The effects of umbilical cord mesenchymal stem cell exosomes and hyperglycemic environment on an In Vitro wound healing model created with human keratinocytes

Both conventional and regenerative treatment modalities are preferred for the treatment of acute and chronic wounds. Compared to acute wounds, the chronic wound treatment process is longer and more challenging, opening the door to regenerative approaches. Diabetic wounds, characterized by impaired healing, pose a challenge in the clinical treatment phase due to their chronic nature and complexity. Regenerative treatment methods, which initially included the application of living cells, have been developed over time and have reached the level of 'cell-free treatment' where only cellular agents are used today. Exosomes, a type of extracellular vesicles secreted by cells, carry the main paracrine properties of the cell from which they originate and have the ability to trigger physiological mechanisms involved in wound healing such as migration and proliferation thanks to their rich cellular material content. In our study, the wound healing effects of human umbilical cord-derived mesenchymal stem cell exosomes (hUC-MSC-Exo) on the scratch wound model created with human keratinocyte cell line (HaCaT) cultured in hyperglycemic and normoglycemic environments were examined in vitro. Increased cellular viability rate in both normoglycemic and hyperglycemic groups mimicking diabetic environment as a result of hUC-MSC-Exo application to HaCaT wound model; migration and proliferation events, thrombospondin 1 (TSP-1), BPAG1e (BP230) related dystonin, interleukin 8 (IL-8) and hydroxyproline (Hyp) levels involved in physiological mechanisms associated with impaired wound healing were analyzed by ELISA. Malondialdehyde and glutathione levels, which indicate the oxidant/antioxidant ratio, were analyzed biochemically. It was determined that hUC-MSC-Exo administration accelerated in vitro wound healing in normoglycemic and hyperglycemic environments in a time-dose dependent manner. 25 million particles/mL and 50 million particles/mL hUC-MSC-Exo concentrations increased metabolic activity in cells, metabolic activity was not affected when these concentrations were exceeded, and 25M p/ml hUC-MSC-Exo dose was found to be the most effective/ideal dose in normoglycemic and hyperglycemic environments in a time-dependent manner. In both normoglycemic and hyperglycemic groups, hUC-MSC-Exo administration increased keratinocyte migration, wound closure rate, BP230, TSP-1 and Hyp levels compared to control groups. This increase was attributed to hUC-MSC exosomes stimulating various cellular signaling pathways and growth factor synthesis in keratinocytes, as well as through the metabolic regulators it contains. In our study, it was also determined that hUC-MSCs triggered the inflammatory phase of wound healing by stimulating IL-8 synthesis from keratinocytes in the acute phase and contributed to the proliferative phase of wound healing by decreasing it in the chronic phase in both normoglycemic and hyperglycemic groups. In this study, we tried to explain the role of molecules involved in the pathophysiological mechanisms associated with impaired wound healing in diabetes with hUC-MSC-Exo treatment. Keywords: Exosome, Hyperglycemic environment, Wound healing, Diabetes, Keratinocyte