J Mater Sci

Biomaterials
BIOMATERIALS

Synthesis and characterization of mechanically strong

carboxymethyl cellulose–gelatin–hydroxyapatite
nanocomposite for load-bearing orthopedic application
Chandrani Sarkar1,2 , Pushpa Kumari1 , Kumar Anuvrat3 , Sumant Kumar Sahu2 ,
Jui Chakraborty4 , and Subhadra Garai1,*

1
Advance Material and Processing Division, CSIR-National Metallurgical Laboratory, Jamshedpur 831007, India
2
Department of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad, Jharkhand 826004, India
3
Centre for Nanotechnology, Central University of Jharkhand, Ranchi 835205, India
4
CSIR-Central Glass and Ceramic Research Institute, 196, Raja S.C. Mullick Road, Jadavpur, 700 032 Kolkata, India

Received: 1 June 2017 ABSTRACT

Accepted: 29 August 2017 Novel three-dimensional hybrid polymer–hydroxyapatite nanocomposites have
been developed as load-bearing synthetic bone graft through in situ mineral-
Ó Springer Science+Business ization process, using natural polymers carboxymethyl cellulose (CMC) and
Media, LLC 2017 gelatin (Gel) as matrix. This process is simple and does not involve any chemical
cross-linker. Detailed structural and physicochemical characterization of the
samples disclosed that incorporation of gelatin with CMC assists the formation
of CMC-Gel polymeric network of new conformational structure through non-
covalent interactions (H-bond). The formation of hydroxyapatite (HA) in this
polymeric network was occurred in such a fashion that the HA serves as
bridging molecule which strengthen the polymeric network more and formed a
mechanically strong three-dimensional CMC-Gel-HA nanocomposite. The
synthesized CMC-Gel-HA nanocomposites have compressive strength and
modulus in the range of 40–86 MPa and 0.4–1.2 GPa, respectively, analogous to
human cancellous as well as cortical bone. In vitro cell interaction of the syn-
thesized nanocomposites with osteoblast-like MG-63 cells has been evaluated.
Results showed that synthesized CMC-Gel-HA nanocomposite promote cells for
high alkaline phosphatase activity and extracellular mineralization. Extracellu-
lar mineralization ability of nanocomposite was investigated by alizarin red
staining and von Kossa staining. Biodegradable nature and bone apatite for-
mation ability of CMC-Gel-HA nanocomposite under simulated physiological
environment were investigated by different characterization processes. Results
indicated that the synthesized CMC-Gel-HA nanocomposite has great potential
to be used as regenerative bone graft in major load-bearing region.

Address correspondence to E-mail: [email protected]

