EFFECT OF CHANGE OF TISSUE INTERFACE STIFFNESS ON OSTEOGENIC DIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHENLin , XUMingqing , LIUYang , DUANHao , HUAWenda ,  HEFei

Department of Orthopedics, First Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650000, P. R. China;

Corresponding author：

HEFei, Email: drhefei@sina.com

Keywords：

Bone marrow mesenchymal stem cells; Interface stiffness; Bionics; Osteogenic differentiation; Rat

DOI：

10.7507/1002-1892.20160315

Video：

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Objective To investigate the effect of tissue interface stiffness change on the spreading, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and to find the suitable stiffness range for stem cell differentiation. Methods Bone marrow of male Sprague Dawley rats (4 weeks old) were selected to isolate and culture BMSCs by whole bone marrow cell adherent method. The third generation BMSCs (1×10⁵ cells/mL) were inoculated into the ordinary culture dishes covered with polyacrylamide hydrophilic gel (PA) which elastic modulus was 1, 4, 10, 40, and 80 kPa (cells seeded on PA), and ordinary culture dish (75 MPa extreme high elastic modulus) as control. Spreading of cells in different stiffness of PA was observed under light microscope. The elastic modulus values of 4, 10, and 40 kPa PA were selected as groups A, B, and C respectively; the ordinary culture dish (75 MPa extreme high elastic modulus) was used as control group (group D). Cell counts was used to detect the growth conditions of BMSCs, alkaline phosphatase (ALP) kit to detect the concentration of ALP, alizarin red staining technique to detect calcium deposition status, and real-time quatitative PCR technique to detect the expressions of bone gla protein (BGP), Runx2, and collagen type I mRNA. Results With increased PA stiffness, BMSCs spreading area gradually increased, especially in 10 kPa and 40 kPa. At 1 and 2 days after culture, the growth rate showed no significant difference between groups (P > 0.05); at 3-5 days, the growth rate of groups B and C was significantly faster than that of groups A and D (P < 0.05), but difference was not statistically significant between groups A and D (P < 0.05); at 5 days, the proliferation of group C was significantly higher than that of group B (P < 0.05). ALP concentrations were (53.69±0.89), (97.30±1.57), (126.60±14.54), and (12.93±0.58) U/gprot in groups A, B, C, and D respectively; groups A, B, and C were significantly higher than group D, and group C was significantly higher than groups A and B (P < 0.05). Alizarin red staining showed that the percentages of calcium nodules was 20.07%±4.24% in group C; group C was significantly higher than groups A, B, and D (P < 0.05). The expression levels of BGP and collagen type I mRNA were significantly higher in groups A, B, and C than group D, and in group C than groups A and B (P < 0.05). The expression level of Runx2 mRNA was significantly higher in groups B and C than group D, and in group C than group B (P < 0.05), but no significant difference was found between groups A and D (P > 0.05). Conclusion PA elastic modulus of 10-40 kPa can promote the proliferation and osteogenic differentiation of BMSCs, and the higher the stiffness, the stronger the promoting effect.

Citation： CHENLin, XUMingqing, LIUYang, DUANHao, HUAWenda, HEFei. EFFECT OF CHANGE OF TISSUE INTERFACE STIFFNESS ON OSTEOGENIC DIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(12): 1524-1531. doi: 10.7507/1002-1892.20160315 Copy

