RADA-16自组装多肽的合成表征及负载BMP-2的细胞相容性研究

Synthesis and characterization of self-assembling peptide RADA-16 and cytocompatibility of BMP-2 loaded thereon

目的将RADA-16、珊瑚和BMP-2组成复合材料体系,研究其超微结构及与BMSCs细胞相容性。方法将RADA-16溶解于10%无菌蔗糖溶液中,浓度为1%(10 mg/mL,m/v),将RADA-16再用等体积细胞培养基DMEM/F12溶液触发多肽自组装,浸润珊瑚,进行电镜扫描。等比例混合RADA-16与BMP-2溶液后,把珊瑚在混合液中浸没,用相同体积的DMEM/F12培养基触发自组装的发生,扫描电镜检测。分离培养兔BMSCs,细胞活性染色观察细胞相容性。结果 RADA-16自组装能够形成凝胶,在50倍电镜下观察珊瑚呈多孔状结构,大小均匀,孔隙在200~500μm范围内,且孔隙紧密排布;130倍扫描电镜下观察,RADA-16自组装多肽凝胶均匀附着于珊瑚孔隙中;1 000倍扫描电镜显示,RADA-16自组装形成片层与网状结构,其直径从几微米到几十微米不等;10 000扫描电镜显示,在珊瑚孔隙中BMP-2黏附于RADA-16,成功合成复合材料体系(RADA-16、珊瑚、BMP-2),BMSCs培养过程中,实验1组(RADA-16、珊瑚、BMP-2)、实验2组(RADA-16、珊瑚)与对照组(珊瑚)中BMSCs都未见明显死亡细胞,差异无统计学意义(P>0.05)。结论多肽RADA-16在特定的条件下可以自组装复合材料体系(自组装多肽、珊瑚、BMP-2),与BMSCs有良好的相容性,可以作为骨修复组织工程的生物支架材料。

Objective To investigate the ultrastructure of the composite system comprising RADA16,coral and BMP-2 and its cytocompatibility with BMSCs. Methods RADA-16 was dissolved in 10% sterile sucrose solution at a concentration of 1% （ 10 mg/mL, m/v） , the self-assembly of the peptide RADA-16 was triggered by the isovolumetric cell culture medium DMEM/F12 solution, and the coral was infiltrated for scanning electron microscope. After RADA-16 and BMP-2 solutions were mixed in equal proportions, the coral was immersed in the resulting mixture, and the self-assembly of peptide was triggered by the same volume of DMEM/F12 medium and examined under a scanning electron microscope. Then, the BMSCs of rabbit were isolated and cultivated, aiming to observe cytocompatibility based on cellular activity staining. Results RADA-16 could be self-assembled into a gel. Under a 50X electron microscope,the coral had a porous structure with uniform size,and the pores were densely arranged in the range of 200 -500 μm;under a 130X scanning electron microscope,the self-assembling peptide gel of RADA-16 was evenly attached to the coral pores;under a 1000X scanning electron microscope,the self-assembling of RADA-16 had the laminar and reticular structures ranging from a few micrometers to tens of micrometers in diameter; under a 10000X scaxming electron microscope,BMP-2 adhered to RADA-16 in coral pores, and was successfully synthesized into a composite system（ RADA-16, coral and BMP-2 ）. During the cultivation of BMSCs, no obvious dying cells were seen in the experimental group 1 （ RADA-16, coral and BMP-2 ） , the experimental group 2 （RADA-16 and coral） and the control group（ coral）, and the differences among these groups were not statistically significant. Conclusion The peptide RADA-16 can achieve a self-assembled composite system（ self-assembling peptide, coral and BMP-2 ） under specific conditions and can be used as a biological scaffold material in the field of bone repair and tissue engineering due to good compatibility with BMSCs.