DOI 10.1007/s10853-017-1528-1
 J Mater Sci

Introduction in the fabrication of orthopedic biomaterial/scaffolds

[28–30]. Moreover, the combination of cellulose and
Nowadays, the treatment of diseased and damaged gelatin has been utilized by many researchers to
bone by the suitable bone substitute is a great chal- develop mechanically and thermally stable cellulose–
lenge for clinicians. It needs to replace defected tis- gelatin complex/film based on non-covalent interac-
sues with a viable functioning alternative substitute tion [31–33]. The cellulose–gelatin complex is being
[1]. Globally, an autograft is considered as the gold potentially used in drug delivery and tissue engi-
standard for bone regeneration/bone defect repair- neering [34, 35]. Several studies have shown that the
ing. But due to its limitation of accessibility and risk incorporation of hydroxyapatite with polysaccha-
of donor site morbidity, an alternative strategy for rides–gelatin complex provides good mechanical
bone grafting is required in the form of synthetic strength to scaffolds due to excellent mechanical
bone grafts that can augment bone regeneration [2–6]. properties of nanosized hydroxyapatite [36–39]. Fur-
Therefore, researchers focused on the development of thermore, the presence of polysaccharides such as
polymer–hydroxyapatite nanocomposites as bone chitosan [40] and alginates [41] induce the orderly
graft materials/scaffolds [7–9]. In most of the articles, deposition of HA crystals in gelatin–polysaccharide
in situ mineralization process is adopted for the system with controlled morphology as same as in
synthesis of polymer–hydroxyapatite nanocompos- natural bone.
ites. It is a bioinspired process in which mineraliza- In our previous report, we have synthesized bio-
tion occurs in polymer matrix, similar to the mimetic carboxymethyl cellulose–HA nanocomposite
biomineralization of hydroxyapatite (HA) in associ- for load-bearing application [42]. In this study, we
ation with extracellular matrix (ECM) of natural bone have aimed to develop self-assembled hybrid poly-
[10, 11]. However, the poor mechanical strength of mers–HA nanocomposite with enhanced mechanical
polymer–HA composite restricts its application as strength and bioactivity by mimicking phenomenal
non-load-bearing powder material. To circumvent science of nature. In nature, sophisticated functional
this problem, extensive research is focused to match materials are developed through hierarchical self-
the structure–mechanical properties of polymer–HA assembly of molecules into structurally stable ar-
composites with natural bone [12–15]. rangements by the driving force of non-covalent
Natural bone is an anisotropic complex of collagen interactions, including hydrogen bonds, ionic bonds
(COL) and hydroxyapatite. From last decades, and electrostatic interaction [43–45]. Pei et al. [46]
researchers have developed collagen–HA nanocom- demonstrated an efficient strategy to fabricate cellu-
posites in order to mimic the structure and compo- lose/gelatin sponge for wound dressing based on
sition of natural bone [16–18]. But the application of intermolecular H-bonding between cellulose and
type-1 COL in bone tissue engineering field is limited gelatin. Therefore, we blended gelatin with car-
due to its high price and poor availability. Hence, boxymethyl cellulose to form a self-assembled CMC-
gelatin has been recognized as an alternative option Gel matrix. The present study also demonstrated the
by many researchers [19–23]. Gelatin is a denatured in situ mineralization of hydroxyapatite on
form of collagen having Arg-Gly-Asp (RGD) stable CMC-Gel matrix by electrostatic interaction
sequence and exhibits good biocompatibility, low and formed a mechanically strong three-dimensional
immunogenicity and low cost. Further, the associa- CMC-Gel-HA nanocomposite. The structural and
tion of gelatin with polysaccharides forms a complex mechanical properties of synthesized nanocompos-
similar to extracellular matrix (ECM) and facilitates ites were thoroughly characterized. In vitro biocom-
the attachment, growth and proliferation of human patibility, cell attachment and cell differentiation
osteoblasts cells, making it most suitable complex for study with osteoblast-like MG-63 cell were carried
tissue engineering [24–27]. Generally, this type of out on the ternary nanocomposites. We have also
association in polysaccharides and gelatin was fab- studied the in vitro assessment of apatite formation
ricated by using chemical cross-linker which may and degradation under simulated physiological con-
cause safety problems in body. ditions. The synthesized CMC-Gel-HA nanocom-
Among other polysaccharides, cellulose is an posite has few advantageous properties compared
abundant and cheapest viscous polymer, widely used with other reported hybrid polymer–HA
J Mater Sci

