Journal: bioRxiv

Article Title: Progressive matrix stiffening of tyramine-modified silk fibroin hydrogels governs stage-specific pulmonary fibroblast activation

doi: 10.64898/2026.06.01.729382

Figure Lengend Snippet: A) Hydrogels were composed of either 0% or 50% mixture of tyramine-modified silk and were exposed to media without or with 5 ng/mL TGFβ. After hydrogels were cast, 25,000 NHLF cells were seeded on the surface. B) SEM imaging of the surface of the silk hydrogels incubated without cells for Day 0 and Day 14. C) Live/Dead imaging of Calcein AM for live cells (green) and Ethidium Homodimer-1 for dead cells (red) indicated cytocompatibility. D) Alamar Blue Assay for metabolic activity indicated that 0% gels showed an increase in activity over 14 days, while 50% gels plateaued from day 7 to 14. E) Mammalian β-Galactosidase Assay for senescence revealed no change for any timepoints tested over 14 days. N=3-4, Error bars = SEM.TCP = Tissue Culture Plate, T-= without TGFβ, T+ = 5 ng/mL of TGFβ. Two-way ANOVA, Tukey’s: ns = not significant p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Normal human lung fibroblasts (NHLFs, PCS-201-013, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium, high glucose (DMEM, ThermoFisher) supplemented with 10% Gibco Fetal Bovine Serum, Premium (FBS, ThermoFisher), 1% Gibco non-essential amino acids (ThermoFisher), and 1% Gibco antibiotic–antimycotic (ThermoFisher).

Techniques: Modification, Imaging, Incubation, Alamar Blue Assay, Activity Assay

Journal: bioRxiv

Article Title: Progressive matrix stiffening of tyramine-modified silk fibroin hydrogels governs stage-specific pulmonary fibroblast activation

doi: 10.64898/2026.06.01.729382

Figure Lengend Snippet: Top row: NHLFs on 0% SF-TA hydrogels at Day 2, Day 7, and Day 14, exhibiting a proliferative, inflammatory phenotype consistent with early fibroblast activation, characterized by secretion of IL-6, IL-8, and MCP-1 (dots) and progressive collagen deposition (fibers). Bottom row: NHLFs on 50% SF-TA hydrogels at Day 2, Day 7, and Day 14, exhibiting metabolic plateau and lower inflammatory cytokine secretion, gradual collagen secretion, with TGFβ-dependent myofibroblast activation. Together, these two substrate conditions recapitulate distinct stages of the fibrotic disease continuum.

Article Snippet: Normal human lung fibroblasts (NHLFs, PCS-201-013, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium, high glucose (DMEM, ThermoFisher) supplemented with 10% Gibco Fetal Bovine Serum, Premium (FBS, ThermoFisher), 1% Gibco non-essential amino acids (ThermoFisher), and 1% Gibco antibiotic–antimycotic (ThermoFisher).

Techniques: Activation Assay