molecules

Article
Mechanical, Structural, and Biological Properties of
Chitosan/Hydroxyapatite/Silica Composites for Bone
Tissue Engineering
Robert Adamski and Dorota Siuta *

Faculty of Process and Environmental Engineering, Lodz University of Technology, 90-924 Lodz, Poland;
[email protected]
* Correspondence: [email protected]; Tel.: +48-42631-3744

Abstract: The aim of this work was to fabricate novel bioactive composites based on chitosan and
non-organic silica, reinforced with calcium β-glycerophosphate (Ca-GP), sodium β-glycerophosphate
pentahydrate (Na-GP), and hydroxyapatite powder (HAp) in a range of concentrations using the
sol–gel method. The effect of HAp, Na-GP, and Ca-GP contents on the mechanical properties,
i.e., Young’s modulus, compressive strength, and yield strain, of hybrid composites was analyzed.
The microstructure of the materials obtained was visualized by SEM. Moreover, the molecular
interactions according to FTIR analysis and biocompatibility of composites obtained were examined.
The CS/Si/HAp/Ca-GP developed from all composites analyzed was characterized by the well-
developed surface of pores of two sizes: large ones of 100 µm and many smaller pores below 10 µm,
the behavior of which positively influenced cell proliferation and growth, as well as compressive
strength in a range of 0.3 to 10 MPa, Young’s modulus from 5.2 to 100 MPa, and volumetric shrinkage





 below 60%. This proved to be a promising composite for applications in tissue engineering, e.g.,
Citation: Adamski, R.; Siuta, D. filling small bone defects.
Mechanical, Structural, and Biological
Properties of Chitosan/ Keywords: chitosan; silica; hydroxyapatite; bone regeneration; calcium β-glycerophosphate; sodium
Hydroxyapatite/Silica Composites β-glycerophosphate pentahydrate
for Bone Tissue Engineering.
Molecules 2021, 26, 1976. https://
doi.org/10.3390/molecules26071976
1. Introduction
Academic Editor: Katarína Valachová
The development of innovative techniques and new biomaterials to fabricate porous,
osteogenic, osteoconductive, osteoinductive, non-toxic, and biodegradable implants [1,2]
Received: 9 March 2021
with adequate mechanical strength is a challenge for many scientists, doctors, and engineers
Accepted: 28 March 2021
Published: 31 March 2021
in the repair and treatment of bone tissue damaged by cancer, osteomyelitis, congenital
defects, or accidents [3,4]. Composites based on ceramics (e.g., bioglasses, silica, hydroxya-
patite, and titian), polymers (both natural and synthetic such as chitosan, collagen, fibrin,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
elastin, alginate, hyaluronic acid, polylactic acid) and hybrid bio-composites [5–13] are
published maps and institutional affil-
mainly used in bone tissue engineering applications. Chitosan is an ideal, inexpensive, and
iations. readily available copolymer of d-glucosamine and N-acetyl-d-glucosamine [14] derived
from the deacetylation of chitin [15] that can be used for the repair and treatment of dam-
aged bone tissue [16]. This polymer is bioadhesive, biodegradable, biocompatible, and
cytocompatible [17,18], and supports the immune system by activating macrophages to
produce anti-inflammatory cytokines [19,20]. Its low toxic effects and biological inertness
Copyright: © 2021 by the authors.
have been confirmed in studies, both in vitro and in vivo [21]. However, scaffolds made
Licensee MDPI, Basel, Switzerland.
This article is an open access article
only with chitosan as the base polymer are characterized by poor mechanical properties
distributed under the terms and
including poor tensile strength and low fracture stiffness, fast degradation rate, and low
conditions of the Creative Commons osteoconductivity [4]. Moreover, it is difficult to control pore size during the fabrication of
Attribution (CC BY) license (https:// chitosan implants, which play a key role in osteoblast survival, growth, and differentia-
creativecommons.org/licenses/by/ tion [22] as well as transport of essential components necessary for bone regeneration, gas
4.0/). diffusion, and removal of metabolism products. These disadvantages have been overcome

Molecules 2021, 26, 1976. https://doi.org/10.3390/molecules26071976 https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1976 2 of 15

by modifying chitosan-based implants with different substances (such as hydroxyapatite,

sodium alginate, hyaluronic acid, calcium phosphate, collagen).
Hydroxyapatite (calcium hydroxyphosphate, Ca10 (PO4 )6 (OH)2 ) is a bioceramic ma-
terial that exhibits chemical and mineralogical similarity to the inorganic component of
bones and teeth [23,24]. It is recognized as one of the better implantable materials in bone
surgery and dentistry due to its biocompatible, osteoinductive, non-inflammatory, and
bioresorbable nature [25]. The structural properties of hydroxyapatite significantly affect
the mechanical and biological properties of bone in both in vitro and in vivo studies [26].
However, the degradation rate of synthetic hydroxyapatite is too slow, and the degradation
rate of the material substrate may not match the growth rate of the tissue [3]. The addition
of hydroxyapatite to the chitosan matrix can improve implant properties such as porosity,
water retention, and osteoinductivity [27]. Qiaoling Hu et al. [28] have proved that the
higher the hydroxyapatite content in the composite, the more brittle the composite is.
Currently, another promising component that exhibits properties ideal for grafting
and scaffolding bone implants is silicon dioxide SiO2 or non-organic silica [29,30]. Three
organic compounds are very often used as a source of silicate ions: tetraethylorthsilicate
(TEOS) [6,31,32], ICPTES (3-isocyanatopropyl triethoxysilane) [31,32], and tetraacetoxysi-
lane (TAS) [5]. Biomaterials based on polymers with silica have higher biomineralization
capability than pure polymer. Silica also increases the stiffness of polymer material without
significantly causing the mechanical strength of the composite to deteriorate. Silicon plays
a significant role in the early stage of cartilage and bone growth and collagen synthesis by
osteoblast cells. In addition, the silanol group also promotes the growth of new apatite
on the bone substitute surface. Beck et al. [33] proved that silica stimulate bone-forming
osteoblasts, suppress bone resorbing osteoclasts, and enhance bone mineral density in vivo.
Based on the above properties, it can be expected that silica is an ideal component for
improving mechanical properties, cell adhesion, proliferation, and biocompatibility of
composite implants for bone regeneration. To our knowledge, biocomposites based on
chitosan, hydroxyapatite, inorganic silica, and calcium β-glycerophosphate or sodium
β-glycerophosphate pentahydrate have not been studied yet by researchers. Calcium
β-glycerophosphate is used for the formation of calcium and phosphorus compounds
(mineralization) in implants, which plays a crucial role in the development of both single
cells and functional connections between them [34].
The present research is intended to fabricate a porous hybrid bio-composite having
optimal bioactive and mechanical strength for bone implants, based on chitosan (CS) and
non-organic silica (SI), reinforced with calcium β-glycerophosphate (Ca-GP) or sodium β-
glycerophosphate pentahydrate (Na-GP), and hydroxyapatite powder (HAp). The sol–gel
method and the convection drying method were applied to the fabrication of implants. The
sol–gel method was used because it is highly adaptable and allows control of important
properties of implants such as geometry, porosity, and the degree of pores in order to mimic
the topological and microstructural characteristics of the extracellular matrix. It is widely
known that after drying and rewetting, the initial moisture content of the composite does
not return to its original internal structure [35,36]. The reason for this is that during drying
the original structure is partially or totally destroyed by the action of surface tension of the
solvent removed. Some chemical changes also take place such as the removal of hydroxyl
groups from the surface of pores. Nevertheless, drying strengthens the solid, allowing for
its storage, and provides the possibility of impregnation with bioactive ingredients. The
study also investigated the influence of the drying temperature on the properties of com-
posites. The composites obtained were characterized in terms of chemical and mechanical
properties using an Instron universal testing machine, scanning electron microscopy (SEM),
and Fourier-transform infrared (FTIR) spectroscopy. The biocompatibility of composites
was also assessed.
Molecules 2021, 26, 1976 3 of 15

