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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18d,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40h,18d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。
BACKGROUND: The rapid development of tissue engineering brings new hope for complete regeneration of bone defects. The rapid expansion of seed cells is one of the key issues. OBJECTIVE: To observe the effect of cell density on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) during passage culture of human mesenchymal stem cells. Design: repeat measurement design. Unit: PLA Third Military Medical University cell culture room and testing room. PARTICIPANTS: The experiment was performed at the State Key Laboratory of Traumatic, Burn and Combined Injury at the Third Military Medical University of Chinese PLA from January 2003 to March 2004. The expanded stem cells were isolated from iliac bone marrow of 5 male healthy volunteers. Methods: The second generation of mesenchymal stem cells were inoculated at the density of 8 × 103 / cm2, 3 × 103 / cm2 and 8 × 102 / cm2. The growth curve was analyzed and expanded for 18 days. Cell growth curve and osteoinduction ability after amplification. MAIN OUTCOME MEASURES: ① Cell growth curve under different planting densities. ② alkaline phosphatase staining and osteocalcin immunohistochemical staining results. Results: The human bone marrow mesenchymal stem cells negatively expressed alkaline phosphatase and CD34. Under the density of 8 × 10 3 / cm 2, the cell doubling time was 40 h and the number of cells was 51 ± 13 after 18 d. At 3 × 103 / cm2 (28 ± 6) fold at the planting density, and the total number of cells increased only by 5 ± 3 times at the density of 8 × 102 / cm2. The cell proliferation ability obtained at low density was stronger than that of high density group, and both cells had osteogenic ability. Conclusion: The density of mesenchymal stem cells has a significant effect on the proliferation of mesenchymal stem cells. The mesenchymal stem cells, as the seed cells of bone tissue engineering, should be rapidly expanded to 8 × 103 / cm2 planting density.