nanocomposites [36–38, 47–52]: (a) three-dimensional was slowly added to polymer solution with continu-
nanocomposite has been developed by self-assembly ous stirring. The pH was maintained at 10–11 by
of CMC, gelatin and HA, without using any cross- adding aqueous ammonia solution and kept for 24 h of
linker; (b) synthesized nanocomposite has high aging at 30 °C. Thirty-seven grams of diammonium
mechanical strength being in the range of human hydrogen phosphate was dissolved in 500 mL DI
cancellous as well as cortical bone; and (c) CMC-Gel- water and made alkaline with ammonia solution. The
HA nanocomposite promotes proliferation and dif- prepared diammonium hydrogen phosphate (0.56 M)
ferentiation of MG-63 cells. solution was slowly added to calcium–polymer solu-
tion. Milky white coloration was observed instanta-
neously, and the total volume of the slurry was made
Experimental procedures up to 3500 mL by adding DI water, allowed to age for a
week at 30 °C. After aging, the slurry was washed with
Materials DI water to neutralize. Neutralized slurry was trans-
ferred to Teflon beaker and dried in an oven at 50 °C
All the chemicals like analytical grade diammonium for 72 h to obtain the three-dimensional nanocom-
hydrogen phosphate [(NH4)2HPO4], calcium nitrate posite (50 g). For comparative study, three-dimen-
tetra-hydrate [Ca (NO3)2 4H2O] (with 98% assay), sional CMC-HA nanocomposite was also prepared
liquid ammonia (with 25% assay), gelatin (pH and labeled as HA-0 [42].
3.8–7.6) and sodium salt of carboxymethyl cellulose
(CMC) (degree of substitution 0.9) were procured Characterization
from Merck, Mumbai, India. Deionized (DI) water
was used in all the experiment. The infrared (IR) spectra of CMC, Gel and
nanocomposites were acquired by using Fourier
Synthesis of three-dimensional CMC-Gel- transform infrared (FTIR) [JASCO-FTIR, Model 410]
HA nanocomposites spectrometer. IR spectrum of the mixture of poly-
mers was also taken to observe the effect of
In this study, CMC-Gel-HA nanocomposites were
hybridization. Gelatin and CMC were dissolved in
synthesized in three different compositions with the
DI water, kept for 24 h and then dried. Solid-state
variation of CMC and gelatin content (Table 1). These
carbon-13 nuclear magnetic resonance (13C-NMR)
nanocomposites were labeled as HA-5, HA-10 and
measurement of CMC, Gel, CMC-HA and CMC-
HA-15, respectively. For the synthesis of each com-
Gel-HA nanocomposite was carried out by using
posite, desired amount of CMC was dissolved in
ECX400-Jeol 400 MHz high-resolution multinuclear
different vessels containing 1200 mL of DI water with
Fourier transform nuclear magnetic resonance (FT-
gentle heating and continuous stirring by using
NMR) spectrometer. The mineral phase and its
magnetic stirrer for 5 h. Subsequently, desired
crystallite size in synthesized nanocomposites were
amount of gelatin was also dissolved in different
identified by using X-ray diffractometer (XRD)
vessels containing 500 mL of DI water and stirred for
(Bruker, D8 Discover CuK radiation with
1 h at room temperature. Then, it is slowly added to
0.154 nm, 40 kV, 40 mA). For each nanocom-
CMC solution and stirred well for half an hour. One
posite, data were collected over the 2 range 20–80°
hundred and eighteen grams of calcium nitrate tetra-
with a scan speed 0.4 s per step. For microstructural
hydrate was dissolved in 500 mL of deionized water analysis, the synthesized nanocomposites were
(DI). The prepared calcium nitrate (0.99 M) solution crushed and powdered. These powder samples
were dispersed in ethanol by ultra-probe sonication
Weight (%) of CMC and gelatin for 20 min and dropped on carbon-coated Cu-grid.
Samples CMC (wt%) Gelatin (wt%) After drying the grids under IR lamp, the
microstructural features of nanocomposites were
HA-0 30 0 captured by using transmission electron microscopy
HA-5 25 5 (TEM cm200, CX Philips JEOL 2100) at an acceler-
HA-10 20 10 ating voltage of 120 kV. Thermogravimetric analysis
HA-15 15 15
(TGA) was carried out in thermogravimetric
 J Mater Sci

