周期性流体应力刺激对组织工程软骨分化影响的实验研究

Influence of Dynamic Fluid Stimulation on the in Vitro Cartilage Differentiation

目的研究周期性流体应力刺激对组织工程软骨体外分化的影响,确立良好的动态培养方案.方法穿刺吸取成年兔骨髓,密度梯度离心法分离扩增骨髓间充质干细胞(Bone marrow derived mesenchymal stem cell,MSC).以2.0×107/ml密度复合于纤维蛋白胶,制成圆柱形人工组织.实验组材料经受周期为0.2 Hz流体应力刺激,于转壁生物反应器中培养2周,对照组静止培养,两组均于条件培养基内诱导软骨组织形成.2周后观察大体形态、和组织学形态、Ⅰ型和Ⅱ型胶原免疫组织化学表达,测量细胞活力和胶原、蛋白多糖含量等生化指标.结果应力刺激组材料的大体形态完整,而对照组材料破碎回缩.实验和对照组均可以诱发材料中MSC分化成为软骨细胞,但流体刺激组表达更高水平的II型胶原和蛋白多糖,细胞活力明显高于对照静止培养组(P＜0.01).结论周期性流体应力刺激明显促进MSC体外软骨分化,转壁生物反应器培养优于单纯静止培养.

Objective To investigate the influences of dynamic fluid strain on the in vitro chondrogenesis of tissue engineering cartilage, establish reliable dynamic culture methods of cartilage differentiation. Methods Bone marrow derived mesenchymal stem cell (MSC) of aspirates from adult rabbits were separated by gradient-density centrifugation and amplified in plates, mixed in fibrin sealant gel at final concentration of 2 × 107/ml in cylinder shape and then experienced 0.2 Hz fluid stimulation in rotating wall vessels (RWVs) bioreactor at 37℃ , 5 % CO2 for two weeks. The static cultures were performed as control group. Both groups experienced inducing cultures for chon-drocyte differentiation in condition medium. Gross appearance, histological section, immunohistochemistry, cell vitality, collagen and proteoglycan contents were examined after two weeks. Results Dynamic fluid strain can preserve better gross appearance of compounded material than control static cultures. Although both groups had induced chondrogenesis, the dynamic fluid stimulation cultures showed higher cell vitality and more collagen and proteoglycan than controls (P<0.01). Conclusion Dynamic fluid stimulation can improve the quality of MSC differentiation into chondrocytes in vitro, and the inducing cultures in RWVs bioreactor was more efficient than static cultures.