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Local nascent protein deposition and remodelling guide mesenchymal stromal cell mechanosensing and fate in three-dimensional hydrogels

Abstract

Hydrogels serve as valuable tools for studying cell–extracellular matrix interactions in three-dimensional environments that recapitulate aspects of native extracellular matrix. However, the impact of early protein deposition on cell behaviour within hydrogels has largely been overlooked. Using a bio-orthogonal labelling technique, we visualized nascent proteins within a day of culture across a range of hydrogels. In two engineered hydrogels of interest in three-dimensional mechanobiology studies—proteolytically degradable covalently crosslinked hyaluronic acid and dynamic viscoelastic hyaluronic acid hydrogels—mesenchymal stromal cell spreading, YAP/TAZ nuclear translocation and osteogenic differentiation were observed with culture. However, inhibition of cellular adhesion to nascent proteins or reduction in nascent protein remodelling reduced mesenchymal stromal cell spreading and nuclear translocation of YAP/TAZ, resulting in a shift towards adipogenic differentiation. Our findings emphasize the role of nascent proteins in the cellular perception of engineered materials and have implications for in vitro cell signalling studies and application to tissue repair.

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Data availability

All the data generated or analysed during this study are included within this article and its Supplementary Information. Additional information is available from the corresponding author on request.

References

  1. 1.

    Kim, S. H., Turnbull, J. & Guimond, S. Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor. J. Endocrinol. 209, 139–151 (2011).

  2. 2.

    Guvendiren, M. & Burdick, J. A. Engineering synthetic hydrogel microenvironments to instruct stem cells. Curr. Opin. Biotechnol. 24, 841–846 (2013).

  3. 3.

    Tibbitt, M. W. & Anseth, K. S. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng. 103, 655–663 (2009).

  4. 4.

    Drury, J. L. & Mooney, D. J. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 24, 4337–4351 (2003).

  5. 5.

    Wells, R. G. The role of matrix stiffness in regulating cell behavior. Hepatology 47, 1394–1400 (2008).

  6. 6.

    Khetan, S. et al. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels. Nat. Mater. 12, 458–465 (2013).

  7. 7.

    Schultz, K. M., Kyburz, K. A. & Anseth, K. S. Measuring dynamic cell-material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels. Proc. Natl Acad. Sci. USA 112, E3757–E3764 (2015).

  8. 8.

    Chaudhuri, O. et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat. Mater. 15, 326–334 (2016).

  9. 9.

    Wang, H. & Heilshorn, S. C. Adaptable hydrogel networks with reversible linkages for tissue engineering. Adv. Mater. 27, 3717–3736 (2015).

  10. 10.

    Rosales, A. M. & Anseth, K. S. The design of reversible hydrogels to capture extracellular matrix dynamics. Nat. Rev. Mater. 1, 15012 (2016).

  11. 11.

    Unlu, G., Levic, D. S., Melville, D. B. & Knapik, E. W. Trafficking mechanisms of extracellular matrix macromolecules: insights from vertebrate development and human diseases. Int. J. Biochem. Cell Biol. 47, 57–67 (2014).

  12. 12.

    Gattazzo, F., Urciuolo, A. & Bonaldo, P. Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim. Biophys. Acta 1840, 2506–2519 (2014).

  13. 13.

    Kadler, K. E., Hill, A. & Canty-Laird, E. G. Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators. Curr. Opin. Cell Biol. 20, 495–501 (2008).

  14. 14.

    Gjorevski, N. & Nelson, C. M. Bidirectional extracellular matrix signaling during tissue morphogenesis. Cytokine Growth Factor Rev. 20, 459–465 (2009).

  15. 15.

    McLeod, C. M. & Mauck, R. L. High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation. Sci. Rep. 6, 38852 (2016).

  16. 16.

    Bian, L., Guvendiren, M., Mauck, R. L. & Burdick, J. A. Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis. Proc. Natl Acad. Sci. USA 110, 10117–10122 (2013).

  17. 17.

    Nicodemus, G. D., Skaalure, S. C. & Bryant, S. J. Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels. Acta Biomater. 7, 492–504 (2011).

  18. 18.

    Huebsch, N. et al. Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation. Nat. Mater. 14, 1269–1277 (2015).

  19. 19.

    Cai, R., Nakamoto, T., Kawazoe, N. & Chen, G. Influence of stepwise chondrogenesis-mimicking 3D extracellular matrix on chondrogenic differentiation of mesenchymal stem cells. Biomaterials 52, 199–207 (2015).

  20. 20.

    Ferreira, S. A. et al. Bi-directional cell–pericellular matrix interactions direct stem cell fate. Nat. Commun. 9, 4049 (2018).

  21. 21.

    Kubow, K. E. et al. Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix. Nat. Commun. 6, 8026 (2015).

  22. 22.

    Li, B., Moshfegh, C., Lin, Z., Albuschies, J. & Vogel, V. Mesenchymal stem cells exploit extracellular matrix as mechanotransducer. Sci. Rep. 3, 2425 (2013).

