(546c) Mechanically Dynamic Silk Protein-Based Hydrogels for Studying Fibrosis

Fibrosis is a ubiquitous disease implicated in approximately 45% of deaths worldwide. The disease is marked by exaggerated wound healing in response to injury with an accumulation of extracellular matrix (ECM) by activated myofibroblasts and an amplified recruitment of immune cells. This fibrotic ECM alters biochemical signatures of diseased tissues and results in mechanical stiffening. These factors stimulate wound healing responses perpetuating a feedback loop that ultimately results in the loss of tissue function and organ failure. Here, we use silk protein-based hydrogels to develop biomaterials that exhibit time-dependent stiffening at: rapid (hours), moderate (days), and slow (weeks) rates. Dynamic mechanical changes were established by chemically modifying silk to alter hydrophobicity which alters the propensity for β-sheet (crystal) formation in the (GAGA)-rich regions of the protein. The gradual formation of these nanoscale β-sheet structures results in the formation of physically crosslinked networks increasing the stiffness of the material. Carboxylic acid residues along the silk backbone were reacted via carbodiimide coupling of primary amines with tyramine groups (Figure 1A). This changed the hydrophobicity of the protein and accelerated network formation by increasing phenol motifs which undergo enzymatic crosslinking in the presence of horseradish peroxidase (HRP) and hydrogen peroxide. Varying the tyramine-modified silk (SF-TA) composition provided control over stiffening rates, which initially represent healthy tissue (1-10 kPa) and stiffen to a compressive modulus of fibrotic tissue (>50 kPa). Hydrogels composed of 100% SF-TA exhibited a 30-fold increase in stiffness from ~3-100 kPa over a week, whereas hydrogels composed of 75% SF-TA/25% native silk achieved a similar outcome over two weeks (Figure 1B i). The mechanism of stiffening was attributed to the formation of β-sheet secondary structures by correlating compressive modulus with β-sheet content calculated through deconvolution of FTIR spectra (Figure 1B ii-iii). The materials supported pulmonary fibroblasts viability and induced increased synoviocyte, a fibroblast-like phenotype of the synovium, activation through the hydrogel stiffening process; increased expression of ⍺-smooth muscle actin (⍺SMA) (Figure 1C). The biological and mechanical impact of incorporating other biomolecules into the system, such as hyaluronic acid and collagen is hypothesized to provide further control over the mechanical profiles and the bioactivity due to hydrophilicity and chemistry. This preliminary work is an important step towards developing in vitro tissue models capable of recapitulating tissue dynamics during disease and tissue development.