# Chemical Synthesis, Characterization, and Biocompatibility Study of Hydroxyapatite/Chitosan Phosphate Nanocomposite for Bone Tissue Engineering Applications

Authors: Nabakumar Pramanik; Debasish Mishra; Indranil Banerjee; Tapas Kumar Maiti; Parag Bhargava; Panchanan Pramanik
Journal: International Journal of Biomaterials
 (2009)

---

## Abstract

A novel bioanalogue hydroxyapatite (HAp)/chitosan phosphate (CSP) nanocomposite has been synthesized by a solution-based chemical methodology with varying HAp contents from 10 to 60% (w/w).  The interfacial bonding interaction between HAp and CSP has been investigated through Fourier transform infrared absorption spectra (FTIR) and x-ray diffraction (XRD). The surface morphology of the composite and the homogeneous dispersion of nanoparticles in the polymer matrix have been investigated through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. The mechanical properties of the composite are found to be improved significantly with increase in nanoparticle contents. Cytotoxicity test using murine L929 fibroblast confirms that the nanocomposite is cytocompatible. Primary murine osteoblast cell culture study proves that the nanocomposite is osteocompatible and highly in vitro osteogenic. The use of CSP promotes the homogeneous distribution of particles in the polymer matrix through its pendant phosphate groups along with particle-polymer interfacial interactions. The prepared HAp/CSP nanocomposite with uniform microstructure may be used in bone tissue engineering applications.


---

## Body

## 1. Introduction

Hydroxyapatite (Ca10(PO4)6(OH)2) (HAp) is an interesting biomaterial with potential orthopedic, dental, and maxillofacial applications due to its excellent biocompatibility, bioactivity, and osteoconductivity  [1].  HAp, being chemically
and structurally similar to the inorganic component of bone, enamel, and dentin
has received considerable attention from the biologists and biomaterial
scientists. It has been successfully used as bone fillers, aesthetic
restorative, coating of orthopedic implants, filler of inorganic/polymer
composites, cell-culture carriers, and so on. It is, however, worth-mentioning
that the application of pure HAp is being limited, due to its brittleness. In recent years, the development of bioactive ceramic-polymer
composites commonly known as bioanalogue has gained a phenomenal impetus in the
orthopedic field for their bone analogue design as well as good biological and
mechanical performances to meet specific clinical requirements [2–4]. The idea
is to use a ceramic-polymer composite material that can develop a considerable
anisotropic character by means of adequate orientation techniques reinforced
with a ceramic that simultaneously assures the mechanical reinforcement and the
bioactive character of the implant [4–7]. In fact, natural bone tissue
is a biocomposite, composed of nano-hydroxyapatite (n-HAp) crystals dispersed
in a collagen matrix.In
the design of hydroxyapatite-based bioanalogue composites, the most commonly
used matrices include polymers like poly(methyl methacrylate) (PMMA), high-density
polyethylene (HDPE), poly-L-lactide (PLA), and many other.  However, the natural biopolymers have
received much attention in the fields of orthopedic and other biomedical
applications, due to their excellent biocompatibility and biodegradability [8].
Chitosan (poly-1,4-D-glucosamine), a partially deacetylated form of chitin, is
structurally similar to glycosaminoglycan, and has many desirable properties as
tissue engineering scaffolds [9].The mechanical properties of an
HAp/polymer composite can be significantly improved by controlling the
interfacial bonding between matrix and the reinforcement; and without proper interface control, a brittle,  polyphase material results rather than a toughened composite. Various methodologies
have been developed to improve the interfacial bonding between the same [10, 11].
Nevertheless, the use of judiciously chosen coupling/anchoring agents has been
proved as a reliable method to strengthen the interfacial bonding between the
filler and polymer matrix with an appreciable enhancement of the compatibility
by making the chemical bridges between the same. Organosilane- and organotitanate-based
coupling agents have already been reported to tailor the particle surface
properties by lowering their specific surface energy [12, 13]. Recently,
grafting of organophosphorus coupling agents (OPCAs) on preformed inorganic supports
or in-situ formation of the inorganic part in presence of organophosphorus reagents
offers a potential alternative to the silicon- or titanate-based coupling
agents. Based on the ability of phosphonate ions to exchange with the phosphate
ions on HAp crystals, any polymer containing a number of phosphate or
phosphonic acid groups is expected to have higher affinity for the HAp
particles [14]. Moreover, the phosphate-containing polymers are expected to
have higher mechanical properties and biocompatibility [15]. In the preparation
of bioanalogue composites, organophosphonate- or phosphate-based
coupling/anchoring agents are being extensively used by the researchers to
improve the compatibility between inorganic nanoparticles and polymer matrix
[16–20]. Greish and Brown have developed a biocompatible HAp-Ca poly(vinyl
phosphonate) composite for clinical applications  [14]. Varma et al. have reported the
preparation of calcium phosphate-coated phosphorylated chitosan film by a
biomimetic method [18]. Tanaka et al. have reported the synthesis of surface-modified
calcium hydroxyapatite with pyrophosphoric acid for use as bioceramics,
particularly for orthopedic applications [19]. Choi et al. have reported the
preparation of surface-modified hydroxyapatite nanocrystals by grafting of
polymers containing phosphonic acid groups [20]. Phillips et al. have grafted
allyl phosphonic acid on calcium phosphate to produce a chemically bonded
composite with superior mechanical properties [21]. All these studies
unequivocally suggest that phosphonate/phosphoric acid-based coupling agents
can be successfully employed to enhance the interfacial bonding between the
particles and the polymer matrix, and hence to improve the mechanical
properties of the resulting composites.In
the present paper, we have attempted to prepare hydroxyapatite (HAp)/chitosan
phosphate (CSP) nanocomposite through a simple solution-based chemical method,
which has the potential of providing much better dispersion of n-HAp particles
in the polymer matrix yielding a composite with uniform microstructure. CSP has
been chosen as polymer matrix because phosphate groups of CSP can act as
coupling/anchoring agent, which is a good cation absorber and has higher
affinity towards the n-HAp particles [15]. Moreover, it is easy to prepare from
cheaper ingredients. We have also extensively studied the dispersion,
morphology, nanoparticle-polymer matrix bonding interactions, mechanical
properties, as well as in vitro biocompatibility and osteogenicity of the
synthesized HAp/CSP nanocomposite.


## 2. Materials and Methods

### 2.1. Chemicals

Calcium
nitrate (Ca(NO3)2⋅4H2O) (99%), diammonium
hydrogen phosphate ((NH4)2HPO4) (DAHP) (99%),
ammonia solution (25%), ammonium chloride (98%), triethanolamine (TEA), and
orthophosphoric acid (H3PO4) were procured from Merck, Mumbai, India.
Chitosan, methyl-thiotetrazole (MTT), trypsin, dexamethasone, sodium beta-glycerol
phosphate, and SEM grade gulteraldehyde were purchased from Sigma-Aldrich Chemical, St Louis, Mo, USA.
Collagenase type I, 0.25% trypsin-EDTA, and Dulbecco’s modified Eagle’s medium (DMEM)
were procured from Invitrogen, USA. Fetal calf
serum and alpha-MEM were supplied by Hyclone and Pan-Biotech, respectively.


### 2.2. Preparation of Nano-Hydroxyapatite (n-HAp)

A
concentration of 0.5 M stock solution each of Ca(NO3)2⋅4H2O
and (NH4)2HPO4 was prepared in distilled
water. Both solutions were taken in such amounts that Ca : P molar ratio was maintained
at 1.67. Triethanolamine (TEA) was used in conjunction with Ca(NO3)2⋅4H2O
solution (Ca2+ : TEA  =  1:0.5) as a capping agent to control the
particle growth during precipitation. The pH of both calcium nitrate and DAHP
solutions was maintained at ~11-12. (NH4)2HPO4 was added drop-wise to the mixture of Ca(NO3)2⋅4H2O
and TEA and vigorously stirred at room temperature using a mechanical stirrer
(2500 rpm). The pH of the reacting mixture was also maintained in the range of
11-12 by adding NH4OH
solution gradually. The process was continued up to 6 hours. The gelatinous precipitate thus obtained was filtered by a
centrifugal filtration process. The precipitate was washed with water
thoroughly as well as with NH4Cl solution to remove the excess
residual TEA, and dried at 90°C for 15 hours.


