Impact of diabetic (diabetes mellitus) patients immune factors on the skin cell viability in vitro

Abstract

At the moment, a lot of scientific research focused on the role of immune mechanisms in diabetic foot ulcers development and impaired healing. A 3D skin culture system as a relevant skin model may prove valuable in investigating these mechanisms and may be a useful tool to study interactions between different cell types such as keratinocytes, fibroblasts, and immune cells. The aim of our research was to study keratinocytes and fibroblasts viability in co-culture with immune factors of patients with diabetes mellitus type 2 (DM2) and patients with diabetes and chronic foot ulcers in a 3D skin culture system. In this study, the multilayer 3D immunocompetent model of human skin comprising keratinocytes, fibroblasts, and mononuclears in an agarose-fibronectin gel was used. The human immortalized keratinocyte cell line, HaCaT, and primary fibroblast cell culture isolated from skin samples of healthy man in abdominal surgery were used for the 3D system. For the experiment 20 % serum of 9 patients with chronic diabetic foot ulcers (without active inflammation signs), 9 diabetic type 2 patients and 9 healthy people, and mononuclears of the same groups of patients were used. 9 experimental series with 3 repeats were carried out. Mononuclears of patients with DM2 and DM2 and diabetic foot syndrome (DFS) had a greater inhibitory effect on fibroblasts, significantly inhibiting their proliferation to a level of 83.78 [79.03; 89.53] % vs 70.18 [66.38; 72.10] % vs 95.40 [91.75; 99.05] %, H = 21.259, p <0.001 – DM2, DFS, and the control group, respectively. There was no significant difference in the cytoinhibitory effect of mononuclears on keratinocytes between different groups: 96.40 [92.82; 100.50] % vs 93.61 [86.80; 97.10] % vs 92.87 [85.15; 95.25] %, H = 4.459, p = 0.108 – control, DM2 and DFS group, respectively. Adding serum to the culture system influenced significantly the viability of neither keratynocytes – 99.40 [95.35; 102.05] % vs 98.60 [90.55; 100.40] % vs 94.79 [91.65; 98.16] %, H = 3.030, p = 0.220 nor of fibroblasts – 95.61 [92.39; 100.19] % vs 95.80 [88.99; 102.15] % vs 96.20 [99.69; 88.70] %, H = 0.353, p = 0.838, control, DM2 and DFS group, respectively. It was determined that the fibroblasts vialability significantly decreases after introducing mononuclears of patients with DM and patients with DM and chronic diabetic foot ulcers to the co-culture system. Adding serum of these patient groups to the culture system doesn’t influence significantly the viability of skin cells.