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BIOMAC 5092 111 ARTICLE IN PRESS

International Journal of Biological Macromolecules xxx (2015) xxxxxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

1 Nano-hydroxyapatite/chitosanstarch nanocomposite as a novel bone

2 construct: Synthesis and in vitro studies
3 Q1 Mohammad Shakir a, , Reshma Jolly a , Mohd Shoeb Khan a , Noor e Iram a , Haris M. Khan b
a
4 Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
b
5 Department of Microbiology, J.N. Medical College, A.M.U., Aligarh, India
6

7
21 a r t i c l e i n f o a b s t r a c t
8
9 Article history: A novel ternary nanocomposite system incorporating hydroxyapatite, chitosan and starch (n-HA/CSST)
10 Received 5 December 2014 has been synthesized by co-precipitation method at room temperature, addressing the issues of biocom-
11 Received in revised form 29 April 2015 patibility, mechanical strength and cytotoxicity required for bone tissue engineering. The interactions,
12 Accepted 3 May 2015
crystallite size, surface morphology and thermal stability against n-HA/CS nanocomposite have been
13 Available online xxx
obtained by comparing the results of FTIR, SEM, TEM, DLS, XRD and TGA/DTA. A comparative study of
14
bioactivity and thermal stability of n-HA/CS and n-HA/CSST nanocomposites revealed that the incorpo-
15 Keywords:
ration of starch as templating agent enhanced these properties in n-HA/CSST nanocomposite. A lower
16 Starch
17 Chitosan swelling rate of n-HA/CSST relative to n-HA/CS indicates a higher mechanical strength supportive of
18 Hydroxyapatite bone tissue ingrowths. The MTT assay on murine broblast L929 and human osteoblasts-like MG-63
19 In vitro bioactivity cells and in vitro bioactivity of n-HA/CSST matrix referred superior non-toxic nature of n-HA/CSST
20 Biomaterial nanocomposite and greater possibility of osteointegration in vivo respectively. Furthermore n-HA/CSST
exhibited improved antibacterial property against both Gram-positive and Gram-negative bacteria rela-
tive to n-HA/CS.
2015 Published by Elsevier B.V.

22 1. Introduction Bone is the most typical triphasic calcied tissue in mam- 41

Q2 mals that consist of cellular components, hydrated extracellular 42

23 In continuation to our previous work on polymer nanocompos- organic matrix and extracellular inorganic phase which is chiey 43

24 ites [1,2], an attempt has been made to synthesize a biomaterial a major reservoir for calcium and phosphate ions needed for 44

25 signicant in the eld of bone tissue engineering employing renew- diverse metabolic activities. It has a wide spectrum of mechani- 45

26 able, bioactive and naturally abundant polymers [3]. The bone cal properties, depending on its type, humidity, density, porosity, 46

27 repair is a prevalent and challenging clinical issue in orthopedic mineral content and interfacial bonding between constituents. 47

28 surgery. In the fast changing dynamics of the world order, a large Hydroxyapatite (HA) [Ca10 (PO4 )6 (OH)2 ], is the fundamental inor- 48

29 number of people being aficted with bone defects these days. ganic component of bone and is a biologically active calcium 49

30 The autogenic and allogenic procedures are commonly in practice phosphate ceramic that is employed in surgery to replace and 50

31 to deal with bone defects. It has been observed that autogenic mimic bone. The shape of HA crystals in a natural bone is needle 51

32 bones reduce the risk of immune rejection though involve multiple like or rod-like with length and width of 4060 nm and 1020 nm, 52

33 surgeries and associated with donor site morbidity while allogenic respectively [69]. Several reports appeared regarding the synthe- 53

34 bones bear risk of infections and immune feedbacks. Therefore it sis of nanometer size HA (n-HA) having various shapes viz. the 54

35 has been realized to design and develop the materials that can serve needle like HA has been synthesized by organic gel system and 55

36 as efcacious bone grafts substitutes and as articial prosthesis to homogeneous precipitation while rod like HA has been synthe- 56

37 address the patient requirements [4,5]. In view of the serious limi- sized by precipitating calcium nitrate tetrahydrate and ammonium 57

38 tation in traditional therapies, tissue engineering provides a novel dibasic phosphate in the presence of polyacrylic acid followed 58

39 platform in bone reconstruction incorporating therapies that mimic by hydrothermal treatment [10]. The HA thus formed displayed 59

40 the critical aspects of natural biological processes. bone-bonding properties, has been extensively used in hard tissue 60

replacement in view of their biocompatibility and osteoconductiv- 61

ity features, but the brittleness of HA materials limits its application 62

Corresponding author. Tel.: +91 9837430035. in bone tissue engineering. Therefore an intense and immedi- 63

E-mail address: shakir078@yahoo.co.in (M. Shakir). ate development of nanocomposite materials with controllable 64

http://dx.doi.org/10.1016/j.ijbiomac.2015.05.009
0141-8130/ 2015 Published by Elsevier B.V.

