【作者】 刘敏；

【导师】 鲁雄；

【作者基本信息】 西南交通大学 ， 材料工程（专业学位）， 2015， 硕士

【摘要】 用于骨修复的组织工程支架需要有较大的表面积、合适的孔隙率和孔尺寸、高度互连的多孔结构,从而可以支持细胞粘附和增殖,还需要具备可以调控和介导细胞活性的能力。本研究通过冷冻干燥法制备壳聚糖(CS)/双相磷酸钙陶瓷(BCP)复合支架。其中,CS不但可以用于支架成形,还可以可促进骨细胞粘附;BCP具有羟基磷灰石(HA)的生物活性和β磷酸三钙(β-TCP)的可降解特性,进一步提高支架的骨诱导性。然后在支架表面同时接枝精氨酸、甘氨酸、天冬氨酸三肽(RGD)和装载骨形态发生蛋白(BMP-2)纳米颗粒,促进细胞的增殖和分化。首先,利用冷冻干燥法制备CS与BCP质量比为8:0、8:1、4:1、2:1的复合支架。通过SEM观察支架微观结构,结果表明多孔支架孔径约为10 μm,BCP颗粒在支架中分散均匀。体外细胞培养表明,CS和BCP质量比为4/1时,能有效促进骨髓间充质干细胞(BMSCs)的增殖和向成骨细胞的分化。其次,通过两种方式将RGD接枝在CS/BCP复合支架表面。一种是将海藻酸钠(ALG)邻二羟基氧化成醛基,通过醛基与RGD的氨基发生希夫碱反应,得到RGD接枝的氧化海藻酸钠(OALG-RGD),并进一步通过希夫碱反应,将OALG-RGD固定在支架表面。另外一种是EDC/NHS反应,将RGD末端的羧基活化,与支架表面暴露的CS的氨基反应,从而将RGD接枝在支架表面。考察不同浓度RGD在复合支架表面的接枝率以及对BMSCs形态,增殖和分化的影响。结果表明,采用第一种方法时,当RGD溶液浓度为1 μg/ml时,支架表面浓度为2.3 nmol/cm2,有利于BMSCs在支架表面的粘附,增殖和向成骨细胞分化。而通过第二种方法,当RGD溶液浓度为度为100 μg/ml,支架表面浓度为60.1 nmol/cm2时最优,RGD浓度太低或太高都不利于细胞活性。最后,将骨生长因子BMP-2和牛血清蛋白BSA混合溶液通过去溶剂法形成包裹BMP-2的纳米颗粒。将CS和OALG层层组装在颗粒表面形成BSA+CS+OALG纳米颗粒,最外层的OALG的醛基与CS的氨基共价结合,从而将纳米颗粒负载到CS/BCP支架上,有效地保护了生长因子的活性并且实现生长因子缓释。通过BMSCs的培养实验,表明RGD和BMP-2在促进细胞粘附,增殖和分化有一定的协同作用。动物实验结果表明同时接枝RGD和装载BMP-2纳米颗粒的支架具有一定的骨修复能力。更多还原

【Abstract】 Development of bone tissue engineering requires the improvement of tissue scaffolds,which need not only large surface area and highly inter-connected porous structure with suitable porosity and pore size to support cell attachment and proliferation,but also signaling molecules to mediate cell activity.In this study,BMP-2 encapsulated nanoparticles and arginine-glycine-aspartic acid(RGD)are immobilized on the chitosan(CS)/biphasic calcium phosphate(BCP)scaffolds for synergistic enhancement of bone marrow stromal cell activity.The study includes three parts:First,CS/BCP scaffolds with different mass ratios of 8:0,8:1,4:1,2:1 were prepared by physical blend and subsequent lyophilization.SEM micrographs revealed that the micropores ranged from 10 μm to 100 μm.The BCP microparticles are uniformly distributed in CS.BMSC culture showed that CS/BCP with ratio of 4:1 leaded to cell proliferation and differentiation best.Secondly,RGD peptide was conjugated to the CS/BCP scaffolds via two methods.One is RGD grafted the oxidized sodium alginate(OALG)by the Schiff base reaction to form OALG-RGD,which was fixed on the scaffolds also through Schiff base reaction.The other was that RGD was directly immobilized on scaffold surfaces through EDC/NHS reaction.The results indicated that RGD peptide immobilized CS/BCP scaffolds can regulate cell proliferation and differentiation.The cell activity was related to the surface density of RGD,which could not be too low or too high.Finally,BMP-2 loaded BSA nanoparticles were prepared by a desolvation technique,and CS and OALG layers were assembled on the surface of nanoparticles to protect the activity of growth factors and achieve sustained release.Then BSA+CS+OALG nanoparticles were immobilized on the scaffolds.BMSCs culture indicated that RGD and BMP-2 had synergistically enhanced cell activity by providing an integrin binding and facilitating osteogenetic differentiation,respectively.In vivo study demonstrated that the RGD and BMP-2 immoblized scaffold has good bone repairing ability.更多还原