T. J. Prajapati1, A. Kaul1, B. W. Padon1, 3, F. Faruk1, 3, W. D. Short1, 3, O. Olutoye1, 3, L. Yu1, 3, H. Li1, 3, O. Jung2, S. Jimenez2, J. Jung2, S. Balaji1, 3 1Baylor College Of Medicine, Pediatric General Surgery, Houston, TX, USA 2Louisiana State University, Biological Engineering, Baton Rouge, LA, USA 3Texas Children’s Hospital, Pediatric General Surgery, Houston, TX, USA
Introduction:
Impaired wound healing and scar formation have far-reaching socioeconomic effects. Excessive reactive oxygen species (ROS) cause oxidative stress in the wounds and delay healing by hampering vascularization and promoting inflammatory macrophage and myofibroblast differentiation resulting in increased inflammation and scarring. Engineered biomaterials capable of scavenging ROS and facilitating controlled release of oxygen can circumvent the challenge of oxygen diffusion in situ to promote cell infiltration and survival, which is not achieved with current biomaterials. We hypothesize that the application of novel lignin (an antioxidant from lignocellulose)-based composites with ROS-scavenging and oxygen-releasing properties will enhance neovascularization and attenuate inflammation to promote wound healing.
Methods:
We photo-crosslinked thiolated lignosulfonate (TLS) in methacrylated gelatin (GelMA) via thiol-ene chemoselective ligation. We developed calcium peroxide (CPO)-incorporated lignosulfonate/poly(lactic-co-glycolic acid) microparticles, where the dissociation of CPO into oxygen is facilitated by lignosulfonate, and added CPO particles to the GelMA-TLS composites and tested its antioxidant capacity (DPPH assay) and oxygen release (pseudo 1st order model). Full thickness 6mm wounds were made in C57BL/6N mice using silicone stents to control for contraction and divided into 4 groups: Untreated (UNTX), GelMA-TLS (TLS), GelMA-TLS with carriers but no oxygen release capacity (CPOc), and GelMA-TLS with carriers+oxygen release (CPO). Wounds were harvested at 7d and examined for epithelial gap, granulation tissue (H&E), myofibroblasts (αSMA), endothelial cells and vessels (CD31), and macrophages (CD206). Data presented as mean+-SD, n=3-6 wounds/group; p-value by ANOVA.
Results:
Morphometric wound analysis showed small differences in epithelial gap; however, treated wounds had greater granulating tissue area with marked increase in CPO (UNTX 1.9+-0.9 mm2 vs TLS 1.9+-0.8 vs CPOc 1.7+-0.4 vs CPO 1.5+-0.4, p<0.05). Notably, the CPO matrix was completely infiltrated and fully integrated with a granulating wound bed, unlike other groups which showed areas of non-infiltrated gel. CPO wounds had greater concentrations of αSMA in the middle of wound beds (UNTX 16.6+-8.7 % vs TLS 10.5+-8.7 vs CPOc 8.8+-2.4 vs CPO 17.5+-9.7, p=ns). Both CPOc and CPO wounds had greater vessel density (UNTX 14.0+4.7 lumens/40x field vs TLS 18.9+-3.9 vs CPOc 27.9+-7.2 vs CPO 25.2+-7.6, p<0.05). The CPOc and CPO wounds had fewer macrophages (UNTX 32.7+-12.4 % cells/40 field vs TLS 24.9+-14.3 vs CPOc 14.6+-4.4 vs CPO 19.3+-5.5, p<0.05).
Conclusion:
Our data demonstrates that the synergistic antioxidation and oxygen production capacity of lignin composites improved wound healing associated with reduced inflammation and enhanced neovascularization, representing new potential therapeutics for attenuating fibrosis and improving wound healing with engineered biomaterials.