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65 bioactivity and satisfactory mechanical properties for bone tissue Table 1

Resemblance of ion concentration of SBF with human blood plasma.
66 engineering is demanded making this eld a fertile research area
67 and attempts have been made to overcome this problem by com- Concentration (mM)
68 bining HA with different polymeric additives such as poly(vinyl
Na+ K+ Ca2+ Mg2+ HCO3 Cl HPO4 2 SO4 2
69 alcohol), poly(lactic acid), poly(acrylic acid), etc.
SBF 142.0 5.0 2.5 1.5 4.2 148.5 1.0 0.5
70 In search of environmentally favorable material from natu-
Blood plasma 142.0 5.0 2.5 1.5 27.0 103.0 1.0 0.5
71 ral and renewable resources as an alternative for bone tissue
72 replacement, the researches have been directed toward the com-
73 bination of HA with natural polymers in the past few years such
74 as starch which has garnered interest due to its attractive fusion stirring for 24 h. The product thus obtained was allowed to ripe for 126
75 of availability, low price and biocompatibility proving it a potential another 24 h without stirring. The product settled on aging, was 127
76 material in medicine, agriculture and packaging industries [1114]. ltered and washed several times with distilled water until the l- 128
77 On the other hand chitosan (CS) which is a randomly partially trate became neutral. The product thus isolated was dried in oven at 129
78 deacetylated form of chitin, the (1,4)-linked polymer of N-acetyl- 85 C. The synthesis of n-HA/CS nanocomposite was also carried out 130
79 d-glucose-2-amine, extracted from crustaceans exoskeletons, has adopting the similar approach to compare the various properties of 131
80 been adopted in different biomedical researches such as bone tis- n-HA/CSST and n-HA/CS nanocomposite scaffolds. 132
81 sue engineering, nerve, retinal tissue engineering, drug carriers and
82 blood vessels, is a biocompatible polymer that can be degraded
83 by enzymes in human body leading to non-toxic degradation 2.2. Immersion in simulated body uid (SBF) study: in vitro 133
84 products [1520]. analysis 134
85 While starch being polar, dissolves in ecofriendly solvent, water,
86 is composed of linear amylose (poly--1,4-d-glucopyranoside) and The in vitro bioactivity of n-HA/CSST and n-HA/CS nanocom- 135
87 branched amylopectin (poly--1,4-d-glucopyranoside and poly-- posites was investigated on the pellets (6 mm in diameter and 136
88 1,6-d-glucopyranoside) [21]. The OH group on starch framework 2 mm in thickness) made from dried samples of nanocomposites 137
89 may possibly be the facile centers for interaction with amino groups immersed into a tube containing 20 ml of SBF solution (having 138
90 of CS and Ca2+ ions of HA facilitating crystallization process of n-HA ion concentrations similar to human blood plasma) oscillating at 139
91 [6]. 37.0 0.5 C in the water bath to allow the soaking of SBF solu- 140
92 Herein we report the synthesis of n-HA/CSST and n-HA/CS tion (Table 1) [22,23]. The SBF solution was prepared by dissolving 141
93 nanocomposite systems via co-precipitation approach, their char- reagent chemicals of NaCl (7.996 g), NaHCO3 (0.350 g), KCl (0.224 g), 142
94 acterization and a comparative study of n-HA/CSST against K2 HPO4 3H2 O (0.228 g), MgCl2 6H2 O (0.305 g), CaCl2 (0.278 g) and 143
95 n-HA/CS nanocomposite in terms of crystallinity, antibacterial Na2 SO4 (0.071 g) into 900 ml distilled water. The uid was buffered 144
96 activity, mechanical and thermal stability. The in vitro evaluation at physiological pH 7.40 at 37 C with tri-(hydroxyl-methyl) amino 145
97 of the physical and biological properties of the proposed nanocom- methane [TRIS] (6.057 g) and HCl, and the solution was made up to 146
98 posite n-HA/CSST has been done after immersion in simulated 1000 ml with additional water. The pellets were withdrawn from 147
99 body uid. SBF after soaking for 2, 4 and 8 weeks period and gently rinsed with 148

