脂肪基质干细胞小肠黏膜下层双面复合的体外成软骨诱导

Chondrogenic differentiation of adipose-derived stromal cells seeded on small intestinal submucosa in vitro

背景:使用体外构建的组织工程软骨治疗软骨损伤是目前的研究热点,材料与支架材料的选择仍有较多问题尚待解决。目的:观察脂肪基质干细胞-小肠黏膜下层复合物在体外经成软骨诱导培养基诱导分化的效果。方法:将第3代脂肪基质干细胞接种于复水后的小肠黏膜下层双面,加入成软骨诱导培养基,于体外诱导培养7d和14d后,使用实时荧光定量RT-PCR检测Ⅱ型胶原mRNA,免疫组织化学染色检测Ⅱ型胶原蛋白,甲苯胺蓝染色观察细胞外基质,扫描电镜观察体外成软骨诱导14d后细胞在支架材料上的生长状况。结果与结论:Ⅱ型胶原mRNA实时荧光定量RT-PCR检测提示,体外成软骨诱导后7,14d的脂肪基质干细胞-小肠黏膜下层复合物Ⅱ型胶原mRNA的标化值与未诱导的脂肪基质干细胞-小肠黏膜下层复合物差异有显著性意义(P<0.05),诱导14d与诱导7d相比,差异亦有显著性意义(P<0.05)。脂肪基质干细胞-小肠黏膜下层复合物成软骨诱导后14d,Ⅱ型胶原免疫组织化学染色为阳性,甲苯胺蓝染色可见基质异染,未诱导的复合物Ⅱ型胶原免疫组织化学染色为阴性。扫描电镜检测显示诱导14d时,细胞长满支架材料的双面。提示脂肪基质干细胞复合至小肠黏膜下层后,在体外经成软骨诱导培养基成软骨诱导后,能够向成软骨细胞分化。

BACKGROUND： Treating cartilage defects with tissue engineered cartilage constructed in vitro is a research hot point, but there are still many problems to be resolved. OBJECTIVE： To observe chondrogenic differentiation effect of chondrogenic medium on adipose-derived stromal cells （ADSCs） and small intestinal submucosa （SIS） composite in vitro. METHODS： The 3rd passage of ADSCs were seeded onto both sides of SIS, and induced by chondrogenic medium in vitro, 7 days and 14 days after being induced, real-time fluorescent quantitative RT-PCR was used to analyze mRNA level of type Ⅱ collagen, type Ⅱ collagen was examined by histoimmunologic staining, toluidine blue staining was used to observe extracellular matrix. Scanning electron microscope （SEM） was used to observe cellular morphology after 14 days of chondrogenic differentiation. RESULTS AND CONCLUSION： Standardized quantity of type Ⅱ collagen mRNA in ADSCs-SIS composites which were induced for 7 days and 14 days were larger than that in uninduced ADSCs-SIS composites （P 0.05）, standardized quantity of type Ⅱ collagen mRNA in ADSCs-SIS composites which were induced for 14 days was larger than that in ADSCs-SIS composites which were induced for 7 days （P 0.05）. Histoimmunologic staining of type Ⅱ collagen was positive in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium, but was negative in non-induced ADSCs-SIS composites. Extracellular matrix was metachromatic stained by toluidine blue in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium. Plenty of ADSCs adhered to both sides of SIS after 14 days of chondrogenic differentiation were observed by SEM. ADSCs could differentiate into chondrogenic cells after being induced by chondrogenic medium in SIS scaffold.