Effect of Low-Level Diode Laser and Red Light-Emitting Diode on Survival and Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells.

Background: This investigation set out to compare the impacts of low-level diode laser (LLDL) and red light-emitting diode (LED) on the survival of human dental pulp stem cells (hDPSCs) and osteogenic/odontogenic differentiation. Methods and materials: In this ex vivo experimental study, the experimental groups underwent the irradiation of LLDL (4 J/cm2 energy density) and red LED in the osteogenic medium. Survival of hDPSCs was assessed after 24 and 48 h (n = 9) using the methyl thiazolyl tetrazolium (MTT) assay. The assessment of osteogenic/odontogenic differentiation was conducted using alizarin red staining (ARS; three repetitions). The investigation of osteogenic and odontogenic gene expression was performed at two time points, specifically 24 and 48 h (n = 12). This analysis was performed utilizing real-time reverse-transcription polymerase chain reaction (RT-PCR). The groups were compared at each time point using SPSS version 24. To analyze the data, the Mann-Whitney U test, analysis of variance, Tukey's test, and t-test were utilized. Results: The MTT assay showed that LLDL significantly decreased the survival of hDPSCs after 48 h, compared with other groups (p < 0.05). The qualitative results of ARS revealed that LLDL and red LED increased the osteogenic differentiation of hDPSCs. LLDL and red LED both upregulated the expression of osteogenic/odontogenic genes, including bone sialoprotein (BSP), alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP), in hDPSCs. The LLDL group exhibited a higher level of gene upregulation (p < 0.0001). Conclusions: The cell survival of hDPSCs was reduced, despite an increase in osteogenic/odontogenic activity. Clinical relevance: Introduction of noninvasive methods in regenerative endodontic treatments.

Carbon Dioxide Fractional Laser Treatment Induces Lgr5+ Stem Cell Activation and Hair Regrowth Through the Canonical Wnt/ß-Catenin Pathway.

BACKGROUND: Different types of alopecia have negative impacts on patients. Recently, some kinds of laser or light therapies have been reported to effectively alleviate hair loss. Carbon dioxide fractional laser (CO2FL) treatment is one of the most effective laser treatments, but its beneficial effects and exact mechanism in hair regrowth have not been reported in detail. The purpose of this study was to investigate the effect and molecular mechanism further. METHODS: C57 and Lgr5-Cre: Rosa-mTmG mouse models of hair regrowth were established by CO2FL treatment, and the parameters that induced the best effect were determined. Tissues were harvested on the day prior to the treatment day and on days 3, 5, 7, 10 and 14 after CO2FL. H&E and immunofluorescence staining, RNA sequencing (RNA-seq), quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and related inhibitor were used to determine the molecular mechanism underlying the effect of CO2FL treatment on the hair cycle and hair regrowth. In clinical trial, five participants were treated three sessions at 1-month intervals to obverse the effects. RESULTS: Hair regrew and covered the treatment area on the tenth day after CO2FL treatment with the best parameters, while the control group showed signs of hair growth on the 14th day. H&E and immunofluorescence staining showed that the transition of hair follicles (HFs) from telogen to anagen was accelerated, and the rapid activation and proliferation of Lgr5+ hair follicle stem cells (HFSCs) were observed in the treatment group. The RNA-seq, qPCR and WB results indicated that the Wnt pathway was significantly activated after CO2FL treatment. Improvement achieved with CO2FL treatment in clinical trial. CONCLUSIONS: The results of this study suggest that CO2FL treatment can promote hair regrowth by activating Lgr5+ HFSCs and upregulating the Wnt/ß-catenin pathway. Clinical trial results demonstrated that CO2FL treatment will be a promising therapeutic regimen for alopecia. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

NIR irradiation of human buccal fat pad adipose stem cells and its effect on TRP ion channels.

