Abstract
BACKGROUND:Chitosan and gelatin have good biocompatibility and biodegradable properties. More and more attentions have been given to the application of the composite membrane of chitosan/ hydroxyapatite (HA) and gelatin/ HA as the tissue scaffolds.
OBJECTIVE: To prepare a biocompatible composite membrane of chitosan/ HA/gelatin having nanometer multilayer HA, and to investigate whether such a membrane might be a candidate of the tissue scaffolds for the bond repair and regeneration.
DESIGN: Controlled observation.
SETTING: College of Chemistry and Environmental Engineering, Jianghan University
MATERIALS: The experiments were performed at the Laboratory of Fine Chemistry, College of Chemistry and Environmental Engineering, Jianghan University from February to August 2007. Chitosan (Yuhuan Ocean Biochemistry Co.,Ltd, Zhengjiang, China, Mw 13.44×104, 93% degree of deacetylation), and gelation(provided by Shanghai Chemical Reagent Co., Shanghai, China) were used in the present study.
METHODS: The mixture chitosan/CaCl2 solution was casted on a glass plate to give the membrane of chitosan/CaCl2. Then, the membrane was soaked in KH2PO4 solution, 0.1 mol/L NaOH solution, and 3 wt% gelatin aqueous solution, respectively. The resulted membrane was washed and dried to obtain the chtiosan/ HA /gelatin composite membrane. The morphological observation of the composite membrane and HA crystal were carried on a scanning electron microscope. The mechanical properties of the composite membrane were measured using the universal testing machine. The tensile strength (σb) and the elongation at break (ε) were calculated. The thermal stability of composite membrane was determined using a WCT-2C thermobalance.
MAIN OUTCOME MEASURES: The morphologies, the mechanical properties, and the thermal stability of the composite membrane and HA crystal were observed.
RESULTS: The multilayer HA crystal with 400 nm thickness in the composite membrane was observed. The tensile strength (σb) and the elongation at break (ε) of the composite membrane were 3.83-10.25 MPa and 3.97%-10.14%, respectively. The decomposition temperature of HA-treated membrane was 310 ℃, which was higher than that of chitosan/gelatin membrane (305 ℃).
CONCLUSION: The composite chitosan/ HA/gelatin membrane was prepared by using chitosan/CaCl2, KH2PO4 and gelatin solutions. Such a biocompatible composite membrane with multilayer HA might be a promising tissue scaffold.



INTRODUCTION

It is well known that natural bone is an inorganic/organic composite materials which has the hydroxyapatite (Ca10(PO4)6(OH)2, HA). HA has been found wide application in the field of tissue scaffolds[1-2]. HA precipitates have been used as popular implant materials in the field of dentistry, orthopedics and plastic surgery [3]. However, some disadvantages of HA limit its application in the field of artificial bone materials. As reported, HA with polymer is the effective formulation technology to gain the intelligent artificial bone materials[4-6]. Chitosan has been found wide application in biomedical fields as a result of its biocompatibility and nontoxicity, such as drug delivery, wound dressing and the tissue replacement material, etc [7-10]. Gelatin has good biocompatibility and biodegradable properties. The composite membrane of gelatin/HA has been used as the replacement materials [11]. Moreover, natural bone is a typical HA/collagen composite material, which has HA particles with 20 nm in diameter and 50 nm in length [12-13]. In addition, chitosan and gelatin templates have been used to control the nucleation and growth of HA crystals in vitro [14-18]. Hence, it is important to investigate the nanometer multilayer HA with the large interfaces.
In this work, the chitosan/ HA/gelatin was prepared by alternate soaking in the chitosan/CaCl2 solution, KH2PO4 solution and gelatin solution. The multilayer HA has oriented crystal growth with the chitosan/gelatin networks; There are limited reports available. Such biocompatible composite membrane with multilayer hydroxyapatite may be a promising tissue scaffold.

MATERIALS AND METHODS

Materials
Chitosan having Mw of 13.44×104 and 93% degree of deacetylation was purchased from Yuhuan Ocean Biochemistry Co., Ltd., Zhejiang province, China. Gelatin was supplied by Shanghai Chemical Reagent Co., Shanghai, China. All chemicals were of analytical grade.

