(B-46) Injectable Lignin Composites with Sustained Oxygenation and Antioxidant Properties Improve Neovascularization and Healing of Diabetic Wounds.

Introduction:: Impaired neovascularization is one of the main causes of the diabetic phenotype of delayed wound healing. Engineered biomaterials with multiple wound healing-promotive functions, including pro-angiogenesis, sustained oxygenation and ROS (reactive oxygen species)–scavenging can promote wound healing. We engineered novel lignin (a natural antioxidant from lignocellulose)-based composites with ROS-scavenging and oxygen-releasing properties and hypothesized that they enhance neovascularization and attenuate inflammation and fibrosis to promote diabetic wound healing.


Materials and Methods:: The Weighted Gene Co-expression Network Analysis (WGCNA) package in R was used to perform correlation network analysis on gene sets from a fibrosis PCR array data collected from human dermal fibroblasts cultured on lignin composites. Injectable lignin composites¹ were prepared and four treatment groups were tested: untreated (UNTX), antioxidant (TLS), antioxidant with O₂ generation (CPO) and its control (CPOc). Full thickness excisional 6 mm wounds were made in db/db (type II diabetic; 8- to 10-week-old, female and male) mice and wounds were harvested at d7 and 14 post wounding, and examined for epithelial gap, granulation tissue, endothelial cells and vessels (CD31), VEGF and HIF1-α, leucocyte (Ly6g, CD45) and macrophage (F4/80, CD206).


Results, Conclusions, and Discussions:: ClueGO functional enrichment analysis revealed thyroid stimulating hormone as a key hub gene. Since thyroid hormone receptors regulates TGF-β signaling, specifically, the binding of thyroid hormone triiodothyronine (T3) through its nuclear receptors regulate the TGF-β/SMAD pathway, our findings indicate a role for lignin composites in governing TGF-β signaling to attenuate fibroblast fibrotic responses². In vivo, in full thickness db/db skin wounds, CPO lignin composites promoted granulation tissue deposition and CD31⁺ capillary lumen density at d7. While histological staining for VEGF showed reduced VEGF expression in the hyperproliferative leading epidermis of CPO-treated wounds at d7, quantification of VEGF expression in the homogenized wound tissue using immunoblotting with ProteinSimple showed an increase in the VEGF expression at d7, suggesting dermal angiogenesis is promoted by CPO lignin composites in diabetic wounds. Interestingly, decreased HIF-1a expression was noted in the leading wound epithelium, with reduced expression in wound homogenates in CPO wounds. CPO lignin composite-treated wounds had reduced macrophage infiltration. To determine the effect of the lignin composite treatment on remodeling in db/db wounds, we tested the effect of treatment at d14 post wounding. We noted visibly improved healing in CPO wounds from gross images, which was supported by the presence of a robust granulating wound bed in representative hematoxylin and eosin stained wounds sections, along with an increase in the CD31⁺ lumens. Conclusions: Our data showed that the dual function of antioxidation and oxygen production capacity of the lignin composites improved wound healing associated with enhanced neovascularization, representing new frontiers in improving diabetic wound healing by engineered biomaterials.




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References (Optional): : 1. Balaji et al. ACS Appl Mater Interface (2023). 2. Alonso-Merino et al. PNAS (2016)