Phototherapy up-regulates dentin matrix proteins expression and synthesis by stem cells from human-exfoliated deciduous teeth

doi:10.1016/j.jdent.2014.07.014

Journal of Dentistry

Volume 42, Issue 10, October 2014, Pages 1292-1299

https://doi.org/10.1016/j.jdent.2014.07.014 Get rights and content

Abstract

Objectives

The aim of this study was to evaluate the effects of infrared LED (850 nm) irradiation on dentin matrix proteins expression and synthesis by cultured stem cells from human exfoliated deciduous teeth (SHED).

Methods

Near-exfoliation primary teeth were extracted (n = 3), and SHED cultures were characterized by immunofluorescence using STRO-1, CD44, CD146, Nanog and OCT3/4 antibodies, before experimental protocol. The SHEDs were seeded (3 × 10⁴ cells/cm²) with DMEM containing 10% FBS. After 24-h incubation, the culture medium was replaced by osteogenic differentiation medium, and the cells were irradiated with LED light at energy densities (EDs) of 0 (control), 2, or 4 J/cm² (n = 8). The irradiated SHEDs were then evaluated for alkaline phosphatase (ALP) activity, total protein (TP) production, and collagen synthesis (SIRCOL™ Assay), as well as ALP, collagen type I (Col I), dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein (DMP-1) gene expression (qPCR). Data were analyzed by Kruskal–Wallis and Mann–Whitney tests (α = 0.05).

Results

Increased ALP activity and collagen synthesis, as well as gene expression of DSPP and ALP, were observed for both EDs compared with non-irradiated cells. The ED of 4 J/cm² also increased gene expression of COL I and DMP-1.

Conclusions

In conclusion, infrared LED irradiation was capable of biostimulating SHEDs by increasing the expression and synthesis of proteins related with mineralized tissue formation, with overall better results for the energy dose of 4 J/cm².

Clinical significance

Phototherapy is an additional approach for the clinical application of LED in Restorative Dentistry. Infrared LED irradiation of the cavity's floor could biostimulate subjacent pulp cells, improving local tissue healing.

Introduction

Pulp tissue inflammation may be exacerbated during cavity preparation and following cavity restoration with non-biocompatible dental materials.¹ Therefore, specific procedures and biomaterials should be clinically applied to enhance pulpal healing by up-regulating collagen-rich dentin matrix production and its mineralization by pulp cells.¹ It has been shown that light-emitting diode (LED) irradiation may improve the repair of different tissues, including pulp.2, 3, 4 Furthermore, previous studies have demonstrated that LED irradiation can biostimulate cultured odontoblast-like MDPC-23 cells⁵ and human dental pulp cells (HDPCs).⁶

It has been reported that LED irradiation in the infrared range of the spectrum (from 700 nm to 1 mm wavelength) is able to pass through the dentin barrier⁷ and photobiostimulate underlying cells,⁸ increasing the cell viability and alkaline phosphatase activity of odontoblast-like cells as well as up-regulating collagen type I (Col I) expression.5, 9, 10 Additionally, infrared light can improve healing of bone defects mechanically created in rats¹¹ and reduce pain in temporomandibular disorders.¹²

Based on these data, it is reasonable to consider that phototherapy technology with LED represents an interesting alternative for clinical application in restorative dentistry, particularly for stimulation of pulp cells subjacent to the cavity floor. Therefore, the authors hypothesized that infrared LED irradiation, at specific parameters, could enhance dentin matrix and dentin mineralization protein expression/synthesis by pulp cells. The aim of this study was to evaluate the effects of infrared LED (850 nm) irradiation on dentin matrix protein gene expression and synthesis by cultured SHEDs.

Section snippets

Primary culture obtained from deciduous teeth

The present study was approved by the Ethical Committee of UNESP—Univ. Estadual Paulista (protocol 63/11). Three near-exfoliation sound primary teeth were donated by the child's legal guardian, who provided a signed consent form. Pulp cells were isolated by enzymatic digestion with collagenase type I and dispase (Worthington Biochemical Corp., Lakewood, NJ, USA).¹³ Cells were cultured in 75 cm² flasks in monolayer on DMEM supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY, USA),

Cell characterization by immunofluorescence

Representative immunofluorescence images of SHEDs from primary teeth identified by CD 146, CD 44, STRO-1, Nanog, and OCT 3/4 antibodies, for the three individuals participating in the experiments (P1, P2, and P3) are shown in Fig. 1. The percentages of labelled cells for P1, P2, and P3 were: (CD146) 84.2%, 71.4%, and 69.0%; (CD 44) 81.8%, 89.2%, and 92.6%; (STRO-1)76.6%, 76.0%, and 75.1%; (Nanog) 78.8%, 80.5%, and 86.2%; and (OCT 3/4) 83.5%; 82.2%, and 83.4%, respectively.

