皮质骨连续植块培养成骨细胞的生物学特性比较

Comparison of biological properties of osteoblasts from continuous explant culture of cortical bone

[目的]通过比较皮质骨2次连续植块培养所获细胞的体外培养和成骨特性,探讨皮质骨连续植块培养的可行性。[方法]取3个月龄新西兰大白兔上肢皮质骨,用组织块法对皮质骨块进行连续植块培养。取第4代细胞进行实验,倒置相差显微镜下观察和结晶紫染色比较细胞形态、噻唑蓝(MTT)法测定细胞增殖曲线、对硝基酚法测碱性磷酸酶(ALP)活性、酶免法测定骨钙素(BGP)活性及观察钙结节的产生等。[结果]普通显微镜下2次连续植块培养的细胞在形态学上无显著差异,呈长梭形、三角形或者不规则形;2次植块培养的细胞在增殖能力方面无明显差异,在ALP、BGP和钙结节等成骨特性上有明显的表达,二者成骨特性亦无明显差异。[结论]皮质骨来源的成骨细胞成分相对单纯,2次连续植块细胞的增殖和成骨能力相当,可以进行连续植块培养以重复利用骨块,达到节省经费、节约时间的目的。

[Objective]To compare the in vitro culture and osteogenic properties of osteoblasts from the two continuous explants culture of cortical bone,and investigate the feasibility of continuous explant culture in the cortical bone.[Method]The experiments were conducted at bioengineer centure of Xiamen University.The cortical bone of upper limbs from 3-month-old New Zealand white rabbits was cultured by continuous explant culture methods.The obtained cells of the fourth generation were studied under the inverted phase contrast microscope and dyed by crystal violet morphologically.The cell proliferation curve was determined by MTT,alkaline phosphatase（ALP） activity was detected with nitrophenol method,and osteocalcin（BGP） was examined by ELISA.[Result]There was no significant morphological difference between the cells from two continuous explant cultures under plain microscope.Most of them were like long shuttle,triangle or irregular shape.There was no different proliferation capacity between the cells from two cortical explant cultures.There was obvious expression of ALP,BGP and calcified nodules in the cells,but no different osteogenic capacity between them.[Conclusion]The osteoblasts from cortical bone was comparatively pure.No significant difference of proliferation and osteogenic ability were noted between the cells from the two cortical explant cultures.The bone chips can be repeatedly used for continuous explant culture.