgical augmentation treatment is mandatory. Indeed,
and the gradual vertical and horizontal resorption of the the displacement of the alveolar nerve is technically tough,
mandibular bone crest in both partially and totally edentu- and this procedure may be associated with certain degree
lous patients can be treated by several prosthetic and surgical of permanent loss of nerve sensitivity [1, 6–9]. Different
options [1–5]. Patients can be rehabilitated with conventional surgical techniques are currently being used to augment
partial removable dentures, but often this treatment does the posterior mandible: guided bone regeneration (GBR)
not meet the expectations of the patients. Regarding implant and alveolar distraction osteogenesis onlay bone grafting;
supported treatment options, vertical ridge augmentation, however, only few of these have been tested in randomized
surgical displacement of the inferior alveolar nerve, and, clinical trial (RCT) [10, 11]. Several surgical bone augmenta-
finally, the placement of short implants (8 mm or less) could tion techniques are related to an unpredictable resorption of
be necessary for the correction of the atrophic posterior the grafted material. Vascularity seems to be the main factor
2 BioMed Research International

in determining whether such a graft can be maintained in posterior mandible, and (15) under chronic treatment with
situ. Traditional distraction osteogenesis aims to maintain the steroids or nonsteroidal anti-inflammatory drugs. Twelve
majority of the vascularity to the transported bone segment. patients were considered eligible and were enrolled in the trial
The drawbacks of distraction osteogenesis include patient (mean age was 57 years, 9 females and 3 males).
cooperation, technique sensitivity, and the possibility of a
second surgery to remove the device [1, 3, 7].
2.1. Augmentation Procedure. Two weeks before bone aug-
Another possible approach is to use an interpositional
mentation and implant placement, all patients underwent
bone graft [1, 4, 8, 11]. The rationale of the interpositional
oral hygiene instructions and professional debridement,
techniques is based on the theory that biomaterial placed
when necessary. On the day of the augmentation proce-
between 2 pieces of pedicled bone with internal cancellous
dure, the envelopes containing the randomized codes were
bone will undergo rapid and complete healing and graft
opened. All patients received antibiotics prior to the surgery.
incorporation with a lower percentage of resorption. The
Antimicrobial prophylaxis was obtained with the use of
sandwich osteotomy allows for the positioning of the graft
1 gr of amoxicillin + clavulanic acid (Augmentin, Glaxo-
in a well-delimited area as well as offering adequate blood
SmithKline, Brentford, Middlesex, UK) (or erythromycin
supply to maintain new bone growth. This procedure enables
500 mg if allergic to penicillin), starting one day before
the simultaneous correction of the sagittal intermaxillary
surgery and for the following 4 days. All patients were
relationship and the vertical dimension. This technique has
treated under local anesthesia with intravenous sedation. A
been used in a variety of maxillary areas including both the
paracrestal incision was made through the buccal mucosa
anterior and posterior mandible and maxilla. When perform-
respecting the emergence of the mental nerve, and, as the
ing the sandwich osteotomy in the posterior mandible, great
full thickness flap was retracted, tension on the mental nerve
surgical precision is required to avoid damage to the inferior
was carefully avoided. The horizontal osteotomy was made at
alveolar nerve. For these reasons and for the few results
4 mm from the mandibular canal using conventional surgical
available in the literature, it is necessary to carry out further
micromotor. Two oblique cuts were made in the coronal third
research to validate the predictability of this regenerative
of the mandibular bone with the mesial cut at least 2 mm
technique [1, 3, 8–11].
distal to the last tooth in the arch Figures 1(a), 1(b), and 1(c).
The aim of the present study is to compare the histological
The osteotomies were completed with the use of bone chisels.
aspects of bone formation in atrophic posterior mandibles
The height of the osteotomized segment was at least 3 mm
augmented by autologous bone block from chin area (control
to allow the insertion of a stabilizing screw without risking
group) to Puros bone block allograft (test group) used as
the fracture of the distracted bone segment. The segment was
inlays with the sandwich technique.
elevated preserving the lingual periosteum, and according to
the outcome of the randomization, the graft materials were
modelled to the desired height and shape to fill the site and
2. Materials and Methods interposed between the raised fragment and the mandibular
basal bone. Titanium miniplates and miniscrews (Gebruder
Between November and April 2010, nineteen patients with
Martin GmbH & Co., KG, Tuttlingen, Germany) were used
bilateral partial edentulism in the posterior mandible were
to fix the osteotomized crestal bone to the basal bone. The
selected for the present study. They all showed a residual
grafted area was covered with a resorbable barrier of peri-
bone height ranging between 5 and 7 mm from the inferior
cardium (Copios Pericardium Membrane Zimmer Dental,
alveolar nerve, which was firstly measured by computed
Switzerland) Figures 2(a), 2(b), and 2(c). Periosteal incisions
tomography scans. All patients required the placement of
were made to release the flaps coronally as needed and
at least 3 implants. The protocol of the study was approved
were sutured with Vicryl 5.0 sutures until the incisions were
by the Ethical Committee of the Second University of
perfectly sealed. Patients were instructed to use Corsodyl
Naples, Naples, Italy, and all the patients signed a written
gel 1% twice a day for 2 weeks and then 0.2 chlorhexidine
informed consent form. All patients were treated in the
mouthwashes twice a day for up to the second month, to
Department of Oral and Maxillofacial Surgery, Second Uni-
avoid brushing and trauma on the surgical sites. Removable
versity of Naples, Naples, Italy. Exclusion criteria were (1)
prostheses were not allowed. Patients were seen after 10 days
general contraindications to implant surgery, (2) irradiation,
for follow-up examinations and sutures removal. Patients
chemotherapy, or immunosuppressive therapy over the past
were recalled for additional postoperative check-ups 1, 2, and
5 years, (3) poor oral hygiene and motivation, (4) active
4 months after the augmentation procedure. Four months
periodontitis, (5) uncontrolled diabetes, (6) pregnancy or
after augmentation, a CT scan was taken to plan implant
lactation, (7) substance abusers, (8) smoking more than 10
placement.
cigarettes per day, (9) psychiatric problems or unrealistic
expectations, (10) acute infection in the area intended for
implant placement, (11) positive to HIV and hepatitis B and 2.2. Implant Placement. Six months after the augmenta-
C, (12) autoimmune diseases such as rheumatoid arthritis, tion procedure, at the moment of dental implant surgery,
systemic lupus erythematosus, scleroderma, Sjogren’s syn- miniplates were removed and the bone core biopsies were
drome, and dermatomyositis/polymyositis, (13) treated or retrieved by using 2.9 mm diameter trephine bur (Komet
under treatment with intravenous aminobisphosphonates, 227b, Italy), and 72 implants (Spline Zimmer Dental, Switzer-
(14) previously subjected to reconstructive procedures of the land) were inserted in situ, as shown in Figure 3. Drills with
BioMed Research International 3

(a) (b)

(c)

Figure 1: Sample of the two oblique cuts performed in the coronal third portion of the mandibular bone with the mesial cut at least 2 mm
distal to the last tooth in the arch (a, b, c).

(a) (b) (c)

Figure 2: Sample of the grafted area covered with a resorbable barrier of pericardium (a, b, c).

increasing diameters were used to prepare the implant sites. System (Assing, Rome, Italy). The specimens were dehy-
The surgical unit was settled with a torque of 25 Ncm. After drated in a graded series of ethanol rinses and embedded
the dental implant placement, the cover screws were placed in a glycol methacrylate resin (Technovit 7200 VLC, Kulzer,
and the flap closure was obtained with Vicryl 4.0. Patients Wehrheim, Germany). After polymerization, the specimens
were instructed to use 0.2% chlorhexidine mouthwash for were sectioned, along their longitudinal axis, with a high-
1 min twice a day for 2 weeks, to have a soft diet for 1 week, precision diamond disc at about 150 𝜇m and ground down
and to avoid brushing and trauma on the surgical sites. No to about 30 𝜇m with a specially designed grinding machine.
removable prosthesis was allowed. Sutures were removed The slides were stained with acid fuchsin and toluidine blue
after 10 days. and examined in normal transmitted light under a Leitz
Laborlux microscope (Laborlux S, Leitz, Wetzlar, Germany).
Histomorphometry of the percentages of newly formed bone,
2.3. Histological Procedure. Bone cores were retrieved, imme- residual grafted material, and marrow spaces was carried
diately stored in 10% buffered formalin, and processed to out using a light microscope (Laborlux S, Leitz, Wetzlar,
obtain thin ground sections using the Precise 1 Automated Germany) connected to a high resolution video camera
4 BioMed Research International

Figure 4: Grafted bone, almost completely surrounded by newly

formed bone can be observed. Acid fuchsin-toluidine blue; original
magnification 60x.
Figure 3: Sample of the bone core biopsies that were retrieved by
using 2.9 mm diameter trephine bur.

(3CCD, JVC KY-F55B, JVC, Yokohama, Japan) and interfaced

to a monitor and PC. This optical system was associated
with a digitizing pad (Matrix Vision GmbH, Oppenweiler,
Germany) and a histometry software package with image
capturing capabilities (Image-Pro Plus 4.5, Media Cybernet-
ics Inc., Immagini & Computer Snc, Milan, Italy). The same
investigator made all the measurements.

