Effects of 970 nm diode laser irradiation on morphology, proliferation, and differentiation of gingival mesenchymal stem cells / Efectos de la irradiación con láser de diodo de 970 m sobre la morfología, la proliferación y la diferenciación de las células madre mesenquimales gingivales

Objective: The objective of this study was to investigate the morphology, proliferation, and differentiation of gingival mesenchymal stem cells (GMSCs) irradiated with a 970 nm Diode Laser (LLLT). It is essential to validate the efficacy of treatment, optimize irradiation conditions and guarantee the safety and quality of stem cells for future use in dental applications. Materials and Methods: GMSCs were cultured in standard conditions and irradiated with a Diode laser (970 nm, 0.5W) with an energy density of 9J/cm2. Cell proliferation was assessed with the WST-1 proliferation kit. GMSCs were differentiated into chondrogenic and osteogenic lineages. Cell morphology was performed with Hematoxylin/eosin staining, and quantitative nuclear analysis was done. Cell viability was monitored with trypan blue testing. Results: GMSCs subjected to irradiation demonstrated a significant increase in proliferation at 72 hours compared to the non-irradiated controls (p=0.027). This indicates that the 970 nm diode laser has a stimulatory effect on the proliferation of GMSCs. LLLT-stimulated GMSCs exhibited the ability to differentiate into chondrogenic and osteogenic lineages. A substantial decrease in cell viability was observed 24 hours after irradiation (p=0.024). However, after 48 hours, the cell viability recovered without any significant differences. This indicates that there might be a temporary negative impact on cell viability immediately following irradiation, but the cells were able to recover and regain their viability over time. Conclusions: This study support that irradiation with a 970 nm diode laser could stimulate the proliferation of GMSCs, maintain their ability to differentiate into chondrogenic and osteogenic lineages, and has minimal impact on the mor- phological characteristics of the cells. These results support the potential use of NIR Lasers in combination with GMSCs as a promising strategy for dental treatments.

Objetivo: El objetivo de este estudio fue investigar la morfología, proliferación y diferenciación de las células madre mesenquimatosas (GMSC) irradiadas con un láser de diodo de 970 nm (LLLT). Es fundamental validar la eficacia del tratamiento, optimizar las condiciones de irradiación y garantizar la seguridad y calidad de las células madre para su uso futuro en aplicaciones dentales.Materiales y Métodos: Las GMSC se cultivaron en condiciones estándar y se irradiaron con un láser de diodo (970 nm, 0,5 W) con una densidad de energía de 9 J/cm2. La proliferación celular se evaluó con el kit de proliferación WST-1. Las GMSC se diferenciaron en linajes condrogénicos y osteogénicos. La morfología celular se realizó con tinción de hematoxilina/eosina y se realizó un análisis nuclear cuantitativo. La viabilidad celular se controló con prueba de azul de tripano. Resultados: Las GMSC sometidas a irradiación demostraron un aumento significativo en la proliferación a las 72 horas en comparación con los controles no irradiados (p=0,027). Esto indica que el láser de diodo de 970 nm tiene un efecto estimulante sobre la proliferación de GMSC. Las GMSC estimuladas con LLLT exhibieron la capacidad de diferenciarse en linajes condrogénicos y osteogénicos. Se observó una disminución sustancial de la viabilidad celular 24 horas después de la irradiación (p=0,024). Sin embargo, después de 48 horas, la viabilidad celular se recuperó sin diferencias significativas. Esto indica que podría haber un impacto negativo temporal en la viabilidad de las células inmediatamente después de la irradiación, pero las células pudieron recuperarse y recuperar su viabilidad con el tiempo. Conclusión: En conclusión, este estudio respalda que la irradiación con un láser de diodo de 970 nm podría estimular la proliferación de GMSC, mantener su capacidad para diferenciarse en linajes condrogénicos y osteogénicos y tiene un impacto mínimo en las características morfológicas de las células. Estos resultados respaldan el uso potencial de láseres NIR en combinación con GMSC como una estrategia prometedora para tratamientos dentales.

Hemolasertherapy: A Novel Procedure for Gingival Papilla Regeneration-Case Report.