2. Materials and Methods

2.1. Materials
Medical grade chitosan powder (CS) from chitin of crab shells with molecular weight
of 680 kDa and degree of deacetylation of 80.4% (Sigma-Aldrich, Germany) was used for
the preparation of composites. Acetic acid, calcium β-glycerophosphate (Ca-GP), and
sodium β-glycerophosphate pentahydrate (Na-GP) were purchased from Sigma-Aldrich
(Germany), and hydroxyapatite (HAp, nanopowder, <200 nm particle size) was acquired
from Merck KGaA (Germany). Reagents except CS used in experiments were of analytical
grade and used as received without further purification.
The source of inorganic silicon was sol of silica (Si, trade name Sizol 030) a commercial
product of the “Rudniki” Chemical Works (Poland), essentially composed of 30% SiO2 and
0.36% Na2 O in water, pH ~9.

2.2. Methods
2.2.1. Preparation of CS/Si/HAp Composites
This fabrication method of CS/Si/HAp composites intended for bone substitutes is
based on the sol–gel technique. Briefly, 0.4 g of chitosan was dissolved in 10 g of 4.0% acetic
acid. The solution obtained was stirred (under slow rotations) until complete dissolution
for 1 h in a water bath at 55 ◦ C. Next, 3.2–12.7 wt.% of hydroxyapatite was added gradually
to the solution and stirred in an ultrasonic bath for 15 minutes to break down powder
agglomerates. A total of 5.5 g of CS/HAp mixture was added dropwise to 11 g of silica
sol and vigorously stirred. During the synthesis, the pH values of the solutions oscillated
between 4 and 8. Then, the paste obtained was poured into cylindrical PE molds with a
diameter of 3 cm and height of 3 cm and aged for 24 h. Finally, CS/Si/HAp composites
were dried in one day at 50 and 100 ◦ C in the oven at atmospheric pressure. After drying,
the cylindrical samples were weighed and measured. Following the fabrication process, the
composites obtained were subjected to chemical, mechanical, and biological assessment.

2.2.2. Preparation of CS/Si/HAp/Ca-GP and CS/Si/HAp/Na-GP Composites

The stages for preparation of CS/Si/HAp compositions described in Section 2.2.1.
were repeated to obtain different CS/Si/HAp/Ca-GP and CS/Si/HAp/Na-GP composites
with various weight ratios of Ca-GP and Na-GP. Briefly, 3.2–12.7 wt.% of hydroxyapatite
and 4.0–9.1 wt.% of Na-GP were added gradually to the chitosan–acetic acid solution and
stirred in an ultrasonic bath for 15 minutes. Then, CS/HAp/Na-GP mixtures were added
to silica sol. The content of SiO2 was c.a. 20 wt.%. Next, the pastes obtained were poured
into cylindrical PE molds and aged for 24 h. Finally, CS/HAp/Si/Na-GP composites were
dried for 24 h at 50 and 100 ◦ C in the oven at atmospheric pressure. In the case of the
CS/Si/HAp/Ca-GP composites, the Ca-GP content was 3.3–8.2 wt.%. After drying, the
cylindrical samples were weighed, measured, and finally ground to obtain the desired
dimensions for the next analysis. To maintain the stability of properties, samples were kept
in desiccators with silica gel.
In the studies, the chitosan–acetic acid solution was prepared on the same day as
the synthesis of samples. This eliminated the possible impact of degradation of chitosan
on the properties of the composites obtained. For a comparison of mechanical properties
of the obtained biocomposites, reference samples containing CS/Si, CS/Si/Ca-GP, and
CS/Si/Na-GP were also prepared according to proportions set out above, but the content
of SiO2 was between 22.0 and 26.0 wt.%.

2.2.3. Determination of Density and Volumetric Shrinkage

Densities of dry samples (ρs ) of CS/Si, CS/Si/HAp, CS/Si/HAp/Ca-GP, CS/Si/Ca-
GP, CS/Si/HAp/Na-GP, CS/Si/Na-GP were determined based on mass (ms ) and volume
of dry gels (Vs ), according to Equation (1):
ms
ρs = (1)
Vs
Molecules 2021, 26, 1976 4 of 15

Skeletal densities were also determined using a helium pycnometer (AccuPyc 1330,
Micromeritics Instrument Corporation, Norcross, GA, USA).
The volumetric shrinkage coefficient (σ) as a percentage (%) for each sample was
calculated using the formula (Equation (2)),

σ = (1 − Vs /Vm )·100% (2)

where Vs is the volume of the dry cylindrical sample and Vm is the volume of the wet sample.

2.2.4. Mechanical Testing of Composites

Mechanical properties of CS/Si, CS/Si/HAp, CS/Si/HAp/Ca-GP, CS/Si/Ca-GP,
CS/Si/HAp/Na-GP, and CS/Si/Na-GP biocomposites were investigated at room tem-
perature using a computer-controlled universal testing machine (Instron 2519-107, USA)
at maximum load 5 kN and constant speed of crosshead displacement 0.2 mm/min. Be-
fore the tests, each cylindrical sample was ground after drying to align the upper and
lower surface. The initial length of the specimens was 15 ± 0.1 mm. A mean value of at
least five different measurements was obtained, and a standard deviation was calculated.
Young’s modulus values were determined from the slope of the linear part of stress–strain
curves, and compressive strength was obtained from the first maximum of stress visible in
the curves.

2.2.5. Surface Analysis of Composites

The surface morphology of CS/Si, CS/Si/HAp, CS/Si/HAp/Ca-GP, CS/Si/Ca-GP,
CS/Si/HAp/Na-GP, and CS/Si/Na-GP nanocomposites was investigated using scanning
electron microscopy (FEI model Quanta 200F, Thermo Fisher Scientific, Hillsboro, OR,
USA), coupled with a field emission gun (FEG) and energy dispersive spectroscopy (EDS)
equipment (EDAX model Genesis 4000). Experiments were performed under a nitrogen
atmosphere at a pressure of 100 Pa (low vacuum operating mode). This mode avoids
coating the sample with a thin conductive layer, such as gold or carbon, which is important
in order not to distort the surface topography.

2.2.6. Structure Characterization of Composites

Attenuated total reflection Fourier-transform infrared (ATR–FTIR) spectroscopy was
applied to characterize intermolecular interactions between components included in the
composites. The spectra of the pure components used in synthesis and spectra for the
prepared composites were recorded. The ATR–FTIR spectra were recorded in the 4000 to
650 cm−1 range using a Jasco FT/IR 6200 spectrometer (JASCO Inter. Co., Ltd., Tokyo,
Japan) equipped with an MCT M detector cooled by liquid nitrogen (77K) and a MIRacle
ATR sampling accessory (diamond/ZnSe) (PIKE Technol., Fitchburg, WI, USA). The whole
spectrometric system was purged by dry argon. The interferometer scanning rate was
0.1 cm/s. Signal accumulation from 300 scans was taken with a resolution of 1 cm− 1 . The
test specimens were in the form of powder, without previous compression.

