Examination of the potential of different cell types to form cell sheet in the presence of ascorbic acid and photobiomodulation

This study was funded by Hacettepe University Scientific Research Projects Coordination Unit (BAP) graduate project entitled "Examination of the potential of different cell types to form cell sheet in the presence of ascorbic acid and photobiomodulation" (FYL-2020 18707). In this study, it is aimed to investigate the formation of cell sheets in the presence of ascorbic acid and polychromatic light (600-1200 nm) with cells in different morphologies. For this purpose, three types of cells have been selected, human umbilical cord-derived mesenchymal stem cells (HUC-MSCs), human dermal fibroblast cells (BJ) and human keratinocyte epithelial cells (HS2). In the first part of the study, characterization studies of HUC-MSCs were performed. Then, in order to obtain a cell sheets from these cells, 20 µg/mL vitamin C (VC) was added to the culture medium, the appropriate photobiomodulation (PBMT) parameters were determined and cell sheets were obtained at 5-7 days in VC group and 6-7 days in PBMT and PBMT+VC groups. In characterization studies, the extracellular matrix (ECM) structure, cell morphology, nucleus structure and different gene expression levels of cell sheets were examined. In this context, immunofluorescence and Hematoxylin-Eosin (H&E) staining results and macroscopic/microscopic observations showed that the thickest and opaque cell sheet was obtained in the PBMT+VC group. Real-time polymerase chain reaction (RT-PCR) results showed that while VC and PBMT are causing to increase of ECM, they did not significantly affect stem cell gene expressions. However, PBMT upregulates osteogenic genes (OCN, RUNX2) expression levels. Hydroxyproline assays results were also regulated upwards in the presence of VC and PBMT. The similar studies were also carried out for BJ fibroblasts and HS2 cells. While no cell sheets were obtained in the PBMT group for the BJ fibroblast cell line, no significant differences were observed between the cell sheets obtained in the VC and PBMT+VC group on the 6th-7th days. RT-PCR and hydroxyproline assay results also supported that ECM increased in the presence of VC and PBMT. In the VC and PBMT+VC group for the HS2 cell line, the cell sheets were obtained in the 8th-9th days. In conclusion, cell sheets could be obtained effectively in the presence of vitamin C for different cell types. Photobiomodulation with polychromatic light was found to have an increasing effect on cell proliferation at the studied conditions. Photobiomodulation provided sheet formation in HUC-MSCs and HS2 cells, and it was also effective in osteogenic differentiation in HUC-MSCs. The results are promising for clinical applications.