2.4. Statistical Analysis. Data were evaluated by the Shapiro-

Wilk test. All the data are presented as mean +/− stan- Figure 5: The autogenous bone block presents marked staining
dard deviations (SD); statistically significant differences were differences from the host trabecular bone and specifically, it shows
accepted as 𝑃 < 0.05. a lower affinity for the stains. The block is surrounded by newly
formed bone. Acid fuchsin-toluidine blue; original magnification
40x.
3. Results
The failures and complications that occurred during the
entire study period were limited. In one patient treated 3.1.2. Test Group. In all the analyzed samples, a good amount
with the Puros bone block, exposure of a titanium plate 2 of newly formed bone could be observed (Figure 8). A tight
months after surgery occurred; it was treated by removing contact between the grafted material and the regenerated
the plaque, and then a satisfactory healing was achieved. bone, without any interposition of fibrous tissue, was found
In two patients treated with autologous bone from mental (Figure 9). The newly formed bone had a high affinity for dyes
symphysis, a temporary paresthesia of the anterior region of and was acid fuchsine positive; therefore, a highly stained
the mandible was appreciated and treated by drug solution, line was observed at the grafting material-new bone interface.
Dobetin 5000 mcg 1 time per day for 1 week and 3 doses in In many fields, it was possible to observe the presence of
the second week. large osteocytes lacunae in contact with the grafted material
(Figure 10). Some trabeculae of grafted material were bridged
3.1. Histological Results by newly formed bone, which was observed both in the inner
and outer portions of some biomaterial particles (Figure 11).
3.1.1. Control Group. In the control group, a significant Marrow stromal cells and blood vessels were found inside the
amount of grafted bone, almost completely surrounded by marrow spaces. In some fields, there was a modest amount of
newly formed bone, was observed (Figure 4). The autologous inflammatory infiltrate. No osteoclasts were observed around
grafted bone showed irregularly shaped margins, proba- the graft particles.
bly due to the remodeling process. The demarcation line The histomorphometric results are summarized in Tables
(cementing line) between grafted bone and newly formed 1, 2, and 3.
bone was evident (Figure 5). In some areas, bone remodeling There was no statistically significant difference between
was conceivable with a rim of osteoblasts depositing osteoid the two groups in terms of amount of new bone, 31.47 ± 2.2
matrix (Figure 6). Osteons in the vicinity of grafted bone versus 30.6±3.7% (𝑃 = 0.5362 has been recorded, while there
could be observed (Figure 7). No signs of inflammatory was a statistically significant difference in the percentage of
infiltrate were present. residual grafted material higher in test group (Table 2).
BioMed Research International 5

Figure 6: A rim of osteoblasts depositing osteoid matrix is evident. Figure 8: A good amount of newly formed bone can be observed.
Acid fuchsin-toluidine blue; original magnification 200x. Acid fuchsin-toluidine blue; original magnification 6x.

Figure 7: An osteon in the vicinity of grafted bone can be seen. Acid Figure 9: The bovine bone block is surrounded by newly formed
fuchsin-toluidine blue; original magnification 200x. bone. A tight contact between the grafted material and the regen-
erated bone without any interposition of fibrous tissue can be
observed. Acid fuchsin-toluidine blue; original magnification 40x.
Table 1
Group Obs. Mean Std. error Std. deviation 95% conf. I. Table 3
Group Ctrl 12 31.47 7155495 2.262.766 29.85131
Group Obs Mean Std. error Std. deviation 95% conf. I.
33.08868
Group Ctrl 12 48.97 1.878.241 5.939.519 44.72112
Group test 12 30.6 117.936 3.729.462 27.9321
53.21888
33.2679
Grouptest 12 41.28 1.888.491 5.971.934 41.84335
Newly formed bone, t value = 0.6307, and P value = 0.5362. There is no
statistically significant difference.
48.40665
Marrow space, t value = 2.8872, and P value = 0.0098. There is statistically
significant difference.
Table 2
Group Obs. Mean Std. error Std. deviation 95% conf. I.
16.57178 representing a valuable and predictable surgical alternative
Group Ctrl 12 19.56 1.320.959 417.724 technique for posterior mandible atrophic ridge.
22.54822
25.28039
In 2006, Jensen retrospectively evaluated the crestal
Group test 12 28.9 1.600.069 5.059.864 stability of alveolar augmentation using an interpositional
32.51961
bone graft for dental implant restorations and found a good
Residual graft material, t value = −4.5015, and P value = 0.0003. There is
statistically significant difference. stability after 4-year follow-up. In 2008, Felice et al. produced
a series of clinical investigations for analyzing the effective-
ness of this technique in relation to the use of biomaterial,
4. Discussion and also in this case the results appeared satisfactory [12–15].
Another main aspect is the choice of the graft material
This study is designed to evaluate how a bone substitute to be used. In 2009, Felice et al. performed a randomized
material may offer some advantages in the place of auto- controlled clinical trial to evaluate two different kinds of graft
genous bone grafts harvested from the mental symphysis materials: bone form iliac crest and bovine anorganic bone.
in the treatment of atrophic posterior mandibles. Moreover, There were ten selected partially edentulous patients having
the authors proposed a novel technique for localized vertical 5–7 mm of residual crystal height above the mandibular
bone augmentation using an interpositional bone block canal. Four months after bone grafting, a bone core was
6 BioMed Research International

it later stated that bone grafting may form wider bone. No

mention was made of the advantage gained with the copious
blood supply by the sandwich osteotomy technique [14–19].
Other studies have shown that fewer cases of dehiscence
were observed with the sandwich osteotomy than with
techniques using only graft or titanium mesh. Laviv et al.
recently reported that in 10 patients treated with a similar
technique, vertical gain ranged from 3 to 6 mm over a 4-
year period [16]. Authors stated that efforts to displace the
segment greater than 5 mm “may not only risk the potential
for vascular embarrassment by detaching periosteal blood
supply, but also can excessively rotate the segment palatally,
Figure 10: Large osteocyte lacunae in contact with the grafted mate- compromising aesthetic gingival projection.” In the anterior
rial are present. Acid fuchsin-toluidine blue; original magnification maxilla, it has been shown that one of the disadvantages could
200x. be the reduced extensibility of the palatal mucoperiosteum
that does not allow a vertical increase of more than 10 mm
to be done [6, 18–20]. In a letter to the editor, Robiony et
al. suggest that the vertical movement could be extended
more than the 10 mm proposed by Jensen, but only in the
canine and premolar zones. They described their experience
with 25 patients and demonstrated that the technique can
be successful without compromising vascular supply and
esthetics [20–24]. All those studies clearly demonstrated,
even with some limitations, how the use of this technique,
by expert surgeons, might be safe and predictable giving less
discomfort for the patients [14, 22, 25].
In the present study, in order to offer our patients a
less invasive surgery, a biomaterial has been compared to
Figure 11: New bone can be seen in the inner and outer portions autologous bone and the difference in newly formed bone
of a residual grafted particle. Acid fuchsin-toluidine blue; original percentages was not statistically significant. During the his-
magnification 100x. tomorphometric evaluation, the percentage of newly formed
bone was found to be lower in the test group (28.9/19.5); this
meant a slower integration of the grafted material, which is
retrieved from each side using a 3 mm external diameter not clinically appreciable; therefore, the use of autologous
trephine for the histological evaluation. The histomorphome- bone blocks does not seem to provide particular advantages.
tric data showed the only statistically significant difference
in the mean percentage of residual graft (between 10% and
13%, 𝑃 values between 0.008 and 0.009) that was greater in 5. Conclusion
the Bio-Oss group, while there were no statistically significant
The results of the present investigation encourage further
differences in the percentage of newly formed bone and in
studies on the present topic. However, other clinical trials
the marrow space between the two groups. Also the clinical
are needed, with a greater number of patients. The results
outcomes present in the literature are very interesting [6, 12,
reported in the literature and this preliminary study under-
14–18]. In 2006, Jensen published a retrospective study to
line how the interposition technique seems to be a valid
evaluate the crystal stability of alveolar augmentation using
therapeutic option in the treatment of vertical atrophy of the
an interpositional bone graft for dental implant restorations.
posterior mandible. Both graft materials gave good results in
Eight patients with 10 graft sites were followed from 1 to 4
relation to this type of surgical technique; the use of Puros
years with panographic evaluation to determine if dimension
bone block allograft represents a less invasive alternative for
changes of the alveolar graft sites had occurred. The author
the patients. In the future, it would be interesting to compare
described a little loss of crystal height and 20 of the 22
this technique with short implants and to record those results
implants confirmed high stability at the follow up evalua-
over the long term.
tion. These results were later confirmed by various studies
conducted by the group of Felice et al. (2008, 2009) and the
results of this study confirmed the possibility of considered Conflict of Interests
this surgical technique like predictable one [2, 4, 7, 12, 16, 18].
The sandwich osteotomy can be considered an alterna- As the corresponding author, Marco Cicciù declares that
tive to other bone augmentation techniques presented in all the authors have no conflict of interests regarding the
the recent literature. Although one study determined that devices used for this study and that the current research is
interpositional bone grafting and alveolar distraction yielded not influenced by any secondary interests, such as financial
statistically similar results in regard to buccolingual width, gain.
BioMed Research International 7

