Incorporation of Lignin in Natural and Synthetic Biomaterials to Alter Mechanical and Biochemical Properties for Enhanced Wound Healing

It is estimated that chronic, non-healing wounds affect more than 6.5 million Americans annually, with an estimated healthcare cost beyond $14 billion. Here, we attempted to create composites of natural (collagen type I or gelatin-methacrylate) or synthetic (poly(ethylene glycol) polymers incorporating a natural plant component, lignin, to combat the costs and limitations current wound healing methods face. Three-dimensional matrices of collagen type I (Col I) are widely used in tissue engineering applications for its abundance in many tissues, bioactivity with many cell types, and excellent biocompatibility. Inspired by the structural role of lignin in plant tissue, we found that sodium lignosulfonate (SLS) and an alkali-extracted lignin from switchgrass (SG) increased the stiffness of Col I gels from 52 to 670 Pa and 52 to 320 Pa, respectively, and attenuated shear thinning properties, with the formulation of 1.8 mg/mL Col I and 5.0 mg/mL SLS or SG. In 2D cultures, no cytotoxicity of collagen-SLS to adipose-derived stromal cells was observed and viability was maintained over 7 days. Inspired by thiol-ene chemistry, we hypothesized that thiol functionalization of lignin would allow for chemoselective addition to typical biomaterials, such as gelatin-methacrylate (GelMA) and poly(ethylene glycol (PEG). Addition of TLS, to either biomaterial, showed no significant changes in their viscoelastic properties with the addition of TLS significantly increasing the antioxidant % for both PEG, which contained native antioxidant capacity, and GelMA, essentially of zero antioxidant capacity in its native form. Incorporation of fish GelMA, into PEG-TLS composites, enhanced cell adhesion for HS and LS human dermal fibroblasts (hdFBs) and showed a significant reduction in COL1A1, ACTA2, TGFB1, HIF1A, and HSP27 for HS when compared to LS. GelMA-TLS composites seeded with HS and LS hdFBs showed similar results for COL1A1 and ACTA2 with overexpression leading to fibrosis and reduced wound healing. Additionally, we determined Hsp27 is a critical marker in excess fibrosis, via a fibrosis array, and both gene and protein expression were significantly attenuated when seeded onto GelMA-TLS composites, compared to controls. The work presented here valorizes two waste by-products, lignin and fish gelatin engineered into composites suited for enhanced wound healing applications