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Effects of HyStem™-HP Hydrogel Elasticity on Osteogenic Differentiation of Human Mesenchymal Stromal Cells


The biological, chemical, and mechanical properties of the extracellular matrix (ECM) are important for adhesion, proliferation, and osteogenic differentiation of mesenchymal stromal cells (MSC). Scaffolds prepared for tissue engineering approaches should imitate the properties of the native ECM of the target tissue. Here, we used the synthetic hydrogel HyStem™-HP with different elasticity as a substrate for human bone marrow derived MSC (hBMMSC) and determined the influence of elasticity on morphology, adhesion, proliferation, and osteogenic differentiation of the cells. hBMMSC cultured on HyStem™-HP with a crosslinking of 1.6% (low elasticity) were well-spread, revealed an organized actin cytoskeleton and many focal adhesion (FA) contacts in comparison cells cultured on HyStem™-HP with a crosslinking of 0.1% (high elasticity) showed less spreading, less FAs, and a less organized actin cytoskeleton. Following osteogenic differentiation markers, like the activity of tissue-non-specific alkaline phosphatase, bsp II expression, and calcium accumulation were more pronounced on HyStem™-HP 1.6% hydrogels compared to HyStem™-HP 0.1%. These findings indicate that osteogenic differentiation of hBMMSC is better promoted by HyStem™-HP with low elasticity and might therefore be a useful substrate for bone tissue engineering.

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  1. 1.

    Aubin, J. Regulation of osteoblast formation and function. Rev. Endocr. Metab. Disord. 2:81–94, 2001.

    Article  Google Scholar 

  2. 2.

    Augello, A., and C. De Bari. The regulation of differentiation in mesenchymal stem cells. Hum. Gene Ther. 21:1226–1238, 2010.

    Article  Google Scholar 

  3. 3.

    Bastow, E., S. Byers, S. Golub, C. Clarkin, A. Pitsillides, and A. Fosang. Hyaluronan synthesis and degradation in cartilage and bone. Cell. Mol. Life Sci. 65:395–413, 2008.

    Article  Google Scholar 

  4. 4.

    Benoit, D. S. W., and K. S. Anseth. Heparin functionalized PEG gels that modulate protein adsorption for hMSC adhesion and differentiation. Acta Biomater. 1:461–470, 2005.

    Article  Google Scholar 

  5. 5.

    Benoit, D. S. W., A. R. Durney, and K. S. Anseth. The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation. Biomaterials 28:66–77, 2007.

    Article  Google Scholar 

  6. 6.

    Boskey, A. L., and B. L. Dick. Hyaluronan interactions with hydroxyapatite do not alter in vitro hydroxyapatite crystal proliferation and growth. Matrix 11:442–446, 1991.

    Article  Google Scholar 

  7. 7.

    Buxboim, A., I. L. Ivanovska, and D. E. Discher. Matrix elasticity, cytoskeletal forces and physics of the nucleus: how deeply do cells ‘feel’ outside and in? J. Cell Sci. 123:297–308, 2010.

    Article  Google Scholar 

  8. 8.

    Chen, D., M. Zhao, and G. R. Mundy. Bone morphogenetic proteins. Growth Factors 22:233–241, 2004.

    Article  Google Scholar 

  9. 9.

    Discher, D. E., P. Janmey, and Y. L. Wang. Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139–1143, 2005.

    Article  Google Scholar 

  10. 10.

    Engler, A. J., S. Sen, H. L. Sweeney, and D. E. Discher. Matrix elasticity directs stem cell lineage specification. Cell 126:677–689, 2006.

    Article  Google Scholar 

  11. 11.

    Ganss, B., R. H. Kim, and J. Sodek. Bone sialoprotein. Crit. Rev. Oral Biol. Med. 10:79–98, 1999.

    Article  Google Scholar 

  12. 12.

    Hamadi, A., M. Bouali, M. Dontenwill, H. Stoeckel, K. Takeda, and P. Rondé. Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397. J. Cell Sci. 118:4415–4425, 2005.

    Article  Google Scholar 

  13. 13.