References [14] A. Bianchi, P. Felice, G. Lizio, and C. Marchetti, “Alveolar

distraction osteogenesis versus inlay bone grafting in posterior
[1] F. D. Das Neves, D. Fones, S. R. Bernardes, C. J. do Prado, and A. mandibular atrophy: a prospective study,” Oral Surgery, Oral
J. F. Neto, “Short implants—an analysis of longitudinal studies,” Medicine, Oral Pathology, Oral Radiology and Endodontology,
The International Journal of Oral & Maxillofacial Implants, vol. vol. 105, no. 3, pp. 282–292, 2008.
21, no. 1, pp. 86–93, 2006. [15] P. Felice, G. Cannizzaro, V. Checchi et al., “Vertical bone aug-
[2] H. Li, C. R. Zhou, M. Y. Zhu, J. H. Tian, and J. H. Rong, “Prepa- mentation versus 7-mm-long implants in posterior atrophic
ration and characterization of homogeneous hydroxyapatite/ mandibles. Results of a randomised controlled clinical trial
chitosan composite scaffolds via in-situ hydration,” Journal of of up to 4 months after loading,” European journal of oral
Biomaterial and Nanobiotechnology, vol. 1, pp. 42–49, 2010. implantology, vol. 2, no. 1, pp. 7–20, 2009.
[3] K. A. Zimmermann, J. M. Leblanc, K. T. Sheets, R. W. Fox, [16] A. Laviv, O. T. Jensn, E. Tarazi, and N. Casap, “Alveolar sandwich
and P. Gatenholm, “Biomimetic design of a bacterial cellu- osteotomy in resorbed alveolar ridge for dental implants: a
lose/hydroxyapatite nanocomposite for bone healing applica- 4 year prospective study,” Journal of Oral and Maxillofacial
tions,” Materials Science and Engineering C, vol. 31, no. 1, pp. Surgery, vol. 72, pp. 292–303, 2014.
43–49, 2011. [17] M. S. Tonetti and C. H. F. Hämmerle, “Advances in bone aug-
[4] B. Rosenquist, “Implant placement in combi- nation with nerve mentation to enable dental implant placement: consensus
transpositioning: experiences with the first 100 cases,” The report of the sixth european workshop on periodontology,”
International Journal of Oral & Maxillofacial Implants, vol. 9, pp. Journal of Clinical Periodontology, vol. 35, no. 8, pp. 168–172,
522–531, 1994. 2008.
[5] A. S. Herford, R. Tandon, T. W. Stevens, E. Stoffella, and [18] P. Felice, C. Marchetti, A. Piattelli et al., “Vertical ridge augmen-
M. Cicciù, “Immediate distraction osteogenesis: the sandwich tation of the atrophic posterior mandible with interpositional
technique in combination with rhBMP-2 for anterior maxillary block grafts: bone from the iliac crest versus bovine anorganic
and mandibular defects,” Journal of Craniofacial Surgery, vol. 24, bone,” European Journal of Oral Implantology, vol. 1, no. 3, pp.
no. 4, pp. 1383–1387, 2013. 183–198, 2008.
[6] M. Chiapasco, M. Zaniboni, and L. Rimondini, “Autogenous [19] B. Fang, Y.-Z. Wan, T.-T. Tang, C. Gao, and K.-R. Dai, “Prolifer-
onlay bone grafts vs. alveolar distraction osteogenesis for the ation and osteoblastic differentiation of human bone marrow
correction of vertically deficient edentulous ridges: a 2–4-year stromal cells on hydroxyapatite/bacterial cellulose nanocom-
prospective study on humans,” Clinical Oral Implants Research, posite scaffolds,” Tissue Engineering A, vol. 15, no. 5, pp. 1091–
vol. 18, no. 4, pp. 432–440, 2007. 1098, 2009.
[7] A. S. Herford, M. Lu, L. Akin, and M. Cicciù, “Evaluation of a [20] Y. Zhang and M. Zhang, “Synthesis and characterization of
porcine matrix with and without platelet-derived growth factor macroporous chitosan/calcium phosphate composite scaffolds
for bone graft coverage in pigs,” The International Journal of Oral for tissue engineering,” Journal Biomedical Material Research,
& Maxillofacial Implants, vol. 27, no. 6, pp. 1351–1358, 2012. vol. 55, pp. 304–312, 2001.
[8] M. Merli, F. Bernardelli, and M. Esposito, “Horizontal and ver- [21] H. M. Hashemi and B. Javidi, “Comparison between interpo-
tical ridge augmentation: a novel approach using osteosynthesis sitional bone grafting and osteogenic alveolar distraction in
microplates, bone grafts, and resorbable barriers,” International alveolar bone reconstruction,” Journal of Oral and Maxillofacial
Journal of Periodontics and Restorative Dentistry, vol. 26, no. 6, Surgery, vol. 68, no. 8, pp. 1853–1858, 2010.
pp. 581–587, 2006. [22] M. Politi and M. Robiony, “Localized alveolar sandwich oste-
otomy for vertical augmentation of the anterior maxilla,” Jour-
[9] M. Simion, S. A. Jovanovic, C. Tinti, and S. P. Benfenati, “Long-
nal of Oral and Maxillofacial Surgery, vol. 57, no. 11, pp. 1380–
term evaluation of osseointegrated implants inserted at the time
1382, 1999.
or after vertical ridge augmentation: a retrospective study on
I23 implants with 1–5 year follow-up,” Clinical Oral Implants [23] O. T. Jensen, L. Kuhlke, J.-F. Bedard, and D. White, “Alveolar
Research, vol. 12, no. 1, pp. 35–45, 2001. segmental sandwich osteotomy for anterior maxillary vertical
augmentation prior to implant placement,” Journal of Oral and
[10] J. H. Fu, T. J. Oh, E. Benavides, I. Rudek, and H. L. Wang, “A
Maxillofacial Surgery, vol. 64, no. 2, pp. 290–296, 2006.
randomized clinical trial evaluating the efficacy of the sand-
wich bone augmentation technique in increasing buccal bone [24] K.-H. Bormann, M. M. Suarez-Cunqueiro, C. von See et al.,
thickness during implant placement surgery: I. Clinical and “Forty sandwich osteotomies in atrophic mandibles: a retro-
radiographic parameters,” Clinical Oral Implants Research, vol. spective study,” Journal of Oral and Maxillofacial Surgery, vol.
25, no. 4, pp. 458–467. 69, no. 6, pp. 1562–1570, 2011.
[11] S.-H. Park, K.-W. Lee, T.-J. Oh, C. E. Misch, J. Shotwell, and H.- [25] J. A. Elo, A. S. Herford, and P. J. Boyne, “Implant success in
L. Wang, “Effect of absorbable membranes on sandwich bone distracted bone versus autogenous bone-grafted sites,” The Jour-
augmentation,” Clinical Oral Implants Research, vol. 19, no. 1, pp. nal of Oral Implantology, vol. 35, no. 4, pp. 181–184, 2009.
32–41, 2008.
[12] O. T. Jensen, “Alveolar segmental “sandwich” osteotomies for
posterior edentulous mandibular sites for dental implants,”
Journal of Oral and Maxillofacial Surgery, vol. 64, no. 3, pp. 471–
475, 2006.
[13] C. Marchetti, S. Trasarti, G. Corinaldesi, and P. Felice, “Interpo-
sitional bone grafts in the posterior mandibular region: a report
on six patients,” The International Journal of Periodontics and
Restorative Dentistry, vol. 27, no. 6, pp. 547–555, 2007.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 410627, 10 pages
http://dx.doi.org/10.1155/2014/410627

Research Article
Surface Characteristics and Bioactivity of a Novel Natural
HA/Zircon Nanocomposite Coated on Dental Implants

Ebrahim Karamian,1 Amirsalar Khandan,2

Mahmood Reza Kalantar Motamedi,3 and Hesam Mirmohammadi4,5
1
Department of Materials Engineering, Najafabad Branch, Islamic Azad University, Isfahan, Iran
2
Young Researchers and Elite Club, Khomeinishahr Branch, Islamic Azad University, Isfahan, Iran
3
Dental Students Research Center, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
4
Dental Materials Research Center, Department of Restorative Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
5
Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA),
Universiteit van Amsterdam and Vrije Universiteit, Amsterdam 1081 JD, The Netherlands

Correspondence should be addressed to Hesam Mirmohammadi; [email protected]

Received 5 February 2014; Accepted 1 April 2014; Published 16 April 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Ebrahim Karamian et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

The surface characteristics of implant which influence the speed and strength of osseointegration include surface chemistry, crystal
structure and crystallinity, roughness, strain hardening, and presence of impurities. The aim of this study was to evaluate the
bioactivity and roughness of a novel natural hydroxyapatite/zircon (NHA/zircon) nanobiocomposite, coated on 316L stainless steel
(SS) soaked in simulated body fluid (SBF). NHA/zircon nanobiocomposite was fabricated with 0 wt.%, 5 wt.%, 10 wt.%, and 15 wt.%
of zircon in NHA using ball mill for 20 minutes. The composite mixture was coated on 316L SS using plasma spray method. The
results are estimated using the scanning electron microscopy (SEM) observation to evaluate surface morphology, X-ray diffraction
(XRD) to analyze phase composition, and transmission electron microscopy (TEM) technique to evaluate the shape and size of
prepared NHA. Surfaces roughness tester was performed to characterize the coated nanocomposite samples. The maximum average
𝑅𝑎 (14.54 𝜇m) was found in the NHA 10 wt.% of zircon coating. In addition, crystallinity (𝑋𝑐 ) was measured by XRD data, which
indicated the minimum value (𝑋𝑐 = 41.1%) for the sample containing 10 wt.% of zircon. Maximum bioactivity occurred in the sample
containing 10 wt.% of zircon, which was due to two reasons: first, the maximum roughness and, second, the minimum crystallinity
of nanobiocomposite coating.

1. Introduction on or into the titanium oxide layer) or physically (by increas-

ing the level of roughness) [2]. There are some advantages
Dental implants are well-accepted and predictable treatment regarding surface modified implants, including (a) providing
modalities for rehabilitation of patients with partial and a better stability between bone and implant during healing
complete edentulism. Dental implantation has become an
process, established by a greater contact area, (b) providing a
established treatment method since its appearance for over
surface configuration that may retain the blood clot, and (c)
40 years. The future would probably see bioactive surfaces
and additives that stimulate the bone growth. According to stimulating the bone formation and healing process [3]. There
Albrektsson et al. several factors can affect the osseointegra- are several methods to improve the quality of implant surface;
tion, including implant material, implant design, implant sur- one of them is adding bioactive materials to the surface of the
face characteristics, status of the bone, surgical technique, and dental implant to induce osteoconductivity.
implant loading conditions [1]. Nowadays, there is an effort to Hydroxyapatite (HA), [Ca10 (PO4 )6 (OH)2 ], is a calcium
speed up the osseointegration by improving the implant-to- phosphate bioceramic material, with osteoconductivity and
bone interface chemically (by incorporating inorganic phases excellent biocompatibility. However, apatite layers coating
2 BioMed Research International