2.2.7. Biocompatibility Analysis of Compositions

KYOU DXR0109B human induced pluripotent stem (IPS) cells [201B7] (ATCC®
ACS1023™; Manassas, VA, USA) derived from fibroblasts obtained from a healthy donor
were used for the study. Prior to testing, all culture vessels and samples of the tested
composites were coated with CellMatrix™ Basement Membrane Gel, (ATCC ACS3035) to
enable cell adhesion, according to the guidelines provided by the vendors.
The culture was carried out in Eppendorf tubes (cell culture plates, size 12 wells,
surface treatment TC treated, flat bottom clear wells) on 12-well plates. An implant was
placed from each type of test composite used in standard culture vessels, and then a
20,000 cells/cm2 area was flooded with 2 mL of the Pluripotent Stem Cell SFM XF/FF
culture medium (ATCC ACS3002). The culture was carried out in an incubator (PHCBI
CytoGrow incubator, Panasonic) at 37 ◦ C, 95% humidity, in the presence of 5% CO2 . Cells
 able cell adhesion, according to the guidelines provided by the vendors.
The culture was carried out in Eppendorf tubes (cell culture plates, size 12 wells, sur-
face treatment TC treated, flat bottom clear wells) on 12-well plates. An implant was
placed from each type of test composite used in standard culture vessels, and then a 20,000
Molecules 2021, 26, 1976 cells/cm2 area was flooded with 2 mL of the Pluripotent Stem Cell SFM XF/FF culture 5 ofme-
15
dium (ATCC ACS3002). The culture was carried out in an incubator (PHCBI CytoGrow
incubator, Panasonic) at 37 °C, 95% humidity, in the presence of 5% CO2. Cells were incu-
bated for 7 daysfor
were incubated (according to the ATCC
7 days (according
® recommendation, KYOU DXR0109B cell culture
to the ATCC® recommendation, KYOU DXR0109B
time between passages) in the presence of the
cell culture time between passages) in the presence test biomaterials
of the test relative to a control
biomaterials culture
relative to a
under standard conditions. The culture medium was changed daily, and
control culture under standard conditions. The culture medium was changed daily, and the suspended
cells
the present incells
suspended the present
mediuminwere harvested,
the medium counted,
were and evaluated
harvested, counted, and for viability.
evaluatedCellsfor
were harvested
viability. using
Cells were a stem cell
harvested usingdissociation
a stem cell reagent (ATCC
dissociation ACS3010),
reagent (ATCCand the number
ACS3010), and
of viable
the number cells
of was assessed
viable using
cells was a Trypan
assessed Blue
using a assay
Trypan (BioRad) and(BioRad)
Blue assay a TC20 ™and Automated
a TC20
Cell Counter (BioRad). Statistical analysis was performed using Student’s
™ Automated Cell Counter (BioRad). Statistical analysis was performed using Student’s t-tests for inde-
pendent
t-tests for samples and the
independent ANOVA
samples andmethod.
the ANOVA Testsmethod.
were repeated three
Tests were times for
repeated eachtimes
three sam-
ple and control.
for each sample and control.

3.3.Results
Resultsand
andDiscussion
Discussion
3.1.
3.1. MorphologicalCharacteristics
Morphological Characteristics
The
The sol–gel
sol–gel method
methodapplied
appliedallows
allowsbone
bonecomposites
compositestotobe
beobtained.
obtained.Examples
Examplesofof
CS/Si/HAp, CS/Si/HAp/Na-GP, CS/Si/Na-GP, and CS/Si/HAp/Ca-GP samples can
CS/Si/HAp, CS/Si/HAp/Na-GP, CS/Si/Na-GP, and CS/Si/HAp/Ca-GP samples can be seen be
seen in Figure
in Figure 1. 1.

Figure1.1. Example
Figure Example photograph
photographof ofcomposites
compositesfabricated
fabricatedbyby
the sol–gel
the method
sol–gel (a) (a)
method CS/Si/HAp; (b)
CS/Si/HAp;
CS/Si/HAp/Na-GP; (c) CS/Si/Na-GP; (d) CS/Si/HAp/Ca-GP.
(b) CS/Si/HAp/Na-GP; (c) CS/Si/Na-GP; (d) CS/Si/HAp/Ca-GP.

Theresulting
The resultingcomposites
composites had had aa three-dimensional
three-dimensional structure,
structure, but
but the
the color,
color, porosity,
porosity,
and stiffness were different. The color of the implants varied; the hydroxyapatite-based
and stiffness were different. The color of the implants varied; the hydroxyapatite-based
implants were
implants werewhiter
whiterthan
thanthethenon-hydroxyapatite
non-hydroxyapatiteimplants.
implants.AAhigher
higherconcentration
concentrationof of
hydroxyapatite resulted
hydroxyapatite resulted in
in aa greater
greater porosity
porosity ofofthe
thestructure
structureand andthus
thusaamore morecomplex
complex
externaltopography.
external topography.Appropriate
Appropriatesurface
surfacechemistry
chemistryandandaahighly
highlyporous
porousstructure
structureofofthe
the
implantsare
implants areneeded
neededto tosupport
supportcell
cellgrowth,
growth,adhesion,
adhesion,proliferation,
proliferation,oxygen,
oxygen,and andnutrient
nutrient
diffusion
diffusion and remove
remove metabolic
metabolicwaste
wasteproduced
produced during
during thethe bone
bone tissue
tissue regeneration
regeneration pro-
process. Porosity
cess. Porosity more
more significantthan
significant than75%75%isisrequired
requiredtoto obtain
obtain osteoconductive
osteoconductiveproperties
properties
of
ofcomposites
composites[3].
[3].In
Inorder
ordertotoassess
assessthe
thestructural
structuraldifferences
differencesof ofthe
theimplants,
implants,photos
photoswere
were
taken
taken using
using SEM.
SEM. Figure
Figure22presents
presentsexample
exampleSEM SEMimages
imagesof ofthe
thestructures
structuresobtained
obtainedofof
CS/Si/HAp/Ca-GP, CS/Si/HAp/Na-GP,
CS/Si/HAp/Ca-GP, CS/Si/HAp/Na-GP, CS/Si/HAp,
CS/Si/HAp, and CS/Si
and CS/Si composites
composites that
that can becan
ap-
be applied
plied for bone
for bone tissue
tissue regeneration.
regeneration. TheThe SEM SEM image
image control
control showed
showed thatthat
thethe surface
surface of
of CS/Si (Figure 2d) was smooth, while those of CS/Si/HAp/Na-GP
CS/Si (Figure 2d) was smooth, while those of CS/Si/HAp/Na-GP (Figure 2b) and (Figure 2b) and
CS/Si/HAp (Figure 2c)
CS/Si/HAp (Figure 2c) were
were rough
rough with
with slots
slots with
with pores
pores size
size below
below 1010µm.μm.However,
However,thethe
surface of the CS/Si/HAp/Ca-GP (Figure 2a) composite was rough, with corrugation and
cornification with a well-developed microstructure. This composite is characterized as
having pores of two sizes: large ones of 100 µm and many smaller pores below 10 µm, the
behavior of which should positively influence cell proliferation and growth.
 Molecules 2021, 26, x FOR PEER REVIEW 6 of

surface of the CS/Si/HAp/Ca-GP (Figure 2a) composite was rough, with corrugation an
Molecules 2021, 26, 1976 cornification with a well-developed microstructure. This composite is characterized
6 of 15 a
having pores of two sizes: large ones of 100 μm and many smaller pores below 10 μm, th
behavior of which should positively influence cell proliferation and growth.

