自体软骨微粒复合COLⅡ-HA-CS仿生基质溶胶修复局灶性关节软骨缺损再生机制研究

Articular cartilage focal defect is a common disease in clinical medicine. If not promptly treated, it causes arthritis.The repaired tissue treated by traditional microfracture technique is always fibrous cartilage with poor wear resistance. In recent years, autologous chondrocyte implantation (ACI) is proved to be a new method. Chondrocytes always needed to be expanded in vitro in the past. Due to lack of the immune monitoring system, chondrocyte phenotype changes before transplantation. This is a key reason why the repaired tissue always has defect in quality. In view of these problems, combined with the latest progress, our group present a new strategy- - regeneration and repair of the articular cartilage focal defect with the composition of autologous cartilage microparticle and COL II-HA-CS biomimetic matrix sol. The in vitro expansion of chondrocyte will be translated into the body by implanting cartilage microparticle. Chondrocytes proliferate in vivo and generate high quality cartilage. Cartilage microparticles will be evenly dispersed and fixed by COL II-HA-CS biomimetic matrix sol. This new strategy only needs one operation.We plan to prepare cartilage microparticle with mechanical pulverization at low temperature, then evaluate the proliferation ability of chondrocyte in cartilage microparticle and elucidate the dose-effect relationship between the particle size and the proliferation ability. The biomimetic matrix sol will be prepared using the theory and technique of temperature controlled phase change. The optimal component ratio and gelation condition will be decided by the gelling experiment. We will also elucidate the dose-effect relationship between the planting density and the new generated cartilage quality and clarify the formation mechanism of regenerative cartilage.This strategy will provide a new principle and new technique for treating the cartilage focal defect and has a wide application prospect.

关节软骨局灶性缺损常见，治疗不及时将引发关节炎。传统微骨折术最终形成耐磨性较差纤维软骨。新近自体软骨细胞移植术为其再生修复提供了新思路，以往均需对软骨细胞进行体外扩增，由于缺乏免疫监控，植入的细胞多已发生表型改变，导致再生软骨质量缺陷。针对上述问题，结合最新进展，项目组提出：应用自体软骨微粒复合仿生基质（COLⅡ-HA-CS）溶胶再生软骨新策略。以软骨微粒移植方式将体外扩增过程转移至体内，实现软骨细胞体内增殖、再生高质量软骨；辅以溶胶进行分散与固定，一次手术即可完成。本项目拟利用低温粉碎法制备软骨微粒，评价微粒中软骨细胞增殖能力、阐明粒径与增殖能力的量效关系；应用温控相变原理研制仿生基质溶胶，通过成胶实验确定组分配比和成胶参数；应用体外培育和体内动物实验，评价微粒接种密度和新生软骨质量之间的量效关系、阐明新生软骨形成机制；为临床应用该策略治疗软骨缺损提供新原理和新技术，具有广泛应用前景。

关节软骨局灶性缺损常见，治疗不及时将引发关节炎。传统微骨折术最终形成耐磨性较差纤维软骨。新近自体软骨细胞移植术为其再生修复提供了新思路，以往均需对软骨细胞进行体外扩增，由于缺乏免疫监控，植入的细胞多已发生表型改变，导致再生软骨质量缺陷。针对上述问题，项目组提出：应用自体软骨微粒复合仿生基质（COLⅡ-HA-CS）溶胶再生软骨新策略。以软骨微粒移植方式将体外扩增过程转移至体内，实现软骨细胞体内增殖、再生高质量软骨；辅以溶胶进行分散与固定，一次手术即可完成。现已按计划完成任务书中所有研究内容：建立贵州小香猪动物实验基地。研制出软骨微粒获取专用装置，采用该装置可有效获得不同粒径软骨微粒，且对软骨细胞损伤小；粒径在100-150µm之间的软骨微粒中的软骨细胞增殖能力最强，体外培养1天，即可见软骨细胞从软骨微粒上脱落，培养3天可见克隆形成，培养7天仍然可见有细胞从软骨微粒中脱落。建立了仿生基质溶胶制备技术，醛基化的硫酸软骨素(OCS)能够与COLⅡ-HA中的胺基发生Schiff‘s Base反应并形成凝胶，从而将软骨微粒固定于缺损；此外，可以通过控调醛基化程度及凝胶各组分比例调整成胶时间和强度。将软骨微粒（粒径为125um左右）与仿生基质溶胶（质量比为COLII:HA:OCS=3:1:1）按照一定比例（60mg:360mg）复合后约5min即可形成具有一定强度的凝胶块；培养1周后颜色与正常软骨相似，组织切片见软骨微粒在凝胶中分布均匀，微粒中的软骨细胞形态结构正常，凝胶基质中可见大量增殖、脱落的软骨细胞；培养1月后凝胶块的大小和形状未发生明显改变，凝胶块硬度和粘弹性有所增加。动物实验结果表明，术后6个月实验组缺损区修复组织表面光滑，平整，边缘与周围正常软骨整合良好，钙化层连续，软骨下骨结构完整，新生修复组织的颜色、成分与周围正常软骨类似，主要为II型胶原蛋白和软骨细胞；对照组缺损区表面毛糙，钙化层不连续，软骨下骨塌陷；组织学评分实验组修复效果明显好于对照组（p<0.05）。本研究的相关成果将为临床治疗局灶性软骨缺损提供新思路。