Fotostimülasyonun 2 ve 3 Boyutlu in vitro Hiperglisemik Modellerde İnsan Dermal Fibroblast, Keratinosit ve Endotel Hücreleri Üzerindeki Etkinliğinin İncelenmesi

Abstract

This study is supported by the Scientific Research Projects Coordination Unit (BAP) of Hacettepe University as part of the comprehensive research project titled "Investigation of the Effectiveness of Photostimulation in 2-Dimensional and 3-Dimensional in vitro Diabetic Ulcer Models" (FBA-2022-20162). In the present study aims to investigate the biological effects of photostimulation on human dermal fibroblasts, keratinocytes, and endothelial cells in 2D and 3D in vitro hyperglycemic models that mimic diabetic conditions. Chronic wounds commonly encountered in diabetic patients are associated with many factors, particularly reduced cellular proliferation and migration due to hyperglycemia, inadequate angiogenesis, re-epithelialization, and increased oxidative stress. In this context, the aim is to investigate the effects of photostimulation on cellular and molecular processes in wound healingthrough in vitro studies and to reveal its potential use in the treatment of diabetic wounds, while also developing in vitro hyperglycemic models where treatment approaches can be tested. Human dermal fibroblast (HDF), keratinocyte (HS2), and endothelial cells (HUVEC) were cultured initially for in vitro cell culture study. A hyperglycemic condition has been determined by examining the proliferation and morphology characteristics of cells in using different glucose concentrations. The effects of photostimulation applied under established hyperglycemic conditions and normal conditions on cell morphology, proliferation, ECM synthesis, migration, and angiogenesis have been investigated using various analytical methods. Cell proliferation was evaluated comparatively through MTT analysis, cell migration through scratch analysis, ECM synthesis through immunocytochemical analyses, and angiogenesis through tube formation and gene expression analyses. Later, a 3-dimensional hyperglycemia model was created by producing a tissue scaffold of polycaprolactone (PCL) / chitosan-gelatin using fibroblast and keratinocyte cells. In the created model, photostimulation was applied using a plasma arc light source that provides polychromatic light in the near-infrared (NIR) wavelength range of 600–1200 nm to investigate the effect of photostimulation on wound healing. The effect of this application on cell viability, morphology, and ECM density has been examined using SEM and MTT analyses. The analyses conducted in the study revealed that photostimulation supports cellular proliferation, migration, ECM synthesis, and the formation of tubules, which play a critical role in the process of angiogenesis, in both 2D and 3D models. Furthermore, considering the biological responses of the cells, keratinocytes have been identified as the most responsive cells to photostimulation, while endothelial cells are the most affected by hyperglycemic conditions. These results suggest that photostimulation could be used as a potential treatment method in the healing of diabetic wounds. Nonetheless, it is assumed that further refinement of this 3-dimensional hyperglycemic model through additional analyses will represent a crucial preliminary phase prior to advancing to in vivo and clinical investigations.