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The Role of Rho GTPases During Fibroblast Spreading, Migration, and Myofibroblast Differentiation in 3D Synthetic Fibrous Matrices

  • 2021 CMBE Young Innovators
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Abstract

Introduction

Connective tissue repair and mechanosensing are tightly entwined in vivo and occur within a complex three-dimensional (3D), fibrous extracellular matrix (ECM). Typically driven by activated fibroblasts, wound repair involves well-defined steps of cell spreading, migration, proliferation, and fibrous ECM deposition. While the role of Rho GTPases in regulating these processes has been explored extensively in two-dimensional cell culture models, much less is known about their role in more physiologic, 3D environments.

Methods

We employed a 3D, fibrous and protease-sensitive hydrogel model of interstitial ECM to study the interplay between Rho GTPases and fibrous matrix cues in fibroblasts during wound healing.

Results

Modulating fiber density within protease-sensitive hydrogels, we confirmed previous findings that heightened fiber density promotes fibroblast spreading and proliferation. The presence of matrix fibers furthermore corresponded to increased cell migration speeds and macroscopic hydrogel contraction arising from fibroblast generated forces. During fibroblast spreading, Rac1 and RhoA GTPase activity proved crucial for fiber-mediated cell spreading and contact guidance along matrix fibers, while Cdc42 was dispensable. In contrast, interplay between RhoA, Rac1, and Cdc42 contributed to fiber-mediated myofibroblast differentiation and matrix contraction over longer time scales.

Conclusion

These observations may provide insights into tissue repair processes in vivo and motivate the incorporation of cell-adhesive fibers within synthetic hydrogels for material-guided wound repair strategies.

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Acknowledgments

We thank Dr. Eric S. White (University of Michigan) for providing the human fibroblasts employed in these studies.

Author Contributions

D.L.M and B.M.B conceived the experiments, supervised the project, and wrote the manuscript. D.L.M. designed and performed the experiments. A.L. conducted data analysis for Figs. 2, 3 and 4. H.L.H created and aided in the use of a custom Matlab script for cell migration analysis. All authors edited and approved the manuscript.

Funding

This work was supported in part by the National Institutes of Health (HL124322, EB030474). DLM acknowledges financial support from the National Science Foundation Graduate Research Fellowship Program (DGE1256260).

Conflict of interest

D.L.M, A.T.L, H.L.H, and B.M.B declare that they have no conflicts of interest.

Data Availability

Datasets are available on request: the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, by request.

Ethical Standards

No human or animal studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA

    Daniel L. Matera & Brendon M. Baker

  2. Department of Biology, University of Michigan, Ann Arbor, MI, 48109, USA

    Alexander T. Lee

  3. Department of Biomedical Engineering, University of Michigan, 2174 Lurie BME Building, 1101 Beal Avenue, Ann Arbor, MI, 48109, USA

    Harrison L. Hiraki & Brendon M. Baker

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  1. Daniel L. Matera
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Matera, D.L., Lee, A.T., Hiraki, H.L. et al. The Role of Rho GTPases During Fibroblast Spreading, Migration, and Myofibroblast Differentiation in 3D Synthetic Fibrous Matrices. Cel. Mol. Bioeng. 14, 381–396 (2021). https://doi.org/10.1007/s12195-021-00698-5

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