Inhibitory effect of red LED irradiation on fibroblasts and co-culture of adipose-derived mesenchymal stem cells

Abstract

The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm²/5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-β1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4^th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-β1, whereas TGF-β1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-β1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf, VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair.

Keywords: Biological sciences; Biomedical engineering; Cell biology; Cell migration; Ctgf; Molecular biology; Proteins; Regenerative medicine; Repair; Scratch assay; TGF-β1; Tenomodulin.

Figure 1

(A) Adipogenic differentiation of ASC (5P) evidenced by intracellular lipid droplets (arrow) stained with Sudan IV, and (B) osteogenic differentiation evidenced by extracellular calcification points (arrow) stained with Alizarin Red-S. (C) Undifferentiated ASC (arrow). (D) Histograms showing the x-axis fluorescence scale considered positive when the cell peak is above 10¹ (CD90), 10² (CD 105) or 10¹ (CD45), and controls for -APC, -PE and -FITC, corresponding to non-marked cells due very low fluorescence (in blue). (E) Flow cytometry of ASC (5P): Observe high expression of CD90, CD105 markers and low expression of CD45. Values represented by the mean ± standard deviation.

Figure 2

(A) Cell viability analysis of FL group 24h after application of 3 different times: 40s, 5min6s and 9min33s. Observe no differences between the times analyzed. Dashed line represents positive control (B) On the 4^th day after the scratch, no differences were observed between the groups. Values represented by the mean ± standard deviation.

Figure 3

qRT-PCR for Reck and Ctgf expression: note higher Ctgf expression in group FC compared to group F. a = equals letters indicate significant differences between groups (p < 0.05). Values represented by the mean ± standard deviation.

Figure 4

Western blotting and band densitometry for VEGF, TGF-β1, tenomodulin and collagen types I and III. a, b, c, d = equal letters indicate significant differences between the groups (p < 0.05). Values represented by mean ± standard deviation (see supplemental data).

Figure 5

Zymography for MMP-9 and MMP-2: note the absence of MMP-9 and the presence of latent (72 kDa) and active (62 kDa) isoforms of MMP-2 (A). Band densitometry represented in the graphics (B and C), showing no differences between groups. Values represented by mean ± standard deviation.

Figure 6

(A) Cell migration analysis: scratch cell count. Note a marked difference for the FC group between the 2^nd and 4^th days, in relation to groups F, FL and FLC. a, b, c, d, e, f, g, h, i = equal letters represent significant differences between groups (p < 0.05). Values represented by the mean ± standard deviation. (B) Representative images of cell migration for scratch closure on days 0, 1, 2, 3 and 4 of all groups. Migration of NIH/3T3 fibroblasts (red arrow) and ASC (yellow arrow) in the FC and FLC groups. Note that the FC group presented higher migration between the 2^nd and 4^th days compared to the other groups, with total scratch closure. In addition, a lower amount of ASC was observed in the scratch region in the FLC group compared to the FC group between the 2^nd and 4^th days.