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PLoS One. 2017; 12(1): e0168937.
Published online 2017 Jan 3. doi:  10.1371/journal.pone.0168937
PMCID: PMC5207507

A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK

Yoshiaki Shingyochi, Shigeyuki Kanazawa, Satoshi Tajima, Rica Tanaka, Hiroshi Mizuno, andMorikuni Tobita*
Michael Hamblin, Editor
Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo, Japan
Massachusetts General Hospital, UNITED STATES
Competing Interests: This research was received funding support from The Yoshida Dental MFG. Co., Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
  • Conceptualization: YS MT.
  • Formal analysis: SK.
  • Investigation: YS.
  • Methodology: YS SK ST.
  • Supervision: RT HM.
  • Writing – original draft: YS.
  • Writing – review & editing: HM MT.
Author information ? Article notes ? Copyright and License information ?
Received 2016 Oct 7; Accepted 2016 Dec 8.

Abstract

Background

Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration.

Materials and Methods

Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser.

Results

In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration.

Conclusions

These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts.

Introduction

Wound healing is a complex biological process that involves a cascade of events, including blood coagulation, inflammation, new tissue formation, and tissue remodeling. This process requires the collaborative efforts of several cell types such as keratinocytes, fibroblasts, endothelial cells, and immune cells []. Cell migration, proliferation, differentiation, and extracellular matrix deposition are activated during wound healing. In particular, the proliferation and migration of fibroblasts play crucial roles in the formation of granulation tissue and lead to wound closure. During fibroblast migration and proliferation, several intracellular and intercellular pathways are activated and coordinated [].

Recent studies showed that low-level laser therapy (LLLT) with various types of lasers promotes wound healing through tissue repair and reduces inflammation. The most common methods of irradiation in LLLT include lasers such as helium neon (He-Ne; 632.8 nm), ruby (694 nm), argon (488 and 514 nm), krypton (521, 530, 568, and 647 nm), gallium-aluminum-arsenide (805 and 650 nm), and gallium arsenide (904 nm) []. In the term of LLLT, several terms were introduced such as Photobiomodulation (PBM) therapy, Biostimulation, Cold/Cool Laser, Soft Laser and Low Power Laser Therapy.

Meanwhile, the carbon dioxide (CO2) laser is a long pulsed infrared laser with a wavelength of 10,600 nm, which is absorbed strongly by water []. A high-power CO2laser is widely used in a variety of surgical procedures, including oral surgery and dermatologic surgery, as an alternative to a traditional scalpel []. Recent studies focused on oral fibroblasts in the dental field also reported that LLLT with a CO2 laser promotes wound healing []. In basic research of LLLT with a CO2 laser for wound healing, Kenneth et al. showed that a super-pulsed CO2 laser decreases transforming growth factor-?1 secretion and increases basic fibroblast growth factor secretion of both normal and keloid dermal fibroblasts in vitro, and, as a result, promotes cell replication and may provide the ability to balance collagen organization against fibrosis []. Furthermore, regarding the role of fibroblasts in the process of wound healing, LLLT with other lasers promotes both proliferation and migration []. Moreover, LLLT activates fibroblast signaling pathways such as the extracellular signal-regulated kinase (ERK)/FOXM1 pathway [].

However, the effects of LLLT with a CO2 laser on dermal fibroblast proliferation, migration, and signaling pathways at the cellular level are still not clearly understood.

In the present study, we investigated the effects of LLLT with a CO2 laser on dermal fibroblast proliferation and migration, and examined the involvement of the Akt, ERK, and Jun N-terminal kinase (JNK) pathways in dermal fibroblast proliferation and migration.

Materials and Methods

Cell culture

Human dermal fibroblasts (HDFs) (American Type Culture Collection, VA, USA) were prepared. HDFs (3.0 × 105 cells) were cultured on a 100 mm dish in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, MI, USA) containing 10% fetal bovine serum (FBS) (Gibco BRL), 100 U/mL penicillin, and 100 mg/mL streptomycin (Wako, Tokyo, Japan) in a humidified incubator at 37°C with a 5% CO2 atmosphere.

CO2 laser irradiation

A CO2 laser system (Opelaser Pro, Yoshida Dental Mfg., Tokyo, Japan) operating at a wavelength of 10.6 ?m was used. The CO2 laser system was equipped with a homogenizer attached to the end of the articulated arm in order to equalize the profile of the laser beam. Power densities were generated in a round homogeneous spot with a diameter of 35 mm (Fig 1). The CO2 laser system was used with continuous wave mode and following irradiation power (irradiance, irradiation time); 0.1 J/cm2 (52.08 mW/cm2, 2 sec), 0.5 J/cm2 (52.08 mW/cm2, 10 sec), 1.0 J/cm2 (52.08 mW/cm2, 20 sec), 2.0 J/cm2 (52.08 mW/cm2, 40 sec), and 5.0 J/cm2 (520.83 mW/cm2, 10 sec).

Fig 1

The CO2 laser machine equipped with a homogenizer.