    Hass, R., C. Kasper, S. Böhm, and R. Jacobs. Different populations and sources of human mesenchymal stem cells (MSC): a comparison of adult and neonatal tissue-derived MSC. Cell Commun. Signal. 9:12, 2011.

    Article  Google Scholar 

  14. 14.

    Hempel, U., T. Hefti, P. Dieter, and F. Schlottig. Response of human bone marrow stromal cells, MG-63, and SaOS-2 to titanium-based dental implant surfaces with different topography and surface energy. Clin. Oral Implants Res. 24:174–182, 2013.

    Article  Google Scholar 

  15. 15.

    Huang, Z., E. R. Nelson, R. L. Smith, and S. B. Goodman. The sequential expression profiles of growth factors from osteoprogenitors [correction of osteroprogenitors] to osteoblasts in vitro. Tissue Eng. 13:2311–2320, 2007.

    Article  Google Scholar 

  16. 16.

    Khatiwala, C. B., S. R. Peyton, and A. J. Putnam. Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells. Am. J. Physiol. Cell Physiol. 290:C1640–C1650, 2006.

    Article  Google Scholar 

  17. 17.

    Kundu, A. K., and A. J. Putnam. Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells. Biochem. Biophys. Res. Commun. 347:347–357, 2006.

    Article  Google Scholar 

  18. 18.

    Liu, Y., X. Z. Shu, and G. D. Prestwich. Osteochondral defect repair with autologous bone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix. Tissue Eng. 12:3405–3416, 2006.

    Article  Google Scholar 

  19. 19.

    Nakamura, T., K. Hanada, M. Tamura, T. Shibanushi, H. Nigi, M. Tagawa, S. Fukumoto, and T. Matsumoto. Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats. Endocrinology 136:1276–1284, 1995.

    Google Scholar 

  20. 20.

    Oswald, J., S. Boxberger, B. Jørgensen, S. Feldmann, G. Ehninger, M. Bornhäuser, and C. Werner. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 22:377–384, 2004.

    Article  Google Scholar 

  21. 21.

    Parekh, S. H., K. Chatterjee, S. Lin-Gibson, N. M. Moore, M. T. Cicerone, M. F. Young, and C. G. Simon, Jr. Modulus-driven differentiation of marrow stromal cells in 3D scaffolds that is independent of myosin-based cytoskeletal tension. Biomaterials 32:2256–2264, 2011.

    Article  Google Scholar 

  22. 22.

    Pelham, Jr., R. J., and Y. Wang. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. U.S.A. 94:13661–13665, 1997.

    Article  Google Scholar 

  23. 23.

    Pike, D. B., S. Cai, K. R. Pomraning, M. A. Firpo, R. J. Fisher, X. Z. Shu, G. D. Prestwich, and R. A. Peattie. Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF. Biomaterials 27:5242–5251, 2006.

    Article  Google Scholar 

  24. 24.

    Pittenger, M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti, S. Craig, and D. R. Marshak. Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147, 1999.

    Google Scholar 

  25. 25.

    Riew, K. D., N. M. Wright, S. L. Cheng, L. V. Avioli, and J. Lou. Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model. Calcif. Tissue Int. 63:357–360, 1998.

    Google Scholar 

  26. 26.

    Ruppert, R., E. Hoffmann, and W. Sebald. Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur. J. Biochem. 237:295–302, 1996.

    Google Scholar 

  27. 27.

    Salasznyk, R. M., R. F. Klees, A. Boskey, and G. E. Plopper. Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5. J. Cell. Biochem. 100:499–514, 2007.

    Google Scholar 

  28. 28.

    Salasznyk, R. M., W. A. Williams, A. Boskey, A. Batorsky, and G. E. Plopper. Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. J. Biomed. Biotechnol. 24–34:2004, 2004.

    Google Scholar 

  29. 29.

    Stevens, M. M. Biomaterials for bone tissue engineering. Mater. Today 11:18–25, 2008.

    Google Scholar 

  30. 30.

