The Effects of Photobiomodulation Delivered by Light-Emitting Diode on Stem Cells from Human Exfoliated Deciduous Teeth: A Study on the Relevance to Pluripotent Stem Cell Viability and Proliferation

Abstract

Objective: Photobiomodulation (PBM) can modulate the proliferation of some types of stem cells. However, few reports have addressed the effects of PBM delivered by light-emitting diode (LED) on stem cells obtained from the pulp tissue of deciduous teeth. The aim of the present study was to investigate the effect of PBM delivered by red LED (630 nm, 75 mW, 37 mW/cm²) with different radiant exposures on the cell cycle, mitochondrial membrane potential, and senescence of stem cells from human exfoliated deciduous teeth (SHED). Materials and methods: Cultures were irradiated with LED (2, 4, 8, 16, and 32 J/cm²). After 24 h, the cell cycle and mitochondrial membrane potential of the cultures were evaluated using flow cytometry. Nonirradiated cultures served as control. Results: Cultures irradiated with 16 J/cm² had higher percentages of cells in the synthesis phase than control cultures (p < 0.05), and no significant differences were found regarding the percentage of cells with viable mitochondria between irradiated and control cultures. No significant difference in cell senescence was found between control cultures and cultures irradiated with 2 or 16 J/cm². Conclusions: LED irradiation at 630 nm (37 mW/cm², 75 mW) with radiant exposure of 16 J/cm² was capable of inducing a proliferative response in stem cells from the pulp tissue of deciduous teeth without affecting mitochondrial function or inducing senescence.

Introduction

The aim of tissue engineering and regenerative medicine (TERM) is the development of biological replacements that enable the reestablishment of the shape and function of injured or lost tissues and organs. However, the morphological and functional complexity of some tissues offers a substantial challenge to the replacement process. Thus, the use of stem cells has considerable importance in such therapeutic strategies.^1,2

The pulp tissue of permanent and primary or deciduous teeth houses mesenchymal stem cells.^3
–5 Stem cells from human exfoliated deciduous teeth (SHED) were first mentioned by Miura et al.³ and have adequate proliferation capacity along with differentiation into odontoblasts, osteoblasts, nerve cells, adipose cells, chondrocytes, and myoblasts, as well as the expression of neural cell markers.^{3

–15} Studies on the therapeutic applications of SHED demonstrate the capacity of such cells to contribute to the repair process of some tissues, including pulp tissue when cultivated in matrices,^5,14 bone tissue,^16,17 and the epithelium of the cornea.¹⁸ SHED have also demonstrated an immunomodulatory capacity, with good results reported in the treatment of adverse conditions of the immune system, such as systemic lupus erythematosus⁷ and rheumatoid arthritis.¹⁹ Different studies have sought to establish conditions that favor high degrees of stem cell proliferation, viability, and plasticity.^20
–22

Photobiomodulation [PBM, often referred as low-level laser therapy (LLLT)] using laser or light-emitting diodes (LEDs) has demonstrated the ability to modulate the viability and proliferation of different cell types, and the effect of these light sources has also been evaluated in stem cell cultures.^23

–26 The effect of PBM on SHED has recently been described and it has been demonstrated that irradiation with infrared LED (850 nm and 40 mW/cm²) is capable of increasing the expression and activity of alkaline phosphatase and collagen synthesis and the expression of dentin sialophosphoprotein in SHED cultivated in a medium that induces osteogenic differentiation.²⁷ Moreover, PBM has been found to increase the viability, proliferation, and production of mineralized tissue in stem cells from deciduous teeth that remain for 12 h in a state of nutritional deprivation before irradiation.²⁸

The evaluation of PBM using LED on stem cells is important, as this resource is an efficient low-cost alternative to PBM using low-level laser.^27

–32 Thus, the aim of the present study was to evaluate the effects of red LED, using dosimetric parameters similar to those recently described, in cell cycle, mitochondrial membrane potential, and cellular senescence of SHED.

Materials and Methods

Patients

For the acquisition of SHED, deciduous teeth were obtained from two healthy patients: one male (10 years of age; tooth 63) and one female (11 years of age; tooth 65). The teeth were sound (with no carious lesions or inflammatory/infectious processes) and had had a clinical indication of extraction due to mobility related to the natural exfoliation process. The methods used in this study received approval from the human research ethics committee (protocol no. 133.407 on May 17, 2012 and no. 1249061 on September 29, 2015).

