X11 - Tunable Hydrogel Platform for Modeling Mechanosensitive Fibroblast Activation

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Devin Johnson

Undergraduate Student
University of California, Irvine, United States

Co-Author(s)

DB

David Berlin

Graduate Student Researcher
University of California, Irvine, United States

Last Author(s)

Quinton Smith

Assistant Professor
University of California, Irvine
Irvine, California, United States

Primary Investigator(s)

Quinton Smith

Assistant Professor
University of California, Irvine
Irvine, California, United States

Introduction: : Fibrosis is a chronic, progressive disorder in which repeated injury and dysregulated wound healing induce excessive extracellular matrix (ECM) deposition and a progressive increase in tissue stiffness [1-5]. This mechanical remodeling disrupts organ function and is propelled by dietary factors, sedentary lifestyles, and genetic predispositions [6]. Conventional tissue-culture plastic exceeds the stiffness of native tissue by several orders of magnitude, confounding the interpretation of mechanosensitive signaling pathways[7-9]. Likewise, animal models introduce species-specific variables and carry substantial ethical and financial burdens. To overcome these limitations, this project engineered a two-dimensional polyacrylamide (PA) hydrogel platform with independently tunable stiffness that recapitulates stiffnesses to replicate that of healthy and early-fibrotic tissue. Human dermal fibroblasts (HDFs), chosen for their robust mechanosensitivity and practical accessibility, serve here as a tractable surrogate for stromal cells [10-13]. We hypothesize that substrate stiffening alone can prime fibroblasts for myofibroblast transdifferentiation, thereby sensitizing them to canonical profibrotic cues such as transforming growth factor-β (TGF-β) [14]. Furthermore, we interrogate the mechanistic hierarchy of this response by disrupting actomyosin contractility, YAP/TEAD transcriptional activity, and actin polymerization. By integrating both biomaterial and cell biology studies, this study establishes a controllable microenvironment for dissecting the reciprocal interplay of mechanical and chemical stimuli in mechanosensitive fibroblast activation and lays groundwork for future stiffness-informed drug-screening strategies.

Materials and

Methods: : Polyacrylamide (PA) hydrogels were cast on glass coverslips with tunable acrylamide: bis-acrylamide ratios, UV-activated with sulfo-SANPAH, and coated with 0.1 mg mL-1 collagen I [15]. Oscillatory rheometry confirmed “soft” (1.5 kPa) and “stiff” (4.5 kPa) moduli within their linear viscoelastic range (2 % strain, 6 rad s-1). Human dermal fibroblasts (HDFs) were seeded at 10000 cells cm-2 in low-serum DMEM (1% FBS, 1% Pen-Strep). Three basal media were used: control, vehicle, and 5ng mL-1 TGF-β to induce myofibroblast transdifferentiation. Media were changed daily for five days. To probe mechanotransductive pathways, inhibition trials were performed where cultures received Verteporfin (0.5 µM, YAP/TEAD inhibitor), Y-27632 (1 µM, ROCK inhibitor), or Latrunculin A (10 nM, actin-depolymerizing agent), refreshed daily for four days.

