Effects of long time different negative pressures on osteogenic differentiation of rabbit bone mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHAO Bowen ,  ZHANG Hongwei , XU Qiang , GE Quanhu , LI Bolong ,  PENG Xinyu , WU Xiangwei

No.1 Department of General Surgery, The First Affiliated Hospital, College of Medicine, Shihezi University, Shihezi Xinjiang, 832008, P.R.China;

Corresponding author：

ZHANG Hongwei, Email: zhw0108@163.com; PENG Xinyu, Email: pengxinyu2000@sina.com

Keywords：

Bone marrow mesenchymal stem cells; negative pressure; osteogenic differentiation; cell proliferation; rabbit

DOI：

10.7507/1002-1892.201701095

Video：

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Objective To investigate the effects of long time different negative pressures on osteogenic diffe-rentiation of rabbit bone mesenchymal stem cells (BMSCs). Methods The rabbit BMSCs were isolated and cultured by density gradient centrifugation. Flow cytometry was used to analyze expression of surface markers. The third passage cells cultured under condition of osteogenic induction and under different negative pressure of 0 mm Hg (control group), 75 mm Hg (low negative pressure group), and 150 mm Hg (high negative pressure group) (1 mm Hg=0.133 kPa), and the negative pressure time was 30 min/h. Cell growth was observed under phase contrast microscopy, and the growth curve was drawn; alkaline phosphatase (ALP) activity was detected by ELISA after induced for 3, 7, and 14 days. The mRNA and protein expressions of collagen type I (COL-I) and osteocalcin (OC) in BMSCs were analyzed by real-time fluorescence quantitative PCR and Western blot. Results The cultured cells were identified as BMSCs by flow cytometry. The third passage BMSCs exhibited typical long shuttle and irregular shape. Cell proliferation was inhibited with the increase of negative pressure. After induced for 4 days, the cell number of high negative pressure group was significantly less than that in control group and low negative pressure group (P<0.05), but there was no significant difference between the low negative pressure group and the control group (P>0.05); at 5-7 days, the cell number showed significant difference between 3 groups (P<0.05). The greater the negative pressure was, the greater the inhibition of cell proliferation was. There was no significant difference in ALP activity between groups at 3 days after induction (P>0.05); the ALP activity showed significant difference (P<0.05) between the high negative pressure group and the control group at 7 days after induction; and significant difference was found in the ALP activity between 3 groups at 14 days after induction (P<0.05). The greater the negative pressure was, the higher the ALP activity was. Real-time fluorescence quantitative PCR and Western blot detection showed that the mRNA and protein expressions of COL-I and OC protein were significantly higher in low negative pressure group and high negative pressure group than control group (P<0.05), and in the high negative pressure group than the low negative pressure group (P<0.05). Conclusion With the increase of the negative pressure, the osteogenic differentiation ability of BMSCs increases gradually, but the cell proliferation is inhibited.

Citation： ZHAO Bowen, ZHANG Hongwei, XU Qiang, GE Quanhu, LI Bolong, PENG Xinyu, WU Xiangwei. Effects of long time different negative pressures on osteogenic differentiation of rabbit bone mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(5): 594-599. doi: 10.7507/1002-1892.201701095 Copy

1.	Fleischmann W, Strecker W, Bombelli M, et al. Vacuum sealing as treatment of soft tissue damage in open fractures. Unfallchirurg, 1993, 96(9): 488-492.
2.	Saxena V, Hwang CW, Huang S, et al. Vacuum-assisted closure:microdeformations of wounds and cell proliferation. Plast Reconstr Surg, 2004, 114(5): 1086-1096.
3.	黄米娜, 刘堃, 梅晰凡, 等. 负压封闭引流技术在大面积皮肤缺损中的应用及护理. 军医进修学院学报, 2012, 33(8): 867-868.
4.	Bassetto F, Lancerotto L, Salmaso R, et al. Histological evolution of chronic wounds under negative pressure therapy. J Plast Reconstr Aesthet Surg, 2012, 65(1): 91-99.
5.	Cozart RF, Atchison JR, Lett ED, et al. The use of controlled subatmospheric pressure to promote wound healing in preparation for split-thickness skin grafting in a fourth degree burn. Tenn Med, 1999, 92(10): 382-384.
6.	Ando J. Shear stress and vascular formation. Nihon Yakurigaku Zasshi, 1996,107(3): 141-152.
7.	Bosnakovski D, Mizuno M, Kim G, et al. Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells. Cell Tissue Res, 2005, 319(2): 243-253.
8.	杨楠, 何惠宇, 胡杨, 等. 复合骨髓间充质干细胞同种异体支架骨修复羊髂骨极限缺损. 中国组织工程研究, 2013, 17(16): 2859-2868.
9.	王峰, 付志厚. 骨髓间充质干细胞复合异体骨修复松质骨缺损. 中国组织工程研究, 2013, 17(27): 4966-4973.
10.	Yang Z, Yao JF, Xu P, et al. Functions and mechanisms ofintermittent negative pressure for osteogenesis in human bone marrow mesenchymal stem cells. Mol Med Rep, 2014, 9(4): 1331-1336.
11.	杨治, 朱养均, 程延, 等. 体外负压培养对骨髓间充质干细胞成骨活性的影响. 中国骨伤, 2011, 24(12): 1024-1027.
12.	Zhang YG, Yang Z, Zhang H, et al. Effect of negative pressure on human bone marrow mesenchymal stem cellsin vitro. Connect Tissue Res, 2010, 51(1): 14-21.
13.	Zhu J, Yu A, Qi B, et al. Effects of negative pressure wound therapy on mesenchymal stem cells proliferation and osteogenic differentiation in a fibrin matrix. PLoS One, 2014, 9(9): e107339.
14.	De R, Zemel A, Safran SA. Theoretical concepts and models of cellular mechanosensing. Methods Cell Bio, 2010, 98(2): 143-175.
15.	Gong T, Zhao K, Yang G, et al. The control of Mesenchymal stem cell differentiation using dynamically tunable surface microgrooves. Adv healthc Mater, 2014, 3(10): 1608-1619.
16.	雷晓华, 邓智利, 宁立娜, 等. 机械力及力学信号转导影响干细胞命运的研究进展. 中国科学: 生命科学, 2014, 44(7): 639-648.
17.	Han SJ, Sniadecki NJ. Simulations of the contractile cycle in cell migration using a biochemical mechanical model. Comput Methods Biomech Biomed Engin, 2011, 14(5): 459-468.
18.	高莺, 李继华, 韩立赤, 等. 张应力诱导大鼠骨髓间充质干细胞骨向分化及其差异基因表达分析. 华西口腔医学杂志, 2009, 27(2): 213-216.
19.	Ding H, Chen S, Yin JH, et al. Continuous hypoxia regulates the osteogenic potential of mesenchymal stem cells in a time-dependent manner. Mol Med Rep, 2014, 10(4): 2184-2190.
20.	Prado-Lòpez S, Duffy MM, Baustian C, et al. The influence of hypoxia on the differentiationcapacities and immunosuppressive properties of clonalmouse mesenchymal stromal cell lines. Immunol Cell Biol, 2014, 92(7): 612-623.

