- Email: [email protected]
silk fibroin composite scaffolds
Materials Letters 157 (2015) 85–88
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Fabrication and characterization of POM/ZrO2/silk fibroin composite scaffolds Abbas Teimouri a,n, Leila Ghorbanian b, Hossien Salavati a, Alireza Najafi Chermahini c a
Chemistry Department, Payame Noor University, 19395-4697 Tehran, Iran Torabinejad Dental Research center and Department of Oral and Maxillofacial Pathology, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran c Department of Chemistry, Isfahan University of Technology, Isfahan 841543111, Iran b
ar t ic l e i nf o
a b s t r a c t
Article history: Received 24 January 2015 Received in revised form 21 April 2015 Accepted 16 May 2015 Available online 23 May 2015
A novel porous polyoxometalates (POM)/zirconia/silk fibroin nanocomposite (POM/ZrO2/SF) was prepared by the freeze-drying method. XRD, FT-IR and SEM characterized the physicochemical structures of the products, demonstrating the formation of the scaffold with nano-architecture. The immersion of POM/ZrO2/SF scaffolds in the simulated body fluid (SBF) showed in vitro bone-bioactivity. The developed POM/ZrO2/SF scaffolds could be a promising candidate in preparing scaffolds for tissue engineering application. & 2015 Elsevier B.V. All rights reserved.
Keywords: Composite materials Nanocomposites Freeze drying technique
1. Introduction Silk fibroin is the structural protein obtained from the cocoon of the silk worm Bombyx mori. Silk proteins represent a unique choice in biomaterial selection for tissue engineering and regenerative medicine applications [1–3]. Due to its excellent biological properties, including good biocompatibility, biodegradability, and minimal inflammatory reaction [4], it can be used as a biomaterial in various forms [5], such as films [6,7], membranes [8], gels [9], sponges [10] and powders [11]. During recent years, nanocomposites based on inorganic nanoparticles and polymers have attracted considerable interest due to the unique properties resulting from the combination of inorganic nanoparticles and the polymeric matrix. [12]. It is believed that the assembly of POMsbased composites on the nanometer scale can provide a path for exhibiting their unique properties in many fields [13]. Because of their unique structural variety and exciting properties, as well as numerous potential applications in catalysis, biology and medicine, Polyoxometalates (POMs) are a significant class of inorganic compounds that have received considerable attention in the area of solid-state material chemistry [14]. Zirconia (ZrO2), due to its remarkable mechanical properties, chemical stability and biocompatibility, is a promising candidate for the production of bone scaffolds [15,16]. Zirconia has also been n
Corresponding author. E-mail address: [email protected] (A. Teimouri).
http://dx.doi.org/10.1016/j.matlet.2015.05.064 0167-577X/& 2015 Elsevier B.V. All rights reserved.
investigated for dentistry application [17,18]. Very recently, we have developed a convenient procedure for the synthesis of diopside/silk fibroin nanocomposite scaffolds for potential applications in maxillofacial bone regeneration [19]. Herein, we report a new route consisting of the synthesis of POM/ZrO2/silk fibroin nano-composite powder using the freeze-drying technique.
2. Materials and methods 2.1. Preparation of regenerated fibroin solution According to the protocol described previously, Silk fibroin (SF) was extracted from silk cocoons by some modifications [20]. Briefly, to remove the glue-like sericin proteins, the cocoons were boiled in aqueous solutions of 0.02 M Na2CO3 several times and then they were dried. After that, the dry degummed silk fibers were dissolved in LiBr, and the resulting solution was dialyzed by distilled water continuously for 3 days. Undissolved particles were removed by centrifugation. The concentration of the final fibroin solution, as determined by weighting the remaining solid after drying, was about 3.5% (w/w). 2.2. Preparation of the POM/ZrO2 nanocomposite According to the described method, the typical POM–ZrO2 substrate was prepared by the sol–gel method. [21] For the
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Fig. 1. (A)-XRD patterns and (B)-FTIR spectra of (a) POM-ZrO2, (b) POM, (c) pure SF, and (d) POM/ZrO2/SF scaffold.
