Abstract
BACKGROUND:It has been reported that bioactivity is found to be favored by the co-operative behavior of silanol (Si-OH) or Ti-OH etc. groups on the material surface and the involved calcium ions. To confirm the hypothesis that a new family of organic-inorganic hybrid materials, which incorporate gelatin chains covalently into Si-O-Ti network, is synthesized through sol-gel procedure.

OBJECTIVE: To synthesize a hybrid of gelatin and CaO-SiO2-TiO2 system which is used for bone repairing, and observe its structure and bioactivity.

DESIGN: Randomized control observation.

SETTING: the laboratory, School of Material Science and Engineering, Shaanxi University of Science and Technology.

MATERIALS: Gelatin (Sinopharm Chemical Reagent Co., Ltd), titanic acid isopropyl ester (Gu'an Hengye Fine Chemicals Co., Ltd), γ-glycidoxy propyl trimethoxy silane (Jingzhou Jianghan Fine Chemicals Co., Ltd), calcium nitrate (Tianjin Bodi Chemicals Co., Ltd)

METHODS: The experiment was carried out in the laboratory, School of Materials Sciences and Engineering, Shaanxi University of Science and Technology between April and August in 2006. A new type of bioactive organic-inorganic hybrid was synthesized through sol-gel processing starting from gelatin, γ-(2,3-glycidoxypropyl)trimethoxysilane (GPSM), Tetraisopropyltitanate (TiPT) and calcium nitrate.①Structure of the hybrid: The structures of the products were investigated with Fourier transformed infrared (FT-IR) diffusive reflection spectroscope, X-ray diffraction (XRD) instrument and scanning electron microscope (SEM) respectively.②Bioactivity: The products Ti15Ca0 and Ti15Ca20 were soaked in a stimulated body fluid (SBF) to evaluate the morphology of the surfaces by SEM and thin-film XRD.

MAIN OUTCOME MEASURES: The structure and bioactivity of the hybrid.

RESULTS: ①The hybrid was completely amorphous and its surface was almost homogenous, which implied the covalent bonding between the organic component and inorganic component. FT-IR spectra result verified the occurrence of Si-OH group and Si-O-Ti bond, as well as the addition of TiPT supplied Si-O-Ti bond.②There were lots of apatite crystallines formed on the surface of hybrid Ti15Ca20 after soaked in SBF for 7 days, which confirmed their in vitro bioactivity. These apatite particles were similar to the bioglass and other bioactive materials in the patterns.

CONCLUSION: A new type of organic-inorganic hybrid material, which incorporates gelatin chains covalently into Si-O-Ti network, is synthesized through sol-gel procedure. There are lots of apatite and brushite crystals formed on the surface of the product Ti15Ca20 after soaking in SBF for 7 days, which obviously proves the bioactivity of the hybrid.

INTRODUCTION

Some ceramics, such as bioglass, sintered hydroxyapatite and glass-ceramic A-W, spontaneously bond to living bone. They are already clinically used as important bone repairing materials[1-3]. It is also revealed subsequently that even metals, such as titanium and its alloys, can bond to living bone, if they are previously subjected to alkali and heat treatment[4,5]. Recently, much attention has been focused on the development of organic-inorganic hybrids as a new type of bioactive material because of their unique properties, such as high flexibility and low elastic modulus combined with high mechanical strength[6-12]. Natural polymers like chitosan, chitin or gelatin have been employed in clinics by virtue of their biosorption and flexibility, but they are not bioactive[13]. Since some ceramics have bioactivity as referred above but are neither biodegradable nor flexile, hybrid materials derived from the integration of those polymers with the ceramics can construct a group of artificial material appropriate for bone repairing. It has been reported that bioactivity is found to be favored by the co-operative behavior of silanol groups (Si-OH) or Ti-OH groups on the material surface and the involved calcium ions, which may release from the implanted material into the body fluid[14,15].
In the present work, hybrid of gelatin and CaO-SiO2-TiO2 system is synthesized by using GPSM as coupling agent through sol-gel processing.