analyzer (Netzsch STA 449 C), over 25–1200 °C [0.2 mg mL 1 in DMEM containing 10% phosphate
temperature range at a heating rate of 10 °C min 1. buffer solution (PBS)] solution was added into each
The surface morphology of the as-synthesized well and incubated in 5% CO2 incubator at 37 °C for
nanocomposites was observed by field-emission 3 h. Then, MTT solution was removed and dimethyl
scanning electron microscope (FESEM Leo s4302 sulphoxide (DMSO) was added to dissolve the for-
UK) for which samples were coated with silver. The mazan crystals. Colored solution was transferred to
porosity percentage of nanocomposites was deter- 96-well plate, and the absorbance of developed color
mined by conventional liquid displacement method was recorded at 595 nm by using ELISA plate reader
[38, 42]. The ethanol was used as a displacing liquid (i-Mark, Bio-Rad, USA).
because it penetrates the pores easily. The pore size For cell adhesion study, sterilized nanocomposites
distribution in each composite was determined by of size 2 mm 2 mm 0.2 mm were placed in
mercury intrusion porosimetry (Model PASCAL 6-well plate. After that, MG-63 cell suspension at a
140/440). The compressive strength and elastic density of 6 104 cells mL 1 was dropped onto the
modulus of the nanocomposite of dimension samples of well plate and incubated for 1, 4 and
(16 mm 8 mm 8 mm) were determined using a 7 days, respectively. Before proceeding to fixation
universal testing machine (UTM, QMAT 3.75, and procedure, images of cells in the presence of
ASTM D695). To check the reproducibility, at nanocomposites were captured by using inverted
least three specimens were tested for each phase contrast microscope (MOTIC AE31). Then,
nanocomposite. samples were rinsed three times with PBS and fixed
with 4% paraformaldehyde for 10 min. The fixative
In vitro biocompatibility and cell adhesion was removed, and samples were washed with PBS
studies and dehydrated with series of graded ethanol (30, 50,
75, 90, 95 and 100%). The morphological character-
Osteoblast-like MG-63 cells were used for in vitro cell istic of adhered cells on the surface of nanocompos-
study. MG-63 cells were derived from osteosarcoma, ites was observed using scanning electron
though they retained important osteogenic cell dif- microscope (SEM).
ferentiation markers, such as the activity of alkaline
phosphatase and osteocalcin production; thus, these In vitro study of cell differentiation
cells are considered as good in vitro model for testing
the interaction of materials with bone cells [53, 54]. Osteoblast-like MG-63 cells at a density of 6 104
Here, cells were grown to subconfluency in Dul- cells per well were seeded into 6-well plate. After
becco’s modified Eagle medium (DMEM) supple- 24 h, the sterilized nanocomposites of size
mented with 10% fetal bovine serum (FBS), 2 mm 2 mm 0.2 mm were carefully placed over
2 mg mL 1 NaHCO3, 1 g mL 1 penicillin G, the cells and maintained in standard culture condi-
1 mg mL 1 streptomycin, in a humidified incubator tion (DMEM) for 7 and 14 days according to pub-
at 37 °C with 5% CO2 (HF90 Heal Force, China). lished method [23, 55]. Cells cultured in osteogenic
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium media (DMEM with osteogenic supplements) and
bromide (MTT) assay of the samples were performed normal media (DMEM) were set as positive and
by indirect method as per the guidelines of ISO negative control, respectively. At the end of desired
10993-5. The small pieces of nanocomposites of period, alkaline phosphatase (ALP) activity of MG-63
dimension 2 mm 2 mm 0.2 mm were steam cells was estimated by using alkaline phosphatase
sterilized for 20 min, incubated in 1 mL of DMEM for activity estimation kit of HiMedia (Product Code—
24 h in humidified incubator at 37 °C with 5% CO2 CCK035) according to manufacturer’s instruction.
and filtered with 0.22- m membrane filter. Extracts The rate of increased absorbance was measured at
were added on pre-seeded (6 104 cells per well) 405 nm for determining the ALP activity of cell.
cells in 6-well plates and kept under humidified Finally, ALP activity was normalized with total pro-
incubator at 37 °C with 5% CO2 for 1, 4 and 7 days. tein content of cell lysates which was determined by
Media and samples extract of well plate were using protein assay kit of HiMedia (Bradford assay,
replenished with fresh ones on every second day. Product Code—HTBC005). Results were representa-
After incubation for specified period, MTT tive of three different experiments.
J Mater Sci