the surface of the implant substrates improve the bone Table 1: Chemical composition of NHA (XRF results).
response. For this reason, it has been used successfully
Compound Concentration (w/w %)
in dentistry for many years. HA products are well known
as implantable ceramics for hard tissue reconstitution. An Ca 75.91
example of such application is a coating applied onto dental P 20.09
implants, providing enhanced fixation of the implant for the Na 1.25
human bone [4]. HA-coated implants have often been used in F 1.20
load-bearing applications, because HA directly bonds to the Mg 0.708
bone and promotes the new bone formation, necessary for Sr 0.151
the implant osseointegration [5–8]. Despite advantages of HA Cl 0.200
coatings, it has some disadvantages. Enhanced susceptibility Si 0.150
to bacterial plaque colonization and HA coating integrity
S 0.073
are two major concerns related to HA-coated implants [9].
Al 0.061
Such bacterial accumulation can start a chain reaction leading
to lesions, and then other risk factors may combine to Cu 0.056
worsen the condition [10]. An exposed rough surface can lead Zn 0.053
to increased bacterial plaque accumulation and eventually Fe 0.052
peri-implantitis [11]. Therefore, the susceptibility of peri- K 0.038
implantitis should be taken into account when using HA- Zr 0.008
coated implants. It is known that the HA surface degrades LOI∗ 0.376
and in some instances separates; hence, it has been a trend Total 100
to substitute the coatings with roughened surface dental ∗
Loss on ignition (1000∘ C, 2 h).
implants, which also incidentally presents better and faster
integration. Although HA can be a good choice to be applied
on the surface of the dental implants as a bioactive and different parameters, while 𝑅𝑎 (absolute value) is by far the
biocompatible material, its mechanical properties (mainly most common. Other common parameters include 𝑅𝑧 and
the strength, roughness, and fracture toughness) need to be 𝑅max .
improved for application in load-bearing parts, since they The aim of this study was to evaluate the bioactivity
have limited its usage to nonload-bearing parts due to their and roughness of novel natural HA/zircon (NHA/zircon)
poorness [12–16]. This goal can be achievable by adding some nanobiocomposite coating, soaked in the SBF solution.
materials to the HA coating, which has already been done to
improve its final coating characteristics. These additives aim
to enhance various properties of the coating, including bioac- 2. Materials and Methods
tivity [17], thermal stability, and its mechanical properties.
Zircon is a tetragonal mineral, consisting of a silicate of 2.1. NHA Extraction. The present research was an experi-
zirconium. The group of silicate biomaterials has the ability mental study. Bovine bones were boiled for 12 h to remove the
to release silicate ions at a definite concentration which helps attached flesh and fat. Afterwards, bones were dried at 110∘ C
the osteoblasts to grow and differentiate [18], leading to bone for 2 h to shed the moisture. To prevent blackening with soot
formation. Therefore, this characteristic of zircon could be during heating, bones were cut into small pieces with 10 mm
beneficial for the surface usage of dental implants. Consid- thickness and heated at 500∘ C (bone ash) for 2 h in air to
ering several studies evaluating the additives in the dental allow evaporation of organic substances. The resulting black
implant coatings (such as zirconia (ZrO2 ), ZrO2 -Al2 O3 , etc.), bone ash was heated for 3 h at 850∘ C. This synthesis is called
we sought to find a new additive with better mechanical thermal decomposition of bone resource to create NHA. The
and biological characteristics. Therefore, the bioactivity and elemental chemical analysis of NHA using X-ray fluorescence
roughness of zircon (ZrSiO4 ) added to the HA coating in (XRF) (Philips PW1606) is shown in Table 1.
simulated body fluid (SBF) were evaluated in this study. SBF
is an inorganic solution with a similar composition to human 2.2. Fabrication of NHA on the Implant Cores. In this work,
blood plasma without organic components. Several studies 316L stainless steel (SS) was used as substrate. The elemental
performed on nanobioceramic coatings include monitoring analysis and the elemental composition (wt.%) of SS were
the behavior of a material when submersed in the saline C 0.03, Si 0.8, Mn 1.3, Cr 17.55, Ni 13, Mo 3.1, 𝑃 ≤ 0.04,
solution [19] or SBF [20]. 𝑆 ≤ 0.03, and Fe as the balance. Specimens with dimensions
Plasma spraying technique is the most commonly used 20 × 10 mm (diameter × thickness) were cut with CNC Wire
method for the HA coatings application. This is a thermal Cut EDM Machine (DK7732F-china suppliers). The test was
spraying process in which powder particles are melted in performed on cylindrical porous and all the specimens were
a high temperature plasma flame and propelled towards a immersed in Analar grade H2 SO4 (specific gravity = 1.84)
substrate material to form a coating. The advantages of this for 1 h in different volume concentrations varying from 5%
process include high coating adhesion strength and also high to 25% at ambient temperatures. The samples were polished
deposition rate, which allow rapid formation of the coatings with 100–1200 grit silicon carbide (SiC) paper. In order to
[21]. Surface roughness can be described using a number of produce a scratch-free, mirror-finish surface, final polishing
BioMed Research International 3

was performed using 4,000 grit silicon carbide papers. The Table 2: Nominal ion concentrations of SBF in comparison with
polished specimens were investigated by the optical micro- those in human blood plasma.
scope to ensure the absence of pits or scratches on the
Ion concentrations (m mol)
surface. Specimens were cleaned with acetone and thoroughly Ion
washed with distilled water. Afterward, specimens were SBF Human blood plasma
surface treated by grit blasting in order to obtain a desired Na+ 142.0 142.0
roughness of surface for better adhesion of coating to the K+ 5.0 5.0
substrate. After the surface treatment process, the specimens Mg2+ 1.5 1.5
were cleaned with distilled water and ultrasonic device as Ca2+ 2.5 2.5
a cleaner technique. NHA/zircon nanobiocomposite was Cl− 147.8 103.0
fabricated with 0 (control), 5, 10, and 15 wt.% of zircon in HCO3 − 4.2 27.0
NHA and coated on the surface of the 316L SS cores using HPO4 2− 1.0 1.0
plasma spray technique. SO4 2− 0.5 0.5
pH 7.4 7.2–7.4
2.3. Phase and Composition Analysis. Phase structure anal-
ysis was performed by X-ray diffraction (XRD) (Philips
X’Pert-MPD diffractometer with Cu K𝛼 radiation (𝜆 1 = 3. Results
0.15418 nm) over the 2𝜃 range of 20–80). The obtained exper-
imental patterns were compared to the standards compiled by 3.1. Phase and Composition Analysis of the XRD Results.
the Joint Committee on Powder Diffraction and Standards Figure 1 shows the XRD patterns and SEM images of the NHA
(JCDPS) which involved card number 09-432 for HA. The 0% and 10% zircon coatings. Figure 1(a) indicates that the
crystallite size of prepared powders was determined using only existing phase is for HA (all the peaks belong to HA).
XRD patterns and modified Scherrer equation. Scanning However, the peaks observed in the XRD pattern of NHA
electron microscopy (SEM) analysis evaluations were per- 10% zircon had a decrease in intensity and an increase in
formed using a Philips XL30 (Eindhoven, The Netherlands) width compared to NHA 0% zircon (Figures 1(a) and 1(c)). In
to investigate the morphology. The Ca/P ratio was deter- addition, the XRD peaks corresponding to NHA 10% zircon
mined using energy-dispersive X-ray spectroscopy (EDX) shifted slightly in comparison with NHA 0% zircon (Figures
microanalysis (FEI Quanta 200 ESEM equipped with an EDX 1(a) and 1(c)). The crystallite sizes of the prepared NHA
EDS device). Samples were coated with Au using spraying, samples with different degrees of zircon content calculated
high vacuum, and 25 kV accelerating voltage. Ca and P using XRD data are shown in Table 5. The crystallite size
ions contents were measured from four spots (Figure 3), of the obtained nanopowders is in the range of 25.5–32 nm.
and consequently the average was calculated. Transmission Determination of crystallite sizes from XRD peak widths
electron microscopy (TEM) technique (Philips CM 200 FEG: makes assumptions on crystallite shape and crystallite size.
Eindhoven, The Netherlands) was utilized to evaluate the
shape and size of prepared HA. 3.2. Scherrer Equation. The modified Scherrer equation is
advantageous for decreasing the sum of absolute values of
2.4. Surface Roughness Evaluation. The roughness (i.e., 𝑅𝑎 ) errors, ∑ (±Δ ln 𝛽)2 , and producing a single line through the
of each sample was measured in three directions. Tour points to give a single value of intercept ln(𝑘𝜆/𝐿) [22]. At
measurements were taken for each sample and then their this sample, shown in Figure 2, the linear regression plot is
average was determined. obtained as 𝑦 = 0.9267𝑥 − 5.225. This is equivalent to ln 𝛽 =
ln(1/ cos 𝜃) + ln(𝑘𝜆/𝐿). From this line, the intercept is −5.225,
2.5. In Vitro Bioactivity Evaluation. In vitro bioactivity was 𝑒−5.225 = 𝑘𝜆/𝐿, and 𝐿 = 25.5 nm. Thus, HA crystallite size was
investigated by soaking the samples in SBF solution. The SBF 25.5 nm.
solution was prepared according to the procedure described
by Kokubo and Takadama [20]. Ion concentrations of SBF are 3.3. SEM, EDX, and TEM Microstructures. Figure 3 shows
similar to those in human blood plasma. Coated samples were the SEM micrographs of NHA powder. In all samples,
soaked in the cell SBF solution (pH 7.4) at 37∘ C for 1, 7, and 14 we found nanoparticles of HA crystals, agglomerated with
days at a solid/liquid ratio of 1 mg/mL, without refreshing the different dimensions. Morphology of particles is sphere and
soaking medium. Element analysis of SBF and physiological semisphere. Microchemical composition of the NHA sample
saline are shown in Table 2. After the predicted soaking time was determined using EDX (Figure 3).
finished, the disc samples were rinsed with deionized water TEM technique was utilized to evaluate the shape and size
and dried in an oven at 110∘ C for 1 h. Samples weight loss was of prepared NHA (Figure 4). The TEM image reveals that the
calculated by the following equation: powders are formed by agglomerates of irregular particles in
𝑊 − 𝑊0 200 nm, consistent with values calculated from XRD data.
Weight Loss percentage (WL.%) = × 100, (1) As shown in Figure 1, NHA crystal sizes are in micron
𝑊0
size, some particles are spherical, and the others are in sharp
where 𝑊 and 𝑊0 are primary and secondary weights, respec- shape. The agglomerated particles are composed of very fine
tively. particles.
4 BioMed Research International

∗ HA-PLA ∼ 1.SD

100 ∗∗
∗ ∗∗ ∗
∗
∗ ∗ ∗
Intensity

∗ ∗ ∗
∗ ∗ ∗ ∗
∗ ∗
25

0
20 30 40 50 60 70 80
2𝜃 (deg)
∗: hydroxyapatite
(a) (b)
HAZ10.SD
∗
∗
∗∗
100
∗ ∗∗ ∗ ∗ ∗ ∗∗
∗ ∗ ∗∗∗∗
∗ ∗ ∗ ∗∗∗∗∗ ∗ ∗∗∗
Intensity

25

0
20 30 40 50 60 70 80
2𝜃 (deg)
∗: hydroxyapatite
(c) (d)

Figure 1: (a) XRD pattern and (b) SEM micrograph of the NHA 0% coated by plasma spraying. (c) XRD pattern and (d) SEM micrograph of
the NHA 10% zircon coated by plasma spraying.

3.4. Surfaces Roughness Results. The results are given in Table 3: Surfaces roughness results.
Table 3. Each sample was measured in three directions.Tour
Experimental Composite Average 𝑅𝑎 (𝜇m)
measurements were taken for each sample and then their 𝑅𝑎 (𝜇m)
name (zircon %)
average was determined. 𝑅𝑎 was found to vary between 12.63
and 14.85 𝜇m. Coatings with four degrees of average rough- N1 0 12.92
ness (𝑅𝑎 : 12.63, 14.30, 14.54, and 12.85 𝜇m for NHA 0%, 5%, N2 0 13.97 12.63
10%, and 15% of zircon, resp.) were created. The maximum N3 0 11
average 𝑅𝑎 was found in the NHA 10% zircon sample. N4 5 13.59
N5 5 15.93 14.30
3.5. In Vitro Bioactivity Evaluation. NHA/ZrSiO4 nanobio- N6 5 13.39
composite powder with different ZrSiO4 contents was pre-
pared and coated by plasma spray method. In vitro bioactivity N7 10 15.55
N8 10 13.77 14.54
of nanopowders was investigated by soaking the powders in
the SBF solution. SEM observations of the NHA 0% and 10% N9 10 14.30
zircon coatings soaked in SBF for 1 and 2 weeks are illustrated N10 15 11.86
in Figure 5. Results indicated that during 1, 7, and 14 days of N11 15 13.44 12.85
soaking, shown in Table 4, calcium ions were released in the
N12 15 13.24
solution. As shown in Figure 6(a), there was a clear increase
BioMed Research International 5

Table 4: The HA biodegradation properties of all the samples in the SBF.