The effect of near infrared (NIR) laser irradiation on proliferation and osteogenic differentiation of buccal fat pad-derived stem cells and the role of transient receptor potential (TRP) channels was investigated in the current research. After stem cell isolation, a 940 nm laser with 0.1 W, 3 J/cm2 was used in pulsed and continuous mode for irradiation in 3 sessions once every 48 h. The cells were cultured in the following groups: non-osteogenic differentiation medium/primary medium (PM) and osteogenic medium (OM) groups with laser-irradiated (L +), without irradiation (L -), laser treated + Capsazepine inhibitor (L + Cap), and laser treated + Skf96365 inhibitor (L + Skf). Alizarin Red staining and RT-PCR were used to assess osteogenic differentiation and evaluate RUNX2, Osterix, and ALP gene expression levels. The pulsed setting showed the best viability results (P < 0.05) and was used for osteogenic differentiation evaluations. The results of Alizarin red staining were not statistically different between the four groups. Osterix and ALP expression increased in the (L +) group. This upregulation abrogated in the presence of Capsazepine, TRPV1 inhibitor (L + Cap); however, no significant effect was observed with Skf96365 (L + Skf).

A Comparative Analysis of Photobiomodulation-Mediated Biological Effects of Single Versus Double Irradiation on Dental Pulp Stem Cells: An In Vitro Study.

Objective: In recent years, fractionated irradiation protocols, rather than a simple plan of exposure, have been proposed as a more effective method in the field of tissue regeneration. Thus, this study aimed at a comparative analysis of single versus double irradiation of an 808-nm diode laser, in terms of dental pulp stem cells' (DPSCs) viability and proliferation in vitro. Methods: Subcultured DPSCs were either irradiated, or not (control group), with energy densities of 3, 7, and 12 J·cm-2 in a single- or double-session manner (24 h apart). On 0, 12, 24, 48, and 72 h postirradiation, cell viability and proliferation were evaluated through Trypan Blue and alamarBlue assays, respectively. Results: During the first 48 h postirradiation, the highest rates of DPSC proliferation were assigned to double irradiation at 3 or single exposure to 7 J⋅cm-2, with no cytotoxic effects on cell viability. Inversely, single irradiation at 12, or a double session of exposure to 7 or 12 J⋅cm-2, led to a significant descent in the rates of proliferation and cell viability. Conclusions: Within the limitations of this study, evidence suggests a positive impact on the biological responses of DPSCs following double session of exposure to lower energy densities as well as a single irradiation at a higher energy dosage.

Near Infrared Laser Photobiomodulation of Periodontal Ligament Stem Cells.

OBJECTIVE: To determine the effect of different energy densities of near infrared diode lasers with wavelengths of 810 or 940 nm on the proliferation and survival of periodontal ligament derived stem cells (PDLSCs). METHODS: After isolation and characterisation, PDLSCs were cultured in clear 96-well plates. Each well was irradiated by either 810 nm (L1) or 940 nm (L2) lasers, with energy densities of 0.5, 1.5 and 2.5 J/cm2 and an output power of 100 mW. A non-irradiated well was used as a control. Cellular viability was measured 24 hours after irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and proliferation was measured 24, 48 and 72 hours after irradiation using trypan blue staining and counting. Propidium iodide (PI) staining was used to identify any pyknotic nuclei or nuclear fragmentation 72 hours after irradiation. RESULTS: An increase in viability was observed only in the group with the 940 nm laser irradiation at energy density of 2.5 J/cm2 (P < 0.001). The proliferation of cells was significantly increased in the group with 940 nm laser irradiation at energy density of 2.5 J/cm2 at all the time points examined in comparison to other groups (P < 0.001). PI staining showed no change in cell nuclei in any of the groups. CONCLUSION: Irradiation of PDLSCs with a 940 nm laser at an energy density of 2.5 J/cm2 could promote efficient cell proliferation.

Pulsed Electromagnetic Fields in Bone Healing: Molecular Pathways and Clinical Applications.

In this article, we provide an extensive review of the recent literature of the signaling pathways modulated by Pulsed Electromagnetic Fields (PEMFs) and PEMFs clinical application. A review of the literature was performed on two medical electronic databases (PubMed and Embase) from 3 to 5 March 2021. Three authors performed the evaluation of the studies and the data extraction. All studies for this review were selected following these inclusion criteria: studies written in English, studies available in full text and studies published in peer-reviewed journal. Molecular biology, identifying cell membrane receptors and pathways involved in bone healing, and studying PEMFs target of action are giving a solid basis for clinical applications of PEMFs. However, further biology studies and clinical trials with clear and standardized parameters (intensity, frequency, dose, duration, type of coil) are required to clarify the precise dose-response relationship and to understand the real applications in clinical practice of PEMFs.

