Effect of Electrical Stimulation on Diabetic Human Skin Fibroblast Growth and the Secretion of Cytokines and Growth Factors Involved in Wound Healing

Abstract

Diabetic foot ulcers are indicative of an impaired wound healing process. This delay may be resolved through electrical stimulation (ES). The goal of the present study was to evaluate the effect of ES on diabetic fibroblast adhesion and growth, and the secretion of cytokines and growth factors. Diabetic human skin fibroblasts (DHSF) were exposed to various intensities of direct current ES (100, 80, 40 and 20 mV/mm). The effect of ES on fibroblast adhesion and growth was evaluated using Hoechst staining, MTT and trypan blue exclusion assays. The secretion of cytokine and growth factor was assessed by cytokine array and ELISA assay. The long-term effects of ES on DHSF shape and growth were determined by optical microscopy and cell count. We demonstrated that ES at 20 and 40 mV/mm promoted cell adhesion, viability and growth. ES also decreased the secretion of pro-inflammatory cytokines IL-6 and IL-8 yet promoted growth factor FGF7 secretion during 48 h post-ES. Finally, the beneficial effect of ES on fibroblast growth was maintained up to 5 days post-ES. Overall results suggest the possible use of low-intensity direct current ES to promote wound healing in diabetic patients.

Keywords: cytokines; diabetics; electrical stimulation; fibroblasts; foot ulcers; growth factors; wound healing.

Figure 1

Diagram of the experimental protocol.

Figure 2

Validation of the conductive PPy/HE/PLLA membrane. Thickness (a1), elasticity (a2) and electrical conductivity in culture medium (b). Membrane biocompatibility with DHSF by cells cultured for 24 h (c) and 72 h (d) (Hoechst staining).

Figure 3

Selection of non-toxic ES intensities for DHSF exposed for 6 or 24 h to ES at various intensities. The cells were cultured for an additional 48 h without ES prior to MTT assay. Data are presented as means ± SD (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001. The control (no ES) and ES-exposed cells were compared, as well as the different ES intensities.

Figure 4

LDH activity of DHSF exposed to different intensities of ES. Data are presented as means ± SD (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001. The control (no ES) and ES-exposed cells were compared, as well as the different ES intensities.

Figure 5

Effect of low-intensity ES on DHSF adhesion and growth. (A) Cells on the conductive membrane after 6 or 24 h of ES, with a morphology similar to that in the control. (B) Following exposure to ES for 6 or 24 h, then culture for an extra 48 h, the cells were detached from the membranes by means of a 0.05%-trypsin-0.01 EDTA solution and cell suspensions were subjected to a trypan blue exclusion assay to count non-stained (viable) cells using a hemocytometer. * p < 0.05. Note the high number of live cells showing the beneficial effect of ES on cell adhesion/growth, basically at 6 h.

Figure 6

Effect of ES on FGF-7 secretion by the DHSF. ES at 20 mV/mm for 6 h significantly increased FGF-7. Values are means ± SD (n = 4). *** p < 0.001. The control (no ES) and ES-exposed cells were compared, as well as the different ES intensities.

Figure 7

Effect of ES on DHSF subcultures. The ES-stimulated DHSF exhibited the same morphology as that observed in the control (A) but proliferated faster than those in the controls (B) up to 5 days post-ES. Results are means ± SD (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001.