脱细胞半月板细胞外基质对半月板细胞的影响

摘要

Objective We explore the effect of porcine decellularized meniscal extracellular matrix (dMECM) on the proliferation, cell activity and redifferentiation of the rabbit passaged meniscal fibrochondrocytes. Methods The novel dMECM biomaterial was prepared using waterproof pulverization and differential centrifugation approach. CCK-8 was used to quantitatively evaluate the cell proliferation of dMECM. The rabbit inner meniscus fibrochondrocytes (P3) were seeded in the dMECM modified growth surface in order to compare with the untreated growth surface (control group). The cell activity was observed by live/dead staining after 7, 14 d culture, the GAG and collagen content werer determined according to kits, and RT-PCR analysis was used to evaluate mRNA expression level of collagens. Results CCK-8 results demonstrated cells proliferation capacity in dMECM group was significantly more potent than that in the control group at 3, 6 d (P<0.05). In comparison with the control group, the live/dead staining results confirmed dMECM surface maintained favorable cell activity. GAG and collagen content assessment results revealed that the fibrochondrocytes in the dMECM group secreted significantly more GAG and collagen than that in the control group at 7, 14 d (P<0.05). RT-PCR results showed that the expression of type II collagen mRNA was significantly up-regulated than the dMECM group at 7, 14 d (P<0.05). Conclusion dMECM enhances the cellular proliferation, viability and redifferentiation of the rabbit passaged meniscal fibrochondrocytes, which shows that dMECM may be one of the ideal candidate biomaterial for meniscal tissue engineering applications in future.%目的探讨脱细胞半月板细胞外基质（dMECM）对传代半月板细胞增殖、细胞活性以及细胞表型的影响。　　方法用 CCK-8法检测 dMECM 对半月板细胞增殖的影响；将 P3代的内侧半月板纤维软骨细胞种植在 dMECM修饰盖玻片上，以未修饰盖玻片为对照，体外培养7、14 d后进行细胞活性检测，糖胺多糖、胶原分泌含量测定并用RT-PCR 检测半月板细胞特异性基因 mRNA 表达变化。　　结果 CCK-8结果证实，与对照组比较，生长在 dMECM修饰盖玻片上的 P3代兔半月板纤维软骨细胞具有更好的细胞增殖特性（P<0.05）；细胞死/活染色结果证实 dMECM组可维持更好的细胞活性；糖胺多糖和胶原定量检测结果证实 dMECM 组在第7、14天时，比对照组分泌更多的糖胺多糖和胶原（P<0.05）。RT-PCR 的检测结果证实体外培养7、14 d 时，dMECM 组 II 型胶原 mRNA 表达显著上调（P<0.05）。　　结论 dMECM 可以很好地促进半月板纤维软骨细胞的增殖、分化以及细胞活性的维持，可能是未来半月板组织工程领域非常有前景的支架材料之一。