Effect of substrate stiffness on pulmonary fibroblast activation by TGF-β

Peptide crosslinkers containing the sequence C-X-CG (X represents various adhesive peptides) were incorporated into poly(ethylene glycol) (PEG) hydrogel networks with different mechanical properties. Pulmonary fibroblasts (PFs) exhibit increased adhesion to rigid hydrogels modified with X = RGDS, DGEA and IKVAV (0.5 and/or 5 mM) compared with a scrambled control (X = HRPNS). PFs exhibit increased adhesion to softer hydrogels when X = DGEA at low (0.5 mM) peptide concentration. PFs seeded onto hydrogels modified with X = RGDS produce alpha-smooth muscle actin (α-SMA), a myofibroblast marker, and form an extensive cytoskeleton with focal adhesions. Decreasing substrate stiffness (achieved through hydrolytic degradation) results in down-regulation of α-SMA expression by PFs. Substrate stiffness increases the sensitivity of PFs to exogenously applied transforming growth factor beta (TGF-β1); PFs on the most rigid gels (E = 900 kPa) express α-SMA when treated with low concentrations of TGF-β1 (1 ng ml−1), while those on less rigid gels (E = 20-60 kPa) do not. These results demonstrate the importance of both mechanical and chemical cues in studying pulmonary fibroblast activation, and establish PEG hydrogels as a viable material for further study of IPF etiology.