Physical and Biological Properties of a Chitosan Hydrogel Scaffold Associated to Photobiomodulation Therapy for Dental Pulp Regeneration: An In Vitro and In Vivo Study.

BACKGROUND: The regeneration of dental pulp, especially in cases of pulp death of immature teeth, is the goal of the regenerative endodontic procedures (REPs) that are based on tissue engineering principles, consisting of stem cells, growth factors, and scaffolds. Photobiomodulation therapy (PBMT) showed to improve dental pulp regeneration through cell homing approaches in preclinical studies and has been proposed as the fourth element of tissue engineering. However, when a blood clot was used as a scaffold in one of these previous studies, only 30% of success was achieved. The authors pointed out the instability of the blood clot as the regeneration shortcoming. Then, to circumvent this problem, a new scaffold was developed to be applied with the blood clot. The hypothesis of the present study was that an experimental injectable chitosan hydrogel would facilitate the three-dimensional spatial organization of endogenous stem cells in dental pulp regeneration with no interference on the positive influence of PBMT. METHODS: For the in vitro analysis, stem cells from the apical papilla (SCAPs) were characterized by flow cytometry and applied in the chitosan scaffold for evaluating adhesion, migration, and proliferation. For the in vivo analysis, the chitosan scaffold was applied in a rodent orthotopic dental pulp regeneration model under the influence of PBMT (660 nm; power output of 20 mW, beam area of 0.028 cm2, and energy density of 5 J/cm2). RESULTS: The scaffold tested in this study allowed significantly higher viability, proliferation, and migration of SCAPs in vitro when PBMT was applied, especially with the energy density of 5 J/cm2. These results were in consonance to those of the in vivo data, where pulp-like tissue formation was observed inside the root canal. CONCLUSION: Chitosan hydrogel when applied with a blood clot and PBMT could in the future improve previous results of dental pulp regeneration through cell homing approaches.

Influência da fotobiomodulação na viabilidade e proliferação de células-tronco do ligamento periodontal humano cultivadas na superfície de filmes de ácido polilático / Influence of photobiomodulation on the viability and proliferation of human periodontal ligament stem cells cultured on the surface of polylactic acid films

O ácido polilático (PLA) é um biomaterial com diversas aplicações biomédicas e tem se destacado como um arcabouço promissor na engenharia de tecidos principalmente devido à sua biocompatibilidade, fácil manipulação e baixo custo. O laser de baixa intensidade (LBI) tem se mostrado uma ferramenta útil para promover a bioestimulação in vitro de vários tipos celulares. O objetivo deste estudo foi avaliar o efeito da fotobiomodulação com LBI na viabilidade e proliferação de células-tronco do ligamento periodontal humano (hPDLSC) cultivadas em arcabouços de PLA. Filmes de PLA foram produzidos e a topografia da superfície foi avaliada por microscopia eletrônica de varredura (MEV) e microscopia de força atômica (AFM). As hPDLSC foram isoladas, caracterizadas e cultivadas nos filmes de PLA e os espécimes foram divididos em dois grupos: Controle ­ não irradiado; e Laser ­ submetido à irradiação com laser diodo (InGaAIP) com comprimento de onda de 660 nm, potência de 30 mW, e dose única de 1 J/cm², de modo contínuo. As análises de viabilidade celular foram realizadas 24 e 48 horas após a irradiação através do ensaio bioquímico Alamar Blue e do ensaio Live/Dead. Os eventos do ciclo celular foram avaliados por citometria de fluxo e a morfologia da interação célula-biomaterial foi avaliada por MEV. Os filmes produzidos exibiram uma superfície plana e regular, com presença eventual de pequenos poros e rugosidade média de 59,381 nm. Os resultados do ensaio Alamar Blue mostraram uma maior atividade metabólica celular no grupo irradiado em relação ao controle em 24 (p<0,05) e 48 h (p<0,001), o que foi confirmado no ensaio Live/Dead por uma maior densidade de células viáveis no grupo Laser. Na análise do ciclo celular o grupo Laser apresentou um aumento de células na fase G2/M comparado com o grupo Controle (p<0,001). As imagens da MEV mostraram uma maior densidade celular no grupo irradiado, com manutenção da morfologia. Em conjunto, os achados deste estudo demonstraram que fotobiomodulação tem a capacidade de aumentar a viabilidade e proliferação das células-tronco periodontais cultivadas em arcabouços de PLA, o que pode contribuir para o desenvolvimento de novos estudos utilizando este protocolo na engenharia tecidual periodontal (AU).