Extracellular mineralization ability of samples Statistical analysis

was investigated by alizarin red staining and von
Kossa staining. As stated above, 6 104 cells per Each experiment was carried out in triplicate, and
well were seeded in 6-well plate and similar culture measured data were expressed as mean standard
condition was maintained for 7, 14 and 21 days. deviation. The data were statistically analyzed by
After culturing for desired period, samples were using GraphPad Prism with one-way ANOVA; the
removed from well plate. After that, cells of the well value of 0.05 is considered statistically significant
plate were fixed with 4% paraformaldehyde for (Scheme 1).
15 min and washed with biological water. Finally,
1 mL of 2% alizarin solution was added into each
well and incubated for 30 min. Excess dye was Results and discussion
removed by washing with biological water. Red- 13
stained images of mineralized nodules were cap- FTIR and C-NMR analysis
tured by using inverted phase contrast microscope
The formation of three-dimensional CMC-Gel-HA
(MOTIC AE31). For von Kossa staining, same pro-
nanocomposite with the effective association of CMC,
cedure was followed except 1 mL of 1% silver
Gel and HA was established by FTIR analysis. The
nitrate solution was added into each well after cell
characteristics FTIR spectra of CMC, Gel and CMC-
fixation with 4% paraformaldehyde and kept under
Gel mixture are shown in Fig. 1a. In CMC-Gel spec-
UV light for 30 min. Residual silver nitrate was
trum, we found that the OH– band of CMC and NH–
removed by washing with biological water. Brown-
of Gel were overlapped and appeared at 3430 cm 1
ish–blackish images of mineralized nodules were
due to H-bonding between CMC and gelatin
captured by using inverted phase contrast micro-
[31, 32, 46]. Noticeably, the COO (sym. str.) band at
scope (MOTIC AE31).
1429 cm 1 of CMC in the presence of gelatin was
shifted toward lower wave number at 1384 cm 1
In vitro study in simulated body fluid (SBF)
confirming the association of CMC with gelatin
The assessment of apatite formation and biodegrad- through intermolecular H-bonding [46]. Due to these
ability of samples was evaluated by SBF treatment H-bonding, gelatin and CMC macromolecules form a
[9, 50, 55]. SBF was prepared by using graded NaCl, stable polymer network of new conformational
NaHCO3, KCl, K2HPO4 3H2O, MgCl2 6H2O, CaCl2- structure for in situ mineralization of HA.
2H2O, Na2SO4, Tris (hydroxyl methyl) methyl amine, According to biomineralization process, early
1 M HCl proposed by Kokubo [56]. Prepared SBF had mineralization of HA was initiated at the anionic sites
ion concentrations (Na 142.0, K 5.0, Mg2 1.5, Ca2 of polymer by electrostatic interaction between Ca2
2.5, Cl 147.8, HCO3 4.2, HPO42 1.0, SO42 0.5 mM) ions and COO groups, depicted by many research-
and pH (7.4) nearly equal to human body fluid. ers [42, 50–52]. Then, phosphate ions react with cal-
Samples of dimension 16 mm 8 mm 8 mm were cium ions to form HA molecule, depending on the
immersed in 30 ml SBF at 37 °C for a maximum pH (10–11) of the reaction mixture. In the spectra of
period of 1 month. Samples were periodically (1, 2, 3, nanocomposites (Fig. 1b), we found that the charac-
4 weeks) withdrawn from SBF solution, rinsed gently teristics phosphate bands of HA at 1034, 603 and
with DI water and soaked in blotting paper for 564 cm 1 correspond to P–OH, O–P–O and P–O–P
removing excess water. Biodegradability of samples vibrations, respectively, confirming the formation of
was evaluated from weight loss (%) calculated by HA in hybrid polymers matrix [40, 57]. In the com-
following formula, weight loss (%) [( 0 )/ posite formation stage, the pH of the reaction mixture
] 100, where is the weight of sample before is higher than the isoelectric point of gelatin where
0 0
SBF treatment, while is the weight of sample after COOH groups of gelatin exist as COO ions and
SBF treatment at time ( ). FTIR spectra of SBF-dipped electrostatically bind with Ca2 ions of HA [40, 58]. In
samples were also acquired after incubation for Fig. 1b, we observed that the band which corre-
desired period (7, 14, 21 and 28 days). Apatite sponds to COO group of polymers was shifted at
deposition on the surface of nanocomposites after 1639 cm 1, suggesting electrostatic interaction
immersion in SBF was confirmed by FESEM. between Ca2 ions of HA and COO groups of
 J Mater Sci

Schematic presentation of synthetic procedure of mechanically strong three-dimensional CMC-Gel-HA nanocomposite.

CMC/gelatin. Such interactions might be considered gelatin in HA-15 with change of chemical environ-
as an evidence for involvement of Ca2 in the for- ment. This change attributed to the interactions
mation of bridging structure with CMC and gelatin between polymers and HA nanoparticles. One inter-
[52, 59]. The band at 1384 cm 1 of CMC-Gel matrix esting observation is that peaks of all carbon atoms in
was not shifted in the spectra of CMC-Gel-HA HA-15 shifted downfield more than that in HA-0.
nanocomposites, indicating that the formation of HA These results confirm the strong molecular interac-
at bridging position did not affect the intermolecular tions occurred among CMC, gelatin and HA in HA-
linking of CMC-Gel matrix rather strengthened the 15 nanocomposite.
polymeric network. Evidently, the sharpness of this
band increases with gelatin concentration. It may be XRD and TEM analysis
due to involvement of more –NH groups (amide II
and amide III) in cross-linking [60] that can be further XRD patterns of synthesized four nanocomposites are
confirmed with the observance of an additional band shown in Fig. 2a. All the patterns showed character-
at 3200–3160 cm 1, indicating the enhanced inter- istics peaks of hydroxyapatite indexed as (002), (102),
molecular H-bonding with the participation of more (211), (202), (310), (222), (004), (213), (511) as JCPDS
number of –NH group of gelatin [60]. These results file (Card No.—09-0432). The crystallite size D (nm)
confirmed that the interaction/cross-linking among of HA was calculated from the most prominent peak
CMC, gelatin and HA in HA-15, HA-10 composite is in XRD by following Debye–Scherer formula
stronger than HA-0, HA-5. / 1/2cos , where is Scherer constant (0.9),
The solid-state 13C-NMR of CMC, gelatin, CMC- wavelength (in angstrom) and diffraction angle
HA and CMC-Gel-HA nanocomposites is shown in [61]. 1/2 is the full width of the diffraction peak at
Fig. S1. In CMC pattern, the significant peaks at 182, half the maximum intensity. It is observed that the
108, 86, 78 and 65 ppm correspond to COO , C1, C2, crystallite size of nanocomposites (inset Fig. 2a)
C3 and C6 of CMC [42, 46]. The peaks of gelatin at decreases from 30 to 10 nm with gelatin content,
172.80, 69.80, 58.63, 41.85, 28.77 and 24.38 ppm were which is of the order of crystallite size found in nat-
assigned to COO , Hyp , Pro- , Gly , Pro- and ural bone [62]. There may be two factors responsible
Pro- , in accordance with earlier report [46]. Com- for modulating the crystallite size of HA in CMC-Gel-
paring the spectra of HA-15 with polymers and HA- HA nanocomposites: (1) the intermolecular interac-
0, it can be observed that the peaks of CMC and tion of organic molecules with the surface of
gelatin appeared in the spectrum of HA-15 with hydroxyapatite suppresses the crystal growth [63]
downfield shifts confirming the presence of CMC and and (2) the space/gap available for growth of bridged
J Mater Sci

