Skip to main content
SpringerLink
Log in

Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells

  • Original Paper
  • Published:
International Orthopaedics Aims and scope Submit manuscript

Abstract

Purpose

The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs.

Methods

Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining.

Results

Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs.

Conclusions

Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147

    Article  CAS  PubMed  Google Scholar 

  2. Lee JH, Park HK, Kim KS (2015) Intrinsic and extrinsic mechanical properties related to the differentiation of mesenchymal stem cells. Biochem Biophys Res Commun 473(3):752–757

    Article  PubMed  Google Scholar 

  3. Beederman M, Lamplot JD, Nan G, Wang J, Liu X, Yin L, Li R, Wei S, Zhang H, Kim SH (2013) BMP signaling in mesenchymal stem cell differentiation and bone formation. J Biomed Sci Eng 6(8A):32–52

    Article  PubMed  PubMed Central  Google Scholar 

  4. Varga AC, Wrana JL (2005) The disparate role of BMP in stem cell biology. Oncogene 24(37):5713–5721

    Article  CAS  PubMed  Google Scholar 

  5. Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P (2003) Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg (Am Vol) 85-A(8):79–80

    Google Scholar 

  6. Kang Q, Song WX, Luo Q, Tang N, Luo J, Luo X, Chen J, Bi Y, He BC, Park JK (2009) A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. Stem Cells Dev 18(4):545–559

    Article  CAS  PubMed  Google Scholar 

  7. Sun L, Qu L, Zhu R, Li H, Xue Y, Liu X, Fan J, Fan H (2016) Effects of mechanical stretch on cell proliferation and matrix formation of mesenchymal stem cell and anterior cruciate ligament fibroblast. Stem Cells Int 2016:1):1–1)10

    Google Scholar 

  8. Luo J, Deng ZL, Luo X, Tang N, Song WX, Chen J, Sharff KA, Luu HH, Haydon RC, Kinzler KW (2007) A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2(5):1236–1247

    Article  CAS  PubMed  Google Scholar 

  9. Huang E, Zhu G, Jiang W, Yang K, Gao Y, Luo Q, Gao JL, Kim SH, Liu X, Li M (2012) Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells. J Bone Miner Res 27(7):1566–1575

    Article  CAS  PubMed  Google Scholar 

  10. Zhang W, Deng ZL, Chen L, Zuo GW, Luo Q, Shi Q, Zhang BQ, Wagner ER, Rastegar F, Kim SH (2010) Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. PLoS One 5(7):58–72

    CAS  Google Scholar 

  11. Chen L, Jiang W, Huang J, He BC, Zuo GW, Zhang W, Luo Q, Shi Q, Zhang BQ, Wagner ER (2010) Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation. J Bone Miner Res 25(11):2447–2459

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hu N, Jiang D, Huang E, Liu X, Li R, Liang X, Kim SH, Chen X, Gao JL, Zhang H (2013) BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells. J Cell Sci 126(2):532–541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Iura A, Mcnerny EG, Zhang Y, Kamiya N, Tantillo M, Lynch M, Kohn DH, Mishina Y (2015) Mechanical loading synergistically increases trabecular bone volume and improves mechanical properties in the mouse when BMP signaling is specifically ablated in osteoblasts. PLoS One 10(10):e0141345

    Article  PubMed  PubMed Central  Google Scholar 

  14. Xiao E, Yang HQ, Gan YH, Duan DH, He LH, Guo YB, Wang SQ, Zhang Y (2014) TRPM7 senses mechanical stimulation inducing Osteogenesis in human bone marrow Mesenchymal stem cells. Stem Cells 33(2):615–621

    Article  Google Scholar 

  15. Xiao E, Chen C, Zhang Y (2016) The mechanosensor of mesenchymal stem cells: mechanosensitive channel or cytoskeleton? Stem Cell Res Ther 7(1):140

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ehrlicher AJ, Nakamura F, Hartwig JH, Weitz DA, Stossel TP (2011) Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin a. Nature 478(7368):260–263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Konstantinidis G, Moustakas A, Stournaras C (2011) Regulation of myosin light chain function by BMP signaling controls actin cytoskeleton remodeling. Cell Physiol Biochem 28(5):1031–1044

    Article  CAS  PubMed  Google Scholar 

  18. Das RK, Gocheva V, Hammink R, Zouani OF, Rowan A (2015) Stress-stiffening-mediated stem-cell commitment switch in soft responsive hydrogels. Nat Mater 15(3):318–325

    Article  PubMed  Google Scholar 

  19. Wu Y, Zhang P, Dai Q, Yang X, Fu R, Jiang L, Fang B (2013) Effect of mechanical stretch on the proliferation and differentiation of BMSCs from ovariectomized rats. Mol Cell Biochem 382(1):273–282