double distilled water and dried. 149

100 2. Materials and methods

3. Characterization 150
101 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide)
102 (MTT), chitosan (CS) with degree of deacetylation (>85%) and
The dried products before and after immersion in SBF were char- 151
103 Dulbeccos modied Eagles medium (DMEM) were purchased
acterized by different techniques. The size of the particles was 152
104 from SigmaAldrich (USA) and Invitrogen, USA, respectively.
determined by transmission electron microscopy (TEM, Hitachi 153
105 Extra pure water soluble wheat starch, [Ca(NO3 )2 4H2 O] (99%),
H-7500, Japan) 120 kV. The hydrodynamic size of the particles 154
106 (NH4 )2 HPO4 (DAHP) (99%), CH3 COOH (99.8%), NaOH (>97%),
was evaluated using dynamic light scattering measurements using 155
DMSO, NaCl, NaHCO3 , KCl, K2 HPO4 3H2 O, MgCl2 6H2 O, CaCl2 ,
Laser-Spectroscatter 201 (RiNA) at 25 C. Data was analyzed using
107
156
108 Na2 SO4 , tri-(hydroxylmethyl) aminomethane [TRIS], HCl and
PMgrv 3.01p17 software supplied with the instrument. SEM images 157
109 ammonia solution (25%) have been procured from Merck, Mumbai,
at different magnications were recorded using Scanning elec- 158
110 India. All chemicals were used without further purication. Double
tron microscope JEOL-JAPAN, equipped with an energy dispersive 159
111 distilled water was used in all the experiments.
X-Ray spectroscope EDAX. The FTIR spectra of n-HA/CSST and n- 160

HA/CS were recorded on (FTIR; Interspec 2020, Spectrolab, U.K.) 161

112 2.1. Experimental procedures in KBr in frequency range of 4000400 cm1 . The crystallinity and 162

phase of the samples was studied by X-ray diffraction (XRD) data 163

113 The synthesis of n-HA/CSST nanocomposite was carried out recorded on Philips PW1710 diffractometer with Cu K radiation 164

114 via co-precipitation approach at room temperature. A solution of at 1.540 A in the range of 20 60 at 40 kV. The thermal stability of 165

115 starch (2 g) prepared in 100 ml of distilled water was slowly added the samples was investigated by thermogravimetric analysis (TGA) 166

116 to (2 g) solution of CS dissolved in 100 ml of 2 wt% aqueous acetic and differential thermal analysis (DTA) studies of the samples car- 167

117 acid. The mixture was kept on magnetic stirring at 1200 rpm at ried out on Shimadzu DTG-60H system (Japan). The samples were 168

118 room temperature until the contents were thoroughly mixed. This heated from 30 C to 800 C at the rate of 10 C/min in the nitrogen 169

119 was followed by addition of 0.1 M [Ca(NO3 )2 4H2 O] and 0.3 M DAHP atmosphere. The shore hardness of the samples was determined 170

120 solutions in drop-wise manner to CSST mixture kept on stirring using a shore hardness instrument prepared by Coats Machine 171

121 maintaining Ca/P stoichiometric ratio of 1.67. The overall mixture Tool Co. Ltd, London. The compressive strength of the prepared 172

122 turned opaque and the pH of the mixture was adjusted to about 11 nanocomposites was measured using a universal mechanical test- 173

123 by using 0.5 M NaOH solution in order to accelerate the nucleation ing machine (INSTRON 4505). Cylindrical specimens were prepared 174

124 of n-HA expected at high pH value leading to a milky white product with dimensions 1 cm 1 cm. The testing conditions were at room 175

125 which ultimately changed to creamish white material on constant temperature. The crosshead speed was set at 10 mm/min and the 176

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177 load was applied until the sample was fractured. The compressive from the pus/wounds samples of the registered patients at JNMC. 233
178
strength was calculated from the relationship: The identied and characterized isolates have been stored as glyc- 234

erol cultures at 20 C. The stock glycerol cultures of the clinical 235

4P
179 CS = isolates were sub cultured in Luria Bertaini (LB) (Hi-Media, Mum- 236
d2
bai, India) broth and maintained as pure cultures until used for 237
180 where P is the load at the fracture point and d is the diameter (mm) testing [26]. S. aureus ATCC 25923 and E. coli ATCC 25922 (Hi-Media, 238
181 of the cylindrical specimen. Three parallel samples were tested for Mumbai, India) were used as standard control strains. 239
182 every scaffold, and the mean value of the compressive strength and
183 shore hardness of different scaffolds were given. 3.3.1. Assays for MIC and MBC of n-HA/CS and n-HA/CSST 240

nanocomposites 241
184 3.1. Swelling test 3.3.1.1. Minimal inhibitory concentration (MIC). S. aureus and E. coli 242

isolates were grown overnight on MHA plates at 35 C before being 243

185 To determine the percentage of water absorption, swelling stud- used. The antibacterial activity of n-HA/CS and n-HA/CSST were 244
186 ies were performed in simulated body uid (SBF) at pH 7.4 and examined using the standard agar dilution method [27]. The MIC 245
187 temperature of 37 C. Nanocomposite scaffolds were made into was determined on MHA plates using serial two-fold dilutions of 246
188 small pellets and dry weights of the pellets (Wd ) and the weights of both the nanocomposites in concentration range from 3200 to 247
189 soaked pellets for (1, 7, 14, 21 and 28 days) (Ww ) in SBF solution at 62 g/ml. The initial bacterial inoculum of 2.5 105 CFU/ml, and 248
190 pH 7.4 were taken after removing the adsorbed water on the sur- the temperature and time of incubation at 37 C and 24 h, respec- 249
191 face of the soaked pellets by lter paper. The swelling percentage tively were maintained throughout the experiments. The MIC is 250
192
was determined using the following formula: dened as the lowest concentration of the nanocomposites sam- 251

