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In vitro bioactivity and MG63 cell compatibility of chitosan silicate hybrid

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  • Save International Journal of M aterials and Chemistry 2013, 3(3A): 1-7 DOI: 10.5923/s.ijmc.201303.01 In Vitro Bioactivity and MG63 Cytocompatibility of Chitosan-Silicate Hybrids Yuki Shirosaki1,*, Tomoyuki Okayama2, Kanji Tsuru3, Satoshi Hayakawa2, Akiyoshi Osaka2 1Frontier Research Acad emy for Young Researchers, Hibik ino, Wak amatsu-ku, Kitaky ushu, 8080196, Jap an 2Graduate School of Natural Science and Technology, Okayama University, Tsushima, Kita-ku, Okayama, 7008530, Japan 3Faculty of Dental Science, Kyushu University, M aidashi, Higashi-ku, Fukuoka, 8128582, Japan Abstract Th is paper describes in vitro bioactivity and cytocompatibility of ch itosan-silicate hybrids derived fro m chitosanand γ-glycido xypropyltrimetho xysilane (GPTMS) through sol-gel processing. Chitosan, one of the biodegradable polymers, was selected as a hybrid matrix whereas GPTMS was employed as crosslinking agent as well as bioactive inorganic species. GPTM S was hydrolyzed co mpletely in acetic-acid solution to yield Si-OH groups, which in turn were condensed to form Si-O-Si netwo rks. Cytotoxicity of GPTM S mono mer was much lower than that of glutaraldehyde. Fro m MG63 osteoblastic cell cu lture, the ch itosan-GPTMS hybrids were cytocompatible better than the hybrids crosslinked with glutaraldehyde, tetraethoxysilane or g lycidylmethaacrylate. The hybrids incorporated with Ca ions deposited apatite on their surface under a simu lated body fluid (SBF, Kokubo solution). Freeze-dry ing lead to porous hybrids with about 90% in porosity and about 100 μm in size. When soaked in SBF, the porous deposited apatite not only on the outer surface but also on the pore wall. The porous hybrids hence are considered to be appropriate materials for scaffo lds. Keywords Chitosan, γ-glycido xypropyltrimetho xysilane (GPTMS), Organic-Inorganic Hybrid, Scaffold, Cytocompatibility, Apatite, Simu lated Body Flu id 1. Introduction The success of tissue engineering primarily depends on the scaffolds that serve to support and reinforce the reg ener ating t issue. The scaffo lds should not on ly p ro mote cell adhesion, cell pro liferat ion, and cell differentiation, but also sh ou ld b e b io co mp at ib le, b io deg rad ab le, an d h igh ly porous[1]. Chitosan consists of polysaccharide chains, and is one of the candidate materials for scaffolds since they are bioresorbable as well as biocompatib le, non-antigenic and non-toxic[2]. Yet their inferiority in mechanical properties and too high in vivo biodegradability are disadvantageous for most medical applicat ions. Hence, mechanical strength, and biodegradation are to be controlled within proper allowance or an acceptab le range befo re ch itosan is emp loyed for scaffolds. For such purposes, cross-lin king thepolysacch ari de chains with a non-to xic agent to form a networked is essential. Various cross-linking agents have been attempted such as epoxy compounds and aldehydes[3, 4], whereas they are all h ighly cytotoxic and may spoil the b iocompatib ility of ch itosan . Sh irosaki et al .