Figure 2. Example SEM

Figure 2. imagesSEM
Example of (a)images
CS/Si/HAp/Ca-GP; (b) CS/Si/HAp/Na-GP;
of (a) CS/Si/HAp/Ca-GP; (c) CS/Si/HAp; (d)(c)
(b) CS/Si/HAp/Na-GP; CS/Si composites with
CS/Si/HAp;
a similar concentration of HAp. with a similar concentration of HAp.
(d) CS/Si composites

3.2. Mechanical3.2.
Assessment
Mechanical Assessment
The mechanical The mechanical
properties properties
of the implantsof after
the implants
drying at after and 100at◦ 50
50 drying and 100
C were °C were inve
investi-
gated at ambienttigated at ambientThe
temperature. temperature. TheHAp,
effect of the effectNa-GP,
of the HAp,
and Na-GP, and Ca-GP
Ca-GP contents oncontents
the on th
mechanical
mechanical properties properties
of hybrid of hybridwas
composites composites
analyzed. was analyzed.
Young’s Young’sstrain,
modulus, modulus,
andstrain, an
compressive
compressive strength strength ofcomposites
of CS/Si/HAp CS/Si/HApare composites
presented areinpresented
Figure 3. in Figure
The 3. The mechanic
mechanical
properties of CS/Si/HAp composites in terms of Young’s modulus significantly increased increase
properties of CS/Si/HAp composites in terms of Young’s modulus significantly
when increasing when increasing the of
the concentration concentration
HAp in the of HAp in
sample and the sampleaand
reached reachedconcen-
maximum a maximum co
centration of 1.0 wt.%. The value of modulus decreased, suggesting
tration of 1.0 wt.%. The value of modulus decreased, suggesting a poor interfacial bonding a poor interfaci
bonding between HAp particles and CS/Si molecules. The
between HAp particles and CS/Si molecules. The results of compression tests showed that results of compression tes
showed that the addition of HAp to CS/Si composite
the addition of HAp to CS/Si composite promoted compressive strength while causing promoted compressive streng
while causing
a decrease in elongation a decrease
at break. Theinhighest
elongation
value at break. The highest
of Young’s modulusvalueof of
25Young’s
MPa formodulus
25 MPa for CS/Si/HAp composites was obtained for
CS/Si/HAp composites was obtained for drying samples at 100 ◦ C and 71 MPa for drying samples at 10050°C and 71 MP
◦ C.
for 50 °C.
Molecules2021,
Molecules 26,x1976
2021,26, FOR PEER REVIEW 77of
of15
15

Figure 3. Dependence of Young’s modulus (a), compressive strength (b), strain (c) vs. concentration of
Figure 3. Dependence
hydroxyapatite with 20ofwt.%
Young’s
SiO2modulus (a),wt.%
and 0.7–1.1 compressive
chitosan.strength (b),ofstrain
The drying (c) vs.
samples at aconcentra-
temperature
tion of hydroxyapatite
◦ with 20 wt.% SiO
◦ 2 and 0.7–1.1 wt.% chitosan. The drying of samples at a
of 100 C is marked in blue and at 50 C in red.
temperature of 100 °C is marked in blue and at 50 °C in red.
In the study, the influence of drying temperature on the mechanical properties of the
In the study,
developed the influence
composites was notedof and
drying temperature
is shown on3.the
in Figure mechanical
The properties
results obtained of the
of Young’s
developed composites was noted and is shown in Figure 3. ◦The results
modulus in case of drying the sample at a temperature of 100 C were lower compared obtained of
Young’s◦ modulus in case of drying the sample at a temperature of 100
to 50 C. In the bar graph in Figure 4, the Young’s modulus values for CS/Si/Na-GP °C were lower
compared to 50 °C. In the bar
and CS/Si/HAp/Na-GP graph in Figure
composites 4, the Young’s
are compared. modulus
Analyzing values
results for samples
for the CS/Si/Na-of
GP and CS/Si/HAp/Na-GP
CS/Si/Na-GP (1–4), we cancomposites
see that thearehigher
compared. Analyzing
concentration results for
of Na-GP in athe samples
sample, the
of CS/Si/Na-GP
lower the value(1–4), we can
of Young’s see that the higher concentration of Na-GP in a sample, the
modulus.
lower the value of Young’s modulus.
Molecules 2021, 26, 1976 8 of 15
Molecules 2021, 26, x FOR PEER REVIEW 8 of 15

Molecules 2021, 26, x FOR PEER REVIEW 8 of 15

Influence
Figure4.4.Influence
Figure of HAp
of HAp and Na-GP
and Na-GP concentrations
concentrations onmodulus
on Young’s Young’s(labels
modulus
above(labels
each above each
composite)
composite)for fordrying
dryingsamples at 100
samples °C. ◦ C.
at 100
Figure 4. Influence of HAp and Na-GP concentrations on Young’s modulus (labels above each
composite) for drying
AAsimilar
similar samples
situation was
situation at
was 100 °C. for for
observed
observed CS/Si/HAp/Na-GP composites,
CS/Si/HAp/Na-GP but this case
composites, is case is
but this
more
morecomplicated.
complicated. During
During thethe
fabrication of CS/Si/HAp/Na-GP
fabrication of CS/Si/HAp/Na-GP and CS/Si/Na-GP compo-
and CS/Si/Na-GP com-
sites, A
thesimilar situation of
concentrations wasSiOobserved for CS/Si/HAp/Na-GP
2 and chitosan were at the same composites,
level, but this caseof
but concentrations is
posites, the concentrations of SiO2 and chitosan were at the same level, but concentrations
moreand
HAp complicated.
Na-GP During
were the fabrication
different. Though of CS/Si/HAp/Na-GP
the CS/Si/HAp/Na-GP composites
and CS/Si/Na-GP compo-
of HAp and Na-GP were different. Though the CS/Si/HAp/Na-GP exhibited composites bet-exhibited
sites,
ter the concentrations
mechanical propertiesofthan and chitosan
SiO2CS/Si/HAp or were at the same
CS/Si/Na-GP, thelevel, but concentrations
presence of Na-GP weak- of
better
HAp the
mechanical
andcomposite.
Na-GP were
properties
different.
than CS/Si/HAp
Though
or CS/Si/Na-GP, the presence of Na-GP
ened Based on the resultsthe CS/Si/HAp/Na-GP
presented in Figure 4,composites exhibited that
it can be concluded bet-
weakened
tersamples
mechanical the composite. Based on the results presented in Figure 4, it can be concluded
in withproperties than CS/Si/HAp
higher concentrations or CS/Si/Na-GP,
of HAp, Young’s modulus the presence of Na-GP
was higher than inweak-
sam-
that
ples in
enedwiththesamples
composite. with higher
Based
higher concentrations on the concentrations
of results
Na-GP.presented
The peak of HAp,
in Young’s
Figure
value of 4, it canmodulus
Young’s be concluded
modulus wasthese
for higher than
that
in samples
samples with
with higher
higher concentrations
concentrations
composites was 66.44 ± 3.32 MPa. of of
HAp, Na-GP.
Young’s The peak
modulus value
was of
higher Young’s
than in modulus
sam- for
these
ples A composites
with higher was 66.44
concentrations ± of3.32 MPa.
Na-GP. The peak value of Young’s
completely different trend was observed for the composites doped with Ca-GP. modulus for these
composites
The values was
A completely
of 66.44different
Young’s ±modulus
3.32 MPa. trend was
increased observed
after mixing withfor the
Ca-GP,composites doped
but this trend with Ca-GP.
heavily
A completely
The values
depended onofthe different
Young’s
concentrationtrend
modulus of was observed
increased
both Ca-GPafter
andformixing
the composites
HAp as with doped
Ca-GP,
illustrated in butwith
Figure Ca-GP.
this5.trend
For heavily
The values
samples withof Young’s
Ca-GP modulus
without increased
HAp, a after
twofold mixing
increase with
in Ca-GP,
the
depended on the concentration of both Ca-GP and HAp as illustrated in Figure 5. For but this
concentration trendof heavily
Ca-GP
depended
causes
samples on the
a slight
with concentration
increase
Ca-GP in Young’s
without of modulus.
HAp, both Ca-GP
a twofold Onandthe HAp in
other
increase as the
illustrated
hand, for in Figure
composites
concentration of5.Ca-GP
with For
7.7 causes
samples
wt.% of with Ca-GP
Ca-GP and 5.0 without
wt.% of HAp, the
a twofold
peak increase
value of in the modulus
Young’s concentration
of of Ca-GP
100.75 ± 4.99
a slight increase in Young’s modulus. On the other hand, for composites with 7.7 wt.% of
causes
MPa wasa slight increase in
determined Young’s modulus. On the other hand, for composites with 7.7
Ca-GP and 5.0 wt.%in ofthe
HAp,grouptheofpeak
all tested
valueconfigurations.
of Young’s modulus of 100.75 ± 4.99 MPa was
wt.% of Ca-GP and 5.0 wt.% of HAp, the peak value of Young’s modulus of 100.75 ± 4.99
determined in the group of all tested configurations.
MPa was determined in the group of all tested configurations.