Ca2+ release in SBF Ca2+ release in SBF WL. % of samples in WL. % of samples in
Samples
after 1 week (ppm) after 2 weeks (ppm) SBF after 1 week (mg) SBF after 2 weeks (mg)
NHA 0% zircon 36 78 1.13 0.28
NHA 5% zircon 40 90 2.40 2.80
NHA 10% zircon 62 115 3.31 4.02
NHA 15% zircon 48 96 0.25 1.11

ln(1/cos𝜃) containing 10 wt.% of zircon. Maximum bioactivity occurred

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 in the sample containing 10 wt.% of zircon.
0
According to Kokubo et al. [23–25], in vitro immer-
sion of bioactive materials in SBF is thought to make in
vivo surface structure changes in materials such as bioac-
−1
tive glass/ceramic. Therefore, to evaluate this, we soaked
NHA/zircon samples in SBF solution. The grown layer is
−2 sometimes called a bone-like apatite because of the XRD
pattern similar to that of bone apatite with broad peaks at
2𝜃 angles of HA, which indicates a superfine grain of apatite
ln 𝛽

−3 crystallite [26]. Overall conclusion is that zircon is a very

complex material. It is totally different from titanium, a metal
used in dentistry in its pure (or commercially pure) form.
−4 We reached interesting findings from the present work that
NHA/zircon (a) proved to be bioactive and (b) had high
y = 0.9267x − 5.225 roughness and interesting microstructural properties.
−5
There is a consensus regarding HA to be osteoconductive
[27, 28] and biocompatible [29, 30]. The biocompatibility [31,
−6
32], osteoconductivity, and osseointegration characteristics
of zirconia are reported in several studies [33]. In addition,
Figure 2: Plot of ln𝛽 versus ln(1/cos 𝜃) of the HA sample heated at zirconia has a high flexural strength and fracture toughness.
850∘ C for 3 h. Accordingly, the combination of these characteristics could
be found in the prepared coating, the novel NHA/zircon. In
fact, this coating due to its bioactivity might speed up the
Table 5: Crystallographic Parameters of the HA Phase of all the
Samples.
osseointegration. However, this claim should be proved by
future cell culturing and animal studies.
Samples Crystallinity (𝑋𝑐 %) Crystallite Size ± 1 (nm) It is reported that topography and surface roughness
NHA 0% Zircon 44.8 25.5 positively affect healing process [34–36]. Dohan Ehrenfest
NHA 5% Zircon 43.2 28 et al. reported that possible physical improvement of the
NHA 10% Zircon 41.1 32
interface between bone and implant by increasing the level
of roughness, leading to speed up osseointegration and
NHA 15% Zircon 42.9 28.2
its quality [2]. Human and animal studies have shown
significantly greater bone and implant contact at rough
surface implants compared to machined surface implants
in calcium releasing in the sample containing 10% zircon. [37]. In 2009, a consensus report reached a conclusion that
It depends on two reasons: (1) increase of the roughness “moderately rough and rough surfaces provided enhanced
(Figure 6(b)) and (2) decrease of the crystallinity of sample bone integration compared with smooth and minimally
containing 10% wt. ZrSiO4 (𝑋𝑐 = 41.1%) (Table 5). rough surfaces” [38]. In this regard, as zircon increased the
roughness of HA coating, the positive role of NHA/zircon
4. Discussion coating for a fast and better healing might be supported. On
the other hand, classic HA-coated implants were reported
In the present study, bioactivity of NHA/zircon coatings to have a higher occurrence of complications [39]. In fact,
with various amounts of zircon (0 wt.%, 5 wt.%, 10 wt.%, and the surface roughness may have a significant impact on the
15 wt.%) within SBF solution was evaluated using XRD and contamination [2], amount and quality of plaque formation
SEM interpretations. The maximum average 𝑅𝑎 (14.54 𝜇m) [40]. Especially in patients with poor oral hygiene, an exposed
was found in the NHA 10 wt.% of zircon coating. In addi- rough surface can lead to increased bacterial plaque accumu-
tion, crystallinity (𝑋𝑐 ) was measured by XRD data, which lation and eventually peri-implantitis [11]. However, evidence
indicated the minimum value (𝑋𝑐 = 41.1%) for the sample for the influence of the implant surface characteristics as a risk
6 BioMed Research International

10 C O Na Mg P Ca
Ca
8

(cps/eV)
6

0
2 4
(KeV)
(a) (b)

Element Series (wt.%)

Carbon K-series 4.011786
Oxygen K-series 28.93848
Sodium K-series 0.798011
Magnesium K-series 0.918738
Phosphate K-series 14.36486
Calcium K-series 37.31573
Sum 86.34761

(c)

Figure 3: (a) SEM micrographs of the NHA powder, (b) EDX spectrum of the NHA powder, and (c) microchemical composition of the NHA
powder determined by EDX.

200 nm facilitate the stability of the blood clot during the early phase
of healing.
The advantages of rough-surfaced dental implants could
be described by provided support for the coagulum devel-
opment and facilitating greater bone healing and better
qualified osseointegration. Perhaps hybrid implants should
be considered in individuals that are highly predisposed to
periodontitis [42]. In such cases, inflammation due to a
rough or badly designed superstructure causes bone loss and
exposed smooth threads. If in such patients the smooth treads
become exposed in the oral cavity, the threads may be less
plaque retentive, increasing the possibility of arresting the
progression of peri-implantitis.
Figure 4: TEM micrograph of prepared NHA at 850∘ C for 3 h.
In the current study, crystallinity was measured accord-
ing to the procedure described by Karamian et al. [44].
As observed in the 𝑋𝑐 results, NHA 10% zircon has less
indicator for peri-implantitis is very limited [41, 42]. In crystallinity and more amorphous structure. The dissolution
overall, there is controversy in the literature regarding the and precipitation behaviour of apatites are the principle
effect of surface characteristics and peri-implantitis. Based on factors governing their bioactivity [45]. The obtained SEM
two systematic reviews in 2008 and 2011, due to limited data micrographs (Figure 5) show the formation and growth of
available in the literature, there is no evidence that implant apatite crystals on the surfaces of the NHA 10% zircon coating
surface characteristics can have a significant influence on the after soaking in the SBF solution during several periods
initiation of peri-implantitis [41, 42]. Moreover, Persson et of time. SEM observations showed amorphous and glassy
al. investigated the effect of surface roughness on the healing surface structure, leading to more biodegradation of NHA
following peri-implantitis treatment in the beagle dog [43]. 10% zircon coating compared to classic HA-coated implants.
They found further amount of reosseointegration in implants Dissolution of NHA/zircon coating may have significant
with a rough surface, likely because the rough surface can advantages, such as an increase in calcium and phosphate.
BioMed Research International 7

(a) (b) (c)

(d) (e) (f)

(g) (h)

Figure 5: SEM observations of the coatings soaked in SBF for (a), (b) 1 week, NHA 10% zircon, (c), (d) 2 weeks, NHA 10% zircon, (e), (f) 1
week, NHA 0% zircon, and (g), (h) 2 weeks, NHA 0% zircon.

The presence of calcium is considered to regulate initiation type IV bone, in fresh extraction sites, and in grafted
of messenger ribonucleic acid (mRNA) transcription and maxillary or nasal sinuses, or when using shorter implants
protein synthesis and therefor lead to cellular differentiation (less than or equal to 10 mm) [9]. Thus, prepared NHA/zircon
[46]. It was reported that more soluble phases on the coatings coating compared to classic HA coating could be more useful
might be more favourable for a stable interface with the in the aforementioned situations due to the higher bioactivity
biological environment [47]. It might be due to the release and roughness.
of calcium ions necessary for bone formation. As shown, the Zircon is a nonresorbable metal which provides good
amount of calcium release in the NHA 10% zircon was three mechanical properties, superior to other ceramic materials,
times more than that in classic HA coating (Figure 6(a)). such as high bioactivity and roughness. Zircon seems to
On the other hand, delamination or biodegradation of the be suitable for dental application due to its good chemical
coatings might be partly responsible for the failure in the and material stability, high strength, and proper roughness.
implant-coating interface [48, 49]. These contradictions need However, considering all the limitations of current work,
to be evaluated by further future studies. more studies are needed about the novel NHA/zircon coating.
Clinical data recommend that HA-coated implants may Besides, NHA/zircon as a new material needs extensive in
be valuable treatment indications when placing implantsin vivo and long-term controlled studies.
8 BioMed Research International

140
Calcium concentration (ppm)
120 16

)
14

Roughness number (𝜇m

100
12
80 10
60 8
6
40
4
20 2
0
0 15
0 2 4 6 8 10 12 14 16 10
5
0
Zircon (%) Zircon (%)
(a) (b)

Figure 6: (a) Calcium concentration (Ca2+ released) after 2 weeks, coatings in SBF solution. (b) Average amount of roughness of the samples.

