Ministry of higher Education

And Scientific Research

Al-Ain University
College of Dentistry

Stem Cell and Tooth Regeneration

A Graduation Project
Submitted to the Committee of Scientific Affairs of the College
of Dentistry in Partial Fulfillment of the Requirements for the
Degree of B.Sc in Dentistry

By
Khitam Kataa Sharhan
Aya ahmad wahaab
Ahmed Jabar Naeem
Kawthar Abbas Hussein

Supervised by

Lecturer Dr. Mr. Ferdos Alaa El din

2023AD 1444AH

i
 ‫ِبسْ ِم اللَّـ ِه الرَّ حْ َم ٰـ ِن الرَّ ِحيم‬

‫َف َب َدَأ ِبَأ ْو ِع َي ِت ِه ْم َق ْب َل ِو َعا ِء َأ ِخي ِه ُث َّم اسْ َت ْخ َر َج َها ِمنْ "‬
‫ان لِ َيْأ ُخ َذ َأ َخاهُ‬
‫ُف َما َك َ‬‫ك ِك ْد َنا لِيُوس َ‬ ‫ِو َعا ِء َأ ِخي ِه َك َذلِ َ‬
‫ت َمنْ‬‫ين ْال َملِ ِك ِإاَّل َأنْ َي َشا َء هَّللا ُ َنرْ َف ُع َد َر َجا ٍ‬ ‫ِفي ِد ِ‬
‫﴾ َن َشا ُء َو َف ْو َق ُك ِّل ِذي ِع ْل ٍم َعلِي ٌم"﴿‪٧٦‬‬
‫صدق هللا العلي العظيم‬
‫سورة یوسف االیة (‪)٧٦‬‬

‫‪ii‬‬
 Acknowledgment

Our faithful thanks to my god for granting us the will

and strength with which this research was
accomplished. We wish to express our sincere
appreciation to our supervisor, Dr. Ferdos Alaa El din
, who has guided and encouraged us to be
professionals and do the right thing. We are deeply
indebted to deanship of the college for persistent help,
patience and motivation. Without their support, the
goal of this project would not have been realized..

iii
 Dedication

To my parents that support and help me in every step

of my life and give me the greatest love…

To all my previous teachers and any one that give me

any information to reach for this level…

To my supervisor for his guidance helpness and

endless support throughout this project…

iv
 LIST OF CONTENTS
Contents Pages No.
ACKNOWLEDGMENT iii
DEDICATION iv

LIST OF CONTENTS v

LIST OF FIGURES vi

LIST OF TABLES vi

ABSTRACT vii

INTRODUCTION 1
AIM OF STUDY 3

STEM CELL 4
DENTIN AND PULP REGENERATION 8
BONE REGENERATION 10
PDL REGENERATION 14
CEMENTIUM REGENERATION 15
GUIDED TISSUE REGENERATION (GTR) 19
DISCUSSION 20

CONCLUSION 22

v
REFERENCES 23

LIST OF FIGURES

Figure No Title Pages

1 Stem cell-based tooth and periodontal 1
regeneration
2 Schematic representation of the 3
periodontium containing the intact
periodontal complex
3 Regenerative strategy of whole tooth 9
and bioroot.
4 CPC-stem cells construct for bone 12
regeneration

LIST OF TABLE
Table No. Title Pages
1 Grading of periodontists
4
2 Different types of stem cell 7

vi
Abstract
Periodontitis is a prevalent infectious disease worldwide, causing the
damage of periodontal support tissues, which can eventually lead to tooth
loss. The goal of periodontal treatment is to control the infections and
reconstruct the structure and function of periodontal tissues including
cementum, periodontal ligament (PDL) fibers, and bone. The
regeneration of these three types of tissues, including the re-formation of
the oriented PDL. The regeneration of lost/damaged sup- port tissue in
the periodontium, including the alveolar bone, periodontal ligament, and
cemen- tum, is an ambitious purpose of periodontal regenerative therapy
and might effectively reduce periodontitis-caused tooth loss. The use of
stem cells for periodontal regeneration is a hot field in translational
research and an emerging potential treatment for periodontitis.

