Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 1.

Department of Embryology and Stem cells, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 2. Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran 3. Nanomedicine and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 4. Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran 6. Department of Biotechnology and Cellular and Molecular Research Center, Faculty of Allied Medicine, Tehran University of Medical Sciences, Tehran, Iran Running title: silk scaffolds for bone tissue engineering

Bioactivity and biocompatibility studies on a novel silk-based scaffold for bone tissue engineering
Sahba Mobini1, 2, Mehran Solati-Hashjin*2, Habibollah Peirovi3, Mazaher Gholipourmalekabadi4, Mahmoud Barati5, Ali Samadikuchaksaraei6

* Corresponding Author: Mehran Solati-Hashjin E-Mail: solati@aut.ac.ir Tel: +98-21 64542360 Fax: +98-21 66468186

Abstract Utilizing novel materials with promising properties is one of the approaches to achieve scaffold based tissue engineering goals. Natural silk polymer has remarkable biomedical and mechanical properties as material for bone tissue engineering scaffolds. In this study fabrication of silk-based composite in which, natural silk (NS) and regenerated silk (RS) were combined to achieve better mechanical properties in the 3D porous form, is explained. Furthermore, biocompatibility and bioactivity of these scaffolds are evaluated. RS has been made using mulberry-silk cocoons. RS/NS composite scaffolds have been fabricated by the freeze-drying technique. Silk protein 1

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 extract was evaluated by FTIR, which showed sharp amide peaks in 1655 cm-1 and 1530 cm-1 wavelength in FTIR spectrum pattern confirming the existence of fibroin. The fabricated 3D scaffolds were morphologically analyzed by scanning electron microscopy and showed an inter-connective spongy structure. Mechanical characterizations were also carried out by a universal testing machine. Results showed that the RS/NS composites have improved mechanical properties compared with scaffolds fabricated with RS alone. In addition, In vitro tests including cell viability and adhesion have been carried out with Osteoblast cells by MTT assay with new calculation approach which confirmed biocompatibility. Bioactivity potential of fibers, RS and composites has been surveyed by the introduction of scaffolds to normal simulated body fluid (SBF) during 21 days. EDX and SEM analysis which accomplished afterwards, proved the existence of CaP crystals on both configurations. Thus, reinforced silk composite with superior mechanical properties is found to be a bioactive and biocompatible alternative for bone tissue engineering applications. Keywords: Bone tissue engineering; biocompatible materials; Scaffold; Silk

1. Introduction Bone is a complex and highly specialized connective tissue [1,2]. As, provision of structural support for the body is the bones main role, any structural bone defect that leads to functional deficits can dramatically affect individuals well-being and quality of life [3]. Some structural bone defects are amenable to autologous or allogenic bone graft or bone replacement by an artificial substitute. Tissue engineering can provide an alternative to traditional treatment protocols by replacing living tissue with the tissue grown in vitro to meet each individual patients needs [4,5]. Tissue engineering employs three-dimensional porous matrices for mechanical stability and support of cell adhesion and expansion. A synthetic bone scaffold must be biocompatible, biodegradable to allow native tissue integration, and mimic the multidimensional hierarchical structure of native bone. Various materials and scaffolds fabrication techniques have been investigated over the past two decades for bone tissue engineering with the aim to increase mechanical stability and improve scaffold-tissue interactions [6]. Satisfaction of mechanical properties, biocompatibility as well as bioactivity requirements play an important role in scaffold utility, particularly when considered for bone tissue regeneration [7,8]. Silks are natural fibers produced by various insects of which mulberry silkworms (Bombyx mori) are of high economic importance, as they could be reared in captivity [9,10]. Recently, it has been shown that silks can be employed for a variety of biomedical applications including support of osteoblast cell growth and differentiation for bone tissue engineering purposes [11-13]. Silkworms silk contains a fibrous protein termed fibroin (composed of heavy 2

