Investigation of photostimulation effect on wound healing in in-vitro diabetic ulcer model

This study was supported by Hacettepe University Scientific Research Projects Coordination Unit (BAP) within the scope of the PhD Thesis Project (FDK-2019-18072) titled "Examination of the Effect of Photostimulation on Wound Healing in an In Vitro Diabetic Ulcer Model " and the comprehensive research project (FBA-2021-19013) titled " Investigation of the Effect of Photoactivated Platelet-Rich Plasma (PRP) on Wound Healing". The aim of the presented doctoral thesis is to develop an effective treatment method for chronic wound healing in which PRP (Platelet-Rich Plasma) and photostimulation are used together. PRP contains many growth factors that stimulate cell proliferation, migration and differentiation. Photostimulation, on the other hand, is applied with a polychromatic light source that presents rays with a wavelength of 600-1200 nm, both activating platelets and stimulating the cellular activities of fibroblasts. In the first stage of the study, chitosan/gelatin scaffolds with different volumetric ratios (1:1, 1:3 and 3:1) were produced by freeze-drying method. The scaffolds produced were crosslinked with genipin (GP) or EDC/NHS, followed by neutralization/stabilization with ethanol or sodium hydroxide. During the characterization phase of the scaffolds, it was determined by SEM analysis that the scaffolds exhibited a porous structure with internal connections, and it was found that the pore diameters were approximately 125 μm and the porosity was around 90%. The presence of polymers and crosslinking agents in scaffolds was proven by ATR-FTIR analysis. In the culture study carried out with the human dermal fibroblast cell (BJ, CRL-2522) line, the CHT/GEL3:1 scaffold cross-linked with genipin was found to be the most suitable in terms of cellular activity and it was decided to use this scaffold in the experiments within the scope of the thesis. After the approval of Hacettepe University Non-Interventional Clinical Research Ethics Committee dated 05.05.2020 and decision number GO 20/372, the isolation of platelet-rich plasma (PRP) with blood taken from volunteers was carried out according to the kit protocol. Tissue scaffolds (CHT/GEL3:1) were loaded with PRP, then fibroblast cells were seeded and both PRP and BJ cells were activated by applying photostimulation (PAC+) under the determined optimum conditions. Considering the results of cell culture studies, the efficiency of cell migration has been demonstrated in the model created under in vitro conditions. In the last step of the study, 2 and 3 dimensional in vitro diabetic wound (ulcer) models were created with BJ fibroblasts exposed to hyperglycemic ambient conditions, and the migration of cells from the wound area was investigated in the presence and absence of light. With F-actin/DAPI staining and SEM analysis, it was determined that cell migration occurred to cell-free regions and that cells continued their metabolic activities in the presence of light in a hyperglycemic environment. The results of the study carried out within the scope of the thesis showed that tissue scaffolds containing autologous PRP were successfully activated by photo-biostimulation without the use of any chemical agent, and the cellular activity in the environment accelerated with the effect of long-term beneficial agents released from PRP and light. Experimental findings are clinically important for the treatment of chronic wounds after in-vivo experiments.