Polylactic acid (PLA) is a biomaterial with diverse biomedical applications and has been a promising scaffold in tissue engineering mainly due to its biocompatibility, easy manipulation and low cost. Low-level laser irradiation (LLLI) has been shown to be a powerful tool to promote in vitro biostimulation in several cell types. The aim of this study was to evaluate the effectiveness of photobiomodulation with LLLI on the viability and proliferation of human periodontal ligament stem cells (hPDLSC) cultured on PLA scaffolds. PLA films were produced by solvent casting method and the surface topography was evaluated by scanning electronic microscopy (SEM) and atomic force microscopy (AFM). hPDLSC were isolated, characterized and cultured on the PLA films. Two groups were evaluated: Control - non irradiated; and Laser - irradiated with diode laser (InGaAIP) with wavelength of 660 nm, power of 30 mW, and a single dose of 1 J/cm² with radiation emitted continuously. Cell viability analyzes were performed 24 and 48 hours after irradiation using the the Alamar Blue biochemical assay and Live/Dead assay. Cell cycle events were assessed by flow cytometry and cell-biomaterial morphological interaction was evaluated by SEM. The films produced showed a flat and regular surface, with the occasional presence of small pores and an average roughness of 59.381 nm. The results of Alamar Blue assay showed a greater cell metabolic activity in irradiated group compared to control at 24 (p<0.05) and 48 h (p<0.001), which was confirmed in the Live/Dead assay by a higher density of viable cells in the Laser group. In the analysis of the cell cycle, the Laser group showed an increase of cells in the G2/M phase compared to the Control group (p <0.001). SEM images showed a higher cell density in the irradiated group, with maintenance of cell morphology. Taken together, the findings of this study demonstrated that photobiomodulation has the ability to increase the viability and proliferation of periodontal stem cells cultured on PLA scaffolds, which may contribute to the development of new studies using this protocol in periodontal tissue engineering (AU).

Comparison of responses of melanocyte lineages from p75(+) and p75(-) human scalp-derived neural crest stem cells under phototherapy.

Phototherapy is an effective therapeutic option in the treatment of vitiligo; however, responses varied among the different types. The underlying mechanism has scarcely been investigated. To investigate and compare the effects of phototherapy on the mutation of melanocyte lineage differentiated from human scalp-derived neural crest stem cells (HS-NCSCs) with p75 neurotrophin receptor expression positive and p75 neurotrophin receptor expression negative group in vitro, the HS-NCSCs were isolated from fetal scalp tissue, which is identified by immunofluorescent staining. The p75(+) and p75(-) cells from HS-NCSCs were isolated by magnetic cell sorting, respectively. The embryonic neural crest stem cell biomarkers were detected by RT-PCR. Narrow-band UVB (NB-UVB) was used to irradiate the cells. Cell proliferation was evaluated by cell count. Tyrosinase, Tyrp1, and Tyrp2 gene expression were measured by quantitative RT-PCR. Tyrosinase and GRCR protein levels were investigated by Western blot analysis. The electrophoretic strip showed that Sox2, Oct4, Sox10, and Nestin of p75(+) HS-NCSCs were brighter than the p75(-) HS-NCSCs. After the same dose radiation with NB-UVB, the cell proliferation of p75(+) group showed less inhibitory rate compared with the p75(-) HS-NCSCs. The tyrosinase mRNA and protein expression of differentiated melanocytes increased significantly in the group of p75(+) HS-NCSCs compared with the p75(-) group. The melanocytic mutation of p75(+) HS-NCSCs increased significantly compared with the p75(-) HS-NCSCs under NB-UVB, which indicated there were more melanocyte precursors in the differentiated cells from p75(+) HS-NCSCs. This may provide new insights for the different repigmentation efficacy of segmental and non-segmental vitiligo.

