In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts

Abstract

The aim of this study was to determine adequate energy doses using specific parameters of LLLT to produce biostimulatory effects on human gingival fibroblast culture. Cells (3 × 10(4) cells/cm(2)) were seeded on 24-well acrylic plates using plain DMEM supplemented with 10% fetal bovine serum. After 48-hour incubation with 5% CO(2) at 37°C, cells were irradiated with a InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) with energy doses of 0.5, 1.5, 3, 5, and 7 J/cm(2). Cells were irradiated every 24 h totalizing 3 applications. Twenty-four hours after the last irradiation, cell metabolism was evaluated by the MTT assay and the two most effective doses (0.5 and 3 J/cm(2)) were selected to evaluate the cell number (trypan blue assay) and the cell migration capacity (wound healing assay; transwell migration assay). Data were analyzed by the Kruskal-Wallis and Mann-Whitney nonparametric tests with statistical significance of 5%. Irradiation of the fibroblasts with 0.5 and 3 J/cm(2) resulted in significant increase in cell metabolism compared with the nonrradiated group (P < 0.05). Both energy doses promoted significant increase in the cell number as well as in cell migration (P < 0.05). These results demonstrate that, under the tested conditions, LLLT promoted biostimulation of fibroblasts in vitro.

Figure 1

Photomicrographs showing human gingival fibroblast cultures seeded in 24-well plates after LLLT. The control group exhibits a large cell-free area on acrylic surface. The group irradiated with 0.5 J/cm² exhibits cell proliferation and migration, with consequent reduction of the “in vitro wound” size. The group irradiated with 3.0 J/cm² presented more intense cell proliferation and migration, resulting in almost complete closure of the “in vitro wound.”

Figure 2

SEM micrograph showing cells with normal morphology that migrated through the transwell membrane. SEM ×500.