Fig. 2. SEM images of (a) SF, (b) POM-ZrO2, and (c) POM-ZrO2/SF scaffold.
A. Teimouri et al. / Materials Letters 157 (2015) 85–88
87
Fig. 3. N2 adsorption–desorption isotherm of POM-ZrO2/SF nanocomposite, Inset: Pore size distribution using the BJH method.
Fig. 4. SEM images of apatite formation on POM-ZrO2/SF 14 days.
preparation of zirconia-supported POMs, a solution of zirconium (IV) n-butoxide (100 mmol, 38.35 g) in n-BuOH (30 ml) was stirred at 80 °C; then the mixture was slowly cooled at room temperature. Afterward, the pH of the mixture was reached to about 2 by using HCl. A similar solution of polyoxometalate was prepared by dissolving H3PW12O40 (3.08 g) in 25 ml ethanol and 10 ml of deionized water and then it was stirred. After that, the resultant solution was added to the zirconia solution drop wise. The obtained solid phase was filtered, washed with hot water several times and
Fig. 5. Light microscopy photographs (100 ) of the cell viability of HGF cells on POM-ZrO2/SF scaffold after they were cultured for 3 days.
dried at 120 °C for 24 h. The dried gel was calcined in vacuum at 350 °C for 4 h. 2.3. Preparation of POM/ZrO2/silk fibroin nanocomposite powder The POM/ZrO2/silk fibroin composites were prepared through the freeze-drying method. Initially, POM/ZrO2 powder was added into the SF solution to obtain the samples. This was followed by
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A. Teimouri et al. / Materials Letters 157 (2015) 85–88
ultrasonification for 30 min. The mixture was poured into a precooled 24-well plate and put at the temperature of 20 °C for an hour; then it was transferred to a freezer at the temperature of 80 °C for 12 h and finally, freeze-dried in a freeze-dryer for 3 days. 2.4. Evaluation of polyoxometalate/silk fibroin nano-composite powder properties The structural morphology of the samples was evaluated using scanning electron microscope (SEM). The samples were analyzed by X-ray diffraction (XRD) using a Philips X’PERT MPD X-ray diffractometer (XRD). A JASCO FT/IR-680 PLUS spectrometer was used to record IR spectra using KBr pellets. 2.5. Bioactive and cell adhesion study in vitro The composite scaffolds were soaked in SBF according to the procedure described in Kokubo et al. [22]. At the designed interval time, scaffolds were taken out and the formation of apatite was observed by SEM. Fibroblast cells were cultivated in culture flasks for 2 weeks, and cell suspension 2 105 cells/cm2 was seeded in scaffolds and cultured in 24-well plates. Cell-scaffolds were cultured in a humidified incubator. After 72 h, the cell-scaffolds were taken out and observed by SEM.
3. Results and discussion The XRD patterns of the POM, POM–ZrO2, pure SF and POM/ ZrO2/SF nanocomposite are shown in Fig. 1A. The presence and dispersion of POM on the silk fibroin were investigated using the XRD technique. The sharp diffraction lines at 2θ ¼28°, 34°, 51° and 60° corresponded to the tetragonal ZrO2 phase. Loading of POM species caused the appearance of new peaks at 2θ ¼ 16°, 28°, 31.5° and 35°. The XRD studies on POM/ZrO2/SF shown in 1Bd revealed the characteristic peaks of both silk fibroin (2θ ¼8.9° and 21.8°) and POM/ZrO2 (2θ ¼16°, 28°, 31.5°, 35°, 51° and 60°) and suggested the presence of both in the scaffold [23]. The crystallite size of the crystalline phase was determined from the peak of maximum intensity using the Debye-Scherrer equation, D¼Kλ/β cos θ [24]. Fig. 1B shows the FTIR spectra of the prepared samples. The broad and strong band at about 600 cm 1 could be attributed to the Zr–O stretching vibration of tetragonal ZrO2[25] (Fig. 1Ba). The FTIR spectrum of the pure POM (Fig. 1Bb) showed four characteristic bands in the region of 1100–700 cm 1: 1080, 980, 890 and 802 cm 1. The positions of these bands could be assigned, in turn, to the stretching