### 2.3. Synthesis of Chitosan Phosphate (CSP)

A
mixture of approximately 2 g chitosan powder, 100 mL of 2% acetic acid solution,
6 g of orthophosphoric acid was taken in a three naked-round bottomed flask equipped
with a stirrer, condenser, thermometer, and nitrogen gas inlet tube. The mixture was heated up to 80°C under constant
stirring until dissolution of chitosan powder and then heated to reflux.
After 2 hours, the resultant solution was cooled and precipitated in excess
methanol. Again, the precipitated gel was dissolved in water and all unreacted phosphate
(H3PO4) and acetic acid were removed by repeated
reprecipitation in excess methanol. Finally, the gel was collected and dried under
vacuum oven at 80°C over night.


### 2.4. Synthesis of HAp/CSP Nanocomposite

At
first, the CSP was dissolved in hot water, and then the nanoparticles were added
slowly with vigorous stirring with the help of a mechanical stirrer at 3000 rpm. After addition of entire nanoparticles to the
polymer solution, the resulting solution mixture was kept in a vacuum desiccator
to remove the bubbles. Then, the mixture was heated in a water bath to
evaporate water. The resulting slurry was poured into a glass petri dish and
dried to make a film, keeping it in a vacuum oven at 90°C over
night. After that, the film was cut into dumbbell-shaped samples. Six sets of
HAp/CSP nanocomposites were prepared by adding 10 to 60 wt.% of n-HAp particles
as filler.


### 2.5. Characterization

#### 2.5.1. Physicochemical Characterization

(1) XRD Study
The
phase analysis of the HAp powder and composite samples was done by XRD (Model PW 1729,
Philips, Holland)
using 35 milliamps, and 40 kV current, with a monochromatic CuKα (target) radiation (λ=1.5405Å) with a step size of 0.04°2θ, a scan rate of 0.02°2θ/s, and a scan range from 2θ=10 to 60°.(2) FTIR Study
The
identification of functional groups in the HAp powder, matrix, and composite
samples as well as the interfacial modification (i.e., nature of bonding
between particles and the polymeric matrix) were analyzed by FTIR analysis
(Model NEXUS870, FTIR, Thermo Nicolet,
USA) within the
scanning range 4000–400 cm-1.(3) TEM Study
The morphology, particle size of HAp powder,
and dispersion of particles in the polymer matrix were observed through a
Phillips CM 200 transmission electron microscope (TEM) with an acceleration
voltage 200 kV.(4) SEM Study
The
surface roughness of the nanocomposite samples was investigated by scanning
electron microscopy (SEM) (Model JSM-5800, JEOL, scanning electron microscope, Japan).(5) Mechanical Testing
The
tensile testing of the composite films was carried out on a universal
mechanical testing machine (Model H10KS, HOUNSFIELD, UK) at a crosshead
rate of 50 mm/min.


#### 2.5.2. Biocompatibility Study

(1) Cell Viability Study
In vitro cytotoxicity study of the material was
carried out by culturing murine fibroblast L929 cells in a contact mode.
Briefly, L929 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and seeded on the sheets/films at their
exponential phase of growth at a density of 105 cells/cm2.
The cells were allowed to attach to the films surface for 3 hours in 5% CO2 incubator at 37°C. Fresh DMEM medium supplemented with 10% fetal
calf serum (FCS) was added to each well to keep the cell containing films
submerged. The plates were incubated for 24 hours at 37°C in a
humidified atmosphere of 5% CO2 in air.  After 24-hour incubation, MTT (4 mg/mL) was
added to each well at a strength of 10% (v/v) and incubated for further 4 hours
at 37°C. Subsequently, the media containing MTT was removed, and 200 μL of DMSO
was added to dissolve the formazan crystals. The absorbance was measured using
an ELISA plate reader (Biorad, USA) at 595 nm.
Student paired t-test was performed to test for statistical significance, and a P-value of
<.05 was determined to represent a significant difference.(2) Primary Murine Osteoblast Culture
Primary
murine osteoblast cells were obtained by serial enzymatic digestion of neonatal
mice calvariae. Briefly, calvariae were dissected from 3-4 days old neonatal
mice. After removing adherent mesenchymal tissue and periosteum, calvariae were
subjected to five sequential 15-minute digestions in an enzyme mixture
containing 0.05% trypsin and 0.1% collagenase type I at 37°C on a rocking
platform. The first two fractions were discarded, and fractions 3–5 were collected
and immediately chilled by the addition of cold DMEM containing 10% FCS.
Released cells were pooled, centrifuged, resuspended in medium, and filtered
through a 70-micron mesh. Cells were plated at 104 viable cells/cm2 in six-well culture plates in DMEM containing 20% FCS. Then, 24 hours
later, the medium was changed to Dulbecco's modified Eagle's medium with 10%
FCS (basal medium), and cells were fed again after 3 days. The cells from
third to fifth passages were used for the differentiation studies.(3) In Vitro Osteoblast Differentiation
Confluent
monolayers of osteoblastic cells were enzymatically lifted from the flasks
using 0.25% trypsin in 4 mM EDTA. The cells were concentrated by centrifugation
at 300 g for 10 minutes and resuspended in a known amount of media [22]. Cells
were counted using hemocytometer and diluted to the desired concentration of
cells in complete media containing alpha-MEM supplement with 10% FBS, 1%
antibiotic-antimycotic solution, 10 μM sodium beta-glycerol phosphate, 50 μg/mL L-ascorbic acid, and 100 nM
dexamethasone. Aliquots of 20 μL
of cell suspension were seeded onto the top of the films prewetted and
equilibrated with complete media placed in the wells of 24-well plates
resulting in a seeding density of 104 cells/cm2. The
films were left undisturbed in an incubator for 3 hours at 37°C to
allow for cell attachment to the films, after which an additional 1 mL of
complete media was added to each well. Medium was changed every 3 days.
Cultures were maintained in a humidified atmosphere consisting of 5% CO2 at 37°C. Osteoblast proliferation was determined at 3, 7, 14 and 21
days. The films were gently washed using fresh medium to remove unattached
cells and exhausted medium followed by MTT assay as described above.(4) Alkaline Phosphatase Assay
Culture
medium was removed from the films and the cells were washed twice with PBS. The
cells were lysed with 250μL of
Triton X-100 (0.01% in PBS) for 30 minutes at 4°C. The obtained homogenate was
used for the measurement of alkaline phosphatase (AP) activity and total
protein concentration. AP activity was determined by an assay based on the
hydrolysis of p-nitrophenyl phosphate to p-nitrophenol. About 50 μL of the Triton lysate was added to 125 μL of active reagent containing
0.012 M p-nitrophenyl phosphate in 0.05 M diethanolamine, pH 9.8, and incubated
for 30 minutes at 37°C. The reaction was stopped with 50 μL of 2.5 M sodium hydroxide and the AP
activity was determined by measuring the absorbance of p-nitrophenol at 405 nm
using a Microplate Reader (Bio-Rad).(5) Osteoblasts Morphology and Their Nodule Formation
Osteoblasts
morphology and their nodule formation were investigated through scanning
electron microscope (SEM) study. For SEM studies, the attached cells on the
composite films were rinsed twice with PBS, and fixed with 250 mL of 2.5%
glutaraldehyde in PBS for 30–60 minutes. After
washing with PBS, dehydration was performed by slow water replacement using
series of ethanol solutions (30%, 50%, 70%, and 90%) for 15 minutes with final
dehydration in absolute ethanol for 30 minutes, allowing samples to dry at room
temperature and under vacuum. The films were mounted on stubs and coated in
vacuum with gold. Cells were examined with a JSM-5800, JEOL, scanning electron microscope, Japan.


## 2.1. Chemicals

Calcium
nitrate (Ca(NO3)2⋅4H2O) (99%), diammonium
hydrogen phosphate ((NH4)2HPO4) (DAHP) (99%),
ammonia solution (25%), ammonium chloride (98%), triethanolamine (TEA), and
orthophosphoric acid (H3PO4) were procured from Merck, Mumbai, India.
Chitosan, methyl-thiotetrazole (MTT), trypsin, dexamethasone, sodium beta-glycerol
phosphate, and SEM grade gulteraldehyde were purchased from Sigma-Aldrich Chemical, St Louis, Mo, USA.
Collagenase type I, 0.25% trypsin-EDTA, and Dulbecco’s modified Eagle’s medium (DMEM)
were procured from Invitrogen, USA. Fetal calf
serum and alpha-MEM were supplied by Hyclone and Pan-Biotech, respectively.