Figure 5. Influence of hydroxyapatite and Ca-GP on Young’s modulus (labels above each compo-
site) for drying samples at 100 °C.
Figure 5.5.Influence
Figure Influenceofof
hydroxyapatite
hydroxyapatite andand
Ca-GP on Young’s
Ca-GP modulus
on Young’s (labels(labels
modulus above each compo-
above each composite)
site) for
Fordrying samples
comparison, at
the
for drying samples at 100 C. 100 °C.
work
◦ of Sowjanya [37] presented composites based on chitosan,
reinforced with nanosilica powder and sodium alginate, which were characterized by
For
Forcomparison,
compressive strength the
comparison, awork
in the ofofSowjanya
range
work 0.59
of [37]MPa
to 0.66
Sowjanya presented
[37]and composites
Young’s
presented based
modulus ofon
composites chitosan,
8.16 to 8.99
based on chitosan,
reinforced with nanosilica powder and sodium alginate, which were characterized by
reinforced with nanosilica powder and sodium alginate, which were characterized by
compressive strength in a range of 0.59 to 0.66 MPa and Young’s modulus of 8.16 to 8.99
compressive strength in a range of 0.59 to 0.66 MPa and Young’s modulus of 8.16 to
8.99 MPa. Composites obtained in the present work were characterized by compressive
strength in a range of 0.3 to 15 MPa, while the cancellous bone is characterized by a
modulus of 0.8 to 1000 MPa, and the compressive strength of human bone is 1 to 210 MPa.
Molecules 2021, 26, 1976 9 of 15

The Young’s modulus results obtained for CS/Si/HAp, CS/Si/HAp/Ca-GP, CS/Si/Ca-

GP, CS/Si/HAp/Na-GP, CS/Si/Na-GP were compared to CS/Si reference composites.
The composition and properties of CS/Si are presented in Table 1. These samples were
characterized by a very low Young’s modulus and compressive strength.

Table 1. Properties of references samples of CS/Si.

Chitosan SiO2 Young’s Compressive Strain rs Vs /Vm

Sample
(wt.%) (wt.%) Modulus (MPa) Strength (MPa) (mm/mm) (g/cm3 ) (%)
CS/Si 1 0.66 24.84 0.39 ± 0.04 0.016 ± 0.004 0.04 ± 0.001 0.40 ± 0.04 3.5 ± 0.32
CS/Si 2 0.76 24.07 1.55 ± 0.13 0.003 ± 0.001 0.002 ± 0.001 0.35 ± 0.03 12.9 ± 0.04
CS/Si 3 0.96 22.53 2.06 ± 0.15 1.13 ± 0.15 0.55 ± 0.09 0.38 ± 0.04 0.8 ± 0.02
CS/Si 4 1.12 21.27 0.36 ± 0.09 0.009 ± 0.001 0.02 ±0.001 0.37 ± 0.04 27.1 ± 1.44

The content of HAp and/or x-GP (where x is Na or Ca) to chitosan has a strong effect
on the mechanical properties of implants. All the above results suggest that alteration of
mechanical properties is a consequence of the interaction between chitosan, silica sol, and
components based on calcium. Better mechanical properties can be a result of crosslinking
reactions within chains of chitosan and molecular interaction between biopolymer and
silica, which stiffens the skeleton of the composite.

3.3. Density and Volumetric Shrinkage

Densities of dry samples of CS/Si, CS/Si/HAp, CS/Si/HAp/Ca-GP, CS/Si/Ca-GP,
CS/Si/HAp/Na-GP, and CS/Si/Na-GP were calculated using Equation (1), and volumetric
shrinkages were estimated using Equation 2. Densities obtained of CS/Si, CS/Si/HAp,
CS/Si/HAp/Ca-GP, CS/Si/Ca-GP, CS/Si/HAp/Na-GP, and CS/Si/Na-GP implants were
in the range of 320 to 820 kg/m3 , which is comparable to density of cancellous bone which
ranges from 300 to 2100 kg/m3 depending on age of the person. The drying temperature
had an impact on the results of the density and volumetric shrinkage of the materials
obtained. Drying samples at 100 ◦ C reduces the volume shrinkage by 50% as shown in
Figure 6 and increases the density of the samples compared to drying at 50 ◦ C. In addition,
the volumetric shrinkage of samples also depends on content and composition and is
within a range of 5% to 45%. The experimental data obtained are difficult to describe by
explicit functional dependence. An additional series of measurements to characterize the
trend in the course of the volumetric shrinkage needs to be performed. In most cases,
composites with Na-GP characterized the lowest volumetric shrinkage
Molecules 2021, 26, x FOR PEER REVIEW 10 of 15 compared with
composites of CS/Si, CS/Si/HAp, CS/Si/HAp/Ca-GP, and CS/Si/Ca-GP.

Figure 6. Volumetric shrinkage of CS/Si/HAp, CS/Si/Ca-GP, CS/Si/HAp/Ca-GP composites at dry-

Figure 6. Volumetric shrinkage of CS/Si/HAp, CS/Si/Ca-GP, CS/Si/HAp/Ca-GP composites at
ing temperature of 50 and 100 °C.
drying temperature of 50 and 100 ◦ C.
CS/Si/HAp, CS/Si/Ca-GP, CS/Si/HAp/Ca-GP implants were chosen as the most
CS/Si/HAp,
promising compositesCS/Si/Ca-GP, CS/Si/HAp/Ca-GP
for further FTIR analysis implants were chosen
and biological studies. as the most
promising composites for further FTIR analysis and biological studies.
3.4. FTIR Analysis
The molecular interactions of CS/Si/HAp, CS/Si/Ca-GP, and CS/Si/HAp/Ca-GP im-
plants were studied by FTIR analysis. Figure 7 shows spectra of composites obtained and
represents the range of individual components used in fabrication. Differences are ob-
served between pure components and composites.
 Figure 6. Volumetric shrinkage of CS/Si/HAp, CS/Si/Ca-GP, CS/Si/HAp/Ca-GP composites at dry-
ing temperature of 50 and 100 °C.
Molecules 2021, 26, 1976 10 of 15
CS/Si/HAp, CS/Si/Ca-GP, CS/Si/HAp/Ca-GP implants were chosen as the most
promising composites for further FTIR analysis and biological studies.