vibrations of P–O, W¼O and W–O–W, and the bending vibration of P–O [26]. These groups were connected to each other by cornersharing oxygens [27]. Pure silk fibroin showed absorption bands at 1650 (amide I), 1530 (amide II) and 1250 cm 1, which were attributed to the β-sheet conformation of silk fibroin [28]. Fig. 2 shows the SEM images of SF, POM/ZrO2 and POM/ZrO2/silk fibroin nano-composites. Pure SF exhibited a macroporous structure with interconnected open pores, and pore sizes varied from 100 to 200 μm (Fig. 2a). The SEM image of the POM/ZrO2 showed that particles had sizes in the range of nanometers (Fig. 2b). SEM image also revealed that POM/ZrO2/SF nanocomposite was composed of aggregated extremely fine particles (Fig. 2c). Specific surface area and the pore size distribution of POM/ ZrO2/SF nanocomposite were determined from N2 adsorption–desorption isotherms (Fig. 3). The surface area was calculated by applying the BET equation to the isotherm [29]. The pore size distribution was calculated by BJH method [30]. The surface area and pore volume of the POM-ZrO2/SF nanocomposite catalysts were in the range of 40–49 m2 g 1 and 0.59–0.71 cm3/g, respectively.
POM/ZrO2/SF scaffold was mesoporous, with N2 adsorption–desorption isotherms of type IV according to the IUPAC classification. Fig. 4 shows that the SEM images, after incubation and formation of an apatite-like layer, can be viewed on the surface of the POM-ZrO2/SF scaffolds. Precipitation started at individual granules gradually developed on the specimen surface and the inner pore after 2 weeks. Fig. 5 shows the morphological features of fibroblasts cultured on the scaffold for 3 days. The results showed an increase of cell activity in culture media containing POM-ZrO2/SF scaffold during incubation, thereby indicating no cytotoxic effect on cell survival and growth.
4. Conclusions In this article, POM-ZrO2/SF scaffolds were fabricated successfully using the freeze-drying method. The physicochemical properties of the composites were characterized by XRD, FT-IR and SEM. The POM–ZrO2/SF scaffolds also showed biocompatibility with the fibroblast cells. The results indicated the prepared POM-ZrO2/SF scaffold could be suitable for using in tissue engineering.
Acknowledgment Supports from the Payame Noor University in Isfahan Research Council (Grant # 62370) and contribution from Isfahan University of Technology are gratefully acknowledged.
References [1] Arai T, Freddi G, Innocenti R, Tsukada M. J Appl Polym Sci 2004;91:2383–90. [2] Jin HJ, Park J, Karageorgiou V, Kim UJ, Valluzzi R, Cebe P. Adv Funct Mater 2005;15:1241–7. [3] Meinel L, Hofmann S, Karageorgiou V, Kirker-Head C, McCool J, Gronwicz G. Biomaterials 2005;26:147–55. [4] Vepari C, Kaplan DL. Prog Polym Sci (Oxford) 2007;32(8–9):991–1007. [5] Jyh-Ping Chen, Shih-Hsien Chen, Guo-Jyun Lai. Res Lett 2012;7:170. [6] Acharya C, Ghos SK, Kundu SC. J Mater Sci Mater Med 2008;19:2827–36. [7] Kundu J, Dewan M, Ghoshal S, Kundu SC. J Mater Sci Mater Med 2008;19:2679–89. [8] Minoura N, Tsukada M, Nagura M. Biomaterials 1990;11:430–4. [9] Fini M, Motta A, Torricelli P, Giavaresi G, Aldini NN, Tschon M, Giardino R, Migliaresi C. Biomaterials 2005;26:3527–36. [10] Li M, Ogiso M, Minoura N. Biomaterials 2003;24:357–65. [11] Hino T, Tanimoto M, Shimabayashi S. J Colloid Interface Sci 2003;266:68–73. [12] Kango S, Kalia S, Celli A, Njuguna J, Habibi Y, Kumar R. Prog Polym Sci 2013;38:1232–61. [13] Kulesza PJ, Chojak M, Karnicka K, Miecznikowski K, Palys B, Lewera A, Wieckowski A. Chem Mater 2004;16:4128–34. [14] Zhang L, Shi Z, Li Zhang, Zhou Y, Hassan S. Mater Lett 2012;86:62–4. [15] Kim HW, Kim HE, Knowles JC. J Biomed Mater Res B: Appl Biomater 2004;70B (2):270–7. [16] Majeti NVR. React Funct Polym 2000;46:1–27. [17] Zhensheng L, Ramay HR, Hauch KD, Xiao D, Zhang M. Biomaterials 