## 2.2. Preparation of Nano-Hydroxyapatite (n-HAp)

A
concentration of 0.5 M stock solution each of Ca(NO3)2⋅4H2O
and (NH4)2HPO4 was prepared in distilled
water. Both solutions were taken in such amounts that Ca : P molar ratio was maintained
at 1.67. Triethanolamine (TEA) was used in conjunction with Ca(NO3)2⋅4H2O
solution (Ca2+ : TEA  =  1:0.5) as a capping agent to control the
particle growth during precipitation. The pH of both calcium nitrate and DAHP
solutions was maintained at ~11-12. (NH4)2HPO4 was added drop-wise to the mixture of Ca(NO3)2⋅4H2O
and TEA and vigorously stirred at room temperature using a mechanical stirrer
(2500 rpm). The pH of the reacting mixture was also maintained in the range of
11-12 by adding NH4OH
solution gradually. The process was continued up to 6 hours. The gelatinous precipitate thus obtained was filtered by a
centrifugal filtration process. The precipitate was washed with water
thoroughly as well as with NH4Cl solution to remove the excess
residual TEA, and dried at 90°C for 15 hours.


## 2.3. Synthesis of Chitosan Phosphate (CSP)

A
mixture of approximately 2 g chitosan powder, 100 mL of 2% acetic acid solution,
6 g of orthophosphoric acid was taken in a three naked-round bottomed flask equipped
with a stirrer, condenser, thermometer, and nitrogen gas inlet tube. The mixture was heated up to 80°C under constant
stirring until dissolution of chitosan powder and then heated to reflux.
After 2 hours, the resultant solution was cooled and precipitated in excess
methanol. Again, the precipitated gel was dissolved in water and all unreacted phosphate
(H3PO4) and acetic acid were removed by repeated
reprecipitation in excess methanol. Finally, the gel was collected and dried under
vacuum oven at 80°C over night.


## 2.4. Synthesis of HAp/CSP Nanocomposite

At
first, the CSP was dissolved in hot water, and then the nanoparticles were added
slowly with vigorous stirring with the help of a mechanical stirrer at 3000 rpm. After addition of entire nanoparticles to the
polymer solution, the resulting solution mixture was kept in a vacuum desiccator
to remove the bubbles. Then, the mixture was heated in a water bath to
evaporate water. The resulting slurry was poured into a glass petri dish and
dried to make a film, keeping it in a vacuum oven at 90°C over
night. After that, the film was cut into dumbbell-shaped samples. Six sets of
HAp/CSP nanocomposites were prepared by adding 10 to 60 wt.% of n-HAp particles
as filler.


## 2.5. Characterization

### 2.5.1. Physicochemical Characterization

(1) XRD Study
The
phase analysis of the HAp powder and composite samples was done by XRD (Model PW 1729,
Philips, Holland)
using 35 milliamps, and 40 kV current, with a monochromatic CuKα (target) radiation (λ=1.5405Å) with a step size of 0.04°2θ, a scan rate of 0.02°2θ/s, and a scan range from 2θ=10 to 60°.(2) FTIR Study
The
identification of functional groups in the HAp powder, matrix, and composite
samples as well as the interfacial modification (i.e., nature of bonding
between particles and the polymeric matrix) were analyzed by FTIR analysis
(Model NEXUS870, FTIR, Thermo Nicolet,
USA) within the
scanning range 4000–400 cm-1.(3) TEM Study
The morphology, particle size of HAp powder,
and dispersion of particles in the polymer matrix were observed through a
Phillips CM 200 transmission electron microscope (TEM) with an acceleration
voltage 200 kV.(4) SEM Study
The
surface roughness of the nanocomposite samples was investigated by scanning
electron microscopy (SEM) (Model JSM-5800, JEOL, scanning electron microscope, Japan).(5) Mechanical Testing
The
tensile testing of the composite films was carried out on a universal
mechanical testing machine (Model H10KS, HOUNSFIELD, UK) at a crosshead
rate of 50 mm/min.


### 2.5.2. Biocompatibility Study

(1) Cell Viability Study
In vitro cytotoxicity study of the material was
carried out by culturing murine fibroblast L929 cells in a contact mode.
Briefly, L929 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and seeded on the sheets/films at their
exponential phase of growth at a density of 105 cells/cm2.
The cells were allowed to attach to the films surface for 3 hours in 5% CO2 incubator at 37°C. Fresh DMEM medium supplemented with 10% fetal
calf serum (FCS) was added to each well to keep the cell containing films
submerged. The plates were incubated for 24 hours at 37°C in a
humidified atmosphere of 5% CO2 in air.  After 24-hour incubation, MTT (4 mg/mL) was
added to each well at a strength of 10% (v/v) and incubated for further 4 hours
at 37°C. Subsequently, the media containing MTT was removed, and 200 μL of DMSO
was added to dissolve the formazan crystals. The absorbance was measured using
an ELISA plate reader (Biorad, USA) at 595 nm.
Student paired t-test was performed to test for statistical significance, and a P-value of
<.05 was determined to represent a significant difference.(2) Primary Murine Osteoblast Culture
Primary
murine osteoblast cells were obtained by serial enzymatic digestion of neonatal
mice calvariae. Briefly, calvariae were dissected from 3-4 days old neonatal
mice. After removing adherent mesenchymal tissue and periosteum, calvariae were
subjected to five sequential 15-minute digestions in an enzyme mixture
containing 0.05% trypsin and 0.1% collagenase type I at 37°C on a rocking
platform. The first two fractions were discarded, and fractions 3–5 were collected
and immediately chilled by the addition of cold DMEM containing 10% FCS.
Released cells were pooled, centrifuged, resuspended in medium, and filtered
through a 70-micron mesh. Cells were plated at 104 viable cells/cm2 in six-well culture plates in DMEM containing 20% FCS. Then, 24 hours
later, the medium was changed to Dulbecco's modified Eagle's medium with 10%
FCS (basal medium), and cells were fed again after 3 days. The cells from
third to fifth passages were used for the differentiation studies.(3) In Vitro Osteoblast Differentiation
Confluent
monolayers of osteoblastic cells were enzymatically lifted from the flasks
using 0.25% trypsin in 4 mM EDTA. The cells were concentrated by centrifugation
at 300 g for 10 minutes and resuspended in a known amount of media [22]. Cells
were counted using hemocytometer and diluted to the desired concentration of
cells in complete media containing alpha-MEM supplement with 10% FBS, 1%
antibiotic-antimycotic solution, 10 μM sodium beta-glycerol phosphate, 50 μg/mL L-ascorbic acid, and 100 nM
dexamethasone. Aliquots of 20 μL
of cell suspension were seeded onto the top of the films prewetted and
equilibrated with complete media placed in the wells of 24-well plates
resulting in a seeding density of 104 cells/cm2. The
films were left undisturbed in an incubator for 3 hours at 37°C to
allow for cell attachment to the films, after which an additional 1 mL of
complete media was added to each well. Medium was changed every 3 days.
Cultures were maintained in a humidified atmosphere consisting of 5% CO2 at 37°C. Osteoblast proliferation was determined at 3, 7, 14 and 21
days. The films were gently washed using fresh medium to remove unattached
cells and exhausted medium followed by MTT assay as described above.(4) Alkaline Phosphatase Assay
Culture
medium was removed from the films and the cells were washed twice with PBS. The
cells were lysed with 250μL of
Triton X-100 (0.01% in PBS) for 30 minutes at 4°C. The obtained homogenate was
used for the measurement of alkaline phosphatase (AP) activity and total
protein concentration. AP activity was determined by an assay based on the
hydrolysis of p-nitrophenyl phosphate to p-nitrophenol. About 50 μL of the Triton lysate was added to 125 μL of active reagent containing
0.012 M p-nitrophenyl phosphate in 0.05 M diethanolamine, pH 9.8, and incubated
for 30 minutes at 37°C. The reaction was stopped with 50 μL of 2.5 M sodium hydroxide and the AP
activity was determined by measuring the absorbance of p-nitrophenol at 405 nm
using a Microplate Reader (Bio-Rad).(5) Osteoblasts Morphology and Their Nodule Formation
Osteoblasts
morphology and their nodule formation were investigated through scanning
electron microscope (SEM) study. For SEM studies, the attached cells on the
composite films were rinsed twice with PBS, and fixed with 250 mL of 2.5%
glutaraldehyde in PBS for 30–60 minutes. After
washing with PBS, dehydration was performed by slow water replacement using
series of ethanol solutions (30%, 50%, 70%, and 90%) for 15 minutes with final
dehydration in absolute ethanol for 30 minutes, allowing samples to dry at room
temperature and under vacuum. The films were mounted on stubs and coated in
vacuum with gold. Cells were examined with a JSM-5800, JEOL, scanning electron microscope, Japan.