Comparative evaluation of low-level laser therapy on proliferation of long-term cryopreserved human dental pulp cells isolated from deciduous and permanent teeth.

The aim of the current study was to evaluate the proliferative effect of low-level laser therapy on long-term cryopreserved dental pulp stem cells (DPSCS) and stem cells from human exfoliated deciduous teeth (SHEDS). The DPSCS and SHEDS were divided into 2 main groups according to gallium aluminum arsenide (GaAIAs) diode laser irradiation densities as 5 J/cm2 and 7 J/cm2. Each main group was further divided into 4 groups according to laser irradiation periods as 0, 24, 48, 72 h groups. During the incubation periods, cells received laser irradiation in every 24 h according to their groups and were put into incubator after irradiation. Cell groups that were not subjected to laser irradiation were served as control groups. Viabilities of cells were determined via MTT assay at the end of all incubation periods, and data were statistically analyzed. Laser irradiation demonstrated significant effects on proliferation rate of DPSCs and SHEDs in comparison with control. Intragroup comparison data of DPSCS revealed that repetitive laser irradiation for long term (72 h) increased the cellular viability significantly in comparison with all other treatment groups; however, no significant differences were found when energy densities were compared within each time interval, except for 48 h group at which irradiation with 7 J/cm2 provided significantly higher cell viability rates of SHEDS. DPSCs showed significantly higher cellular viability than SHEDs only for the 7 J/cm2 energy density in 72 h. Longer term (72 h) repetitive laser irradiation with energy densities of 5 and 7 J/cm2 (wavelength of 980 nm) may be recommended to induce the proliferative effect on long-term cryopreserved DPSCS and SHEDS.

Ghrelin reverts intestinal stem cell loss associated with radiation-induced enteropathy by activating Notch signaling.

BACKGROUD: Exposure to high-dose radiation, such as after a nuclear accident or radiotherapy, elicits severe intestinal damage and is associated with a high mortality rate. In treating patients exhibiting radiation-induced intestinal dysfunction, countermeasures to radiation are required. In principle, the cellular event underlying radiation-induced gastrointestinal syndrome is intestinal stem cell (ISC) apoptosis in the crypts. High-dose irradiation induces the loss of ISCs and impairs intestinal barrier function, including epithelial regeneration and integrity. Notch signaling plays a critical role in the maintenance of the intestinal epithelium and regulates ISC self-renewal. Ghrelin, a hormone produced mainly by enteroendocrine cells in the gastrointestinal tract, has diverse physiological and biological functions. PURPOSE: We investigate whether ghrelin mitigates radiation-induced enteropathy, focusing on its role in maintaining epithelial function. METHODS: To investigate the effect of ghrelin in radiation-induced epithelial damage, we analyzed proliferation and Notch signaling in human intestinal epithelial cell. And we performed histological analysis, inflammatory response, barrier functional assays, and expression of notch related gene and epithelial stem cell using a mouse model of radiation-induced enteritis. RESULTS: In this study, we found that ghrelin treatment accelerated the reversal of radiation-induced epithelial damage including barrier dysfunction and defective self-renewing property of ISCs by activating Notch signaling. Exogenous injection of ghrelin also attenuated the severity of radiation-induced intestinal injury in a mouse model. CONCLUSION: These data suggest that ghrelin may be used as a potential therapeutic agent for radiation-induced enteropathy.

Interplay among p21Waf1/Cip1, MUSASHI-1 and Krüppel-like factor 4 in activation of Bmi1-CreER reserve intestinal stem cells after gamma radiation-induced injury.

Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

Preconditioning adipose-derived stem cells with photobiomodulation significantly increased bone healing in a critical size femoral defect in rats.

