Development of a lactoferrin and lysozyme-containing pluronic-based hydrogel for wound healing

The wound healing process consists of three overlapping phases: Inflammation, proliferation and remodeling. Although these stages generally occur spontaneously following an injury, some malfunctioning mechanisms in this process may sometimes cause problems like non-healing wounds or extensive scar formation and the process needs to be supported by a drug formulation in order to accelerate healing. Therefore, hydrogel formulations are of great interest as they may help regulate the mechanisms laying behind the wound healing phases and make great carriers for bioactive molecules, as well as promoting or mimicking extracellular matrix, while providing an antimicrobial environment to prevent infections. It was aimed in this study to develop such a hydrogel formulation made up of Pluronic® F127 and hyaluronic acid as the carrier, containing lactoferrin from human milk and lysozyme from chicken egg white as the bioactive molecules. In order to reveal the combinatorial effects of these two proteins on fibroblast proliferation and migration in vitro, MTT, migration and scratch wound healing assays were performed on NIH/3T3 mouse fibroblast cells treated with lactoferrin and/or lysozyme. Moreover, in vivo wound healing activity of the generated hydrogels containing lactoferrin and/or lysozyme were also tested on cutaneous skin wounds in rats. According to the results obtained from the MTT assay, the best concentrations of lactoferrin and lysozyme were determined and they were used in migration assays. As a result, it was shown that lactoferrin and lysozyme can be used and combined for inducing fibroblast migration. On the other hand, in vivo wound healing results revealed that even though lactoferrin and lysozyme can be formulated in a poloxamer-based hydrogel for wound healing, lactoferrin and/or lysozyme-containing hydrogels showed no significant difference in terms of wound contraction when compared to the blank hydrogel. However, histopathological examination unveiled that the hydrogels containing lactoferrin and lysozyme lead to better wound healing than the hydrogel itself. So, it was concluded that although the concentrations of lactoferrin and lysozyme in question can be used in wound healing hydrogels, more characterization and further testing in the concentrations may end up with a hydrogel with a better healing effect.