体外壳聚糖-明胶-生长因子缓释支架对骨髓间充质干细胞增殖的影响

Effect of In Vitro Slow-release Scaffolds of Chitosan-Gelatin-Growth Factor Composite on Proliferation of Bone Marrow Mesenchymal Stem Cells

制备具有缓释功能的壳聚糖-明胶-碱性成纤维细胞生长因子(bFGF)-肝细胞生长因子(HGF)三维大孔支架,探讨两种生长因子在体外对骨髓间充质干细胞(BMSCs)增殖的协同作用。方法:采用冷冻干燥法,将不同比例的壳聚糖、明胶、bFGF和HGF依次混合,使其成为具有一定孔径的三维缓释支架。取SD大鼠乳鼠股骨和胫骨骨髓,分离、培养BMSCs。取生长状态良好的第三代BMSCs,将其接种于96孔板后,加入支架混合培养,5d后进行MTT细胞增殖测定。结果:在与含1μg/mL的bFGF支架混合培养,以及与同时含1μg/mL的bFGF、1μg/mL的HGF支架混合培养后,BMSCs增殖明显(P<0.05),但这二组间无统计学差异(P>0.05)。分别与含0.1μg/mL的bFGF支架、0.01μg/mL的bFGF支架、1μg/mL的HGF支架混合培养后,细胞增殖无统计学意义(P>0.05)。结论:壳聚糖-明胶可作为生长因子缓释支架材料;bFGF具有促进BMSCs增殖的作用,促进作用的大小与加入bFGF的量有关;HGF对BMSCs不具有增殖作用;在实验浓度范围内bFGF和HGF体外促进BMSCs增殖上不具有协同性。

Three-dimensional chitosan-gelatin porous scaffolds were made for constantly releasing basic fibroblast growth factor（bFGF） and/or hepatocyte growth factor（HGF）.The combined effect of both growth factors on bone marrow stromal cell（BMSC） proliferation in vitro were discussed.Chitosan,gelatin,bFGF and HGF were mixed in varied proportions and dry frozen to form porous 3D scaffolds.BMSCs were isolated from SD rat femur bone marrow and expanded in vitro.Third passage of favourable BMSCs were seeded in 96-well plate,then mixed with scaffolds.Cell proliferation was then analyzed by MTT proliferation analysis 5 days later.BMSC growth is significantly increased（P0.05） due to the constant release of growth factors from the scaffold with 1μg/mL bFGF alone or 1μg/mL bFGF combined with 1μg/mL HGF.There is no significant difference between these two groups.BMSC growth is slightly increased（P0.05） by the constant release growth factors from the scaffold with 0.1μg/mL bFGF,0.01μg/mL bFGF or 1μg/mL HGF alone.Chitosan-glutin forms a 3D scaffold that can slowly but constantly release growth factors;while bFGF promotes BMSCs proliferation in vitro which is not enhanced by HGF.The more bFGF involved,the more BMSCs proliferate.There is no cooperativity between bFGF and HGF within the experimental concentration range in promoting BMSCs proliferation.