Beagle犬骨髓基质细胞体外成骨诱导分化的实验研究

Experimental study on osteogenic potentiality and induced differentiation of Beagles' bone marrow stromal cells in vitro

目的观察犬骨髓基质细胞在体外培养的条件及生物学特性,并向成骨方向定向诱导培养,为骨组织工程提供适宜的种子细胞。方法分离纯化犬骨髓基质细胞,并利用条件培养液将其向成骨方向诱导培养、扩增;采用倒置相差显微镜观察细胞形态;用Von kossa及碱性磷酸酶(alkaline phosphatase,ALP)染色的方法鉴定其成骨性能。通过分光光度仪及MTT染色法描绘标准曲线及标准细胞浓度-光密度值(optical density,OD)曲线。结果原代培养18d后可分离得到骨髓基质细胞,经成骨条件培养液诱导培养后,形态相对稳定,具有成骨性能。结论可以通过在体外分离纯化骨髓基质细胞并诱导培养的方法,获得足够数量的具有成骨潜能的细胞作为骨组织工程的种子细胞。

Objective To prepare seed cells for bone tissue engineering, establish the culturing condition and biological characteristics of canine bone marrow stromal cells （BMSCs）, and proliferate them into osteoblasts in vitro. Methods Bone marrow was drew out from Beagle dogs and cultured with differentiation culture medium to harvest BMSCs. The morphology of the cells was studied with a phase contrast microscope. Von Kossa staining and alkaline phosphatase （ALP） activity test were employed to assess BMSCs osteoblastic differentiation and the generation of calcified extracellular matrix. The standard curves of cell-amount vs OD-value were drawn through spectrophotometer test and MTI＂ assay. Results BMSCs could be observed after 18 days of primary culture. The viable characteristics of BMSCs were relatively stable, and these cells possessed bone formation potentials. Conclusion BMSCs can be cultured, differentiated and proliferated with active osteogenic function with differentiation culture medium in vitro, thus they can be candidate cells for bone tissue engineering.