## 2.5.1. Physicochemical Characterization

(1) XRD Study
The
phase analysis of the HAp powder and composite samples was done by XRD (Model PW 1729,
Philips, Holland)
using 35 milliamps, and 40 kV current, with a monochromatic CuKα (target) radiation (λ=1.5405Å) with a step size of 0.04°2θ, a scan rate of 0.02°2θ/s, and a scan range from 2θ=10 to 60°.(2) FTIR Study
The
identification of functional groups in the HAp powder, matrix, and composite
samples as well as the interfacial modification (i.e., nature of bonding
between particles and the polymeric matrix) were analyzed by FTIR analysis
(Model NEXUS870, FTIR, Thermo Nicolet,
USA) within the
scanning range 4000–400 cm-1.(3) TEM Study
The morphology, particle size of HAp powder,
and dispersion of particles in the polymer matrix were observed through a
Phillips CM 200 transmission electron microscope (TEM) with an acceleration
voltage 200 kV.(4) SEM Study
The
surface roughness of the nanocomposite samples was investigated by scanning
electron microscopy (SEM) (Model JSM-5800, JEOL, scanning electron microscope, Japan).(5) Mechanical Testing
The
tensile testing of the composite films was carried out on a universal
mechanical testing machine (Model H10KS, HOUNSFIELD, UK) at a crosshead
rate of 50 mm/min.


## 2.5.2. Biocompatibility Study

(1) Cell Viability Study
In vitro cytotoxicity study of the material was
carried out by culturing murine fibroblast L929 cells in a contact mode.
Briefly, L929 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and seeded on the sheets/films at their
exponential phase of growth at a density of 105 cells/cm2.
The cells were allowed to attach to the films surface for 3 hours in 5% CO2 incubator at 37°C. Fresh DMEM medium supplemented with 10% fetal
calf serum (FCS) was added to each well to keep the cell containing films
submerged. The plates were incubated for 24 hours at 37°C in a
humidified atmosphere of 5% CO2 in air.  After 24-hour incubation, MTT (4 mg/mL) was
added to each well at a strength of 10% (v/v) and incubated for further 4 hours
at 37°C. Subsequently, the media containing MTT was removed, and 200 μL of DMSO
was added to dissolve the formazan crystals. The absorbance was measured using
an ELISA plate reader (Biorad, USA) at 595 nm.
Student paired t-test was performed to test for statistical significance, and a P-value of
<.05 was determined to represent a significant difference.(2) Primary Murine Osteoblast Culture
Primary
murine osteoblast cells were obtained by serial enzymatic digestion of neonatal
mice calvariae. Briefly, calvariae were dissected from 3-4 days old neonatal
mice. After removing adherent mesenchymal tissue and periosteum, calvariae were
subjected to five sequential 15-minute digestions in an enzyme mixture
containing 0.05% trypsin and 0.1% collagenase type I at 37°C on a rocking
platform. The first two fractions were discarded, and fractions 3–5 were collected
and immediately chilled by the addition of cold DMEM containing 10% FCS.
Released cells were pooled, centrifuged, resuspended in medium, and filtered
through a 70-micron mesh. Cells were plated at 104 viable cells/cm2 in six-well culture plates in DMEM containing 20% FCS. Then, 24 hours
later, the medium was changed to Dulbecco's modified Eagle's medium with 10%
FCS (basal medium), and cells were fed again after 3 days. The cells from
third to fifth passages were used for the differentiation studies.(3) In Vitro Osteoblast Differentiation
Confluent
monolayers of osteoblastic cells were enzymatically lifted from the flasks
using 0.25% trypsin in 4 mM EDTA. The cells were concentrated by centrifugation
at 300 g for 10 minutes and resuspended in a known amount of media [22]. Cells
were counted using hemocytometer and diluted to the desired concentration of
cells in complete media containing alpha-MEM supplement with 10% FBS, 1%
antibiotic-antimycotic solution, 10 μM sodium beta-glycerol phosphate, 50 μg/mL L-ascorbic acid, and 100 nM
dexamethasone. Aliquots of 20 μL
of cell suspension were seeded onto the top of the films prewetted and
equilibrated with complete media placed in the wells of 24-well plates
resulting in a seeding density of 104 cells/cm2. The
films were left undisturbed in an incubator for 3 hours at 37°C to
allow for cell attachment to the films, after which an additional 1 mL of
complete media was added to each well. Medium was changed every 3 days.
Cultures were maintained in a humidified atmosphere consisting of 5% CO2 at 37°C. Osteoblast proliferation was determined at 3, 7, 14 and 21
days. The films were gently washed using fresh medium to remove unattached
cells and exhausted medium followed by MTT assay as described above.(4) Alkaline Phosphatase Assay
Culture
medium was removed from the films and the cells were washed twice with PBS. The
cells were lysed with 250μL of
Triton X-100 (0.01% in PBS) for 30 minutes at 4°C. The obtained homogenate was
used for the measurement of alkaline phosphatase (AP) activity and total
protein concentration. AP activity was determined by an assay based on the
hydrolysis of p-nitrophenyl phosphate to p-nitrophenol. About 50 μL of the Triton lysate was added to 125 μL of active reagent containing
0.012 M p-nitrophenyl phosphate in 0.05 M diethanolamine, pH 9.8, and incubated
for 30 minutes at 37°C. The reaction was stopped with 50 μL of 2.5 M sodium hydroxide and the AP
activity was determined by measuring the absorbance of p-nitrophenol at 405 nm
using a Microplate Reader (Bio-Rad).(5) Osteoblasts Morphology and Their Nodule Formation
Osteoblasts
morphology and their nodule formation were investigated through scanning
electron microscope (SEM) study. For SEM studies, the attached cells on the
composite films were rinsed twice with PBS, and fixed with 250 mL of 2.5%
glutaraldehyde in PBS for 30–60 minutes. After
washing with PBS, dehydration was performed by slow water replacement using
series of ethanol solutions (30%, 50%, 70%, and 90%) for 15 minutes with final
dehydration in absolute ethanol for 30 minutes, allowing samples to dry at room
temperature and under vacuum. The films were mounted on stubs and coated in
vacuum with gold. Cells were examined with a JSM-5800, JEOL, scanning electron microscope, Japan.