W w Wd ples that resulted in no visible growth of the bacterial strains. The 252
193 %S = 100 MIC measurements were done in triplicate to ascertain the value 253
Wd
of MIC for each tested bacteria. 254
194 where %S is the swelling percentage. All samples were triplicated
195 in the experiment. The experimental values were analyzed using 3.3.1.2. Minimal bactericidal concentration (MBC). After the MIC 255
196 Students t-test and p-value of <0.05 was considered statistically determination of the nanocomposites an aliquots of 25 l from 256
197 signicant. all tubes in which no visible bacterial growth was observed were 257

seeded on to MHA plates not supplemented with the nanocompos- 258

198 3.2. Cytotoxicity assay ites and incubated for 24 h at 37 C. The MBC endpoint is dened 259

as the lowest concentration of antibacterial agent that kills 100% of 260

199 The cellular toxicity assay of nanocomposites, n-HA/CSST the initial bacterial population. 261

200 and n-HA/CS, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

201 tetrazolium bromide) was carried out by culturing murine 4. Results and discussion 262
202 broblast L929 cells (NCCS, Pune, India) in a contact mode as
203 reported earlier [24]. In brief, L929 cells were cultured in Dulbeccos 4.1. TEM and DLS studies 263
204 modied Eagles medium (DMEM) and seeded on the lms at their
205 exponential phase of growth at a density of 105 cells/cm2 . Vari- The TEM micrographs of n-HA/CS and n-HA/CSST nanocom- 264
206 ous concentrations (from 0 to 50 mg/ml) of the nanocomposites posites (Fig. 1) display that in n-HA/CS scaffold the particles were 265
207 n-HA/CSST and n-HA/CS were added to the monolayer. The cells found to be severely agglomerated as compared to that seen in 266
208 were allowed to attach on the lms surface for 3 h in 5% CO2 incu- n-HA/CSST scaffold where particles were relatively in homoge- 267
209 bator at 37 C. The fresh DMEM medium supplemented with 10% neous dispersed state indicating that the presence of ST inhibited 268
210 fetal calf serum (FCS-GIBCO, USA) was added to each well to keep the aggregation of n-HA/CS particles in n-HA/CSST nanocomposite 269
211 the cell containing lms submerged. The plates were incubated similar to that reported by Yang et al. [28]. The rod shaped parti- 270
212 for 24 h at 37 C in a humidied atmosphere of 5% CO2 in air. The cles of n-HA/CSST with an average size in the range of 1217 nm 271
213 MTT (4 mg/ml) was added to each well at strength of 10% (v/v) fol- which were comparatively lower than the average size of n-HA/CS 272
214 lowed by incubation for another 4 h at 37 C. The media containing particles (2030 nm). These results were further conrmed by 273
215 MTT was removed and 200 l of DMSO was added to dissolve the DLS studies (Supplementary information, Fig. S.1) and an aver- 274
216 formazan crystals. The absorbance was measured using an ELISA age particle size in the range of 1530 nm and 3050 nm have 275
217 plate reader (Biorad, USA) at 595 nm. The resulting absorbance val- been observed in the case of n-HA/CSST and n-HA/CS nanocom- 276
218 ues recorded gave a quantitative measure of viable cells in terms posites respectively complementing the TEM results. However, the 277
219 of the cell population. The %proliferation was calculated and plot slightly greater value of average sizes for both the nanocompos- 278
220 was generated using excel (Microsoft word-2007). The cytotox- ites compared to TEM arises from the fact that DLS calculates the 279
221 icity of both the nanocomposites was also evaluated on human hydrodynamic size of the particles involving the solvent layer at 280
222 osteoblasts-like MG-63 cells (NCCS Pune) (Supplementary infor- the interface. Therefore, these ndings suggested that the incorpo- 281
223 mation under Section 3.2.1.). The experiments were triplicated for ration of starch in n-HAP/CS matrix appears to control the size of 282
224 both the nanocomposites samples. The experimental values were the particles. 283
225 analyzed using Students t-test and p-value of <0.05 was considered
226 statistically signicant. 4.2. SEM 284

227 3.3. Bacterial strains The comparison of the SEM micrographs of the n-HA/CS and n- 285