BACKGROUND: Interdental papilla is of major importance to patients' orofacial aesthetics, especially regarding anterior teeth as part of the smile's harmony. Loss of gingival tissue, which constitutes interdental papilla, forms what in odontology is called black spaces. This loss, besides affecting the smile's aesthetics, also provokes phonetic and functional damage. OBJECTIVE: The objective of the authors is to present the result of three clinical cases treated with an innovative technique called hemolasertherapy, which stimulates growth of gingival papilla and thus permanently fills in the black spaces. METHODS: The photobiomodulation therapy (PBMT) used a 660 nm diode laser (Laser Duo, MMO-São Carlos, SP, Brazil), punctual, contact mode in two steps: before the bleeding (first PBMT) and immediately after bleeding (second PBMT). Parameters used were power output: 100 mW, CW; diameter tip: 5 mm; spot area: 0.19 cm2; irradiation exposure time per point: 20 sec; 14 points per daily session; total of 2 sessions, with a 1-week interval; E: 2 J per point; E: per daily session, 28 J; irradiance per point: 0.52 W/cm2; fluence per point: 10.4 J/cm2. Total in two daily sessions: total energy: 56 J; total fluence: 294.75 J/cm, 560 sec total time. An in vitro preliminary study was simultaneously carried out to demonstrate what could happen at cellular level in hemotherapy clinical cases associated with PBMT laser application. RESULTS: This initial study demonstrated that the blood clot originated from the bleeding provoked in the gingival area is rich in mesenchymal stem cells. PBMT enables preservation, viability, and further differentiation, stimulating the return of gingival stem cells, which would support their survival and differentiation in the blood clot, thus favoring interdental papilla regeneration. CONCLUSIONS: Follow-up was done for a time span of 4-5 years and considered excellent with regard to papilla preservation.

Reversal of drug-induced gingival overgrowth by UV-mediated apoptosis of gingival fibroblasts - an in vitro study.

Gingival overgrowth (GO) is an undesirable result of certain drugs like Cyclosporine A (CsA). Histopathology of GO shows hyperplasia of gingival epithelium, expansion of connective tissue with increased collagen, or a combination. Factors such as age, gender, oral hygiene, duration, and dosage also influence onset and severity of GO. One of the mechanisms behind uncontrolled cell proliferation in drug-induced GO is inhibition of apoptotic pathways, with a consequent effect on normal cell turnover. Our objective was to determine if UV photo-treatment would activate apoptosis in the gingival fibroblast component. Human gingival fibroblast cells (HGF-1) were exposed to 200ng/ml or 400ng/ml CsA and maintained for 3, 6, and 9 days, followed by UV radiation for 2, 5, or 10min (N=6). Naïve (no CsA or UV), negative (UV, no CsA), and positive controls (CsA, no UV) were designated. Prior to UV treatment, growth media was replaced with 1M PBS to prevent absorption of UV radiation by serum proteins, and cells were incubated in growth media for 24h post-UV before processing for TUNEL assay, cell proliferation assays, or immunofluorescence. Data showed a temporal increase in proliferation of HGF-1 cells under the influence of CsA. The 200ng/ml dose was more effective in causing over-proliferation. UV treatment for 10min resulted in significant reduction in cell numbers, as evidenced by counts and proliferation assays. Our study is a first step to further evaluate UV-mediated apoptosis as a mechanism to control certain forms of GO.

Effectiveness of laser adjunctive therapy for surgical treatment of gingival recession with flap graft techniques: a systematic review and meta-analysis.

