氨基修饰的纳米纤维对人和大鼠 MSCs增殖分化的影响

目的:探讨氨基修饰后的静电纺丝纳米纤维对大鼠和人骨髓来源的间充质干细胞(Rat and human bone marrow mesenchymal stem cells, r MSCs and hMSCs)增殖及成骨分化的影响。方法:采用静电纺丝法制备聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)纳米纤维,用氨气等离子体处理其表面来接枝氨基;通过测量PLGA纳米纤维(NF)及氨基修饰后的纳米纤维(NF-NH_2)接触角来证明修饰效果;将r MSCs和hMSCs分别接种于NF和NF-NH_2,用CCK-8试剂盒检测接种后1, 3 (4), 7天的细胞增殖;接种后的21天,用茜素红S染色(ARS)法检测细胞成骨分化情况。结果:氨气等离子体处理后纳米纤维接触角从81.28±0.33降低至53.99±0.79,说明氨基修饰后的PLGA NF亲水性增加;CCK-8结果显示氨基修饰增加了r MSCs的黏附,接种24 h后r MSCs在NF和NF-NH_2上的检测吸光值分别为0.096±0.011和0.175±0.014(P0.001),而对hMSCs黏附和增殖没有影响,接种24 h后hMSCs在NF和NF-NH_2上的检测吸光值分别为0.237±0.004和0.238±0.006(P0.05);ARS染色结果显示氨基修饰后r MSCs成骨分化增多(在NF和NF-NH_2表面ARS染色区域比例分别13.147±3.223%和36.677±5.230%),而hMSCs在修饰前后的纳米纤维上均有表达(修饰前后ARS染色比例分别为50.283±2.942%和38.254±3.272%)。结论:氨基修饰的NF可以促进大鼠来源的MSCs黏附增殖以及成骨分化,而对人骨髓来源的MSCs没有显著影响,这提示我们MSCs的增殖分化行为可能具有种属依赖性。

The Effect of Amino-group Modified Nanofibers on Proliferation and Differentiation of MSCs derived from Human and Rat Bone Marrow

ABSTRACT Objective: To investigate the effects of ammine group modified electrospinning nanofibers on the proliferation and osteogenic differentiation of rat and human bone marrow derived mesenchymal stem cells (rMSCs, hMSCs). Methods:Electrospun technique was employed to fabricate poly(lactic-co-glycolic acid) (PLGA) nanofibers, whose surface was grafted with amino-group through ammonia plasma treatment; Contact angle was measured on the PLGA nanofibers (NF) surface and amino-group modified (NF-NH2) surface; Cell Counting Kit-8 (CCK-8) was used to test the proliferation of rMSCs and hMSCs after seeded onto NF and NF-NH2 for 1, 3(4), and 7 days; Alizarin Red S (ARS) was used to detect the osteogenesis of rMSCs and hMSCs after seeded 21 days. Results:The contact angle was reduced from 81.28 ± 0.33 to 53.99 ± 0.79 by ammonia plasma treatment, which indicated that after modified by amino-group the surface of NF became hydrophilic. CCK-8 results showed that more rMSCs attached to NF-NH2 surface. After 24 h seeded onto surfaces, the absorbance data of rMSCs on PLGA NF and NF-NH2 was 0.096 ± 0.011 and 0.175 ± 0.014 respectively (P <0.001). However the NF-NH2 had no effects on the adhesion and proliferation of hMSCs. After 24 h seeded onto surfaces, the absorbance data of hMSCs on PLGA NF and NF-NH2 was 0.237 ± 0.004 and 0.238 ± 0.006 respectively (P >0.05). It can be seen from the ARS staining results that hMSCs produced calcium deposit equally on both NF and NF-NH2 surfaces, while NF-NH2 enhanced osteogenic differentiation of rMSCs (the ARS staining area was increased from 13.147 ± 3.223% to 36.677 ± 5.230%). Conclusion:NF modified by amino-groups can enhance rMSCs'' adhesion, proliferation and osteogenic differentiation. The same phenomenon didn''t happened on hMSCs, indicating that MSCs have a species dependent response.