【作者】 李继彦；

【导师】 叶建东；

【作者基本信息】 华南理工大学 ， 材料学， 2013， 博士

【摘要】 因外伤、感染和肿瘤等造成的骨组织缺损是临床上的常见病症，自体骨和异体骨是常用的骨修复材料。但自体骨来源有限以及异体骨存在的免疫排斥反应限制了它们的应用。所以如何构建具有良好的生物相容性、生物应答特性、基因激活特性和促进新生组织形成功能的生物材料受到广泛的关注。微球具有良好的流动性，并且微球堆积体的孔隙连通性非常优异，有利于骨缺损部位的填充和修复。传统的陶瓷微球需要经高温烧结，不利于负载药物和生物活性成分，以高分子为基体的微球缺乏骨传导性和骨诱导性。针对上述问题，本研究以磷酸钙骨水泥为原料，分别采用液滴冷凝法和挤出-滚圆法制备了粒径可控的多孔磷酸钙微球，并对微球进行改性处理，提高微球的强度、抗崩解性、降解性、细胞相容性和骨诱导性等方面的性能。为了提高本研究制备的微球的细胞生物学性能，本研究首先采用聚乙二醇（PEG）水溶液对磷酸钙骨水泥固化体进行表面改性。经PEG处理后，骨水泥固化体表面被规则的片状结晶覆盖，形成规则片状结晶的原因主要是PEG的加入一方面提高了溶液的离子浓度，从而减缓溶液中离子的扩散速度；另一方面PEG与钙离子螯合，减慢了羟基磷灰石的结晶速度。MC3T3-E1细胞实验结果表明这种规则片状拓扑结构有利于细胞粘附、增殖和分化。通过液滴冷凝法制备了粒径在0.3～3mm可控的磷酸钙微球。通过滴重法，从理论上探讨了各因素对微球粒径的影响。随着液固比的变化，微球的总孔隙率介于40%～60%，显孔隙率介于20%～60%，孔隙之间连通性良好。由于研究发现硅酸钙具有优异的矿化性和骨诱导性，所以，在微球中添加一定量的硅酸钙以改善微球的理化性能和成骨能力。微球强度随着硅酸钙添加量的增大先提高后降低，当硅酸钙添加量为20%时，微球的强度是不添加硅酸钙的微球2倍。复合硅酸钙的微球在PBS中浸泡2周仍未出现明显的崩解现象，比不含硅酸钙的微球的抗崩解时间提高了10倍。细胞实验结果表明，添加20%硅酸钙的微球比不含硅酸钙的微球细胞相容性好，可以促进细胞的增殖。兔子股骨缺损修复实验结果表明，微球的组织相容性良好，植入4周时微球被纤维组织和骨组织包裹，并且周围有血管生成；随着植入时间延长至8周，微球内部出现纤维组织和少量的新骨，微球完全被骨组织包裹；16周时各微球样品内外都有大量的骨组织，骨母细胞和骨细胞数量明显下降，这说明骨的成熟度提高。兔子背部竖脊肌8周的植入实验结果表明，微球（d=2mm）间的孔隙有利于血管化。微球的体内植入实验结果还发现硅酸钙的添加促进了微球的体内降解，但添加量过大会使微球因在体内降解过快从而影响微球的骨传导性。适当的添加量是利用硅酸钙改善微球强度和成骨性能的关键。挤出-滚圆技术很少被用于无机复合微球的制备，本研究以壳聚糖为赋形剂，磷酸钙骨水泥为主要原料，通过挤出-滚圆的方法制备了粒径均一，抗崩解性良好的磷酸钙微球。挤出-滚圆法可以制备粒径在0.3～3mm可控的微球，效率高，微球圆度好。挤出-滚圆过程中在机械力的作用下，微球内磷酸钙原料颗粒之间接触更加紧密，赋性剂只是填充在磷酸钙原料颗粒之间，不影响磷酸钙骨水泥的水化结晶，所以水化后以针棒状结晶为主。微球的抗崩解性优异，在PBS中浸泡2周仍没有明显的崩解现象；微球强度接近松质骨。通过PEG溶液浸泡后，微球表面被规则片状结晶所覆盖，微球内小于100nm的微孔所占比例增大，100～1000nm的微孔所占比例减小，结晶形貌的变化改变了微球内孔径分布的分布。细胞实验结果表明规则片状形貌促进了细胞的增殖。为了满足大块骨缺损的填充修复，通过真空浸渍的方法，以PLGA为粘结剂，制备了磷酸钙微球支架。CT结果显示磷酸钙微球支架的平均孔隙率为35.36%±1.18%，孔隙连通性良好，微球之间孔隙基本保留下来。采用过程法对微球堆积过程进行模拟，模型的平均孔隙率是40.3%±0.11%，孔隙完全连通。连通的孔隙结构有利于新骨组织的长入，同时也有利于营养物质的输送和代谢物的排出。更多还原