Cell proliferation after LLLT

Cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96®Aqueous One Solution Cell Proliferation Assay, Promega, WI, USA). HDFs at passage 3–5 were plated at a density of 2000 cells per well in 96-well plates, incubated in DMEM containing 10% FBS for 24 h, and then incubated in DMEM containing 1.0% FBS for 24 h. After 48 h cells were plated, HDFs were washed twice with phosphate-buffered saline (PBS), which was aspirated before laser irradiation. HDFs in each well were evenly irradiated at an irradiation power of 0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2 in continuous wave mode at 25°C (room temperature). The non-irradiated group served as the control. HDFs were incubated in DMEM containing 1.0% FBS for 48 h. Thereafter, 20 ?L of CellTiter 96® One Solution Reagent was added to each well of the 96-well assay plate containing HDFs in 100 ?L of culture medium. HDFs were incubated for an additional 2 h at 37°C in a 5% CO2 atmosphere. The production of formazan by viable HDFs was measured as absorbance at 490 nm using a 96-well plate reader. To observe the effects of Akt, ERK, or JNK inhibition, HDFs were treated with an inhibitor of each signaling molecule, namely, 10 mM LY294002 (Jena Bioscience, Berlin, Germany), 10 mM U-0126 (Calbiochem, CA, USA), or 10 mM SP600125 (Calbiochem), respectively, for 60 min before irradiation. After treatment with each inhibitor, the MTS assay was conducted. Nine replicate samples were prepared in each assay. And the assay was repeated three times.

Cell migration after LLLT

HDFs were plated at a density of 8000 cells per well in 96-well plates, incubated in DMEM containing 10% FBS for 24 h, and then incubated in DMEM containing 1.0% FBS for 24 h. Confluent HDFs were wounded using the WoundMaker device (Essen BioScience, MI, USA). Then, HDFs were washed twice with PBS, which was aspirated before laser irradiation. Thereafter, HDFs were irradiated under the same conditions as described for the MTS assay. Images of the wounded cell monolayers were monitored and quantified with the IncuCyte live-cell imager (Essen BioScience) at 0, 6, 12, 18, and 24 h after wounding. The migration rate was expressed as migration distance/time (?m/h). In addition, HDFs were treated with the inhibitors of each signaling molecule (LY294002, U-0126, or SP600125 [10 mM]) for 60 min before wounding. After each inhibitor treatment, the treated HDFs were wounded and monitored for 24 h. Nine replicate samples were prepared in each assay. And the assay was repeated three times.

Western blot analysis

HDFs were plated at a density of 2.0 × 105 cells per 100mm dish, incubated in DMEM containing 10% FBS for 24 h, and then incubated in DMEM containing 1.0% FBS for 24 h. Confluent HDFs were washed twice with PBS, which was aspirated before laser irradiation, and then irradiated with the laser at 1.0 J/cm2. At specified time points after irradiation (5, 15, or 30 min), HDFs were lysed in RIPA buffer, which contained 100 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% deoxycholic acid sodium salt monohydrate (Nacalai Tesque, Kyoto, Japan), and 1.0% Nonidet P-40 (Wako, Osaka, Japan), incubated for 20 min at 4°C, and centrifuged at 15,000 × g for 15 min at 4°C. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane using an iBlot 2 Dry Blotting System (Invitrogen, MA, USA). The membranes were incubated in Tris-buffered saline containing 5% skimmed milk and 0.05% Tween-20 for 60 min and blotted with primary antibodies at 4°C overnight. Anti-p-Akt (Ser473, 1:1000; Cell Signaling Technology, MA, USA), anti-Akt (1:1000; Cell Signaling Technology), anti-p-ERK (Thr202/Tyr204, 1:1000; Cell Signaling Technology), anti-ERK (1:1000; Cell Signaling Technology), anti-p-stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185, 1:1000; Cell Signaling Technology), anti-SAPK/JNK (1:1000; Cell Signaling Technology), and anti-GAPDH (1:2000; Abcam, Cambridge, UK) antibodies were used as primary antibodies. The membranes were incubated for 1 h with an anti-mouse or anti-rabbit horseradish peroxidase-linked secondary antibody (1:10,000; Cell Signaling Technology). Reaction products were visualized by detection of chemiluminescence using ImmunoStar LD (Wako, Osaka, Japan). Relative band densities were quantified using Multi Gauge software (Fujifilm, Tokyo, Japan). For inhibitor studies, HDFs were treated with the inhibitors of each signaling molecule (LY294002, U-0126, or SP600125 [10 mM]) for 60 min before irradiation. After each inhibitor treatment, western blot analyses were conducted using the treated HDFs. Three replicate samples were prepared in each assay. And the assay was repeated three times.

Statistical analysis

Statistical analyses were performed using GraphPad Prism6 software (Graphpad software). All data are presented as the mean ± standard deviation. One-way ANOVA with unpaired samples was used to determine whether there are any statistically significant differences between the groups. A Tukey-Kramer test was then performed to compare groups. Significance was considered at p < 0.05.

Results

LLLT with a CO2 laser stimulated HDF proliferation

HDFs were irradiated under various conditions (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) and incubated for 48 h. The non-irradiated group served as the control. Cell viability was then assessed with the MTS assay. LLLT with a CO2 laser statistically significantly promoted the proliferation of HDFs at doses of 0.5, and 1.0 J/cm2. LLLT with a CO2laser at a dose of 1.0 J/cm2 most effectively promoted cell proliferation in this study (Fig 2).

Fig 2

Effects of LLLT with a CO2 laser on HDF proliferation.