[5] hav e already po inted out thatγ-glycidoxypropyltrimethoxysilane (GPTMS) is a good * Corresponding author: (Yuki Shirosaki) Published online at Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved cross-lin king agent for chitosan, and that the chitosanGPTM S hybrids are far better than chitosan in cytocompati bility with hu man osteosarcoma cell M G63. Moreover, they might induce apatite nucleation and growth under the body environment or in a simulated body fluid (Kokubo solution,) since they involve inorganic species (silanol groups, Ca2+) that are considered essential for inducing the apatite nucleat ion[6]. Such ability of spontaneous apatite deposition is often denoted as bioactivity. If they are bioactive, they might be fixed in tissue defects with forming direct bonds. That is, they are employable as scaffolds, tissue substitutes, or filler for tissue defects. In this study, we examined in vitro bioacti vity and cytocompatibility of chitosan-GPTMS hybrids as well as the structure of their hybrids. Moreover, freeze drying technique is used to prepare porous chitosan -GPTMS hybrids and their applicability to tissue engineering scaffolds is explored. 2. Materials and Methods 2.1. Preparati on of the Hybri ds An appropriate amount of chitosan (high mo lecular weight, deacetylation: 79.0%, Ald rich®, USA) was dissolved in 0.25M acetic acid aqueous solution to attain a concentration of 2% (w/v), to wh ich γ-glycido xypropyltrimetho xysilane (GPTMS; Ch isso, Tokyo, Japan , CH2OCHCH2OCH2CH2Si 2 Yuki Shirosaki et al.: In Vitro Bioactivity and M G63 Cytocompatibility of Chitosan-Silicate Hybrids (OCH3)3) and calciu m chlo ride (CaCl2, Nacalai Tesque, Kyoto, Japan) were added so that the solutions with compositions in Table 1 were obtained. Here, 1mole chitosan stands for 1 mole deacetylated amino groups of their unit. Table 1 also listed the samples codes. Tetraethoxysilane (TEOS), g lutaraldehyde (GA), and glycidylmethacrylate (GMA) were used as another crosslinking reagents for comparison. After stirring at room temperature for 1h, the resultant solutions were poured into polypropylene containe rs. The resultant solutions were aged in the shielded polypropylene containers at 60°C for 2 days. Then, the gel-like hybrids were dried at 60°C for 2 days to yield hybrid memb ranes. The obtained membranes were soaked in 0.25N sodium hydroxide to neutralize and wash out the remaining acetic acid, and rinsed well with d istilled water. In addit ion, free ze-drying was used to derive porous hybrids: A fraction of each resultant sol was poured into a polystyrene container, and fro zen at -20°C for 24h in the refrigerator befo re vacuum drying. Table 1. Starting composition of the hybrids (molar ratio) Sample Chitosan GPT MS CaCl2 GA TEOS GMA Ch 1.0 ChCa05 1.0 0.5 ChG10 1.0 1.0 ChG10Ca005 1.0 1.0 0.05 ChG10Ca01 1.0 1.0 0.1 ChG10Ca025 1.0 1.0 0.25 ChG10Ca05 1.0 1.0 0.5 ChGA05 1.0 0.5 ChT10 1.0 1.0 ChGMA10 1.0 1.0 2.2. Structural Characterizati on of the Hybri ds Fourier-transform infrared (FT-IR) spectra were measured with a FT-IR spectrometer (Nexus470, Thermo Nicolet) using the KBr method, where the signals fro m 128 scans were accu mulated with a resolution of 4 cm-1. The pore diameter was evaluated with the optical microscope images. Outlines of the pore were traced and the maximu m diameter was derived using Image-Pro Plus software (Planetron, Tokyo, Japan). In each procedure, at least twenty pores were picked up fro m three different areas of a sample. The density of the scaffolds was derived fro m the weight and geometric volume. 