Although cell–matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.
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Nuttelman, C. R., Tripodi, M. C. & Anseth, K. S. Synthetic hydrogel niches that promote hMSC viability. Matrix Biol. 24, 208–218 (2005).
Ruoslahti, E. & Reed, J. C. Anchorage dependence, integrins, and apoptosis. Cell 77, 477–478 (1994).
VandeVondele, S., Vörös, J. & Hubbell, J. A. RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion. Biotechnol. Bioeng. 82, 784–790 (2003).
Docheva, D., Popov, C., Mutschler, W. & Schieker, M. Human mesenchymal stem cells in contact with their environment: Surface characteristics and the integrin system. J. Cell Mol. Med. 11, 21–38 (2007).
Meredith, J. E. Jr, Fazeli, B. & Schwartz, M. A. The extracellular matrix as a cell survival factor. Mol. Biol. Cell 4, 953–961 (1993).
Guilak, F. et al. Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5, 17–26 (2009).
Reilly, G. C. & Engler, A. J. Intrinsic extracellular matrix properties regulate stem cell differentiation. 43, 55–62 (2010).
Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).
Guvendiren, M. & Burdick, J. A. Stiffening hydrogels to probe short- and long-term cellular responses to dynamic mechanics. Nature Commun. 3, 792 (2012).
Fu, J. et al. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nature Methods 7, 733–736 (2010).
Gilbert, P. M. et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).
Guvendiren, M. & Burdick, J. A. The control of stem cell morphology and differentiation by hydrogel surface wrinkles. Biomaterials 31, 6511–6518 (2010).
Kilian, K. A., Bugarija, B., Lahn, B. T. & Mrksich, M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc. Natl Acad. Sci. USA 107, 4872–4877 (2010).
McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K. & Chen, C. S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell 6, 483–495 (2004).
Ruiz, S. A. & Chen, C. S. Emergence of patterned stem cell differentiation within multicellular structures. Stem Cells 26, 2921–2927 (2008).
Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nature Mater. 9, 518–526 (2010).
Benoit, D. S., Schwartz, M. P., Durney, A. R. & Anseth, K. S. Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. Nature Mater. 7, 816–823 (2008).
Ferreira, L. S. et al. Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells. Biomaterials 28, 2706–2717 (2007).
Ifkovits, J. L. & Burdick, J. A. Photopolymerizable and degradable biomaterials for tissue engineering applications. Tissue Eng. 13, 2369–2385 (2007).
Mann, B. K., Gobin, A. S., Tsai, A. T., Schmedlen, R. H. & West, J. L. Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: Synthetic ECM analogs for tissue engineering. Biomaterials 22, 3045–3051 (2001).
Nicodemus, G. D. & Bryant, S. J. Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng. 14, 149–165 (2008).
Khetan, S., Katz, J. S. & Burdick, J. A. Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels. Soft Matter 5, 1601–1606 (2009).
Burdick, J. A., Chung, C., Jia, X., Randolph, M. A. & Langer, R. Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules 6, 386–391 (2005).
Erickson, I. E. et al. Macromer density influences mesenchymal stem cell chondrogenesis and maturation in photocrosslinked hyaluronic acid hydrogels. Osteoarthritis Cartilage 17, 1639–1648 (2009).
Legant, W. R. et al. Measurement of mechanical tractions exerted by cells in three-dimensional matrices. Nature Methods 7, 969–971 (2010).
Hahn, M. S., Miller, J. S. & West, J. L. Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Adv. Mater. 18, 2679–2684 (2006).
Khetan, S. & Burdick, J. A. Patterning network structure to spatially control cellular remodeling and stem cell fate within 3-dimensional hydrogels. Biomaterials 31, 8228–8234 (2010).
West, J. L. & Hubbell, J. A. Polymeric biomaterials with degradation sites for proteases involved in cell migration. Macromolecules 32, 241–244 (1999).
Miller, J. S. et al. Bioactive hydrogels made from step-growth derived PEG-peptide macromers. Biomaterials 31, 3736–3743 (2010).
Marklein, R. A. & Burdick, J. A. Spatially controlled hydrogel mechanics to modulate stem cell interactions. Soft Matter 6, 136–143 (2010).
Hudson, J. E. et al. A synthetic elastomer based on acrylated polypropylene glycol triol with tunable modulus for tissue engineering applications. Biomaterials 31, 7937–7947 (2010).
Maekawa, M. et al. Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285, 895–898 (1999).
Vardouli, L., Moustakas, A. & Stournaras, C. LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor- β. J. Biol. Chem. 280, 11448–11457 (2005).
Shih, Y. R., Tseng, K. F., Lai, H. Y., Lin, C. H. & Lee, O. K. Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells. J. Bone Miner. Res. 26, 730–738 (2011).
Duxbury, M. S., Ashley, S. W. & Whang, E. E. Inhibition of pancreatic adenocarcinoma cellular invasiveness by blebbistatin: A novel myosin II inhibitor. Biochem. Biophys. Res. Commun. 313, 992–997 (2004).
Even-Ram, S. et al. Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. Nature Cell Biol. 9, 299–309 (2007).
Bennett, K. P. et al. Preoteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells. BMC Genom. 8, 380 (2007).
Williams, C. G., Malik, A. N., Kim, T. K., Manson, P. N. & Elisseeff, J. H. Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing hydrogels and cell encapsulation. Biomaterials 26, 1211–1218 (2005).
Singer, V. L., Jones, L. J., Yue, S. T. & Haugland, R. P. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal. Biochem. 249, 228–238 (1997).
Gao, Y. & Kilfoil, M. L. Accurate detection and complete tracking of large populations of features in three dimensions. Opt. Express 17, 4685–4704 (2009).
This work was supported by funding from a Fellowship in Science and Engineering from the David and Lucile Packard Foundation (J.A.B.), a CAREER award (J.A.B.) and Graduate Research Fellowship (S.K.) from the National Science Foundation, and grant GM74048 from the National Institutes of Health (C.S.C.). The authors would like to thank R. Marklein and C. Choi for helpful discussions and experimental assistance.
The authors declare no competing financial interests.
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Khetan, S., Guvendiren, M., Legant, W. et al. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels. Nature Mater 12, 458–465 (2013). https://doi.org/10.1038/nmat3586
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