【作者】 王健；

【导师】 赵良启；

【作者基本信息】 山西大学 ， 微生物学， 2013， 硕士

【摘要】 组织工程技术在上个世纪80年代就已经有人开始研究,它代表着医学研究已经开始进入到器官克隆新时代。骨组织工程的研究是组织工程的一个重要分支技术,为当今骨科医学解决关节软骨损伤修复的研究热点方向,并取得了很多研究成果,但在研发过程中也伴随着新的问题亟待解决。修复损伤器官的组织工程技术从工程支架,种子细胞和生长因子三大方面进行研究。生长活性因子在人体内的半衰期短,马上就会被代谢稀释,完全不能满足组织自身修复再生的需要。研制生长因子缓释微球,其目的在于延长生长因子在体内的存活时间,为应用组织工程技术修复软骨损伤奠定物质基础。生长因子属蛋白类物质容易被体内蛋白酶降解,导致其半衰期短,难以满足组织工程修复损伤的需要。制备载生长因子缓释纳米微球,旨在延长其在体内的存活时间,以一定的缓释率释放,诱导种子细胞分化并形成骨或软骨组织。我们创建聚羟基丁酸与羟基辛酸(PHBHOx)载骨形成蛋白(rBMP)纳米微球制备方法,研发rBMP-PHBHOx缓释纳米微球,为关节一体化支架硬骨区细胞提供生长因子。分别采用溶剂挥发法、静电纺丝法、W1O/W2超声乳化法制备微球,并以载药量、包封率为技术指标经正交试验优化了超声乳化法制备纳米微球的操作条件,以磷酸盐缓冲液作为释放介质(pH7.4)考察微球的体外缓释性能,并通过真空泵产生负压将纳米微球胶体液吸纳到骨组织工程支架的空隙中去,用电镜扫描,观察两者的粘附性和相容性。在以上三种方法中,静电纺丝法制备的微球,颗粒较大且在高压电流作用下可使其rBMP失活；溶剂挥发法制备的微球易粘连并会形成较大颗粒群。超声乳化法制备的微球粒径小,颗粒圆润,分散性较好。经制备条件优化,主要制备条件为PHBHOx浓度为6.5%,乳化剂Tween80为5%及Span80为1%,生长因子浓度10%,PVA浓度为3%,有机相水相体积比为3：1。制备的纳米微球平均粒径为(564.75±53.46)nm,载药量为(1.172±0.262)×10-2%,包封率为(83.50±2.82)%,体外释药率13天可达87.89%。微球负压法纳入骨组织工程支架,可见微球在支架上粘附性好且分布均匀。本文建立了超声乳化制备rBMP-PHBHOx新方法,研发出rBMP-PHBHOx生长因子缓释纳米微球制剂,为一体化支架的骨关节损伤修复研究奠定了一定基础。更多还原

【Abstract】 Tissue engineering technology which made modern medicine step out of the scope of organ transplantion and enterd into a new era of organ reconstruction emerged in the1980s of last century. The technology of cartilage tissue engineering which serves as a important branch of tissue engineering developed fast and made progress in recent years. How ever, many key issues still needed to be resolved.Tissue engineering repair tissue organ damage technology including carrier support, seed cells, biological activity factor three basic elements. Active growth factor in the body short half-life, will soon be metabolism and dilution, and can’t meet the need of regeneration tissue repair. We developed the growth factor slow release microspheres, the aim is to extend the growth factor in the body, survival time for the application of tissue engineering cartilage damage repair lay the material foundation.Growth factors which are belong to protein substances are easy to be degraded by protease in vivo and cannot satisfy the need of tissue engineering for short half-life.It is necessary to prepare the sustained-release nanospheres containing a growth factor is to prolong their survival time in body, induce seed cell to differentiate and form bone or cartilage tissue. The purpose of this article is to create poly(hydroxybutyrate-co-hydroxyoctanoate) containing bone morphogenetic protein (rBMP) and make rBMP-PHBHOx sustained-release nanospheres which can provide growth factors for the bone layer of the joint integrative scaffold. Microspheres were prepared by electrospinning, solrent eraporatin and ultranic emulsification, respectively. Phacoemulsification nano microspheres were investigated by orthogonal test method for optimizing conditions preparation. The release properties of microspheres were investigated with PH7.4phosphate buffer as a medium in vitro. Nanosphere colloid was added into the bone tissue engineering scaffold by negative pressure method and the adhesion and compatibility were observed by electron microscopy. Microspheres prepared by Electrospinning method were larger and easy to make rBMP degenerate. The microspheres prepared by solvent evaporation method were easy to adhere together and form larger particles. The microspheres prepared by phacoemulsification were not only round but also smaller with good distribution. Optimized preparation conditions were as follows:density of PHBHOx was6.5%, Tween80was5%and Span80was1%, concentration of growth factors was10%, density of PVA was3%, the volume ratio of organic phase and aqueous phase was3tol. Microspheres technical parameters were as follows:average diameter was (564.75±53.46)nm, drug loading was (1.172±0.262) x10-2%, the encapsulation efficiency was (83.50±2.82)%, the in vitro release rate of13days up to87.89%. Microspheres were added to bone tissue engineering scaffold by negative pressure method. The results showed that microspheres were visible and had good property of adhesion on the scaffolds. rBMP-PHBHOx slow-release nano microspheres contained growth factor were prepared by phacoemulsification method in this paper. Results of these studies lay a certain foundation for the research of repairing the joint injury with integration stents.更多还原