Objective: To investigate the biocompatibility of type I collagen scaffold with rat bone marrow mesenchymal stem cell (BMSCs) and its role on proliferation and differentiation of BMSCs so as to explore the feasibility of collagen scaffold as neural tissue engineering scaffold.
Methods: Type I collagen was used fabricate collagen scaffold. BMSCs were isolated by density gradient centrifugation. The 5th passage cells were used to prepare the collagen scaffold-BMSCs complex. The morphology of collagen scaffold and BMSCs was observed by scanning electron microscope (SEM) and HE staining. The cell proliferation was measured by MTT assay at 1, 3, 5, and 7 days after culture in vitro. After cultured on collagen scaffold for 24 hours, the growth and adhesion of green fluorescent protein positive (GFP +) BMSCs were observed by confocal microscopy and live cell imaging.
Results: The confocal microscopy and live cell imaging results showed that GFP + BMSCs uniformly distributed in the collagen scaffold; cells were fusiform shaped, and cell process or junctions between the cells formed in some cells, indicating good cell growth in the collagen scaffold. Collagen scoffold had porous fiber structure under SEM; BMSCs could adhered to the scaffold, with good cell morphology. The absorbance ( A) value of BMSCs on collagen scaffold at 5 and 7 days after culture was significantly higher than that of purely-cultured BMSCs ( t=4.472, P=0.011; t=4.819, P=0.009). HE staining showed that collagen scaffold presented a homogeneous, light-pink filament like structure under light microscope. BMSCs on the collagen scaffold distributed uniformly at 24 hours; cell displayed various forms, and some cells extended multiple processes at 7 days, showing neuron-like cell morphology.
Conclusion: Gelatinous collagen scaffold is easy to prepare and has superior biocompatibility. It is a promising scaffold for neural tissue engineering.
目的: 研究胶原凝胶支架对大鼠 BMSCs 增殖和分化的影响，探讨其作为神经组织工程支架的可行性。.
方法: 利用Ⅰ型胶原制备胶原凝胶支架。采用密度梯度离心法分离培养大鼠 BMSCs，取第 5 代细胞制备胶原凝胶-BMSCs 复合体。采用扫描电镜、HE 染色观察胶原凝胶支架的形态结构及复合培养后细胞形态；MTT 检测该支架对 BMSCs 增殖的影响。取绿色荧光蛋白（green fluorescent protein， GFP）阳性（GFP +）BMSCs 于胶原凝胶支架中培养 24 h，通过激光共聚焦显微镜及活细胞工作站观察细胞生长及与材料的黏附情况。.
结果: 激光共聚焦显微镜及活细胞工作站观察示，GFP +BMSCs 均匀分布于胶原凝胶支架内，大部分 GFP +BMSCs 呈梭形，部分细胞伸出突起，部分细胞间形成连接，提示 BMSCs 在三维空间中生长良好。扫描电镜示胶原凝胶支架为多孔纤维网状结构，BMSCs 可黏附于该支架材料上，细胞形态良好。MTT 检测示，BMSCs 于胶原凝胶支架中培养后 3、5、7 d 的吸光度（ A）值均高于单纯 BMSCs 培养，其中 5、7 d 时组间差异有统计学意义（ t=4.472， P=0.011； t=4.819， P=0.009）。HE 染色示，胶原凝胶支架呈均质淡粉染细丝样物质。BMSCs 在胶原凝胶内培养 24 h 后均匀分布其中；7 d 时 BMSCs 形态多样，部分细胞伸出细长突起，具有体外培养的神经元样形态。.
结论: 胶原凝胶支架制备简便，具有良好的生物相容性，可作为神经组织工程支架材料。.
Keywords: Neural tissue engineering; biocompatibility; bone marrow mesenchymal stem cells; gelatinous collagen; rat; scaffold material.