HA/CSST nanocomposites depicted from Fig. 2(a, b) revealed that 286

228 A total of 35 isolates of Staphylococcus aureus (Gram-positive n-HA/CSST has relatively rough and porous surface as compared 287

229 bacteria) and Escherichia coli (Gram-negative bacteria) were to smoother and packed surface of n-HA/CS suggesting that the 288

230 obtained from the stocks culture, Department of Microbiology, addition of ST inuenced the surface morphology by modifying the 289

231 Jawaharlal Nehru Medical College (JNMC), Aligarh Muslim Univer- n-HA/CS matrix, an important requirement favoring the tissue in- 290

232 sity (AMU), Aligarh, India [25]. The isolates were originally isolated growth, bone formation and biological xation with surrounding 291

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Fig. 1. TEM micrographs of (a) n-HA/CS and (b) n-HA/CSST nanocomposites.

292 tissue [29]. The above discussion suggested that there is an ample potential bonding ability of n-HA/CSST nanocomposite that facil- 328

293 possibility for interaction of OH of ST and NH2 of CS with each itates bone ingrowth formation and good osteointegration in vivo. 329

294 other and also with n-HA in n-HA/CSST scaffold leading to changes
295 in physical properties of this scaffold relative to n-HA/CS scaffold 4.4. Energy dispersive X-ray spectroscopy (EDX) 330
296 [6]. This has been conrmed by comparing the mechanical prop-
297 erties of these two scaffolds where n-HA/CSST has shown greater The comparative study of EDX spectra of n-HA/CS, n-HA/CSST 331
298 hardness as compared to n-HA/CS. and their respective SBF scaffolds kept for 2, 4 and 8 weeks shown 332

in Fig. 3(ah) has been made. The observed semiquantitative ratio 333

299 4.3. In vitro bioactivity evaluation of n-HA/CSST and n-HA/CS of Ca/P of 1.00 against the expected range of 1.67 0.67 in natural 334

300 nanocomposite scaffolds bone has been found in the EDX spectra of n-HA/CSST nanocom- 335

posite scaffolds kept in SBF for 2 weeks (Fig. 3(ad)) [30]. However 336

301 Generally, it is believed that the in vitro calcication ability of n-HA/CSST scaffold kept for 8 weeks (Fig. 3(g, h)) in SBF gave Ca/P 337

302 biomaterials has a correlation with the bone-bonding ability in value of 1.58 much closer to the theoretical stoichiometric ratio of 338

303 vivo. Bioactivity is a result of the chemical reactions occurring at hydroxyapatite (Ca/P = 1.67) [31] as compared to the value of 1.84 339

304 the surface of a material exposed to body uids in order to the obtained in case of n-HA/CS scaffold kept in SBF for 8 weeks that 340

305 form a surface layer of hydroxyl carbonated apatite (HCA) upon could be attributed to an appreciable increase in elemental concen- 341

306 implantation which is an essential criterion for establishing bond- trations of Ca and P (precursors for HA formation) along with other 342

307 ing with natural bone. Thus, investigating the biological behavior elements. 343

308 of bioceramics in SBF is considered to be the most efcient method

309 to authenticate their bioactivity in the body environment. In vitro 4.5. FTIR analysis 344

310 bioactivity of the nanocomposites n-HA/CSST and n-HA/CS scaf-

311 folds has been monitored by SEM microphotographs of the two The possible interaction between the various components in 345

312 scaffolds (Fig. 2(ch)) soaked in SBF solution with ionic concen- n-HA/CSST has been graphically displayed in Scheme 1. The pre- 346

313 tration analogous to blood plasma at pH 7.40 and 36.5 C for 2, liminary information regarding the interaction of different phases 347

314 4 and 8 weeks. It has been observed that there was considerable in n-HA/CS and n-HA/CSST scaffolds has been obtained by com- 348

315 biomimetic deposition of HA in both the matrix surfaces but n- paring the FTIR spectra. The FTIR spectra of n-HA/CS, n-HA/CSST 349

316 HA/CSST matrix exhibited greater deposition in the form of thick and n-HA/CSST-SBF (8 weeks) exhibit bands characteristic of n- 350

317 layer as compared to n-HA/CS. The comparison of SEM images of HA, CS and ST moieties (Fig. 4) in their respective scaffolds. In the 351

318 both the scaffolds soaked in SBF for 8 weeks (Fig. 2(g, h)) revealed FTIR spectrum of n-HA/CS, the presence of HA in CS matrix can be 352

319 that a thick apatite layer deposited throughout the n-HA/CSST identied by its characteristic bands of phosphate group at 467, 563 353

320 nanocomposite as compared to n-HA/CS scaffold. In addition, a crit- and 602 cm1 assigned to phosphate bending modes of vibrations. 354