Key world

Periodontal regeneration ; Bone-PDL-Cementum ; scaffolds; stem cells;

growth factors ; tissue engineering.

vii
Introduction
The stem cells used for tooth and periodontal regeneration are both dental
and non-dental mesenchymal stem cells (MSCs). The concepts in
restoring damage tissues have undergone significant change, from
substitution to restoration or replacement, and finally regeneration. Since
stem cell-based tissue engineering regenerative medicine emerged two
decades ago, these novel therapeutic strategies have been evaluated for
their potential to replace, repair, maintain, and enhance tissue or organ
function [1]. Current investigations of stem cell-based tissue engineering
for tooth and periodontal tissue include regeneration of dentin–pulp, tooth
root, whole tooth, and periodontal tissue (Figure 1)

Figure 1.Stem cell-based tooth and periodontal regeneration. Current

investigations of stem cell-based tooth and periodontal regeneration
including dentin–pulp, tooth root, whole tooth, and periodontal tissue
regeneration.

1
Periodontitis, an oral disease with a high prevalence worldwide, affects
the function of teeth and constitutes one of the main oral health burdens
[2]
Periodontitis is a widespread infectious oral disease, characterized by
irreversible damage in the tooth-supporting tissues, which include the
alveolar bone, periodontal ligament (PDL), and cementum (Figure 2).
This eventually leads to tooth loss with serious functional and aesthetic
problems for the patients [3]. An epidemiological survey has suggested
that > 50% of all adults in the world are affected by periodontal diseases.
Furthermore, periodontal disease occurrence is increasing with time; in
the 10-year period from 2005 to 2015, the prevalence rates have risen
rapidly compared to earlier periods [4]. Periodontal disease pathogenesis
involves complicated interactions between the host’s immune response
with the microbial colony in the periodontal pocket, as well as other
factors including smoking and genetics. The sub-gingival biofilms on the
teeth, which are extracted from patients with periodontitis, consist of an
aggregation of bacterium that is attached and embedded into a matrix on
the tooth surface. The inflammatory reaction of the periodontal tissues
has been verified by the large numbers of leukocytes which are mobilized
in the vicinity of inflammation [5]. The treatment of periodontist by
patient's home care plaque control is well recognised. The debridement of
bacterial deposits coating the surface of the root, deep within the
periodontal pocket is also essential and is achieved in the first instance,
by scaling techniques. In addition, periodontal surgery is used where the
depth of the deposits within the pocket, prevents adequate access for
debridement.

2
Figure 2. Schematic representation of the periodontium containing the intact

periodontal complex (i.e., bone-PDL-cementum apparatus). As a result of disease

(e.g., periodontitis), damage to the periodontium leads to the loss of multiple

periodontal tissues surrounding and supporting the tooth. Abbreviation: PDL,

periodontal ligament.

Aim of study

To improve the knowledge of how to regeneration of periodontium

tissue and to understand some diseases that affect the tissues of the

mouth, such as periodontists.

Table 1: Grading of periodontists

3
Stem cell

The stem cells used for tooth and periodontal regeneration are both dental

and non-dental mesenchymal stem cells (MSCs). The features of these

stem cells have been identified and characterized [6;Table 1]. MSCs,

which were first found in bone marrow, are multipotential stromal cells

that can differentiate into a variety types of cells, such as osteoblasts,

chondrocytes, myocytes, and adipocytes. Dental pulp stem cells (DPSCs),

the first isolated MSCs from human teeth, have the potential to

differentiate into odontoblasts, osteoblasts, chondrocytes, myocytes, and

adipogenic, and neurogenic differentiation patterns as DPSCs, but with

more proliferative activity than bone marrow MSCs or DPSCs [7]. Stem

cells from the dental apical papilla (SCAPs) were reported in apical

papillae of the developing tooth root apex, with the characteristics of

highly proliferative, migratory, and regenerative potential, and are able to

form dentin in vivo [8] Stem cells from the dental fol- licle (DFSCs),

which were first isolated from dental follicle of human third molar teeth,

and reported as the progenitor cells or precursor cells (PCs) of

cementoblasts, periodontal ligament cells, and osteoblasts, are able to

differentiate into osteoblasts/cementoblasts, adipocytes, and neurons[9].