TP production, ALP activity, and collagen synthesis

Percentages of TP

Discussion

Both gene expression and synthesis of proteins involved in tertiary dentin formation were up-regulated in SHEDs irradiated with an infrared LED source delivering 2 or 4 J/cm² (40 mW/cm²), with an overall greater biostimulation for the latter. A major struggle in the phototherapy field is to find the optimal irradiation set of parameters for each different clinical treatment and specific tissue,¹⁶ because there are many factors involved in phototherapy success, such as wavelength, energy density,

Conclusion

In conclusion, infrared LED irradiation, at selected parameters, is able to up-regulate gene expression and synthesis of proteins related to the formation and mineralization of dentin matrix by human dental pulp cells. Further in vivo studies should be developed to ensure the clinical use of this phototherapy.

Acknowledgments

The authors acknowledge the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (grants: 2011/13895-0 and 2013/17758-3) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (grant: 301291/2010-1) for financial support. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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Specific parameters of infrared LED irradiation promote the inhibition of oxidative stress in dental pulp cells
2021, Archives of Oral Biology
Citation Excerpt :
Between the diodes and the bottom of the wells of the 24-well plates, lenses are positioned so that the light is simultaneously delivered to the whole bottom area (2 cm2) on which the cells are attached. The radiant exposures were delivered at 0 (control), 4, 15, and 30 J/cm2 (Montoro et al., 2014; Turrioni et al., 2014, 2015b, 2017), and the exposure times for each radiant exposure were determined based on the following formula: density of energy = power × exposure time × area−1; where power (80 and 160 mW) and area (2 cm2) were the fixed parameters, and the exposure times were 1 min 40 s, 3 min 20 s, 6 min 15 s, 12 min 30 s, and 25 min Mitochondrial activity was assessed using the methyl tetrazolium (MTT) assay, performed 24 h after irradiation, to analyze cell viability.
The present study aimed to assess the oxidative stress and the viability of dental pulp cells stimulated by lipopolysaccharide (LPS) and submitted to photobiomodulation (PBM) with infrared light-emitting diode (LED, 850 nm).
Three healthy primary teeth (n = 3) were collected and seeded in 24-well plates with 10 µg/mL of LPS to induce inflammatory mediator formation. The cells were irradiated (850 nm, 40 mW/cm² and 80 mW/cm²) at the proposed radiant exposures of 0 (control), 4, 15, and 30 J/cm² shortly after LPS supplementation. The tests were performed 24 h after irradiation to assess mitochondrial activity (MTT assay), the number of viable cells (Trypan Blue), cell morphology (Scanning Electron Microscopy - SEM), and the quantification of Nitric Oxide (NO) and Reactive Oxygen Species (ROS). The data were analyzed using Kruskal–Wallis and Dunn’s tests (p < 0.05).
The irradiated groups showed larger viable cells number than the non-irradiated group with LPS (p < 0.0001). All irradiation parameters decreased ROS concentrations after LPS application compared to the non-irradiated group (p < 0.05). All irradiation parameters enhanced the NO values compared to those of the control group (p < 0.05). The SEM images showed cells with regular morphology that adhered to the substrate.
According to the parameters used in this study, the radiant exposure of 15 J/cm² and irradiance of 40 mW/cm² were the most effective irradiation parameters to stimulate and modulate oxidative stress in the primary teeth-derived dental pulp cells.
“Metabolism of Odontoblast-like cells submitted to transdentinal irradiation with blue and red LED”
2017, Archives of Oral Biology
Citation Excerpt :
However, the two wavelengths did not increase the production of this protein, irrespective of the energy doses delivered to the cells. The physical parameters capable of photo-bio-stimulating immortalized pulp or mesenchymal cells have been investigated (Turrioni et al., 2014; Holder et al., 2012; Oliveira et al., 2011). However, the lack of uniformity among studies (i.e.: not standardized irradiance, energy dose, area of irradiated surface and even the cell cultivation regimen) is held responsible for the controversial results for phototherapy, in spite of being promising.
The present study evaluated the trans-dentinal effect of light emitting diodes (LEDs) irradiation on the metabolism of odontoblast-like cells.
Seventy-two dentin discs (0.2 mm thick) were obtained from human molar teeth. MDPC-23 cells (20,000 cells/disc) were seeded on the pulpal side of the discs using DMEM, supplemented with 10% fetal bovine serum (FBS). After 12 h, the culture medium was replaced with DMEM containing 0.5% FBS. After additional 12 h, blue (455 ± 10 nm) or red (630 ± 10 nm) LEDs were used at irradiances of 80 and 40 mW/cm², respectively, to irradiate the occlusal side of the discs. The energy doses were fixed at 2 or 4 J/cm². Cell viability, alkaline phosphatase activity (ALP), total protein production and collagen synthesis were evaluated 72 h after irradiation. Data were submitted to Kruskal-Wallis and Mann-Whitney tests (α=0.05).
Red light promoted proliferative effects at the energy dose of 4 J/cm²_. Conversely, cell cultures irradiated with 2 J/cm² emitted by the blue light showed reduced viability. ALP production was stimulated by red light in comparison with blue light at 4 J/cm². Total protein production was reduced after exposure to blue light at 4 J/cm², while no effect was observed on collagen production.
Irradiation with red LED at 4 J/cm² bio-stimulated the viability of odontoblast-like cells, whilst blue light had unfavorable effects on the cellular metabolism.
Effect of low-level laser irradiation on proliferation of human dental mesenchymal stem cells; a systemic review
2016, Journal of Photochemistry and Photobiology B: Biology
Citation Excerpt :
Four studies were excluded; one reported on the effect of light-emitting diode (LED) exposure on dental pulp cells [63]. Two studies were excluded as they investigated the cell differentiation and not the proliferation [64,65]; and another one investigated the survival of DMSCs in response to the dental adhesives [66]. Overall, 6 studies evaluated the effect of LLLT on proliferation of DMSCs [57–62].
Identification of factors that enhance the proliferation of human dental mesenchymal stem cells (DMSCs) is vital to facilitate tissue regeneration. The role of low-level laser irradiation (LLLI) on proliferation of human DMSCs has not been well established.
To assess the effect of LLLI on proliferation of human DMSCs when applied in-vitro.
Electronic search of literature was conducted (2000–2016) on PubMed, Web of Science, and Scopus databases. Search terms included low-level light therapy, low-level laser irradiation, low-level light irradiation, LLLT, humans, adolescent, adult, cells, cultured, periodontal ligament, dental pulp, stem cells, dental pulp stem cells, mesenchymal stem cells, periodontal ligament stem cell, deciduous teeth, cell proliferation, adult stem cells, radiation, and proliferation.
The literature search identified 165 studies with 6 being eligible for inclusion; all used diode lasers; 5 studies used InGaAIP diode lasers; 4 used 660 nm, and the other two applied 810 nm or 980 nm wavelength LLLI. The distance between the DMSCs and the laser spot ranged between 0.5 mm to 2 mm. The time intervals of cell proliferation analysis ranged from 0 h to 7 days after LLLI. After 660 nm LLLI, an increase in the DMSC's proliferation was reported [DMSCs extracted from dental pulp of deciduous teeth (two irradiations, 3 J/cm², 20 mW was more effective than 40 mW), adult teeth (two irradiations, 0.5 and 1.0 J/cm², 30 mW), and from adult periodontal ligament (two irradiations, 1.0 J/cm² was more effective than 0.5 J/cm², 30 mW)]. Similarly, an increase in the proliferation of DMSCs extracted from dental pulp of adult teeth was reported after 810 nm LLLI (7 irradiations in 7 days, 0.1 and 0.2 J/cm², 60 mW) or 980 nm LLLI (single irradiation, 3 J/cm², 100 mW). However, 660 nm LLLI in one study did not increase the proliferation of DMSCs (single irradiation, energy densities of 0.05, 0.30, 7, and 42 J/cm², 28 mW).
There is limited evidence that in-vitro LLLI (660/810/980 nm, with energy densities of 0.1-3 J/cm²) increases the proliferation of DMSCs. Considering the limited evidence and their method heterogeneity it is difficult to reach a firm conclusion. Further research is necessary to identify the optimal characteristics of the LLLI setting (wave length, energy density, power output, frequency/duration of irradiations, distance between the cells and the laser spot/probe) to increase proliferation of DMSCs, and assess its impact on replicative senescence, as well as determine feasibility of the use in the clinical setting.
Photobiomodulation-induced osteogenic differentiation of mesenchymal stem cells
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The Role of Photobiomodulation on Dental-Derived Stem Cells in Regenerative Dentistry: A Comprehensive Systematic Review
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Effect of diode low level laser and red light emitting diode irradiation on cell proliferation and osteogenic/odontogenic differentiation of stem cells from the apical papilla
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View all citing articles on Scopus