## 3. Results and Discussion

### 3.1. Physicochemical Study

#### 3.1.1. XRD Study

Figure1(b) shows the X-ray diffraction pattern of the synthesized apatite powder. The
d-values correspond to that of calcium hydroxyapatite (Ca10(PO4)6(OH)2)
(JCPDS card no.
74-0566). Taking into account the broadening of each peak in XRD, mean crystallite
size has been calculated using Scherrer’s equation, that is, D=0.9λ/βcosθ,
where D is the average crystallite size in Å, β is
the peak broadening of the diffraction line measured at half of its maximum
intensity in “radian,” λ is the wavelength of X-rays, and θ is the Bragg’s
diffraction angle. The mean crystallite size is found to be 15 nm. The
approximate particle size of HAp powder is found to be 6–10 nm in diameter
by 26–56 nm in length
with needle-like acicular crystals as has been observed from TEM micrograph.
The use of synthesized n-HAp powder with the CSP polymer as a matrix thus
provides an effective means to produce nanocomposites.Figure 1
X-ray diffraction patters of (a) CSP, (b) HAp, and (c) HAp/CSP nanocomposite.Figure1(c) shows the X-ray diffraction pattern of the HAp/CSP composite. The
crystallite size of HAp in composite is found to be 12 nm. The crystallinity
(Xc) of the pure HAp and HAp/CSP composite is determined by an empirical
relation between Xc and β002 (i.e., β002×Xc3=KA) [23], where Xc is the crystallinity degree, β002 is full width of the peak at half intensity of (002) plane in degree −2θ, and KA is a constant (0.24). The Xc for pure HAp powder is 0.15, and in the HAp/CSP
composite, it is found to be 0.11. The crystallinity of the n-HAp particles as
well as CSP polymer has decreased after composite formation as shown in Figure 1
indicating that crystal structures of both HAp and CSP have changed after
composite formation, which may be resulted from the interface binding between
particles and matrix. The XRD peaks, (002) and (211) have shifted to higher 2θ values in case of HAp/CSP composite
as compared to pure HAp (i.e., from 2θ=25.98 to 26.57 and 31.89 to
33.46, resp.), which is possibly due to compression from the contracting
polymeric matrix through interfacial bonding. Simultaneously, the crystalline peak of CSP polymer at 2θ=20.8 has shifted
to 21.6, whereas, another crystalline peak of CSP
at around 2θ=10.5 has disappeared in the composite (Figure 1(c)). The
shift and decrease in crystallinity of each peak of the polymer as well as HAp
after composite formation clearly indicate the presence of bonding between HAp
particles and polymer matrix. The peak at 32.49 [300] of n-HAp is
weakened after composite formation, which also indicates the participation of
n-HAp in bonding with the polymer.


#### 3.1.2. FTIR Study

Figure2(b) shows the FTIR spectrum of CSP having characteristic peaks at 2934 and 2850 cm-1 for asymmetric and symmetric stretching of methylene (–CH2–)
groups, respectively. On the other hand, the bands at around 1544 and 1620 cm-1 are for N–H stretching in CSP. The bands at around 1088 and 1047 cm-1 are attributed to the C–O–P stretching and phosphorylated hydroxyl group, whereas,
the characteristic peaks at around 991 and 493 cm-1 are attributed
for P–OH groups in CSP polymer. The bands at around 1100 to 1250 cm-1 are due to P–O, P=O stretching of phosphate group. The bands at 632 and 3571 cm-1 are for structural OH groups in the n-HAp crystals (Figure 2(c)). The bands at
1092, 1030, and 604 cm-1 indicate the presence of PO43− group, and the spectrum also indicates the presence of H2O (3422 and
1641 cm-1) in HAp crystals. The peaks at 1047 and 493 cm-1 for P–OH groups in CSP have disappeared as shown in the Figure 2(d) for HAp/CSP
composite, which may be due to the formation of polyphosphonate salt [14]. The
peak intensity of P–OH at 991 and 1088 cm-1 in CSP polymer has
decreased after composite formation, which evidences that the P–OH groups of
CSP polymer have taken part in bonding with hydroxyapatite. Similar trend is
seen in case of OH (632, 3571 cm-1) groups of HAp crystals, which is
an indication of participation in bonding of HAp with polymer. Most of the
peaks either in polymer or in HAp have shown clear shift after composite
formation (Table 1). Thus, a comparison of the FTIR analysis has indicated that
there is a chemical bonding at the nanoparticle-polymer interface.Table 1
FTIR peak positions of various functional groups before and after composite formation.
Functional groupsIR peak positions (cm-1)CSCSPHAp particlesHAp/CSPPO43−——1092, 1030, 6041079, 1025, 602P–OH—1088, 1047, 991, 493—1070, disappeared, 984, disappearedO–H bending——632, 3571625, 3563O–H stretching3430342734223402N–H3467, 1622, 15443465, 1620, 1544—3462, 1620, 1543Figure 2
FTIR spectra of (a) chitosan, (b) chitosan phosphate, (c) HAp powder, and (d) HAp/CSP nanocomposite.


#### 3.1.3. TEM Study

Figure3(a) shows the TEM micrograph of the synthesized n-HAp powder. The micrograph
depicts the acicular needle-like crystals of HAp powder in nanometer range,
having 6–10 nm in diameter
by 26–56 nm in length.
The micrograph shows the presence of agglomerations among the particles, which
are due to high specific surface energy of n-HAp particles resulting in their
aggregations. Nanosized HAp particles with homogeneous dispersion are well
identified in case of HAp/CSP composite (Figure 3(b)). The particle size of HAp
is also controlled by the polymer (CSP) in composite as depicted in the TEM
micrograph.TEM micrographs of (a) HAp powders and (b) HAp/CSP nanocomposite (40% w/w).
(a)(b)


#### 3.1.4. SEM Study

Figure4 shows the SEM micrographs of fracture surface of the composite samples with
different contents of n-HAp particles. The micrographs depict that with
increase in amount of HAp particles loading, the surface roughness for the
composites is increased.SEM micrographs of HAp/CSP nanocomposite samples with various contents of n-HAp particles (a) 20% (w/w), (b) 30% (w/w), (c) 40% (w/w), and (d) 50% (w/w).
(a)(b)(c)(d)


#### 3.1.5. Mechanical Properties Study

The
tensile strength and modulus of the composites are found to be increased with
increase in amount of HAp contents (Table2). In 30% (w/w) n-HAp content, the
increase in tensile
strength and modulus has been found to be 100.4% and 190.8% than pure CSP polymer,
respectively. In 40% (w/w) HAp loading, the tensile strength and modulus are
increased by 140.8% and 225.5%, respectively. With 50% (w/w) HAp content, the
tensile strength started to decrease, whereas, modulus is increased by 381.5%
over pure CSP polymer. The results are summarized in Table 2. From the results,
it is clear that the mechanical properties have been improved significantly
with high nanoparticle loading capability, which means that the composite tends
to be used as a biomaterial with high osteoconductivity [24]. The enhancement
in tensile strength and modulus in case of HAp/CSP nanocomposite could be
attributed to the excellent bonding at the nanoparticle-polymer matrix
interface through the pendant phosphate groups of CSP.Table 2
Mechanical properties of HAp/CSP nanocomposite samples. Number after  ±  sign corresponds to the standard deviation.
n-HAp particles loaded (w/w)Tensile strength (MPa)Young’s modulus (MPa)Elongation at break (%)0%6.37±0.5574.45±0.9472.87±2.6610%7.57±0.29147.87±1.2347.99±2.1720%10.43±0.87184.89±2.7733.95±0.6630%12.77±1.06216.56±3.4530.84±1.8440%15.34±0.47242.38±2.7622.28±2.5850%11.89±0.78358.54±4.8520.77±1.4260%10.65±0.49302.68±3.9718.76±0.95


### 3.2. Biocompatibility Study

#### 3.2.1. Cell Viability Study

A
cell line of murine fibroblast (L929) has been selected for the MTT assay test.
The difference in cell viability index is shown in Figure5 of the composite
samples of different concentrations, as compared to the control tissue culture
plate. The statistical analysis (Student’s t-test) has indicated that
the difference in cell viability index is insignificant. Hence, the developed
composite is cytocompatible.Figure 5
Cell viability test using murine fibroblast cell line L929 on CSP and
HAp/CSP films, CS was taken as control (n=3).


#### 3.2.2. In Vitro Osteoblast Differentiation Study

The
neonatal mouse calvarial osteoblasts have been used to study the development of
bone cells on CSP and HAp/CSP nanocomposite film surfaces. The results of cell
proliferation of murine calvarial osteoblasts cultured on CSP and HAp/CSP
nanocomposite films show a cell proliferation during the first 7 days (Figure6).
The proliferation phase follows a growth retardation after 7 days due to high
confluence of cells on the film surfaces. The results show that HAp/CSP
nanocomposite is an appropriate biomaterial for osteoblast proliferation.Figure 6
Osteoblast cell proliferation on CSP and HAp/CSP films, CS was taken as control. Significant increase in cell number (P<.001) was found at day 7 in all the variations followed by retardation of growth phase.