5. Conclusion [5] R. Garcia and R. H. Doremus, “Electron microscopy of the

bone-hydroxylapatite interface from a human dental implant,”
The maximum 𝑅𝑎 (14.54 𝜇m) was found in the sample con- Journal of Materials Science: Materials in Medicine, vol. 3, no. 2,
taining 10% wt. ZrSiO4 . In addition, the maximum calcium pp. 154–156, 1992.
released (115 ppm) was found in this sample, which depends [6] Y. Harada, “Experimental studies of healing process on com-
on two reasons: (a) increase of roughness and (b) decrease of pound blocks of hydroxyapatite (HAP) particles and tricalcium
crystallinity (to 41.1%). NHA/zircon nanocomposite coating phosphate (TCP) powder implantation in rabbit mandible—
possessed a good bioactivity and roughness and could be comparison of HAP/TCP ratios and plastic methods,” Shika
suitable for hard tissue formation in the field of biomedical Gakuho. Dental Science Reports, vol. 89, no. 2, pp. 263–297, 1989.
implants. Further studies are needed to validate the results of [7] L. L. Hench, “Bioceramics: from concept to clinic,” The Ameri-
this survey. can Ceramic Society Bulletin, vol. 74, no. 7, pp. 1487–1510, 1991.
[8] T. J. Webster, C. Ergun, R. H. Doremus, and R. Bizios,
“Hydroxylapatite with substituted magnesium, zinc, cadmium,
Conflict of Interests and yttrium. II. Mechanisms of osteoblast adhesion,” Journal of
Biomedical Materials Research, vol. 59, no. 2, pp. 312–317, 2002.
The authors declare that there is no conflict of interests
regarding the publication of this paper. [9] A. R. Biesbrock and M. Edgerton, “Evaluation of the clini-
cal predictability of hydroxyapatite-coated endosseous dental
implants: a review of the literature,” The International journal
Acknowledgment of oral & maxillofacial implants, vol. 10, no. 6, pp. 712–720, 1995.
[10] J. Mouhyi, D. M. Dohan Ehrenfest, and T. Albrektsson, “The
The authors would like to extend their gratitude for the sup- peri-implantitis: implant surfaces, microstructure, and physic-
port provided by Najafabad Branch, Islamic Azad University, ochemical aspects,” Clinical Implant Dentistry and Related
Isfahan, Iran. Research, vol. 14, no. 2, pp. 170–183, 2012.
[11] L. Zetterqvist, S. Feldman, B. Rotter et al., “A prospective,
References multicenter, randomized-controlled 5-year study of hybrid and
fully etched implants for the incidence of peri-implantitis,”
[1] T. Albrektsson, P. I. Branemark, H. A. Hansson, and J. Lind- Journal of Periodontology, vol. 81, no. 4, pp. 493–501, 2010.
strom, “Osseointegrated titanium implants. Requirements for [12] J. W. Choi, Y. M. Kong, H. E. Kim, and I. S. Lee, “Reinforcement
ensuring a long-lasting, direct bone-to-implant anchorage in of hydroxyapatite bioceramic by addition of Ni3Al and Al2O3,”
man,” Acta Orthopaedica Scandinavica, vol. 52, no. 2, pp. 155– Journal of the American Ceramic Society, vol. 81, no. 7, pp. 1743–
170, 1981. 1748, 1998.
[2] D. M. Dohan Ehrenfest, P. G. Coelho, B. S. Kang, Y. T. [13] L. L. Hench and J. Wilson, An Introduction to Bioceramics,
Sul, and T. Albrektsson, “Classification of osseointegrated World Scientific, River Edge, NJ, USA, 1993.
implant surfaces: materials, chemistry and topography,” Trends [14] S. Kim, Y. M. Kong, I. S. Lee, and H. E. Kim, “Effect
in Biotechnology, vol. 28, no. 4, pp. 198–206, 2010. of calcinations of starting powder on mechanical properties
[3] A. Wennerberg and T. Albrektsson, “Effects of titanium surface of hydroxyapatite-alumina bioceramic composite,” Journal of
topography on bone integration: a systematic review,” Clinical Materials Science: Materials in Medicine, vol. 13, no. 3, pp. 307–
Oral Implants Research, vol. 20, no. 4, pp. 172–184, 2009. 310, 2002.
[4] C. V. M. Rodrigues, P. Serricella, A. B. R. Linhares et al., [15] Y. M. Kong, S. Kim, H. E. Kim, and I. S. Lee, “Reinforcement
“Characterization of a bovine collagen-hydroxyapatite compos- of hydroxyapatite bioceramic by addition of ZrO2 coated with
ite scaffold for bone tissue engineering,” Biomaterials, vol. 24, Al2O3,” Journal of the American Ceramic Society, vol. 82, no. 11,
no. 27, pp. 4987–4997, 2003. pp. 2963–2968, 1999.
BioMed Research International 9

[16] I. S. Lee, C. N. Whang, H. E. Kim, J. C. Park, J. H. Song, and S. [33] P. Assal, “The osseointegration of zirconia dental implants,”
R. Kim, “Various Ca/P ratios of thin calcium phosphate films,” Schweizer Monatsschrift für Zahnmedizin, vol. 123, no. 7-8, pp.
Materials Science and Engineering C, vol. 22, no. 1, pp. 15–20, 644–654, 2012.
2002. [34] G. E. Romanos and C. B. Johansson, “Immediate loading
[17] T. J. Levingstone, Optimisation of Plasma Sprayed Hydroxyap- with complete implant-supported restorations in an edentulous
atite Coatings, Dublin City University, 2008. heavy smoker: Histologic and histomorphometric analyses,”
[18] C. Wu, J. Chang, S. Ni, and J. Wang, “In vitro bioactivity of International Journal of Oral and Maxillofacial Implants, vol. 20,
akermanite ceramics,” Journal of Biomedical Materials Research no. 2, pp. 282–290, 2005.
A, vol. 76, no. 1, pp. 73–80, 2006. [35] R. Crespi, P. Capparé, E. Gherlone, and G. E. Romanos,
[19] T. M. Sridhar, U. Kamachi Mudali, and M. Subbaiyan, “Sintering “Immediate versus delayed loading of dental implants placed
atmosphere and temperature effects on hydroxyapatite coated in fresh extraction sockets in the maxillary esthetic zone: a
type 316L stainless steel,” Corrosion Science, vol. 45, no. 10, pp. clinical comparative study,” International Journal of Oral and
2337–2359, 2003. Maxillofacial Implants, vol. 23, no. 4, pp. 753–758, 2008.
[20] T. Kokubo and H. Takadama, “How useful is SBF in predicting [36] V. Borsari, G. Giavaresi, M. Fini et al., “Physical characterization
in vivo bone bioactivity?” Biomaterials, vol. 27, no. 15, pp. 2907– of different-roughness titanium surfaces, with and without
2915, 2006. hydroxyapatite coating, and their effect on human osteoblast-
[21] S. Jinawath, D. Pongkao, and M. Yoshimura, “Hydrothermal like cells,” Journal of Biomedical Materials Research B: Applied
synthesis of hydroxyapatite from natural source,” Journal of Biomaterials, vol. 75, no. 2, pp. 359–368, 2005.
Materials Science: Materials in Medicine, vol. 13, no. 5, pp. 491– [37] R. J. Lazzara, T. Testori, P. Trisi, S. S. Porter, and R. L. Weinstein,
494, 2002. “A human histologic analysis of osseotite and machined surfaces
[22] A. Monshi and S. S. Attar, “A new method to measure nano using implants with 2 opposing surfaces,” International Journal
size crystals by scherrer equation using XRD,” Majlesi Journal of Periodontics and Restorative Dentistry, vol. 19, no. 2, pp. 117–
of Materials Engineering, vol. 2, no. 3, 2010. 129, 1999.
[23] T. Kokubo, S. Ito, Z. T. Huang et al., “Ca, P-rich layer formed on [38] N. P. Lang and S. Jepsen, “Implant surfaces and design (Working
high-strength bioactive glass-ceramic A-W,” Journal of Biomed- Group 4),” Clinical Oral Implants Research, vol. 20, supplement
ical Materials Research, vol. 24, no. 3, pp. 331–343, 1990. 4, pp. 228–231, 2009.
[24] T. Kokubo, H. Kushitani, C. Ohtsuki, S. Sakka, and T. Yama- [39] A. Piattelli, F. Cosci, A. Scarano, and P. Trisi, “Localized chronic
muro, “Chemical reaction of bioactive glass and glass-ceramics suppurative bone infection as a sequel of peri-implantitis in a
with a simulated body fluid,” Journal of Materials Science: hydroxyapatite-coated dental implant,” Biomaterials, vol. 16, no.
Materials in Medicine, vol. 3, no. 2, pp. 79–83, 1992. 12, pp. 917–920, 1995.
[25] P. Li, C. Ohtsuki, T. Kokubo et al., “Effects of ions in aqueous [40] W. Teughels, N. Van Assche, I. Sliepen, and M. Quirynen,
media on hydroxyapatite induction by silica gel and its rel- “Effect of material characteristics and/or surface topography on
evance to bioactivity of bioactive glasses and glass-ceramics,” biofilm development,” Clinical Oral Implants Research, vol. 17,
Journal of Applied Biomaterials, vol. 4, no. 3, pp. 221–229, 1993. supplement 2, pp. 68–81, 2006.
[26] A. S. Posner, “The mineral of bone,” Clinical Orthopaedics and [41] L. J. A. Heitz-Mayfield, “Peri-implant diseases: diagnosis and
Related Research, vol. 200, pp. 87–99, 1985. risk indicators,” Journal of Clinical Periodontology, vol. 35,
[27] K. Soballe, H. Brockstedt-Rasmussen, E. S. Hansen, and C. supplement 8, pp. 292–304, 2008.
Bunger, “Hydroxyapatite coating modifies implant membrane [42] S. Renvert, I. Polyzois, and N. Claffey, “How do implant
formation: controlled micromotion studied in dogs,” Acta surface characteristics influence periimplant disease?” Journal
Orthopaedica Scandinavica, vol. 63, no. 2, pp. 128–140, 1992. of Clinical Periodontology, vol. 38, supplement 11, pp. 214–222,
[28] K. Soballe, E. S. Hansen, B. H. Rasmussen, P. H. Jorgensen, and 2011.
C. Bunger, “Tissue ingrowth into titanium and hydroxyapatite- [43] L. G. Persson, T. Berglundh, L. Sennerby, and J. Lindhe, “Re-
coated implants during stable and unstable mechanical condi- osseointegration after treatment of peri-implantitis at different
tions,” Journal of Orthopaedic Research, vol. 10, no. 2, pp. 285– implant surfaces,” Clinical Oral Implants Research, vol. 12, no. 6,
299, 1992. pp. 595–603, 2001.
[29] D. P. Rivero, J. Fox, A. K. Skipor, R. M. Urban, and J. O. [44] E. Karamian, A. Khandan, M. Eslami, H. Gheisari, and N. Rafi-
Galante, “Calcium phosphate-coated porous titanium implants aei, “Investigation of HA vanocrystallite size crystallographic
for enhanced skeletal fixation,” Journal of Biomedical Materials characterizations in NHA, BHA and HA pure powders and
Research, vol. 22, no. 3, pp. 191–201, 1988. their influence on biodegradation of HA,” Advanced Materials
[30] C. A. van Blitterswijk, S. C. Hesseling, J. J. Grote, H. K. Koerten, Research, vol. 829, pp. 314–318, 2014.
and K. de Groot, “The biocompatibility of hydroxyapatite [45] Q. Zhang, J. Chen, J. Feng, Y. Cao, C. Deng, and X. Zhang,
ceramic: a study of retrieved human middle ear implants,” “Dissolution and mineralization behaviors of HA coatings,”
Journal of Biomedical Materials Research, vol. 24, no. 4, pp. 433– Biomaterials, vol. 24, no. 26, pp. 4741–4748, 2003.
453, 1990. [46] M. Gregoire, I. Orly, and J. Menanteau, “The influence of
[31] Y. Ichikawa, Y. Akagawa, H. Nikai, and H. Tsuru, “Tissue calcium phosphate biomaterials on human bone cell activities.
compatibility and stability of a new zirconia ceramic in vivo,” An in vitro approach,” Journal of Biomedical Materials Research,
The Journal of Prosthetic Dentistry, vol. 68, no. 2, pp. 322–326, vol. 24, no. 2, pp. 165–177, 1990.
1992. [47] C. Van Blitterswijk, H. Leenders, J. van der Brink, Y. Bovell, J.
[32] T. Albrektsson, H. A. Hansson, and B. Ivarsson, “Interface anal- Flach, J. de Bruijn et al., “Degradation and interface reactions
ysis of titanium and zirconium bone implants,” Biomaterials, of hydroxyapatite coatings: effect of crystallinity,” Transactions
vol. 6, no. 2, pp. 97–101, 1985. of the Society for Biomaterials, vol. 16, p. 337, 1993.
10 BioMed Research International