  23. 23.

    Scott, L. E., Mair, D. B., Narang, J. D., Feleke, K. & Lemmon, C. A. Fibronectin fibrillogenesis facilitates mechano-dependent cell spreading, force generation, and nuclear size in human embryonic fibroblasts. Integr. Biol. 7, 1454–1465 (2015).

  24. 24.

    Daley, W. P., Peters, S. B. & Larsen, M. Extracellular matrix dynamics in development and regenerative medicine. J. Cell Sci. 121, 255–264 (2008).

  25. 25.

    Jansen, K. A., Atherton, P. & Ballestrem, C. Mechanotransduction at the cell–matrix interface. Semin. Cell Dev. Biol. 71, 75–83 (2017).

  26. 26.

    Dieterich, D. C. et al. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc. 2, 532–540 (2007).

  27. 27.

    Caliari, S. R., Vega, S. L., Kwon, M., Soulas, E. M. & Burdick, J. A. Dimensionality and spreading influence MSC YAP/TAZ signaling in hydrogel environments. Biomaterials 103, 314–323 (2016).

  28. 28.

    Doyle, A. D. & Yamada, K. M. Mechanosensing via cell–matrix adhesions in 3D microenvironments. Exp. Cell Res. 343, 60–66 (2016).

  29. 29.

    Hytonen, V. P. & Wehrle-Haller, B. Protein conformation as a regulator of cell–matrix adhesion. Phys. Chem. Chem. Phys. 16, 6342–6357 (2014).

  30. 30.

    Tuckwell, D., Calderwood, D. A., Green, L. J. & Humphries, M. J. Integrin alpha 2 I-domain is a binding site for collagens. J. Cell Sci. 108, 1629–1637 (1995).

  31. 31.

    Connelly, J. T., Petrie, T. A., Garcia, A. J. & Levenston, M. E. Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels. Eur. Cells Mater. 22, 168–177 (2011).

  32. 32.

    Keselowsky, B. G., Collard, D. M. & Garcia, A. J. Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation. Proc. Natl Acad. Sci. USA 102, 5953–5957 (2005).

  33. 33.

    Massia, S. P. & Hubbell, J. A. Vascular endothelial cell adhesion and spreading promoted by the peptide REDV of the IIICS region of plasma fibronectin is mediated by integrin alpha 4 beta 1. J. Biol. Chem. 267, 14019–14026 (1992).

  34. 34.

    Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).

  35. 35.

    Brusatin, G., Panciera, T., Gandin, A., Citron, A. & Piccolo, S. Biomaterials and engineered microenvironments to control YAP/TAZ-dependent cell behaviour. Nat. Mater. 17, 1063–1075 (2018).

  36. 36.

    Fogerty, F. J., Akiyama, S. K., Yamada, K. M. & Mosher, D. F. Inhibition of binding of fibronectin to matrix assembly sites by anti-integrin (alpha 5 beta 1) antibodies. J. Cell Biol. 111, 699–708 (1990).

  37. 37.

    McDonald, J. A. et al. Fibronectin’s cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix. J. Biol. Chem. 262, 2957–2967 (1987).

  38. 38.

    Lee, H. P., Gu, L., Mooney, D. J., Levenston, M. E. & Chaudhuri, O. Mechanical confinement regulates cartilage matrix formation by chondrocytes. Nat. Mater. 16, 1243–1251 (2017).

  39. 39.

    Cameron, A. R., Frith, J. E., Gomez, G. A., Yap, A. S. & Cooper-White, J. J. The effect of time-dependent deformation of viscoelastic hydrogels on myogenic induction and Rac1 activity in mesenchymal stem cells. Biomaterials 35, 1857–1868 (2014).

  40. 40.

    Rodell, C. B., Dusaj, N. N., Highley, C. B. & Burdick, J. A. Injectable and cytocompatible tough double-network hydrogels through tandem supramolecular and covalent crosslinking. Adv. Mater. 28, 8419–8424 (2016).

  41. 41.

    Loebel, C., Rodell, C. B., Chen, M. H. & Burdick, J. A. Shear-thinning and self-healing hydrogels as injectable therapeutics and for 3D-printing. Nat. Protoc. 12, 1521–1541 (2017).

  42. 42.

    Rodell, C. B., Kaminski, A. L. & Burdick, J. A. Rational design of network properties in guest–host assembled and shear-thinning hyaluronic acid hydrogels. Biomacromolecules 14, 4125–4134 (2013).

  43. 43.

    Dooling, L. J., Buck, M. E., Zhang, W. B. & Tirrell, D. A. Programming molecular association and viscoelastic behavior in protein networks. Adv. Mater. 28, 4651–4657 (2016).

  44. 44.

    McKinnon, D. D., Domaille, D. W., Cha, J. N. & Anseth, K. S. Biophysically defined and cytocompatible covalently adaptable networks as viscoelastic 3D cell culture systems. Adv. Mater. 26, 865–872 (2014).

  45. 45.