We assessed the combined impacts of human demineralized bone matrix (hDBM) scaffold, adipose-derived stem cells (hADS), and photobiomodulation (PBM) on bone repair of a critical size femoral defect (CSFD) in 72 rats. The rats were divided into six groups: control (group 1); ADS (group 2 - ADS transplanted into hDBM); PBM (group 3 - PBM-treated CSFDs); ADS + PBM in vivo (group 4 - ADS transplanted into hDBM and the CSFDs were treated with PBM in vivo); ADS + PBM in vitro (group 5 - ADS were treated with PBM in vitro, then seeded into hDBM); and ADS + PBM in vitro+in vivo (group 6 - PBM-treated ADS were seeded into hDBM, and the CSFDs were treated with PBM in vivo. At the anabolic phase (2 weeks after surgery), bone strength parameters of the groups 5, 6, and 4 were statistically greater than the control, ADS, and PBM in vivo groups (all, p = 0.000). Computed tomography (CT) scans during the catabolic phase (6 weeks after surgery) of bone healing revealed that the Hounsfield unit (HU) of CSFD in the groups 2 (p = 0.000) and 5 (p = 0.019) groups were statistically greater than the control group. The groups 5, 4, and 6 had significantly increased bone strength parameters compared with the PBM in vivo, control, and ADS groups (all, p = 0.000). The group 5 was statistically better than the groups 4, and 6 (both, p = 0.000). In vitro preconditioned of hADS with PBM significantly increased bone repair in a rat model of CSFD in vivo.

The effect of photobiomodulation on human dental pulp-derived stem cells: systematic review.

This systematic review assessed if photobiomodulation of human dental pulp tissue improved cell viability, proliferation, and/or differentiation compared with a placebo. This systematic review was conducted in line with PRISMA. PICO question was established; inclusion and exclusion criteria were established before a search had begun. A literature search was conducted through PubMed, Scopus, and Cochrane. Studies were included if published within the last 20 years in English language, or where translation was available; laser parameters were mentioned; human dental pulp tissue was studied in vitro. Studies were excluded if non-human dental pulp tissue was studied and where the study was an in vivo study. Out of the total 121 studies found, 109 were excluded. Of the twelve included studies, three full-text articles were not available despite attempts made to contact the respective authors, leaving nine studies. Four of the included studies reported the use of stem cells derived from human deciduous teeth (SHEDs), and five used those from human permanent teeth (DPSCs). Most included studies utilized InGaAlP laser with wavelengths 660 nm, and one study with 610 nm. Other types of lasers included LED InGaN, and GaAlAs. Out of all included studies, two had a moderate risk of bias, and the rest had a low risk of bias. All studies confirmed positive effects on proliferation. One study also found improved osteogenic differentiation of the stem cells derived from stem cells of deciduous teeth. After assessing SHEDs and DPSCs separately, it is found that photobiomodulation improved cell proliferation in both subgroups. Due to heterogeneity in design protocols and laser parameters, it was not possible to compare the studies together. However, this study indicated that cell viability and proliferation did improve with photobiomodulation.

Biological Responses of Stem Cells to Photobiomodulation Therapy.

BACKGROUND: Stem cells have attracted the researchers interest, due to their applications in regenerative medicine. Their self-renewal capacity for multipotent differentiation, and immunomodulatory properties make them unique to significantly contribute to tissue repair and regeneration applications. Recently, stem cells have shown increased proliferation when irradiated with low-level laser therapy or Photobiomodulation Therapy (PBMT), which induces the activation of intracellular and extracellular chromophores and the initiation of cellular signaling. The purpose of this study was to evaluate this phenomenon in the literature. METHODS: The literature investigated the articles written in English in four electronic databases of PubMed, Scopus, Google Scholar and Cochrane up to April 2019. Stem cell was searched by combining the search keyword of "low-level laser therapy" OR "low power laser therapy" OR "low-intensity laser therapy" OR "photobiomodulation therapy" OR "photo biostimulation therapy" OR "LED". In total, 46 articles were eligible for evaluation. RESULTS: Studies demonstrated that red to near-infrared light is absorbed by the mitochondrial respiratory chain. Mitochondria are significant sources of reactive oxygen species (ROS). Mitochondria play an important role in metabolism, energy generation, and are also involved in mediating the effects induced by PBMT. PBMT may result in the increased production of (ROS), nitric oxide (NO), adenosine triphosphate (ATP), and cyclic adenosine monophosphate (cAMP). These changes, in turn, initiate cell proliferation and induce the signal cascade effect. CONCLUSION: The findings of this review suggest that PBMT-based regenerative medicine could be a useful tool for future advances in tissue engineering and cell therapy.