【Abstract】 The current replacement procedures for bone defect therapy mainly depended onautologous tissue which is the golden standard. Unfortunately, the origination of autologousbone is often limited in supply, and the allogenous bone takes an increased risk of diseasetransmission. Microparticulate forms have been widely used to treat defective bones. Asmicrospheres were good at flowability, the defect of bone can be filled well by them.Completely-connected hole among microspheres is good for bone repair.Calcium phosphate cement (CPC) is highly promising for wide clinical applicationsbecause of its good biocompatibility, excellent bioactivity, low heat release during theself-setting reaction, adequate stiffness, and easy shaping for any complicated geometry. Inrecent years, new strategies that exploit the intrinsic properties of CPCs have been envisaged,and pre-set CPC scaffolds or granules by various processing techniques have been extensivelyput forward.In order to improve the cytocompatibility of microspheres, the surface modification ofhardened calcium phosphate cement has been study first. After immersed inpolyethyleneglycol solution the surface of modified sample was covered by regular blade-likecrystalline structure. The results of cell experiments exhibited that the samples with regularblade-like crystalline structure had better cell response (cell attachment, viability, proliferationand differentiation) compared to those with irregular blade-like crystalline structure.In this study, calcium phosphate microspheres(0.3-3mm) with apparent porosity of47%or more were prepared by dripping-freezing procedure. The mechanical strength, resistance todisintegration, resorbability, and bioactivity of the microspheres has been improvedsignificantly as calcium silicate added.No remarkably disintegration has been found duringtwo weeks because of the addition of calcium silicate. The microspheres with differentcontent of calcium silicate were implante in femoral bone defects and sarcotheca of back ofrabbits. New bone can be seen not only in the gaps between microspheres but also internal ofmicrospheres at8weeks. The microspheres contain40%calcium silicate have been degradedmost part at16weeks. The number of osteoblasts, osteocytes and multinuclear cells wasdecreased as time extension. Blood vessels can be found around new bones. These results indicated that calcium silicate improved the degradation and osteogenic capability ofmicrospheres. The pore structure among microspheres was good for vascularization.Another method used to produce porous calcium phosphate microspheres(0.3-3mm) isextrusion–spheronisation. Different with the microspheres which were prepared bydripping-freezing procedure, the main crystal structure is rodlike, no disintegration was foundmore than two weeks. After immersed in the polyethyleneglycol solution, the surface ofmicrospheres was covered by regular blade-like structure. The result of MTT showed that theregular blade-like structure is good for cell proliferation.The microspherical scaffolds were prepared by vacuum impregnation, PLGA was usedas binder. The porosity(35.36%±1.18%) of scaffold was tested by micro-CT. The strength ofscaffolds improved as the content of PLGA increased, but the porosity decreased as manypores were filled by PLGA. Process based simulation was used to simulation the process ofmicrospheres accumulation. The result indicated the pores among microspheres are fullconnected. This structure is good for transportation of nutrition and elimination of metabolin.更多还原