2006;26:3919–28. [18] An Sang-Hyun, Matsumoto T, Miyajima H, Nakahira A, Kim Kyo-Han, Imazato S. Dent Mater 2012;28:1221–31. [19] Ghorbanian L, Emadi R, Razavi SM, Shin H, Teimouri A. Int J Biol Macromol 2013;58:275–80. [20] Rockwood DN, Preda RC, Yucel T, Xiaoqin Wang, Lovett ML, Kaplan DL. Nat Protoc 2011;10:1612–31. [21] Farhadi S, Zaidi M. Appl Catal A: Gen 2009;354:119–26. [22] Kokubo T, Takadama H. Biomaterials 2006;27:2907–15. [23] Wenk E, Merkle HP, Meinel L. J Control Release 2011;150(2):128–41. [24] Jermy BR, Pandurangan A. Appl Catal A: Gen 2005;295:185–92. [25] Nakamoto K. Infrared and Raman spectra of inorganic and coordination compounds. 4th ed.. New York: Wiley; 1986. [26] Rocciccioli-Deltcheff C, Frank M, Thouvenot R. Inorg Chem 1983;22:207–16. [27] Okuhara T, Mizuno N, Misono M. Adv Catal 1996;41:113–252. [28] Ni S, Chou L, Chang J. Ceram Int 2007;33:83–8. [29] Brunauer S, Emmett PH, Teller EJ. Am Chem Soc 1938;60:309–19. [30] Gregg SJ, Sing KSW. Adsorption, surface area and porosity. second edition. New York: Academic Press; 1982.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Fabrication and characterization of POM/ZrO2/silk fibroin composite scaffolds Abbas Teimouri a,n, Leila Ghorbanian b, Hossien Salavati a, Alireza Najafi Chermahini c a
Chemistry Department, Payame Noor University, 19395-4697 Tehran, Iran Torabinejad Dental Research center and Department of Oral and Maxillofacial Pathology, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran c Department of Chemistry, Isfahan University of Technology, Isfahan 841543111, Iran b
ar t ic l e i nf o
a b s t r a c t
Article history: Received 24 January 2015 Received in revised form 21 April 2015 Accepted 16 May 2015 Available online 23 May 2015
A novel porous polyoxometalates (POM)/zirconia/silk fibroin nanocomposite (POM/ZrO2/SF) was prepared by the freeze-drying method. XRD, FT-IR and SEM characterized the physicochemical structures of the products, demonstrating the formation of the scaffold with nano-architecture. The immersion of POM/ZrO2/SF scaffolds in the simulated body fluid (SBF) showed in vitro bone-bioactivity. The developed POM/ZrO2/SF scaffolds could be a promising candidate in preparing scaffolds for tissue engineering application. & 2015 Elsevier B.V. All rights reserved.
Keywords: Composite materials Nanocomposites Freeze drying technique
1. Introduction Silk fibroin is the structural protein obtained from the cocoon of the silk worm Bombyx mori. Silk proteins represent a unique choice in biomaterial selection for tissue engineering and regenerative medicine applications [1–3]. Due to its excellent biological properties, including good biocompatibility, biodegradability, and minimal inflammatory reaction [4], it can be used as a biomaterial in various forms [5], such as films [6,7], membranes [8], gels [9], sponges [10] and powders [11]. During recent years, nanocomposites based on inorganic nanoparticles and polymers have attracted considerable interest due to the unique properties resulting from the combination of inorganic nanoparticles and the polymeric matrix. [12]. It is believed that the assembly of POMsbased composites on the nanometer scale can provide a path for exhibiting their unique properties in many fields [13]. Because of their unique structural variety and exciting properties, as well as numerous potential applications in catalysis, biology and medicine, Polyoxometalates (POMs) are a significant class of inorganic compounds that have received considerable attention in the area of solid-state material chemistry [14]. Zirconia (ZrO2), due to its remarkable mechanical properties, chemical stability and biocompatibility, is a promising candidate for the production of bone scaffolds [15,16]. Zirconia has also been n
Corresponding author. E-mail address: [email protected] (A. Teimouri).