MATERIALS AND METHODS

Materials
The experiment was carried out in the laboratory, School of Materials Sciences and Engineering, Shaanxi University of Science and Technology between April and August in 2006.The main materials included Gelatin (C102H151O39N31), chemical purity, Sinopharm Chemical Reagent Co., Ltd; Titanic acid isopropyl ester ([(CH3)2CHO]4Ti), Mr =284.26, chemical purity, Gu'an Hengye Fine Chemicals Co., Ltd; Calcium nitrate [Ca(NO3)2·4H2O], Mr =236.15, purity ≥99.0%, Tianjin Bodi Chemicals Co.,Ltd; Isopropyl alcohol [(CH3)2CHOH], Mr =60.10, analytical purity, Tianjin NO.6 Chemical Reagent Factory; γ-glycidoxy propyl trimethoxy silane [CH2OCHCH2O(CH2)3Si(OCH3)3], Mr =236, chemical purity, Jingzhou Jianghan Fine Chemicals Co., Ltd; Hydrochloric acid (HCl), Mr =36.46, analytical purity 36-37%, Kaifeng Chemical Fertilizer & Industry Group Reagent Factory; Distilled water (H2O), Mr =18.00, School of Chemical Industry, Shaanxi University of Science and Technology.

Methods
Material synthesis
Appropriate amounts of gelatin were dissolved in 0.1 mol/L HCl solution for the mass fraction of 12.5%. Then GPSM (CH2OCHCH2O(CH2)3Si(OCH3)3) was added to the solution while stirring at 40 ℃ so that the mass ratio of gelatin/GPSM was 1. Tetraisopropyltitanate (TiPT) (Ti(OCH(CH3)2)4), isopropyl (IPA, (CH3)2CHOH) and H2O were added when the solution became clear and homogeneous. The molar ratio of TiPT/(GPSM+TiPT) was described by mTi as in Table 1. Two hours later, calcium nitrate-etrahydrate (Ca(NO3)2·4H2O) whose molar ratios with respect to (GPSM+TiPT) was figured by mCa was finally mixed into the former solution.

The precursor sols were further stirred for additional 30 minutes after homogeneity. The solution was aged in a polytetrafluoroethylene (PTFE) container covered with a preservative film having several pinholes under room ambient conditions for 1 day with partial evaporation of the solvents. They were kept in the same film-covered container at 40 ℃ in a oven for gelation. Gelation time was defined as the duration of time until the precursor sol lost fluidity. The obtained gel was dried at 60 ℃ for 7 days.

Characterization of the hybrids
The structures of the products were investigated with Fourier transformed infrared (FT-IR) diffusive reflection spectroscope, X-ray diffraction (XRD) instrument and scanning electron microscope (SEM) respectively.

Evaluation on bioactivity
The products Ti15Ca0 and Ti15Ca20 after heat treatment were made block samples and at 36.5 ℃ soaked in a stimulated body fluid (SBF) with PH value of 7.25 and ion concentrations (Na+ 142.0 mmol/L, Ca2+ 2.5 mmol/L, K+ 5.0 mmol/L, Mg2+ 1.5 mmol/L, Cl－ 147.8 mmol/L, HCO3－ 42.0 mmol/L, HPO42－ 1.0 mmol/L, SO42－ 0.5 mmol/L) nearly equal to those of human blood plasma[16]. Their bioactivities were evaluated by examining the apatite formation on their surfaces in the SBF. The surfaces of the obtained specimens were analyzed by thin-film XRD (TF-XRD). The morphology of the surfaces was observed by SEM.

RESULTS AND DISCUSSION

Characterization of the hybrids
Figure 1 showed FT-IR spectra of the products with different TiPT contents. The absorption peaks at 1 030, 690 and 480 cm－1 were assigned to antisymmetric stretching vibration, symmetric stretching vibration and bending vibration of Si-O-Si bond, respectively[17]. This indicated that the methoxy silane groups of GPSM were polymerized to yield Si-O-Si bridging bonds. The occurrence of peaks at 930 cm－1, which denoted the Si-O-Ti bond, showed that the addition of TiPT supplied Si-O-Ti bond to reinforce the network. The intensity of the peak at 870 cm－1 ascribed to Si-OH bonds was increased with the increasing TiPT content. The calcium ion might be ionically bonded to the network, as in conventional calcium silicate glasses.

Figure 2 and 3 showed hybrid was completely amorphous and its surface was almost homogenous, which implied again the covalent bonding between the organic component and inorganic component.

Bioactivity of the hybrids
The TF-XRD patterns of Figure 4 showed that the hybrid containing Ca soaked in SBF exhibited obvious diffraction peaks at 26.0° and 32.0° in 2θ, locations assigned to each crystal plane of apatite and confirming their in vitro bioactivity. There was also some brushite appeared. The Ca-free product didn't show ability to form apatite. It was reported that hybrids releasing Ca ion could form lots of Si-OH and Ti-OH groups by exchanging with H3O+ ion and increasing the ionic activity product of the apatite in the surrounding SBF.