Dental epithelial stem cells (DESCs), which were isolated from the

4
developing third molar or epithelial rests of Malassez, were also studied

for tooth and periodontal regeneration [10]

and can generate enamel–dentin-like complex structures in vivo after

being combined with dental mesenchymal cells[11]. The non-dental stem

cells used for tooth and periodontal regeneration are primarily bone

marrow mesenchymal stem cells (BMMSCs), adipose-derived stem cells

(ADSCs), embryonic stem cells (ESCs), and induced pluripotent stem

cells (iPSCs). BMMSCs were the first MSCs discovered and have shown

a capacity for osteogenic, adipogenic, chondrogenic, and myogenic

differentiation. For tooth and periodontal regeneration, BMMSCs can

upregulate the expression of odontogenic genes and contribute to tooth

regeneration after being recombined with embryonic oral epithelium [12].

Adipose-derived stem cells are stem cells derived from adipose tissues

and are, thus, abundant and have been widely used for regenerative

medicine. Transplanting ADSCs in periodontal tissue defects can

promote cementum and organize periodontal ligament fibers and

periodontal vessel regeneration[13]. In many cases, stem cell sheets are

transplanted into the damaged periodontium with bone substitutes, such

as hydroxy- apatite/tricalcium phosphate TCP [14,15]), bovine bone

[16], synthetic hydrogels [17], and their composites[18], to increase the

stability of the cells within the defect. .

5
Table 2 : Different types of stem cells [19]

6
The Dentin-Pulp Regeneration

Dental pulp is a complex organized tissue with various types of cells and

structures, providing nutrition, sensation, and defense against the various

pathogens; additionally, it produces dentin and maintains the biological

7
and physiological vitality of the dentin. Pulpitis, which is one of the most

common diseases related to pulp, usually caused by dental trauma and

caries. Due to the complex structure, small volume, and insufficient blood

supply of dental pulp, it is difficult to eradicate the infection and initiate

self-healing in pulpitis. The traditional therapy for pulpitis is root canal

therapy, by removing the inflamed pulp and replacing it with inorganic

material. However, this treatment usually leads to tooth fragility and

fracture [20]. As a result, keeping or regenerating vital pulps is a better

choice. There are primarily two approaches for dental pulp regenera- tion:

pulp revascularization; and scaffold and/or stem cell-based pulp

regeneration. Dental pulp revascularization usually depends on inducing

host cells from the apical region to migrate into the root canal and

differentiate into a vascularized pulp tissue. The root canal space should

be disinfected, and the resulting blood clots induce cell migration and

initiate pulp tissue formation. Another approach is scaffold and/or stem

cell-based pulp regeneration[21]. A combination of dental pulp cells and

platelet-rich plasma could increase dentin–pulp regeneration within the

root canals of miniature dogs[22]. Cell homing induced by a series of

molecules (vascular endothelial growth factor, platelet- derived growth

factor, or basic fibroblast growth factor combined with nerve growth

factor and bone morphogenic protein 7 [BMP7]) is sufficient for the

8
regeneration of dental-pulp-like tissue in endodontically treated human

teeth [23].

Figure 3:Regenerative strategy of whole tooth and bioroot. Fully

functional teeth can be regenerated in vivo by transplanting

bioengineered tooth germs reconstituted from epithelial and

mesenchymal cells via the organ germ method. The functional

regeneration of bioroot in vivo by transplanting bioroot complex

reconstituted from scaffold, mesenchymal stem cells, and mesenchymal

stem cells sheets

9
Bone Regeneration

Bone loss is a major hallmark of periodontitis. Pathogenic

microorganisms in the biofilm, genetic factors, and environmental issues

such as tobacco use, can all contribute to periodontitis and bone loss.

Losing the supporting bone around a tooth results in tooth movement and

dislocation, eventually leading to tooth loss [24]. Various techniques have

been developed to enhance the osteogenesis process, including bone

grafts, [25], scaffolds [26], stem cells [27] and growth factors. Bone

grafts can be conveniently divided into four groups [28,29]. First,

autogenous bone grafts are generally viewed as the “gold standard” for

bone replacement [30]. Clinical applications showed that new bone and

new periodontal connective tissue attachment were obtained [31].