http://dx.doi.org/10.1016/j.matlet.2015.05.064 0167-577X/& 2015 Elsevier B.V. All rights reserved.
investigated for dentistry application [17,18]. Very recently, we have developed a convenient procedure for the synthesis of diopside/silk fibroin nanocomposite scaffolds for potential applications in maxillofacial bone regeneration [19]. Herein, we report a new route consisting of the synthesis of POM/ZrO2/silk fibroin nano-composite powder using the freeze-drying technique.
2. Materials and methods 2.1. Preparation of regenerated fibroin solution According to the protocol described previously, Silk fibroin (SF) was extracted from silk cocoons by some modifications [20]. Briefly, to remove the glue-like sericin proteins, the cocoons were boiled in aqueous solutions of 0.02 M Na2CO3 several times and then they were dried. After that, the dry degummed silk fibers were dissolved in LiBr, and the resulting solution was dialyzed by distilled water continuously for 3 days. Undissolved particles were removed by centrifugation. The concentration of the final fibroin solution, as determined by weighting the remaining solid after drying, was about 3.5% (w/w). 2.2. Preparation of the POM/ZrO2 nanocomposite According to the described method, the typical POM–ZrO2 substrate was prepared by the sol–gel method. [21] For the
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A. Teimouri et al. / Materials Letters 157 (2015) 85–88
Fig. 1. (A)-XRD patterns and (B)-FTIR spectra of (a) POM-ZrO2, (b) POM, (c) pure SF, and (d) POM/ZrO2/SF scaffold.
Fig. 2. SEM images of (a) SF, (b) POM-ZrO2, and (c) POM-ZrO2/SF scaffold.
A. Teimouri et al. / Materials Letters 157 (2015) 85–88
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Fig. 3. N2 adsorption–desorption isotherm of POM-ZrO2/SF nanocomposite, Inset: Pore size distribution using the BJH method.
Fig. 4. SEM images of apatite formation on POM-ZrO2/SF 14 days.
preparation of zirconia-supported POMs, a solution of zirconium (IV) n-butoxide (100 mmol, 38.35 g) in n-BuOH (30 ml) was stirred at 80 °C; then the mixture was slowly cooled at room temperature. Afterward, the pH of the mixture was reached to about 2 by using HCl. A similar solution of polyoxometalate was prepared by dissolving H3PW12O40 (3.08 g) in 25 ml ethanol and 10 ml of deionized water and then it was stirred. After that, the resultant solution was added to the zirconia solution drop wise. The obtained solid phase was filtered, washed with hot water several times and
Fig. 5. Light microscopy photographs (100 ) of the cell viability of HGF cells on POM-ZrO2/SF scaffold after they were cultured for 3 days.
dried at 120 °C for 24 h. The dried gel was calcined in vacuum at 350 °C for 4 h. 2.3. Preparation of POM/ZrO2/silk fibroin nanocomposite powder The POM/ZrO2/silk fibroin composites were prepared through the freeze-drying method. Initially, POM/ZrO2 powder was added into the SF solution to obtain the samples. This was followed by
88
A. Teimouri et al. / Materials Letters 157 (2015) 85–88
ultrasonification for 30 min. The mixture was poured into a precooled 24-well plate and put at the temperature of 20 °C for an hour; then it was transferred to a freezer at the temperature of 80 °C for 12 h and finally, freeze-dried in a freeze-dryer for 3 days. 2.4. Evaluation of polyoxometalate/silk fibroin nano-composite powder properties The structural morphology of the samples was evaluated using scanning electron microscope (SEM). The samples were analyzed by X-ray diffraction (XRD) using a Philips X’PERT MPD X-ray diffractometer (XRD). A JASCO FT/IR-680 PLUS spectrometer was used to record IR spectra using KBr pellets. 2.5. Bioactive and cell adhesion study in vitro The composite scaffolds were soaked in SBF according to the procedure described in Kokubo et al. [22]. At the designed interval time, scaffolds were taken out and the formation of apatite was observed by SEM. Fibroblast cells were cultivated in culture flasks for 2 weeks, and cell suspension 2 105 cells/cm2 was seeded in scaffolds and cultured in 24-well plates. Cell-scaffolds were cultured in a humidified incubator. After 72 h, the cell-scaffolds were taken out and observed by SEM.
