组织工程中应变对骨髓间充质干细胞增殖及成骨分化的影响
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摘要
骨髓间充质干细胞具有自我复制、未分化的特点,并可在不同条件下分化为中胚层起源的多种细胞,这些特点决定了其在组织工程中的重要地位。此外,机体内几乎所有的细胞都会受到力学因素的影响,所以有必要研究力学因素对骨髓间充质干细胞的作用,为其体外扩增、诱导分化提供一种新途径。
本实验首先探索了分离、培养骨髓间充质干细胞的方法,并了解其一般生物学特性。其次,选取传至第3代(P_3)的骨髓间充质干细胞进行实验。加载大小为1000微应变、频率为0.5Hz(1秒拉伸,1秒缓解)的单向循环拉伸应变,每12小时拉伸30分钟,连续72小时。测定了未加载及应变加载后不同时间点碱性磷酸酶的活性;应变和(或)成骨条件培养基对骨髓间充质干细胞增殖及碱性磷酸酶活性的影响;加载组与未加载组细胞基质钙盐沉积情况;骨钙素的基因表达情况。最后,筛选适宜骨组织工程的支架材料。
结果显示:应变加载组较非加载组碱性磷酸酶活性明显增强;应变加载后6h的碱性磷酸酶活性最强;应变能有效地促进骨髓间充质干细胞向成骨细胞分化,且诱导效能强于普遍采用的诱导条件培养基,但抑制了细胞的增殖速度。在几种材料中,生物陶瓷更适宜用作骨组织工程的支架。
Bone marrow-derived mesenchymal stem cells (BMSCs) are a population of cells capable of replicating as undifferentiated cells and that have the potential to differentiate to lineages of mesoblastic origin. And the characters of BMSCs have suggested the great value of this specific cell population for tissue engineering. Furthermore, complex mechanical forces are clearly involved in cell development in vivo. Therefore, it is necessary to investigate the effect of strain on proliferation and differentiation of BMSCs, and may provide increased insight in the role of strain on proliferation and osteogenic differentiation of BMSCs in vitro and lead to improved strategies in bone tissue engineering.In this study, the method of cell isolation and culture was mastered, and its general biology characteristics were realized. Next, BMSCs for the following experiments were at the same third passage. Using a four-point bending loads system, a uniform 1000με cyclic tension strain was applied to the cells at 0.5Hz (1s on, 1s off) for 30 minutes per 12 hours, and lasted out 72 hours. We assessed the ALP activity of unstrained plates and strained plates at different times, the proliferation and ALP activity of BMSCs under strain and/or osteogenic supplement medium, the matrix-deposited calcium, gene expression of osteocalcin (OCN). Lastly, four kinds of materials served as bone tissue-engineering scaffold was compared.The results suggested that strained cells expressed higher ALP activity than unstrained cells, and the ALP levels expressed by BMSCs is highest at the time of the 6th hour after being applied strain. We demonstrated that BMSCs, stretched by cyclic deformation of the cell substrate, inhibited their growth and accelerated their osteogenic differentiation. And we detected that p-TCP can serve as bone tissue-engineering scaffold materials more appropriately.
本实验首先探索了分离、培养骨髓间充质干细胞的方法,并了解其一般生物学特性。其次,选取传至第3代(P_3)的骨髓间充质干细胞进行实验。加载大小为1000微应变、频率为0.5Hz(1秒拉伸,1秒缓解)的单向循环拉伸应变,每12小时拉伸30分钟,连续72小时。测定了未加载及应变加载后不同时间点碱性磷酸酶的活性;应变和(或)成骨条件培养基对骨髓间充质干细胞增殖及碱性磷酸酶活性的影响;加载组与未加载组细胞基质钙盐沉积情况;骨钙素的基因表达情况。最后,筛选适宜骨组织工程的支架材料。
结果显示:应变加载组较非加载组碱性磷酸酶活性明显增强;应变加载后6h的碱性磷酸酶活性最强;应变能有效地促进骨髓间充质干细胞向成骨细胞分化,且诱导效能强于普遍采用的诱导条件培养基,但抑制了细胞的增殖速度。在几种材料中,生物陶瓷更适宜用作骨组织工程的支架。
Bone marrow-derived mesenchymal stem cells (BMSCs) are a population of cells capable of replicating as undifferentiated cells and that have the potential to differentiate to lineages of mesoblastic origin. And the characters of BMSCs have suggested the great value of this specific cell population for tissue engineering. Furthermore, complex mechanical forces are clearly involved in cell development in vivo. Therefore, it is necessary to investigate the effect of strain on proliferation and differentiation of BMSCs, and may provide increased insight in the role of strain on proliferation and osteogenic differentiation of BMSCs in vitro and lead to improved strategies in bone tissue engineering.In this study, the method of cell isolation and culture was mastered, and its general biology characteristics were realized. Next, BMSCs for the following experiments were at the same third passage. Using a four-point bending loads system, a uniform 1000με cyclic tension strain was applied to the cells at 0.5Hz (1s on, 1s off) for 30 minutes per 12 hours, and lasted out 72 hours. We assessed the ALP activity of unstrained plates and strained plates at different times, the proliferation and ALP activity of BMSCs under strain and/or osteogenic supplement medium, the matrix-deposited calcium, gene expression of osteocalcin (OCN). Lastly, four kinds of materials served as bone tissue-engineering scaffold was compared.The results suggested that strained cells expressed higher ALP activity than unstrained cells, and the ALP levels expressed by BMSCs is highest at the time of the 6th hour after being applied strain. We demonstrated that BMSCs, stretched by cyclic deformation of the cell substrate, inhibited their growth and accelerated their osteogenic differentiation. And we detected that p-TCP can serve as bone tissue-engineering scaffold materials more appropriately.
引文
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