    Thompson, L. D., M. W. Pantoliano, and B. A. Springer. Energetic characterization of the basic fibroblast growth factor-heparin interaction: identification of the heparin binding domain. Biochemistry 33:3831–3840, 1994.

    Google Scholar 

  31. 31.

    Tsai, K.-S., S.-Y. Kao, C.-Y. Wang, Y.-J. Wang, J.-P. Wang, and S.-C. Hung. Type I collagen promotes proliferation and osteogenesis of human mesenchymal stem cells via activation of ERK and Akt pathways. J. Biomed. Mater. Res. A 94A:673–682, 2010.

    Google Scholar 

  32. 32.

    Uygun, B. E., S. E. Stojsih, and H. W. Matthew. Effects of immobilized glycosaminoglycans on the proliferation and differentiation of mesenchymal stem cells. Tissue Eng. Part A 15:3499–3512, 2009.

    Google Scholar 

  33. 33.

    Wang, L.-S., C. Du, J. E. Chung, and M. Kurisawa. Enzymatically cross-linked gelatin-phenol hydrogels with a broader stiffness range for osteogenic differentiation of human mesenchymal stem cells. Acta Biomater. 8:1826–1837, 2012.

    Google Scholar 

  34. 34.

    Wiwatwongwana, F., and S. Pattana. Characterization on properties of modification gelatin films with carboxymethylcellulose. In: The First TSME International Conference on Mechanical Engineering, 2010.

  35. 35.

    Xiao, G., D. Jiang, R. Gopalakrishnan, and R. T. Franceschi. Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, cbfa1/runx2. J. Biol. Chem. 277:36181–36187, 2002.

    Google Scholar 

  36. 36.

    Yue, B., B. Lu, K. Dai, X. Zhang, C. Yu, J. Lou, and T. Tang. BMP2 gene therapy on the repair of bone defects of aged rats. Calcif. Tissue Int. 77:395–403, 2005.

    Google Scholar 

  37. 37.

    Zemel, A., F. Rehfeldt, A. E. Brown, D. E. Discher, and S. A. Safran. Optimal matrix rigidity for stress fiber polarization in stem cells. Nat. Phys. 6:468–473, 2010.

    Google Scholar 

  38. 38.

    Zhang, J., A. Skardal, and G. D. Prestwich. Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery. Biomaterials 29:4521–4531, 2008.

    Google Scholar 

  39. 39.

    Zou, L., X. Zou, L. Chen, H. Li, T. Mygind, M. Kassem, and C. Bünger. Effect of hyaluronan on osteogenic differentiation of porcine bone marrow stromal cells in vitro. J. Orthop. Res. 26:713–720, 2008.

    Google Scholar 

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The authors thank Carolin Preißler and Christine Kupke for excellent technical assistance, Prof. Bornhäuser and colleagues of the Bone Marrow Transplantation Center of the University Hospital Dresden for providing the hBMMSC and Rudi Hoetzel (Institute of Pharmacy, University of Leipzig) for his support with rheological measurements. This work was supported by a grant of BMBF to PD and by grants of Deutsche Forschungsgemeinschaft to UH (TR67B1) and MCH (TR67A1).

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Correspondence to Peter Dieter.

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Associate Editor Chwee Teck Lim oversaw the review of this article.

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Supplementary Fig. 1. Viscoelastic properties of HyStem™-HP 0.1% and HyStem™-HP 1.6% as determined during a frequency sweep (0.1–10 Hz) at a strain amplitude of 2% by oscillation rheology. The data illustrates that HyStem™-HP 0.1% hydrogels lose strength at lower shear frequency than HyStem™-HP 1.6% hydrogels. Onsets of loss of hydrogel structure are indicated by arrows. Data represent mean ± SD (n = 4). Supplementary material 1 (TIFF 1834 kb)

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Mai, M., Hempel, U., Hacker, M.C. et al. Effects of HyStem™-HP Hydrogel Elasticity on Osteogenic Differentiation of Human Mesenchymal Stromal Cells. Cel. Mol. Bioeng. 7, 155–164 (2014).

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  • Substrate flexibility
  • Synthetic extracellular matrix
  • PEG
  • Gelatin
  • Heparin