Probing Fibroblast Activation in Response to Extracellular Cues with Whole Protein- or Peptide-Functionalized Step-Growth Hydrogels

Abstract

Synthetic hydrogels with well-defined mechanical properties have become invaluable tools for probing cell response to extracellular cues including matrix stiffness and integrin binding. These synthetic matrices are often decorated with either proteins or integrin-binding peptides to promote cell adhesion and to direct or probe cell behavior. For example, both collagen I-functionalized polyacrylamide and peptide-functionalized poly(ethylene glycol) hydrogels have been instrumental in elucidating the role of the elasticity or 'stiffness' of the matrix in promoting fibroblast activation in wound healing and fibrosis. However, the two methods of promoting integrin binding are not often directly compared in the same system, partly owing to differences in material designs, despite the potential differences in the way cells interact with whole proteins and protein mimetic peptides. We hypothesized that such a comparison could provide insight into the ways integrin binding affects fibroblast activation within commonly utilized in vitro cell culture models, and more broadly, to inform the design of materials to modulate fibroblast activation in studies of wound healing and disease. To enable this comparison, we developed a method to conjugate whole proteins to step-growth poly(ethylene glycol) (PEG) hydrogels and investigated fibroblast response to protein-peptide pairs: fibronectin and PHSRN(G) 10 RGDS or collagen I and (POG) 3 POGFOGER(POG) 4 , which are important in matrix remodeling and relevant to fibroblast activation. With this approach, we observed that human pulmonary fibroblasts adopted a similar morphology on fibronectin and PHSRN(G) 10 RGDS, although with a slight increase in the percentage of alpha smooth muscle actin (αSMA) expressing cells on PHSRN(G) 10 RGDS. Interestingly, we observed that fibroblasts formed activated clusters on the collagen mimic (POG) 3 POGFOGER(POG) 4 while exhibiting less activation on collagen I. This cell activation and clustering is reminiscent of fibroblast foci that are observed in lung fibrosis, suggesting the relevance of these well-defined polymer-peptide hydrogels for investigating fibrosis and decoupling biochemical and biophysical cues.

Emerging Biomimetic Materials for Studying Tumor and Immune Cell Behavior

“…90 PEG, when modified with methacrylate and dimethacrylate, is photocrosslinkable under UV light and can easily be conjugated with active protein sites for cell adhesion to better evaluate cell morphology and production of growth factors. 55,117 This material has been used to fabricate biologically active microenvironments by conjugating PEG to different protein binding sites such as RGD, IKVAV, and GFOGER to mimic the cellular binding sites in the fibrous structure of the ECM. 82,115,141 Gill and coworkers used PEG conjugated with RGDS (PEG-RGDS) for cell adhesion and GGGPQGIWGQGK (PQ) for degradation by matrix metalloproteinases.…”

Self-Healing Boronic Acid-Based Hydrogels for 3D Co-cultures

“…We next examined if the mechanism of hydrogel formation was permissive to the encapsulation of human cells. To promote cell adhesion, fibronectin was included within the pregelation solution, where we hypothesized that the boronic acid moiety may interact with free alcohol groups presented by fibronectin for retention of the protein within the hydrogel. , A fibronectin concentration of 300 nM was selected, which is in the range of concentrations reported for encapsulation in synthetic hydrogels to promote cell adhesion (typically, 100–300 nM). − To examine fibronectin retention over time, we encapsulated rhodamine-labeled fibronectin along with human lung fibroblasts (CCL-151, 5000 cells/μL) during hydrogel formation and used confocal microscopy to measure fluorescence within the hydrogels. Fibronectin was observed throughout the hydrogel over the course of 7 days, where over 50% of the fibronectin originally encapsulated was estimated to be retained at day 7 (Figure S2).…”

Tumor microenvironment in non-melanoma skin cancer resistance to photodynamic therapy

“…Nevertheless, CAF populations have turned out to be heterogeneous, coexisting within the same tumor (such as inflammatory or myofibroblastic phenotypes) (16,17). Because of this diversity, recent investigations have focused their efforts to study the broad amount of cytokines implicated in these changes (such as transforming growth factor beta (TGFb), Interleukin-1 (IL-1) or integrins), as they are not only morphological, but also functional (18)(19)(20)(21).…”