Annals of Biomedical Engineering

, Volume 44, Issue 10, pp 2971–2983 | Cite as

The Distinct Effects of Estrogen and Hydrostatic Pressure on Mesenchymal Stem Cells Differentiation: Involvement of Estrogen Receptor Signaling

  • Ying Zhao
  • Fei-Zhou Yi
  • Yin-Hua Zhao
  • Yong-Jin Chen
  • Heng MaEmail author
  • Min ZhangEmail author


This study aimed to investigate the differential and synergistic effects of mechanical stimulation and estrogen on the proliferation and osteogenic or chondrogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs) and the roles of estrogen receptor (ER) in them. BMSCs were isolated and cultured using the whole bone marrow adherence method, and flow cytometry was used to identify the surface marker molecules of BMSCs. Cells were pre-treated with 1 nM 17β-estradiol or 1 nM of the estrogen receptor antagonist tamoxifen. Then, the cells were stimulated with hydrostatic pressure. Assessment included flow cytometry analysis of the cell cycle; immunofluorescent staining for F-actin; protein quantification for MAPK protein; and mRNA analysis for Col I, OCN, OPN and BSP after osteogenic induction and Sox-9, Aggrecan and Col-II after chondrogenic induction. Hydrostatic pressure (90 kPa/1 h) and 1 nM 17β-estradiol enhanced the cellular proliferation ability and the cytoskeleton activity but without synergistic biological effects. Estrogen activated ERKs and JNKs simultaneously and promoted the osteogenic differentiation, whereas the pressure just caused JNK-1/2 activation and promoted the chondrogenic differentiation of BMSCs. Estrogen had antagonism effect on chondrogenic promotion of hydrostatic pressure. Mechanobiological effects of hydrostatic pressure are closely associated with ERα activity. MAPK molecules and F-actin were likely to be important mediator molecules in the ER-mediated mechanotransduction of BMSCs.


Hydrostatic pressure Estrogen Bone marrow mesenchymal stem cells (BMSCs) Differentiation Estrogen receptor (ER) Mitogen-activated protein kinase (MAPK) 



Bone marrow mesenchymal stem cells




Estrogen receptor




Collagen I






Bone sialoprotein


Collagen II


Mitogen-activated protein kinase


c-Jun N-terminal kinase


External signal-regulated kinase



This research was supported by the National Natural Science Foundation of China (Nos. 81371188, 31570951).

Author contributions

YZ. and F-Z.Y. made the acquisition of data and most of the analysis of the data, and were also involved in the drafting of the manuscript. Y-H.Z. and Y-J.C. helped with the interpretation of the data. H.M. and M.Z. was involved in the conception and design of the study, and did the most of the manuscript drafting and revising. All authors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no financial or non-financial competing interests related to this study.


