细胞-凝胶复合物促进聚己内酯内核与细胞膜片组织整合的可行性研究

The Feasibility of Using Cell-Hydrogel to Integrate PCL Inner Support with Cell-Sheet

目的探讨运用软骨膜片包裹聚己内酯(Polycaprolactone,PCL)内核,在裸鼠体内构建组织工程软骨的可行性;探讨Pluronic F-127凝胶和软骨细胞混合物促进PCL内核与软骨膜片组织整合的可行性。方法常规分离培养乳猪耳软骨细胞,取第1代软骨细胞高密度接种,体外培养4周后形成软骨膜片。软骨膜片包裹PCL内核形成"三明治"模型作为未处理对照组(NC组);模型内注射Pluronic F-127凝胶作为对照组(PC组);模型内注射Pluronic F-127凝胶和软骨细胞混合物作为实验组(Exp组)。3组植入同一裸鼠皮下(n=8),12周后取材比较各组软骨再生情况。结果软骨细胞高密度培养可形成软骨样组织,组织学证实有典型软骨陷窝结构,且高表达蛋白聚糖和Ⅱ型胶原。"三明治"模型植入体内后,可见软骨膜片形成了更为成熟的软骨样组织,表面光滑,质地柔韧。各组湿重、体积及厚度测量结果均有显著性差异,但各组力学检测结果无显著性差异。组织学证实,NC组和PC组仅外周软骨膜片形成了成熟的软骨样组织,PCL内核与膜片间未见软骨形成;Exp组PCL内核与软骨膜片间可见大量软骨再生,新生软骨与外周软骨膜片再生软骨实现了良好整合。结论软骨膜片包裹PCL内核可构建具有一定力学强度的组织工程软骨,注射Pluronic F-127凝胶和软骨细胞混合物后可有效促进PCL内核与软骨膜片的组织整合。

Objective To explore the feasibility of using sandwich model, PCL as an inner support encapsulated by chondrocyte sheet, to create tissue-engineered （TE） cartilage; To explore the feasibility of integrating PCL with chondrocyte sheet by injecting the mixture of Pluronic F-127 hydrogel and chondrocytes. Methods The isolated auricular ehondrocytes of passage 1 were seeded in six-well plate at a high density. Chondrocyte sheet formed after 4 weeks in vitro. PCL inner support encapsulated by chondrocyte sheet was set up as non-treated control group （NC group）. Sandwich model injected with Pluronie F-127 hydrogel was set up as control group （PC group）. Sandwich model injected with the mixture of Plnronic F-127 hydrogel and chondrocytes was set up as the experiment group （Exp group）. The above constructs were implanted subcutaneously into the same nude mice （n=8）. The samples were harvested 12 weeks after implantation for the evaluation of cartilage formation. Results After in vitro high cell-density culture, ehondrocyte sheet formed into cartilaginous tissue. Histologically, sheet showed specific cartilage characters including cartilage lacunae and expressed GAG and collagen II. After in vivo culture, sheet from the ＂sandwich＂ structure formed more mature cartilaginous tissue with smooth surface and flexible texture. The results of wet weight, thickness and volume from everv zroun were significantly different. However, there was no significant difference for Young＇s modulus. Histologically, an abundance of cartilage-like tissue was found between PCL inner support and chondrocyte sheet in Exp group, strongly expressing GAG. Although sheet developed into mature cartilage tissue in the NC and PC group, none of cartilaginous tissue was found between the PCL inner support and chondrocyte sheet. Conclusion PCL inner support encapsulated by chondrocyte sheet could construct TE cartilage with high mechanical strength. The approach of injection with the mixture of Pluronic F-127 hydrogel and chondrocytes could realize the cartilaginous integration of PCL inner support and chondrocyte sheet.