#### 3.2.3. Alkaline Phosphatase Enzyme Activity Study

The
differentiation process of the osteoblast cells on CSP and HAp/CSP
nanocomposite has been analyzed by the alkaline phosphatase (AP) enzyme
activity, which is an early osteogenic marker. The highest enzyme activity is
achieved at day 14 in both materials (Figure7).  The AP activity shown by osteoblast cells
growth on HAp/CSP nanocomposite is significantly higher than that on CSP
surface.Figure 7
AP activity of osteoblast cells grown on CSP and HAp/CSP films, CS was taken as control. Both in case of CSP and HAp/CSP enzyme activity increased up to day 14 (P<.001). HAp/CSP nanocomposite is proven a better support for osteoblast differentiation than CSP alone (P<.01).


#### 3.2.4. Osteoblasts Morphology and Their Nodule Formation

Further,
SEM examination has been conducted at day 14 to observe the bone formation
process. Osteoblast cultured on the chitosan films reveals a round shape and
stayed segregated. Secreted extracellular matrix or mineralization is not
observed there (Figure8(a)). Whereas, osteoblast cultured on chitosan phosphate
films has shown tight aggregation and some mineralization (Figure 8(b)). However,
osteoblast cultured on HAp/CSP nanocomposite shows a characteristic growing in multiple-layer
patterns and tends to form a nodular cell aggregation (Figure 8(c)). Nodules
contain densely packed cells are embedded within highly mineralized extracellular matrix secreted by them. SEM
and AP enzyme activity studies indicate that incorporation of hydroxyapatite
increases in vitro osteogenicity of chitosan phosphate films.SEM images of osteoblasts grown on different matrices at day 14 on (a) chitosan (CS), (b) chitosan phosphate (CSP), (c) HAp/CSP nanocomposite. Scale bar of each image is 10μm. Arrow “1” shows a nodular cell aggregate, arrow “2” shows the extracellular matrix secreted by the cells in around themselves, and arrow “3” shows a visible mineral nodule.
(a)(b)(c)


## 3.1. Physicochemical Study

### 3.1.1. XRD Study

Figure1(b) shows the X-ray diffraction pattern of the synthesized apatite powder. The
d-values correspond to that of calcium hydroxyapatite (Ca10(PO4)6(OH)2)
(JCPDS card no.
74-0566). Taking into account the broadening of each peak in XRD, mean crystallite
size has been calculated using Scherrer’s equation, that is, D=0.9λ/βcosθ,
where D is the average crystallite size in Å, β is
the peak broadening of the diffraction line measured at half of its maximum
intensity in “radian,” λ is the wavelength of X-rays, and θ is the Bragg’s
diffraction angle. The mean crystallite size is found to be 15 nm. The
approximate particle size of HAp powder is found to be 6–10 nm in diameter
by 26–56 nm in length
with needle-like acicular crystals as has been observed from TEM micrograph.
The use of synthesized n-HAp powder with the CSP polymer as a matrix thus
provides an effective means to produce nanocomposites.Figure 1
X-ray diffraction patters of (a) CSP, (b) HAp, and (c) HAp/CSP nanocomposite.Figure1(c) shows the X-ray diffraction pattern of the HAp/CSP composite. The
crystallite size of HAp in composite is found to be 12 nm. The crystallinity
(Xc) of the pure HAp and HAp/CSP composite is determined by an empirical
relation between Xc and β002 (i.e., β002×Xc3=KA) [23], where Xc is the crystallinity degree, β002 is full width of the peak at half intensity of (002) plane in degree −2θ, and KA is a constant (0.24). The Xc for pure HAp powder is 0.15, and in the HAp/CSP
composite, it is found to be 0.11. The crystallinity of the n-HAp particles as
well as CSP polymer has decreased after composite formation as shown in Figure 1
indicating that crystal structures of both HAp and CSP have changed after
composite formation, which may be resulted from the interface binding between
particles and matrix. The XRD peaks, (002) and (211) have shifted to higher 2θ values in case of HAp/CSP composite
as compared to pure HAp (i.e., from 2θ=25.98 to 26.57 and 31.89 to
33.46, resp.), which is possibly due to compression from the contracting
polymeric matrix through interfacial bonding. Simultaneously, the crystalline peak of CSP polymer at 2θ=20.8 has shifted
to 21.6, whereas, another crystalline peak of CSP
at around 2θ=10.5 has disappeared in the composite (Figure 1(c)). The
shift and decrease in crystallinity of each peak of the polymer as well as HAp
after composite formation clearly indicate the presence of bonding between HAp
particles and polymer matrix. The peak at 32.49 [300] of n-HAp is
weakened after composite formation, which also indicates the participation of
n-HAp in bonding with the polymer.


### 3.1.2. FTIR Study

Figure2(b) shows the FTIR spectrum of CSP having characteristic peaks at 2934 and 2850 cm-1 for asymmetric and symmetric stretching of methylene (–CH2–)
groups, respectively. On the other hand, the bands at around 1544 and 1620 cm-1 are for N–H stretching in CSP. The bands at around 1088 and 1047 cm-1 are attributed to the C–O–P stretching and phosphorylated hydroxyl group, whereas,
the characteristic peaks at around 991 and 493 cm-1 are attributed
for P–OH groups in CSP polymer. The bands at around 1100 to 1250 cm-1 are due to P–O, P=O stretching of phosphate group. The bands at 632 and 3571 cm-1 are for structural OH groups in the n-HAp crystals (Figure 2(c)). The bands at
1092, 1030, and 604 cm-1 indicate the presence of PO43− group, and the spectrum also indicates the presence of H2O (3422 and
1641 cm-1) in HAp crystals. The peaks at 1047 and 493 cm-1 for P–OH groups in CSP have disappeared as shown in the Figure 2(d) for HAp/CSP
composite, which may be due to the formation of polyphosphonate salt [14]. The
peak intensity of P–OH at 991 and 1088 cm-1 in CSP polymer has
decreased after composite formation, which evidences that the P–OH groups of
CSP polymer have taken part in bonding with hydroxyapatite. Similar trend is
seen in case of OH (632, 3571 cm-1) groups of HAp crystals, which is
an indication of participation in bonding of HAp with polymer. Most of the
peaks either in polymer or in HAp have shown clear shift after composite
formation (Table 1). Thus, a comparison of the FTIR analysis has indicated that
there is a chemical bonding at the nanoparticle-polymer interface.Table 1
FTIR peak positions of various functional groups before and after composite formation.
Functional groupsIR peak positions (cm-1)CSCSPHAp particlesHAp/CSPPO43−——1092, 1030, 6041079, 1025, 602P–OH—1088, 1047, 991, 493—1070, disappeared, 984, disappearedO–H bending——632, 3571625, 3563O–H stretching3430342734223402N–H3467, 1622, 15443465, 1620, 1544—3462, 1620, 1543Figure 2
FTIR spectra of (a) chitosan, (b) chitosan phosphate, (c) HAp powder, and (d) HAp/CSP nanocomposite.


### 3.1.3. TEM Study

Figure3(a) shows the TEM micrograph of the synthesized n-HAp powder. The micrograph
depicts the acicular needle-like crystals of HAp powder in nanometer range,
having 6–10 nm in diameter
by 26–56 nm in length.
The micrograph shows the presence of agglomerations among the particles, which
are due to high specific surface energy of n-HAp particles resulting in their
aggregations. Nanosized HAp particles with homogeneous dispersion are well
identified in case of HAp/CSP composite (Figure 3(b)). The particle size of HAp
is also controlled by the polymer (CSP) in composite as depicted in the TEM
micrograph.TEM micrographs of (a) HAp powders and (b) HAp/CSP nanocomposite (40% w/w).
(a)(b)


### 3.1.4. SEM Study

Figure4 shows the SEM micrographs of fracture surface of the composite samples with
different contents of n-HAp particles. The micrographs depict that with
increase in amount of HAp particles loading, the surface roughness for the
composites is increased.SEM micrographs of HAp/CSP nanocomposite samples with various contents of n-HAp particles (a) 20% (w/w), (b) 30% (w/w), (c) 40% (w/w), and (d) 50% (w/w).
(a)(b)(c)(d)