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 and light chains) that forms the structural core of the silk and glue-like proteins, termed Sericins, which surround the fibroin fibers. Fibroin is an insoluble protein in which Glycine, Alanine, and serine compose 90% of its amino acids that form crystalline -pleated [14]. Silk exists in various polymorphs including silk I (-helix structure) and silk II (-sheet structure). Silk I structure is the water-soluble state and converts to a -sheet structure which is insoluble in water when exposed to methanol, potassium chloride or sodium chloride [11,15-17]. As the crystalline system does not completely regenerate in reprocessed fibroin, it does not retrieve all the properties of the native fibroin [18,19]. In this study we have explored the idea of engineering of silk composite in which natural silk (NS) and regenerated silk (RS) were combined to achieve better mechanical properties in the 3D porous form. We also investigate the biomimitics capability of fibroin in both conformations. In addition biocompatibility of the RS and RS/NS has been investigated by MTT method using the new calculation to overcome the inaccuracy due to stain absorption of 3D scaffolds. 2. Materials and Methods 2.1 Materials Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) obtained from GibCo and Penicillin-streptomycin (Pen-Strep), Trypsin/EDTA, MTT substrate [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrasodium bromide] from Sigma Chemical Co. Cell culture plastics were from Nunch (Denmark). Osteoblast cell line (G 292) was purchased from National Cell bank of Iran, Pasteur Institute. LiBr, Na2CO3, Methanol and all other salts for SBF were supplied by Merck (Germany). Cellulose dialysis tube (cut off value of 12000 Da) was from Sigma (Germany) and finally, Bombix.Mori cocoons were the generous gift from Iranian Silkworm Research Center.

2.2 Scaffolds Preparation Fibroin extraction was performed using the protocol reported by D. Kaplan et al. [20]. Briefly, Bombyx mori cocoons were boiled for 1 h in a 0.02M aqueous sodium carbonate solution and then rinsed thoroughly with cold and hot water to extract the sericin proteins. The degummed silk was dissolved in 9M LiBr at 55C for 4 h. Then the fibroin solution was filtered and dialyzed against distilled water for 3 days to yield the fibroin water solution. The solution lyophilized for 3 days to get the dried storable fibroin. The fibroin solutions with 4 %w/v, 8 %w/v concentrations were prepared through resolving the above dried fibroin with stirring. These solutions were put either into the Teflon molds or 24-well polystyrene plates and then frozen at 20C for 4 h and then at 80C for 1h. The ice/silk composites were then lyophilized for 12 h leaving a porous matrix. After drying, porous matrixes were obtained, and immersed in methanol 99.9% for about 1h to induce crystallization and transforming to -sheet structure and insolubility in water. The insoluble RS 3D-scaffolds were then prepared after removing methanol and additional lyophilizing. Natural continuous silkworm silk fibers (NS) were used as reinforcements for regenerated silk fibroin to fabricate composite 3D-scaffolds. In order to examine an optimal amount of silk fibers, three different fiber concentrations of 50, 30 and 90 % v/v were used. NS and RS 4 % w/v solution were mixed together in proper molds then frozen and lyophilized just like RS samples. Dried scaffolds were also treated with methanol to make them insoluble and for transformation to -sheet structure. Samples' cods and composition are 3