Photobiomodulation therapy improves human dental pulp stem cell viability and migration in vitro associated to upregulation of histone acetylation.

This in vitro study evaluated the role of photobiomodulation therapy (PBMT) on viability and migration of human dental pulp stem cells (hDPSCs) and its association to epigenetic mechanisms such as histone acetylation. The hDPSCs were characterized and assigned into control and PBMT groups. For the PBMT, five laser irradiations at 6-h intervals were performed using a continuous-wave InGaAlP diode laser. Viability (MTT), migration (scratch), and histone acetylation H3 (H3K9ac immunofluorescence) were evaluated immediately after the last irradiation. PBMT significantly increased the viability (P = 0.004). Also, PBMT group showed significantly increased migration of cells in the wound compared to the control in 6 h (P = 0.002), 12 h (P = 0.014) and 18 h (P = 0.083) being faster than the control, which only finished the process at 24 h. PBMT induced epigenetic modifications in hDPSC due to increased histone acetylation (P = 0.001). PBMT increased viability and migration of hDPSCs, which are related with the upregulation of histone acetylation and could be considered a promising adjuvant therapy for regenerative endodontic treatment.

Does photobiomodulation change the synthesis and secretion of angiogenic proteins by different pulp cell lineages?

This study aimed to compare the synthesis and secretion of VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, and FGF-2 between pulp fibroblasts from human primary teeth (HPF) and stem cell from human deciduous teeth (SHED) before and after photobiomodulation. HPF were obtained from explant technique and characterized by immunohistochemistry, while SHED were obtained from digestion technique and characterized by flow cytometry. HPF (control group) and SHED were plated, let to adhere, and put on serum starvation to synchronize the cell cycles prior to photobiomodulation. Then, both cell lineages were irradiated with 660-nm laser according to the following groups: 2.5 and 3.7 J/cm2. MTT and crystal violet assays respectively verified viability and proliferation. ELISA Multiplex Assay assessed the following proteins: VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, FGF-2, at 6, 12, and 24 h after photobiomodulation, in supernatant and lysate. Two-way ANOVA/Tukey test evaluated cell viability and proliferation, while angiogenic production and secretion values were analyzed by one-way ANOVA (P < .05). Statistically similar HPF and SHED viability and proliferation patterns occurred before and after photobiomodulation (P > .05). HPF exhibited statistically greater values of all angiogenic proteins than did SHED, at all study periods, except for FGF-2 (supernatant; 12 h); VEGFR1 (lysate; non-irradiated; 12 h); and VEGFR1 (lysate; non-irradiated; 24 h). Photobiomodulation changed the synthesis and secretion of angiogenic proteins by HPF. HPF produced and secreted greater values of all tested angiogenic proteins than did SHED before and after irradiation with both energy densities of 2.5 and 3.7 J/cm2.

Modulatory effect of photobiomodulation on stem cell epigenetic memory: a highlight on differentiation capacity.

Differentiation potential of stem cells into various lineages makes these cells as promising sources to treat multiple diseases. In this regard, the use of different strategies and protocols to increase differentiation capacity is highly demanded. Low-level laser therapy, a relatively noninvasive technique, has the capacity to accelerate the healing of numerous injuries and a portion of restorative capacity could be correlated with the stem cell activation and differentiation. Several mechanisms have been diagnosed to participate in orientation of stem cells to functional mature cells. Among them, the status of DNA methylation orchestrates the maintenance of tissue-specific gene expression during the differentiation procedure. DNA methylation is a momentous event in embryogenesis and functional maturation. This review article highlighted the potency of laser irradiation (low-level intensities) in the differentiation of stem cells by modulation of methylation. The analysis of these modalities could help us to understand the underlying mechanisms participating in the therapeutic effects of photobiomodulation.

