【作者】 黄伟；

【导师】 王迎军；

【作者基本信息】 华南理工大学 ， 材料学， 2010， 硕士

【摘要】 传统医学通常依靠自体骨和异体骨移植进行骨修复,但它们各自存在明显的缺点。人工骨修复材料在近年来得到了广泛关注,尤其是针对骨组织工程中存在的关键问题之一——构建功能化的、特别是能介导蛋白的富集和细胞的粘附、迁移、增殖,诱导细胞特异性分化、维持细胞表型的骨组织工程支架,已成为该领域的热点。本研究结合组织工程与药物缓释技术以构建功能化的骨修复支架,在为细胞提供生长的三维环境的同时,可控缓释有特定作用的药物分子,刺激细胞响应,促进细胞的生长。本文以PLGA和PHBV为主要基体,首次完成PHBV微球改性PLGA基体多孔支架的多级构建,并进一步将该支架系统中的PHBV微球功能化——即负载药物,最终构建了具有多级结构和多级成分的功能化骨组织工程支架。它一方面可以为细胞的生长提供三维空间,支持其生长;另一方面能缓释药物,刺激细胞响应并促进其增殖。本研究根据PLGA与PHBV在丙酮中的溶解度差异,采用预制的PHBV微球改性PLGA基体,构建了PHBV微球增强PLGA基体的具有多级结构的复合支架(简称PLGA/m-PHBV)。通过合理地调控PHBV微球的粒径大小、氯化钠和PHBV微球的比例,可以控制PLGA/m-PHBV支架的孔隙率及孔径分布。当PHBV微球的质量达到PLGA基体质量的50%时,支架孔隙率超过83%,抗压强度约1.4MPa。采用人骨髓间质干细胞(hMSCs)对PLGA/m-PHBV支架的细胞相容性进行评价。结果表明,hMSCs在该支架上粘附良好,细胞生长形态正常。其增殖实验表明该支架体系能够很好地支持hMSCs在其上增殖;Live/Dead实验显示细胞大多分布在PLGA基体和微球交界处。在成骨诱导液中分别培养14天和21天后,对支架上的细胞分别进行碱性磷酸酶染色和茜素红染色,结果均呈阳性,表明hMSCs被诱导成了成骨细胞,并开始形成钙结节。利用HA与AL之间的螯合作用预制HA-AL螯合体并采用单乳液法将其包入PHBV制得PHBV/HA-AL释药微球,探讨了多个因素对微球粒径的影响。研究表明,随着搅拌速度的增加、表面活性剂浓度的增高和PHBV浓度的降低,微球的粒径逐渐减小。由于HA与AL之间的螯合作用,使得药物包封率达到80%以上;体外28天累积释放药物70%,并且累积释放率与时间遵循指数关系。体外60天的降解实验中,该系统没有引起明显酸性。细胞毒性试验表明,该释药微球系统的细胞毒性极小。将PHBV/HA-AL微球粒径优化,用该释药微球改性PLGA基体,构建了具有多级结构的功能化的PLGA/PHBV-HA-AL载药型骨组织工程支架。通过控制氯化钠、PHBV/HA-AL微球与PLGA的比例,得到了孔隙率超过81%的多孔支架。该支架的抗压强度超过1.1MPa,具有连通孔并且微球在基体上分布均匀。采用人骨髓间质干细胞(hMSCs)评价了PLGA/ PHBV-HA-AL支架的细胞相容性。结果表明,hMSCs在该支架上粘附良好,细胞形态正常。此外,MTT实验表明该支架系统能较好地支持hMSCs增殖;Live/Dead实验显示细胞大多集中在PLGA基体和微球交界处。在成骨诱导液中分别培养14天、21天后,对支架上的细胞进行碱性磷酸酶(ALP)和茜素红染色,其结果呈阳性,表明hMSCs被成功诱导成了成骨细胞。对支架上的hMSCs原位诱导,结果表明I型胶原免疫组化染色呈阳性,说明该载药支架能将hMSCs诱导为成骨细胞。更多还原

【Abstract】 Although autogenous and allogeneic bone transplantation for bone repair has their drawbacks separately, they were widely used traditionally. Man-made bone repair materials have gained extensive attention in late years. Scholars paid especially attention to one of the key problems, which is establishing a functional bone repair scaffold with the ability to promote the adhesion, migration, proliferation of cells and seduce them toward specified ones. This study combined tissue engineering and control release of drugs together to establish a functional bone repair scaffold in the hope of providing a 3-D environment as well as controlled releasing certain drug moleculars to stimulate the cells and promote them to grow. This research employed PLGA and PHBV as the matrix, and then fabricated a multilevel structured PLGA scaffold modified by PHBV microspheres. Furthermore, we functionalized the PHBV mcirospheres by loading them with alendronate to make the whole scaffold with the property of sustained releasing drugs. This new scaffold with special structure can provide the 3-D space for the cells to grow on the one hand, on the other, it releases drugs with controlled manner to stimulate the cells to response and proliferate.According to the different solubility in acetone, PLGA and PHBV have been employed to fabricate PLGA/m-PHBV scaffold using particle-leaching method. The PHBV microspheres were used to enhance the PLGA scaffold. By adjusting the amount of PHBV microspheres, NaCl and PLGA concentration, we can control the porosity and pore size distribution of the scaffold. In this study, we made a scaffold with the porosity above 83% and the compressive strength of 1.4MPa. hMSCs were employed to evaluate the cyto-property. The result demonstrated that hMSCs adhered and proliferated well on the PLGA/m-PHBV scaffold. Live/dead experiment showed that most of the visible cells were distributed between PHBV microspheres and PLGA matrix. After induced in the osteogenic medium (OGM) for 21d, hMSCs on the scaffolds were turned into osteoblast according to the positive result of alizarin red dyeing.This study prepared PHBV/HA-AL microspheres with controllable diameter using single emulsion method and investigated the influential factors of microsphere diameter. The results showed that increasing the stirring speed and the concentration of surfactant could reduce the diameter of the microspheres. Owing to the chelation between hydroxyapatite (HA) and AL, this drug delivery system had a high encapsulation efficiency which exceeds 80% and only released 70% of AL within 28 days. During the in vitro degradation experiment, this system did not cause obvious acid environment. Meanwhile, the in vitro cytotoxicity experiment showed that this system did little harm to the hMSCs.The diameter of PHBV/HA-AL microspheres was tailored and they were used to fabricate PLGA/PHBV-HA-AL scaffold by the same method used above. This scaffold possessed a multi-level structure with the porosity of 81% and the compressive strength of 1.1MPa. hMSCs adhered and proliferated well on the scaffolds. The same cells distribution profile was found in the live/dead experiment. After induced in the osteogenesis inducing solution (OS) for 14d and 21d separately, the results of alkaline phosphatase and alizarin red dyeing were positive, which meant that hMSCs had turned into osteoblasts. After been cultured in the L-DMEM for 14d, the result of immunohistochemistry of collagenⅠon the scaffold was positive, which confirmed that this functional scaffold could induce the hMSCs to osteoblasts.更多还原