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

作　　者：刘天一[1] 周广东[1] 苗春雷[1] 陈付国[1] 魏娴[1] 殷德明[1] 曹颖[1] 崔磊[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学第九人民医院整复外科上海市组织工程重点实验室,上海200011

出　　处：《上海第二医科大学学报》2004年第4期262-266,共5页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：国家高技术研究发展计划项目资助("863"计划)(2002AA205021);上海市"重中之重"重点学科基金资助.

摘　　要：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.

关 键 词：骨髓基质干细胞 组织工程 软骨 地塞米松 聚合蛋白多糖 胶原 

分 类 号：Q813[生物学—生物工程]