321 ical comparison of SEM micrographs of n-HA/CS and n-HA/CSST However, the stretching mode of vibration of phosphate group in 355

322 nanocomposites taken after time period of 8 weeks (Fig. 2(g, h)) n-HA overlaps with the C O C stretching vibration of CS discerns 356

323 revealed curbed rod like structures possibly of n-HA irregularly as a broad band in the region of 9501034 cm1 [32], while OH 357

324 embedded in SEM micrograph of n-HA/CSST with length of about stretching band of HA gets overlapped with the OH stretching band 358

325 2025 nm in CSST matrix which could not be observed in n-HA/CS of CS and a broad peak at about 3430 cm1 appeared. The peaks at 359

326 micrograph suggesting that the presence of ST induced higher 1458 and 2927 cm1 may reasonably be assigned to C H stretch- 360

327 growth of HA nanoparticles. These results suggested a promising ing of chitosan [33], while bands at 1540 cm1 and 1655 cm1 361

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Fig. 2. SEM micrographs of (a) n-HA/CS, (b) n-HA/CSST and their respective SBF study after 2, 4 and 8 weeks (ch). Rod shape n-HA.

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Fig. 3. EDX micrographs of micrographs of (a) n-HA/CS, (b) n-HA/CSST and their respective SBF study after 2, 4 and 8 weeks (ch).

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800

700

600

Intensity (a.u)
500

400

300

200

100
a
0
b
-100 c
-200 d
15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
0
Scheme 1. Possible interaction between different components in n-HA/CSST
2Theta( )
nanocomposite.
Fig. 5. X-ray diffractograms of (a) n-HA/CS, (b) n-HA/CSST, (c) n-HA/CSST-SBF (8
weeks) and (d) bone.

band at 1037 cm1 and small shifts in PO4 3 stretching modes may 387

arise in view of the distortion of HA crystalline structure due to 388

replacing phosphate groups by carbonate groups [36]. 389

4.6. X-ray diffraction (XRD) studies 390

The X-ray diffraction patterns of the nanocomposites n-HA/CS 391

(a), n-HA/CSST (b), n-HA/CS-SBF (8 weeks) (c) and original human 392

bone (d) are shown in Fig. 5(ad). The average crystallite sizes of 393

nanocomposites and the human bone were calculated using Scher- 394
395
rers equation:

K
L= 396
Fig. 4. FTIR spectra of n-HA/CS, n-HA/CSST and n-HA/CSST-SBF (8 weeks). cos 

362 represented N-H bending (amide II) and C O stretching (amide where L is the average crystallite size, is the full width of the 397

363 I), respectively [34]. The FTIR spectrum of n-HA/CSST shows all peak at half of maximum intensity (rad) (FWHM) [1,37],  is the 398

364 characteristic bands corresponding to n-HA and CS at expected wavelength of monochromatic X-ray beam radiation Cu radiation 399

365 positions along with a wide band at 3436 cm1 attributable to  is the peak diffraction angle (Braggs angle), K is
( = 1.5406 A), 400

366 O H stretching of amylopectin with its width warranting the for- a Scherrer constant dened as the crystallite shape and is approx- 401

367 mation of inter and intra-molecular hydrogen bonding. A slight imately equal to 0.9. The characteristic peaks of n-HA appears at 402

368 modication in CS band appearance characteristic of aliphatic C H 2 = 26, 29.3, 32.3, etc. which conrms the presence of n-HA crys- 403

369 stretching band at 2927 cm1 may be due to C H asymmetric tallites. In Fig. 5(ac), the presence of these characteristic peaks 404

370 stretching band of starch expected to appear in this range. The conrmed the presence of n-HA in all the scaffolds which matches 405

371 bands at 1432 and 1385 cm1 may arise due to the angular defor- well with the XRD peaks of original human bone displayed in 406

372 mation of C H bonds in ST molecule. A positive shift in (amide Fig. 5(d), indicating that crystallization of n-HA still existed after 407

373 II) in n-HA/CS from 1540 cm1 to 1620 cm1 in n-HA/CSST refers nanocomposite formation which could be resulted from interface 408

374 to the possible H-bonding between OH of starch and amino group binding between n-HA particles and polymers matrix [38]. The 409

375 of CS [34]. Moreover slight shifts in the phosphate group vibra- average crystallite size of all the four scaffolds were calculated by 410

376 tions of HA in n-HA/CSST scaffold indicate that the presence of Scherer equation conrming the nanostructure of the nanocompos- 411

377 ST incited the dissociation and interaction of polymer with nucle- ites and were found to be 24.1 nm for n-HA/CSST (SBF-8 weeks) 412

378 ating crystal [35]. The FTIR spectrum of n-HA/CSST kept in SBF and 20.0 nm for n-HA/CS as compared to 12.9 nm for n-HA/CSST 413