Second, tissue banks provide different types of allogeneic bone graft.

[32], including freeze-dried bone allografts (FDBAs) and demineralized

freeze-dried bone allografts (DFDBAs). Clinical trials showed that

periodontal bone fillings of 1.3 to 2.6 mm were obtained. DFDBAs

generated significantly more vital bone at 38.4%, compared to that via

FDBAs at 24.6% [33]. Third, xenografts have been used, for example,

Bio-Oss [34]. One study investigated the effects of employing titanium

mesh in conjunction with Bio-Oss for localized alveolar ridge

augmentation. Radiographic analyses showed that a 2.86 mm vertical and

10
3.71 mm buccolabial ridge augmentation was obtained, while

histomorphometry analysis showed that 36.4% of the grafted region had

new bone [35]. Forth, synthetic alloplastic materials have been

developed, for example, hydroxyapatite (HA) [36-38], tricalcium

phosphates (TCP) [30], a calcium-layered-polymer of polymethyl

methacrylate and hydroxyethyl methacrylate (PMMA and HEMA

polymer [31] and bioactive glass [32].

Injectable and absorbable scaffolds were developed for bone regeneration

applications [33,34].

11
12
Figure 4 CPC-stem cells construct for bone regeneration. (a)

Representative hematoxylin-eosin (HE). . CPC-stem cells construct for

bone regeneration. (a) Representative hematoxylin-eosin (HE) images of

the CPC-MF-hBMSC group at 12 weeks post-surgery. Bone bridging was

achieved in the images of the CPC-MF-hBMSC group at 12 weeks post-

surgery. Bone bridging was achieved in the critical-sized defects. The

defect was closed with newly woven bone and trabecular bone. (c) and

critical-sized defects. The defect was closed with newly woven bone and

trabecular bone. (c) and (d) (d) were high magnification images of the

dotted-line rectangle in (b); (e) quantification of new bone were high

magnification images of the dotted-line rectangle in (b); (e) quantification

of new bone and and residual CPC area fraction. (f–h) Representative HE

images at 12 weeks. The cell-seeded groups residual CPC area fraction.

(f–h) Representative HE images at 12 weeks. The cell-seeded groups

showed more new bone than CPC control. The greatest amount of new

bone was observed in the showed more new bone than CPC control. The

greatest amount of new bone was observed in the tri-culture group; (i)

high magnification images of new bone from dotted rectangles in the tri-

culture culture group; (i) high magnification images of new bone from

dotted rectangles in the tri-culture group (h). New blood vessels in the

macropores of CPC scaffolds; (j) quantification of the new bone group

13
(h). New blood vessels in the macropores of CPC scaffolds; (j)

quantification of the new bone area and vessel[39-43]

PDL Regeneration

The PDL fibers connect the cementum on the tooth root surface to the

alveolar bone and fix the tooth in the alveolar socket to attenuate the

occlusal stresses. The regeneration of PDL is an important requirement

for periodontal regeneration. The ideal outcome would be that the

regenerated highly-organized collagen fibers could re-insert

perpendicularly and firmly attached to the regenerated cementum and

new bone [44]

Inflammation in the periodontal pocket can change the cell biology in the

pathological periodontium. Once it is damaged, the periodontium has an

only limited capacity for regeneration, which relies on the availability of

MSCs. Several types of MSCs remain and are responsible for tissue

homeostasis, serving as a source of renewable progenitor cells to generate

other required cells throughout adult life. In addition, studies to date have

shown that periodontal stem cells can be transplanted into periodontal

defects with no adverse immunologic or inflammatory consequences.

Therefore, periodontal regeneration relies on the successful recruitment

of locally-derived renewable progenitor cells to the lesion site for tissue

homeostasis and subsequent differentiation into PDL, cementum and

14
bone-forming cells [45]. New PDL-like tissues were successfully formed

via the delivery of stem cells to the defect sites, including the delivery of

periodontal ligament stem cells (PDLSCs) BMMSCs [46], adipose-

derived stem cells (ADSCs) [47], and induced pluripotent stem cells

(iPSCs) [48]. PDLSCs were cultured and osteogenically induced and then

seeded on a biphasic calcium phosphate scaffold (BCP). Then the

PDLSC-seeded scaffolds were transplanted into six dogs. The results

showed that the transplantation of PDLSC-seeded BCP promoted

effective periodontal regeneration, including new bone formation and

PDL with reorganized and reborn collage fibers inserting into adjacent

cementum and bone at the right angle, along with abundant blood vessels

at 12 weeks. Therefore, PDLSC-seeded scaffolds were a promising

method for periodontal regeneration.