similar to natural bone where HA crystals grow in

parallel with the collagenous fibers along the (002)
c-axis [62]. Moreover, the diffused rings of (004), (211)
and (002) planes of HA were found in SAED pattern,
among which the brightest one represents (211) plane
which is consistent with XRD spectra. These results
emphasized the role of self-assembled CMC-Gel
matrix on the morphology of HA crystals as well as
its oriental arrangement. In FTIR, we have already
illustrated the formation of new conformational
CMC-Gel matrix through H-bonding. We also
assumed that the nucleation of HA has been occurred
in the bridging position of CMC and gelatin. Initially,
the coordination of Ca2 ion with polymeric network
stabled the stereochemical arrangement more and
HA is grown into the space provided by this
arrangement. So, the stereochemical geometry in
CMC-gelatin-Ca complexes is supposed to control
the mineralization process. The hydroxyapatites
mineralized at these sites gradually grow along the
extended direction of these macromolecules to the
critical size of nucleation and eventually to form an
elongated HA particles [41] where HA crystals are
formed with the development of preferred orienta-
tion [(002) plane]. These results revealed that the
assembled CMC-gelatin matrix modulates the mor-
phology of HA particles from globular (in CMC-HA)
to needlelike in CMC-Gel-HA nanocomposite and
also controlled the size.

a FTIR spectra of CMC, Gel and CMC-Gel mixture.

Thermogravimetric analysis
b FTIR spectra of four synthesized nanocomposites.
The TGA thermograms of synthesized nanocompos-
HA crystal is reduced due to enhanced cross-linking ites are presented in Fig. S2. There are three stages of
between CMC and gelatin in CMC-Gel-HA weight loss in the nanocomposites: First stage is in
nanocomposite [40]. the temperature range of 50–200 °C assigned the loss
Bright-field TEM images of CMC-HA nanocom- of adsorbed water molecule. The second stage at
posite [HA-0] and CMC-Gel-HA nanocomposite about 200–600 °C corresponds to the thermal
[HA-15] are shown in Fig. 2b, c. In the image of decomposition of the polymeric CMC-Gel matrix
CMC-HA nanocomposite, we found that the particles [31], and the third stage (600–1000 °C) attributes the
are 30 nm in size with globular shape, whereas the dehydroxylation of HA [64]. The weight loss due to
HA particles in CMC-Gel-HA nanocomposite decomposition of organic components (CMC and
(Fig. 2c) are smaller in size ( 20 nm) with elongated Gel) in HA-0, HA-5, HA-10 and HA-15 was 26, 27, 27
morphology. The selected area electron diffraction and 29%, respectively, which is slightly less than the
(SAED) pattern of CMC-Gel-HA nanocomposite actual materials taken in synthesis process that indi-
(Fig. 2d) shows slightly crescent-shaped diffraction cates the loss of unbound polymers during washing
ascribed to (002) plane of HA which indicates that the step. The remaining mass in HA-0, HA-5, HA-10 and
distribution of HA particles in the composite is HA-15 was 62, 62, 60 and 58%, respectively, which
slightly oriented with respect to that plane [19], represents the thermally stable HA content.
 J Mater Sci

a XRD of as-synthesized nanocomposites [plot of crystallite size is in ]. b TEM image of CMC-HA nanocomposite. c TEM
image of CMC-Gel-HA nanocomposite. d SEAD pattern of CMC-Gel-HA nanocomposite.

