兔脂肪间充质干细胞膜片促进关节软骨损伤修复

Adipose- derived mesenchymal stem cell sheet for promoting the repair of articular cartilage defect in rabbit

摘要:

［目的］探讨兔脂肪间充质干细胞膜片对兔关节软骨损伤的修复作用。［方法］体外分离培养兔脂肪间充质干细胞， 通过多系分化对细胞进行鉴定。通过化学方法构建兔脂肪间充质干细胞膜片（添加 50 μg/ml 抗坏血酸），膜片构建成功后对其进行检测：分别在倒置相差显微镜下观察膜片的结构，大体观以及扫描电镜观察。制备兔膝关节骨软骨缺损模型（直径为 3.5 mm，深度为 3 mm），然后将实验分为两组：对照组和细胞膜片组。术后 3 个月取标本进行检测，观察软骨的修复情况。［结果］原代兔脂肪间充质干细胞呈短梭形、三角形贴壁生长，经过传代培养后细胞逐渐呈涡旋状生长，第 3 代兔脂肪间充质干细胞具有多系分化能力；兔脂肪间充质干细胞膜片具有多层结构，细胞分泌大量的细胞外基质，将细胞镶嵌在其中。术后 3 个月取标本观察检测提示：细胞膜片组骨软骨缺损处可见大量新生的软骨组织再生，新生的软骨组织形态和颜色均接近于正常软骨；而对照组几乎没有新生软骨的形成。O’Driscoll 评分结果也显示细胞膜片组的评分明显高于对照组，差异有统计学意义 （P<0.05）。 ［结论］兔脂肪间充质干细胞膜片能够促进兔关节软骨的再生，为临床软骨损伤的修复提供了新的思路。

Abstract:

[Objective] To explore the repair effect of adipose-derived mesenchymal stem cell sheet on articular cartilage defect in rab- bit. [Methods] In vitro, rabbit adipose-derived mesenchymal stem cells were isolated and cultured, and then were identified by multi-line differentiation. Subsequently, the adipose-derived mesenchymal stem cell sheet was constructed by chemical method with adding 50μg/ml ascorbic acid. The sheet structure of the cell was investigated by inverted phase contrast microscope, in addition, the gross observation and scanning electron microscope. In vivo, the osteochondral defect models with 3.5mm in diameter and 3mm in depth was prepared on the fem- oral condyles in rabbits, and divided into two groups, the blank control group and cell sheet group with a cell sheet implanted on the defect. Three months later, specimens were harvested for gross evaluation and histological observation. [Results] In vitro, the primary rabbit adi- pose- derived mesenchymal stem cells grew adherently in a short spindle shape and triangle shape. After subculture, the cells gradually grew in a vortex shape. The third-generation rabbit adipose-derived mesenchymal stem cells had multi-lineage differentiation capacity. The rabbit adipose-derived mesenchymal stem cells sheet had a multi-layer structure, and the cells secreted a large amount of extracellu- lar matrix, with cells embedded in it. In vivo, the osteochondral defect had a large amount of new cartilage tissue regenerated in the cell sheet group at 3 months after implantation, which looked like normal cartilage in shape and color, whereas no new cartilage was seen in the blank control group. The cell sheet group was marked significantly higher O' Driscoll score than the blank control group (P＜0.05) . [Conclusion] This adipose-derived mesenchymal stem cell sheet does promote the articular cartilage regeneration in rabbit, which might provide a new idea for the repair of cartilage damage in clinical setting.