Cementum Regeneration

The cementum occurred as a thin acellular layer around the root neck,

with thicker cellular cementum covering the lower part of the root up to

the apex [49–51]. Hertwig’s epithelial root sheath (HERS) cells were

suggested to secrete acellular cementum in the initial stage of

cementogenesis. Later on, cellular and reparative cementum was

produced by the dental follicle-derived cementoblast. Several diseases

15
such as periodontitis usually affected the acellular cementum. However,

the predictability and quality of cementum regeneration in an everyday

clinical situation appeared to be low. Ideally, the regenerated cementum

should closely resemble the acellular extrinsic fiber cementum (AEFC),

because it contributed most to the attachment function. In most

periodontal regeneration studies, the quality of the attachment function

was questionable, because the newly-formed cementum was cellular

intrinsic fiber cementum (CIFC), instead of the desired AEFC. The

numerical density of the inserting fibers in CIFC was low, and the

interfacial tissue bonding appeared to be weak [49-51].

Several cementum-specific proteins were shown to promote new

cementum and bone formation for the damaged periodontal tissues [52].

These proteins included cementum-derived growth factor (CDGF),

cementum attachment protein (CAP) and cementum protein-1 (CEMP1).

They could induce several signaling pathways associated with

mitogenesis, increase the concentration of cytosolic Ca2+, activate the

protein kinase C cascade, and promote the migration and preferential

adhesion of progenitor cells. These actions could result in the

cementoblast and osteoblast differentiation and the production of a

mineralized extracellular matrix resembling the cementum [52]. Indeed,

the addition of CEMP1 to the 3D PDLCs cultures increased the ALP

specific activity by 2-fold and induced the expression of cementogenic

16
and osteogenic markers, forming new tissues that mimicked bone and

cementum [53].

The stem cells in the PDL, gingiva, and alveolar bone served as sources

for cementoblast progenitors [54], producing cementum-specific markers

and cementum-like mineralized nodules in culture [55]. Indeed, PDLSCs,

stem cells from the dental follicle (DFSCs), and ADSCs were all able to

differentiate into cementoblasts and regenerate the periodontium to form

cementum-like tissue, as well as PDL fibers and periodontal vessel

regeneration in vivo [56,57]. In another study, DFCSs were combined

with the treated dentin matrix (TDM) and implanted subcutaneously into

the dorsum of mice. Histological examination revealed a whirlpool-like

alignment of the DFCs in multiple layers that were positive for

collagenase I (COLI), integrin β1, fibronectin and ALP, suggesting the

formation of a rich extracellular matrix. TDM could induce and support

DFCSs to develop new cementum-periodontal complexes and dentin-pulp

like tissues, implying successful root regeneration [58]. Therefore,

transplanting these types of stem cells into periodontal defects would be

an effective technique for cementum regeneration [59].

Furthermore, co-culturing several types of cells could also promote

cementoblast differentiation. The biological effects of the conditioned

medium from the developing apical tooth germ cells (APTG-CM) on the

differentiation and cementogenesis of PDLSCs were investigated. The

17
PDLSCs were cultured together with APTG-CM and demonstrated

properties of the cementoblast lineages. This included morphological

changes, greater proliferation, elevated ALP activity, and the expression

of cementum-related genes and mineral nodules. An

immunocompromised mice model was tested, and after transplantation in

vivo, the induced PDLSCs demonstrated tissue-regenerative ability and

generated new cementum and periodontal ligament-like structures. This

structure contained a layer of cementum-like mineralized tissues with

periodontal ligament-like collagen fibers which were attached to the new

cementum. In comparison, the untreated PDLSC transplant control

generated only connective tissues [60]. Therefore, APTG-CM

demonstrated the capability of providing a cementogenic

microenvironment and promoting the differentiation of PDLSCs into the

cementoblastic lineage, thereby enhancing periodontal tissue engineering.