Surface morphology of synthesized such a way that the optimal packing efficiency was
nanocomposites achieved in the composites [65].
Pore size distribution of nanocomposites inset in
The microstructure of all the four nanocomposites is their corresponding FESEM images (Fig. 3). HA-0,
shown in Fig. 3. It is observed that the HA HA-5, HA-10 and HA-15 nanocomposites exhibited
nanoparticles aggregated with the formation of a bimodal distribution of pore size in the broad range
bead-like structure and embedded in a polymeric of 0.01–100 m with a corresponding total porosity
template. Bead-like morphology is orderly knitted 55, 47, 36 and 15%, respectively. However, CMC-
over polymeric network with uniform distribution of Gel-HA nanocomposites (HA-5, HA-10 and HA-15)
irregular shaped interconnected pores. From these possess major pores in the range of 0.01–0.1 m,
FESEM micrographs, we noticed that the size of while in CMC-HA nanocomposite (HA-0) pores in
agglomerates was increased with gelatin content with the range of 0.01–100 m. Result showed that the
the formation of a nearly compact structure in HA-15. overall porosity (%) and pore volume for macropores
It indicates that the molecular interaction causes the was decreased with gelatin content which is in good
accretion of nanoparticles and organized them in agreement with corresponding FESEM images of
J Mater Sci

FESEM images of a HA-0, b HA-5, c HA-10, d HA-15, and pattern of pore size distribution of nanocomposites is in of
their respective images.

nanocomposites where compactness increased with nanocomposites are evaluated and represented in
gelatin content. As we explained that the cross-link- Fig. 4. It is observed that the CMC-Gel-HA
ing was increased with gelatin concentration, this nanocomposites have strength and modulus in the
cross-linking offers more intermolecular association range of 40–85 MPa and 0.4–1.2 GPa, respectively,
forming more number of junction points; as a result, analogous to human cancellous and cortical bone
the size of pores was reduced [33]. Thus, the [66]. The mechanical strength of the CMC-Gel-HA
nanocomposite (HA-15) with highest cross-linking nanocomposites was improved mainly by the strong
appeared to be more compact with the smallest pore interaction between CMC-HA, gelatin–HA and
size. CMC-gelatin [42, 46]. All these interactions facilitated
cross-linking among polymers and HA, which is
Mechanical characterization of synthesized responsible for the formation of mechanically strong
nanocomposites bone graft. It also showed that the strength and
modulus of HA-15 were drastically enhanced, almost
The bone graft to be used in load-bearing application tenfold of HA-0 composite. It depicts that the high
must have optimum strength and modulus to match intermolecular interactions driven the compact
with natural bone. To check the potentiality of syn- microstructure of nanocomposite has additional
thesized nanocomposites in load-bearing application, contribution in compressive strength and elastic
the compressive strength and elastic modulus of modulus. The compressive stress–strain curves of
 J Mater Sci

a Compressive strength and b elastic modulus of nanocomposites; c stress versus strain curves of nanocomposites.

nanocomposites are given in Fig. 4c. The variation in In vitro biocompatibility and proliferation
curve nature was observed, and HA-0 (without study
gelatin) deformed permanently after yield point due
to comparatively weak interaction in CMC-HA and The in vitro biocompatibility and proliferation of
highly porous nature of nanocomposite. But, HA-5 MG-63 cells were studied in the presence of
composite showed viscoelastic deformation because nanocomposites extracts using MTT assay (Fig. 5a).
the molecular interactions have been improved after In MTT assay, the absorbance due to metabolic
incorporation of gelatin. The plastic region of stress– activity of mitochondrial dehydrogenase of live cells
strain curves of HA-5 reflects the porous nature of was measured. The absorbance produced in HA-0,
composites, whereas compact HA-10 and HA-15 HA-5, HA-10 and HA-15 extracts showed the non-
nanocomposites exhibited higher strength with lot of toxic nature of nanocomposites, and they are statis-
elastic strain energy indicating that the support- tically significant to the control, which corresponds to
able load per unit surface area was maximum, with a each time period (1, 4 and 7 days). Furthermore, the
low strain index. This observance manifested a rule cells proliferated to higher extent as the culture per-
of direct proportionality of stress–strain behavior of iod is increased from 1 to 7 days in control as well as
nanocomposites as a function of microstructure. in samples extracts.
J Mater Sci

a MTT assay represented metabolic activity of MG-63 photographs of MG-63 cells cultured on HA-15 for 1, 4 and 7 days
cells after cultured with nanocomposites extract (HA-0, HA-5, ( in SEM 30 m, in phase contrast magnifica-
HA-10 and HA-15) for 1, 4 and 7 days ( represent the tion 10 , denotes cell and denotes sample).
standard deviation, 3). b SEM micrograph and phase contrast