Figure 5 showed the apatite particles covering the surface of hybrid Ti15Ca20 after soaking for 7 days. The pattern of these particles was similar to those of bioglass and other bioactive materials.

Conclusions
In the present study, a new family of organic-inorganic hybrid materials, which incorporated gelatin chains covalently into Si-O-Ti network, was synthesized through sol-gel procedure. Homogeneous products were obtained and tamed in vitro testes. There were lots of apatite and brushite crystals formed on the surface of the product Ti15Ca20 after soaking in SBF for 7 days, which obviously proved the bioactivity of the hybrid. This kind of hybrid would be hopefully applied for bone repairing after further study on their mechanical properties.

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应用于骨修复的明胶－CaO-SiO2-TiO2 生物
活性杂化材料的合成及其性能☆

殷海荣1, 2，武丽华2，丁纪根3，赵高扬1，张光华4
1西安理工大学材料科学与工程学院，陕西省西安市 710048；2陕西科技大学材料科学与工程学院，陕西省咸阳市 712081; 3江苏扬阳化工设备制造有限公司，江苏省靖江市 214500; 4陕西科技大学化学与化工学院，陕西省咸阳市 712081
殷海荣☆，男，1962年生，陕西省合阳市人，汉族，1984年西北轻工业学院毕业，教授，在读博士，主要从事生物材料、功能玻璃的研究。
摘要
背景：有研究表明，材料生物活性是由材料表面的Si-OH或Ti-OH等基团和体液中的钙离子共同作用的结果, 可否将明胶链与Si-O-Ti无机网络共价结合合成一种新的有机－无机杂化材料？
目的：合成一种能应用于骨修复的明胶-CaO-SiO2-TiO2 生物活性杂化材料，并观察其结构及生物活性。
设计：随机对照观察。
单位：陕西科技大学材料科学与工程学院实验室。
材料：明胶（国药集团上海化学试剂有限公司），钛酸异丙酯（固安恒业精细化工有限公司），γ－环氧丙基氧丙基三甲氧基硅烷（荆州市江汉精细化工有限公司），硝酸钙（天津市博迪化工有限公司）。
方法：实验于2006－04/08在陕西科技大学材料科学与工程学院实验室完成，通过溶胶－凝胶法以明胶、γ-（2，3环氧丙基氧丙基）三甲氧基硅烷、钛酸四异丙酯及硝酸钙为初始原料合成了一种新的有机－无机杂化材料。① 材料的结构分析：材料的结构用傅立叶红外光谱、X射线衍射及扫描电子显微镜观察分别进行分析。② 生物活性评价：样品Ti15Ca0和Ti15Ca20进行模拟体液浸泡实验，通过薄膜X射线衍射分析，扫描电子显微照片分析其表面组或和形貌。
主要观察指标：材料的结构分析及其生物活性。
结果：①材料呈完全无定形态，表面近乎均一，有机成分与无机成分之间以共价键结合，傅立叶红外光谱分析证明了Si-OH基团和Si-O-Si键的存在，并且钛酸四异丙酯的加入引入了Si-O-Ti键。② 该材料在SBF中浸泡后产生磷灰石晶体，其形貌与生物玻璃和其它生物活性材料相似，记明其具有体外生物活性。
结论：通过溶胶－凝胶法将明胶链与Si-O-Ti无机网络共价结合合成了一种新的有机－无机杂化材料。样品Ti15Ca20在模拟体液中浸泡后其表面生成大量磷灰石和透钙磷石晶体，充分证明了材料的生物活性。
关键词：明胶－CaO-SiO2-TiO2；生物活性；溶胶－凝胶；骨修复材料
中图分类号:R318.08 文献标识码:A 文章编号:1673-8225(2007)22-04440-03
殷海荣，武丽华，丁纪根，赵高扬，张光华.应用于骨修复的明胶－CaO-SiO2-TiO2 生物活性杂化材料的合成及其性能[J].中国组织工程研究与临床康复，2007，11(22):4440-4442 [www.zglckf.com/zglckf/ejournal/upfiles/07-22/22k-4440(ps).pdf]
(Edited by: Li XK/Yang Y/Wang L)