Photobiomodulation with 630 plus 810 nm wavelengths induce more in vitro cell viability of human adipose stem cells than human bone marrow-derived stem cells.

The goal of the current experiment is to explore the influence of combined and/or single applications of red and near infrared (NIR) photobiomodulation (PBM) at different wavelengths, energy densities and times on cell viability, population doubling time (PDT), and apoptosis of in vitro cultures of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and h adipose-derived stem cells (hASCs). Both in vitro hBM-MSCs and hASCs were irradiated with 36 protocols using two different laser types (helium­neon [He-Ne] and diodes), four different laser wavelengths (HeNe laser, 630 nm, 810 nm, 630 + 810 nm); three different energy densities (0.6 J/cm2, 1.2 J/cm2, 2.4 J/cm2); and three different PBM times (1, 2, and 3). One-way ANOVA analysis showed that PBM with the 630 nm red laser significantly stimulated cellular viability of both hBM-MSCs and hASCs. The 630 nm red laser significantly decreased PDT of hBM-MSCs. One-way ANOVA demonstrated that the 630 + 810 laser significantly stimulated cellular viability, and significantly decreased PDT and apoptosis of hBM-MSCs and hASCs. Two-way ANOVA analysis showed that PBM with the 630 nm red laser and 630 + 810 nm laser significantly stimulated cellular viability of hASCs compared to the control hASCs, and experimental and control hBM-MSCs. Our study demonstrated that PBM with the combined 630 + 810 nm lasers significantly stimulated cell viability, and significantly decreased PDT and apoptosis of hBM-MSCs and hASCs in vitro. We reported new in vitro evidence where PBM administered at 630 nm (one and two times, 0.6 and 1.2 J/cm2) and 630 + 810 nm (three times, 2.4 J/cm2) significantly increased hASC cell viability compared to its control and the PBM-treated hBM-MSC groups.

Effect of single and multiple doses of low-level laser therapy on viability and proliferation of stem cells from human exfoliated deciduous teeth (SHED).

The present study aimed to evaluate in vitro whether the low-level laser (LLL) delivering fractionated total energy (multiple irradiation) or single irradiation stimulates regeneration-associated events (viability and proliferation) in stem cells from human exfoliated deciduous teeth (SHED). Cells received LLL irradiation (InGaAlP-660 nm), according to the following experimental groups: G1 (single irradiation 2.5 J/cm2, 10 mW, 10 s, 0.10 J), G2 (single irradiation 5.0 J/cm2, 10 mW, 20 s, 0.20 J), G3 (single irradiation 7.5 J/cm2, 10 mW, 30 s, 0.30 J), G4 (two irradiations 2.5 J/cm2, 10 mW, 10 s; total energy 0.20 J), G5 (three irradiations 2.5 J/cm2, 10 mW, 10 s; total energy 0.30 J), and G6 (non-irradiated). Cell viability was assessed by MTT and trypan blue exclusion (TBE) methods, while cell proliferation was evaluated by crystal violet (CV) and sulforhodamine B (SRB) assays after 24, 48, and 72 h after the first irradiation. By MTT, there was no difference between groups at 24 and 72 h. At 48 h, the groups subjected to multiple irradiation (G4 and G5) presented higher cell viability rates. The average percentages of viable cells for all groups by TBE method were 91.04%, 96.63%, and 97.48% at 24, 48, and 72 h, respectively. By CV, there was no significant difference between groups at 24 and 48 h; at 72 h, G2, G3, and G4 presented higher cell proliferation. By SRB, G1 and G4 presented lower proliferation rates in all the periods. When the groups presenting the same total energy were compared, G2 (0.20 J) presented lower cell viability rates and higher cell proliferation rates in comparison with G4; G3 (0.30 J) presented similar results to those of G5, with higher cell viability and proliferation. The application of laser delivering fractionated total energy (two or three applications of 2.5 J/cm2) induced higher cell viability at 48 h, while the single irradiation with 2.5 J/cm2 did not stimulate metabolic activity in such period and the proliferation over time. The 5.0 and 7.5 J/cm2 single doses and the three applications of 2.5 J/cm2 maintained cell viability and stimulated proliferation of SHED at 72 h.