LLLT with a CO2 laser promoted HDF migration

To examine the effect of LLLT with a CO2 laser on dermal fibroblast migration, we monitored wounded HDFs for 24 h upon irradiation with various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2). The non-irradiated group served as the control. Irradiated (0.5 and 1.0 J/cm2) HDFs showed a statistically significant increase in the migration rate at 24 h (14.13363095 and 15.97625 ?m/h, respectively) (Fig 3A). Representative images demonstrated that the migration of irradiated (1.0 J/cm2) HDFs to the site of wounded cell monolayers was promoted at 12 and 24 h compared with the non-irradiated group (Fig 3B).

Fig 3

Effects of LLLT with a CO2 laser on HDF migration.

Activation of Akt, ERK, and JNK was involved in CO2 laser-induced cell proliferation and migration

To examine the molecular mechanisms responsible for the effects of LLLT on fibroblasts, we investigated the involvement of Akt, ERK, and JNK in LLLT-induced fibroblast proliferation and migration. First, we measured the activation of these signaling molecules in response to LLLT stimulation using western blot analysis. Before western blotting, HDFs were irradiated with a CO2 laser at 1.0 J/cm2 and incubated for different durations (0–30 min). Second, we performed MTS and cell migration assays with LY294002, U-0126, or SP600125 (an Akt, ERK, and JNK inhibitor, respectively) to examine how inhibition of these proteins affects fibroblast proliferation and migration.

LLLT stimulation significantly increased levels of p-Akt and p-JNK at 5–15 min and of p-ERK at 15 min (Fig 4). LY294002 significantly inhibited CO2 laser-induced Akt phosphorylation, U-0126 significantly inhibited CO2 laser-induced ERK phosphorylation, and SP600125 significantly inhibited CO2 laser-induced JNK phosphorylation (Fig 5). In the MTS assay, Akt-inhibited and irradiated (1.0 J/cm2) HDFs showed a statistically significant reduction in proliferation after 48 h of incubation compared with irradiated HDFs without Akt inhibition. ERK-inhibited HDFs and JNK-inhibited HDFs showed similar results (Fig 6). In the cell migration assay, Akt-inhibited and irradiated (1.0 J/cm2) HDFs showed a statistically significant reduction in migration at 24 h (10.48365 ?m/h) compared with irradiated HDFs without Akt inhibition (18.12677222 ?m/h). ERK-inhibited and irradiated (1.0 J/cm2) HDFs showed a statistically significant reduction in migration at 24 h (14.74557857 ?m/h) compared with irradiated HDFs without ERK inhibition (18.12677222 ?m/h). JNK-inhibited and irradiated (1.0 J/cm2) HDFs showed a statistically significant reduction in migration at 24 h (8.905873016 ?m/h) compared with irradiated HDFs without JNK inhibition (18.12677222 ?m/h) (Fig 7.) Thus, inhibition of Akt, ERK, and JNK significantly blocked CO2 laser-induced cell proliferation and migration.

Fig 4

Effects of LLLT with a CO2 laser on the activities of Akt, ERK, and JNK.

Fig 5

Effect of inhibition of Akt, ERK, or JNK on LLLT-induced activation of signaling molecules in HDFs.

Fig 6

Effect of inhibition of Akt, ERK, or JNK on LLLT-induced HDF proliferation.

Fig 7

Effect of inhibition of Akt, ERK, or JNK on LLLT-induced HDF migration.

Discussion

High- or low-power laser therapies are used for therapeutic purposes. Low-power (non-surgical) lasers, which are defined as LLLT, are widely used to promote granulation and improve wound repair []. In addition, they also have anti-inflammatory and analgesic effects []. Although LLLT does not have an ablative or thermal mechanism like other medical laser procedures, a photochemical effect causes chemical changes in several tissues []. Mester et al. first reported LLLT as a therapeutic modality, showing that low-energy (1 J/cm2) irradiation with a ruby laser promotes wound healing []. Many reports suggest that LLLT such as gallium arsenide, He-Ne, argon, and ruby lasers, as well as a red light-emitting diode, stimulate wound healing []. Consistent with this, LLLT with a CO2 laser induces proliferation of fibrochondrocytes and gingival fibroblasts []. However, the mechanisms of action underlying the effects of LLLT with a CO2 laser on skin wound healing are not clearly understood [].

Wound healing is a dynamic and complex biological process. Proliferation and migration of dermal fibroblasts have important roles in skin wound repair. Fibroblasts proliferate and migrate to the wound area, compose the new extracellular matrix, and conduce wound healing []. In this study, we focused on the activation of dermal fibroblasts and performed experiments to evaluate the wound healing effect of LLLT with a CO2 laser in vitro.

First, we demonstrated that HDFs irradiated with a CO2 laser at various power levels exhibited increased proliferation and migration. Treatment with a CO2 laser at 1.0 J/cm2promoted fibroblast proliferation the most. Similar results were obtained for fibroblast migration. These data indicate that CO2 laser irradiation at 1.0 J/cm2 is most effective for fibroblast activation in this experiment. From these results, we adopted an irradiation power of 1.0 J/cm2 for subsequent western blotting to analyze the activation of various signaling molecules.

Akt is an important signaling factor for cell survival, proliferation, and migration []. In the present study, we demonstrated that LLLT with a CO2 laser-induced activation of Akt signaling and promoted fibroblast proliferation and migration. These findings suggested that Akt is an important factor for CO2 laser-induced fibroblast activation because the effects of the CO2 laser were inhibited after Akt signaling inhibition.