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 demonstrated in table1. 2.3 Characterization 2.3.1 Fourier transforms infrared spectroscopy (FTIR) Fibroin protein was analyzed by FTIR on an EQUINOX 55FTIR spectrophotometer. Freeze-dried fibroin was mixed with KBr and pressed under the state of ASTM E1252-07standard. The pellets were analyzed in the range of 500 to 4000 cm -1 and resolution of 4cm-1 to determine the functional groups which represent fibroin protein. 2.3.2 Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis The surface, cross-section morphologies and pore distributions, sizes and interconnectivity of scaffolds, morphology of fibers as well as cell adhesion on scaffolds were observed by Philips scanning electron microscopy, using XL 30/ESEM with a field emission gun. The specimens were sputter coated with gold operating at voltage of 15 or 20 keV. Additionally, the chemical composition of crystals in biomimatics experiments was semi-quantitvely explored via EDX (Rontec, Germany) directly connected to SEM. 2.3.3 Compressive mechanical properties Compression properties of 3D cylinder-shaped scaffolds were measured using Dynamic Testing Machine (HCT 25/400, servo Hydrolic PID controller, Zwick/Roell, Germany).The setup which was applied in this study had 0.01 N and 0.001 m axial resolution. Cylindrical samples, 7 mm 28.26 mm2, were examined at a crosshead speed of 1 mm/min according to a modification based on ASTM method F451-95 [21-23]. The compressive strength was determined from the stress-strain curve and calculations were performed using toolkit 1998 software. 2.3.4 Porosity (13) Hexane which is a non-solvent agent for silk was used as the displacement (23) liquid. It permeates easily through the interconnected scaffold pores and causes no swelling or shrinkage. The silk scaffold was then immersed in a known volume (V1) of hexane in a graduated cylinder for 5 min. The total volume of hexane impregnated scaffold along with hexane was recorded as (V2). The residual hexane volume in the cylinder after removal of hexane saturated scaffold was recorded as (V3). The porosity of the scaffold () was obtained by: (%) = 100[23] 2.4 Cell culture experiments 2.4.1 Osteoblast seeding on scaffolds The human osteoblast cell line G 292 was expanded in DMEM low glucose containing 10U/ml penicillin and 100 g/ml streptomycin, 10% FBS (normal medium) at 37C in humidified, 5%CO2/95% air incubator. Cultivation volume and duration depend on the number of cells through the calculation: 5105 cells considered for each sample. The disc-shape scaffolds were rinsed with alcohol 70% v/v in a biological laminar hood and washed several times with sterile PBS before cell seeding. Then, the scaffolds were pre-incubated in a normal culture medium for 2h before seeding. The scaffolds were seeded with high-concentration cell suspension and incubated for 2 hours. Then the volume of the medium for each sample of scaffolds was increased to 1 ml to cover the scaffold completely. Polystyrene surface of cell 4

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 culture plates were used as control. 2.4.2 Cell response analysis Cell viability and biocompatibility assessments were carried out using MTT assay after 48h of osteoblasts culture. As due to absorption of the dye, these 3D scaffolds affect the degree of color change in MTT assay, control scaffolds were placed into the polystyrene control wells for normalization. These scaffolds were soaked in the culture medium for 30 minutes before running the test. The growth medium of each scaffold samples as well as controls was replaced with MTT solution with 0.02 ml of MTT solution with concentration of 5 mg/ml. Then, 0.1 ml serum-free medium was added to each well for covering the entire surface. The wells containing MTT solution were incubated for 4 h in a humidified atmosphere of 5%CO2 in the air. At this stage, the MTT was removed and 0.1 ml of dimethyl sulphoxide (DMSO) was added into each well in order to dissolve the Formazan crystals. In this stage the stain from one sample of each type was transferred to its own control (those scaffolds which soaked in the culture medium). The plate was agitated for 20 min in the dark and the viable cells in the colored solution were quantified using a scanning multi-well spectrophotometer at 540 nm. 2.4.3 Cell morphology One cell-seeded sample of each type after 4 days of the experiment was fixed by 2.5% glutaraldehide in PBS for 2h and washed truly with PBS. Then samples submerged in Osmium tetroxide 0.1% in 0.1M PBS for 30min and then, washed carefully with PBS. Dehydration was accomplished by using a gradation series of Acetone/distilled water solutions. Freeze drying was performed later in -40C to -75C for 12h. Finally, samples prepared for SEM imaging by coating with gold spattering machine and imaged using the same apparatus. 2.4.4 Statistical Analysis All experiments were performed in sixth replicate. The results were given as means Standard deviation (SD). Statistical analysis was performed by using T test; P<0.05 was considered significant. 2.5 Biomimetic Bio-mineralization study was performed through placing samples in SBF for 21 days. SBF was made using the protocol reported by Ayako Oyane et al. [24]. Briefly, HEPES, NaOH as well as salts (weigh amounts are described in table2) were mixed and the total volume was adjusted to 1000ml by adding ultra-pure water. Penicillin/streptomycin (in the same proportion of cell culture experiments) was added in concentration of 1% to keep away from the contamination during the test. Finally, the pH was adjusted to 7.40 at 36.5 C by adding adequate amount of HCl. Two disc-shape scaffold samples of each type as well as natural fibers were placed in the filtered SBF and incubated at 37C/5% CO2 for the time points mentioned above (recommended by M.Bohner et al. [25]). SBF was changed every 3 days to introduce fresh ions to scaffolds. Samples were dried in the air at room temperature and investigated by SEM and EDX to depict the probable crystal formation and chemical composition respectively. 3. Result and discussion 3.1 FTIR analysis of extracted fibroin There are four different types of distinguishable vibration peaks which are related to 5

