Matrix stress relaxation regulates fibroblast activation in a 3D microenvironment

Introduction: : Fibroblasts, the cells responsible for building and maintaining the ECM, are shown to be sensitive to changes in their mechanical environment. Significant changes in matrix mechanics accompany the progression of fibrosis as excess ECM is produced [1]. For example, increased matrix stiffness drives fibroblast activation, a phenotypic change that occurs in response to injury or inflammation [2,3]. While fibrosis results in drastic increases in tissue stiffness, concurrent changes in time-dependent properties such as viscoelasticity are less studied. Viscoelastic materials, which exhibit stress relaxing properties, have been shown to curtail fibroblast spreading, a behavior associated with activated fibroblasts [4]. However, the role that matrix stress relaxation plays in fibroblast activation in a 3D microenvironment remains an open question. We hypothesize that in slower stress-relaxing matrices, fibroblasts will demonstrate markers of activation. To assess this, we employ 3D hydrogel matrices that offer independent tunability of physical properties, including stiffness, stress relaxation, and ligand presence (Fig. 1A).

Materials and

Methods: : Interpenetrating networks (IPN) of alginate and collagen I were used to mimic healthy or fibrotic ECM. We varied calcium concentration to achieve soft or stiff IPN (3 kPa vs. 8 kPa), and stress relaxation rate was varied by using alginates of different molecular weights. The mechanical properties of the matrices were assessed via shear rheology using time sweeps at 0.5% strain and 1 Hz, and by stress relaxation tests with 10% constant strain. Matrix stiffness was reported using the Young’s modulus, and stress relaxation rate was reported as the time taken for the IPN to relax to half the maximum stress (Fig. 1B, 1C).
Primary human dermal fibroblasts (HDF) were encapsulated in alginate-collagen IPN and cultured for 7 days. For these experiments, the IPNs mimicked the stiffness of healthy or fibrotic tissue and exhibited fast or slow stress relaxation rates. Following a week of culture, the IPNs were fixed, sectioned, and stained for α-smooth muscle actin (α-SMA), collagen I and fibronectin to assess fibroblast activation.

Results, Conclusions, and Discussions:: HDF exhibited increased markers of activated fibroblasts when cultured in stiff matrices with slow stress relaxation compared to fast-relaxing matrices of similar stiffnesses (Fig. 2A). In soft IPNs, we observed no significant difference in α-SMA or collagen expression between fast- and slow-relaxing conditions (Fig. 2A-D). However, the expression of fibronectin was increased in slow-relaxing conditions irrespective of matrix stiffness (Fig. 2F). The expression of α-SMA, collagen and fibronectin are significantly increased in stiff, slow-relaxing matrices compared to fast-relaxing matrices. We observed that fast stress relaxation attenuates the expression of α-SMA and ECM proteins, even in stiff matrices that mimic fibrotic tissue, which aligns with previous results [4,5].

Our results show that fibroblast activation is regulated by stress relaxation in a 3D environment. We have shown using alginate-collagen IPNs that fibroblasts exhibit changes in α-SMA, collagen, and fibronectin expression in slow-relaxing matrices with the stiffness of fibrotic tissue. The tunability of this hydrogel platform allows us to mimic the increased stiffness and altered stress relaxation of fibrotic tissue, adding physiological relevance to our study. Based on the phenotypic results observed, we will interrogate the mechanisms by which fibroblasts sense and respond to stress relaxation. Thus, the aim of our research is to explore the biophysical signals underpinning the activation of fibroblasts and the onset of fibrotic diseases.

Acknowledgements and/or References (Optional)::

References: [1] DeLeon-Pennel, Matrix Biology (2020). [2] Schwager, American Journal of Physiology (2018). [3] Tiskatrok, Scientific Reports (2023). [4] Hui, Cellular and Molecular Bioengineering (2021). [5] Charrier, Nature Communications (2018).