1.	Henkel J, Woodruff MA, Epari DR, et al. Bone regeneration based on tissue engineering conceptions-a 21st century perspective. Bone Res, 2013, 1(3):216-248.
2.	张志强, 黄向华, 赵林远.微环境对细胞的影响以及仿生学在组织工程支架中的应用.中国生物工程杂志, 2014, 34(4):101-109.
3.	Crowley C, Wong JM, Fisher DM, et al. A systematic review on preclinical and clinical studies on the use of scaffolds for bone repair in skeletal defects. Curr Stem Cell Res Ther, 2013, 8(3):243-252.
4.	华闻达, 韩东, 吴迪, 等.骨髓间充质干细胞对不同时长应力刺激的力学响应.医用生物力学, 2015, 30(1):43-49.
5.	Azeem A, English A, Kumar P, et al. The influence of anisotropic nano-to micro-topography on in vitro and in vivo osteogenesis. Nanomedicine (Lond), 2015, 10(5):693-711.
6.	Gang EJ, Jeong JA, Hong SH, et al. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. Stem Cells, 2004, 22(4):617-624.
7.	何学令, 姚晓玲, 冯贤, 等.力学刺激和成骨化学诱导剂对大鼠骨髓间充质干细胞成骨分化能力的影响.医用生物力学, 2011, 26(2):116-120.
8.	Jiang J, Papoutsakis ET. Stem-cell niche based comparative analysis of chemical and nano-mechanical material properties impacting ex vivo expansion and differentiation of hematopoietic and mesenchymal stem cells. Adv Healthc Mater, 2013, 2(1):25-42.
9.	Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs stem cell lineage specification. Cell, 2006, 126(4):677-689.
10.	Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science, 2005, 310(5751):1139-1143.
11.	Even-Ram S, Artym V, Yamada KM. Matrix control of stem cell fate. Cell, 2006, 126(4):645-647.
12.	Beningo KA, Lo CM, Wang YL. Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions. Methods Cell Biol, 2002, 69:325-339.
13.	Chen P, Chen L, Han D, et al. Wetting behavior at micro-/nanoscales:direct imaging of a microscopic water/air/solid three-phase interface. Small, 2009, 5(8):908-912.
14.	Stroh C, Wang H, Bash R, et al. Single-molecule recognition imaging microscopy. Proc Natl Acad Sci U S A, 2004, 101(34):12503-12507.
15.	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C (T)) Method. Methods, 2001, 25(4):402-408.
16.	Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 2004, 363(9419):1439-1441.
17.	Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering:state of the art and future trends. Macromol Biosci, 2004, 4(8):743-765.
18.	Park K, Millet LJ, Kim N, et al. Measurement of adherent cell mass and growth. Proc Natl Acad Sci U S A, 2010, 107(48):20691-20696.
19.	Tee SY, Fu J, Chen CS, et al. Cell shape and substrate rigidity both regulate cell stiffness. Biophys J, 2011, 100(5):L25-27.
20.	Gaharwar AK, Mihaila SM, Swami A, et al. Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells. Adv Mater, 2013, 25(24):3329-3336.
21.	Caplan AI. Review:mesenchymal stem cells:cell-based reconstructive therapy in orthopedics. Tissue Eng, 2005, 11(7-8):1198-1211.
22.	Seong JM, Kim BC, Park JH, et al. Stem cells in bone tissue engineering. Biomed Mater, 2010, 5(6):062001.
23.	Winer JP, Janmey PA, McCormick ME, et al. Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. Tissue Eng Part A, 2008, 15(1):147-154.
24.	陈晨, 张毅. Notch信号通路与间充质干细胞分化.中国实验血液学杂志, 2010, 18(2):510-514.
25.	Matthews BD, Overby DR, Mannix R, et al. Cellular adaptation to mechanical stress:Role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels. Cell Sci, 2006, 119(Pt 3):508-518.
26.	Xue R, Li JY, Yeh Y, et al. Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation. J Orthop Res, 2013, 31(9):1360-1365.
27.	Asparuhova MB, Gelman L, Chiquet M. Role of the actin cytoskeleton in tuning cellular responses to external mechanical stress. Scand J Med Sci Sports, 2009, 19(4):490-499.
28.	McBeath R, Pirone DM, Nelson CM, et al. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell, 2004, 6(4):483-495.
29.	Park JS, Chu JS, Tsou AD, et al. The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β. Biomaterials, 2011, 32(16):3921-3930.
30.	Du J, Chen X, Liang X, et al. Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity. Proc Natl Acad Sci U S A, 2011, 108(23):9466-9471.