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 amide groups in proteins. Protein shows characteristic vibration bands between 1630cm-1 _1650cm-1 for amide I (C=O stretching), 1540cm-1_1520cm-1 for amide II (secondary NH bending), and 1270cm-1_1230cm-1 for amide III (CN and NH functionalities) in their FTIR spectra [26]. In 1960 Miyazawa and Blout [27] studied the infrared spectra of polypeptides in various configurations. They demonstrated that except some special proteins, polypeptides show amid I bands in -helix form at 1655cm-1 and amid II in -sheet form at 1540cm-1 while in the extended configuration they show these bands at 1630cm-1 and 1520cm-1 respectively. Conformation of extracted fibroin was investigated by FTIR spectra which revealed pure fibroin with silk I structure, for the same pattern for fibroin reported several times by other groups [19,28-32]. Figure 1 shows the spectra of extracted fibroin which is divided to four zones for easier clarification. Picks positioned at 1655cm-1, 1530cm-1 and 1239cm-1 representing amid I, amide II and amid III respectively. Moreover, the pick at 699cm -1 is also a validation for amid II. 3.2 Morphology of scaffolds Freeze-drying of fibroin solution with or without fiber, filled in appropriate molds, leads to porous 3D scaffolds which can be produced in any size or shape. Samples were made in two different shapes, disc-shape and cylindrical form for mechanical and cell culture analysis respectively, Figure 2(a) SEM images of RS and NS/RS scaffolds in different concentration are shown in Fig. 2(b)-(f). The more concentration of fibroin solution produced the smaller pore size in RS scaffolds structure. Composite samples (RS/NS) however, reveal a completely different structure. Fibers dispense homogenously and support special porous structure in combination with freeze-dried spongy structure. The porous structure of this blend has been shown in Fig. 2(d) with less fiber which gradually becomes more compact by accumulation more fiber in Fig.2(e)-(f). 3.3 Mechanical properties of scaffolds Mechanical properties obtained from static compressive tests of both RS and RS/NS composite samples have been illustrated in Figure 3. For RS samples, both mechanical strength and module decrease by reducing fibroin concentration. This trend is reasonable due to ice/fibroin composite in freeze-drying method enriched by ice in lower concentration of fibroin (4 %w/v). Therefore less fibroin as well as much bigger pores can be obtained in F4 samples in compression with F8 samples, which also make less strong structure. In composite samples the E module and compressive strength both rise up gradually by accumulation more fiber. This trend can reveal that fibers are functioning as the reinforcement component in spongy composite. However, more fiber in the system could be a limitation for desirable pore size and structure, Fig. 2(f). Fiber reinforcement makes a significant increase in compressive mechanical properties, although there is a slight drop in C1F4 samples. Figure 4 shows the results from the liquid displacement test for measurement of porosity. All of the scaffolds show porosity between 83 to 89% in which, C3F4 has the highest porosity with 89% and F8 has the less porosity with 83%. In fact, there is an inverse relationship between the fibroin concentration and percentage of porosity. However, no prediction could be drawn from the introduction of fibers to the composite system. As it is summarized in Table 3, C3F4 scaffold has the most porous structure in a same time has the highest compressive module and strength. This can be due to grouping effect. 3.4 Cell seeding and cell response