2.3. Cell Culture 2.3.1. Cytotoxicity of the Cross-linking Agents On degradation under the body environment, the hybrids may release several frag ments into plas ma. Since, chitosan frag ments have been proved non-toxic. It should be the first thing to examine cytotoxicity of debris fro m cross-linking reagents: it was evaluated in vitro using osteoblastic cells MG63 cells (DAINIPPON PHA RMACEUTICA L Co., LTD, Japan). M G63 cells were seeded in 96-well p lates at 6400 cells per well (2x104 cells·c m-2) in 250 μl D-M EM with 10% FBS, 1 % penicillin and streptomycin solution and 2.5 μg ml-1 fungizone and supplemented with 50 μg ml-1 ascorbic acid, 1% M EM-non essential amino acids and 2.0 mM ml-1 L-glutamine. GA and GPTMS were added to the culturing med ia up to 100 pp m and 1000 pp m, respectively. The cell culture system was maintained in a humidified incubator at 36.5°C with 5% CO2 in air. After 24h of culture, surviving cell nu mbers were determined indirectly by MTT assay. MTT50 represents the concentration of each cross-linking agent where the absorbance were half that of the cells without agent. 2.3.2. Cytocompatib ility of the So lid Memb rane The solid membranes were soaked in PBS (pH7.4) solution and sterilized by autoclaving at 121°C for 20 min. MG63 cells were cu ltured (104 cells·cm-2) under control conditions (absence of materials, standard plastic culture plates) and on the surface of the solid membranes. The cultures were evaluated throughout the incubation time at days 1,3, 5, and 7 for cell v iability and observed by scanning electron microscopy (SEM ). The cell v iability was determined by MTT assay. 2.4. Eval uation of i n Vitro Apatite Formati on SBF was prepared as described in the literatures[6]. It has been proved that in vivo apatite format ion on bioactive implants is almost fully reproduced under in vitro experiments in SBF. The solid membranes (30x10x1mm2) and porous scaffolds were soaked in 30 ml SBF held in capped polystyrene bottles at 36.5°C up to 14 days as pH was monitored. Then, they were gently rinsed with distilled water and dried at roo m temperature. Their surfaces were characterized using an X-ray d iffracto meter with a thin-film measuring attachment (TF-XRD): RAD-II AX, CuKα, (Rigaku, To kyo, Japan), operated at 40kV-20mA accelerati on. The surface morphology was observed under a scanning electron microscope (SEM : JEOL, JMSM -6300, To kyo, Japan) equipped with an energy-dispersion X-ray (EDX) analyzer (model DX-4, Phillips). The concentrations of the component elements released into SBF were measured by inductively coupled plasma emission spectroscopy (ICP; ICPS-7500, Sh imazu, Japan). 3. Results and Discussion 3.1. The Soli d Membrane Fig. 1 shows the FT-IR absorption spectra of the specime ns. The bands at 1650, 1565, and 1110-1000 cm-1 were assigned to the constituent bonds of chitosan and GPTMS[5]. Growth of a co mponent (Si-O) in the band at 1100-1000 cm-1 confirmed the format ion of Si-O-Si bridging bonds on hybridizat ion[5]. Difference in IR profile was detected between Ch G10Ca05 and Ch G10 in the peaks assigned to the amino groups. This should be attributed to the presence of Ca(II) in the system, suggesting that the chemical environment around the amino groups is different a mong the International Journal of M aterials and Chemistry 2013, 3(3A): 1-7 3 solid memb ranes with an without Ca(II). Nishi et al.