379 for 8 weeks (Fig. 4) exhibit the bands assignable to OH and H2 O and 14.7 nm for human bone. The increase in average crystallite 414

380 along with a new weak intensity bands at 874 cm1 (v2), 1416 and size of n-HA/CSST immersed in SBF solution for 8 weeks may be 415

381 1462 cm1 (v3) characteristic of CO3 2 indicative of the formation explained in terms of growth in number and size of n-HA particles 416

382 of small amount of CO3 2 moiety in n-HA in presence of ST [6] leading to complete coverage of polymer matrix by apatite layer 417

383 which is advantageous for bone mineral (expected to be 48 wt% [29]. Thus the immersion of n-HA/CSST for sufcient time period 418

384 in the human body), as it enhance the mechanical consistency and in SBF enhances ability of formation and nucleation of apatite layer 419

385 bioactivity of the apatite leading to more osteoconduction and tis- which is triggered by the addition of ST possibly due to greater 420

386 sue in-growth is expected on implantation. A slight broadening in interaction in n-HA/CSST scaffold. 421

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Fig. 6. TGA curves of CS, n-HA/CS, n-HA/CSST and n-HA/CSST (SBF-8 weeks) scaf-
folds.

Fig. 8. Swelling percentage of n-HA/CS and n-HA/CS/ST nanocomposites. *Statistical

signicance level by t-test (p < 0.05).

loss in the range of (3540%) with the incorporation of starch. The 444

TGA curve of n-HA/CSST (SBF-8 weeks) represented that the initial 445

degradation temperature was shifted slightly to lower temperature 446

which indicates lesser total weight loss of about (2530%) in the 447

temperature range of 65300 C attaining stability beyond 300 C 448

suggesting that the total weight loss due to the thermal decom- 449

position of the nanocomposite (n-HA/CSST-8 weeks) decreased, 450

as the inorganic content (HA) in the nanocomposite increased. The 451

higher stability of n-HA/CSST (SBF-8 weeks) comparative to other 452

scaffolds may be explained in terms of increase amount of hydroxy- 453

apatite in the polymer matrix in presence of ST after immersing 454

in SBF solution for 8 weeks which strongly supports superior in 455

vitro bioactivity of n-HA/CSST (soaked in SBF for 2, 4 and 8 weeks) 456

nanocomposite compared to n-HA/CS corroborated from SEM stud- 457

ies [29,39]. Thus comparative study of thermal analysis of the 458

various scaffolds suggested that ST signicantly raised the thermal 459

stability of n-HA/CS nanocomposite in n-HA/CSST [40], possibly 460

due to regular increased interactions. 461

Fig. 7. DTA curves of CS, n-HA/CS, n-HA/CSST and n-HA/CSST (SBF-8 weeks) scaf-
folds. 4.8. Swelling test 462

422 4.7. TGADTA analysis The study of swelling percentage of n-HA/CS and n-HA/CSST 463

nanocomposites in SBF solution for different time intervals (1, 7, 464

423 In order to meet out the biocompatibility of the biomaterials, 14, 21 and 28 days) displayed in Fig. 8, revealed a regular decrease 465

424 it is important to verify the thermal stability via TGA analysis not in swelling capacity of both the scaffolds on increase in time inter- 466

425 only in the temperature range of human body but also in higher vals. However, a comparative analysis of swelling percentages of 467

426 temperature intervals which involves sterilization processes. The n-HA/CS and n-HA/CSST indicate that n-HA/CSST has signi- 468

427 TGA and DTA curves of CS, n-HA/CS, n-HA/CSST and n-HA/CSST- cantly lower swelling capacity for the time period of 1 and 7 days 469

428 SBF-8 weeks have been displayed in Figs. 6 and 7, respectively. The as compared to n-HA/CS (Fig. 8), which may be explained in terms 470

429 TGA curve of CS represented the two-step weight loss in the range of increased interactions between n-HA, CS and ST as compared to 471

430 of 90130 C and 240400 C corresponding to loss of adsorbed n-HA/CS [41,42]. 472

431 water molecule present in the scaffold and the decomposition of CS,
432 respectively as depicted from Fig. 6. The presence of one endother- 4.9. In vitro toxicity of nanocomposite 473

433 mic and one exothermic peak in DTA curve of CS in Fig. 7 further
434 support TGA curve. However the TGA graph of n-HA/CS nanocom- The nanocomposites scaffolds n-HA/CSST and n-HA/CS were 474

435 posite comprises of two step weight loss in the range of 85150 C subjected to cytotoxic studies with murine broblast L929 and 475

436 and 225410 C in consistency with DTA curve, having total weight human osteoblasts-like MG-63 cells. The cells were incubated 476

