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Hydrogels with tunable stress relaxation regulate stem cell fate and activity

Abstract

Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel’s initial elastic modulus, degradation, and cell-adhesion-ligand density. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell–ECM interactions and as an important design parameter of biomaterials for cell culture.

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Figure 1: Modulating the nanoscale architecture of alginate hydrogels to modulate stress relaxation properties independent of initial elastic modulus and matrix degradation to capture the viscoelastic behaviours of living tissues.
Figure 2: Cell spreading and proliferation for fibroblasts encapsulated within gels are enhanced with faster stress relaxation.
Figure 3: MSCs undergo osteogenic differentiation and form an interconnected mineralized collagen-1-rich matrix only in rapidly relaxing gels.
Figure 4: Osteogenic differentiation of MSCs mediated through ECM ligand density, enhanced RGD ligand clustering, and myosin contractility in stiffer hydrogels.
Figure 5: Nuclear localization of YAP is enhanced by faster stress relaxation, but decoupled from MSC fate.
Figure 6: Hypothesis for how initial elastic modulus and stress relaxation properties of matrix regulate cellular behaviours.

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Acknowledgements

The authors acknowledge the help of S. Koshy, M. Mehta, C. Verbeke, X. Zhao (now at MIT), and other members of the Mooney lab. The authors also thank the Weitz lab for use of a rheometer, O. Uzun for help with GPC, S. Reinke (Berlin-Brandenburg Center for Regenerative Therapies) for providing the human bone haematoma samples, and D. Wulsten and S. Reinke for the support in bone fracture haematoma testing. This work was supported by an NIH Grant to D.J.M. (R01 DE013033), an NIH F32 grant to O.C. (CA153802), an Einstein Visiting Fellowship for D.J.M., funding of the Einstein Foundation Berlin through the Charité—Universitätsmedizin Berlin, Berlin-Brandenburg School for Regenerative Therapies GSC 203, ZonMW-VICI grant 918.11.635 (The Netherlands) for D.K., and Harvard MRSEC for D.J.M. (DMR-1420570). This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN).

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O.C., L.G., D.K., M.D., N.H. and D.J.M. designed the experiments. O.C. and L.G. conducted most of the experiments. D.K. helped with experiments involving the MSCs. S.A.B. helped with alginate characterization. J.C.W. helped with EDS experiments and analysis. H.-p.L. assisted with mechanical characterization. E.L. and G.N.D. carried out fracture haematoma measurement. O.C. and L.G. analysed the data. O.C., L.G., D.K. and D.J.M. wrote the manuscript.

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Correspondence to David J. Mooney.

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Chaudhuri, O., Gu, L., Klumpers, D. et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nature Mater 15, 326–334 (2016). https://doi.org/10.1038/nmat4489

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