The MAPK family consists mainly of ERK, JNK, and p38 MAPK. ERK is implicated in the regulation of various cellular processes including cell proliferation, migration, growth, differentiation, and tumor progression []. The JNK pathway is activated by the exposure of cells to several stresses such as heat shock, cytokines, osmotic shock, protein synthesis inhibitors, oxidative stress, ultraviolet radiation, and DNA-damaging agents []. JNK is also involved in many cellular processes [].

This study demonstrated that LLLT with a CO2 laser activated ERK and JNK. Furthermore, their inhibition significantly blocked CO2 laser-induced cell proliferation and migration. These results suggested that CO2 laser-induced fibroblast proliferation and migration also require ERK or JNK activation.

These findings are in line with previous research showing that LLLT with several lasers activates the signaling molecules. Although expression of signaling molecules such as MAPKs differs among different cell types, various studies have shown a correlation between cell proliferation and MAPK stimulation as a reaction to extracellular stimuli []. Miyata et al. reported that MAPK/ERK plays a role in the increased proliferation of human dental pulp cells following low-level diode laser irradiation []. Furthermore, LLLT with a He-Ne laser stimulates Akt activation, which is mediated by PI3K, and activation of the PI3K/Akt signaling pathway is crucial for promoting cell proliferation and migration induced by LLLT [].

A CO2 laser is one of the most commonly used medical lasers, especially in oral surgery and skin surgery, because it is inexpensive compared with other medical lasers and is easy to use. To our knowledge, the present study is the first to investigate the activation of signaling mechanisms in dermal fibroblasts upon LLLT with a CO2 laser. However, further research is required to elucidate the mechanisms upstream or downstream of Akt, ERK, and JNK activation by LLLT with a CO2 laser.

It is not clear how LLLT with a CO2 laser activates the signaling molecules. Some reports suggest that intracellular photobiostimulation of LLLT occurs via the electron transport chain enzymes in mitochondria, which increases cellular metabolism and function []. The photonic energy is converted to chemical energy in the form of ATP, which enhances cellular functions []. However, additional work is needed to elucidate how these mechanisms lead to activation of the signaling molecules.

In conclusion, the present study demonstrated that LLLT with a CO2 laser mediates HDF proliferation and migration by the activation of Akt, ERK, and JNK. Our findings provide novel mechanistic insights into the positive effects of LLLT with a CO2 laser on fibroblast proliferation and migration

Acknowledgments

We are grateful to the Division of Molecular and Biochemical Research, Research Support Center, Juntendo University Graduate School of Medicine for their technical support.

Funding Statement

This work was supported by The Yoshida Dental MFG. Co., Ltd (http://www.yoshida-net.co.jp/en/index.html) Funding to this project and preparation of the carbon dioxide Laser machine to HM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

All relevant data are within the paper.

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Articles from PLoS ONE are provided here courtesy of Public Library of Science
Lasers Med Sci. 2016 Mar 16. [Epub ahead of print]
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Combined effects of low-level laser therapy and human bone marrow mesenchymal stem cell conditioned medium on viability of human dermal fibroblasts cultured in a high-glucose medium.

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Hendudari F1, Piryaei A2,3, Hassani SN4, Darbandi H5, Bayat M6.

Author information

  • 1Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, 1985717443, 19395/4719, Tehran, Iran.
  • 2Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, 1985717443, 19395/4719, Tehran, Iran. piryae@sbmu.ac.ir.
  • 3Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. piryae@sbmu.ac.ir.
  • 4Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
  • 5Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
  • 6Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, 1985717443, 19395/4719, Tehran, Iran. mohbayat@sbmu.ac.ir.

Abstract

Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm2 and energy densities of 0.5, 1, and 2 J/cm2). About 10 % of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm2 was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT?+?HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT?+?hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium.

Lasers Med Sci. 2015 Jan;30(1):375-81. doi: 10.1007/s10103-014-1651-z. Epub 2014 Oct 29.

Effects of 915 nm GaAs diode laser on mitochondria of human dermal fibroblasts: analysis with confocal microscopy.

Belletti S1, Uggeri J, Mergoni G, Vescovi P, Merigo E, Fornaini C, Nammour S, Manfredi M, Gatti R.
Author information
1Unit of Anatomy Histology and Embryology-Department of Biomedical, Biotechnological and Translational Sciences (S.Bi.Bi.T), University of Parma, Parma, Italy.
Abstract
Low-level laser therapy (LLLT) is widely used in tissue regeneration and pain therapy. Mitochondria are supposed to be one of the main cellular targets, due to the presence of cytochrome C oxidase as photo-acceptor. Laser stimulation could influence mitochondria metabolism affecting mainly transmembrane mitochondrial potential. The aim of our study is to evaluate “in vitro” the early mitochondrial response after irradiation with a 915 GaAs laser. Since some evidences suggest that cellular response to LLLT can be differently modulated by the mode of irradiation, we would like to evaluate whether there are changes in the mitochondrial potential linked to the use of the laser treatments applied with continuous wave (CW) in respect to those applied with pulsed wave (PW). In this study, we analyzed effects of irradiation with a 915-nm GaAs diode laser on human dermal fibroblast. We compared effects of irradiation applied with either CW or PW at different fluences 45-15-5 J/cm(2) on transmembrane mitochondrial potential. Laser scanning microscopy (LSM) was used in living cells to detect ROS (reactive oxygen species) using calcein AM and real-time changes of and transmembrane mitochondrial potential following distribution of the potentiometric probe tetramethylrhodamine methyl ester (TMRM). At higher doses (45-15 J/cm(2)), fibroblasts showed a dose-dependent decrement of transmembrane mitochondrial potential in either the modalities employed, with higher amplitudes in CW-treated cells. This behavior is transient and not followed by any sign of toxicity, even if reactive oxygen species generation was observed. At 5 J/cm(2), CW irradiation determined a little decrease (5%) of the baseline level of transmembrane mitochondrial potential, while opposite behavior was shown when cells were irradiated with PW, with a 10% increment. Our results suggest that different responses observed at cellular level with low doses of irradiation, could be at the basis of efficacy of LLLT in clinical application, performed with PW rather than CW modalities.
Avicenna J Med Biotechnol. 2014 Apr-Jun; 6(2): 113–118.

