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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。
OBJECTIVE: To observe the in vitro and in vivo osteogenesis of human umbilical cord mesenchymal stem cells (hUCMSCs) and scaffold material β-tricalcium phosphate (β-TCP). Methods: The complex of hUCMSCs and β-TCP was constructed in vitro. The cells were seeded onto the scaffolds at the concentration of 3 × 105 / mL for complex formation. Electron microscopy, immunofluorescence staining, MTT and ALP assays were performed. The composite was implanted into nude mice to study osteogenic ability. The experiment was divided into 3 groups: hUCMSCs and β-TCP complexes were implanted into the β-TCP scaffolds and implanted into hUCMSCs for 2 weeks in vitro. Two months after implantation, the specimens were removed for general observation, X-ray observation and histological observation. Repeated measurement of experimental data using SPSS16.0 software package ANOVA of randomized block design. RESULTS: After hUCMSCs were implanted into the scaffold for 4h, the cells adhered to the scaffold. After 1 week, the cells proliferated significantly in the scaffold, and β-TCP was osteoinductive. The composite was implanted into nude mice for 2 months, and the X-ray density of hUCMSCs and β-TCP composite group was the highest. Hematoxylin and eosin staining revealed irregular new bone and vascularization after 2 months in hUCMSCs and β-TCP complex group, but no obvious new bone and blood vessel formation in simple β-TCP group. Masson staining showed that after 2 months, a large amount of collagen was formed in the hUCMSCs and the β-TCP complex group, and no obvious collagen formation was observed in the pure β-TCP group. VG staining showed that after 2 months, a large amount of osteoid and a few lacunae were formed in the pores of hUCMSCs and β-TCP complex group, but no osteoid formation was found in the simple β-TCP group. CONCLUSION: The hUCMSCs and β-TCP have good biocompatibility. The complexes constructed by them show new bone formation and vascularization when implanted into nude mice for 2 months.