人骨髓间质干细胞的培养及成骨功能研究

目的　体外扩增人骨髓间质干细胞 (humanbonemarrow derivedmesonchymalstemcells,hBMMSCs) ,研究其生物学特征及体内、外成骨功能。方法　分离培养hBMMSCs,并对其形态、增殖动力学、碱性磷酸酶 (alkalinephosphatase,ALP)表达及体内、外成骨功能进行研究。结果　hBMMSCs可在体外培养扩增 ,群体倍增时间约为 3 5d。ALP检测表明 ,未经矿化诱导的hBMMSCs可表达少量ALP ,诱导后其表达量明显增高。vonkossa染色证实在矿化诱导液作用下hBMMSCs在体外可形成钙结节。体内成骨实验证实 ,1× 10 5个hBMMSCs接种到 30mm3 块状HA/TCP载体移植BALB/C裸小鼠皮下 3个月 ,可观察到层板状骨组织生成。结论　hBMMSCs是一种具有成骨潜能的前体细胞 ,经培养、扩增、诱导后 ,在体外可形成矿化结节 ,体内可在载体表面形成骨组织。

Bone formation in vitro and in vivo by human bone marrow-derived mesenchymal stem cells

Objective To culture and study the osteogenic characteristics of human bone marrow-derived mesenchymal stem cells(hBMMSCs). Methods hBMMSCs were separated and cultured from human iliac crest marrow. Growth kinetics of hBMMSCs was studied by growth curve. Under the osteoinductive culture, osteogenic differentiation of hBMMSCs was tested by alkaline phosphatase (ALP). Osteogenic functions of hBMMSCs in vitro and in vivo were also respectively detected by von Kossa stain and by transplanting hydroxyapatite/tricalcium phosphate ceramics (HA/TCP) with hBMMSCs.Results hBMMSCs were cultured successfully. The growth curve of the second passage of BMMSCs indicated that the time of population doublings was about 3.5 days. The results of ALP stain were evident by the significant increase in ALP activity after hBMMSCs cultured in osteoinductive medium. Some mineralized nodules were detected by von Kossa stain at nineteenth day of osteoinductive culture. In vivo assay, histological evalution showed bone formation in 3 months after grafts of HA/TCP with hBMMSCs.Conclusions Osteoinductive solution can induce hBMMSCs to differentiate osteogenetic cell lines. Mineralized nodules and bone formation were found in vitro and in vivo assay. The results demonstrate that hBMMSCs have the potential for osteogenesis.