Cellular behavior of MG-63 cells in close contact probably due to the large numbers of cell-binding
with CMC-Gel-HA nanocomposite was evaluated peptides is available in CMC-Gel-HA nanocompos-
using SEM and phase contrast microscopy (Fig. 5b) ite. It promotes better cell–material interaction
after 1, 4 and 7 days of culture. In these images, MG- inducing more cellular activity [68, 69]. Thus, the cell
63 cells attached and spread well with cytoplasmic density onto the samples was increased with incu-
extension onto the surface of nanocomposite indi- bation time from 1 to 7 days. The SEM images of cell
cating that the environment of nanocomposite was seeded nanocomposites are consistent with corre-
well suited for cell growth and proliferation [67]. This sponding phase contrast images of surrounding cells.
 J Mater Sci

Evaluation of osteogenic differentiation HA) nanocomposite for this study. We measured

the ALP activity of MG-63 cells (Fig. 6a) cultured
Alkaline phosphatase (ALP) is an early-stage with nanocomposites [HA-0 and HA-15] for 7 and
osteogenic differentiation marker secreted by 14 days and compared with controls. Results
osteoblasts. For evaluating the osteogenic proper- showed that the ALP activity of cells at day 14 was
ties of load-bearing CMC-Gel-HA nanocomposite remarkably increased in positive control and CMC-
and the role of gelatin on the osteogenesis of Gel-HA nanocomposite. But negative control and
nanocomposite, we have chosen the strongest HA- CMC-HA nanocomposite showed less production
15 (CMC-Gel-HA) nanocomposite and HA-0 (CMC- of alkaline phosphatase. The positive effect of

a Quantitative alkaline phosphatase (ALP) activity of in vitro culture with osteogenic media, basal media, CMC-HA
MG-63 cells cultured with osteogenic media, basal media, CMC- nanocomposite (HA-0) and CMC-Gel-HA nanocomposite (HA-
HA nanocomposite (HA-0) and CMC-Gel-HA nanocomposite 15) for 7, 14 and 21 days by, b alizarin red staining and c von
(HA-15) for 7 and 14 days ( represent the standard Kossa staining.
deviation, 3). Matrix mineralization of MG-63 cells after
J Mater Sci

CMC-Gel-HA nanocomposite on cells for high ALP Biodegradation and bone apatite formation
activity was probably due to the presence of gelatin, in SBF
an excellent contributor of osteogensis [68, 69]. The
mineralization of extracellular matrix (ECM) with Degradation of CMC-Gel-HA nanocomposite in SBF
calcium phosphate is a late-stage marker for is shown in Fig. 7a. A continuous loss in weight is
osteogenic differentiation. Alizarin red staining and observed with time, and slight deflection is noticed in
von Kossa staining are commonly used for detect- 28th day where weight of nanocomposite is not fur-
ing calcium phosphate present in the deposited ther reduced. In FTIR spectra of SBF-dipped CMC-
mineral with red and deep brown coloration, Gel-HA nanocomposite (Fig. 7b), we found that the
respectively [23, 55]. Calcium phosphate deposi- band at 3200–3160 cm 1 and 1384 cm 1 of synthe-
tions of MG-63 cells cultured with HA-0 and HA-15 sized nanocomposite (Fig. 1b) was disappeared in
for 7, 14 and 21 days are presented in Fig. 6b, c. At SBF-dipped samples indicating the breakage of cross-
day 21, the remarkable calcium deposition was linking between polymers. Whether few small bands
observed in HA-15 as well as in the positive control appeared at 1550–1300 cm 1 emphasized the break-
(Fig. 6b, c). Negative control does not show calcium age of cross-linking after incubation in SBF and gave
deposition till the day 21, and CMC-HA nanocom- individual polymeric bands. The surface micro-
posite shows very negligible mineralization on graphs of nanocomposite after immersion in SBF for
extracellular matrix. This result reliable with high 7, 14, 21 and 28 days are shown in Fig. 7c. Initially,
ALP activity of cells in positive control and CMC- the compact microstructure of nanocomposite started
Gel-HA nanocomposite promotes more mineral- to break with the deposition of bone apatite on the
ization [70]. surface of nanocomposite. With immersion time, the

a Representative plot of weight remaining (%) versus incubation times. c Representative SEM images of SBF-treated
time of CMC-Gel-HA nanocomposite immersed in SBF. b FTIR CMC-Gel-HA nanocomposite in different incubation periods.
spectra of SBF-treated CMC-Gel-HA nanocomposite in different