可降解重组人骨形态发生蛋白-2复合贴膜的研究

发布时间：2018-04-05 14:33

本文选题：重组人骨形态发生蛋白-2　切入点：壳聚糖　出处：《兰州大学》2015年硕士论文

【摘要】：目的：本研究壳聚糖通过乳化交联形成rhBMP-2壳聚糖微球,复合在可降解的明胶海绵支架上,制备可降解骨形态发生蛋白复合贴膜,提高BMP有效诱导成骨性。研究方法：1、选用H2SO4作为乳化剂,通过乳化交联法制备NaOH/rhBMP-2/壳聚糖微球,观察壳聚糖微球的形态特征、细胞相容性以及其缓释作用。2、制备rhBMP-2/壳聚糖微球/明胶海绵复合贴膜,将其与rhBMP-2/壳聚糖微球及明胶海绵对照研究,通过体外释放实验,观察并分析复合膜的体外释放情况。3、通过动物实验骨缺损模型,观察可降解骨形态发生蛋白复合贴膜缓释能力及诱导成骨作用。将可降解骨形态发生蛋白复合贴膜植入新西兰兔的桡骨缺损区域,在预定时间点,通过影像、组织学检查,以及通过测定缺损区域骨样本骨量、钙量及碱性磷酸酶。观察rhBMP-2/壳聚糖微球/明胶海绵诱导成骨的情况。应用IBM SPSS19.0统计学软件数据包对数据进行分析,组间数据采用均数±标准差(x±s)描述,组间数据的比较,采用单因素方差分析,检测水准a=0.05,p0.05具有统计学意义。结果：以壳聚糖为原料,通过乳化交联负载rhBMP-2成功制备出NaOH-壳聚糖微球,其形态规整,分散均匀,微球的成球性良好,与单纯可吸收明胶海绵复合支架比较,壳聚糖微球复合明胶海绵贴膜前期突释小,中后期可持续缓慢释放。通过MTT法检查壳聚糖微球细胞毒性为1级,细胞相容性良好,以兔桡处建造骨缺损模型,通过大体观察、影像学检查,测试骨量、骨密度、钙含量及碱性磷酸酶活性检测,统计学分析结果提示：可降解骨形态发生蛋白-2复合贴膜可有效促进骨缺损修复,较单纯的明胶海绵及壳聚糖微球控释作用佳。结论：1、以H2SO4为乳化剂,通过乳化交联法制备的NaOH-壳聚糖微球具有良好的生物相容性和蛋白高吸附性。此法工艺简便易行,壳聚糖微球稳定性良好；2、以壳聚糖为原料,复合明胶海绵所制备的可降解重人组骨形态发生蛋白-2复合贴膜,经体外释放实验、植入兔桡骨缺损模型观察检测分析,载rhBMP-2的NaOH-壳聚糖微球与带正电的明胶海绵复合通过稳定的静电作用可有效提高复合膜的控释作用,较单纯的壳聚糖微球及明胶海绵有更好的诱导成骨性。
[Abstract]:Aim: in this study, rhBMP-2 chitosan microspheres were prepared by emulsification and crosslinking of chitosan onto degradable gelatin sponge scaffolds to prepare degradable bone morphogenetic protein composite film, so as to improve the osteogenicity induced by BMP.Methods H2SO4 was used as emulsifier to prepare NaOH/rhBMP-2/ chitosan microspheres by emulsification and crosslinking method. The morphology, cytocompatibility and slow-release of chitosan microspheres were observed. The rhBMP-2/ chitosan microspheres / gelatin sponge composite film was prepared.Compared with rhBMP-2/ chitosan microspheres and gelatin sponge, the in vitro release of the composite membrane was observed and analyzed, and the bone defect model was established by animal experiment.To observe the ability of biodegradable bone morphogenetic protein (BMP) composite membrane to release and to induce osteogenesis.The degradable bone morphogenetic protein composite patch was implanted into the radius defect area of New Zealand rabbits. The bone mass, calcium content and alkaline phosphatase in the defect area were measured by imaging, histological examination and bone samples at a predetermined time point.The osteogenesis of rhBMP-2/ chitosan microspheres / gelatin sponge was observed.IBM SPSS19.0 data package was used to analyze the data. The data between groups were described by mean 卤standard deviation (x 卤s). The comparison of data among groups and single factor analysis of variance (ANOVA) showed statistical significance.Results: NaOH- chitosan microspheres were successfully prepared by emulsifying and crosslinking loaded rhBMP-2 with chitosan as raw material. The microspheres were characterized by regular morphology, uniform dispersion and good spheroidization, compared with simple absorbable gelatin sponge composite scaffolds.Chitosan microspheres combined with gelatin sponge had little sudden release in the early stage and sustained and slow release in the middle and late stage.The cytotoxicity of chitosan microspheres was grade 1 by MTT assay. The rabbit model of bone defect was constructed at the radial of rabbit. Bone mass, bone mineral density, calcium content and alkaline phosphatase activity were measured by gross observation and imaging examination.The results showed that the biodegradable bone morphogenetic protein-2 composite film could effectively promote the repair of bone defect, which was better than that of simple gelatin sponge and chitosan microspheres.Conclusion the NaOH-chitosan microspheres prepared by emulsification and crosslinking with H2SO4 as emulsifier have good biocompatibility and high protein adsorption.The method is simple and convenient, and chitosan microspheres have good stability. The biodegradable heavy human group bone morphogenetic protein-2 composite membrane was prepared from chitosan as raw material and gelatin sponge, and was released in vitro.The observation and analysis of rabbit radial defect model showed that the combination of rhBMP-2 loaded NaOH-chitosan microspheres and positively charged gelatin sponge could effectively improve the controlled release effect of the composite membrane through stable electrostatic interaction.Compared with simple chitosan microspheres and gelatin sponges, the osteogenesis was better than that of chitosan microspheres and gelatin sponges.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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