Various flap graft techniques in the treatment of gingival recession have already been reported in the literatures for root coverage. Laser therapy has effects of ablative, hemostatic, and decontamination. Therefore, we performed a meta-analysis of randomized controlled trials (RCTs) to compare the efficacy of flap surgery combined with laser with surgery alone for treating gingival recession. The studies were searched from PubMed, Embase, Web of science, and the Cochrane Central Register of Controlled Trials by two reviewers up to August 2017. The quality of RCTs was assessed by Cochrane Handbook. Data were extracted from studies and analyzed by Review Manager 5.3. 95% confidence interval (CI) and risk ratio (RR) were calculated for dichotomous data. Seven RCTs with 173 patients and 296 teeth were included in the meta-analysis. We found no statistically significant differences between two groups in GRD (gingival recession depth) (P = 0.21), GRW (gingival recession width) (P = 0.92), RES (root esthetic score) (P = 0.21), and CRC (complete root coverage) (P = 0.09). Statistically significant differences were found between two groups in the WKT (width of keratinized tissue) (P < 0.0001) and 1-year follow-up of PD (probing depth) (P = 0.03) and CAL (clinical attachment level) (P < 0.00001). The meta-analysis found that surgery with laser therapy provided clinical advantages in terms of WKT and 1-year follow-up of PD and CAL. However, flap graft associated with laser did not offer additional benefit to root coverage and esthetics in treating gingival recession. More long-term studies are required to assess these parameters.

Role of the adjunctive antimicrobial photodynamic therapy to periodontal treatment at plasmatic oxidative stress and vascular behavior.

BACKGROUND: To evaluate for the first time in vivo the effects of methylene blue (MB) photosensitizer dissolved in ethanol in antimicrobial photodynamic therapy (aPDT) as adjuvant periodontal treatment, at plasmatic oxidative stress and vascular behavior in rat model. METHODS: Wistar rats were divided into negative control (NC, no periodontitis) and positive control (PC, with periodontitis, without any treatment). The other groups had periodontitis and were treated with scaling and root planing (SRP); SRP+aPDT+MB dissolved in water (aPDT I); SRP+aPDT+MB dissolved in ethanol (aPDT II). The periodontitis was induced by ligature at the mandibular right first molar. At 7/15/30days, rats were euthanized, the plasma was used to determine oxidative stress parameters and gingival tissue for histomorphometric analysis. RESULTS: PC showed higher thiobarbituric acid reactive substances levels in 7/15/30days. aPDT II was able to block the lipid peroxidation, especially between 15th and 30th days. Glutathione reduced levels were consumed in PC, aPDT I and II groups throughout the experiment. aPDT II increased the vitamin C levels which were restored in this group in the 30th day. aPDT II group showed the highest number of blood vessels. CONCLUSION: In summary, the aPDT with MB dissolved in ethanol provides better therapeutic responses in periodontitis treatment.

Effects of two wattages of low-level laser therapy on orthodontic tooth movement.

INTRODUCTION: Mixed outcomes have been found in animal and clinical studies with regard to the use of low-level laser therapy (LLLT) as a modality to accelerate orthodontic tooth movement (OTM). One major reason for the variable findings is the different methodologies and protocols for laser therapy use. OBJECTIVE: The aim of this study was to determine whether orthodontically moved molars exposed to two different wattages at the same energy density of LLLT exhibited differences in the amount of tooth movement and molecular and histological changes in the adjacent periodontal areas. METHODS: An orthodontic force was applied to rat upper first molars exposed to 500mW (EX-500) and 1000mW (EX-1000) of laser application, with a control group (CT) with no laser application. Gene expression in the periodontal ligament (PDL) and histology of the palatal gingiva of the molars were analyzed. RESULTS: There was a statistically significant difference for OTM between EX-500 but not between EX-1000 and CT groups. RANKL and MMP-13 expression levels in the PDL of orthodontically moved molars, however, were increased significantly in laser-exposed groups compared to CT. Early signs of dysplasia were observed in over half of the animals in the EX-1000 group. CONCLUSIONS: Our results provide evidence for molecular changes and the potential dysplastic effects of laser on the surrounding soft tissues. Further studies are needed to better identify an optimum laser protocol to maximize the desired effect.

Effects of laser biostimulation on the epithelial tissue for keratinized layer differentiation: an in vitro study.