### 3.1.5. Mechanical Properties Study

The
tensile strength and modulus of the composites are found to be increased with
increase in amount of HAp contents (Table2). In 30% (w/w) n-HAp content, the
increase in tensile
strength and modulus has been found to be 100.4% and 190.8% than pure CSP polymer,
respectively. In 40% (w/w) HAp loading, the tensile strength and modulus are
increased by 140.8% and 225.5%, respectively. With 50% (w/w) HAp content, the
tensile strength started to decrease, whereas, modulus is increased by 381.5%
over pure CSP polymer. The results are summarized in Table 2. From the results,
it is clear that the mechanical properties have been improved significantly
with high nanoparticle loading capability, which means that the composite tends
to be used as a biomaterial with high osteoconductivity [24]. The enhancement
in tensile strength and modulus in case of HAp/CSP nanocomposite could be
attributed to the excellent bonding at the nanoparticle-polymer matrix
interface through the pendant phosphate groups of CSP.Table 2
Mechanical properties of HAp/CSP nanocomposite samples. Number after  ±  sign corresponds to the standard deviation.
n-HAp particles loaded (w/w)Tensile strength (MPa)Young’s modulus (MPa)Elongation at break (%)0%6.37±0.5574.45±0.9472.87±2.6610%7.57±0.29147.87±1.2347.99±2.1720%10.43±0.87184.89±2.7733.95±0.6630%12.77±1.06216.56±3.4530.84±1.8440%15.34±0.47242.38±2.7622.28±2.5850%11.89±0.78358.54±4.8520.77±1.4260%10.65±0.49302.68±3.9718.76±0.95


## 3.1.1. XRD Study

Figure1(b) shows the X-ray diffraction pattern of the synthesized apatite powder. The
d-values correspond to that of calcium hydroxyapatite (Ca10(PO4)6(OH)2)
(JCPDS card no.
74-0566). Taking into account the broadening of each peak in XRD, mean crystallite
size has been calculated using Scherrer’s equation, that is, D=0.9λ/βcosθ,
where D is the average crystallite size in Å, β is
the peak broadening of the diffraction line measured at half of its maximum
intensity in “radian,” λ is the wavelength of X-rays, and θ is the Bragg’s
diffraction angle. The mean crystallite size is found to be 15 nm. The
approximate particle size of HAp powder is found to be 6–10 nm in diameter
by 26–56 nm in length
with needle-like acicular crystals as has been observed from TEM micrograph.
The use of synthesized n-HAp powder with the CSP polymer as a matrix thus
provides an effective means to produce nanocomposites.Figure 1
X-ray diffraction patters of (a) CSP, (b) HAp, and (c) HAp/CSP nanocomposite.Figure1(c) shows the X-ray diffraction pattern of the HAp/CSP composite. The
crystallite size of HAp in composite is found to be 12 nm. The crystallinity
(Xc) of the pure HAp and HAp/CSP composite is determined by an empirical
relation between Xc and β002 (i.e., β002×Xc3=KA) [23], where Xc is the crystallinity degree, β002 is full width of the peak at half intensity of (002) plane in degree −2θ, and KA is a constant (0.24). The Xc for pure HAp powder is 0.15, and in the HAp/CSP
composite, it is found to be 0.11. The crystallinity of the n-HAp particles as
well as CSP polymer has decreased after composite formation as shown in Figure 1
indicating that crystal structures of both HAp and CSP have changed after
composite formation, which may be resulted from the interface binding between
particles and matrix. The XRD peaks, (002) and (211) have shifted to higher 2θ values in case of HAp/CSP composite
as compared to pure HAp (i.e., from 2θ=25.98 to 26.57 and 31.89 to
33.46, resp.), which is possibly due to compression from the contracting
polymeric matrix through interfacial bonding. Simultaneously, the crystalline peak of CSP polymer at 2θ=20.8 has shifted
to 21.6, whereas, another crystalline peak of CSP
at around 2θ=10.5 has disappeared in the composite (Figure 1(c)). The
shift and decrease in crystallinity of each peak of the polymer as well as HAp
after composite formation clearly indicate the presence of bonding between HAp
particles and polymer matrix. The peak at 32.49 [300] of n-HAp is
weakened after composite formation, which also indicates the participation of
n-HAp in bonding with the polymer.


## 3.1.2. FTIR Study

Figure2(b) shows the FTIR spectrum of CSP having characteristic peaks at 2934 and 2850 cm-1 for asymmetric and symmetric stretching of methylene (–CH2–)
groups, respectively. On the other hand, the bands at around 1544 and 1620 cm-1 are for N–H stretching in CSP. The bands at around 1088 and 1047 cm-1 are attributed to the C–O–P stretching and phosphorylated hydroxyl group, whereas,
the characteristic peaks at around 991 and 493 cm-1 are attributed
for P–OH groups in CSP polymer. The bands at around 1100 to 1250 cm-1 are due to P–O, P=O stretching of phosphate group. The bands at 632 and 3571 cm-1 are for structural OH groups in the n-HAp crystals (Figure 2(c)). The bands at
1092, 1030, and 604 cm-1 indicate the presence of PO43− group, and the spectrum also indicates the presence of H2O (3422 and
1641 cm-1) in HAp crystals. The peaks at 1047 and 493 cm-1 for P–OH groups in CSP have disappeared as shown in the Figure 2(d) for HAp/CSP
composite, which may be due to the formation of polyphosphonate salt [14]. The
peak intensity of P–OH at 991 and 1088 cm-1 in CSP polymer has
decreased after composite formation, which evidences that the P–OH groups of
CSP polymer have taken part in bonding with hydroxyapatite. Similar trend is
seen in case of OH (632, 3571 cm-1) groups of HAp crystals, which is
an indication of participation in bonding of HAp with polymer. Most of the
peaks either in polymer or in HAp have shown clear shift after composite
formation (Table 1). Thus, a comparison of the FTIR analysis has indicated that
there is a chemical bonding at the nanoparticle-polymer interface.Table 1
FTIR peak positions of various functional groups before and after composite formation.
Functional groupsIR peak positions (cm-1)CSCSPHAp particlesHAp/CSPPO43−——1092, 1030, 6041079, 1025, 602P–OH—1088, 1047, 991, 493—1070, disappeared, 984, disappearedO–H bending——632, 3571625, 3563O–H stretching3430342734223402N–H3467, 1622, 15443465, 1620, 1544—3462, 1620, 1543Figure 2
FTIR spectra of (a) chitosan, (b) chitosan phosphate, (c) HAp powder, and (d) HAp/CSP nanocomposite.


## 3.1.3. TEM Study

Figure3(a) shows the TEM micrograph of the synthesized n-HAp powder. The micrograph
depicts the acicular needle-like crystals of HAp powder in nanometer range,
having 6–10 nm in diameter
by 26–56 nm in length.
The micrograph shows the presence of agglomerations among the particles, which
are due to high specific surface energy of n-HAp particles resulting in their
aggregations. Nanosized HAp particles with homogeneous dispersion are well
identified in case of HAp/CSP composite (Figure 3(b)). The particle size of HAp
is also controlled by the polymer (CSP) in composite as depicted in the TEM
micrograph.TEM micrographs of (a) HAp powders and (b) HAp/CSP nanocomposite (40% w/w).
(a)(b)


## 3.1.4. SEM Study

Figure4 shows the SEM micrographs of fracture surface of the composite samples with
different contents of n-HAp particles. The micrographs depict that with
increase in amount of HAp particles loading, the surface roughness for the
composites is increased.SEM micrographs of HAp/CSP nanocomposite samples with various contents of n-HAp particles (a) 20% (w/w), (b) 30% (w/w), (c) 40% (w/w), and (d) 50% (w/w).
(a)(b)(c)(d)


## 3.1.5. Mechanical Properties Study

The
tensile strength and modulus of the composites are found to be increased with
increase in amount of HAp contents (Table2). In 30% (w/w) n-HAp content, the
increase in tensile
strength and modulus has been found to be 100.4% and 190.8% than pure CSP polymer,
respectively. In 40% (w/w) HAp loading, the tensile strength and modulus are
increased by 140.8% and 225.5%, respectively. With 50% (w/w) HAp content, the
tensile strength started to decrease, whereas, modulus is increased by 381.5%
over pure CSP polymer. The results are summarized in Table 2. From the results,
it is clear that the mechanical properties have been improved significantly
with high nanoparticle loading capability, which means that the composite tends
to be used as a biomaterial with high osteoconductivity [24]. The enhancement
in tensile strength and modulus in case of HAp/CSP nanocomposite could be
attributed to the excellent bonding at the nanoparticle-polymer matrix
interface through the pendant phosphate groups of CSP.Table 2
Mechanical properties of HAp/CSP nanocomposite samples. Number after  ±  sign corresponds to the standard deviation.
n-HAp particles loaded (w/w)Tensile strength (MPa)Young’s modulus (MPa)Elongation at break (%)0%6.37±0.5574.45±0.9472.87±2.6610%7.57±0.29147.87±1.2347.99±2.1720%10.43±0.87184.89±2.7733.95±0.6630%12.77±1.06216.56±3.4530.84±1.8440%15.34±0.47242.38±2.7622.28±2.5850%11.89±0.78358.54±4.8520.77±1.4260%10.65±0.49302.68±3.9718.76±0.95


## 3.2. Biocompatibility Study

### 3.2.1. Cell Viability Study

A
cell line of murine fibroblast (L929) has been selected for the MTT assay test.
The difference in cell viability index is shown in Figure5 of the composite
samples of different concentrations, as compared to the control tissue culture
plate. The statistical analysis (Student’s t-test) has indicated that
the difference in cell viability index is insignificant. Hence, the developed
composite is cytocompatible.Figure 5
Cell viability test using murine fibroblast cell line L929 on CSP and
HAp/CSP films, CS was taken as control (n=3).