    Feng, Y. et al. Exo1: a new chemical inhibitor of the exocytic pathway. Proc. Natl Acad. Sci. USA 100, 6469–6474 (2003).

  46. 46.

    von Kleist, L. & Haucke, V. At the crossroads of chemistry and cell biology: inhibiting membrane traffic by small molecules. Traffic 13, 495–504 (2012).

  47. 47.

    Mishev, K., Dejonghe, W. & Russinova, E. Small molecules for dissecting endomembrane trafficking: a cross-systems view. Cell Chem. Biol. 20, 475–486 (2013).

  48. 48.

    Purcell, B. P. et al. Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition. Nat. Mater. 13, 653–661 (2014).

  49. 49.

    Wolf, K. et al. Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J. Cell Biol. 201, 1069–1084 (2013).

  50. 50.

    Sridhar, B. V. et al. Development of a cellularly degradable PEG hydrogel to promote articular cartilage extracellular matrix deposition. Adv. Healthc. Mater. 4, 702–713 (2015).

  51. 51.

    Blache, U. et al. Notch-inducing hydrogels reveal a perivascular switch of mesenchymal stem cell fate. EMBO Rep. 19, e45964 (2018).

  52. 52.

    Cosgrove, B. D. et al. N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells. Nat. Mater. 15, 1297–1306 (2016).

  53. 53.

    Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).

  54. 54.

    Cruz-Acuna, R. et al. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair. Nat. Cell Biol. 19, 1326–1335 (2017).

  55. 55.

    Hezaveh, H. et al. Encoding stem-cell-secreted extracellular matrix protein capture in two and three dimensions using protein binding peptides. Biomacromolecules 19, 721–730 (2018).

  56. 56.

    Gardner, O. F., Alini, M. & Stoddart, M. J. Mesenchymal stem cells derived from human bone marrow. Methods Mol. Biol. 1340, 41–52 (2015).

  57. 57.

    Schoen, R. C., Bentley, K. L. & Klebe, R. J. Monoclonal antibody against human fibronectin which inhibits cell attachment. Hybrid 1, 99–108 (1982).

  58. 58.

    Gramlich, W. M., Kim, I. L. & Burdick, J. A. Synthesis and orthogonal photopatterning of hyaluronic acid hydrogels with thiol-norbornene chemistry. Biomaterials 34, 9803–9811 (2013).

  59. 59.

    Wade, R. J., Bassin, E. J., Rodell, C. B. & Burdick, J. A. Protease-degradable electrospun fibrous hydrogels. Nat. Commun. 6, 6639 (2015).

  60. 60.

    Almany, L. & Seliktar, D. Biosynthetic hydrogel scaffolds made from fibrinogen and polyethylene glycol for 3D cell cultures. Biomaterials 26, 2467–2477 (2005).

  61. 61.

    Bauer, A. et al. Hydrogel substrate stress-relaxation regulates the spreading and proliferation of mouse myoblasts. Acta Biomater. 62, 82–90 (2017).

  62. 62.

    Doube, M. et al. BoneJ: free and extensible bone image analysis in ImageJ. Bone 47, 1076–1079 (2010).

  63. 63.

    Loebel, C. et al. Cross-linking chemistry of tyramine-modified hyaluronan hydrogels alters mesenchymal stem cell early attachment and behavior. Biomacromolecules 18, 855–864 (2017).

  64. 64.

    Tseng, Q. et al. Spatial organization of the extracellular matrix regulates cell–cell junction positioning. Proc. Natl Acad. Sci. USA 109, 1506–1511 (2012).

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Acknowledgements

This work was supported by the Swiss National Foundation through an SNF Early Postdoc Mobility Fellowship (to C.L.), the National Science Foundation (DMR award 1610525, the Center for Engineering MechanoBiology CMMI: 15-48571) and the National Institutes of Health (R01 EB008722). We are grateful for help from the Penn EMRL Electron Microscopy Core for TEM and the Penn CDB Microscopy Core Facility for TFM, and would like to thank D. Seliktar for providing the PEG-DA, and A. Garcia, M. Davidson, R. Daniels, B. Cosgrove and M. D’Este for helpful conversations.

Author information

C.L., R.L.M. and J.A.B. conceived the ideas and designed the experiments. C.L. conducted the experiments and analysed the data. C.L., R.L.M. and J.A.B. interpreted the data and wrote the manuscript.

Correspondence to Jason A. Burdick.

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Further reading

Fig. 1: Nascent protein deposition by encapsulated hMSCs occurs early, independent of hydrogel type.
Fig. 2: Nascent ECM proteins create an adhesive layer at the cell–hydrogel interface.
Fig. 3: Adhesion to nascent proteins controls hMSC mechanosensing in degradable hydrogels.
Fig. 4: Dynamic hydrogel composition modulates viscoelastic properties and cell spreading.
Fig. 5: Nascent protein remodelling is required for cell spreading and osteogenesis in dynamic hydrogels.
Fig. 6: Nascent protein adhesion and remodelling enhance cell spreading in degradable/dynamic hydrogels.