Gingival augmentation techniques proposed in the international literature do not exclude a surgical component, which determines consequent post-surgical discomfort and results are not always predictable. In recent years, the introduction of laser biostimulation has led to a less invasive approach, particularly in the treatment of periodontally compromised patients, limiting the surgical phase to seriously compromised cases, with regeneration techniques for the restoration of a correct periodontal tissue anatomy. The aim of this in vitro study is to establish the validity of laser biostimulation in order to develop the epithelial keratinized layer of the tissue by stimulating fibroblasts-keratinocytes organotypic cultures and fibroblasts and keratinocytes mono-cultures. We created two groups (test and control), each one composed of 3 fibroblast cultures, 3 keratinocyte cultures and 3 organotypic cultures. We performed laser irradiation of test group with Wiser Doctor Smile Lambda, Flat Top Handpiece, at 50 J/cm2 of fluency with one application every 40 h for a total of 5 applications. Forty-eight h after the last laser application, we investigated the presence and amount of keratins 5 and 8 with citofluorymetric and western blotting analyses. Analyses showed an increase in keratin synthesis in test group cultures, showing a remarkable increase in production of keratin 8 in co-cultures test. Laser biostimulation can considerably enhance keratin synthesis when applied with high energy doses and repeated applications to keratinocytes-fibroblasts co-cultures.

Proliferation, migration, and expression of oral-mucosal-healing-related genes by oral fibroblasts receiving low-level laser therapy after inflammatory cytokines challenge.

BACKGROUND AND OBJECTIVES: Increased expression of inflammatory cytokines in the oral cavity has been related to the etiopathogenesis of oral mucositis and to delayed oral mucosal repair. Low-level laser therapy (LLLT) stimulates proliferation and migration of gingival fibroblasts, but the effects of specific inflammatory cytokines on oral mucosal cells and the modulation of these effects by LLLT have not been fully investigated. Therefore, this study investigated the effects of LLLT on oral fibroblasts after being challenged by oral-mucositis-related inflammatory cytokines. METHODS: Human gingival fibroblasts were seeded in plain culture medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours. Then, cells were kept in contact with inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) in serum-free DMEM for 24 hours. After this period, cells were subjected to LLLT with a diode laser device (LaserTABLE, InGaAsP, 780 nm, 25 mW) delivering energy doses from 0.5 to 3 J/cm2 . Irradiation was repeated for 3 consecutive days. Twenty-four hours after the last irradiation, cell migration (wound-healing and transwell migration assays), cell proliferation (BrdU), gene expression of COL-I and growth factors (real-time PCR), and synthesis of COL-I (Sirius Red assay) and VEGF (ELISA) were assessed. Data were subjected to two-way ANOVA and Tukey's tests or Kruskall-Walis and Mann-Whitney tests (P < 0.05). RESULTS: The inflammatory cytokines decreased the migration capacity of gingival fibroblasts. However, a statistically significant difference was observed only for IL-6, detected by transwell assay, where 30% less cells migrated through the pores (P < 0.05) and IL-8, with an increased wound area (116%; P < 0.05), detected by the wound healing method. Cell proliferation was not affected by contact with cytokines, while growth factors and COL-I expression (approximately 80%; P < 0.05), as well as VEGF synthesis (approximately 20%; P < 0.05), were decreased after contact to all tested cytokines. The opposite was seen for total collagen synthesis. LLLT promoted an acceleration of fibroblast migration (30%; P < 0.05) and proliferation (112%; P < 0.05) when delivering 0.5 J/cm2 to the cells previously in contact with the inflammatory cytokines. Gene expression of VEGF (approximately 30%; P < 0.05), and EGF (17%; P < 0.05), was stimulated by LLLT after contact with TNF-α and IL-6. CONCLUSION: LLLT can counteract the negative effects of high concentrations of inflammatory cytokines, especially IL-6 and IL-8 on gingival fibroblast functions directly related to the wound-healing process. Lasers Surg. Med. 48:1006-1014, 2016. © 2016 Wiley Periodicals, Inc.

Evaluation of antimicrobial photodynamic therapy with indocyanine green and curcumin on human gingival fibroblast cells: An in vitro photocytotoxicity investigation.

