Adipose-derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine

Abstract

Chronic wounds represent a major socio-economic problem in developed countries today. Wound healing is a complex biological process. It requires a well-orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose-derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell-migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases-2 (MMP-2) and MMP-9 were analysed by quantitative real-time polymerase chain reaction (qRT-PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b-FGF, VEGF, MMP-2 and MMP-9 in ASC, and b-FGF, VEGF and MMP-9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP-9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP-9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.

Keywords: Adipose-derived stem cells; Chronic wound; Gene expression; Hydroxyectoine; Keratinocytes; Migration; Proliferation; Wound fluid; Wound healing.

Figure 1

(A) Expression of cell surface marker by adipose‐derived stem cells (ASC). Detection of CD73, CD90 and CD105 expression and lacking CD45 expression. (B) Adipogenesis of ASC. Pictures show intracellular lipid droplets under phase contrast microscopy (left side) and after Oil‐Red‐O staining (right side).

Figure 2

Proliferation of adipose‐derived stem cells (ASC, dark grey bars) and keratinocytes (light grey bars) after incubation with chronic wound fluid (CWF) and addition of hydroxyectoine (HyEc) in different concentrations, presented as percentage related to the untreated control. Shown are the results of five independent experiments plotted as mean + SEM. Keratinocyte proliferation significantly decreased after 24 and 48 hours (^* P < 0·001), whereas proliferation of ASC did not significantly change. HyEc did not change these results in none of the tested concentrations.

Figure 3

Migration of adipose‐derived stem cells (ASC, transwell‐assay). Shown is the percentage of migrated (dark grey) and non‐migrated (light grey) ASC after 24 hours. Hydroxyectoine (HyEc) in concentrations of 10 μM, 100 μM and 1 mM, 2% chronic wound fluid (CWF) and 2% CWF plus HyEc in different concentrations were used as chemotactic stimuli. Culture media served as control (ctrl). Shown are the results of three independent experiments as mean ±SEM. Asterisks (*) indicate significance compared with control, plus signs (+) show significance compared with CWF (P < 0·05).

Figure 4

Migration of keratinocytes (scratch‐assay). Diagrammed is the reduction of the initial defect area in the monolayer cell culture under the influence of (A) different concentrations of hydroxyectoine (HyEc): 1 mM (black line), 100 μM (dashed grey line), 10 μM (grey line) and (B) 2% chronic wound fluid (CWF, grey line) and 2% CWF plus 1 mM HyEc (black line). The dashed black line represents the control group (culture medium). Shown are the results of three independent experiments as mean + SEM. *P < 0·05.

Figure 5

Gene expression changes of adipose‐derived stem cells (ASC) and keratinocytes. (A) Keratinocytes (KC, grey lines) and ASC (black lines) were treated with 2% chronic wound fluid (CWF, dashed lines) or 2% CWF plus 1 mM hydroxyectoine (HyEc, continuous lines) for 2, 6, 12, 24 and 48 hours or left untreated. (B) Influence over time of 1 mM HyEc on keratinocyte and ASC gene expression of basic fibroblast growth factor (b‐FGF, black lines), vascular endothelial growth factor (VEGF, dashed black lines), matrix metalloproteinases‐2 (MMP‐2, grey lines) and MMP‐9 (dashed grey lines). Fold‐changes (FC) of gene expression were analysed by quantitative real‐time polymerase chain reaction and plotted as mean + SEM. Asterisks indicate significant differences in gene expression compared with the control group and plus‐signs between CWF/HyEc group and CWF group with P < 0·05 (n = 3).