    Article  CAS  PubMed  Google Scholar 

  20. Song G, Yang J, Shen X, Luo Q, Shi Y, Qin J (2007) Mechanical stretch promotes proliferation of rat bone marrow mesenchymal stem cells. Colloids Surf B Biointerfaces 58(2):271–277

    Article  CAS  PubMed  Google Scholar 

  21. Sun L, Ling Q, Rui Z, Li H, Xue Y, Liu X, Fan J, Fan H (2016) Effects of mechanical stretch on cell proliferation and matrix formation of mesenchymal stem cell and anterior cruciate ligament fibroblast. Stem Cells Int 2016:1):1–1)10

    Google Scholar 

  22. Nakamura A, Dohi Y, Akahane M, Ohgushi H, Nakajima H, Funaoka H, Takakura Y (2009) Osteocalcin secretion as an early marker of in vitro osteogenic differentiation of rat mesenchymal stem cells. Tissue Eng Part C Methods 15(2):169–180

    Article  CAS  PubMed  Google Scholar 

  23. Shenaq DS, Teven CM, Seitz IA, Rastegar F, Greives MR, He TC, Reid RR (2015) Characterization of reversibly immortalized calvarial mesenchymal progenitor cells. J Craniofac Surg 26(4):1207–1213

    Article  PubMed  PubMed Central  Google Scholar 

  24. Gu K, Zhang L, Jin T, Rutherford RB (2004) Identification of potential modifiers of Runx2/Cbfa1 activity in C2C12 cells in response to bone morphogenetic protein-7. Cells Tissues Organs 176(1–3):28

    Article  CAS  PubMed  Google Scholar 

  25. Kupcsik L, Stoddart MJ, Li Z, Benneker LM, Alini M (2010) Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering. Tissue Eng A 16(6):1845–1855

    Article  CAS  Google Scholar 

  26. Beederman M, Lamplot JD, Nan G, Wang J, Liu X, Yin L, Li R, Shui W, Zhang H, Kim SH (2013) BMP signaling in mesenchymal stem cell differentiation and bone formation. J Biomed Sci Eng 6(8A):32–52

    Article  PubMed  PubMed Central  Google Scholar 

  27. Kanazawa T, Furumatsu T, Hachioji M, Oohashi T, Ninomiya Y, Ozaki T (2012) Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells. J Orthop Res 30(3):468–474

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was supported by the National Natural Science Foundation of China (81301551), the Chongqing Research Program of Basic Research and Frontier Technology (cstc2013jcyjA10022), the Visiting Scholar Foundation of Key Laboratory of Biorheological Science and Technology (Chongqing University), the Ministry of Education (CQKLBST-2012-004), the Scientific and Technological Research Program of Chongqing Municipal Education Commission(KJ1702024), the Scientific and Technological Research Program of Chongqing Yubei district (2017 nongshe 42), and the Program for Innovation Team Building at Institutions of Higher Education in Chongqing in 2016 (CXTDG201602006). Mingxing Lei is supported by projects funded by China Postdoctoral Science Foundation (2016M590866) and Special Funding for Postdoctoral Research Projects in Chongqing (Xm2015093).

Author information

Author notes

  1. Yang Song and Yinhong Tang contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing, 400044, People’s Republic of China

    Yang Song, Mingxing Lei & Li Yang

  2. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Stomatological Hospital of Chongqing Medical University, Chongqing, 401147, People’s Republic of China

    Yinhong Tang, Jinlin Song, Panpan Liang, Tiwei Fu, Xudong Su, Pengfei Zhou & Enyi Huang

  3. Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, College of Stomatology, Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Yinhong Tang, Jinlin Song, Panpan Liang, Tiwei Fu, Xudong Su, Pengfei Zhou & Enyi Huang

  4. Integrative Stem Cell Center, China Medical University Hospital, China Medical University, Taichung, 40402, Taiwan

    Mingxing Lei

  5. Institute of New Drug Development, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung, 40402, Taiwan

    Mingxing Lei

Authors
  1. Yang Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yinhong Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinlin Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mingxing Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Panpan Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tiwei Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xudong Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pengfei Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Li Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Enyi Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Yang Song and Yinhong Tang contributed equally to this paper.

Corresponding author

Correspondence to Enyi Huang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Song, Y., Tang, Y., Song, J. et al. Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells. International Orthopaedics (SICOT) 42, 947–955 (2018). https://doi.org/10.1007/s00264-018-3796-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00264-018-3796-z

Keywords

Search

Navigation