UNLABELLED: Recent investigations have suggested that antimicrobial photodynamic therapy (aPDT) can be an alternative treatment for the management of periodontal infections. However, currently there is very limited data regarding the photocytotoxicity of this method on human gingival fibroblast (HuGu) cells. AIM: The in vitro optimal concentrations of indocyanine green (ICG) and curcumin as photosensitizers (PSs) and the irradiation time of diode laser emission were evaluated by assessing the photocytotoxicity of the treatment on HuGu cells. MATERIALS AND METHOD: Monolayers of HuGu cells were incubated with various final concentrations of ICG (500, 750, 1000, 1250, 1500, 1750, and 2000µg/ml) and curcumin (3, 4, 5, 10, and 20mM). Three exposure times of the diode laser (30s, 60s, and 2×30s irradiation with an interval of 1min between each) and one of exposure time of 5min for LED were tested; cell viability was determined using neutral red assay. Chlorhexidine (CHX) as a gold standard antimicrobial agent for periodontal disease was considered as a control group. RESULTS: ICG and curcumin significantly reduced HuGu cell viability at concentrations below 1000µg/ml and 10mM, respectively (P<0.01). Cytotoxicity was higher when the cells were treated for 2×30s irradiation with an interval of 1min and then again exposed to the laser for 30s (2% and 0.1%). CHX demonstrated no significant reduction in HuGu cell survival. CONCLUSION: Photocytotoxicity is influenced by PS concentration, exposure time of PS, and time of irradiation. High doses of ICG and curcumin with lowest exposure time of light source and without cytotoxic effects may be an effective strategy for aPDT as an alternative treatment for periodontal disease.

Short- and Medium-Term Effects of Low-Level Laser Therapy on Periodontal Status in Lingual Orthodontic Patients.

OBJECTIVE: The purpose of this study was to evaluate the short- and medium-term effects of low-level laser therapy (LLLT) applied in repeated doses in adults with a healthy periodontium treated by lingual orthodontic appliances. BACKGROUND DATA: Plaque accumulation, in combination with difficulty in removing it in lingual orthodontic patients, can cause gingival inflammation. METHODS: Twelve orthodontic patients scheduled for fixed lingual orthodontic treatment were selected. Clinical measurements [visible plaque index (VPI), bleeding on probing (BOP), and probing depth (PD), and collection of gingival crevicular fluid (GCF)], which was used to measure the levels of interleukin-1beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), was performed before bonding the lingual device, and at the short-term (1, 2, 3 months) and medium-term (12 months) follow-up appointments. For each patient, quadrant 1 or 2 was randomly chosen for irradiation by a diode laser (λ = 670 nm, 190 mW, 6.05 W/cm(2), 60 sec/ tooth) (Laser Group) and the contralateral quadrant was used as the Control Group. RESULTS: In both studied groups, a slight worsening of the periodontal condition was observed, which was evident at the 3rd month follow-up and which was mainly at the lingual side in the Control Group. The levels of IL-1ß in the GCF were significantly increased in the Control Group compared with the Laser Group at the 2nd and 3rd months after bonding. At the 12th month follow-up, an improvement of the inflammation was observed in both groups in the study. CONCLUSIONS: LLLT showed short-term effects by preventing a substantial increase in IL-1ß levels. At medium-term follow-up, LLLT diminished VPI, BOP, and PD scores.

Efficacy of the ND:YAG laser therapy on EBV and HSV1 contamination in periodontal pockets.

AIM: The aim of this retrospective multicenter study was to verify the efficacy of Nd:YAG laser in the treatment of periodontal pockets infected by Epstein-Barr Virus (EBV) and Herpes Simplex Virus 1 (HSV1). METHODS: Subgingival plaque samples of 291 Italian periodontal patients were analyzed by Real Time PCR to evaluate the frequency of both viruses before and after Nd:YAG laser-assisted periodontal treatment. RESULTS: Before treatment, EBV and HSV1 were observed in 29.9% and in 3.8% of periodontal patients respectively, while co-infection with both viruses was detected in 1.7% of cases. Periodontal Nd:YAG laser treatment ("Periodontal Biological Laser-Assisted Therapy", PERIOBLAST) produced statistical significant benefits, especially in EBV periodontal infection: 78.2% of EBV positive patients became EBV-negative following treatment. CONCLUSIONS: Results of this preliminary study highlight that EBV is found in periodontal pockets more frequently than HSV1, supporting the theory of the potential role of EBV in the onset and progression of periodontal disease. Moreover, our data showed that Nd:YAG laser-assisted periodontal treatment (Perioblast) is also effective in case of viral infection, validating evidences that it represents a successful alternative approach to traditional periodontal protocols.