Cell isolation

Immediately after extraction, each tooth was placed in a tube containing DMEM/F12 medium (Invitrogen, Carlsbad, CA) with the addition of 4% antibiotic (100 U/mL of penicillin and 100 μg/mL of streptomycin sulfate; Invitrogen) and 15% fetal bovine serum (FBS) (HyClone, South Logan, UT). In laminar flow, the pulp tissue was removed with sterile endodontic files, transferred to a Petri dish, washed with phosphate-buffered saline (PBS) 0.01 M, pH 7.4, fragmented, and distributed in six-well culture plates containing DMEM F12 medium (Invitrogen) with the addition of 100 U/mL of penicillin, 100 μg/mL of streptomycin sulfate (Invitrogen), 1% nonessential amino acids (Invitrogen), and 15% FBS (HyClone). The plates were maintained in a laboratory incubator at 37°C with 5% CO₂ for 20 days, with the culture medium changed every 3 days. The cells were then detached (trypsin 0.25%; Invitrogen) and plated (10⁴ cells) in 25-cm² bottles. The cultures were kept in semiconfluence to avoid differentiation. Cultures above three passages on tissue culture plastic were analyzed for surface marker expression. All experiments described henceforth were performed separately for each cell line (n = 3 for each).

Characterization by flow cytometry

The extracted cells were cultured for 48 h, washed thrice with PBS (Invitrogen), and harvested in 0.25% trypsin/ethylene diamine tetraacetic acid (EDTA). Aliquots containing 10⁶ cells were stained with the following fluorescein isothiocyanate (FITC)-conjugated antibodies: CD105 (AbD Serotec, Raleigh, NC), CD45 (Sigma-Aldrich, St. Louis, MO), CD90 (Abcam, Cambridge, MA), CD34 (Santa Cruz Biotechnology, Dallas, TX), CD29 (Santa Cruz Biotechnology), CD73 (Santa Cruz Biotechnology), and CD146 (Abcam) at a concentration of 1 μg/mL at 4°C for 1 h. The corresponding isotope antibody was used as the negative control, and goat anti-Mouse IgG (H/L): FITC (AbD Serotec) was used as a secondary antibody. The cells were pelleted, washed twice with PBS, and fixed with 1% paraformaldehyde. Fluorescence-activated cell sorting (FACS) analysis was performed in a BD Biosciences FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Surface antigen expression was determined with a variety of directly labeled antibodies following the supplier's recommendations.

Cell culture

The cell cultures were kept in Dulbecco MEM medium (DMEM; Vitrocell, Campinas, São Paulo, Brazil) and Ham's/F12 supplement (Vitrocell) at a proportion of 1:1, to which a 1% antibiotic solution (100 U/mL of penicillin and 100 μg/mL of streptomycin sulfate) (Vitrocell), 1% nonessential amino acid solution (100 × ; Sigma-Aldrich, St. Louis, MO), and 15% FBS (Vitrocell) were added. The cultures were maintained in semiconfluence to prevent cell differentiation until reaching a sufficient amount for the experiments (maximum of six passages). The medium was exchanged every 2 days to maintain cell viability. The number of viable cells for the experiments was determined using the exclusion technique of nonvital cells stained with Trypan Blue (Invitrogen). All experiments were performed in triplicate (n = 6).

Photobiomodulation

A single irradiation was performed using a cluster of four LEDs. The characteristics of each LED, as well as the dosimetric parameters used in this study, are described in Table 1.

Table 1.

Dosimetric Parameters Used in Each Group

Parameters	Application technique	Number of applications	Radiant exposure (J/cm²)	Exposure (s)	Total radiant energy per well (J)
Peak wavelength 630 nm Spectral width 15 nm Operating mode (FWHM) continuous Output power per LED 75 mW Polarization random LED aperture diameter 0.9 cm Radiance at output 118 mW/cm² Beam area per well 2 cm² Radiance per well 37 mW/cm² Beam profile top hat	1.3 cm above the surface of the well	1	2	54	4.0
			4	107	8.0
			8	214	16.1
			16	428	32.2
			32	856	64.4

FWHM, full width at half maximum; LED, light-emitting diode.