Results, Conclusions, and Discussions:: Rheometry confirmed discrete elastic regimes where soft gels averaged 1.5kPa and stiff gels averaged 4.5kPa. On soft substrates, HDFs remained relatively quiescent, exhibiting few α-SMA stress fibers and low Collagen1A protein expression. When imaging cells cultured on the stiffer substrate, however, this increase in stiffness elicited a rise in both markers, indicating that substrate mechanics are sufficient to induce fibrotic protein expression. TGF-β further amplified stiffness-induced activation, revealing dense stress-fiber networks and abundant collagen deposition under stiff plus TGF-β conditions. qRT-PCR analysis was also used to confirm these findings, showing that stiff substrates, when combined with TGF-β, produce a significant fold increase in gene expression of ACTA2 and COL1A1.
After the materials for the model and cellular responses were confirmed, an inhibitor trial was conducted to study the mechanosensitive responses that were vital for fibroblast activation. ROCK inhibition (Y-27632) abrogated stiffness-dependent increases in both gene and protein expression, underscoring the necessity of actomyosin contractility. Verteporfin similarly suppressed activation across stiffness conditions, implicating YAP/TEAD signaling as a critical downstream effector of mechanical input. Latrunculin A lowered overall marker expression yet preserved a residual stiffness gradient, suggesting that while F-actin polymerization promotes activation, alternative cytoskeletal or membrane-based sensors may partly compensate when actin dynamics are compromised.
This project emphasizes ECM stiffening heightens fibroblast sensitivity to TGF-β, boosting ACTA2 and COL1A1 at protein and transcript levels. Results align with models where cytoskeletal tension and YAP/TAZ mechanotransduction dictate cell fate. Purely elastic PA gels and single-cue stimulation limit realism; future studies should employ viscoelastic matrices, multiple cytokines, and detailed real-time analyses.
Despite its utility, the model employs purely elastic PA gels that lack the viscoelastic dissipation characteristic of native liver ECM, potentially underestimating time-dependent mechanosignals. Additionally, only TGF-β was examined as a soluble activator; future studies should integrate cytokine cocktails and time-lapse analyses to capture activation kinetics. Incorporating viscoelastic or dynamically stiffening materials will further approximate in vivo conditions. Our findings highlight the primacy of matrix mechanics in fibrotic signaling and provide a tractable platform for screening mechano-targeted antifibrotic therapeutics.

Acknowledgements and/or References (Optional): : [1] Guo, Ting, et al. "Regulators, functions, and mechanotransduction pathways of matrix stiffness in hepatic disease." Frontiers in Physiology 14 (2023): 1098129.
[2] Saneyasu, Takaoki, Riaz Akhtar, and Takao Sakai. "Molecular cues guiding matrix stiffness in liver fibrosis." BioMed research international 2016.1 (2016): 2646212.
[3] Berumen, Jennifer, et al. "Liver fibrosis: Pathophysiology and clinical implications." WIREs mechanisms of disease 13.1 (2021): e1499.
[4] Acharya, Pragyan, et al. "Cellular mechanisms of liver fibrosis." Frontiers in pharmacology 12 (2021): 671640.
[5] Puche, Juan E., Yedidya Saiman, and Scott L. Friedman. "Hepatic stellate cells and liver fibrosis." Compr Physiol 3.4 (2013): 1473-1492.
[6] Zamani, Mohammad, et al. "Global prevalence of advanced liver fibrosis and cirrhosis in the general population: a systematic review and meta-analysis." Clinical Gastroenterology and Hepatology (2024).
[7] Karsdal, Morten A., et al. "Novel insights into the function and dynamics of extracellular matrix in liver fibrosis." American Journal of Physiology-Gastrointestinal and Liver Physiology 308.10 (2015): G807-G830.
[8] Kaur, Savneet, et al. "In vitro models for the study of liver biology and diseases: advances and limitations." Cellular and molecular gastroenterology and hepatology 15.3 (2023): 559-571.
[9] Feng, Jiantao, et al. "Substrate stiffness influences the outcome of antitumor drug screening in vitro." Clinical hemorheology and microcirculation 55.1 (2013): 121-131.
[10] Martino, Fabiana, et al. "Cellular mechanotransduction: from tension to function." Frontiers in physiology 9 (2018): 824.
[11] Kang, Ningling. "Mechanotransduction in liver diseases." Seminars in liver disease. Vol. 40. No. 01. Thieme Medical Publishers, 2020.
[12] Lysy, Philippe A., et al. "Human skin fibroblasts: From mesodermal to hepatocyte‐like differentiation." Hepatology 46.5 (2007): 1574-1585.
[13] Tai, Chenxu, et al. "Human skin dermis-derived fibroblasts are a kind of functional mesenchymal stromal cells: judgements from surface markers, biological characteristics, to therapeutic efficacy." Cell & bioscience 12.1 (2022): 105.
[14] Meindl‐Beinker, Nadja M., and Steven Dooley. "Transforming growth factor‐β and hepatocyte transdifferentiation in liver fibrogenesis." Journal of gastroenterology and hepatology 23 (2008): S122-S127.
[15] Tse, Justin R., and Adam J. Engler. “Preparation of hydrogel substrates with tunable mechanical properties.” Current Protocols in Cell Biology, vol. 47, no. 1, June 2010, https://doi.org/10.1002/0471143030.cb1016s47.