Selective cytotoxic effects of low-power laser irradiation on human oral cancer cells.

BACKGROUND AND OBJECTIVES: Low-power laser irradiation (LPLI) is known to regulate cell proliferation and migration in clinical use. Recent studies have shown that LPLI induces cell death in some certain types of cancer cell lines. However, the cytotoxic selectivity of LPLI for cancer cells is not fully understood. The aim of this study was to compare the cytotoxic effects of LPLI in both human oral cancer OC2 cells and normal human gingival fibroblast (HGF) cells. MATERIALS AND METHODS: LPLI at 810 nm with an energy density from 10 to 60 J/cm(2) was used to irradiate human oral cancer OC2 cells and normal HGF cells. RESULTS: We found that LPLI significantly diminished cell viability of human oral cancer OC2 cells due to cell cycle arrest at the G1 phase and the induction of cell death but that it had no or little effects on cell cycle progression and death in normal HGF cells. Moreover, the production of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP) were elevated in human oral cancer OC2 cells compared with the un-irradiated cells. In contrast, these effects remained unchanged in normal HGF cells after exposure to LPLI. LPLI also induced apoptosis in caspase-3 dependent manner in human oral cancer OC2 cells, a mode of action that could be mediated by ROS and mitochondrial damage. CONCLUSION: Our findings imply LPLI might be a potential therapy for oral cancers.

Blue light irradiation-induced oxidative stress in vivo via ROS generation in rat gingival tissue.

It has been reported that oxidative stress with reactive oxygen species (ROS) generation is induced by blue light irradiation to a living body. Only limited research has been reported in dental field on the dangers of blue light, mostly focusing on cytotoxicity associated with heat injury of dental pulp. We thus performed an in vivo study on oral tissue exposed to blue light. ROS generated upon blue light irradiation of flavin adenine dinucleotide were measured by electron spin resonance spectroscopy. After blue light irradiation, the palatal gingiva of Wistar rats were isolated. Collected samples were subjected to biochemical analysis of lipid peroxidation and glutathione. Singlet oxygen was generated by blue light irradiation, but was significantly quenched in an N-acetyl-L-cysteine (NAC) concentration-dependent manner. Blue light significantly accelerated oxidative stress and increased the oxidized glutathione levels in gingival tissue. These effects were also inhibited by NAC pre-administration. The results suggest that blue light irradiation at clinical levels of tooth bleaching treatment may enhance lipid peroxidation by the induction of oxidative stress and the consumption of a significant amount of intracellular glutathione. In addition, NAC might be an effective supplement for the protection of oral tissues against blue light irradiation-induced oxidative damage.

Anti-inflammatory effect of 635 nm irradiations on in vitro direct/indirect irradiation model.

Low-level laser therapy (LLLT) has been promoted for its beneficial effects on tissue healing and pain relief. As during laser treatment it is possible to irradiate only a small area of the surface body or wound and, correspondingly, of a very small volume of the circulating blood, it is necessary to explain how its photomodification can lead to a wide spectrum of therapeutic effects. To establish the experimental model for indirect irradiation, irradiation with 635 nm was performed on immortalized human gingival fibroblasts (IGFs) in the presence of Porphyromonas gingivalis lipopolysaccharides (LPS). The irradiated medium was transferred to non-irradiated IGFs which were compared with direct irradiated IGFs. The protein expressions were assessed by Western blot, and prostaglandin E2 (PGE2 ) was measured using an enzyme-linked immunoassay. Reactive oxygen species (ROS) were measured by DCF-DA; cytokine profiles were assessed using a human inflammation antibody array. Cyclooxygenase-2 (COX-2) protein expression and PGE2 production were significantly increased in the LPS-treated group and decreased in both direct and indirect irradiated IGFs. Unlike direct irradiated IGFs, ROS level in indirect irradiated IGFs was decreased by time-dependent manners. There were significant differences of released granulocyte colony-stimulating factor (G-CSF), regulated on activated normal T-cell expressed and secreted (RANTES), and I-TAC level observed compared with direct and indirect irradiated IGFs. In addition, in the indirect irradiation group, phosphorylations of C-Raf and Erk1/2 increased significantly compared with the direct irradiation group. Thus, we suggest that not only direct exposure with 635 nm light, but also indirect exposure with 635 nm light can inhibit activation of pro-inflammatory mediators and may be clinically useful as an anti-inflammatory tool.

