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21 a r t i c l e i n f o a b s t r a c t
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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.
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
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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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
nanocomposites 241
184 3.1. Swelling test 3.3.1.1. Minimal inhibitory concentration (MIC). S. aureus and E. coli 242
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
198 3.2. Cytotoxicity assay ites and incubated for 24 h at 37 C. The MBC endpoint is dened 259
199 The cellular toxicity assay of nanocomposites, n-HA/CSST the initial bacterial population. 261
227 3.3. Bacterial strains The comparison of the SEM micrographs of the n-HA/CS and n- 285
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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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
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
(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.
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
which indicates lesser total weight loss of about (2530%) in the 447
suggesting that the total weight loss due to the thermal decom- 449
422 4.7. TGADTA analysis The study of swelling percentage of n-HA/CS and n-HA/CSST 463
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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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
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
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
523 to be most satisfactory when compared to the natural bone 631. 583
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