In vitro Therapeutic Effects of Low Level Laser at mRNA Level on the Release of Skin Growth Factors from Fibroblasts in Diabetic Mice

Nooshafarin Kazemi Khoo,1 Mohammad Ali Shokrgozar,2,* Iraj Ragerdi Kashani,3 Amir Amanzadeh,2 Ehsan Mostafavi,4 Hassan Sanati,2 Laleh Habibi,1 Saeid Talebi,1 Morteza Abouzaripour,3 and Seyed Mohammad Akrami1,*
1Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran
2National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
3Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4Department of Epidemiology, Pasteur Institute of Iran, Tehran, Iran
*Corresponding authors: Mohammad Ali Shokrgozar, Ph.D., National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran. Tel: +98 21 66953311, +98 21 88953005. E-mail:ri.ca.ruetsap@razogrkohsam
Seyed Mohammad Akrami, Ph.D., Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran. Tel: +98 21 66953311, +98 21 88953005. E-mail:ri.ca.smut@msimarka
Author information  Article notes  Copyright and License information
Received October 29, 2013; Accepted January 29, 2014.

Introduction

During the past three decades, lasers have been broadly used in medical fields. Since then, numerous scientific studies have been carried out to evaluate laser effects on various cells including fibroblasts (13). One of the most important functional aspects of laser therapy is photobiostimulation effects of low level lasers, often described as lasers with less than 500 mW average power, on various biological systems especially microcirculation (47). There are numerous reports of LLLT effects on wound healing especially diabetic ulcers (6, 810), and its effect in increasing new capillary formation through the release of growth factors (11). Among the many physiological mechanisms of LLLT, it is important to recognize that laser may affect the release of various growth factors.

The growth factors play a role in epithelial cell formation, formation of new fibroblasts and collagen and neovascularization. The release of growth factors from injured and inflammatory cells is, therefore, an important part of wound healing (2, 7, 11). The basic Fibroblast Growth Factor (bFGF) stimulates the proliferation of all cell types involved in wound healing both in vitro and in vivo (12). VEGF is an extremely potent angiogenic stimulator(11). PDGF regulates cell growth and division and plays a significant role in angiogenesis (7). In most of these studies, visible red light is used which is suitable for monolayer cell cultures in vitro. However, in vivo, infrared lights with higher penetration are more useful, though some reports show inhibitory effects of infrared lasers on fibroblasts (3, 13). The aim of this study was to evaluate the effects of 810 nm infrared laser (which have very good results in our clinical use in diabetic patients) on in vitro expression of FGF, PDGF and VEGF from isolated skin fibroblasts in diabetic and nondiabetic mice.

Materials and Methods

Skin fibroblasts were isolated from 4 week old male Balb/c mice, obtained from Pasteur Institute of Iran. Mice were sacrificed using chloroform according to the ethics committee of Tehran University of Medical Sciences. After shaving the back of mice, 1 cm 2 of full thickness skin was cut by scalpel and placed into 70% ethanol for 2min. Then, it was washed with DMEM (Gibco) plus antibiotic (penicillin and streptomycin) three times and was cut into small pieces using scalpel and transferred into 15 ml round bottomed tube. After adding 2 ml of 0.25% trypsin and brief vortexing, it was incubated for 30 min at 37°C in 95% air with 10% CO2 in humidified incubator and vortexed briefly every 10 min. After spinning at 120 g/5 min, supernatant was removed and 4-5 ml of 0.1% collagenase type 2 (invitrogen) was added and incubated for 4 hr at 37°C and was vortexed briefly every 10 min. After spinning at 120 g/5 min, supernatant was transferred to a well of a 6-well plate and the sediment was added into 5 other wells (instead of discarding the supernatant which contained small number of cells, we transferred it to a well to use the cells later) and 1 ml DMEM supplemented with 10% FBS, 2 mM L-glutamine and 25 µg/mlgentamycin was added to each well. Medium was changed to a fresh one every other day. Upon reaching confluency, cells were detached with trypsin and subcultured in two 12-well culture plates. Prior to irradiation, 12-well plates were microscopically verified to guarantee that the cells were confluent and there was no contamination. Following aspiration of 75% DMEM culture medium, irradiation was started. The remaining 25% medium avoided dehydration of the fibroblasts through irradiation. One plate was irradiated (case) and the other plate was non-irradiated (control).