Preparation of the composite membrane
The chitosan/CaCl2 solution was prepared by dissolving CaCl2 in the 2 wt% chitosan acetic acid solution, the mixture was casted on a glass plate to give a gel sheet, then immerged in KH2PO4 solution, and then coagulated with 0.1 mol/L NaOH solution to generate transparent membranes. The resulted membrane was washed with distilled water. The above-mentioned membrane was immerged in 3 wt% gelatin aqueous solution, and then washed with distilled water and dried to obtain the chtiosan/HA/gelatin
composite membrane. The same process was repeated from 1 to 3 times. The composite membranes were calcined at 800 ℃ for 5 hours to obtain the HA crystal. The content of HA in the membrane were calculated as follow equation: w (HA)＝m2 /m1×100 %,where m1(g) and m2 (g) are the weights of the dried membrane and the initial weight of the composite membrane.

Characterization
The morphological observation of the composite membrane and HA crystal were carried on a scanning electron microscope (Sem, S-570, Hitachi, Japan). The membrane and the HA crystal were frozen in liquid nitrogen, and vacuum dried, and then coated with gold before viewing in SEM.
The mechanical properties of the membrane in the dry state were measured on a universal testing machine (CMT6503, Shenzhen SANS Test Machine Co., Ltd., Shenzhen, China). According to ISO6239-1886 (E), the tensile strength (σb) and the elongation at break (ε) were obtained, respectively. The size of membrane was 70 mm in length, 10 mm in width.
The thermogravimetry(TG), differential thermal analysis(DTG) and differential thermogravimetry(DTA) of composite membrane were performed on a thermobalance (WCT-2C, Beijing Optical Instruments Factory, China) under nitrogen from 20-700 ℃ at a heating rate of 20 ℃/min.

RESULTS

Morphology of composite membrane and HA crystal
The morphological structures of composite membrane (w = 28.0%) and HA crystal are shown in Figure 1. The cross section of the membrane was homogeneous morphologies. The multilayer HA crystal was about 400 nm in thickness.

The mechanical properties of composite membrane
Table 1 shows the results of mechanical properties of composite membrane. As precipitation times (n) increases from 1 to 3, the content of HA increased form 27.6 to 32.0%. The σb andε of the composite membrane were 3.83- 10.25 MPa, and 3.97%-10.14%, respectively.

Thermal properties of composite membrane
Figure 2 shows the TG, DTG and DTA curves of composite membrane. The first loss weight stepwise was finished at 202 ℃, its value was 30.53%. The second step was finished at 352 ℃.

DISCUSSION

There are reports that HA can be formed through the ionic charge of the amino group on chitosan by alternate soaking in CaCl2 and Na2HPO4 solution [1,16]. It is noted that the chitosan/CaCl2 composite membrane shrinks significantly as it is soaked in K2HPO4 solution, indicating that the phosphate ions are adsorbed by the amino groups on chitosan and HA has been formed in the chitosan/CaCl2 membrane[16] HA formation process can be described as the following reaction [19]: 10CaCl2+6Na2HPO4+2H2O→Ca10(PO4)6(OH)2 +12NaCl +8HCl
Usually, the initiation of the mineral crystalline during tissue calcification is caused by heterogeneous nucleation [20]. When the chitosan/HA membrane is immerged in gelatin solution, the composite membrane of chitosan/HA/gelatin has been formed. This can be explained by the strong interaction caused by the negatively charged of gelatin and positively charged of chitosan. As second precipitation times, Ca2+ is enriched on the carboxyl groups of gelatin in the solution, and then PO43- enriches on the amino groups of chitosan, leading to the heterogenous nucleation of HA in the chitosan/gelatin networks. As precipitation times increasing, HA is grown again and again to form the multilayer HA.
The results of the scanning electron microscope suggest that there exists strong molecular interaction between chitosan and gelatin networks, leading HA to be dispersed uniformly in the composite membrane. During the calcined process, the chitosan and gelatin in the membrane have been thoroughly discomposed, leaving only the multilayer HA. Such nanometer HA with a large interface is useful for the application of the bond cell culture.
The mechanical properties of HA/polymer composite membrane usually begin to decrease as the content of HA in the membrane more than 10%. In the present study, the composite membranes have relatively good mechanical properties, the value of σb and ε of membrane are 3.83 MPa and 3.97%, respectively, when the content of HA reaches 32.0%. This result can be explained that the strong interaction of chitosan and gelatin can improve the mechanical properties of chitosan/gelatin.
The first loss weight stepwise at 202 ℃ is assigned to the moisture and the adsorbed water of HA. The second step at 352 ℃ is caused by oxidation and degradation of chitosan and gelatin. As the temperature is higher than 352 ℃, a slight and almost steady sample weight is observed, this suggests that most of the organic composition of membrane has been decomposed. The decomposition peak of chitosan/HA/gelatin (310 ℃) is higher than that of chitosan/geltin (305 ℃), indicating that the hybrid HA in chitosan/gelatin membrane can improve its thermal stability.
The composite membrane of chitosan/ HA/gelatin was prepared by alternate soaking in the chitosan/CaCl2 solution, KH2PO4 solution. There exists multilayer hydroxyapatite crystal with 400 nm thickness in the composite membrane. The decomposition temperature of membrane was 310 ℃, which was higher than that of chitosan/gelatin membrane (305 ℃). Such a biocompatible composite membrane may be a promising tissue scaffold.

