体外培养的半月板细胞软骨形成能力区划差异的分析研究

Regional Variation in Chondrogenesis Potential of in Vitro Cultured Meniscal Cells

目的比较经体外培养的半月板红区和白区细胞在软骨化形成能力方面的差异,验证细胞团粒作为半月板组织工程研究模型的可行性。方法分离半月板红区和白区细胞,进行平面扩增,并分别集结成细胞团粒,在形成细胞团粒后的当日,7,14和21 d收集细胞团粒样本,对样本进行DMMB、I型和II型胶原的免疫组化染色,以及DNA定量,sGAG和II型胶原的ELISA分析。结果在细胞团粒培养阶段,II型胶原、I型胶原和sGAG在细胞团粒中稳步累积,且胶原标志物呈现特异的空间分布形态。红区来源的半月板细胞具备比白区来源的细胞更强的sGAG、I型和II型胶原合成能力(P<0.05)。结论细胞团粒可以成为良好的半月板组织工程研究的微模型,在经过单层扩增和立体培养后,红区来源的半月板细胞较白区细胞具备更强的软骨化形成能力。

Objective To evaluate the chondrogenesis potential variation which might exist between in vitro cultured red zone and white zone meniscal cells,and testify the feasibility of aggregate as a model for tissue engineering research of meniscus.Methods Aggregates were developed from in vitro monolayer cultured meniscal cells with cell origin respectively of red and white zone.At day 0,7,14 and 21 aggregate samples were harvested for assays including DMMB staining,immunostaining for type I and II collagen,DNA quantification and ELISA assay of sGAG and type II collagen.Results Type I,type II collagen and sGAG all steadily accumulated in aggregates.Collagen markers demonstrated a specific spatial distribution pattern.Meniscal cells derived from red zone revealed an increased potential in sGAG,type I and II collagen production in comparison with cells of white zone origin（P〈0.05）.Conclusion Aggregate could be a good micro model for tissue engineering research of meniscus.After being monolayered expanded and 3D cultured,red zone meniscal cells exceed white zone cells in chondrogenesis potential.