Irradiation source

In this study, we used an infrared GaAlAs Laser (Physio laser, RJ, Germany) with a wavelength of 810 nm (13), with an area of 1 cm 2, power output of 10 mW and in continuous mode. In all experiments, the laser probe was fixed with a delivery arm that permitted precise positioning of the fiber tip 1 cm in front of the cell monolayer that allowed the laser beam width to be 1 cm 2. The laser power of 10 mW was used in all experiments. The cell cultures were irradiated with a single dose for 1 min and 40 s at a power density of 10 mW/cm 2 and energy density of 1 J/cm 2. After irradiation, the remaining medium was removed and new DMEM medium was added. Cells were then incubated for 1 hr prior to RNA extraction.

 

PCR

Total RNA was isolated from laser treated and untreated fibroblasts using CinnaPure RNA extraction kit (CinnaGen Co., Iran). One µg of RNA was reverse transcribed to cDNA using PrimeScript cDNA Synthesis Kit (Taka-ra) with 2 µl PrimeScript Buffer, 0.5 µl Prime Script RT Enzyme, 0.5 µl Oligo dT Primer, Random Hexamers and 1.5 µl RNase Free dH2O. The amplification through Polymeraze Chain Reaction (PCR) was carried out using 10 µg/µl of cDNA and primer design master mix and primers. The primers sequences are shown in Table 1. PCR amplification was performed using a 48-well tray (ABI) with 20 µl final reaction mixture containing 10 µl real time master mix (primer design), 7 µl ddH2O, 1 µl forward and reverse primer (10 pmol/µl) and 2 µl cDNA. PCR profile consisted of 95°C for 15 min, 95°C for 30 s, 60°C for 55 s, and 72°C for 20 s. Amplification was carried out for 40 cycles. To avoid any contamination of the preparation, PCR set-up was performed in a separate laboratory under sterile conditions. In every run, real time was performed in duplicate for each sample and one negative control for each primer was included to exclude false-positive results.

Table 1

 

Primers sequences

In this study, we used TATA box binding protein (TBP) as the housekeeping gene (internal control). Data was analyzed using ??CT equation. The fold change for PCR products were calculated in laser treated versus non-treated samples in each group of cells. PCR products were resolved in 1% aga-rose gel electrophoresis, and ethidium bromide-stained specific bands were visualized under UV light and photographed (to check if they have correct PCR product size).

Results

Data were analyzed using the SPSS (SPSS Inc., Chicago, IL, 16th version). A p-value less than 0.05 was considered statistically significant. The descriptive statistics were presented as median and standard deviation. All experiments were repeated at least 2 times with 2 parallel measurements (duplicate) in different wells. The statistical analysis was done by calculating ANOVA, post-hoc (LSD).

The investigated factors before laser irradiation had no significant difference in diabetic and nondiabetic mice. A significant increase (p = 0.017) was seen in the expression of FGF after irradiation in diabetic mice in comparison with nondiabetic mice after laser irradiation (Figure 1). Although the expression of PDGF also increased in both diabetic and nondiabetic mice after LLLT, it was not statistically significant (Figure 2). The expression of VEGF decreased after LLLT in both diabetic and nondiabetic mice, but it was not statistically significant (Figure 3). The investigated factors after laser irradiation had significant increase in diabetic mice in comparison with nondiabetic mice only for FGF after laser irradiation. P-value of growth factors in diabetic and nondiabetic mice in laser group and control group are shown in Table 2.

Figure 1

 

LLLT increases Fgf expression from isolated skin fibroblasts in diabetic mice in comparison with non diabetic mice, significantly [DI(-)LA(-) in comparison with DI(+)LA(+)] The starred column, DI(+)LA(+), shows this significant increase. Fgf expression

Figure 2

 

LLLT increases Pdgf expression in both diabetic and non diabetic mice, but this increase is not significant [DI(-)LA(-) in comparison with DI(-)LA(+) and DI(+)LA(-) in comparison with DI(+)LA(+)]. Pdgf expression between diabetic and non-diabetic mice

Figure 3

 

LLLT decreases Vegf expression in diabetic and non diabetic mice, but this decrease is not significant [DI(-) LA(-) in comparison with DI(-)LA(+) and DI(+)LA(-) in comparison with DI(+)LA(+)]. Vegf expression between diabetic and non-diabetic mice before

Table 2

P-value of growth factors comparison in diabetic and non diabetic mice in laser and control groups:

 

Discussion

There are many clinical and experimental data on the relationship between metabolic disturbance in diabetes and wound healing. Mattin (14), Goldstein (15) and Loots (16) examined skin fibroblasts from diabetics in tissue culture and found that their in vitro replication life span was reduced when compared with the control group. Grazul-Bilska et al suggested a defective FGF receptor or down-regulation of the FGF receptor-mediated cascade in diabetic status (17). Greenhalgh et al showed that PDGF and FGF stimulate wound healing in diabetic patients(18). Recent studies show the potential role of increased VEGF in diabetic retinopathy and nephropathy (19, 20).

Although some studies (1, 2) demonstrate no beneficial effects of low level laser irradiation on fibroblasts, this study showed stimulatory effects of LLLT on some fibroblast growth factors and therefore confirmed previous studies that yielded beneficial stimulating effect on fibroblasts (8, 9, 21, 22). Moore et al (13) reported inhibitory effect of 810 nm infrared laser on fibroblast. Van Breughel et al (3) reported a range of absorption peaks in 420, 445, 470, 560, 630, 690 and 730 nm and a general decrease in absorption at longer wavelengths and concluded that several molecules in fibroblasts serve as photoacceptors and absorb special wavelengths. Karu (23) also showed that the use of appropriate wavelength within the bandwidth of the absorption spectra of photo-acceptor molecules is an important factor. In this study, we observed the inhibitory effect of 810 nm infrared laser only on VEGF expression, but in other measured growth factors, the stimulatory effect was observed, although the increase in PDGF expression was not statistically significant which can be related to the small sample size. In our study, penetration depth can almost be ignored as wavelengths in the visible and infrared spectrum will pass through a monolayer cell culture (24).