Irradiation with a low-level diode laser induces the developmental endothelial locus-1 gene and reduces proinflammatory cytokines in epithelial cells.

We demonstrated previously that low-level diode laser irradiation with an indocyanine green-loaded nanosphere coated with chitosan (ICG-Nano/c) had an antimicrobial effect, and thus could be used for periodontal antimicrobial photodynamic therapy (aPDT). Since little is known about the effects of aPDT on periodontal tissue, we here investigated the effect of low-level laser irradiation, with and without ICG-Nano/c, on cultured epithelial cells. Human oral epithelial cells were irradiated in a repeated pulse mode (duty cycle, 10 %; pulse width, 100 ms; peak power output, 5 W). The expression of the developmental endothelial locus 1 (Del-1), interleukin-6 (IL-6), IL-8, and the intercellular adhesion molecule-1 (ICAM-1) were evaluated in Ca9-22 cells stimulated by laser irradiation and Escherichia coli-derived lipopolysaccharide (LPS). A wound healing assay was carried out on SCC-25 cells irradiated by diode laser with or without ICG-Nano/c. The mRNA expression of Del-1, which is known to have anti-inflammatory activity, was significantly upregulated by laser irradiation (p < 0.01). Concurrently, LPS-induced IL-6 and IL-8 expression was significantly suppressed in the LPS + laser group (p < 0.01). ICAM-1 expression was significantly higher in the LPS + laser group than in the LPS only or control groups. Finally, compared with the control, the migration of epithelial cells was significantly increased by diode laser irradiation with or without ICG-Nano/c. These results suggest that, in addition to its antimicrobial effect, low-level diode laser irradiation, with or without ICG-Nano/c, can suppress excessive inflammatory responses via a mechanism involving Del-1, and assists in wound healing.

The effects of pulsed and sinusoidal electromagnetic fields on E-cadherin and type IV collagen in gingiva: a histopathological and immunohistochemical study.

BACKGROUND: The potential beneficial effects of extremely low frequency pulsed and sinusoidal electromagnetic fields have been shown on many tissues. Gingival epithelium plays an important role in immunosurveillance of the periodontal tissues. The epithelium acts as a mechanical barrier through cell junctions such as E-cadherin. OBJECTIVES: Investigation of the effects of extremely low frequency magnetic fields on gingiva. MATERIAL AND METHODS: Twenty-seven male Wistar albino rats were used. The rats were divided into three groups: control group (n = 9), SEMF group (n = 9), PEMF group (n = 9). The SEMF and PEMF (pulse time: 25 µsn, pulse frequency: 50 Hz) groups were subjected to 1.5 mT, 50 Hz, exposure 6 h a day, 5 days a week for 28 days in methacrylate boxes. The gingival tissue pieces processed for routine histological and immunohistochemical examination and tissue sections were stained with H-E and Masson trichrome. In addition, E-cadherin and type IV collagen expressions were examined immunohistochemically. RESULTS: Intraepithelial lymphocytes and proliferation of epithelial cells increased in both electromagnetic field groups. The over-expressions of E-cadherin on gingival epithelium was detected in the PEMF and SEMF groups. The expression level of type IV collagen was not significant between the control and electromagnetic field treated groups, except for a significant increase in the basal cell layer of the PEMF group, as compared to the control and SEMF groups. CONCLUSIONS: PEMF and SEMF have a local pro-inflammatory effect on gingiva, leading to an increase in E-cadherin level but not type IV collagen. Both PEMF and SEMF can be used as a supportive device in the treatment of gingival diseases, especially those which lead to defects in the epithelial barrier.

Modulation of lipopolysaccharide-induced NF-κB signaling pathway by 635 nm irradiation via heat shock protein 27 in human gingival fibroblast cells.

