TGF-β_1、IGF-I和地塞米松诱导BMSCs体外构建组织工程化软骨

Preliminary study of in vitro cartilage engineering by BMSCs induced with TGF-β1 IGF-I and dexamethasone

目的 探讨体外联合应用TGF-β1、IGF-Ⅰ及地塞米松诱导骨髓基质干细胞(BMSCs)体外构建组织工程化软骨的可行性。方法 抽取8周龄猪股骨骨髓,应用贴壁法分选单个核细胞,体外培养扩增后获得BMSCs,收集第2代细胞,以5×107/cm3的密度接种到聚羟基乙酸(PGA)制成的圆柱形三维支架材料上,7 d后联合应用TGF-β1、IGF-Ⅰ及地塞米松进行诱导和体外培养,未诱导组作为平行对照。4周和8周分别取材进行大体观察、组织学、组织化学及免疫组织化学检测。结果 体外诱导培养4周后,形成的细胞材料复合物表面光滑,质地柔韧,保持原大小形状;组织学显示有较为明显软骨陷窝,并分布有Ⅱ型胶原及聚合蛋白多糖(GAG);8周时外观灰白色,较坚硬,软骨陷窝更为明显,并连接成片,Ⅱ型胶原染色呈强阳性,有更多甲苯胺蓝异染的软骨特异性基质成分。对照组细胞材料复合物逐渐收缩,8周时直径仅约2 mm大小,组织学检测无软骨陷窝结构,Ⅱ型胶原免疫组化结果阴性。结论TGF-β1、IGF-Ⅰ及地塞米松可以体外诱导BMSCs在三维支架上形成组织工程化软骨。

Objective To study the feasibility of engineering cartilage in vitro using bone mesenchymal stem cells (BMSCs) as seed cells, which were chondrogenically induced by TGF-β1、IGF-Ⅰand dexamethasone. Methods Bone marrow was aspirated from femurs of 8-week old pigs. BMSCs wereisolated and in vitro expanded. The cells of passage 2 were collected, counted and seeded onto a cylinder-shaped polyglycolic acid (PGA) scaffoldat a density of 5×107/cm3. In experimental group, the cell-scaffold constructs were cultured in high-glucose DMEM medium with 10% FBS for 7 days. Then the medium was replaced with chondrogenic induction medium of DMEM containing TGFβ1, IGF- Ⅰ , dexamethasone and 10% FBS. Whereas in control group, the constructs were maintained in DMEM medium + 10% FBS. Results were evaluated by histology, histochemistry and immunohistochemistry after 4 and 8 weeks of in vitro culture. Results Grossly, the constructs in experiment group were smooth and relative firm in texture after 4 weeks of cultre. Hematoxylin and eosin staining showed the formation of distinct lacuna structure. Type ⅡCollagen and glycosaminoglycan( GAG) were also detected. At 8 weeks, the experimental constructs became firmer and glossier with a mature lacuna structure distributed in most of the areas. GAG and other extracellular matrix (ECM) expressions were confirmed by Safranine-O staining, Masson staining, Alcian blue staining and Toluidine blue staining. Positive staining of typeⅡcollagen was also detected by immunohistochemistry. In contrast, in the control group, the constructs contracted gradually, with a size decreased to 2 millimeters in diameter at 8weeks. Lacuna was barely detected in histology and type Ⅱ collagen expression was nearly negative. Conclusion TGF-β1、 IGF-Ⅰ and dexamethasone can induce BMSCs to differentiate into chondrocyte in the three-dimensional scaffold, and ultimately formed engineered cartilage in vitro.