1. Henkel J, Woodruff MA, Epari DR, et al. Bone regeneration based on tissue engineering conceptions-a 21st century perspective. Bone Res, 2013, 1(3):216-248.
2. 张志强, 黄向华, 赵林远.微环境对细胞的影响以及仿生学在组织工程支架中的应用.中国生物工程杂志, 2014, 34(4):101-109.
3. Crowley C, Wong JM, Fisher DM, et al. A systematic review on preclinical and clinical studies on the use of scaffolds for bone repair in skeletal defects. Curr Stem Cell Res Ther, 2013, 8(3):243-252.
4. 华闻达, 韩东, 吴迪, 等.骨髓间充质干细胞对不同时长应力刺激的力学响应.医用生物力学, 2015, 30(1):43-49.
5. Azeem A, English A, Kumar P, et al. The influence of anisotropic nano-to micro-topography on in vitro and in vivo osteogenesis. Nanomedicine (Lond), 2015, 10(5):693-711.
6. Gang EJ, Jeong JA, Hong SH, et al. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. Stem Cells, 2004, 22(4):617-624.
7. 何学令, 姚晓玲, 冯贤, 等.力学刺激和成骨化学诱导剂对大鼠骨髓间充质干细胞成骨分化能力的影响.医用生物力学, 2011, 26(2):116-120.
8. Jiang J, Papoutsakis ET. Stem-cell niche based comparative analysis of chemical and nano-mechanical material properties impacting ex vivo expansion and differentiation of hematopoietic and mesenchymal stem cells. Adv Healthc Mater, 2013, 2(1):25-42.
9. Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs stem cell lineage specification. Cell, 2006, 126(4):677-689.
10. Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science, 2005, 310(5751):1139-1143.
11. Even-Ram S, Artym V, Yamada KM. Matrix control of stem cell fate. Cell, 2006, 126(4):645-647.
12. Beningo KA, Lo CM, Wang YL. Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions. Methods Cell Biol, 2002, 69:325-339.
13. Chen P, Chen L, Han D, et al. Wetting behavior at micro-/nanoscales:direct imaging of a microscopic water/air/solid three-phase interface. Small, 2009, 5(8):908-912.
14. Stroh C, Wang H, Bash R, et al. Single-molecule recognition imaging microscopy. Proc Natl Acad Sci U S A, 2004, 101(34):12503-12507.
15. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C (T)) Method. Methods, 2001, 25(4):402-408.
16. Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 2004, 363(9419):1439-1441.
17. Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering:state of the art and future trends. Macromol Biosci, 2004, 4(8):743-765.
18. Park K, Millet LJ, Kim N, et al. Measurement of adherent cell mass and growth. Proc Natl Acad Sci U S A, 2010, 107(48):20691-20696.
19. Tee SY, Fu J, Chen CS, et al. Cell shape and substrate rigidity both regulate cell stiffness. Biophys J, 2011, 100(5):L25-27.
20. Gaharwar AK, Mihaila SM, Swami A, et al. Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells. Adv Mater, 2013, 25(24):3329-3336.
21. Caplan AI. Review:mesenchymal stem cells:cell-based reconstructive therapy in orthopedics. Tissue Eng, 2005, 11(7-8):1198-1211.
22. Seong JM, Kim BC, Park JH, et al. Stem cells in bone tissue engineering. Biomed Mater, 2010, 5(6):062001.
23. Winer JP, Janmey PA, McCormick ME, et al. Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. Tissue Eng Part A, 2008, 15(1):147-154.
24. 陈晨, 张毅. Notch信号通路与间充质干细胞分化.中国实验血液学杂志, 2010, 18(2):510-514.
25. Matthews BD, Overby DR, Mannix R, et al. Cellular adaptation to mechanical stress:Role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels. Cell Sci, 2006, 119(Pt 3):508-518.
26. Xue R, Li JY, Yeh Y, et al. Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation. J Orthop Res, 2013, 31(9):1360-1365.
27. Asparuhova MB, Gelman L, Chiquet M. Role of the actin cytoskeleton in tuning cellular responses to external mechanical stress. Scand J Med Sci Sports, 2009, 19(4):490-499.
28. McBeath R, Pirone DM, Nelson CM, et al. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell, 2004, 6(4):483-495.
29. Park JS, Chu JS, Tsou AD, et al. The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β. Biomaterials, 2011, 32(16):3921-3930.
30. Du J, Chen X, Liang X, et al. Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity. Proc Natl Acad Sci U S A, 2011, 108(23):9466-9471.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF CHANGE OF TISSUE INTERFACE STIFFNESS ON OSTEOGENIC DIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS

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