Polysucrose hydrogel loaded with natural molecules/extracts for multiphase-directed sustainable wound healing

Natural molecules/extracts have numerous beneficial effects on wound healing processes which are challenged by appropriate use and non-toxic dosage. Polysucrose-based (PSucMA) hydrogels have been synthesized with in situ loading of one or more natural molecules/extracts namely Manuka honey (MH), Eucalyptus honey (EH1, EH2), Ginkgo biloba (GK), thymol (THY) and metformin (MET). EH1 presented low amounts of hydroxymethylfurfural and methylglyoxal compared to MH indicating that EH1 was not temperature-abused. It also showed high diastase activity and conductivity. GK was added to PSucMA solution along with other additives including MH, EH1 and MET and crosslinked to form dual loaded hydrogels. The in vitro release profiles of EH1, MH, GK and THY from the hydrogels followed the exponential Korsmeyer-Peppas equation, with a release exponent value of less than 0.5 indicating a quasi-Fickian diffusion mechanism. The IC₅₀ values of these natural products using L929 fibroblasts and RAW 264.7 macrophages indicated that EH1, MH and GK were cytocompatible at relatively high concentrations compared to MET, THY and curcumin used as a control. MH and EH1 induced high IL6 concentration compared to GK. In vitro studies were modelled to mimic the overlapping wound healing phases using human dermal fibroblasts (HDFs), macrophages and human umbilical endothelial cells (HUVECs) in dual culture. HDFs showed a highly interconnected cellular network on GK loaded scaffolds. EH1 loaded scaffolds were seen to induce formation of spheroids which increased in number and size in co-culture studies. The SEM images of HDF/HUVEC seeded GK, GKMH and GKEH1 loaded hydrogels indicated formation of vacuoles and lumen structures. These results indicated that a combination of GK and EH1 in the hydrogel scaffold would accelerate tissue regeneration by acting on the four overlapping phases of wound healing.