View full text

Phototherapy up-regulates dentin matrix proteins expression and synthesis by stem cells from human-exfoliated deciduous teeth

Abstract

Objectives

Methods

Results

Conclusions

Clinical significance

Introduction

Section snippets

Primary culture obtained from deciduous teeth

Cell characterization by immunofluorescence

TP production, ALP activity, and collagen synthesis

Discussion

Conclusion

Acknowledgments

Dental Clinics of North America

Analytical Biochemistry

Dental Materials

Effect of GaAIAs laser on reactional dentinogenesis induction in human teeth

Photomedicine Laser Surgery

Odontoblast responses to GaAIAs laser irradiation in rat molars: an experimental study using heat-shock protein-25 immunohistochemistry

European Journal of Oral Sciences

Effects of red light-emitting diode irradiation on dental pulp cells

Journal of Dental Research

Transdentinal cell photobiomodulation using different wavelengths

Operative Dentistry

Infrared LED irradiation photobiomodulation of oxidative stress in human dental pulp cells

International Endodontic Journal

Correlation between light transmission and permeability of human dentin

Lasers in Medical Science

LED light attenuation through human dentin: a first step toward pulp photobiomodulation after cavity preparation

American Journal of Dentistry

In vitro effect of low-level laser on odontoblast-like cells

Laser Physics Letters