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 Osteoblast cells response to RS scaffolds and RS/NS composite scaffolds were investigated by MTT assay. As it has also been noticed by other group [33] conventional MTT method does not yield consistent results when it is used for measurement of cellular proliferation on scaffolds which tend to absorb Formozan stain. Therefore, we have taken into the account the stain abortion of the scaffolds for interpretation of MTT results. Figure 5 demonstrates the MTT result which reveals C1F4 has the least variance from its control. Also, we have observed the highest stain absorption in C1F4. F4, as a simple fibroin scaffold shows less biocompatibility in comparison with the scaffolds containing fibers. It has been noted that addition of more fibers to the composite (C2F4) decreases the number of the cells on the scaffold. This could be explained by the fact that fibers decrease the porosity of the scaffold and thence, decrease the total surface area available for cell attachment and growth. Figure 6 shows SEM image of seeded scaffolds. Osteoblasts covered the surface of both plane and composite scaffolds. The stretched and typical morphology of cells support the results from MTT assay. 3.5 Boimimetics Mineralization of RS scaffold, RS/NS composite scaffold as well as degummed fibers has been explored by SEM. To test which component plays the main role in mineralization, pure fibers also were immersed in SBF for 21 days. Figure 7 shows the crystal formation on fibers after 21 days in normal SBF and capacity of natural fibers in mineralization in SBF. Element analysis using EDX, in 1 and 21 days after immersing showed CaP needle shape crystals were formed on fibers. NaCl peaks in EDX which is found in both are probably due to the supersaturation of solution. Figure 8 shows the SEM image of composite RS/NS scaffolds in SBF after 21 day. EDX chemical analysis reveals Cap formation on these scaffolds. To test if this formation is only supported by fibers or regenerated fibroin has also the same capacity, F4 scaffolds are also immersed in SBF for 21 days. Figure 9 demonstrates the formation of Cap crystals on RS scaffolds as well. However the morphologies of RS crystals are cubic, which is different from the needle types on the fibers.

4. Conclusion In this study, the structural characteristics and properties of the regenerated silk fibroin/natural silk composite 3D-scaffolds were investigated for the first time. Cell growth and adhesion have also been evaluated on this composite scaffold. Our new RS/NS composite has been synthesized of non-bioengineered silk fibroin protein from silkworms with an easy to follow protocol. High compressive module, compressive strength as well as highly porous structure with a simple fabrication technique make this scaffold a good choice for bone-tissue repair applications. Needle shape CaP crystals reveals on RS/NS scaffolds in SBF shows the bioactive capability of scaffolds. Adherence and growth of established Osteoblaste on the fabricated silk matrices have been satisfactory. As regenerated fibroin cannot form fully -sheet structures, utilization of natural silk fibers as reinforcement would be a suitable strategy for

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 future tissue engineering applications since regenerated silk loses some of its characteristics even after methanol treatment. Acknowledgments This work was partly funded by Nanomedicine and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran. Authors thank Armin Springer, Max Bergman Center, Dresden, for running EDX and taking SEM images. References [1] M. J. Olszta, X. Cheng, S. S. Jee, R. Kumar, Y. Kim, M. J. Kaufman, E. P. Douglas and L. B. Gower, Bone structure and formation: A new perspective, Mater. Sci. Eng., 58: 77-116, 2007. [2] R. Murugan and S. Ramakrishna, Development of nanocomposites for bone grafting, Compos. Sci. Technol., 65: 2385-2406, 2005. [3] J. R. Porter, T. T. Ruckh and K. C. Popat, Bone Tissue Engineering: A Review in Bone Biomimetics and Drug Delivery Strategies, AlChE., 25: 1539-1560, 2009. [4] A. J. Salgado, O. P. Coutinho and R. L. Reis, Bone Tissue Engineering: State of the Art and Future Trends, Macromol. Biosci., 4: 743-765, 2004. [5] J. O. Hollinger, T. A. Einhorn, B. A. Doll and C. Sfeir, Bone tissue engineering Fundamentals, Boca Raton: CRC Press, 2005. [6] D. W. Hutmacher, J. T. Schantz, C. X. Lam, K. C. Tan and T. C. Lim, State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective, J. Tissue. Eng. Regen. Med., 1: 245-260, 2007. [7] K. Rezwan, Q. Z. Chen, J. J. Blaker and A. R. Boccaccini, Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering, Biomaterials, 27: 3413-3431, 2006. [8] L. Yang, M. Hedhammar, T. Blom, K. Leifer, J. Johansson, P. Habibovic and C. A. van Blitterswijk, Biomimetic calcium phosphate coatings on recombinant spider silk fibers, biomed. Mater., 5: 045002, 2010. [9] J. G. Hardy, L. M. Romer and T. R. Scheibel, Polymeric materials based on silk proteins, Polymer, 49: 4309-4327, 2008. [10] M. Yang, J. Kawamura, Z. Zhu, K. Yamauchi and T. Asakura, Development of silk-like materials based on Bombyx mori and Nephila clavipes dragline silk fibroins, Polymer, 50: 117-124, 2009. [11] C. Vepari and D. L. Kaplan, Silk as a biomaterial, Prog. Polym. Sci., 32: 991-1007, 2007. [12] G. H. Altman, F. Diaz, C. Jakuba, T. Calabro, R. L. Horan, J. Chen, H. Lu, J. Richmond and D. L. Kaplan, Silk-based biomaterials, Biomaterials, 24: 401-416, 2003. [13] U. J. Kim, J. Park, H. J. Kim, M. Wada and D. L. Kaplan, Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin, Biomaterials, 26: 2775-2785, 2005. [14] E. S. Sashina, A. M. Bochek, N. P. Novoselov and D. A. Kirichenko, Structure and Solubility of Natural Silk Fibroin, Russ. J. Appl. Chem., 79: 869-876, 2006. [15] C. S. Ki, I. C. Umb and Y. H. Park, Acceleration effect of sericin on shear-induced b-transition of silk fibroin, Polymer, 50: 4618-4625, 2009. [16] Z. H. Zhu, K. Ohgo and T. Asakura, Preparation and characterization of regenerated Bombyx mori silk fibroin fiber with high strength, Express Polym. 8

