The Combined Influence of Viscoelastic and Adhesive Cues on Fibroblast Spreading and Focal Adhesion Organization

Abstract

Tissue fibrosis is characterized by progressive extracellular matrix (ECM) stiffening and loss of viscoelasticity that ultimately results in reduced organ functionality. Cells bind to the ECM through integrins, where av integrin engagement in particular has been correlated with fibroblast activation into contractile myofibroblasts that drive fibrosis progression. There is a significant unmet need for in vitro hydrogel systems that deconstruct the complexity of native tissues to better understand the individual and combined effects of stiffness, viscoelasticity, and integrin engagement on fibroblast behavior. Here, we developed hyaluronic acid hydrogels with independently tunable cell-instructive properties (stiffness, viscoelasticity, ligand presentation) to address this challenge. Hydrogels with mechanics matching normal or fibrotic lung tissue were synthesized using a combination of covalent crosslinks and supramolecular interactions to tune viscoelasticity. Cell adhesion was mediated through incorporation of either RGD peptide or engineered fibronectin fragments promoting preferential integrin engagement via avb3 or a5b1. We showed that preferential avb3 engagement enabled human lung fibroblasts to assume a myofibroblast-like phenotype on fibrosismimicking stiff elastic hydrogels with increased spreading, actin stress fiber organization, and focal adhesion maturation as indicated by paxillin organization. In contrast, preferential a5b1 binding suppressed these metrics. Viscoelasticity, mimicking the mechanics of healthy tissue, largely curtailed fibroblast spreading and focal adhesion organization independent of adhesive ligand type, highlighting its role in preventing fibroblast activation. Together these results provide new insights into how mechanical and adhesive cues collectively guide disease-relevant cell behaviors.

The interplay of fibroblasts, the extracellular matrix, and inflammation in scar formation

“…Despite the simplistic view that soft microenvironments are necessary to reduce scar formation, as in fetal wound healing, the increased signaling and cytoskeletal remodeling on HA gels indicate the importance of the modulation of contractile machinery of fibroblasts in wound repair, as discussed earlier in this section. Further investigation of this phenomenon via studies that take HA viscoelastic substrates into account is also warranted ( 210 ).…”

Hydrogel mechanics regulate fibroblast DNA methylation and chromatin condensation

“…5,8,10,24,25 Recent studies found that viscoelastic hydrogels supported reduced cell spreading and nuclear translocation of mechanoregulatory transcriptional factors as a result of lower cellular contractility compared to elastic hydrogels of similar stiffness. 26–33…”

Current possibilities and future opportunities provided by three-dimensional lung ECM-derived hydrogels

“…Alternatively, natural ECM components have also been used to generate single component hydrogels that readily support cell attachment, but are more limited in the possibilities for tuning their biomechanical properties. Examples of such hydrogels include collagen type I, fibrin, gelatin (methacrylate) and hyaluronan ( Bourke et al, 2011 ; Tjin et al, 2017 ; Sun et al, 2020 ; Martinez-Garcia et al, 2021b ; Hui et al, 2021 ; Blokland et al, 2022 ; Loebel et al, 2022 ; Martinez-Garcia et al, 2022 ). Such hydrogels provide the 3D environment for cells, modelling the dimensionality and possibly the biomechanical mimicry of the in vivo situation, but they are not reflective of the complexity of the ECM components within the tissue microenvironment.…”