1. Fleischmann W, Strecker W, Bombelli M, et al. Vacuum sealing as treatment of soft tissue damage in open fractures. Unfallchirurg, 1993, 96(9): 488-492.
2. Saxena V, Hwang CW, Huang S, et al. Vacuum-assisted closure:microdeformations of wounds and cell proliferation. Plast Reconstr Surg, 2004, 114(5): 1086-1096.
3. 黄米娜, 刘堃, 梅晰凡, 等. 负压封闭引流技术在大面积皮肤缺损中的应用及护理. 军医进修学院学报, 2012, 33(8): 867-868.
4. Bassetto F, Lancerotto L, Salmaso R, et al. Histological evolution of chronic wounds under negative pressure therapy. J Plast Reconstr Aesthet Surg, 2012, 65(1): 91-99.
5. Cozart RF, Atchison JR, Lett ED, et al. The use of controlled subatmospheric pressure to promote wound healing in preparation for split-thickness skin grafting in a fourth degree burn. Tenn Med, 1999, 92(10): 382-384.
6. Ando J. Shear stress and vascular formation. Nihon Yakurigaku Zasshi, 1996,107(3): 141-152.
7. Bosnakovski D, Mizuno M, Kim G, et al. Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells. Cell Tissue Res, 2005, 319(2): 243-253.
8. 杨楠, 何惠宇, 胡杨, 等. 复合骨髓间充质干细胞同种异体支架骨修复羊髂骨极限缺损. 中国组织工程研究, 2013, 17(16): 2859-2868.
9. 王峰, 付志厚. 骨髓间充质干细胞复合异体骨修复松质骨缺损. 中国组织工程研究, 2013, 17(27): 4966-4973.
10. Yang Z, Yao JF, Xu P, et al. Functions and mechanisms ofintermittent negative pressure for osteogenesis in human bone marrow mesenchymal stem cells. Mol Med Rep, 2014, 9(4): 1331-1336.
11. 杨治, 朱养均, 程延, 等. 体外负压培养对骨髓间充质干细胞成骨活性的影响. 中国骨伤, 2011, 24(12): 1024-1027.
12. Zhang YG, Yang Z, Zhang H, et al. Effect of negative pressure on human bone marrow mesenchymal stem cellsin vitro. Connect Tissue Res, 2010, 51(1): 14-21.
13. Zhu J, Yu A, Qi B, et al. Effects of negative pressure wound therapy on mesenchymal stem cells proliferation and osteogenic differentiation in a fibrin matrix. PLoS One, 2014, 9(9): e107339.
14. De R, Zemel A, Safran SA. Theoretical concepts and models of cellular mechanosensing. Methods Cell Bio, 2010, 98(2): 143-175.
15. Gong T, Zhao K, Yang G, et al. The control of Mesenchymal stem cell differentiation using dynamically tunable surface microgrooves. Adv healthc Mater, 2014, 3(10): 1608-1619.
16. 雷晓华, 邓智利, 宁立娜, 等. 机械力及力学信号转导影响干细胞命运的研究进展. 中国科学: 生命科学, 2014, 44(7): 639-648.
17. Han SJ, Sniadecki NJ. Simulations of the contractile cycle in cell migration using a biochemical mechanical model. Comput Methods Biomech Biomed Engin, 2011, 14(5): 459-468.
18. 高莺, 李继华, 韩立赤, 等. 张应力诱导大鼠骨髓间充质干细胞骨向分化及其差异基因表达分析. 华西口腔医学杂志, 2009, 27(2): 213-216.
19. Ding H, Chen S, Yin JH, et al. Continuous hypoxia regulates the osteogenic potential of mesenchymal stem cells in a time-dependent manner. Mol Med Rep, 2014, 10(4): 2184-2190.
20. Prado-Lòpez S, Duffy MM, Baustian C, et al. The influence of hypoxia on the differentiationcapacities and immunosuppressive properties of clonalmouse mesenchymal stromal cell lines. Immunol Cell Biol, 2014, 92(7): 612-623.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of long time different negative pressures on osteogenic differentiation of rabbit bone mesenchymal stem cells

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