Sustained ROS Scavenging and Pericellular Oxygenation by Lignin Composites Rescue HIF-1α and VEGF Levels to Improve Diabetic Wound Neovascularization and Healing

Introduction: Impaired neovascularization is one of the main causes of the diabetic phenotype of delayed wound healing. Engineered biomaterials that can alleviate multiple wound healing impediments in the diabetic environment, i.e. pericellular ischemia and excessive reactive oxygen species (ROS), can promote diabetic wound healing. We engineered novel lignin (a natural antioxidant from lignocellulose)-based composites with ROS-scavenging and sustained oxygen-releasing properties and hypothesized that they enhance neovascularization and attenuate inflammation to promote diabetic wound healing.

Materials and

Methods: We investigated the effect of injectable lignin composites1 on accelerating angiogenesis in oxidative stress conditions (±high glucose). Test groups: 1) untreated control (UNTX), 2) gelatin methacrylate only (GelMA), 3) GelMA+thiolated lignosulfonate-antioxidant nanoparticles (TLS), 4) GelMA+TLS+calcium peroxide based-O2 generating nanoparticles (CPO) and CPOc (CPO control without O2 generation)). After 96 h of culturing MVECs (microvascular endothelial cells), micrographs were captured and analyzed using Angiogenesis Analyzer2. RNA was extracted (PureLink RNA Mini kit) and bulk RNA sequencing was conducted on Illumina NextSeq2000. The Weighted Gene Co-expression Network Analysis (WGCNA) package in R was used to perform correlation network analysis on gene sets from MVECs cultured on lignin composites. qRT-PCR and ELISA were conducted to analyze HIF-1α and VEGF expression. 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 7 day (d7) post wounding, and examined for epithelial gap, granulation tissue, endothelial cells and vessels (CD31), VEGF and HIF-1α, leucocytes (CD45) and macrophages (F4/80, CD206).

Results, Conclusions, and Discussions: MVECs cultured on CPO composites with antioxidant and O2 release showed increased capillary network formation and angiogenic phenotype under high glucose condition (Figure 1). The co-expression profiles of associated genes were further analyzed using unsupervised learning, revealing distinct patterns (Figure 2A and 2B). From these modules, the turquoise module containing 5,073 genes was selected for further investigation. As demonstrated in Figure 2C, CPO composites facilitated the expression of Hif-1α (hypoxia-inducible factor 1α) in the pool of top 25 hub genes. In enrichment analysis, Vegf (vascular endothelial growth factor) associated pathways were significantly upregulated (Figure 2D and E). The results of ELISA revealed that CPO composites rescued the HIF-1α and VEGF levels of MVEC under high glucose conditions to normal levels (Figure 3). In full thickness db/db skin wounds, CPO lignin composites promoted granulation tissue deposition and CD31+ capillary lumen density at d7 (Figure 4A and 4D). Quantification in CPO treated wounds at d7 showed an increase in the VEGF expression (Figure 4B and 4E), suggesting dermal angiogenesis is promoted by CPO lignin composites in diabetic wounds. Interestingly, a decrease in Hif-1α expression was observed in both the leading wound epithelium and wound homogenates from the wound treated with CPO lignin composites (Figure 4C and 4F). Our data showed that the dual function of antioxidation and oxygen production capacity of the CPO lignin composites improved wound healing associated with enhanced neovascularization, representing new frontiers in improving diabetic wound healing by engineered biomaterials.

Acknowledgements (Optional): This research was funded in part by NIH/NIGMS R01 GM141366-01A1 (SB) and NSF EPSCoR (JPJ) and CAREER award (JPJ).