Light absorbance was calculated as described by Silva et al.³³ The absorption coefficient was estimated at about 93%. As the effects of PBM depend on the absorption of energy by the cell or target tissue, the description and calculation of the energy that effectively reaches the target are essential in any study involving PBM.³³

The LED (emitters) was positioned over the upper portion of the plate such that each irradiated a well in continuous mode simultaneously (Fig. 1). The temperature of the medium increase was less than 1°C after the irradiation in all groups, except the group receiving 32 J/cm² where the increase was 1.5°C.

FIG. 1.

SHED irradiation procedure, demonstrating cluster with four LEDs positioned over 24-well culture plate [A—before irradiation; B—after irradiation; I—distance from LED to bottom of well (1.8 cm); II—distance of LED to surface of culture medium (1.3 cm)]. LED, light-emitting diode; SHED, stem cells from human exfoliated deciduous teeth.

Evaluation of PBM effect on cell cycle and mitochondrial membrane potential (ΔΨm)

The cells (10⁵/mL per well) were plated in triplicate (n = 6) in 24-well culture plates (Techno Plastic Products AG, Zollstrasse, Switzerland) in DMEM/F12 medium containing a 1% antibiotic solution and 10% FBS, based on Eduardo et al.³⁴ and irradiated with five different energy densities (2, 4, 8, 16, and 32 J/cm²), as previously described by Turrioni et al.²⁸

After irradiation, the plates were incubated at 37°C for 24 h in an atmosphere with 5% CO₂. The cultures in all groups were then incubated (in triplicate) with trypsin solution at 37°C for 10 min in an atmosphere with 5% carbon dioxide, and the contents of the wells were centrifuged at 293 g for 5 min.

For the cell cycle analysis, the precipitates were resuspended in an RNAse alcohol solution and stored in a freezer at −20°C for 24 h. The samples were then centrifuged for 10 min at 1811 g, resuspended in 200 μL of FACS buffer (with 10% Triton X-100 and 18 μg/mL of propidium iodide) (Sigma Chemical Co., St. Louis, MO), and kept at room temperature for 30 min in the dark. The samples were then transferred to cytometric tubes and analyzed using a FACSCalibur flow cytometry system (Becton Dickinson Biosciences, San Jose, CA) at FL-2 H fluorescence intensity. The histograms were performed with the aid of the CellQuest™ program, version 3.1.

For the analysis of mitochondrial membrane potential, rhodamine 123 (Rh123) (Sigma Chemical Co., St. Louis, MO) was added (5 μg/mL) to the tubes, and the samples were incubated for 30 min at 37°C in an atmosphere with 5% carbon dioxide. The tubes were then centrifuged, the supernatant was discarded, and the precipitates were resuspended in 100 μL of FACS buffer. The reading and analysis of Rh123 were performed in a FACSCalibur flow cytometer with the aid of the CellQuest program, version 3.1.

After the analysis of the data from the dose–response assays, 2 and 16 J/cm² were chosen, as these energy densities achieved the best results in the cell cycle analysis. The justification for this choice is described in the Results section.

Evaluation of PBM on senescence of SHED

The cultures of SHED in the control group and in the irradiated groups with 2 or 16 J/cm² were incubated for 24 h at 37°C in an atmosphere with 5% carbon dioxide. The medium was then removed, and the wells were washed with PBS 1 × buffer solution, fixed, and stained using the β-Galactosidase Staining Kit (Cell Signaling Technology, Inc. Danvers, MA). The plate was then kept at room temperature for 15 min. The wells were washed again with PBS, and the stained cultures were viewed under an inverted microscope (Nikon, Eclipse) with the aid of the NIS-Elements F3.00, SP7, Laboratory Imaging program at a magnification of 80 × . The percentage of cells in senescence was determined by counting blue stained cells on photographs obtained directly from the inverted microscope. Three independent experiments were performed in triplicate.

Data analysis

Analysis of variance (ANOVA) followed by the Tukey–Kramer test was used to identify statistically significant differences among the groups. A significance level of α = 0.05 was assumed. The graphs were created with the aid of the Prism program (version 5.0) and ModFit program (version 3.2).