3.4.
3.4.FTIR
FTIRAnalysis
Analysis
The
Themolecular
molecularinteractions
interactionsofofCS/Si/HAp, CS/Si/Ca-GP,and
CS/Si/HAp, CS/Si/Ca-GP, andCS/Si/HAp/Ca-GP
CS/Si/HAp/Ca-GP im-
implants were studied by FTIR analysis. Figure 7 shows spectra of composites obtained
plants were studied by FTIR analysis. Figure 7 shows spectra of composites obtained and
and represents
represents the the range
range of individual
of individual components
components used
used in fabrication.
in fabrication. Differences
Differences areare
ob-
observed between pure components and composites.
served between pure components and composites.

Figure 7. FTIR
Figure characterization
7. FTIR of implants
characterization made
of implants of CS/Si/HAp,
made CS/Si/Ca-GP,
of CS/Si/HAp, CS/Si/Ca-GP,CS/Si/HAp/Ca-GP.
CS/Si/HAp/Ca-GP.

The spectrum obtained for chitosan powder samples shows typical bands, such as
a broad band in the region of 3500–3000 cm−1 corresponding to an overlap of OH group
vibration and NH group vibration stretching. A characteristic peak at 2919.2 cm−1 is
indicative of the C-H bond vibration antisymmetric stretching. An N-H chemical bond in
the primary amine and secondary amide occurred at 1540 cm−1 . Deforming, antisymmetric
vibrations for the C-H bond were confirmed by a peak at 1458 cm−1 and stretching vibration
for N-C-O by a peak at 1241.5 cm−1 . The band for the C-O group vibration stretching
(amide I band) was confirmed by a peak at 1651.2 cm−1 , and vibration stretching for the
C-O-C group by a peak at 1063.07 cm−1 .
The infrared spectra of raw HAp powder exhibit characteristic absorption bands such
as a broad band in the region of 3500–3000 cm−1 with a peak at 3245 cm−1 corresponding
to the vibration of the OH group as a result of water in hydroxyapatite and adsorbed
by components from the atmosphere. P-O stretching vibration at 1236 cm−1 and P-O
asymmetric stretching vibration (the PO4 3− ν1 mode) appeared at 1026 cm−1 .
For spectra of Sizol, a peak at 3270 cm−1 for the surface OH group can be observed.
The presence of the Si-H stretching bond is confirmed by the peak at 2154 cm−1 and that
at 1062.59 cm−1 , equivalent to Si-O-C stretching bond (open chain) or Si-O-Si symmetric
stretching bond vibration.
The Ca-GP spectrum showed that for a peak at 1250 cm−1 , C-C skeletal vibrations
overlap with the P-O stretching bond. The C-O and P-O-R stretching bands were confirmed
by peaks at 1253 and 1005 cm−1 .
Molecules 2021, 26, 1976 11 of 15

The composite CS/Si/Ca-GP shows several differences compared with the spectra of
pure components. The band at 2020.55 cm−1 is less pronounced, while bands in the region
of 1770–1320 cm−1 become sharper. In spectra for a composite, the peaks at 1241.45 and
at 946.4 cm−1 are less distinct. The band at 2415.89 cm−1 (corresponding to O-H group
stretching vibration) disappears completely, while the band at 2558.59 cm−1 becomes more
pronounced (N-H in amine salts). This could indicate that Si atoms join the group OH
in chitosan. The peak at 1061.14 cm−1 is more significant and smooth. Furthermore, the
band shift from 1063.07 to 1061.14 cm−1 indicates some interaction in symmetric bond
vibration Si-O-Si.
For composites CS/Si/HAp, FTIR spectra show similar differences as described
above for CS/Si/Ca-GP composites. A new peak appears at 2557.15 cm−1 , the peak at
2415.88 cm−1 disappears, and the peak at 1049.57 cm−1 is more significant. Furthermore, a
double peak appears instead of a single peak at 669.41 cm−1 . The band shift from 1063.07 to
1049.57 cm−1 indicates some interaction with the symmetric bond Si-O-Si. The band shift
is also observed for OH group vibration, and the peak for this group in the CS/Si/HAp
composite is at 3261.52 cm−1 (for CS at 3245.61 cm−1 , Si at 3270.68 cm−1 , and HAp at
3244.65 cm−1 ).
The CS/Si/HAp/Ca-GP composite exhibited similar behavior. The band at 2560.56 cm−1
becomes more pronounced. A significant band shift is also observed for OH group vibra-
tion, and the peak is at 3260.56 cm−1 (where for Ca-GP it is at 3240.31 cm−1 and the peak is
flatter than for other pure components and composites). In the case of Si-O-Si, a shift is
observed from 1063.07 to 1035.10cm−1 .
Comparing spectra of composites, for CS/Si/Ca-GP and CS/Si/HAp we can observe
a double peak at 670cm−1 , while for CS/Si/HAp/Ca-GP for the same wave number a
single peak is observed. The bands in the region of 1800–1300 cm−1 are sharper for CS/Si
composites than for other composites analyzed. In each case, the band shift for OH band
vibration and Si-O-Si vibration indicates interaction between the Si atom and the OH group.
The highest value for this shift is obtained when Ca-GP is added to the composite, and
for this sample, the calculated value of Young’s modulus was the highest. The new bond
between Si and the OH group reinforces the composite.
In all probability, a very high concentration of Sizol in composites, in comparison
with other components, and the possibility of precipitating crystals can impair molecular
interactions.

3.5. In Vitro Study

The composite’s essential characteristics, determining its suitability for bone tissue
applications, are its biocompatibility and cytotoxicity, which require both in vitro studies
on isolated, well-defined cells or tissues in laboratory conditions and in vivo testing on
animals. In vitro tests are inexpensive and allow initial results of the cell reaction to
the tested composite to be obtained quickly. In this study, fibroblast cells were used in
cytotoxicity and biocompatibility tests of CS/Si/HAp, CS/Si/Ca-GP, and CS/Si/HAp/Ca-
GP composites. Results obtained of cell growth, proliferation, and viability are displayed
in Figure 8.
 applications, are its biocompatibility and cytotoxicity, which require both in vitro studies
on isolated, well-defined cells or tissues in laboratory conditions and in vivo testing on
animals. In vitro tests are inexpensive and allow initial results of the cell reaction to the
tested composite to be obtained quickly. In this study, fibroblast cells were used in cyto-
Molecules 2021, 26, 1976 toxicity and biocompatibility tests of CS/Si/HAp, CS/Si/Ca-GP, and CS/Si/HAp/Ca-GP 12 of 15
composites. Results obtained of cell growth, proliferation, and viability are displayed in
Figure 8.

Figure 8.
Figure 8. Cell growth (a), viability
viability (b),
(b), and
andproliferation
proliferationresults
results(c)
(c)ofofCS/Si/HAp,
CS/Si/HAp,CS/Si/Ca-GP,
CS/Si/Ca-GP,
CS/Si/HAp/Ca-GP composites.
CS/Si/HAp/Ca-GP composites.