[48] Y. L. Chang, D. Lew, J. B. Park, and J. C. Keller, “Biomechanical

and morphometric analysis of hydroxyapatite-coated implants
with varying crystallinity,” Journal of Oral and Maxillofacial
Surgery, vol. 57, no. 9, pp. 1096–1108, 1999.
[49] J. J. Lee, L. Rouhfar, and O. R. Beirne, “Survival of
hydroxyapatite-coated implants: a meta-analytic review,”
Journal of Oral and Maxillofacial Surgery, vol. 58, no. 12, pp.
1372–1379, 2000.
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 580675, 4 pages
http://dx.doi.org/10.1155/2014/580675

Research Article
Solubility of Two Resin Composites in Different Mouthrinses

Sezin Ozer,1 Emine Sen Tunc,1 Nuray Tuloglu,2 and Sule Bayrak2
1
Faculty of Dentistry, Department of Pediatric Dentistry, Ondokuz Mayıs University, Kurupelit, 55139 Samsun, Turkey
2
Department of Pediatric Dentistry, Faculty of Dentistry, Eskisehir Osmangazi University, Eskisehir, Turkey

Correspondence should be addressed to Sezin Ozer; [email protected]

Received 19 February 2014; Accepted 20 March 2014; Published 7 April 2014

Academic Editor: David M. Dohan Ehrenfest

Copyright © 2014 Sezin Ozer et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Aim. This study aimed to compare the solubility of a universal restorative resin composite (Filtek Z250; FZ250) and a silorane-based
resin composite (Filtek Silorane; FS) after immersion in alcohol-containing mouthrinse, alcohol-free mouthrinse, and artificial
saliva. Methods. 30 discs (10 mm × 1 mm) were prepared from each material and desiccated until a constant mass was obtained.
Specimens were immersed in the test solutions for two days and desiccated again. Solubility was calculated based on the change
in weight of each specimen before and after immersion. Data was analyzed using two-way ANOVA and Tukey’s Post Hoc test
(𝑃 < 0.05). Results. Solubility values for both resin composites were the highest in the alcohol-containing mouthrinse. FZ250
showed greater solubility than FS; the difference was only significant in artificial saliva. Conclusion. Both resin-composite materials
tested exhibited some degree of solubility in each of the test solutions. The use of an alcohol-free mouthrinse may be preferable for
patients with extensive composite restorations.

1. Introduction properties. In addition, leakage of fillers, ions, and organic

substances such as residual monomers, methacrylic acid, and
Dental composites constitute an important group of materi- formaldehyde from resin composite material can occur as
als in modern restorative dentistry [1]. Composites—which a result of exposure to an aqueous environment. Some of
generally consist of a resin matrix, inorganic filler, and these organic substances are potent irritants and may induce
coupling agent—are becoming more and more popular as delayed allergic reactions. Both material degradation and
dental restorative materials because of their strength, rapid leakage have been shown to be dependent upon time and
polymerization, and esthetic appearance [2]. However, poly- material composition [6].
merization shrinkage and shrinkage-related stress are two
major drawbacks of resin composites that still need to be Effective plaque control is crucial for the maintenance
addressed. In order to overcome these shortcomings, dental of periodontal health and control of cariogenic activity. For
siloranes consisting of a new type of organic matrix (i.e., most individuals, toothbrushing with a dentifrice is the
monomers with a ring-opening oxirane) were introduced in most efficient, safe and economical method of removing
2007 [3]. plaque; however, many individuals find it difficult to achieve
Upon intraoral application, resin composites face con- a plaque-free dentition with this method alone. While the use
stant exposure to an aqueous environment. Water diffused of an antiseptic agent such as a mouthrinse as an adjunct to
into the resin matrix may contribute to the relaxation of toothbrushing with a dentifrice may be justified, especially in
polymerization shrinkage stress to some extent [4], and, caries-active patients [7, 8], the detrimental effects of mouth-
by expanding the polymer matrix and increasing the bulk rinses on resin composite restorations must be taken into
volume of the resin composite, water sorption may reduce the account [9].
size of marginal gaps generated by polymerization shrinkage The organic matrix of conventional resin composites is
[5]. On the other hand, water sorption may lead to degra- generally based on methacrylate chemistry, especially cross-
dation of the resin matrix and debonding of the matrix- linking dimethacrylates. The solubility of dimethacrylate-
filler interface, resulting in a deterioration of mechanical based resin composites in various solutions including water
2 BioMed Research International

and mouthrinses has been widely studied [2, 10–12]; however, 𝜇g/mm3 as the change in weight before and after immersion
few studies have been carried out on silorane-based resin using the formula SL = 𝑚0 − 𝑚1 /𝑉, where 𝑉 is the volume of
composites [13–15]. Therefore, this study aimed to measure the sample in mm3 .
the solubility of two different resin composites in three
different solutions: an alcohol-free mouthrinse, an alcohol- 2.4. Statistical Analysis. Statistical analysis was conducted
containing mouthrinse, and artificial saliva [16]. The null using the software SPSS 12.0 (SPSS Software, Munich, Ger-
hypothesis was that solubility values would not differ, regard- many). Means and standard deviations were calculated,
less of the restorative material or solution tested. and multiple comparisons were performed using two-way
ANOVA and Tukey’s Post Hoc test, with a level of 𝑃 < 0.05
2. Materials and Methods considered statistically significant.

2.1. Restorative Materials and Solutions. This study employed

a randomized factorial design conducted with 10 replications
3. Results
of 2 different restorative materials (FZ250 and FS) and 3 dif- Means, standard deviations, and significant differences in
ferent solutions (artificial saliva, an alcohol-free mouthrinse solubility are presented in Table 2. Solubility values of FS were
(Oral-B, Pro-Expert), and an alcohol-containing mouthrinse lower than those of FZ250 in each of the solutions tested;
(Oral-B, Advantage)) (Table 1). however, these differences were only statistically significant
in artificial saliva (𝑃 < 0.05). Although no statistically
2.2. Preparation of Specimens. For each material, a total of 30 significant differences were observed between the solubility
disc-shaped specimens were prepared using a plastic mold values of the same material in different mouthrinses (𝑃 >
(1 ± 0.1 mm × 10 ± 0.1 mm). The mold was filled and pressed 0.05), solubility values for both materials were higher in
by hand between two glass microscope slides to extrude any the alcohol-containing mouthrinse than in the alcohol-free
excess material. Specimens were light-cured on both sides mouthrinse.
and in different positions for a total of 60 seconds using
an LED curing unit (Elipar Free Light II, 3 M/ESPE, St. 4. Discussion
Paul, MN, USA; light intensity: 1000 mV/cm2 ). Specimens
were removed from the molds, and any excess material The current study found the solubility of the universal resin
was removed by gently grinding on both sides with 600- composite FZ250 to be higher than that of the silorane resin
grit silicon carbide paper (Phoenix Beta, Buehler, Germany). composite FS; however, the difference between the two was
Debris was removed with a dust blower. only statistically significant in artificial saliva (𝑃 < 0.05).
Thus, the null hypothesis was accepted, with the exception of
2.3. Solubility Measurements. Solubility measurements were solubility performance in artificial saliva.
conducted in line with ISO 4049 standards [17]. A dig- Excessive solubility of dental restorative materials can
ital caliper (Digimatic Caliper, Mitutoyo, Tokyo, Japan; lead to surface deformation and marginal discrepancies.
accuracy = 0.002 mm) was used to measure diameter and Resin composite materials come into extensive contact with
thickness. The diameter of each specimen was measured at oral fluids, food components, and drinks in the oral envi-
two points at right angles to one another, and the mean ronment [18], and while the solubility of dimethacrylate-
diameter was calculated. The thickness of each specimen based resin composites in different solutions has been widely
was measured at the center of the specimen and at four studied [2, 10–12], to our knowledge, there is very little
equally spaced points on the circumference, and the mean information available regarding the solubility of silorane-
thickness was calculated. The volume (𝑉) of each specimen based resin composites [13–15]. Therefore, the present study
was calculated in mm3 using the formula 𝑉 = 𝜋 × 𝑟2 × ℎ, aimed to evaluate the solubility of FS and FZ250 in 3 different
where 𝑟 is the mean sample radius (diameter/2) and ℎ is the solutions.
mean sample thickness. Solubility mean values presented by the composite resins
For each material, 10 specimens were placed in a glass vial tested varied from 2.3 to 4.2 𝜇g/mm3 ; these values were
containing 10 mL of artificial saliva, 10 in a glass vial contain- lower than the maximum value established by the ISO 4049
ing 10 mL of alcohol-free mouthrinse (Oral-B, Pro-Expert) standard (<7.5 𝜇g/mm3 ) [17]. Although the direct exploration
and 10 in a glass vial containing 10 mL of alcohol-containing is not possible, the results of the present study showed that the
mouthrinse (Oral-B, Advantage). Vials were wrapped in solubility of all materials in all solutions is acceptable for ISO
aluminum foil to exclude light and placed in an incubator at 4049.
37∘ C. With the exception of artificial saliva, the solubility of
Specimens were weighed using an analytical scale accu- FS and FZ250 did not differ significantly (𝑃 > 0.05);
rate up to 0.0001 mg (Precise XB 220A, Switzerland). All however, the solubility of FZ250 was greater than that of
specimens were stored in a vacuum desiccator at 37∘ C until FS. It is likely that the low solubility of FS is related to
a constant weight (𝑚0 ) was obtained. Specimens were then the distinctive polymerization characteristics of the material.
immersed in the solutions for 2 days, removed, and desiccated This finding is consistent with a previous study showing
again until a constant mass was obtained, and the weights silorane-based resin composites to be more hydrophobic than
were recorded again (𝑚1 ). Solubility (SL) was recorded in methacrylate-based resin composites [13, 19]. The solubility
BioMed Research International 3

Table 1: Materials used in the study.

