传代培养密度对人间充质干细胞增殖及成骨分化的影响

背景组织工程技术的快速发展为骨缺损完全再生修复带来了新的希望,种子细胞的快速扩增是其中的关键问题之一.目的观察人间充质干细胞传代培养时,细胞种植密度对间充质干细胞增殖及成骨诱导分化影响.设计重复测量设计.单位解放军第三军医大学细胞培养室和检测分析室.对象实验于2003-01/2004-03在解放军第三军医大学的创伤、烧伤和复合伤国家重点实验室完成.从5例男性健康志愿者髂骨骨髓中分离扩增的干细胞.方法将第2代间充质干细胞以8×103/cm2,3×103/cm2,8×102/cm2密度接种,分析其生长曲线,并扩增培养18 d,记录细胞扩增数量,再分析扩增后细胞的生长曲线和成骨诱导能力.主要观察指标①不同种植密度下细胞生长曲线.②碱性磷酸酶染色和骨钙素的免疫组化染色结果.结果人骨髓间充质干细胞阴性表达碱性磷酸酶、CD34;在8×103/cm2种植密度下,细胞倍增时间40 h,18 d后细胞数量扩增(51±13)倍;在3×103/cm2种植密度下,细胞扩增(28±6)倍;在8×102/cm2种植密度下,细胞总数仅增加(5±3)倍.在低密度下获得的细胞增殖能力强于高密度组,两组细胞均具有成骨诱导能力. 结论种植密度对间充质干细胞增殖有明显影响,快速扩增间充质干细胞作为骨组织工程种子细胞,宜选择8×103/cm2种植密度.

Effects of cell density on proliferation and osteogenic differentiation of human mesenchymal stem cells during subculturing in vitro

BACKGROUND: Rapid development of tissue engineering is the new hope of repairing bone defect so as to obtain complete bone regeneration,and rapid proliferation of seed cells is one of the important factors in it.OBJECTIVE: To evaluate the proliferation and osteogenic differentiation of human mesenchymal stem cells (MSCs) plated at different cell density during subculturing period.DESIGN: Repetitive measurement.PARTICIPANTS: The experiment was conducted in the Trauma, Burn and Combined Injury Laboratory of the Third Military Medical University of Chinese PLA between January 2003 and March 2004. MSCs were obtained from iliac bone bone marrow of the 5 healthy male volunteers by separation and proliferation.METHODS: The MSCs of second generation were inoculated at 8×103/cm2,3×103/cm2, and 8×102/cm2, respectively, and then the growth curve was drawn and analyzed. After 18 days of proliferation and culture, the increased amount of MSCs was recorded to analyze the post-proliferation growth curve and osteogenic ability.MAIN OUTCOME MEASURES: ①Growth curve of cells plated at different cell density; ②Results of alkaline phosphatase(ALP) staining and immunohistochemical staining of osteocalcin.RESULTS: Human MSCs from bone marrow were CD34 negative and expressed low level of ALP. Population doubling time was 40 hours after MSCs were plated at 8×103/cm2 and the number of the cells was expanded (51±13) times after 18 days. MSCs were expanded (28±6) times 18 days after plated at 3×103/cm2. While plated at 8×102/cm2, the number of the cells was only expanded (5±3) times. Proliferative ability of cells cultured at a low density was stronger that that at a high density. MSCs could be induced to differentiate into the osteocytic lineages after plated at not only 8×103/cm2 but also 8×102/cm2.CONCLUSION: Cell density plays an evident role in the proliferation of MSCs. Cell density of 8×103/cm2 is proper to the rapid proliferation of MSCs as seed cells in bone tissue engineering.