三维丝素支架材料制备及其与骨髓间充质干细胞的生物相容性

Preparation of three-dimensional fibrin scaffold and its biocompatibility with bone marrow mesenchymal stem cells

目的:成为组织工程合适的三维支架材料,关键在于支架材料与骨髓间充质干细胞的生物相容性,实验应用天然高分子材料丝纤维制备三维丝素蛋白支架材料,并探索其方法的可行性。方法:实验于2006-07/2007-06在华中科技大学同济医学院协和医院中心实验室和骨科实验室完成。①用全物理相过程制备三维疏松多孔的丝素蛋白支架材料,将鼠骨髓骨髓间充质干细胞与上述载体材料体外复合培养。②观察指标:应用倒置显微镜、扫描电镜观察细胞与材料复合生长情况;复合培养1周后流式细胞仪检测细胞周期了解材料有无致畸性;在培养第2,4,6,8天用MTT法测定细胞增殖情况;诱导培养条件下在第2,4,6,8天测定碱性磷酸酶活性,了解材料对骨髓间充质干细胞分化的影响。结果:①倒置相差显微镜和扫描电镜观察显示材料为疏松多孔结构,孔隙大小较均一,骨髓间充质干细胞能在材料上良好地黏附、增殖和生长。②复合培养条件后流式细胞仪检测显示细胞周期未受材料的影响,未检测到有异倍体细胞。③MTT法检测显示细胞增殖未受材料的影响,诱导培养条件下检测碱性磷酸酶活性显示其未受到材料的影响结论:用全物理相过程可初步制备具有良好的生物相容性和较好孔隙率的三维疏松多孔的天然支架材料,其有望作为组织工程理想支架材料。

AIM： To explore the process to form a porous three-dimensional （3-D） fibrin scaffold and its biocompatibUity with the bone marrow mesenchymal stem cell （BMSC） in order to offer an ideal scaffold for tissue engineering. METHODS： From July 2006 to June 2007, the experiment was carried out in the Central Laboratory and Orthopaedic Laboratory, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology.①The porous 3-D fibrin scaffold was built by means of whole physical phase procession. Rat BMSC were cultured on the scaffold in vitro. ②The morphologic function of the BMSC were observed by inverted phase contrast microscope and scanning electron microscope. The scaffold＇s oncogenicity on cell cycle was detected by flow cytometry after coculturing for a week. The proliferation of the BMSC on the scaffold was measured by MTT assay at days 2, 4, 6 and 8. Meanwhile, the BMSC was cultured on the scaffold with osteogenic supplements, then their alkaline phosphatase （ALP） activity was measured to know its differentiation. RESULTS：① This method could produce a porous 3-D fibrin scaffold with equal pore size as showed by inverted phase contrast microscope and scanning electron microscope. The BMSC adhered, proliferated and grew perfectly on the scaffold.②The cellular activity, cell cycle and ALP activity were not influenced by this scaffold. No heteroploid cells were found. CONCLUSION： The porous 3-D fibrin scaffold prepared by the whole physical phase procession can be applicable as an ideal tissue-engineering scaffold due to its good porosity and good biocompatibUity.