基于羧基化PLGA纳米骨修复材料的制备及性能研究

发布时间：2018-05-01 20:34

本文选题：骨组织工程 + 聚(乳酸-羟基乙酸)聚合物　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：骨缺损或骨不连的治疗一直是骨科领域长期致力解决的临床难题。骨组织工程和再生医学的快速发展为骨缺损和骨不连的修复和治疗提供了希望。支架材料作为骨组织工程最重要的元素之一,其制备及研发受到研究者广泛关注。无机粒子与聚(乳酸-羟基乙酸)聚合物复合制备的骨组织工程支架材料很多,其能够发挥无机粒子和聚(乳酸-羟基乙酸)聚合物的优点。以往无机粒子／聚(乳酸-羟基乙酸)聚合物复合支架的制备方法是将无机粒子在聚(乳酸-羟基乙酸)聚合物溶液或熔体中混合,这就导致无机离子在聚(乳酸-羟基乙酸)聚合物内部分散不均匀,植入机体后不能有效与宿主细胞接触进而发挥生物学作用。因此,制备结构和成分均匀的无机粒子／聚(乳酸-羟基乙酸)聚合物复合支架具有重要的临床意义。目的探讨末端羧基化聚(乳酸-羟基乙酸)聚合物(PLGA-COOH)合成方法；基于羧基化PLGA制备两种纳米骨修复材料；对纳米骨修复材料的相关表征、力学性能、降解性能及生物相容性进行检测；验证纳米骨修复材料的体内异位成骨能力和骨缺损修复性能。材料与方法1、制备羧基化PLGA,分别与纳米级HAP和TCP共混,经搅拌,洗盐和冻干等过程制备PLGA-COOH/HAP和PLGA-COOH/TCP复合支架。研究其相关表征及力学性能和降解性能等。2、复合支架与MC3T3-E1细胞体外共培养定性和定量分析支架对细胞增值、细胞周期、成骨分化及黏附性能的影响。3、复合支架体外负载rhBMP-2,研究体外释放性能。通过SD大鼠臀大肌肌袋实验评价复合支架及负载rhBMP-2支架的异位成骨性能。4、通过兔颅骨临界性骨缺损模型评价支架无负荷情况下骨缺损修复性能。5、通过兔尺骨临界性骨缺损模型评价复合支架及负载rhBMP-2支架长骨大节段骨缺损的修复性能。结果1、核磁氢谱和分子量检测显示,羧基被成功接枝在PLGA聚合物末端,PLGA聚合物分子量120000左右。SEM和Micro CT扫描显示支架具有多孔结构,孔径均一。EDX证实两种支架的特征性元素均为钙、磷和碳。力学性能测试显示两种支架的抗压缩和抗弯曲强度没有明显差异。降解实验显示随着时间延长,两种支架重量均减低；支架中无机成分晶体结构稳定；PLGA聚合物分子量随时间延长而减低。2、CCK-8和流式结果显示,两种支架具有良好生物相容性和促细胞增值作用。ALP测定显示两种复合支架具有一定促成骨分化性能,优于单纯PLGA;扫描电镜和激光共聚焦结果显示,复合支架和羧基化PLGA均有利于细胞的黏附,且细胞形态较好。3、两种复合支架可作为rhBMP-2的缓释载体,有效释放时间可达360小时。将负载rhBMP-2的支架植入SD大鼠臀大肌肌袋中,术后2周和4周可见明显异位骨化发生,新生骨块明显。4、复合支架具有修复无负荷情况下兔颅骨临界性骨缺损的能力,与单纯PLGA聚合物比,更有利于新生骨生成和骨改建。5、复合支架及负载rhBMP-2支架具有修复兔尺骨长节段临界性骨缺损的能力,负载rhBMP-2后增强了复合支架骨修复性能。结论1、成功制备羧基化PLGA聚合物。基于羧基化PLGA聚合物成功制备两种纳米骨修复材料,PLGA-COOH/HAP知PLGA-COOH/TCP。其孔隙率和孔径、力学性能、降解性能基本满足理想骨组织工程材料要求。2、支架材料具有较好的细胞生物相容性及促细胞增值、分化作用,无明显的细胞毒性。3、PLGA-COOH/HAP知PLGA-COOH/TCP可作为rhBMP-2缓释载体；复合支架材料负载rhBMP-2具有较好的生物活性,有利于新生骨生成。单纯支架材料植入体内未导致明显的炎症反应,具有良好的生物安全性,符合国家生物医用材料相关标准。4、兔颅骨临界性骨缺损情况下,PLGA-COOH/HAP知PLGA-COOH/TCP复合支架有较强的骨修复和骨改建能力,优于单纯PLGA-COOH。5、负载rhBMP-2的复合支架能够修复兔尺骨大节段临界性骨缺损,两种复合支架具有潜在的临床应用价值。
[Abstract]:The treatment of bone defect or nonunion of bone has been a long-term problem in the field of Department of orthopedics. The rapid development of bone tissue engineering and regenerative medicine provides hope for the repair and treatment of bone defect and bone nonunion. As one of the most important elements of bone tissue engineering, the preparation and research and development of the scaffold are widely concerned by the researchers. There are many bone tissue engineering scaffolds prepared by particles and poly (lactic acid hydroxy acetic acid) polymer, which can give full play to the advantages of inorganic particles and poly (lactic acid hydroxy acetic acid) polymers. The preparation method of inorganic particles / poly (lactic acid hydroxyacetic acid) polymer composite scaffolds is to use inorganic particles in poly (lactic acid hydroxyacetic acid) polymers. Mixing in the solution or melt leads to the inhomogeneous dispersion of inorganic ions in the poly (lactic acid hydroxy acetic acid) polymer, which can not be effectively contacted with the host cell to play a biological role. Therefore, the preparation of a homogeneous inorganic particle / poly (lactoacetic acid) polymer composite scaffold with homogeneous structure and composition is of great importance. Objective to investigate the synthesis of terminal carboxylated poly (lactate glycolic acid) polymer (PLGA-COOH); to prepare two kinds of nano bone repair materials based on carboxylation PLGA, test the related characterization, mechanical properties, biodegradability and biocompatibility of nanoscale repair materials, and verify the ectopic osteogenesis of nanoscale repair materials in vivo Ability and bone defect repair performance. Materials and methods 1, prepare carboxylation PLGA, blend with nano HAP and TCP respectively, prepare PLGA-COOH/HAP and PLGA-COOH/TCP composite scaffolds in the process of mixing, salt and freeze-drying. The related characterization and mechanical properties and degradation properties of.2 are studied, and the complex scaffold and MC3T3-E1 cells are co cultured and determined in vitro. The effect of stents on cell proliferation, cell