骨髓间充质干细胞复合鸡蛋膜构建组织工程化骨的实验研究

Construction of tissue engineered bone based on rat bone marrow mesenchymal stem cells and eggshell membrane

目的探讨鸡蛋膜作为组织工程支架材料同大鼠骨髓间充质干细胞（bone marrow mesenchy-cmal stem cells,BMSCs）复合培养构建组织工程化骨的可行性。方法采用梯度离心法分离大鼠骨髓间充质干细胞;将第2代BMSCs同鸡蛋膜支架材料复合,利用含0.1μmol/L地塞米松、10mmol/Lβ-甘油酸钠、50μg/ml维生素C的条件培养液诱导培养2周后,分别将复合材料植入Wistar大鼠右侧背部皮下,左侧植入无细胞复合的鸡蛋膜作为对照。28d后处死大鼠收集标本,利用扫描电镜技术、HE染色、免疫组织化学（Ⅰ型胶原、碱性磷酸酶）和茜素红染色进行相应检测。结果细胞同鸡蛋膜复合培养后,细胞在鸡蛋膜上容易贴附生长,7d后鸡蛋膜表面有大量的细胞生长,14d后细胞连接成片且鸡蛋膜网状孔隙中有大量的细胞生长。复合材料植入大鼠28d后,实验组鸡蛋膜内部有大量细胞存在,植入区可见有组织形成,对照组鸡蛋膜内部无细胞,植入区未见组织形成;实验组BMSCs向成骨细胞分化并分泌矿化基质,表达Ⅰ型胶原,碱性磷酸酶,对照组均为阴性。结论鸡蛋膜具有良好的组织生物相容性,以BMSCs作为种子细胞、鸡蛋膜作为支架材料构建组织工程化骨具备一定的可行性。

Objective To investigate the feasibility of tissue engineered bone construted by using eggshell membrane as scaffolds and loaded with bone marrow mesenchymal stem cells（BMSCs） derived from rat bone marrow as seed cell. Methods BMSCs were isolated from rat bone marrow with density gradient centrifugation. The second generation cells were seeded on eggshell membrane, and then induced with osteogenic media（50 μg/ml ascorbic acid, 10 mmol/L β-glycerephosphate, and 0.1 μmol/L dexamethasone） for 2 weeks in vitro. The cellseeded scaffolds were implanted subcutaneously in Wistar rats for 4 weeks. The samples were assessed by scanning electron microscope, HE staining, immuhistochemical staining （type Ⅰ collagen and Alkaline phosphatase （ALP）） as well as alizarin red staining（ARS）. Results Eggshell membrane showed good biocompatibility. Microscopic analysis demonstrated rapid proliferation of subcultured BMSCs in 7 d. The cells grew well in the holes of eggshell membrane scaffold in 14 d. The inoculated BMSCs were well-distributed and attached. Animal experiments showed that there was more osteogenesis in exprimental group than that of in control group, containing rich extracellular matrix. The tissue were stained positively with mineralization nodes, type Ⅰ collagen as well as ALP. Conclusion It was feasible to generate tissue engineered bone with eggshell membrane and BMSCs.