Acknowledgements: This work was supported by the Foundation of Science and Technology Bureau of Wuhan city, No.200751699478-06.

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壳聚糖/纳米多层结构羟基磷灰石/明胶
复合膜的性能☆

彭湘红，万 昆
江汉大学化学与环境工程学院，湖北省武汉市 430056
彭湘红☆，女，1966年生，广东省新宁市人，汉族，2006年武汉大学毕业，博士，副教授，主要从事天然高分子应用的研究。
摘要
背景：壳聚糖和明胶具有良好生物相容性和生物可降解特性。壳聚糖/羟基磷灰石和明胶/羟基磷灰石复合材料作为骨组织支架的应用研究也越来越多得到人们的关注。
目的：制备一种生物相容性好的壳聚糖/纳米多层结构羟基磷灰石/明胶复合膜，探讨其是否可作为潜在的骨修复和再生的组织支架材料。
设计：对比观察。
单位：江汉大学化学与环境工程学院。
材料：实验于2007-02/08在江汉大学化学与环境工程学院精细化工实验室完成。壳聚糖（浙江玉环海洋生物化学有限公司，重均相对分子质量为1.344×105，脱乙酰度为93%），明胶（上海医药公司）。
方法：壳聚糖/氯化钙溶液在玻璃板上通过流延法形成壳聚糖/氯化钙复合膜。壳聚糖/氯化钙复合膜分别浸泡在磷酸二氢钾溶液， 0.1 mol/L氢氧化钠溶液和质量分数为0.03的明胶溶液中，所得的复合膜洗涤并干燥得到壳聚糖/羟基磷灰石/明胶复合膜。重复上述过程，得到具有多层纳米羟基磷灰石的壳聚糖/羟基磷灰石/明胶复合膜。扫描电镜观察复合膜的断裂面及膜内羟基磷灰石的形貌。用GMT6503 型微机控制电子万能试验机测定壳聚糖膜和复合膜的拉伸强度和断裂伸长率。用WCT-2C微机差热天平测定复合膜的热稳定性。
主要观察指标：扫描电镜下复合膜的断裂面及膜内羟基磷灰石的形貌。测定复合膜的力学性能及热稳定性。
结果：①扫描电镜下复合膜内羟基磷灰石晶体为厚400 nm的多层结构。②随着沉积次数逐渐增加，复合膜的拉伸强度和断裂伸长率分别为3.83~10.25 MPa，3.97％~10.14%。③复合膜的热分解温度为310 ℃，高于壳聚糖/明胶复合膜的热分解温度（305 ℃）。
结论：通过在壳聚糖/氯化钙溶液、磷酸二氢钾溶液、氢氧化钠溶液和明胶溶液中交替沉积方法制备的壳聚糖/羟基磷灰石/明胶复合膜具有良好的生物相容性和较大的界面积，可作为一种潜在的组织支架材料。
关键词：壳聚糖；明胶；复合膜；羟基磷灰石；沉析；生物材料
中图分类号: R318.08 文献标识码: B 文章编号: 1673-8225(2008)14-02777-03
彭湘红，万昆.壳聚糖/纳米多层结构羟基磷灰石/明胶复合膜的性能[J].中国组织工程研究与临床康复，2008，12(14):2777-2779
[www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2777(ps).pdf]
(Edited by Usharamamoorthy/Song LP/Wang L)