脐带间充质干细胞复合β-磷酸三钙生物陶瓷体内外成骨能力的初步研究

The study of bone formation of hUCMSCs compounded with β-TCP biological ceramic in vitro and in vivo

目的:观察人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)和支架材料β-磷酸三钙(β-TCP)生物陶瓷复合的体内外成骨情况。方法:在体外构建hUCMSCs和β-TCP的复合物,将细胞以3×105/mL的浓度接种到支架材料上进行复合体构建,并进行电镜观察、特异性免疫荧光染色、MTT、ALP检测。将复合物植入裸鼠体内,进行成骨能力研究。实验分为3组,即植入单纯β-TCP支架组、植入体外成骨诱导2周的hUCMSCs和β-TCP复合物组、单纯植入体外成骨诱导2周的hUCMSCs组。植入后2个月取出标本,进行大体观察、X线片观察和组织学观察。采用SPSS16.0软件包对实验数据进行重复测量随机区组设计的方差分析。结果:hUCMSCs植入支架4h后,即可见细胞在支架上附着,1周后可见细胞在支架中大量增殖,β-TCP具有一定的骨诱导性。复合物植入裸鼠内2个月,hUCMSCs和β-TCP复合组X线密度最高。HE染色显示,2个月后,hUCMSCs和β-TCP复合组见不规则新骨和血管形成,单纯β-TCP组未见明显新骨和血管形成。Masson染色显示,2个月后,hUCMSCs和β-TCP复合组见大量胶原形成,单纯β-TCP组未见明显胶原形成。VG染色显示,2个月后,hUCMSCs和β-TCP复合组孔隙中有大量类骨质及少量骨陷窝形成,单纯β-TCP组未见类骨质形成。结论:hUCMSCs和β-TCP具有良好的生物相容性,两者构建的复合物在植入裸鼠体内2个月时可见新骨及血管化形成。

PURPOSE： To observe the osteogenesis of the construct combining human umbilical cord mesenchymal stem cells（hUCMSCs） with scaffold β-TCP biological ceramic both in vitro and in vivo.METHODS： A composite was constructed combining hUCMSCs with β-TCP.The cellular capability of adhesion and growth on scaffold were investigated visually by scanning electron microscopy（SEM）,specific immunofluorescence staining,MTT and ALP.The constructs were implanted in nude mice for osteogenic potential.The animals were divided into 3 groups： Group 1（pure β-TCP）,Group 2（the construct combining hUCMSCs with scaffold β-TCP was induced to bone in vitro for 2 weeks）,Group 3（simple implantation of the hUCMSCs which was induced to bone in vitro for 2 weeks）.2 months after implantation,the specimens were harvested for gross observation,radiographic examination and histological observation.SPSS 16.0 software package was used for statistical analysis through repeated measures analysis of variances.RESULTS： Cells can be seen attached in the scaffold after 4 hours of the implantation of HUCMSCs,a large number of cells reproduce in the scaffold after 1 week.After 2 months of implantation in the nude mice,the group of hUCMSCs and β-TCP incubation had the highest radiodensity.2 months after implantation,the group of hUCMSCs and β-TCP had irregular new bone and vascular formation,β-TCP group had no obvious new bone and blood vessel formation.2 months after implantation,hUCMSCs and β-TCP group had a lot of collagen formation,while β-TCP group had no obvious formation of collagen by using masson staining.2 months after implantation,hUCMSCs and β-TCP group had a large number of osteoid formation,while β-TCP group had no obvious formation of osteoid by using VG staining.CONCLUSIONS： hUCMSCs and β-TCP have good biocompatibility,the formation of new bone and blood vessels can be seen after 2 months of implantation of the construct into mice.