Material Contents Lot no Manufacturer

Bisphenol A polyethyleneglycol diether dimethacrylate
(bis-EMA) (5–10% by wt), silane treated ceramic (75–85% by
wt), urethane dimethacrylate (UDMA) (5–10% by wt),
bisphenol-A-glycidyl dimethacrylate (Bis-GMA) (<5% by 8FA 3M/ESPE, St. Paul, MN,
Filtek Z250 universal restorative
wt), triethyleneglycol dimethacrylate (TEGDMA) (<5% by USA
wt), and water
Filled to 60% by volume with zircon silica filler, average
particle size = 0.6 𝜇m
Silorane (3,4-epoxycyclohexylethylcyclo-polymethylsiloxane,
Filtek Silorane (posterior bis-3,4-epoxycyclohexylethyl-phenylmethylsilane) N236344 3M/ESPE, St. Paul, MN,
restorative) Fillers: Quarz (silane layer) radiopaque yttrium fluoride USA
Filler loading 76% (wt %)
Aqua, Glycerin, polysorbate 20, Aroma, methylparaben, Procter & Gamble MN
Oral-B, Pro-Expert mouthrinse 99602155
cetylpyridinium chloride, sodium fluoride, sodium saccharin, GmbH, Stra𝛽e e 1, 64521
(alcohol-free mouthrinse)
sodium benzoate, propylparaben, Cl 42051, and Cl 47005 Gross Gerau, Germany
Aqua, glycerin, alcohol, aroma, methylparaben, poloxamer
Oral-B, Advantage mouthrinse 95587215 Procter & Gamble UK,
407, cetylpyridinium chloride, sodium fluoride, sodium
(alcohol-containing mouthrinse) Weybrige, KT13 0XP
saccharin, Propylparaben, Cl 42051, and Cl 47005
NaCl (400 mg/L), KCL (400 mg/L), CaCl2 ⋅2H2 O (795 mg/L),
Artificial saliva NaH2 PO4 ⋅H2 O (690 mg/L) KSCN (300 mg/L), Na2 S⋅9H2 O
(5 mg/L), and urea (1000 mg/L)

Table 2: Mean solubility values of materials tested (𝜇g/mm3 ). resins have been compromised by aging them in alcohol-
containing solutions [9]. In the present study, both materials
Test materials tested exhibited greater solubility in the mouthrinse contain-
Solutions
FZ250 (sd) FS (sd) ing alcohol when compared to the alcohol-free mouthrinse
Alcohol-containing mouth rinse (𝑃 > 0.05). These findings suggest that it may be preferable
4.2 (1,4)(A,1) 3.1 (1,7)(B,1)
(advantage) for patients with extensive restorations to avoid the use of
Alcohol-free mouth rinse mouthrinse containing alcohol as part of their daily oral
3.5 (0,9)(A,1) 3.0 (1,1)(B,1)
(Pro-Expert) hygiene routine so as to prevent the need for recurrent
Artificial saliva 3.4 (1,3)(A,1) 2.3 (0,6)(B,2) restorative treatment.
Differences in superscript letters indicate statistically significant differences As with all in vitro studies, caution must be used when
within columns and differences in superscript numbers indicate significant extrapolating the results of the present study to the oral
differences within rows. environment. Clinical studies are needed to examine the in
vivo solubility behavior of different resin composites.

of resin composites is related to the dissolution and leaching

of various components, particularly unreacted monomers
5. Conclusion
[20]. The organic matrix of conventional resin composites Within the limitations of this in vitro study, the following
is generally based on methacrylate chemistry, especially conclusions can be drawn:
cross-linking methacrylates, as in FZ250. The density of the
links in methacrylate-based resin composites may vary as a (1) the solubility of FS was lower than that of FZ250 in all
result of the polymerization of free radicals, causing spatial the solutions tested;
heterogeneity that may facilitate the entrapment of residual
monomers in microgels, from where they may be easily (2) solubility of both of the restorative materials tested
leached [21]. When compared to methacrylate-based resin was lower in alcohol-free mouthrinse than in alcohol
polymerization, the photoactivated cationic polymerization mouthrinse containing;
process of silorane resins is relatively insensitive to oxygen.
Not only does this reduce polymerization shrinkage, it also (3) alcohol-free mouthrinse may prefer to alcohol con-
increases the degree of conversion [15, 22, 23]. taining mouthrinse in patients with extensive restora-
Ethanol, which is found in many mouthrinses, may tions.
accelerate the hydrolytic degradation of resin-based materials
[24]. In vitro studies have reproduced the subsurface and Conflict of Interests
surface degradation of resin composites by storing them
in ethanol [6], and the mechanical properties of composite The authors declare that there is no conflict of interests.
4 BioMed Research International

References [18] R. Bagheri, M. F. Burrow, and M. Tyas, “Influence of food-

simulating solutions and surface finish on susceptibility to
[1] W. Geurtsen and U. Schoeler, “A 4-year retrospective clinical staining of aesthetic restorative materials,” Journal of Dentistry,
study of class I and class II composite restorations,” Journal of vol. 33, no. 5, pp. 389–398, 2005.
Dentistry, vol. 25, no. 3-4, pp. 229–232, 1997. [19] Y. J. Wei, N. Silikas, Z. T. Zhang, and D. C. Watts, “Diffusion
[2] Y. Zhang and J. Xu, “Effect of immersion in various media on the and concurrent solubility of self-adhering and new resin-matrix
sorption, solubility, elution of unreacted monomers, and flex- composites during water sorption/desorption cycles,” Dental
ural properties of two model dental composite compositions,” Materials, vol. 27, no. 2, pp. 197–205, 2011.
Journal of Materials Science: Materials in Medicine, vol. 19, no. [20] P. L. Fan, A. Edahl, R. L. Leung, and J. W. Stanford, “Alternative
6, pp. 2477–2483, 2008. interpretations of water sorption values of composite resins,”
[3] W. Weinmann, C. Thalacker, and R. Guggenberger, “Siloranes Journal of Dental Research, vol. 64, no. 1, pp. 78–80, 1985.
in dental composites,” Dental Materials, vol. 21, no. 1, pp. 68–74, [21] J. Malacarne, R. M. Carvalho, M. F. de Goes et al., “Water
2005. sorption/solubility of dental adhesive resins,” Dental Materials,
[4] A. J. Feilzer, A. J. de Gee, and C. L. Davidson, “Relaxation vol. 22, no. 10, pp. 973–980, 2006.
of polymerization contraction shear stress by hygroscopic [22] N. Ilie and R. Hickel, “Silorane-based dental composite: behav-
expansion,” Journal of Dental Research, vol. 69, no. 1, pp. 36–39, ior and abilities,” Dental Materials Journal, vol. 25, no. 3, pp.
1990. 445–454, 2006.
[5] T. Hirasawa, S. Hirano, S. Hirabayashi, I. Harashima, and M. [23] W. Lien and K. S. Vandewalle, “Physical properties of a new
Aizawa, “Initial dimensional change of composites in dry and silorane-based restorative system,” Dental Materials, vol. 26, no.
wet conditions,” Journal of Dental Research, vol. 62, no. 1, pp. 4, pp. 337–344, 2010.
28–31, 1983. [24] S. Y. Lee, H. M. Huang, C. Y. Lin, and Y. H. Shih, “Leached
[6] J. L. Ferracane, “Hygroscopic and hydrolytic effects in dental components from dental composites in oral simulating fluids
polymer networks,” Dental Materials, vol. 22, no. 3, pp. 211–222, and the resultant composite strengths,” Journal of Oral Rehabil-
2006. itation, vol. 25, no. 8, pp. 575–588, 1998.
[7] M. Addy, “Chlorhexidine compared with other locally delivered
antimicrobials. A short review,” Journal of Clinical Periodontol-
ogy, vol. 13, no. 10, pp. 957–964, 1986.
[8] M. Addy and J. M. Moran, “Clinical indications for the use of
chemical adjuncts to plaque control: chlorhexidine formula-
tions,” Periodontology 2000, vol. 15, no. 1, pp. 52–54, 1997.
[9] J. E. McKinney and W. Wu, “Chemical softening and wear of
dental composites,” Journal of Dental Research, vol. 64, no. 11,
pp. 1326–1331, 1985.
[10] K. Asaoka and S. Hirano, “Diffusion coefficient of water through
dental composite resin,” Biomaterials, vol. 24, no. 6, pp. 975–979,
2003.
[11] L. Musanje and B. W. Darvell, “Aspects of water sorption from
the air, water and artificial saliva in resin composite restorative
materials,” Dental Materials, vol. 19, no. 5, pp. 414–422, 2003.
[12] H. Koizumi, H. Satsukawa, N. Tanoue, T. Ogino, M. Nishiyama,
and H. Matsumura, “Effect of metal halide light source on
hardness, water sorption and solubility of indirect composite
material,” Journal of Oral Science, vol. 47, no. 4, pp. 165–169,
2005.
[13] W. M. Palin, G. J. P. Fleming, F. J. T. Burke, P. M. Marquis, and
R. C. Randall, “The influence of short and medium-term water
immersion on the hydrolytic stability of novel low-shrink dental
composites,” Dental Materials, vol. 21, no. 9, pp. 852–863, 2005.
[14] J. D. Eick, R. E. Smith, C. S. Pinzino, and E. L. Kostoryz,
“Stability of silorane dental monomers in aqueous systems,”
Journal of Dentistry, vol. 34, no. 6, pp. 405–410, 2006.
[15] N. Ilie and R. Hickel, “Macro-, micro- and nano-mechanical
investigations on silorane and methacrylate-based composites,”
Dental Materials, vol. 25, no. 6, pp. 810–819, 2009.
[16] H. H. Huang, Y. H. Chiu, T. H. Lee et al., “Ion release from
NiTi orthodontic wires in artificial saliva with various acidities,”
Biomaterials, vol. 24, no. 20, pp. 3585–3592, 2003.
[17] EN ISO, 4049, Dentistry—Polymer-Based Filling, Restorative
and Luting Materials, International Organization for Standard-
ization, Geneva, Switzerland, 1988.