软骨微粒复合仿生基质溶胶体外构建组织工程软骨可行性研究

发布时间：2018-06-23 09:26

本文选题：软骨微粒 + 仿生基质溶胶　；参考：《第三军医大学》2015年硕士论文

【摘要】：背景:关节软骨损伤临床发病率越来越高,正被人们高度关注。由于正常透明软骨是一种特殊组织,无血管、神经,损伤后其自我修复能力低下,直径超过4mm的软骨缺损不能完全自我修复。软骨损伤后,会导致关节肿胀和疼痛,影响运动功能,不及时治疗,最终发展为骨性关节炎。传统治疗关节软骨缺损的方法有:微骨折技术、自体/异体骨软骨移植技术等,但都存在一定的缺陷和不足。随着组织工程技术的发展,自体软骨细胞移植(ACI)技术在临床得到应用,并取得明显治疗效果,但还是存在明显缺陷:首先软骨细胞体外培养、扩增,难以模拟在体微环境,缺乏机体免疫系统的监控,植入的软骨细胞通常已经发生表型改变,最终形成的修复组织无论在结构上还是成份上都与天然软骨存在本质差异,其次该方法需要二次手术植入修复缺损,治疗期限跨度长,身体损伤大,给患者带来巨大身体痛苦和经济负担。目的:本研究利用组织工程技术体外构建组织工程软骨,无菌条件下取贵州小香猪膝关节软骨,制作成软骨微粒,体外培养后观察软骨微粒中软骨细胞脱落、增殖情况,筛选最佳增殖效果的粒径范围,然后用增殖效果最佳的软骨微粒作为种子细胞;制备Ⅱ型胶原蛋白、硫酸软骨素、透明质酸(COLⅡ/CS/HA)仿生基质凝胶,加入EDAC交联剂,升温成凝胶后,检测其成胶性能;用软骨微粒与仿生基质溶胶混合后体外共培养,通过大体和组织学检测,评估体外构建组织工程软骨的可行性。本实验成功后,可望为一次手术治疗关节软骨缺损提供依据。方法:取3-5月龄贵州小香猪膝关节,把软骨制成微粒状;将微粒用孔径分别为120μm、212μm、475μm、880μm微粒筛过滤;收集不同粒径的软骨微粒,按粒径由小至大设A、B、C、D组,保证每组质量相同,均匀种植到3个12孔培养板中,用高糖培养基培养;培养1d、3d、7d时,分别取出1板进行细胞计数,筛选脱落、增殖效果最佳的粒径范围。用环钻在离体新鲜猪膝关节上制备直径8mm全厚软骨缺损模型;将COLⅡ/CS/HA配制成终浓度10mg/ml复合仿生基质溶胶,取3ml调至中性,加入EDAC20μl搅拌均匀;用1ml注射器吸入中性复合胶并注入软骨缺损模型,放入37℃孵箱,6min后观察成凝胶情况;设计软骨微粒获取装置,包括主机、刨头、踏板;利用装置获取软骨微粒在100-120μm之间,符合最佳增殖粒径标准;COLⅡ/CS/HA按质量比8:1.5:0.5溶于0.15mol/lHCL中,取3ml调至中性备用;软骨微粒按5×104个/ml与COLⅡ/CS/HA溶胶混合,加入EDAC20μl搅拌均匀,注入软骨缺损模型中,升温6min成凝胶后,放入高糖培养基中培养,1周后进行大体,免疫荧光和组织学观察。结果:制备的软骨微粒即刻显微镜观察软骨细胞分布均匀,呈强折光性,可见部分软骨陷窝被切开;培养1d各组微粒均有少量细胞脱落;3d时可见较多软骨细胞从软骨微粒中脱落、增殖;7d时大量软骨细胞脱落、增殖,几乎长满板底,贴壁并呈长条状。在3个时间点对各组软骨细胞脱落、增殖情况计数,A组较B、C、D组软骨细胞增殖好,差异均有统计学意义(P0.05),最佳增殖粒径为小于120μm的软骨微粒;制备的仿生基质溶胶3ml,加入EDAC20μl交联剂后低温下呈溶胶状,具有流动性,升温一定时间后,变成凝胶状,弹性、强度良好;软骨微粒复合仿生基质溶胶体外共培养1W,大体观察见:外观呈淡红色,培养基充分浸入基质中,触之表面光滑并具有一定的弹性和强度;免疫荧光示:软骨微粒中的软骨细胞及脱落、增殖的软骨细胞成活良好;HE染色示:软骨微粒均匀分散于凝胶中,软骨微粒中软骨细胞存活良好;阿尔新兰染色示:软骨细胞从微粒中脱落、增殖并扩散至周围基质中;天狼猩红染色偏振光示:软骨微粒与凝胶基质融合良好,且成份基本相同。结论:软骨微粒体外培养完全可以存活,不同粒径的软骨微粒均有软骨细胞脱落、增殖。证实粒径小于120μm增殖效果最好,粒径越小切开软骨陷窝的机率大,细胞从微粒中脱落就多,此方法获取的软骨微粒作为组织工程软骨种子细胞来源具有可行性;COLⅡ/CS/HA仿生基质溶胶加入EDAC交联剂后利用温控相变原理,6min能够由溶胶成凝胶,且凝胶弹性、强度好,具备植入体内缺损处的初始稳定性,满足作为支架材料要求;软骨微粒复合仿生基质溶胶体外共培养,混合物中有大量软骨细胞脱落、增殖,软骨微粒与基质融合较好,体外成软骨具有可行性,辅以最佳的在体培养增殖环境,能形成透明软骨,是修复关节软骨缺损一种新方法。
[Abstract]:Background: the clinical incidence of articular cartilage injury is becoming more and more high and is being paid great attention to. Because normal hyaline cartilage is a special tissue, no blood vessel, nerve, its self repair ability is low and the cartilage defect with diameter more than 4mm can not be repaired completely. After the injury of cartilage, it can cause joint swelling and pain and affect motor function. The traditional methods for the treatment of articular cartilage defects are: micro fracture technique, autologous / allograft osteochondral transplantation, but there are some defects and shortcomings. With the development of tissue engineering, autologous chondrocyte transplantation (ACI) technology is used in clinical application, and a significant treatment effect has been obtained. However, there are still obvious defects: first, the culture of cartilage cells in vitro, amplification, is difficult to simulate in the body microenvironment, the lack of the monitoring of the body's immune system, the implanted cartilage cells usually have phenotypic changes, and the final formed tissues are essentially different from the natural cartilage in both the structure and the composition, followed by the method. The two operation was implanted to repair the defect, the duration of the treatment was long, the body damage was large, and the patient had great physical pain and economic burden. Objective: to construct tissue engineering cartilage in vitro by tissue engineering technique, take the cartilage of knee joint of Guizhou small Xiang pig under aseptic condition, make cartilage microparticles, and observe the medium soft cartilage after culture in vitro. Bone cells shedding, proliferation, screening the best size of the proliferation effect, and then using the best cartilage particles as seed cells; preparation of type II collagen, chondroitin sulfate, hyaluronic acid (COL II /CS/HA) bionic matrix gel, adding EDAC crosslinker, after heating the gel to detect the gelation performance; cartilage microparticles and imitation The feasibility of construction of tissue engineered cartilage in vitro was evaluated through gross and histological examination. After the success of this experiment, we hope to provide a basis for a surgical treatment of articular cartilage defects. Methods: the knee joint of 3-5 months old Guizhou miniature pigs was taken to make the cartilage microparticle shape, and the pore size was 120 mu m, respectively. 