The
The growth
growth of of fibroblasts
fibroblasts after
after 77 days
days ofof cell
cellculture
culturewaswascompared
comparedto tocells
cellscultured
cultured
without composites (control, p
without composites (control, p > 0.05). > 0.05). The lowest mean value of cell
cell growth (Figure 8a)
growth (Figure 8a)
among
among thethe composites
composites tested
tested was
wasrecorded
recordedininaawell wellwith
withCS/Si/HAp
CS/Si/HAp (149%),
(149%), but
but the
the
differences
differencesare arenot statistically
not significant
statistically compared
significant compared to thetocontrol. The highest
the control. cell growth
The highest cell
was observed
growth in the wells
was observed in with CS/Si/HAp/Ca-GP
the wells with CS/Si/HAp/Ca-GPcomposite composite
due to its good
due mechanical
to its good
properties,
mechanicalhighly poroushighly
properties, microstructure (greater than 75%),
porous microstructure and surface
(greater roughness.
than 75%), There
and surface
were additional perforations in the walls of the pores CS/Si/HAp/Ca-GP
roughness. There were additional perforations in the walls of the pores composite, which
are necessary for proper
CS/Si/HAp/Ca-GP cell growth,
composite, whichcell
aremigration,
necessary and mass transport.
for proper cell growth,Thecell
cellmigration,
prolifera-
tion assay indicated that none of the CS/Si/HAp, CS/Si/Ca-GP, or
and mass transport. The cell proliferation assay indicated that none of the CS/Si/HAp, CS/Si/HAp/Ca-GP
composites
CS/Si/Ca-GP, caused a cytotoxic effect,composites
or CS/Si/HAp/Ca-GP confirmed caused
by the lack of cell death
a cytotoxic effect,inconfirmed
comparison by
to
thethe control
lack of cell(100%
deathofinviability).
comparison Thetohighest cell viability
the control (100% ofand proliferation
viability). were also
The highest cell
observed in the
viability and CS/Si/HAp/Ca-GP
proliferation were also composite.
observed inThe the results obtained of composite.
CS/Si/HAp/Ca-GP structural, me-
The
chanical, and biological investigation of composites showed that the
results obtained of structural, mechanical, and biological investigation of composites CS/Si/HAp/Ca-GP
composite has promising potential in biomedical applications such as bone regeneration,
and therefore animal tests can be considered.
Molecules 2021, 26, 1976 13 of 15

4. Conclusions
In this study, a new, patented method of fabrication of bio-hybrid Cs/Si, CS/Si/HAp,
CS/Si/HAp/Ca-GP, CS/Si/Ca-GP, CS/Si/HAp/Na-GP, and CS/Si/Na-GP composites
using the sol–gel technique was developed. The basic structural, mechanical, and biological
properties of composites obtained were investigated through FTIR, SEM, and the Instron
universal testing machine. Of all composites analyzed, the CS/Si/HAp/Ca-GP composite
obtained was the most promising for further research in the field of biomaterials for bone
tissue regeneration. It was characterized by the well-developed surface of pores of two
sizes: large ones of 100 µm and many smaller pores below 10 µm, the behavior of which
positively influenced cell proliferation and growth, as well as a density comparable to the
bone density, compressive strength of 0.3–10 MPa, Young’s modulus of 5–100 MPa, and
volumetric shrinkage below 60%. The CS/Si/HAp/Ca-GP composite had an appropriate
morphology and did not show cytotoxicity towards the tested fibroblasts. Positive test
results predispose the material to be suitable for animal testing.
The study showed significant dependence on the drying temperature mainly on the
resulting structure’s physicochemical properties of composites. Drying samples at 100 ◦ C
reduced the volume shrinkage by 50% and increased the density and mechanical properties
of the samples compared to drying at 50 ◦ C. However, it was noticed that the use of high-
temperature convection drying at atmospheric pressure is an advantage because it reduces
the costs of obtaining the biomaterial and can also replace the sterilization process. In
future research, drying strategies should be designed to achieve optimal physicochemical
properties of the implants for bone tissue regeneration.

Author Contributions: Conceptualization, R.A. and D.S.; methodology, R.A.; resources, R.A.; in-
vestigation, R.A. and D.S.; writing—original draft preparation, R.A. and D.S.; writing—review and
editing, D.S. All authors have read and agreed to the published version of the manuscript.
Funding: The study was supported by the Faculty of Process and Environmental Engineering at
Lodz University of Technology.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors thank Anna Adamska for assisting in mechanical and cytotoxic-
ity tests.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Przekora, A.; Palka, K.; Ginalska, G. Chitosan/β-1,3-glucan/calcium phosphate ceramics composites-Novel cell scaffolds for
bone tissue engineering application. J. Biotechnol. 2014, 182–183, 46–53. [CrossRef] [PubMed]
2. Umar Aslam Khan, M.; Haider, S.; Haider, A.; Izwan Abd Razak, S.; Rafiq Abdul Kadir, M.; Shah, S.A.; Javed, A.; Shakir, I.;
Al-Zahrani, A.A. Development of porous, antibacterial and biocompatible GO/n-HAp/bacterial cellulose/β-glucan biocomposite
scaffold for bone tissue engineering. Arab. J. Chem. 2021, 14, 102924. [CrossRef]
3. Keller, L.; Regiel-Futyra, A.; Gimeno, M.; Eap, S.; Mendoza, G.; Andreu, V.; Wagner, Q.; Kyzioł, A.; Sebastian, V.; Stochel, G.; et al.
Chitosan-based nanocomposites for the repair of bone defects. Nanomed. Nanotechnol. Biol. Med. 2017, 13, 2231–2240. [CrossRef]
[PubMed]
4. Vaidhyanathan, B.; Vincent, P.; Vadivel, S.; Karuppiah, P.; AL-Dhabi, N.A.; Sadhasivam, D.R.; Vimalraj, S.; Saravanan, S.
Fabrication and investigation of the suitability of chitosan-silver composite scaffolds for bone tissue engineering applications.
Process Biochem. 2021, 100, 178–187. [CrossRef]
5. Yazdanpanah, Z.; Bahrololoom, M.E.; Hashemi, B. Evaluating morphology and mechanical properties of glass-reinforced natural
hydroxyapatite composites. J. Mech. Behav. Biomed. Mater. 2015, 41, 36–42. [CrossRef]
6. Sathain, A.; Monvisade, P.; Siriphannon, P. Bioactive alginate/carrageenan/calcium silicate porous scaffolds for bone tissue
engineering. Mater. Today Commun. 2021, 26, 102165. [CrossRef]
7. Prasad, A. State of art review on bioabsorbable polymeric scaffolds for bone tissue engineering. Mater. Today Proc. 2021. [CrossRef]
8. Corrales, L.P.; Esteves, M.L.; Vick, J.E. Scaffold design for bone regeneration. J. Nanosci. Nanotechnol. 2014, 14, 15–56. [CrossRef]
Molecules 2021, 26, 1976 14 of 15