Moreover, the irradiance (W/cm2) might have an important influence on the outcome of this study. As other experiments confirmed, lower irradiances are more effective than higher irradiances (3, 10, 24). The weak photo-stimulating effect in this study may be related to short incubation period. Hawkins et al (25) showed that 1-3 hrpost irradiation is sufficient to, measure cellular response to laser therapy. In some studies, this incubation period lasts 24-72 hr. Moreover, some authors proposed longer incubation period for having the response although the photobiostimulation influence may be weakened over time due to decreased vitality and untimely cell death in the irradiated cell cultures as a result of reaching confluence(22, 26).

Although some studies demonstrate FGF and PDGF decrease and VEGF increase in diabetic status (1719), we did not find significant difference between these factors before laser irradiation in diabetic mice in comparison with nondiabetics. It may be due to the small sample size. However, we observed significant increase in FGF expression following LLLT in cultured fibroblasts of diabetic mice in comparison with nonirradiant nondiabetic mice. Previous studies suggest that LLLT is more effective on impaired tissues rather than normal condition (27). In this study, we also found PDGF increase and VEGF decrease following LLLT in diabetic mice compared with nondiabetic mice but it was not statistically significant which can be related to small sample size. These results confirmed our previous studies using LLLT on diabetic patients (2830) and other studies suggesting beneficial effects of LLLT on fibroblast activity (8, 21, 22, 31).

This study suggests potential beneficial effects of LLL irradiation at the cellular level in clinical studies. LLLT application in cutaneous wounds of human skin may be assumed useful at the applied dosimetric parameters, but future investigation with larger sample size is necessary to explain the exact mechanisms of laser biomodulation. Subsequently, resolving the lack of scientific evidence and nullifying the controversial acknowledgements of the effect of low power lasers can bring about a wide spread acceptance for the use of LLLT in clinical settings.

Acknowledgement

The authors are grateful to Dr. Bonakdar for lab supplies and necessary materials provided for this study and also laboratory workers of Cell Bank of Iran affiliated to Pasteur Institute of Iran, Mr. Mehrjo and Mr. Majidi for providing the culture medium and technical support. We also thank Tehran University of Medical Sciences for financial support by grant number 10415.

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J Cosmet Laser Ther. 2013 Dec;15(6):310-7. doi: 10.3109/14764172.2013.803366. Epub 2013 Jul 1.

Effect of low-level laser therapy on the release of interleukin-6 and basic fibroblast growth factor from cultured human skin fibroblasts in normal and high glucose mediums.

Esmaeelinejad M1, Bayat M.

Author information

  • 1Oral and Maxillofacial Surgery Department, Dental Faculty, Shahid Beheshti University of Medical Sciences , Tehran , Iran.

 

Abstract

INTRODUCTION:

This study evaluated the effects of low-level laser therapy (LLLT) on human skin fibroblasts (HSFs) that have been cultured in high glucose concentration media.

MATERIALS AND METHODS:

HSFs were cultured under physiological glucose condition medium, and then cultured in high glucose concentration medium (15 mM/L) for 1 or 2 weeks prior to LLLT. Experimental HSFs were irradiated with three energy densities (0.5, 1, and 2 J/cm(2)) once daily for three consecutive days. Release of interleukin-6 (IL-6) and basic fibroblast growth factor (bFGF) was evaluated using the enzyme-linked immunosorbent assay (ELISA) method.

RESULTS:

Statistical analysis showed three doses of 0.5 (p = 0.049), 1 (p = 0.027), and 2 J/cm(2) (p = 0.004) stimulated the release of IL-6 in HSFs cultured in high glucose concentration medium compared with that of non-irradiated HSFs that were cultured in the same medium. LLLT with 2 J/cm(2) induced the release of bFGF from HSFs cultured in high glucose concentration medium for 1 or 2 weeks (both p = 0.04).

CONCLUSION:

Our study showed that LLLT stimulated the release of IL-6 and bFGF from HSFs cultured in high glucose concentration medium. LLLT was more effective in releasing IL-6 and bFGF while HSFs which were cultured in physiologic glucose concentration medium during laser irradiation.

Int J Dent.  2012;2012:719452. Epub 2012 Jul 15.

In Vitro Wound Healing Improvement by Low-Level Laser Therapy Application in Cultured Gingival Fibroblasts.

Basso FG, Pansani TN, Turrioni AP, Bagnato VS, Hebling J, de Souza Costa CA.

Source

Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (UNICAMP), 13414-903 Piracicaba, SP, Brazil.