[7] though no more define data are availab le at this mo ment, one suggested the presence of good interactions between the might suppose the presence of any calcium-amino group amino groups of chitosan and Ca2+ to form co mplexes. Thus, interactions. Figure 1. FT -IR absorption spectra of (a) ChG10Ca05, (b) ChG10, and (c) Ch Fi gure 2. MTT assay for MG63 cells cult ured in t he media added with GA (a) and GPTMS (b): ~25 and ~500 ppm for MTT50, respect ively. See text for MTT50 Figure 3. Morphology of MG63 cells cultured for 1 day on the solid membranes (a) Ch without cross-linking agent, and cross-linked with (b) GPT MS(ChG10), (c) GA(ChGA05), (d) GMA(ChGMA10), and (e) TEOS(ChT10), respectively 4 Yuki Shirosaki et al.: In Vitro Bioactivity and M G63 Cytocompatibility of Chitosan-Silicate Hybrids The MTT assay results for MG63 were presented in Fig. 2 as a function of the GA and GPTMS concentration in the culturing med ia, together with MTT50. The MTT assay in Fig, 2(a) indicated that the cell viability decreased rapidly with GA , approaching to 25 ppm in MTT50. In contrast, Fig 2(b) showed the cell viability remained up to ~200 pp m as high as that for the media without GPTM S, then it gradually decreased to give 500 pp m in MTT50. Therefore, it is concluded that GPTMS mono mer was about 20 times less cytotoxic than GA. Fig. 3 shows morphology of M G63 cells cultured for 1 day on the solid memb ranes cross-linked with (b) GPTMS (Ch G10), (c) GA (Ch GA05), (d) GMA (Ch GMA10), and (e) TEOS (ChT10), respectively. Few cells were adhered on (a) Ch, (b) Ch G10, and (e) ChT10, were some on (a ) and (e) were spherical but those on ChG10 had many pseudopodia and spread out. In contrast, the cells on (b) Ch GA 05 found burst indicating that the cells were dead. Here, the cytotoxicity of GA was also confirmed. MTT assay results in Fig. 4 presented above tendency for the MG63 cells cultured on the solid me mbranes (a) through (e). The cells grew far better on (b) Ch G10 and (e) ChT10 than on any other memb ranes. In part icular, Ch G10 and ChT10 showed similar effects, while at 7 days Ch G10 exceeded ChT10. Cu ltures (a) Ch and (d) Ch GMA10gave mostly similar results throughout the culture period. It would not be surprising that the cells on (c) Ch GA05 became ext inct completely. The greatest viability of the cells for (b) Ch G10 was in excellent accordance with Fig. 5 that showed the morphology of M G63 cells cultured for 7 days on ChG10 (Fig. 4(b)) and ChT10 (Fig. 4(e)). That is, the cells covered completely the surface o f Ch G10, reaching a confluent stage, but ChT10 showed much in ferior coverage by the cells. Fro m those results, it was concluded that GPTMS is the best cross-lin king agent over GMA and TEOS, while GA is the wo rs t. Figure 4. Cell proliferation (MTTassay) of MG63 cells cultured on the solid membranes (a) Ch without cross-linking agent, and cross-linked with (b) GPT MS(ChG10), (c) GA(ChGA05), (d) GMA(ChGMA10), and (e) TEOS(ChT10), respectively Figure 5. Morphology of MG63 cells cultured for 7d on ChG10 (left) and ChT10 (right) International Journal of M aterials and Chemistry 2013, 3(3A): 1-7 5 Fig. 6(a) thorough (g) shows TF-XRD patterns for the solid membranes after soaked in SBF up to 14 days. The diffractions for the as-prepared membranes were denoted as “ 0d ”. Apatite did not deposit on (a) Ch, (b) Ch CA05, or (c) Ch G10 within 14 days but on the other four Ch G10 samples that involved Ca2+ ions. The calciu m content shortened the induction period, such as ~14d, ~7d, ~7d, and 3d for (d) Ch G10Ca005, (e) Ch G10Ca01, (f) Ch G10Ca025, and (g)Ch G10Ca05, respectively. Fig. 7(a) through (d) shows SEM images of the surface microstructure of the solid memb ranes after soaked in SBF for 14 days. An EDX analysis indicate that the deposits n Ch: 14d and Ch Ca05: 14d involved Ca and Cl whole Si was detected for those on Ch G10: 14d. The spherical deposits densely covering the surface of the Ch G10Ca05 membranes were apatite according to Fig. 6. The ability of apatite formation was enhanced as the amount of CaCl2 increased. Such