3. Results and discussion The XRD patterns of the POM, POM–ZrO2, pure SF and POM/ ZrO2/SF nanocomposite are shown in Fig. 1A. The presence and dispersion of POM on the silk fibroin were investigated using the XRD technique. The sharp diffraction lines at 2θ ¼28°, 34°, 51° and 60° corresponded to the tetragonal ZrO2 phase. Loading of POM species caused the appearance of new peaks at 2θ ¼ 16°, 28°, 31.5° and 35°. The XRD studies on POM/ZrO2/SF shown in 1Bd revealed the characteristic peaks of both silk fibroin (2θ ¼8.9° and 21.8°) and POM/ZrO2 (2θ ¼16°, 28°, 31.5°, 35°, 51° and 60°) and suggested the presence of both in the scaffold [23]. The crystallite size of the crystalline phase was determined from the peak of maximum intensity using the Debye-Scherrer equation, D¼Kλ/β cos θ [24]. Fig. 1B shows the FTIR spectra of the prepared samples. The broad and strong band at about 600 cm 1 could be attributed to the Zr–O stretching vibration of tetragonal ZrO2[25] (Fig. 1Ba). The FTIR spectrum of the pure POM (Fig. 1Bb) showed four characteristic bands in the region of 1100–700 cm 1: 1080, 980, 890 and 802 cm 1. The positions of these bands could be assigned, in turn, to the stretching vibrations of P–O, W¼O and W–O–W, and the bending vibration of P–O [26]. These groups were connected to each other by cornersharing oxygens [27]. Pure silk fibroin showed absorption bands at 1650 (amide I), 1530 (amide II) and 1250 cm 1, which were attributed to the β-sheet conformation of silk fibroin [28]. Fig. 2 shows the SEM images of SF, POM/ZrO2 and POM/ZrO2/silk fibroin nano-composites. Pure SF exhibited a macroporous structure with interconnected open pores, and pore sizes varied from 100 to 200 μm (Fig. 2a). The SEM image of the POM/ZrO2 showed that particles had sizes in the range of nanometers (Fig. 2b). SEM image also revealed that POM/ZrO2/SF nanocomposite was composed of aggregated extremely fine particles (Fig. 2c). Specific surface area and the pore size distribution of POM/ ZrO2/SF nanocomposite were determined from N2 adsorption–desorption isotherms (Fig. 3). The surface area was calculated by applying the BET equation to the isotherm [29]. The pore size distribution was calculated by BJH method [30]. The surface area and pore volume of the POM-ZrO2/SF nanocomposite catalysts were in the range of 40–49 m2 g 1 and 0.59–0.71 cm3/g, respectively.
POM/ZrO2/SF scaffold was mesoporous, with N2 adsorption–desorption isotherms of type IV according to the IUPAC classification. Fig. 4 shows that the SEM images, after incubation and formation of an apatite-like layer, can be viewed on the surface of the POM-ZrO2/SF scaffolds. Precipitation started at individual granules gradually developed on the specimen surface and the inner pore after 2 weeks. Fig. 5 shows the morphological features of fibroblasts cultured on the scaffold for 3 days. The results showed an increase of cell activity in culture media containing POM-ZrO2/SF scaffold during incubation, thereby indicating no cytotoxic effect on cell survival and growth.