Results

Cell phenotype of SHED

The SHED cultures demonstrated adherence to the culture bottles and were positively labeled for CD29, CD73, CD90, CD105, and CD146. No labeling occurred for CD45 and CD34, confirming the characterization of mesenchymal stem cells (see Dominici et al.³⁵).

Effects of PBM on cell cycle phases

No differences were found between the experimental groups and control group in relation to the percentage of cells in the G0/G1 and G2/M phases (Fig. 2). Only the group irradiated with 16 J/cm² had a higher percentage of cells in the S phase in comparison to the control group (p < 0.05). No significant differences in relation to cell cycle phases were found in the groups irradiated with other energy densities in comparison to the control. The groups irradiated with 2, 4, and 16 J/cm² showed higher number of cells in the S phase in comparison to the group irradiated with 32 J/cm² (p < 0.05).

FIG. 2.

Percentage of cells distributed throughout cell cycle (G0/G1, G2/M, Phase S, and fragmented DNA) in all experimental groups; *statistically significant difference in comparison to control (p < 0.05); data expressed as mean ± SD of three independent experiments.

The percentage of cells with fragmented DNA was higher in the groups irradiated with 4 and 32 J/cm² in comparison to the control group (p < 0.05). The group irradiated with 32 J/cm² also had a higher percentage of cells with fragmented DNA in comparison to the group irradiated with 2 J/cm² (p < 0.05). There was no difference in DNA fragmentation between irradiated groups.

Effect of PBM on mitochondrial membrane potential (Δψm)

No differences among the experimental groups were found with regard to the percentage of cells with active mitochondria (Fig. 3).

FIG. 3.

Evaluation of mitochondrial membrane potential: percentage of cells with active mitochondria in each experimental group; data expressed as mean ± SD.

Choice of dosimetric parameters for evaluation of senescence

Irradiation with 16 J/cm² increased the percentage of cells in the S phase, indicating the potential induction of a proliferative response. Cultures irradiated with 4 and 32 J/cm² had higher percentages of cells with fragmented DNA in comparison to control cultures, indicating the induction of apoptosis. Cultures irradiated with 2 and 8 J/cm² exhibited no statistically significant differences in comparison to the control group for any of the phases of the cell cycle. However, cells irradiated with 2 J/cm² demonstrated a nonsignificant increase in the percentage of cells in the S phase in comparison to the control group. Thus, energy densities of 2 and 16 J/cm² were chosen for the evaluation of senescence.

Effect of PBM on senescence

SHED irradiated with 2 and 16 J/cm² showed no difference in the percentage of cells in senescence compared to control (Figs. 4 and 5).

FIG. 4.

Senescence assay in control culture (C) and cultures irradiated with 2 and 16 J/cm². Arrows indicate cells in a senescent state.

FIG. 5.

Percentage of senescent cells in each experimental group; data expressed as mean ± SD.

Discussion

The present study evaluated the effects of red LED (630 nm, 37 mW/cm², 75 mW) using five different energy densities (2, 4, 8, 16, and 32 J/cm²) on the cell cycle, mitochondrial membrane potential, and cellular senescence of SHED. Irradiation with 16 J/cm² was capable of inducing a proliferative response in SHED without affecting mitochondrial function or inducing senescence.

The behavior of stem cells used in TERM is regulated by a number of factors, some of which are linked to the origin of the cells and others are linked to the microenvironment into which the cells are inserted.³⁶ Thus, many researchers have sought scaffolds, biologically active products, and other methods that favor the maintenance of high rates of stem cell proliferation, viability, and plasticity.^21,22,37

The effects of PBM, that is, LLLT and/or LED on stem cell cultures have previously been evaluated.^23

–29 The wavelengths used were red (∼660 nm) and infrared (∼810 nm), as well as difference radiances, energy densities, operating modes, evaluation periods, and methods hinder the direct comparison of the findings.^23

–26 Both LLLT and LED seem to be able to modulate the proliferative response and differentiation of stem cells, depending on the dosimetric parameters.^23,24

The effects of PBM do, however, vary due to the dosimetric parameters and cell type,^{23,24,38
–40} including stem cells derived from the pulp tissue of permanent and deciduous teeth. Four studies addressed the effect of laser irradiation on stem cells from permanent teeth^39