  1. 1.
    Angele, P., J. U. Yoo, C. Smith, J. Mansour, K. J. Jepsen, M. Nerlich, and B. Johnstone. Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. J. Orthop. Res. 21:451–457, 2003.CrossRefPubMedGoogle Scholar
  2. 2.
    Bajada, S., I. Mazakova, J. B. Richardson, and N. Ashammakhi. Updates on stem cells and their applications in regenerative medicine. J Tissue Eng. Regen. Med. 2:169–183, 2008.CrossRefPubMedGoogle Scholar
  3. 3.
    Chen, Y. J., M. Zhang, and J. J. Wang. Study on the effects of mechanical pressure to the ultrastructure and secretion ability of mandibular condylar chondrocytes. Arch. Oral Biol. 52:173–181, 2007.CrossRefPubMedGoogle Scholar
  4. 4.
    Dai, Z., Y. Li, L. D. Quarles, T. Song, W. Pan, H. Zhou, and Z. Xiao. Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation. Phytomedicine 14:806–814, 2007.CrossRefPubMedGoogle Scholar
  5. 5.
    Discher, D. E., D. J. Mooney, and P. W. Zandstra. Growth factors, matrices, and forces combine and control stem cells. Science 324:1673–1677, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Farndale, R. W., D. J. Buttle, and A. J. Barrett. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta 883:173–177, 1986.CrossRefPubMedGoogle Scholar
  7. 7.
    Farrar, E. K., and H. Mitchell. Osteoarthritis and exercise: a review of the literature. J. S. C. Med. Assoc. 105:8–11, 2009.PubMedGoogle Scholar
  8. 8.
    Finger, A. R., C. Y. Sargent, K. O. Dulaney, S. H. Bernacki, and E. G. Loboa. Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure. Tissue Eng. 13:1151–1158, 2007.CrossRefPubMedGoogle Scholar
  9. 9.
    Frenkel, B., A. Hong, S. K. Baniwal, G. A. Coetzee, C. Ohlsson, O. Khalid, and Y. Gabet. Regulation of adult bone turnover by sex steroids. J. Cell. Physiol. 224:305–310, 2010.CrossRefPubMedGoogle Scholar
  10. 10.
    Frost, H. M. On the estrogen-bone relationship and postmenopausal bone loss: a new model. J. Bone Miner. Res. 14:1473–1477, 1999.CrossRefPubMedGoogle Scholar
  11. 11.
    Ghazanfari, S., M. Tafazzoli-Shadpour, and M. A. Shokrgozar. Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells. Biochem. Biophys. Res. Commun. 388:601–605, 2009.CrossRefPubMedGoogle Scholar
  12. 12.
    Hong, L., G. Zhang, H. Sultana, Y. Yu, and Z. Wei. The effects of 17-beta estradiol on enhancing proliferation of human bone marrow mesenchymal stromal cells in vitro. Stem Cells Dev. 20:925–931, 2011.CrossRefPubMedGoogle Scholar
  13. 13.
    Huang, C. Y., P. M. Reuben, and H. S. Cheung. Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading. Stem Cells 23:1113–1121, 2005.CrossRefPubMedGoogle Scholar
  14. 14.
    Imgenberg, J., B. Rolauffs, A. J. Grodzinsky, M. Schunke, and B. Kurz. Estrogen reduces mechanical injury-related cell death and proteoglycan degradation in mature articular cartilage independent of the presence of the superficial zone tissue. Osteoarthr. Cartil. 21:1738–1745, 2013.CrossRefPubMedGoogle Scholar
  15. 15.
    Jeong, J. Y., S. H. Park, J. W. Shin, Y. G. Kang, K. H. Han, and J. W. Shin. Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture. J. Mater. Sci. Mater. Med. 23:2773–2781, 2012.CrossRefPubMedGoogle Scholar
  16. 16.
    Kee, C. C. Osteoarthritis: manageable scourge of aging. Nurs. Clin. North Am. 35:199–208, 2000.PubMedGoogle Scholar
  17. 17.
    Kelly, D. J., and C. R. Jacobs. The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells. Birth Defects Res. C Embryo Today 90:75–85, 2010.CrossRefPubMedGoogle Scholar
  18. 18.
    Leskela, H. V., A. Olkku, S. Lehtonen, A. Mahonen, J. Koivunen, M. Turpeinen, J. Uusitalo, O. Pelkonen, L. Kangas, K. Selander, and P. Lehenkari. Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts. Bone 39:1026–1034, 2006.CrossRefPubMedGoogle Scholar
  19. 19.
    Liao, Q. C., Y. L. Li, Y. F. Qin, L. D. Quarles, K. K. Xu, R. Li, H. H. Zhou, and Z. S. Xiao. Inhibition of adipocyte differentiation by phytoestrogen genistein through a potential downregulation of extracellular signal-regulated kinases 1/2 activity. J. Cell. Biochem. 104:1853–1864, 2008.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Miyanishi, K., M. C. Trindade, D. P. Lindsey, G. S. Beaupre, D. R. Carter, S. B. Goodman, D. J. Schurman, and R. L. Smith. Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro. Tissue Eng. 12:2253–2262, 2006.CrossRefPubMedGoogle Scholar
  21. 21.
    Piette-Brion, B., C. de Bast, E. Chamoun, G. de Dobbeleer, J. Andre, A. Huybrechts, M. Ledoux, and G. Achten. Superficial pemphigus during the treatment of rheumatoid polyarthritis with D-penicillamine and piroxicam (Feldene). Dermatologica 170:297–301, 1985.CrossRefPubMedGoogle Scholar
  22. 22.