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Figures Caption: Figure 1. FTIR spectrum of extracted fibroin. Figure 2. (a) Porous scaffolds in different size and shape, SEM images of scaffolds (b) F8, (c) F4, (d) C1F4, (e) C2F4, (f) C3F4 Figure 3. (a) Compressive strength of scaffolds, (b) E module of scaffolds in compressive test Figure 4. Porosity of scaffolds, (n = 3; mean SD). Figure 5. Viability of cells by MTT assay, (n = 3; mean SD). Figure 6. SEM images of Osteoblast covered, (a) composite RS/NS scaffold, (b) RS scaffold after 4 days of cell seeding Figure 7. (a) and (b) SEM images of natural silk fibers one day after immersing in SBF, (c) and (d) SEM images of natural silk fibers after 21 days incubation in SBF, CaP needle shape crystals form on the fibers, (e) EDX chemical analysis of fibers after one day in SBF, (f) EDX chemical analysis of the fibers after 21 days in SBF, crystals reveals Ca and P peaks. Figure 8. SEM images of (a) C1F4 scaffold after one day incubation in SBF, (b) C1F4 scaffold after 21 days in incubation SBF, (c) EDX chemical analysis of C1F4 scaffold after one day in SBF, (d) EDX chemical analysis of C1F4 scaffold after 21 days in SBF. Figure 9. SEM images of (a) F4 scaffold after 21 days incubation in SBF, (b) F4 scaffold after 21 days incubation in SBF, (c) EDX chemical analysis of F4 scaffold after one day in SBF, (d) EDX chemical analysis of F4 scaffold after 21 days in SBF.

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Figure 3

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Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 582 583 584 585 586 587 588 589 590 591 592 593 594

Figure 4

595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 14

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 632 633 634 635 636 637 638 639 640 641 642 643

Figure 5 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661

662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 15

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697

Figure 6 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726

727 728 729 730 731 16

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764

Figure 7

765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 17

Figure 8

Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 782 783 784 785 786 787 788 789 790 Table1. Code and composition of the samples

791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827

Figure 9

Tables:

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Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 Sample code Composition 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 F4 4wt% Fibroin F8 8wt% Fibroin C1F4 90-10 V% RS/NS Composite C2F4 70-30 V% RS/NS Composite C3F4 50-50 V% RS/NS Composite

Table 2. Molarity of reagents for preparing SBF [24] Reagents Amount NaCl 5.403 g NaHCO3 0.504 g Na2CO3 0.426 g KCL 0.225 g K2HPO4.3H2O 0.230 g MgCl2.6H2O 0.311 g 0.2M-NaOH 100 ml HEPES 17.892 g CaCl2 0.293 g Na2SO4 0.072 g

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Articles in Press, J. Med. Biol. Eng. (Sep 7, 2012), doi: 10.5405/jmbe.1065 Table 3. Summary results of porosity and mechanical properties of 3D scaffolds Sample F4 F8 C1F4 C2F4 C3F4 874 875 Young Modulus (MPa) 1.114 1.87 0.795 2.75 3.42 Compressive strength(MPa) 1.027 1.38 0.75 1.85 1.87 Porosity 87.49% 83.46% 88% 87% 89%

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