–42; all used red laser (660 nm), irradiation of 20–40 mW, and energy densities ranging from 0.5 to 42 J/cm² (with the exception of two groups in the study conducted by Pereira et al.,⁴¹ who reported that PBM using laser crossed the dentinal barrier). These findings varied considerably, which hinders the establishment of the optimal dosimetric parameters for the treatment of dental pulp stem cells. Recent studies have also reported positive effects of PBM using laser radiation on the viability and proliferation of SHED, depending on the dosimetric parameters used.^43,44

The same difficulty is encountered regarding the establishment of the optimal dosimetric parameters for phototherapy involving LED to maintain the viability and proliferation of stem cells derived from the pulp tissue of deciduous teeth. Only two studies addressing this issue were found in the literature.^27,28

Turrioni et al.²⁷ investigated the effects of infrared LED irradiation on the expression and synthesis of proteins related to the formation of mineralized tissue by SHED cultivated in an osteogenic differentiation inducing medium. The cells were irradiated with a set of 24 LEDs (850 ± 10 nm, 40 mW/cm²) for 50 sec (2 J/cm²) or 100 sec (4 J/cm²), and better results were achieved using irradiation with 4 J/cm². The same group of researchers²⁸ investigated the effect of LED (850 ± 10 nm, 40 mW/cm²) irradiation on SHED without the use of a differentiation inducing medium, and the stem cells were kept in a medium with 0.5% FBS for 12 h before the experiments to simulate a state of nutritional deprivation. The authors report that the stem cell cultures irradiated with 4 J/cm² demonstrated greater viability than nonirradiated cells after 12 and 72 h [as evidenced by the MTT (3-(4, 5-dimethylthiazol-2-y)-2, 5-diphenyl tetrazolium bromide) assay]. In addition, all irradiated cultures had a larger number of viable cells in comparison to nonirradiated cells after 12 h, whereas only those irradiated with 4 J/cm² demonstrated an increase in proliferation after 72 h.

Unfortunately, the importance of each physical irradiation parameter (wavelength, power, energy, radiant exposure, number of irradiations, and interval between irradiations) has not yet been fully clarified. However, it is accepted that the beneficial effects of PBM depend on the biphasic dose response; the challenge is obtaining the best dose (radiant exposure/energy density) for each application.^40,45,46 In the present study, irradiation with 16 J/cm² was the only energy density capable of inducing an increase in the percentage of cells in the S phase of the cell cycle, demonstrating a positive effect on cellular proliferation. Moreover, no significant differences in mitochondrial membrane potential or senescence were found with this energy density in comparison to the nonirradiated control group, suggesting that LED has no undesirable effect on SHED.

The divergences between the present findings and those described by Turrioni et al.²⁸ may be linked to the difference in the LED wavelength used, the use of different evaluation methods and periods, and/or the fact that the cells were maintained in a medium with 10% FBS beginning at the time of irradiation. However, both studies indicated that SHED irradiated with LED showed significant increase in cell viability and proliferation, using particular dosimetric parameters.

For the desired results in tissue engineering,^{5,14,16

–19} stem cells must have good proliferation and viability rates,^21–22 as well as a low rate of senescence.⁴⁷ In this context, based on our results, PBM with LED may be an important tool in tissue engineering. Thus, further studies should be conducted to optimize dosimetric parameters⁴⁰ and the metabolic pathways involved in PBM of SHED by LED.

Conclusions

In this study, LED irradiation at 630 nm (37 mW/cm² and 75 mW) at an energy density of 16 J/cm² induced a proliferative response on SHED without affecting mitochondrial function or inducing senescence. Further studies could demonstrate that LED is a useful tool for the modulation of SHED for use in tissue engineering and regenerative medicine.

Footnotes

Acknowledgments

K.P.S.F. (CNPq—National Council for Technological and Scientific Development), Grant No. 311078/2015-0 and FAPESP (São Paulo Research Foundation) Grant No. 2013/07502-1. D.A.M. (CNPq), Grant No. 306124/2015-7. R.A.M.F. (CNPq), Grant No. 305739/2014-0 and FAPESP Grant No. 2014/12381-1, S.K.B. (CNPq), Grant No. 305905/2014-7.

Ethical Statement

All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Author Disclosure Statement

No competing financial interests exist.

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