(562c) Graduate Student Award Session: The Combined Influence of Viscoelastic and Adhesive Cues on Fibroblast Spreading and Focal Adhesion Organization

Background: 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 preferential αv 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 how multiple cues such as stiffness, viscoelasticity, and integrin engagement impact fibroblast behavior. We previously developed hyaluronic acid (HA) hydrogels with spatiotemporally controllable cell-instructive properties (stiffness, viscoelasticity, ligand presentation)¹. In this work, we used a similar system to study the influence of preferential integrin engagement (αvβ3 and α5β1) on fibroblast spreading and focal adhesion organization².

Methods: Elastic norbornene-functionalized HA (NorHA) hydrogels were fabricated via ultraviolet (UV) light-mediated thiol-ene addition using dithiol (DTT) crosslinker and LAP photoinitiator. Viscoelastic hydrogels were fabricated using a combination of NorHA and DTT (covalent crosslinks) with β-cyclodextrin modified HA (CD-HA) and thiolated adamantane (Ad) peptides (non-covalent Ad:CD guest-host interactions) (Fig. 1A). Cell adhesion was mediated through incorporation of either RGD peptide or engineered fibronectin fragments promoting preferential integrin engagement via αvβ3 or α5β1. Hydrogels mechanics were measured via oscillatory shear rheology. Human lung fibroblasts (HLFs) were cultured atop hydrogels and visualized via fluorescence microscopy. Focal adhesion (FA) analysis was conducted via the Focal Adhesion Analysis Server (FAAS) automated imaging processing pipeline.

Results and Discussion: HA hydrogels with mechanics matching normal (G’ ~ 0.5 kPa) or fibrotic (G’ ~ 5 kPa) lung tissue were synthesized using a combination of covalent crosslinks and supramolecular interactions to tune stiffness and viscoelasticity (Fig. 1B). Thiolated RGD peptide or fibronectin fragments designed to preferentially bind αvβ3 (Fn4G) or α5β1 (Fn9*10) integrins were incorporated to dictate cellular adhesion. HLFs were cultured atop hydrogels for three days (Fig. 1C) and cell spread area, cell shape index (CSI), F-actin and a-smooth muscle actin (α-SMA) expression, and FA organization were quantified (area and FA organization shown in Fig. 1D). On fibrosis-mimicking stiff elastic hydrogels, preferential αvβ3 engagement by HLFs led to increased spreading, actin stress fiber organization, and FA maturation as indicated by paxillin organization. In contrast, preferential α5β1 binding suppressed these metrics. Viscoelasticity, mimicking the mechanics of healthy tissue, led to decreased fibroblast spreading and FA organization independent on adhesive ligand type, highlighting its role in reducing fibroblast-activating behaviors.

Conclusions: In this study, we successfully developed a modular hydrogel system enabling independent control of covalent crosslinking, incorporation of supramolecular guest-host interactions, and functionalization with adhesive ligands preferentially engaging integrin heterodimers. Fibroblasts cultured atop hydrogels preferentially engaging αvβ3 (RGD, Fn4G) displayed increased spreading, actin stress fiber formation, and FA size on stiffer elastic hydrogels, but viscoelasticity played a suppressive role regardless of adhesive ligand presentation. In particular, fibrosis-associated αv engagement on Fn4G-modified hydrogels promoted increased spread area and FA size, even on softer elastic materials. Together, these results provide new insights into how mechanical and adhesive cues collectively guide disease-relevant cell behaviors. Ongoing work is looking at light-mediated spatiotemporal control over stiffness, viscoelasticity, and ligand presentation to mimic heterogeneous tissue as well as incorporating enzymatically-cleavable crosslinkers to enable selective cell release.

References: (1) Hui, E., Gimeno, K.I., Guan, G., Caliari, S.R. Biomacromolecules, 2019. (2) Hui, E., Moretti, L., Barker, T.H., Caliari, S.R. Cellular and Molecular Bioengineering, 2021.