富含BMP-2的PLGA材料制备及对兔关节软骨缺损修复的应用研究

发布时间：2018-01-18 12:19

本文关键词：富含BMP-2的PLGA材料制备及对兔关节软骨缺损修复的应用研究　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

更多相关文章： 软骨缺损 修复 PLGA BMP-2 磷脂

【摘要】：目的:骨和关节软骨缺损的修复迄今尚未有一种理想的方法,而对前者的研究较多,对软骨缺损的研究相对较少。临床治疗关节软骨缺损常用软骨成形术、骨髓刺激术等,由于修复的软骨成分以纤维软骨为多、短期效果尚可、但长期随访效果欠佳。间充质干细胞的多方向分化潜能,一直是软骨修复研究的热点,但是对间充质干细胞软骨分化后的问题仍存在着的争论。多能干细胞也用于软骨缺损的修复,由于涉及伦理道德问题及长期使用发生肿瘤的风险,也阻碍了多能干细胞的进一步研究。支架材料同样是研究的热点,特别是天然材料,如水凝胶、胶原等,但存在植入体内后机械强度差,降解时间难以控制等问题;人工合成高分子材料,如聚乳酸(PLA)和聚乙醇酸(PGA),有容易出现局部酸中毒、过量加热引起单体链解聚等问题;羟磷灰石等生物材料由于脆性大、塑性困难,较少用于软骨修复中。组织工程方法特别是模仿自然软骨形成步骤的方法目前是研究的热点,但是对软骨的构建离临床的应用尚有很长一段距离。正是基于目前关节软骨缺损的修复尚无“金标准”的情况下,本实验希望能寻找到更理想的材料和方法,除了能够通过实验证明制备的材料对骨组织工程的应用可行性外,最主要的是希望材料能促进关节软骨缺损的修复,为未来临床的应用夯实基础,这是本论文研究的目的和出发点。方法:1.制备骨形态发生蛋白(BMP-2)与磷脂复合物:BMP-2与磷脂按照不同的比例,溶解在有机化学溶剂中,经过冷冻干燥形成复合物。2.采用溶液浇铸法制备聚乳酸-羟基乙酸共聚物(PLGA)、骨形态发生蛋白(BMP-2)和磷脂的复合物膜。3.检测复合物的理化特征,包括:BMP-2的包封率、复合材料的静态接触角、吸水率、扫描电镜下的形态、能量色谱仪分析等等,并对材料的BMP-2体外释放能力通过透析法进行测定。4.PLGA-BMP-2-磷脂复合物与OCT-1成骨样细胞联合培养,通过细胞粘附试验、细胞增殖试验、免疫组织化学分析、细胞碱性磷酸酶测定、实时定量荧光PCR分析等方法了解并比较制备的复合缓释材料对细胞粘附、增殖、分化特别是长期生长的影响。5.制备新西兰大白兔膝关节软骨缺损实验动物模型,将制备的复合物植入软骨模型内,通过大体标本、组织学染色、核磁共振扫描等方法观察并比较植入物修复关节软骨缺损情况。结果:1.PLGA-BMP-2-磷脂复合物的包封率、静态接触角、吸水性、扫描电镜形态及体外释放试验结果均明显优于试验对照(PLGA-BMP-2)。特别是BMP-2的体外释放,实验组在长达30天观察中仍有BMP-2表达,而对照组的释放时间主要集中在开始释放的最初3天。2.PLGA-BMP-2-磷脂复合物与细胞联合培养结果显示:实验组促进了细胞的粘附与增殖,特别是长时间(7天)观察,实验组对细胞增殖的促进作用更加明显(p0.01);对细胞免疫组化分析显示实验组能够提高OCT-1细胞的Ⅰ型胶原表达;实验组在长期观察中(15天)显示出对细胞碱性磷酸酶活性有更强的影响,与对照组有显著的差异(p0.01);同样在长期观察中(15天),通过RT-PCR测定OCT-1细胞的成骨相关基因:骨钙素(OCN)、胶原蛋白(COL)、骨桥蛋白(OPN)、RUNT相关转录因子2(RUNX2)的表达,均显示实验组与对照组有显著性差异(p0.01),实验组明显上调了这些基因表达。3.动物关节软骨缺损的体内修复实验结果显示在2周、4周、8周、12周,经过大体标本观察、组织学检查、MRI检查均显示出实验组修复软骨缺损的能力更强。特别是经过长时期观察(8周、12周),实验组与对照组之间比较有显著性差异(p0.01)。结论:1.磷脂增强了PLGA与BMP-2的结合能力,解决了既往PLGA/BMP-2复合物中BMP-2释放过快的问题,证明了PLGA-BMP-2-磷脂复合物能够达到BMP-2缓释的目的。2.PLGA-BMP-2-磷脂复合物体外实验证实复合物能够促进OCT-1细胞的粘附、增殖,分化;尤其是对成骨细胞的长期增殖分化有明显影响,证明了PLGA-BMP-2-磷脂复合物有更强的BMP-2缓释能力,有能力应用于骨组织工程。3.PLGA-BMP-2-磷脂复合物植入动物关节缺损模型体内后能够促进关节软骨缺损的修复,实验组效果优于对照组,证实了实验组中BMP-2对软骨修复有缓释作用及动物体内植入复合物的可行性。4.PLGA-BMP-2-磷脂复合物能够为软骨的修复提供一种新颖的复合生物材料。
[Abstract]:Objective: to repair bone and articular cartilage defects has not been an ideal method, and the former researches on cartilage defects are relatively few. Annuloplasty in the clinical treatment of articular cartilage defects commonly used in cartilage, bone marrow stimulation, the cartilage repair by fibrocartilage is much, short-term effect is acceptable, but long term follow-up effect. Mesenchymal stem cell pluripotency, is a research hotspot in the repair of cartilage, but cartilage mesenchymal stem cells after differentiation problems still exist in the debate. Pluripotent stem cells to repair soft bone defect, because of the risk of ethical problems and long-term use of tumor, but also hinder the further study of pluripotent stem cells. The same scaffold is a hotspot of research, especially the natural materials, such as collagen hydrogel, etc., but there is in vivo mechanical strength, degradation Time is difficult to control; synthetic polymer materials, such as polylactic acid (PLA) and polyglycolic acid (PGA), are prone to local acid poisoning, caused by excessive heating of single chain depolymerization problems; hydroxyapatite biomaterials such as brittleness, plastic difficult, less used in cartilage tissue engineering. Methods especially the method of imitating the natural cartilage formation step is currently the research focus, but the construction of cartilage from the clinical application is still a long distance. It is based on the repair of articular cartilage defects is the "gold standard" under the condition of this experiment, hoping to find more ideal materials and methods, in addition to through the experiment proves that the feasibility of preparation of materials for bone tissue engineering, the most important is that material can promote the repair of articular cartilage defects, and lay a solid foundation for future clinical application, this article research The purpose and starting point. Methods: 1. preparation of bone morphogenetic protein (BMP-2) and phospholipid complex: BMP-2 with phospholipid according to different proportion, dissolved in organic chemical solvents, freeze-dried forms.2. complexes were