Osteoblastic differentiation and changes in the redox state in pulp stem cells by laser treatment

Abstract

The aim of this study was to determine the effect of low-level laser therapy (LLLT) on cell proliferation, mitochondrial membrane potential changes (∆Ψm), reactive oxygen species (ROS), and osteoblast differentiation of human dental pulp stem cells (hDPSCs). These cells were irradiated with 660- and 940-nm lasers for 5 s, 50 s, and 180 s. Cell proliferation was assessed using the resazurin assay, cell differentiation by RUNX2 and BMP2 expression, and the presence of calcification nodules using alizarin-red S staining. ROS was determined by the dichlorofluorescein-diacetate technique and changes in ∆Ψm by the tetramethylrhodamine-ester assay. Data were analyzed by a Student's t-test and Mann-Whitney U test. The 940-nm wavelength for 5 and 50 s increased proliferation at 4 days postirradiation. After 8 days, a significant decrease in proliferation was observed in all groups. Calcification nodules were evident in all groups, with a greater staining intensity in cells treated with a 940-nm laser for 50 s, an effect that correlated with increased RUNX2 and BMP2 expression. ROS production and Δψm increased independently of irradiation time. In conclusion, photobiomodulation (PBM) with LLLT induced morphological changes and reduced cell proliferation rate, which was associated with osteoblastic differentiation and increased ROS and Δψm, independent of wavelength and time.

Keywords: Human dental pulp stem cells; Low-level laser therapy; Osteoblast differentiation; Photobiomodulation; Reactive oxygen species.

Fig. 1

Isolation, characterization, and laser irradiation protocol for hDPSCs. Flow cytometry histograms with surface markers positive for CD105 (a), CD90 (b), and CD73 (c) and negative for CD34 (d), CD45 (e), and CD 146 (f). Isolated mesenchymal cells with fibroblastoid appearance (g). Magnification bar: 200 µm. Laser irradiation with Duo laser (MM Optics Ltda, Sao Paulo, Brazil) 660-nm wavelength (h) or Epic X laser (Biolase, CA, USA) 940-nm wavelength (i)

Fig. 2

Changes in cell number and morphological analysis of hDPSCs cultures treated with LLLT. a Irradiation with a 660-nm laser for 5, 50, and 180 s caused a significant reduction in cell number at 6 days postirradiation. The 940-nm laser initially produced an increase in cell number when irradiating these cells for 5 and 50 s at 4 days postirradiation. At 8 days, all experimental groups exhibited a significant reduction in cell number compared with the untreated group (control). p < 0.05 (*), p < 0.01 (**). Data are expressed as averages ± DE differentiation medium (DM). b To determine morphologic changes, cells were treated with 660 nm and 940 nm LLLT for 5, 50, and 180 s and maintained in culture for 8 days postirradiation. The control group corresponded to hDPSCs without irradiation. Differentiation medium (DM). Magnification bar 200 µm

Fig. 3

Mineralization of the extracellular matrix. a The mineralizing capacity of the extracellular matrix secreted by cells was determined by alizarin red staining. The strong intensity of the staining indicated the formation of calcification nodules. b Measured absorbance of alizarin red staining extracted from LLLT-treated cells at different wavelengths (660 nm and 940 nm) and times (5, 50, and 180 s) at 7-, 14-, and 21-days postirradiation. c Quantification of RUNX2 and BMP2 expression in 660-nm and 940-nm laser-treated hDPSCs for 14 and 21 days. Data are expressed relative to GAPDH gene expression levels and cells treated with osteogenic differentiation medium (DM) without laser irradiation were analyzed as a positive differentiation control. Cells without LLLT (control), differentiation medium (DM). p < 0.05 (*); p < 0.01 (**). Data are shown as the mean ± SD

Fig. 4

Effect of LLLT on ROS production and mitochondrial membrane potential in hDPSCs. a ROS production detected by the dichlorofluorescein-diacetate (DCF-DA) technique in cells irradiated with 660-nm and 940-nm lasers for 5, 50, and 180 s. Each treatment was compared in relative fluorescence units (RFU) and quantified by spectrofluorometry (485 nm_exc/535 nm_ems) to the negative control. ROS production corresponding to the positive control, tert-Butyl hydroperoxide (TBPH 1100X), and cells co-incubated with TBPH and 50 μg/mL ascorbic acid (AA), as well as ROS production by irradiation at both wavelengths during the maximum time evaluated (180 s) and AA are shown. b Changes in ROS production were recorded by Axiovert 40 CFL fluorescence microscopy (Carl Zeiss, USA) with a contrast of 5000, range of 1.05, brightness of 13,225, and an exposure time of 89.3 ms. Bar: 100 µm. c To evaluate changes in membrane potential, the cells were irradiated with 660-nm and 940-nm lasers for 5, 50, and 180 s. The fluorescence intensity obtained for each treatment was compared with the negative control (untreated cells), which was designated baseline mitochondrial membrane potential (Δψm). Fluorescence intensity corresponding to the positive control (TBHP) and cells irradiated for the longest exposure time (180 s) at each wavelength and co-incubated with 50 μg/mL ascorbic acid (AA) is shown. Fluorescence intensity was determined with a spectrofluorometer at a wavelength of 549 nm_exc/575 nm_ems. Results represent the mean ± SD of three independent experiments in triplicate (n = 9). Statistically significant differences between treated and untreated control groups are indicated by asterisks (p < 0.05). Lowercase letters (a) show significant differences between test groups (p < 0.05)