18
Guided tissue regeneration (GTR)

Guided tissue regeneration (GTR) is a surgical procedure that specifically

aims to regenerate the periodontal tissues when the disease is advanced

and could overcome some of the limitations of conventional therapy. Is a

dental  surgical procedures that us barrier membrane to direct the growth of

new bone and gingival tissue at sites with insufficient volumes or dimensions

of bone or gingiva for proper function, esthetics or prosthetic restoration[61].

The objectives of GTR are to assess the treatment of periodontal infra‐

bony defects measured against conventional surgery (open flap

debridement (OFD) and factors affecting outcomes.

Current treatments for destructive periodontal (gum) disease are not able

to restore damaged bone and connective tissue support for teeth. There

are therefore limitations in treating patients with advanced disease. The

surgical technique, guided tissue regeneration (GTR) may be able to

achieve regeneration and therefore improve upon conventional surgical

results. The results of this review have shown some advantage to using

GTR in infra‐bony defects but with wide variations in the benefits

achievable compared with conventional surgery. We were unable to

identify conclusively factors responsible for this variability. Therefore,

patients and health professionals need to consider the predictability of the

technique compared with other methods of treatment before making final

19
decisions on use [61]. Adverse effects of treatment were generally minor

and similar between groups although with an increased treatment time for

GTR. We recommend further research to address the issue of variability

and to identify which characteristics of the disease or the patient are more

clearly associated with a beneficial outcome[62].

Discussion

Several kinds of stem cells have been isolated from human adult teeth and

have been used for tooth and periodontal regeneration. Non- dental stem

cells, like BMMSCs, ADSCs, and iPSCs, have also been used for these

studies. Different strategies were investigated for tooth and periodontal

regeneration, such as epithelial–mesenchymal-based whole tooth

regeneration or cell homing strategies, bioroot regeneration, cell

injection, or cell sheet-based periodontal regeneration.

Jaw, face, and mouth tissues are the rich sources of stem cells, which

more accessible than other stem cells, so stem cell and tissue engineering

treatments in dentistry have received much clinical attention in recent

years. This review study examines three essential elements of tissue

engineering in dentistry and clinical practice, including stem cells derived

from the intra- and extra-oral sources, growth factors, and scaffolds.

20
Oral tissues are a rich source of SCs, which have attracted dentists’

attention because of their easy access to other SCs. These cells have

unique capabilities making them of great importance in tissue

engineering,, regeneration, or the replacement of damaged or diseased

tissues. In dentistry, there are problems such as alveolar bone resorption

for patients following tooth extraction or loss due to periodontal disease,

dental caries, and tooth fractures caused by trauma. Moreover, in

individuals losing their teeth, it leads to bone loss, especially in the lower

jaw, thereby making such individuals lose the treatment option of implant

placement. Following such problems, stem cell tissue engineering

therapies to repair large defects in periodontal tissue and alveolar bone to

replace lost teeth seem to be of paramount importance.

The Periodontitis is characterized by progressive destruction of the tooth‐

supporting apparatus. Its primary features include the loss of periodontal

tissue support manifest through clinical attachment loss (CAL) and

radiographically assessed alveolar bone loss, presence of periodontal

pocketing and gingival bleeding. . If untreated, it may lead to tooth loss,

although it is preventable and treatable in the majority of cases.

21
Conclusion
Several kinds of stem cells have been isolated from human adult teeth and

have been used for tooth and periodontal regeneration. Non-dental stem

cells, like BMMSCs, ADSCs, and iPSCs, have also been used for these

studies. Different strategies were investigated for tooth and periodontal

regeneration, such as epithelial–mesenchymal-based whole tooth

regeneration or cell homing strategies, bioroot regeneration, cell

injection, or cell sheet-based periodontal regeneration. However, the fates

and functions of stem cells after transplantation need to be clarified.

Although tooth stem cell banking and clinical trials have been organized,

their beneficial results for patients need to be determined. With improved

efficacy of tooth and periodontal regeneration, stem cell-based tooth and

periodontal regeneration may become widely used for clinical

applications in the future.

22
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