Heat shock protein-27 (HSP27) is a member of the small HSP family which has been linked to the nuclear factor-kappa B (NF-κB) signaling pathway regulating inflammatory responses. Clinical reports have suggested that low-level light therapy/laser irradiation (LLLT) could be an effective alternative treatment to relieve inflammation during bacterial infection associated with periodontal disease. However, it remains unclear how light irradiation can modulate the NF-κB signaling pathway. We examined whether or not 635 nm irradiation could lead to a modulation of the NF-kB signaling pathway in HSP27-silenced cells and analyzed the functional cross-talk between these factors in NF-κB activation. The results showed that 635 nm irradiation led to a decrease in the HSP27 phosphorylation, reactive oxygen species (ROS) generation, I-κB kinase (IKK)/inhibitor of κB (IκB)/NF-κB phosphorylation, NF-κB p65 translocation and a subsequent decrease in the COX-1/2 expression and prostaglandin (PGE(2) ) release in lipopolysaccharide(LPS)-induced human gingival fibroblast cells (hGFs). However, in HSP27-silenced hGFs, no obvious changes were observed in ROS generation, IKK/IκB/NF-κB phosphorylation, NF-κB p65 translocation, nor in COX-1/2 expression, or PGE(2) release. This could be a mechanism by which 635 nm irradiation modulates LPS-induced NF-κB signaling pathway via HSP27 in inflammation. Thus, HSP27 may play a role in regulating the anti-inflammatory response of LLLT.

Irradiation by light-emitting diode light as an adjunct to facilitate healing of experimental periodontitis in vivo.

BACKGROUND AND OBJECTIVE: This study evaluated the biostimulatory effect of 660 nm light-emitting diode (LED) as an adjunct in the treatment of experimental periodontitis. MATERIAL AND METHODS: Ninety-six Sprague-Dawley rats underwent experimental periodontitis by placement of a silk ligature followed with or without additive Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) injection. Irradiation with LED light was performed at varying energy densities of 5, 10 and 15 J/cm2, 1 d after debridement and detoxification. Rats were killed at 3, 7 and 14 d after irradiation with LED light, and the effect of irradiation was evaluated by descriptive histology and quantitative measurements of periodontal bone loss, inflammatory infiltration and cellular proliferation. RESULTS: Reduction of inflammation, accelerated collagen deposition and realignment was noted following irradiation with LED light at densities of 10 and 15 J/cm2, and temporary reduction of periodontal bone loss, as well as bundle bone apposition, was noted at day 3 in rats treated with 10 J/cm2 light. The biomodulatory effect was stronger in sites treated with Pg-LPS injection. In sites without Pg-LPS injection, temporary reduction of inflammation was noted in all LED light-irradiated specimens at day 3. No significant change in cellular proliferation was noted in any LED light-treated group. CONCLUSIONS: LED light (660 nm) with an energy density of 10 J/cm2 appeared suitable as an adjunct modality for periodontitis by temporarily reducing inflammation, facilitating collagen realignment and bundle bone deposition. Future studies will aim to amplify the biostimulatory effect of LED light by adding a supplementary medium or repeated irradiation.

Effect of laser on TNF-alpha expression in inflamed human gingival tissue.

This study sought to evaluate the effect of low-level laser treatment combined with scaling and root planing (SRP) on gingival tissue levels of TNF-alpha in subjects with periodontal disease. Eighty gingival papilla biopsy samples were obtained from 60 patients diagnosed with chronic advanced periodontitis; randomly assigned to three treatment groups (n = 20), as well as 20 subjects with no periodontal disease (group A). Group B received SRP on a single quadrant/day for four consecutive days. On day 5, all quadrants were rescaled. Groups C and D received the same treatment as group B plus laser application with the low-level diode laser (630-670 nm, 1.875 J/cm(2)) for five and ten consecutive days, respectively. Papilla biopsies were obtained from subjects and evaluated by ELISA for levels of TNF-alpha. The values in the control group were 5.2 ± 3.21 pg/mg and baseline values for the examined groups were 46.01 ± 16.69. Significantly decreased level of TNF-alpha for groups C and D was found after treatment, while group B demonstrated reduction of TNF-alpha of 31.34%. The results of this study show suppression of TNF-alpha in gingival tissue after low-level laser treatment as adjunct to SRP. Data may suggest beneficial anti-inflammatory effects of the laser treatment when used as adjunctive periodontal treatment.