gms | German Medical Science

Objectives: The ability of electrical stimulation (EStim) to enhance bone regeneration through stimulation of chondro- and osteogenesis and extracellular matrix deposition has been shown in vivo and in vitro. In a previous study, we found that EStim also modified macrophage response by changing M1 to M2 macrophage ratio in vivo. These changes may occur due to the effects of electricity on macrophage polarization, trans-differentiation, migration and/or metabolic activity. Objective: to analyze if EStim have differential effect on M0, M1 and M2 macrophage metabolic activity and/or polarization in vitro and the relevance of these processes to bone tissue regeneration.

Methods: THP-1 cells were differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), cells were polarized toward M1 (with IFN gamma and LPS) or M2 (with IL4 and IL13) phenotype in the presence (or absence, control group) of 100 mV/mm, 1h/day EStim. Metabolic activity of M0, M1 and M2 EStim-treated and control cells was measured by means of AlamarBlue assay. Expression of cell surface markers CD14, CD11b, CD80, CD86 and CD206 was evaluated by flow cytometry and IL8, IL1b, IL6, IL10 and TNF alpha production was quantified by means of cytometric bead array. Gene expression of macrophage activation and osteogenic differentiation markers was measured by RT-qPCR. A minimum of 4 replicates per group per analysis was considered. T-test or Mann-Whitney test were used as appropriate and significance level was set at p < 0.05.

Results: We found that the response to EStim is variable between the types of macrophages. Whereas M0 and M2 macrophages show increased metabolic activity in response to EStim, the opposite is observed on M1 cells. Surface expression of CD11b and gene expression and secretion of IL8 are increased by EStim in M0 while CD14, CD80 and CD86 surface markers and CD86 gene expression in M1 macrophages are reduced by the treatment. When considering M2 phenotype, EStim induces a higher IL8 secretion with no effects on expression of genes and cell surface markers evaluated in the present study. In addition, electricity down-regulates CCL22 and up-regulates CD163, IL10, TGM2 and osteopontin gene expression in M0 and M1 cells while BMP2 gene expression was increased only in EStim-treated M0 macrophages.

Conclusion: EStim has differential effect on M0, M1 and M2 macrophage metabolic activity and polarization. M2 macrophages are less sensitive to EStim, whereas M0 and M1 macrophages show changes in gene expression, cell surface markers and cytokine secretion in response to treatment. Enhancement of M2 and suppression of M1 metabolic activity together with up-regulation of osteogenic genes in M0 and M1 cells and higher secretion of angiogenic IL8 in M0 and M2 macrophages can be mechanisms underlying the pro-regenerative effect of EStim.