212 micron m, 475 m, 880 micron particle sieves were filtered, and the cartilage particles of different sizes were collected. According to the size of A, B, C, D groups, each group was given the same quality, and 3 12 hole culture plates were planted evenly. The 1 plates were counted for 1D, 3D and 7d respectively. The diameter of 8mm full thickness cartilage defect was prepared on the knee joint of fresh pig. COL II /CS/HA was prepared into a final concentration 10mg/ml composite biomimetic matrix sol, 3ml to neutral, and EDAC20 Mu l to be stirred, and neutral compound glue was inhaled with 1ml syringe and injected into the cartilage defect model, and the gel condition was observed at 37 C, and the gel condition was observed after 6min. A cartilage microparticle acquisition device, including a host, a planer, and a pedal, is used to obtain the cartilage particles between 100-120 mu m and the optimum diameter standard. COL II /CS/HA is dissolved in 0.15mol/lHCL by mass ratio 8:1.5:0.5 to neutral reserve, and the cartilage particles are mixed by 5 x 104 /ml with COL II /CS/HA sols, adding EDAC20 Mu L and even mixing, In the model of injecting cartilage defect, after heating 6min into gelatin, it was cultured in high glucose medium. After 1 weeks, gross, immunofluorescence and histological observation were carried out. Results: the cartilage microscopes were prepared by the immediate microscope to observe the uniform distribution of cartilage cells, strong refraction, and some cartilage lacunae were cut, and a small amount of cells were removed from each group of 1D. At 3D, many chondrocytes were found to fall off from the cartilage particles and proliferate. When 7d, a large number of chondrocytes were dropped, proliferated, almost full of plate and long strip. At 3 time points, the chondrocytes were dropped, the proliferation was counted, and the proliferation of chondrocytes in group A was better than that in B, C and D group, the difference was statistically significant (P0.05), and the best proliferation particle size was the same. The cartilage particles less than 120 mu m; the prepared biomimetic matrix sol 3ml, adding EDAC20 Mu l crosslinking agent, is gelatinous and fluidity at low temperature. After a certain time heating, it becomes gelatinous, elastic and strong, and the cartilage microparticles compound bionic matrix sol is co cultured in vitro for 1W. The general observation is that the appearance is light red, the medium is fully immersed in the matrix. The surface of the contact is smooth and has a certain elasticity and strength; immunofluorescence shows that the cartilage cells in the cartilage particles and the chondrocytes proliferate well; HE staining shows that the cartilage particles are evenly dispersed in the gel, and the cartilage cells in the cartilage microparticles are well alive; alnew blue shows that the cartilage cells fall off the particles and proliferate. Diffused into the surrounding matrix, Sirius red staining polarized light showed that the cartilage particles were well fused with the gel matrix, and the composition was basically the same. Conclusion: the cartilage microparticles can survive in vitro, and the cartilage particles with different particle sizes have the cartilage cells falling off and proliferating. It is proved that the particle size is smaller than 120 m, the smaller the particle size is, the smaller the diameter of the cartilage is cut in the cartilage. The probability of the nests is large and the cells fall off from the particles. The cartilage particles obtained by this method are feasible as the source of the tissue engineering cartilage seed cells. After adding EDAC crosslinker to the COL II /CS/HA bionic matrix sol, the 6min can be formed by the sol gel, and the gel is elastic and has good strength and has the defects in the body. The initial stability is satisfied as the requirement of scaffold material; cartilage microparticle composite biomimetic matrix sol is co cultured in vitro. A large number of chondrocytes in the mixture fall off, proliferate, the fusion of cartilage particles and matrix is better, and the cartilage is feasible in vitro, supplemented with the best in body culture environment, can form hyaline cartilage, is the repair of articular cartilage deficiency. Undermine a new method.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684.3

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