correlation between Ca(II) and apatite formation agree well with the apatite deposition tendency observed for bioactive silicate glasses and glass ceramics[8-10]. Figure 6. T F-XRD patterns for the hybrid membranes before and after being soaked in SBF up to 14 days. (a) Ch, (b) ChCa05, (c) ChG10, (d) ChG10Ca005, (e) ChG10Ca01, (f) ChG10Ca025, and (g) ChG10Ca05. ○:apatite Figure 7. SEM images of the surface microstructure of the solid membranes. Top row: Ch, ChCa05, and ChG10 soaked in SBF for 14d. Bottom row: ChG10Ca05 soaked in SBF for 3 to 14d. Bar : 10 μm 6 Yuki Shirosaki et al.: In Vitro Bioactivity and M G63 Cytocompatibility of Chitosan-Silicate Hybrids Figure 8. The concentration of Ca(II), P(V), and Si(IV) and pH of SBF after soaking selected solid membranes: (a) Ch, (b) ChCa05, (c) ChG10, (d) ChG10Ca05 Fi gure 9. SEM images of t he as-prepared porous scaffold ChG10Ca05 (a), and (b) t he scaffold aft er soaked in SBF for 7d. The XRD profile of insert (c) indicates the flakes of (b) are apatite crystallites Fig. 8 demonstrates the concentration of Si(IV), Ca(II), and P(V) and p H of SBF after soaking selected membrane samples: Ch, ChCa05, Ch G10, and Ch G10Ca05. The profiles for Ch G10Ca01 and Ch G10Ca025 were very similar to those of Ch G10Ca05. SBF was well buffered and pH was kept constant when the solid membranes were soaked up to 14d. The slight decrease in Ca(II) for Ch was exp lained in terms of adsorption of calciu m ions on chitosan, forming complexes with the amino groups[7]. A hump in Ca(II) concentration for Ch Ca05 during 3 and 5d was due to the Ca(II) release into SBF. The decrease in the concentration of Ca(II) in accordance with the decrease in that of P(V) suggested formation of some calciu m phosphates, though no distinct X-ray diffraction signals of calciu m phosphates were detected for Ch Ca05. The release of Si(IV) was detected for all memb ranes that contained GPTMS. A lthough no change in the concentration of Ca(II) and P(V) was observed for Ch G10, the synchronized decrease in both species for Ch G10Ca05 was indicative of calciu m phosphate deposition as the XRD profiles shoed the format ion of apatite in Fig. 6. The FT-IR spectra in Fig. 1 indicated the formation of Si-O-Si bonds, wh ich might be hydroly zed to y ield Si-OH when the solid memb ranes were soaked in SBF. Therefore, the same mechanism of apatite deposition attributed to bioactive silicate glass and glass ceramics was suggested to be applicable to the present membranes of Ch G10Ca series. That is, the mechanism depended on the release of Ca(II) and the presence of Si-OH groups as the key factors[10, 11]. 3.1. The Porous Scaffol ds Fig. 9(a) shows a SEM image o f a porous hybrid, Ch G10Ca05, as a typical porous microstructure of the chitosan-hybrid scaffolds obtained in the present study due to freeze-dry ing. The pore size and porosity of the scaffo lds was approximately 100 μm and about 90%, respectively. Moreover, the pores were interconnected with windows of walls open to adjacent pores ~50 μm, large enough for supply oxygen and nourishment. Those pores were within the range to promote effective ingrowth of bone tissue and to provide mechanical interlock for firm in itial fixat ion, proposed by Helebert et al.