4. Conclusions In this article, POM-ZrO2/SF scaffolds were fabricated successfully using the freeze-drying method. The physicochemical properties of the composites were characterized by XRD, FT-IR and SEM. The POM–ZrO2/SF scaffolds also showed biocompatibility with the fibroblast cells. The results indicated the prepared POM-ZrO2/SF scaffold could be suitable for using in tissue engineering.
Acknowledgment Supports from the Payame Noor University in Isfahan Research Council (Grant # 62370) and contribution from Isfahan University of Technology are gratefully acknowledged.
References [1] Arai T, Freddi G, Innocenti R, Tsukada M. J Appl Polym Sci 2004;91:2383–90. [2] Jin HJ, Park J, Karageorgiou V, Kim UJ, Valluzzi R, Cebe P. Adv Funct Mater 2005;15:1241–7. [3] Meinel L, Hofmann S, Karageorgiou V, Kirker-Head C, McCool J, Gronwicz G. Biomaterials 2005;26:147–55. [4] Vepari C, Kaplan DL. Prog Polym Sci (Oxford) 2007;32(8–9):991–1007. [5] Jyh-Ping Chen, Shih-Hsien Chen, Guo-Jyun Lai. Res Lett 2012;7:170. [6] Acharya C, Ghos SK, Kundu SC. J Mater Sci Mater Med 2008;19:2827–36. [7] Kundu J, Dewan M, Ghoshal S, Kundu SC. J Mater Sci Mater Med 2008;19:2679–89. [8] Minoura N, Tsukada M, Nagura M. Biomaterials 1990;11:430–4. [9] Fini M, Motta A, Torricelli P, Giavaresi G, Aldini NN, Tschon M, Giardino R, Migliaresi C. Biomaterials 2005;26:3527–36. [10] Li M, Ogiso M, Minoura N. Biomaterials 2003;24:357–65. [11] Hino T, Tanimoto M, Shimabayashi S. J Colloid Interface Sci 2003;266:68–73. [12] Kango S, Kalia S, Celli A, Njuguna J, Habibi Y, Kumar R. Prog Polym Sci 2013;38:1232–61. [13] Kulesza PJ, Chojak M, Karnicka K, Miecznikowski K, Palys B, Lewera A, Wieckowski A. Chem Mater 2004;16:4128–34. [14] Zhang L, Shi Z, Li Zhang, Zhou Y, Hassan S. Mater Lett 2012;86:62–4. [15] Kim HW, Kim HE, Knowles JC. J Biomed Mater Res B: Appl Biomater 2004;70B (2):270–7. [16] Majeti NVR. React Funct Polym 2000;46:1–27. [17] Zhensheng L, Ramay HR, Hauch KD, Xiao D, Zhang M. Biomaterials 2006;26:3919–28. [18] An Sang-Hyun, Matsumoto T, Miyajima H, Nakahira A, Kim Kyo-Han, Imazato S. Dent Mater 2012;28:1221–31. [19] Ghorbanian L, Emadi R, Razavi SM, Shin H, Teimouri A. Int J Biol Macromol 2013;58:275–80. [20] Rockwood DN, Preda RC, Yucel T, Xiaoqin Wang, Lovett ML, Kaplan DL. Nat Protoc 2011;10:1612–31. [21] Farhadi S, Zaidi M. Appl Catal A: Gen 2009;354:119–26. [22] Kokubo T, Takadama H. Biomaterials 2006;27:2907–15. [23] Wenk E, Merkle HP, Meinel L. J Control Release 2011;150(2):128–41. [24] Jermy BR, Pandurangan A. Appl Catal A: Gen 2005;295:185–92. [25] Nakamoto K. Infrared and Raman spectra of inorganic and coordination compounds. 4th ed.. New York: Wiley; 1986. [26] Rocciccioli-Deltcheff C, Frank M, Thouvenot R. Inorg Chem 1983;22:207–16. [27] Okuhara T, Mizuno N, Misono M. Adv Catal 1996;41:113–252. [28] Ni S, Chou L, Chang J. Ceram Int 2007;33:83–8. [29] Brunauer S, Emmett PH, Teller EJ. Am Chem Soc 1938;60:309–19. [30] Gregg SJ, Sing KSW. Adsorption, surface area and porosity. second edition. New York: Academic Press; 1982.