RESUMO
INTRODUCTION: Intractable ulcers may ultimately lead to amputation. To promote wound healing, researchers developed a serum-free ex vivo peripheral blood mononuclear cell quality and quantity culture (MNC-QQc) as a source for cell therapy. In mice, pigs, and even humans, cell therapy with MNC-QQc reportedly yields a high regenerative efficacy. However, the mechanism of wound healing by MNC-QQc cells remains largely unknown. Hence, using an in vitro wound healing model, this study aimed to investigate MNC-QQc cells and the migratory potential of dermal fibroblasts. METHODS: After separation from a 50 mL blood sample from healthy individuals, mononuclear cells were cultured for 7 days in a serum-free ex vivo expansion system with five different cytokines (MNC-QQc method). The effects of MNC-QQc cells on human dermal fibroblast migration were observed by scratch assay. An angiogenesis array screened the MNC-QQc cell supernatant for proteins related to wound healing. Finally, fibroblast migration was confirmed by observing the intracellular signal transduction pathways via Western blot. RESULTS: The migration of fibroblasts co-cultured with MNC-QQc cells increased by matrix metallopeptidase-9 (MMP9) secretion, as suggested by the angiogenesis array. Furthermore, the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in fibroblast/MNC-QQc cell co-culture and fibroblast culture with added recombinant human MMP9 protein increased. When fibroblasts were cultured with either an MMP9 inhibitor or a STAT3 inhibitor, both fibroblast migration and STAT3 phosphorylation were significantly suppressed. CONCLUSIONS: MNC-QQc cells promote wound healing by the secretion of MMP9, which induces fibroblast migration via the STAT3 signaling pathway.
RESUMO
Persistent inflammatory environment and abnormal macrophage activation are characteristics of chronic diabetic wounds. Here, we attempted to characterize the differences in macrophage activation and temporal variations in cytokine expression in diabetic and non-diabetic wounds, with a focus on interleukin (IL)-6 mRNA expression and the p38 MAPK and PI3K/Akt signaling pathways. Cutaneous wound closure, CD68- and arginase-1 (Arg-1)-expressing macrophages, and cytokine mRNA expression were examined in non-diabetic and streptozotocin-induced type 1 diabetic mice at different time points after injury. The effect of IL-6 on p38 MAPK and Akt phosphorylation was investigated, and an in vitro scratch assay was performed to determine the role of IL-6 in primary skin fibroblast migration. Before injury, mRNA expression levels of the inflammatory markers iNOS, IL-6, and TNF-α were higher in diabetic mice; however, IL-6 expression was significantly lower 6 h post injury in diabetic wounds than that in non-diabetic wounds. Non-diabetic wounds exhibited increased p38 MAPK and Akt phosphorylation; however, no such increase was found in diabetic wounds. In fibroblasts from non-diabetic mice, IL-6 increased the phosphorylation of p38 MAPK and levels of its downstream factor CREB, and also significantly increased Akt phosphorylation and levels of its upstream factor P13K. These effects of IL-6 were not detected in fibroblasts derived from the diabetic mice. In scratch assays, IL-6 stimulated the migration of primary cultured skin fibroblasts from the non-diabetic mice, and the inhibition of p38 MAPK was found to markedly suppress IL-6-stimulated fibroblast migration. These findings underscore the critical differences between diabetic and non-diabetic wounds in terms of macrophage activation, cytokine mRNA expression profile, and involvement of the IL-6-stimulated p38 MAPK-Akt signaling pathway. Aberrant macrophage activation and abnormalities in the cytokine mRNA expression profile during different phases of wound healing should be addressed when designing effective therapeutic modalities for refractory diabetic wounds.