prepared by solution casting poly lactic acid glycolic acid copolymer (PLGA), bone morphogenetic protein (BMP-2) including physicochemical characteristics, complex membrane phospholipids and detection of.3. complexes: BMP-2 encapsulation efficiency, static contact angle of composite material, water absorption, scanning electron microscope, energy chromatograph analysis and so on, and in vitro BMP-2 on material release ability was determined by.4.PLGA-BMP-2- phospholipid complex OCT-1 and co cultured with osteoblast like cells by dialysis method, through cell adhesion test, cell proliferation assay, immunohistochemistry, cell alkaline phosphatase, real-time quantitative PCR analysis method to investigate and compare the preparation The compound sustained-release materials on cell adhesion, proliferation, differentiation, especially the long-term growth effects of.5. preparation of New Zealand rabbits knee articular cartilage defect animal model, were implanted into cartilage model complex preparation, the specimen, histological staining, nuclear magnetic resonance scanning method to observe and compare the implant repair of articular cartilage defect situation results: 1.PLGA-BMP-2- phospholipid complex encapsulation efficiency, static contact angle, water absorption, scanning electron microscope and the in vitro release test results were significantly better than the control test (PLGA-BMP-2). Especially the release of BMP-2 in vitro, the experimental group during the 30 day observation is still in the expression of BMP-2, while the control group concentrated release time at the beginning of the first 3 days of release of.2.PLGA-BMP-2- phospholipid complex and cell co culture results showed that: the experimental group promoted the adhesion and proliferation of cells, especially the long time (7 days) observation, Effect of experimental group on cell proliferation more obviously (P0.01) on cell; immunohistochemical analysis showed that the experimental group can improve the expression of type I collagen OCT-1 cells; the experimental group in the long-term observation (15 days) showed a stronger effect on cell alkaline phosphatase activity, there is a significant difference with the control group (P0.01); also in long-term observation in (15 days), the determination of OCT-1 cells into bone related genes by RT-PCR: Osteocalcin (OCN), collagen (COL), osteopontin (OPN), RUNT related transcription factor 2 (RUNX2) expression in the experimental group showed a significant difference with the control group (P0.01) the experimental group was significantly increased, the expression of these genes in vivo animal experimental results of.3. repair of articular cartilage defects in 2 weeks, 4 weeks, 8 weeks, 12 weeks, after gross observation, histological examination, MRI examination showed that the repair of cartilage defect in experimental group especially the ability. A long period of observation (8 weeks, 12 weeks), compared with a significant difference between the experimental group and the control group (P0.01). Conclusion: 1. phospholipid enhanced binding ability of PLGA and BMP-2, to solve the BMP-2 PLGA/BMP-2 complex in the previous release too quick, proved to.2.PLGA-BMP-2- phospholipid complex object PLGA-BMP-2- phospholipid composite can reach BMP-2 the release proved complex can promote OCT-1 cell proliferation, adhesion, differentiation; especially has obvious effect on osteoblast proliferation and differentiation of long-term, proved that the BMP-2 release ability of PLGA-BMP-2- phospholipid complexes have stronger ability, application in bone tissue engineering.3.PLGA-BMP-2- phospholipid complex animal implantation joint defect model in vivo can promote the repair of articular cartilage defects, the experimental group is better than the control group, BMP-2 was confirmed in the experimental group have sustained-release effect and animal implantation on cartilage repair The feasibility of the.4.PLGA-BMP-2- phospholipid complex into the complex can provide a novel composite biomaterial for the repair of cartilage.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R687;R318.08

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