437 loss of about (7275%) compared to (8590%) in case of CS suggest- with various concentrations (050 mg/ml). The cellular toxicity of 477

438 ing that the n-HA enhanced the thermal stability of CS. Further, the these nanocomposites was evaluated using MTT assay after 24 h. 478

439 thermal stability of n-HA/CS scaffold got increased by incorpora- MTT assay revealed signicant non-toxic nature of n-HA/CSST to 479

440 tion of ST in n-HA/CSST which shows two step weight loss in the both the cell lines even at higher concentrations (2550 mg/ml) 480

441 range of 80140 C and 260 380 C corresponds to moisture loss as compared to n-HA/CS at similar concentrations (Fig. 9(a, b)). 481

442 and decomposition of organic moieties, respectively which is in In other words n-HA/CSST showed signicant superior biocom- 482

443 agreement with its DTA curve, resulting in decrease in total weight patibility without interfering the cellular machinery over n-HA/CS 483

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Fig. 9. Cellular toxicity of nanocomposites n-HA/CS and n-HA/CSST determined by MTT assay on: (a) L929 cells and (b) MG-63 cells. *Statistical signicance level by t-test
(p < 0.05).

484 conrming excellent in vitro cytocompatibility (Supplementary 4.10. MIC and MBC of n-HA/CS and n-HA/CSST 489

485 information, Tables S.1 and S.2). These comparative results suggest
486 that n-HA/CSST would be a promising candidate for bone tissue The antibacterial activity results suggested that both the scaf- 490

487 engineering in search of a scaffold to be used as bone implant for folds exhibited antibacterial properties for both Gram positive and 491

488 orthopedic applications in mammals [43]. Gram-negative bacteria. However it is found that MIC and MBC 492

Fig. 10. Pellets display: (a) CS, (b) n-HA/CS and (c) n-HA/CSST scaffolds; mechanical properties: (d) compressive strength and (e) shore hardness.

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Table 2 monitored by SEM images of n-HA/CS and n-HA/CSST scaffolds 546

MIC and MBC of n-HA/CSST and n-HA/CS nanocomposites.
suggested a better bioactivity of n-HA/CSST indicating the greater 547

Nanocomposite Bacterial strains MIC (g/ml) MBC (g/ml) ability to facilitate bone ingrowth formation and possibility of 548

n-HA/CSST S. aureus 500 1000 good osteointegration in vivo. The MTT assay studies on n-HA/CS 549

E. coli 1000 2000 and n-HA/CSST revealed higher cell proliferation in n-HA/CSST 550

compared to n-HA/CS warranting a superior non-toxicity. The com- 551

n-HA/CS S. aureus 1000 2000
E. coli 1600 3200 parison of results of antibacterial property of n-HA/CSST and 552

n-HA/CS against both Gram-positive and Gram-negative bacteria 553

revealed an improved antibacterial property of n-HA/CSST. Thus, 554

493 values for n-HA/CSST were lower in comparison to that shown by these ndings on n-HA/CSST nanocomposite would be a step for- 555
494 n-HA/CS (Table 2). The superior antibacterial nature of n-HA/CSST ward in the development of a competent bone construct in the eld 556
495 as compared to n-HA/CS may be due to relatively smaller particle of Bone tissue engineering. 557
496 size of n-HA/CSST [1] enhancing surface area to volume ratio lead-
497 ing to higher antibacterial activity. The greater antibacterial activity
Acknowledgements 558
498 of n-HA/CSST may also be explained by relating higher interaction
499 ability of small sized particles of n-HA/CSST with bacteria resulting
Prof. M. Shakir acknowledges the grant supports from DRS-II, Q3 559
500 higher disruption of cell membranes and destruction of cytoplasm
FIST and PURSE to the Department of Chemistry, A.M.U., Aligarh. 560
501 [44].
The authors acknowledge and thank to JNMC, AMU, Aligarh, for 561

providing human bone material employed for XRD analysis. The 562
502 4.11. Mechanical properties
authors, Mohd Shoeb Khan and Reshma Jolly are thankful to Univer- 563

sity Grants Commission (UGC/UGC-MANF) for nancial assistance. 564

503 4.11.1. Shore hardness and compressive strength of
504 nanocomposites
505 The initial mechanical properties are usually important crite- Appendix A. Supplementary data 565

506 ria in choosing the scaffold materials for bone tissue engineering.
507 Shore hardness measured by a scleroscopic hardness tester is a Supplementary data associated with this article can be found, 566

508 kind of dynamic hardness which measures the height of the bounce in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2015. 567

509 of a diamond tipped hammer dropped from a xed height on the 05.009 568

510 sample to be analyzed [45]. A comparative study of shore hard-

511 ness of CS, n-HA/CS and n-HA/CSST performed on their pellets References 569
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