新西兰兔骨髓间充质干细胞-庆大霉素-藻酸钙三维缓释培养体系的构建及体外评价

Establishment of New Zealand rabbit BMSCs-gentamicin-calcium alginate 3D sustained-release system and in vitro evaluation

目的构建新西兰兔骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)-庆大霉素-藻酸钙三维缓释培养体系,研究BMSCs的生长和分化情况。方法分别构建BMSCs-藻酸钙三维培养体系(W组)及BMSCs-庆大霉素-藻酸钙三维缓释培养体系(U组),在37℃、饱和湿度、5%CO_2培养箱内培养,培养液为高糖DMEM(含15%胎牛血清、10ng/mL转化生长因子β_1),隔日换液,观察细胞形态变化及凝珠形态变化。第2、4、6周对凝珠进行H-E染色、甲苯胺蓝染色及Ⅱ型胶原染色。结果两组三维凝珠培养第10天局部有细胞团簇形成;第21天可见大量细胞团簇形成,BMSCs多保持圆球形或类球形。两组细胞增殖及生长能力差异无统计学意义(P>0.05)。培养2周,两组凝珠甲苯胺蓝染色均呈阳性,但无明显细胞外基质形成,Ⅱ型胶原抗体染色均呈弱阳性;培养4周,两组凝珠甲苯胺蓝染色均显示位于细胞团块外周的细胞呈蓝色,中央的细胞染色不明显,细胞团外周蓝色染色物质较少,中央有较多紫红色物质,Ⅱ型胶原抗体染色均呈强阳性。H-E染色、甲苯胺蓝染色及Ⅱ型胶原抗体染色均未发现两组BMSCs的转化生长有差异,增殖数量差异也无统计学意义。结论适量的庆大霉素局部缓释并未明显影响BMSCs的生长及转化,又可达到最小抑菌浓度;庆大霉素对BMSCs及软骨细胞超微结构的影响有待进一步探讨。

Objective To establish a New Zealand rabbit bone marrow mesenchymal stem cells （BMSCs）-gentamicincalcium alginate 3D sustained-release culture system and to study the growth and differentiation of BMSCs. Methods BMSCscalcium alginate 3D culture system （W group） and BMSCs-gentamicin-calcium alginate 3D sustained-release culture system （U group） were constructed and were cultured with HG-DMEM （15% FBS, 10 ng/mL TGF-β） under saturated humidity, 37℃ and at 5 % CO2 , with the culture medium changed on a daily basis, and the cell morphology and microsphere morphology changes were observed. H-E staining, toluidine blue staining and type lI collagen staining were performed for the microspheres on week 2, 4, and 6. Results Cell clusters were formed locally in the two groups after the 3D microspheres were cultured for 10 days. A large number of cell clusters were formed after 21 days, and BMSCs maintained a spherical or approximate spherical shape. There was no significant difference in cell proliferation or growth between the two groups （P〉0.05）. After a 2-week culture, toluidine blue staining of microspheres showed positive staining in both groups, but with no obvious extracellular matrix formation, and staining for collagen type II antibody was weakly positive. After a 4 week culture, toluidine blue staining was obvious in the periphery of the cell microspheres in both groups, but the staining was unapparent in the center; extracellular matrix around the cell clusters had less blue colored substance, and the central cell clusters had more mauve substance; collagentype Ⅱ staining was strongly positive in both groups. Conclusion Local sustained-release of appropriate amount of gentamicin has no noticeable effect on the growth and transformation of BMSCs while reaching the minimum inhibitory concentration. The influence of Gentamicin on ultrastructure of BMSCs and chondrocytes remains to be further investigated.