[12] and de Groot[13]. When soaked in SBF for 7d, the porous ChG10Ca05 scaffo lds International Journal of M aterials and Chemistry 2013, 3(3A): 1-7 7 deposited apatite not only the outer surface but also on the [4] D. P. Speer, M . Chvapil, C. D. Eskelson, J. Ulreich, pore walls. The inserted XRD profile confirmed that the flaky deposits were apatite. Th is concludes that the present porous scaffolds have good bioactivity as they maintained Biological effects of residual glutaraldehyde in glutaraldehy de -tanned collagen biomaterials, Journal of Biomdedical M aterials Research, 14[6], 753-764, 1980 the original porous configuration and flexibility. Therefore, [5] Y. Shirosaki, K.Tsuru, S. Hayakawa, A. Osaka, M . A. Lopes, the porous scaffolds are promisingly applicable to bone tissue engineering scaffolds. J. D. Santos, M . H. Fernandes, In vitro cytocompatibility of MG63 cells on chitosan-organosiloxane hybrid membranes, Biomaterials, 26[5], 485-493, 2005 [6] T. Kokubo, H. Kushitani, S Sakka, T. Kitsugi, Y. Yamamuro, 5. Conclusions Solution able to reproduce in vitro surface-structure changes in bioactive glass-ceramics A-W., Journal of Biomedical The toxicity of GPTMS monomer was much lower than M aterials Research, 24[6], 721-734, 1990. that of GA. M G63 osteoblastic cell cu ltured on chitosan-GP [7] N. Nishi, Y.M aekita, S. Nishimura, O. Hasegawa, S. Tokura, TMS solid memb ranes showed good cytocompatibility Highly phosphorylated derivatives of chitin, partially compared with the hybrids crosslinked by other reagents such as GA. Ability of the apatite deposition on the solid memb ranes was accelerated not only the release of Ca ions fro m the hybrids but also that of Si ions. Porous hybrids were deacetylated chitin and chitosan as new functional polymers - metal – binding property of the insolubilized materials, International Journal of Biological M acromolecules, 9[2], 109-114, 1987. easily prepared by freeze-d rying technique and deposited [8] M . Ogino, F. Ohuchi, L. L. Hench, Compositional apatite even the walls of the inside pores under the simulated dependence of the formation of calcium-phosphate films on body fluid. Therefore, it has been concluded that Bioglass, Journal of Biomedical M aterials Research, 14[1[, 55-64, 1980 the chitosan-GPTMS porous hybrids are pro mising candidat es of basic materials fro m which tissue engineering scaffolds [9] T. Kokubo, S.Ito, Z. T. Huang, T. Hayashi, S. Sakka, T. are to be derived. Kitsugi, T. Yamamuro, Ca, P-ruch layer formed on high-strength bioactive glass-ceramic A-W, Journal of Biomedical M aterials Research, 24[3], 331-343, 1990 REFERENCES [10] C. Ohtsuki, T. Kokubo, T. Yamamuro, Mechanism of apatite formation on CaO-SiO2-P2O5 glasses in a simulated body fluid, Journal of Non-Crystalline Solids, 143[1], 84-92, 1992. [1] T. Tateishi, G.Chen, T. Ushida, Biodegradable porous [11] P.Li, C. Ohtsuki, , T .Kokubo, K. Nakanishi, N. Soga, T. scaffolds for tissue engineering. Journal of Artificial Organs, Nakamura, T. Yamamuro, Apatite formation induced by 5[2], 77-8, 2002. silica gel in a simulated body fluid, Journal of the American Ceramic Society, 75[8], 2094-2097. 1992 [2] T. Chandy, C. P. Sharma, Chitosan-as a biomaterial., Biomaterials Artificial Cells and Artificial Organs, 18[1], 1-24, 1990. [12] S. F. Hulbert, S. J. Morrison, J. J.Klawitter, Tissue reaction to three ceramics of porous and non-porous structures, Journal of Biomedical M aterials Research, 6[5], 347-74, 1972 [3] S. R Jameela, A. Jayakrishnan, Gluraraldehyde crosslinked chitosan as a long acting biodegradable drug delivery vehicle: [13] K. de Groot, Bioceramics consisting of calcium phosphate studies on the in vitro release of mitoxantrone and in vivo salts, Biomaterials, 1[1], 47-50, 1980. degradation of microspheres in rat muscle, Biomaterials, 16[10], 769-775, 1995

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