cycle, osteogenesis and adhesion properties of.3, the composite scaffold was loaded with rhBMP-2 in vitro, and the release performance was studied in vitro. The ectopic osteogenesis of the composite scaffold and the supported rhBMP-2 scaffold was evaluated through the muscle bag experiment of the gluteus maximus muscle of SD rats. The scaffold was evaluated by the rabbit skull defect model. The repair performance of bone defect repair was.5. The repair performance was evaluated by the rabbit ulna critical bone defect model. Results 1, nuclear magnetic hydrogen spectrum and molecular weight detection showed that the carboxyl group was successfully grafted on the end of PLGA polymer, and the molecular weight of PLGA polymer was about 120000.SEM and Micro CT scanning. The stent has a porous structure, and the pore size homogeneous.EDX confirms that the characteristic elements of the two scaffolds are calcium, phosphorus and carbon. The mechanical properties test showed that the compressive and bending strength of the two kinds of scaffolds were not significantly different. The degradation experiments showed that the weight of the two scaffolds decreased with the prolongation of time, and the crystal structure of the inorganic components in the scaffold was stable; PLGA The molecular weight of the polymer decreased with time and decreased.2, CCK-8 and flow results showed that the two scaffolds had good biocompatibility and cell growth promoting effect.ALP determination showed that two kinds of composite scaffolds had a certain bone differentiation performance, superior to pure PLGA; scanning electron microscopy and laser cofocal results showed that the composite scaffold and carboxyl PLGA were all The cell morphology is good for cell adhesion, and the cell morphology is better.3. The two composite scaffolds can be used as the sustained release carrier of rhBMP-2. The effective release time is up to 360 hours. The stents loaded with rhBMP-2 are implanted in the muscle bag of the gluteus maximus muscle of the SD rats. The ectopic ossification occurs at 2 and 4 weeks after the operation, the new bone mass is.4, and the composite scaffold has no load repair. The ability of the critical bone defect of the rabbit skull, compared with the simple PLGA polymer, is more beneficial to the new bone formation and bone remodeling.5. The composite scaffold and the supported rhBMP-2 scaffold have the ability to repair the long segmental critical bone defect of the rabbit ulna. After the load rhBMP-2, the composite scaffold bone repair ability is enhanced. Conclusion 1, the successful preparation of the carboxyl PLGA polymer. Carboxyl PLGA polymers have successfully prepared two kinds of nanoscale repair materials. PLGA-COOH/HAP knows that the porosity and pore size, mechanical properties and degradation properties of PLGA-COOH/TCP. basically meet the requirements of the ideal bone tissue engineering materials for.2. The scaffold materials have good cellular biocompatibility and cell proliferation, differentiation, and no obvious cytotoxicity.3, PLGA- COOH/HAP know that PLGA-COOH/TCP can be used as the rhBMP-2 sustained-release carrier; the composite scaffold material loaded with rhBMP-2 has good biological activity and is beneficial to the formation of new bone. The simple scaffold material has no obvious inflammatory reaction in the body and has good biological safety. It conforms to the national biological medical material standard.4, the critical bone of the rabbit skull In the case of defect, the PLGA-COOH/HAP PLGA-COOH/TCP composite stent has strong bone repair and bone remodeling, which is superior to PLGA-COOH.5 alone. The composite scaffold with rhBMP-2 can repair the critical bone defect of the rabbit ulna segment, and the two composite scaffolds have potential clinical value.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R687

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