9. Jang, C.; Kim, W.; Kim, G. Effects of fibrous collagen/CDHA/hUCS biocomposites on bone tissue regeneration. Int. J. Biol.
Macromol. 2021, 176, 479–489. [CrossRef] [PubMed]
10. Zhao, W.; Huang, Z.; Liu, L.; Wang, W.; Leng, J.; Liu, Y. Porous bone tissue scaffold concept based on shape memory PLA/Fe3O4.
Compos. Sci. Technol. 2021, 203, 108563. [CrossRef]
11. Woźniak, M.J.; Chlanda, A.; Oberbek, P.; Heljak, M.; Czarnecka, K.; Janeta, M.; John, Ł. Binary bioactive glass composite scaffolds
for bone tissue engineering—Structure and mechanical properties in micro and nano scale. A preliminary study. Micron 2019,
119, 64–71. [CrossRef] [PubMed]
12. Baino, F.; Fiume, E.; Miola, M.; Leone, F.; Onida, B.; Verné, E. Fe-doped bioactive glass-derived scaffolds produced by sol-gel
foaming. Mater. Lett. 2019, 235, 207–211. [CrossRef]
13. John, Ł.; Janeta, M.; Rajczakowska, M.; Ejfler, J.; Łydzba, D.; Szafert, S. Synthesis and microstructural properties of the scaffold
based on a 3-(trimethoxysilyl)propyl methacrylate-POSS hybrid towards potential tissue engineering applications. RSC Adv.
2016, 6, 66037–66047. [CrossRef]
14. Anitha, A.; Sowmya, S.; Kumar, P.T.S.; Deepthi, S.; Chennazhi, K.P.; Ehrlich, H.; Tsurkan, M.; Jayakumar, R. Chitin and chitosan in
selected biomedical applications. Prog. Polym. Sci. 2014, 39, 1644–1667. [CrossRef]
15. Nawrotek, K.; Tylman, M.; Adamus-Włodarczyk, A.; Rudnicka, K.; Gatkowska, J.; Wieczorek, M.; Wach, R. Influence of chitosan
average molecular weight on degradation and stability of electrodeposited conduits. Carbohydr. Polym. 2020, 244, 116484.
[CrossRef]
16. Huang, Y.M.; Lin, Y.C.; Chen, C.Y.; Hsieh, Y.Y.; Liaw, C.K.; Huang, S.W.; Tsuang, Y.H.; Chen, C.H.; Lin, F.H. Thermosensitive
chitosan-gelatin-glycerol phosphate hydrogels as collagenase carrier for tendon-bone healing in a rabbit model. Polymers 2020, 12,
436. [CrossRef]
17. Croisier, F.; Jérôme, C. Chitosan-based biomaterials for tissue engineering. Eur. Polym. J. 2013, 49, 780–792. [CrossRef]
18. Muzzarelli, R.A.A. Chitin Nanostructures in Living Organisms. In Chitin. Topics in Geobiology, 34th ed.; Gupta, N.S., Ed.; Springer:
Dordrecht, The Netherlands, 2011. [CrossRef]
19. Pillai, C.K.S.; Paul, W.; Sharma, C.P. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Prog. Polym. Sci.
2009, 34, 641–678. [CrossRef]
20. Zhou, H.Y.; Jiang, L.J.; Cao, P.P.; Li, J.B.; Chen, X.G. Glycerophosphate-based chitosan thermosensitive hydrogels and their
biomedical applications. Carbohydr. Polym. 2015, 117, 524–536. [CrossRef] [PubMed]
21. Palma, P.J.; Ramos, J.C.; Martins, J.B.; Diogenes, A.; Figueiredo, M.H.; Ferreira, P.; Viegas, C.; Santos, J.M. Histologic Evaluation of
regenerative endodontic procedures with the use of chitosan scaffolds in immature dog teeth with apical periodontitis. J. Endod.
2017, 43, 1279–1287. [CrossRef]
22. Nawrotek, K.; Tylman, M.; Rudnicka, K.; Balcerzak, J.; Kamiński, K. Chitosan-based hydrogel implants enriched with calcium
ions intended for peripheral nervous tissue regeneration. Carbohydr. Polym. 2016, 136, 764–771. [CrossRef] [PubMed]
23. Kong, L.; Gao, Y.; Lu, G.; Gong, Y.; Zhao, N.; Zhang, X. A study on the bioactivity of chitosan/nano-hydroxyapatite composite
scaffolds for bone tissue engineering. Eur. Polym. J. 2006, 42, 3171–3179. [CrossRef]
24. Mohamed, K.R.; Beherei, H.H.; El-Rashidy, Z.M. In vitro study of nano-hydroxyapatite/chitosan-gelatin composites for bio-
applications. J. Adv. Res. 2014, 5, 201–208. [CrossRef]
25. Chauhan, N.; Singh, Y. L-histidine controls the hydroxyapatite mineralization with plate-like morphology: Effect of concentration
and media. Mater. Sci. Eng. C 2021, 120, 111669. [CrossRef]
26. Tredwin, C.J.; Young, A.M.; Georgiou, G.; Shin, S.H.; Kim, H.W.; Knowles, J.C. Hydroxyapatite, fluor-hydroxyapatite and
fluorapatite produced via the sol-gel method. Optimisation, characterisation and rheology. Dent. Mater. 2013, 29, 166–173.
[CrossRef] [PubMed]
27. Rogina, A.; Ivanković, M.; Ivanković, H. Preparation and characterization of nano-hydroxyapatite within chitosan matrix. Mater.
Sci. Eng. C 2013, 33, 4539–4544. [CrossRef]
28. Hu, Q.; Li, B.; Wang, M.; Shen, J. Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite
rods via in situ hybridization: A potential material as internal fixation of bone fracture. Biomaterials 2004, 25, 779–785. [CrossRef]
29. Dong, Y.; Liang, J.; Cui, Y.; Xu, S.; Zhao, N. Fabrication of novel bioactive hydroxyapatite-chitosan-silica hybrid scaffolds:
Combined the sol-gel method with 3D plotting technique. Carbohydr. Polym. 2018, 197, 183–193. [CrossRef]
30. Lai, S.M.; Chen, W.C.; Wu, T.W.; Yang, A.J.M.; Yang, C.H. Properties and preparation of chitosan/silanol quaternary ammonium
modified silica hybrids using sol-gel process. J. Macromol. Sci. Part B Phys. 2011, 50, 1430–1446. [CrossRef]
31. Pipattanawarothai, A.; Suksai, C.; Srisook, K.; Trakulsujaritchok, T. Non-cytotoxic hybrid bioscaffolds of chitosan-silica: Sol-gel
synthesis, characterization and proposed application. Carbohydr. Polym. 2017, 178, 190–199. [CrossRef]
32. Kaliaraj, R.; Gandhi, S.; Sundaramurthi, D.; Sethuraman, S.; Krishnan, U.M. A biomimetic mesoporous silica–polymer composite
scaffold for bone tissue engineering. J. Porous Mater. 2018, 25, 397–406. [CrossRef]
33. Beck, G.R.; Ha, S.W.; Camalier, C.E.; Yamaguchi, M.; Li, Y.; Lee, J.K.; Weitzmann, M.N. Bioactive silica-based nanoparticles
stimulate bone-forming osteoblasts, suppress bone-resorbing osteoclasts, and enhance bone mineral density in vivo. Nanomed.
Nanotechnol. Biol. Med. 2012, 8, 793–803. [CrossRef]
34. Douglas, T.E.L.; Skwarczynska, A.; Modrzejewska, Z.; Balcaen, L.; Schaubroeck, D.; Lycke, S.; Vanhaecke, F.; Vandenabeele, P.;
Dubruel, P.; Jansen, J.A.; et al. Acceleration of gelation and promotion of mineralization of chitosan hydrogels by alkaline
phosphatase. Int. J. Biol. Macromol. 2013, 56, 122–132. [CrossRef]
Molecules 2021, 26, 1976 15 of 15

35. Adamski, R.; Siuta, D.; Kukfisz, B.; Mitkowski, P.T.; Szaferski, W. Influence of process parameters in superheated steam drying on
fire and explosion parameters of woody biomass. Fuel Process. Technol. 2021, 211, 106597. [CrossRef]
36. Pakowski, Z.; Krupinska, B.; Adamski, R. Prediction of sorption equilibrium both in air and superheated steam drying of energetic
variety of willow Salix viminalis in a wide temperature range. Fuel 2007, 86, 1749–1757. [CrossRef]
37. Sowjanya, J.A.; Singh, J.; Mohita, T.; Sarvanan, S.; Moorthi, A.; Srinivasan, N.; Selvamurugan, N. Biocomposite scaffolds containing
chitosan/alginate/nano-silica for bone tissue engineering. Colloids Surfaces B Biointerfaces 2013, 109, 294–300. [CrossRef]