    Potier, E., J. Noailly, and K. Ito. Directing bone marrow-derived stromal cell function with mechanics. J. Biomech. 43:807–817, 2010.CrossRefPubMedGoogle Scholar
  23. 23.
    Rajalin, A. M., H. Pollock, and P. Aarnisalo. ERRalpha regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells. Biochem. Biophys. Res. Commun. 396:477–482, 2010.CrossRefPubMedGoogle Scholar
  24. 24.
    Roman-Blas, J. A., S. Castaneda, R. Largo, and G. Herrero-Beaumont. Osteoarthritis associated with estrogen deficiency. Arthritis Res. Ther. 11:241–248, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Safshekan, F., M. Tafazzoli-Shadpour, M. A. Shokrgozar, N. Haghighipour, R. Mahdian, and A. Hemmati. Intermittent hydrostatic pressure enhances growth factor-induced chondroinduction of human adipose-derived mesenchymal stem cells. Artif. Organs 36:1065–1071, 2012.CrossRefPubMedGoogle Scholar
  26. 26.
    Sakao, K., K. A. Takahashi, Y. Arai, A. Inoue, H. Tonomura, M. Saito, T. Yamamoto, N. Kanamura, J. Imanishi, O. Mazda, and T. Kubo. Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure. Osteoarthr. Cartil. 16:805–814, 2008.CrossRefPubMedGoogle Scholar
  27. 27.
    Simmons, C. A., S. Matlis, A. J. Thornton, S. Chen, C. Y. Wang, and D. J. Mooney. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J. Biomech. 36:1087–1096, 2003.CrossRefPubMedGoogle Scholar
  28. 28.
    Tolomio, S., A. Ermolao, A. Lalli, and M. Zaccaria. The effect of a multicomponent dual-modality exercise program targeting osteoporosis on bone health status and physical function capacity of postmenopausal women. J Women Aging 22:241–254, 2010.CrossRefPubMedGoogle Scholar
  29. 29.
    Uthman, O. A., D. A. van der Windt, J. L. Jordan, K. S. Dziedzic, E. L. Healey, G. M. Peat, and N. E. Foster. Exercise for lower limb osteoarthritis: systematic review incorporating trial sequential analysis and network meta-analysis. BMJ 347:f5555, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Vinardell, T., R. A. Rolfe, C. T. Buckley, E. G. Meyer, M. Ahearne, P. Murphy, and D. J. Kelly. Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells. Eur. Cell Mater. 23:121–132, 2012; (discussion 133-4).CrossRefPubMedGoogle Scholar
  31. 31.
    Wagner, D. R., D. P. Lindsey, K. W. Li, P. Tummala, S. E. Chandran, R. L. Smith, M. T. Longaker, D. R. Carter, and G. S. Beaupre. Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium. Ann. Biomed. Eng. 36:813–820, 2008.CrossRefPubMedGoogle Scholar
  32. 32.
    Wang, Y., J. Wang, D. Bai, J. Song, R. Ye, Z. Zhao, L. Lei, J. Hao, C. Jiang, S. Fang, S. An, Q. Cheng, and J. Li. Cell proliferation is promoted by compressive stress during early stage of chondrogenic differentiation of rat BMSCs. J. Cell. Physiol. 228:1935–1942, 2013.CrossRefPubMedGoogle Scholar
  33. 33.
    Yousefian, J., F. Firouzian, J. Shanfeld, P. Ngan, R. Lanese, and Z. Davidovitch. A new experimental model for studying the response of periodontal ligament cells to hydrostatic pressure. Am. J. Orthod. Dentofac. Orthop. 108:402–409, 1995.CrossRefGoogle Scholar
  34. 34.
    Zeiter, S., P. Lezuo, and K. Ito. Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. Biorheology 46:45–55, 2009.PubMedGoogle Scholar
  35. 35.
    Zhang, M., Y. J. Chen, T. Ono, and J. J. Wang. Crosstalk between integrin and G protein pathways involved in mechanotransduction in mandibular condylar chondrocytes under pressure. Arch. Biochem. Biophys. 474:102–108, 2008.CrossRefPubMedGoogle Scholar
  36. 36.
    Zhang, M., F. M. Chen, A. H. Wang, Y. J. Chen, X. Lv, S. Wu, and R. N. Zhao. Estrogen and its receptor enhance mechanobiological effects in compressed bone mesenchymal stem cells. Cells Tissues Organs 195:400–413, 2012.CrossRefPubMedGoogle Scholar
  37. 37.
    Zhang, M., J. J. Wang, and Y. J. Chen. Effects of mechanical pressure on intracellular calcium release channel and cytoskeletal structure in rabbit mandibular condylar chondrocytes. Life Sci. 78:2480–2487, 2006.CrossRefPubMedGoogle Scholar
  38. 38.
    Zhao, Y. H., X. Lv, Y. L. Liu, Y. Zhao, Q. Li, Y. J. Chen, and M. Zhang. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: the roles of RhoA and Rac1. Stem Cell Res. 14:283–296, 2015.CrossRefPubMedGoogle Scholar

Copyright information

© Biomedical Engineering Society 2016

Authors and Affiliations

  1. 1.State Key Laboratory of Military Stomatology, Department of General Dentistry and Emergency, School of StomatologyFourth Military Medical UniversityXi’anChina
  2. 2.Department of Physiology, School of Basic MedicineFourth Military Medical UniversityXi’anChina
  3. 3.The 105th Hospital of PLAHefeiChina
  4. 4.Department of Pathophysiology, School of Basic MedicineFourth Military Medical UniversityXi’anChina

Personalised recommendations