### 3.2.2. In Vitro Osteoblast Differentiation Study

The
neonatal mouse calvarial osteoblasts have been used to study the development of
bone cells on CSP and HAp/CSP nanocomposite film surfaces. The results of cell
proliferation of murine calvarial osteoblasts cultured on CSP and HAp/CSP
nanocomposite films show a cell proliferation during the first 7 days (Figure6).
The proliferation phase follows a growth retardation after 7 days due to high
confluence of cells on the film surfaces. The results show that HAp/CSP
nanocomposite is an appropriate biomaterial for osteoblast proliferation.Figure 6
Osteoblast cell proliferation on CSP and HAp/CSP films, CS was taken as control. Significant increase in cell number (P<.001) was found at day 7 in all the variations followed by retardation of growth phase.


### 3.2.3. Alkaline Phosphatase Enzyme Activity Study

The
differentiation process of the osteoblast cells on CSP and HAp/CSP
nanocomposite has been analyzed by the alkaline phosphatase (AP) enzyme
activity, which is an early osteogenic marker. The highest enzyme activity is
achieved at day 14 in both materials (Figure7).  The AP activity shown by osteoblast cells
growth on HAp/CSP nanocomposite is significantly higher than that on CSP
surface.Figure 7
AP activity of osteoblast cells grown on CSP and HAp/CSP films, CS was taken as control. Both in case of CSP and HAp/CSP enzyme activity increased up to day 14 (P<.001). HAp/CSP nanocomposite is proven a better support for osteoblast differentiation than CSP alone (P<.01).


### 3.2.4. Osteoblasts Morphology and Their Nodule Formation

Further,
SEM examination has been conducted at day 14 to observe the bone formation
process. Osteoblast cultured on the chitosan films reveals a round shape and
stayed segregated. Secreted extracellular matrix or mineralization is not
observed there (Figure8(a)). Whereas, osteoblast cultured on chitosan phosphate
films has shown tight aggregation and some mineralization (Figure 8(b)). However,
osteoblast cultured on HAp/CSP nanocomposite shows a characteristic growing in multiple-layer
patterns and tends to form a nodular cell aggregation (Figure 8(c)). Nodules
contain densely packed cells are embedded within highly mineralized extracellular matrix secreted by them. SEM
and AP enzyme activity studies indicate that incorporation of hydroxyapatite
increases in vitro osteogenicity of chitosan phosphate films.SEM images of osteoblasts grown on different matrices at day 14 on (a) chitosan (CS), (b) chitosan phosphate (CSP), (c) HAp/CSP nanocomposite. Scale bar of each image is 10μm. Arrow “1” shows a nodular cell aggregate, arrow “2” shows the extracellular matrix secreted by the cells in around themselves, and arrow “3” shows a visible mineral nodule.
(a)(b)(c)


## 3.2.1. Cell Viability Study

A
cell line of murine fibroblast (L929) has been selected for the MTT assay test.
The difference in cell viability index is shown in Figure5 of the composite
samples of different concentrations, as compared to the control tissue culture
plate. The statistical analysis (Student’s t-test) has indicated that
the difference in cell viability index is insignificant. Hence, the developed
composite is cytocompatible.Figure 5
Cell viability test using murine fibroblast cell line L929 on CSP and
HAp/CSP films, CS was taken as control (n=3).


## 3.2.2. In Vitro Osteoblast Differentiation Study

The
neonatal mouse calvarial osteoblasts have been used to study the development of
bone cells on CSP and HAp/CSP nanocomposite film surfaces. The results of cell
proliferation of murine calvarial osteoblasts cultured on CSP and HAp/CSP
nanocomposite films show a cell proliferation during the first 7 days (Figure6).
The proliferation phase follows a growth retardation after 7 days due to high
confluence of cells on the film surfaces. The results show that HAp/CSP
nanocomposite is an appropriate biomaterial for osteoblast proliferation.Figure 6
Osteoblast cell proliferation on CSP and HAp/CSP films, CS was taken as control. Significant increase in cell number (P<.001) was found at day 7 in all the variations followed by retardation of growth phase.


## 3.2.3. Alkaline Phosphatase Enzyme Activity Study

The
differentiation process of the osteoblast cells on CSP and HAp/CSP
nanocomposite has been analyzed by the alkaline phosphatase (AP) enzyme
activity, which is an early osteogenic marker. The highest enzyme activity is
achieved at day 14 in both materials (Figure7).  The AP activity shown by osteoblast cells
growth on HAp/CSP nanocomposite is significantly higher than that on CSP
surface.Figure 7
AP activity of osteoblast cells grown on CSP and HAp/CSP films, CS was taken as control. Both in case of CSP and HAp/CSP enzyme activity increased up to day 14 (P<.001). HAp/CSP nanocomposite is proven a better support for osteoblast differentiation than CSP alone (P<.01).


## 3.2.4. Osteoblasts Morphology and Their Nodule Formation

Further,
SEM examination has been conducted at day 14 to observe the bone formation
process. Osteoblast cultured on the chitosan films reveals a round shape and
stayed segregated. Secreted extracellular matrix or mineralization is not
observed there (Figure8(a)). Whereas, osteoblast cultured on chitosan phosphate
films has shown tight aggregation and some mineralization (Figure 8(b)). However,
osteoblast cultured on HAp/CSP nanocomposite shows a characteristic growing in multiple-layer
patterns and tends to form a nodular cell aggregation (Figure 8(c)). Nodules
contain densely packed cells are embedded within highly mineralized extracellular matrix secreted by them. SEM
and AP enzyme activity studies indicate that incorporation of hydroxyapatite
increases in vitro osteogenicity of chitosan phosphate films.SEM images of osteoblasts grown on different matrices at day 14 on (a) chitosan (CS), (b) chitosan phosphate (CSP), (c) HAp/CSP nanocomposite. Scale bar of each image is 10μm. Arrow “1” shows a nodular cell aggregate, arrow “2” shows the extracellular matrix secreted by the cells in around themselves, and arrow “3” shows a visible mineral nodule.
(a)(b)(c)


## 4. Summary and Conclusions

A
novel bioanalogue HAp/CSP nanocomposite with uniform dispersion of n-HAp
particles has been synthesized successfully following a solution-based chemical
methodology with an appreciable improvement in mechanical properties and
minimal surface defects. XRD and FTIR analyses clearly confirm the presence of
interfacial bonding interaction between the filler and matrix. The use of CSP
acts as a coupling/anchoring agent and provides a significant platform for
better dispersion of nanoparticles in the polymer matrix through its pendant
phosphate groups. Cytotoxicity test confirms that the developed composite is
cytocompatible. Primary murine osteoblast cell culture study proves that the
HAp/CSP nanocomposite is osteocompatible and highly osteogenic in vitro. The
uniform dispersion of HAp particles using CSP through its pendant phosphate
groups followed by improvement in mechanical properties of the composite is an
important parameter in order to improve the bioactivity. Therefore, the
developed HAp/CSP nanocomposite may be potentially used in bone tissue
engineering applications.



---

*Source: 512417-2009-01-25.xml*