Abstract

The aim of this study was to determine adequate energy doses using specific parameters of LLLT to produce biostimulatory effects on human gingival fibroblast culture. Cells (3 × 10(4) cells/cm(2)) were seeded on 24-well acrylic plates using plain DMEM supplemented with 10% fetal bovine serum. After 48-hour incubation with 5% CO(2) at 37°C, cells were irradiated with a InGaAsP diode laser prototype (LASERTable; 780 ± 3?nm; 40?mW) with energy doses of 0.5, 1.5, 3, 5, and 7?J/cm(2). Cells were irradiated every 24?h totalizing 3 applications. Twenty-four hours after the last irradiation, cell metabolism was evaluated by the MTT assay and the two most effective doses (0.5 and 3?J/cm(2)) were selected to evaluate the cell number (trypan blue assay) and the cell migration capacity (wound healing assay; transwell migration assay). Data were analyzed by the Kruskal-Wallis and Mann-Whitney nonparametric tests with statistical significance of 5%. Irradiation of the fibroblasts with 0.5 and 3?J/cm(2) resulted in significant increase in cell metabolism compared with the nonrradiated group (P < 0.05). Both energy doses promoted significant increase in the cell number as well as in cell migration (P < 0.05). These results demonstrate that, under the tested conditions, LLLT promoted biostimulation of fibroblasts in vitro.

Laser Med Sci. 2012 Jul 20. [Epub ahead of print]

Effect of laser and LED phototherapies on the healing of cutaneous wound on healthy and iron-deficient Wistar rats and their impact on fibroblastic activity during wound healing.

Oliveira Sampaio SC, de C Monteiro JS, Cangussú MC, Pires Santos GM, Dos Santos MA, Dos Santos JN, Pinheiro AL.

Source

Center of Biophotonics, School of Dentistry, Federal University of Bahia, Av. Araújo Pinho, 62, Canela, Salvador, BA, 40110-150, Brazil, susanasampaio2006@yahoo.com.br.

Abstract

Iron deficiency impairs the formation of hemoglobin, red blood cells, as well the transport of oxygen. The wound healing process involves numerous functions, many of which are dependent on the presence of oxygen. Laser has been shown to improve angiogenesis, increases blood supply, cell proliferation and function. We aimed to study the effect of ?660 nm laser and ?700 nm light-emitting diode (LED) on fibroblastic proliferation on cutaneous wounds on iron-deficient rodents. Induction of iron anemia was carried out by feeding 105 newborn rats with a special iron-free diet. A 1?×?1 cm wound was created on the dorsum of each animal that were randomly distributed into seven groups: I, control anemic; II, anemic no treatment; III, anemic?+?L; IV, anemic?+?LED; V, healthy no treatment; VI, healthy?+?laser; VII, healthy?+?LED (n?=?15 each). Phototherapy was carried out using either a diode laser (?660 nm, 40 mW, 10 J/cm(2)) or a prototype LED device (?700?±?20 nm, 15 mW, 10 J/cm(2)). Treatment started immediately after surgery and was repeated at 48-h interval during 7, 14, and 21 days. After animal death, specimens were taken, routinely processed, cut, stained with hematoxylin-eosin, and underwent histological analysis and fibroblast counting. Significant difference between healthy and anemic subjects on regards the number of fibroblast between treatments was seen (p?<?0.008, p?<?0.001). On healthy animals, significant higher count was seen when laser was used (p?<?0.008). Anemic subjects irradiated with LED showed significantly higher count (p?<?0.001). It is concluded that the use of LED light caused a significant positive biomodulation of fibroblastic proliferation on anemic animals and laser was more effective on increasing proliferation on non-anemics.

PLoS One.  2011;6(7):e22453. Epub 2011 Jul 21.

Low-Level Laser Therapy Activates NF-kB via Generation of Reactive Oxygen Species in Mouse Embryonic Fibroblasts.

Chen AC, Arany PR, Huang YY, Tomkinson EM, Sharma SK, Kharkwal GB, Saleem T, Mooney D, Yull FE, Blackwell TS, Hamblin MR.

Source

Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

Abstract

BACKGROUND:

Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear.

METHODOLOGY/PRINCIPAL FINDINGS:

In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2) and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration.

CONCLUSION:

We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

J Biomed Opt.  2011 Jul;16(7):075004.

Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode.

Volpato LE, de Oliveira RC, Espinosa MM, Bagnato VS, Machado MA.

Source

University of Cuiaba?, Cuiaba? School of Dentistry, Rua Esteva?o de Mendonc?a, 317 apto 501, Goiabeiras, Cuiaba?, MT, 78065-480 BrazilUniversity of Sa?o Paulo, Bauru School of Dentistry, Al. Dr. Octa?vio Pinheiro Brisolla, 9-75 Bauru, SP, 17012-901 BrazilFederal University of Mato Grosso, Department of Statistics, Avenida Fernando Corre^a, s/n[ordinal indicator, masculine] Coxipo?, Cuiaba?, MT, 78060-900 BrazilUniversity of Sa?o Paulo, Physics Institute of Sa?o Carlos, Av. Trabalhador Sa?o-Carlense, 400 Sa?o Carlos, SP, 13566-590 Brazil.

Abstract

Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm(2)), infrared laser (780 nm, 40 mW, 1 W/cm(2)), and red LED (637 ± 15 nm, 40 mW, 1 W/cm(2)). All applications were punctual and performed with a spot with 0.4 mm(2) of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p ? 0.05) were used for statistical analysis. After 72 h, phototherapy with low-intensity laser and LED showed no toxicity at the cellular level. It even stimulated methylthiazol tetrazolium assay (MTT) conversion and neutral red uptake of fibroblasts cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p < 0.001 for neutral red uptake). Considering the parameters and protocol of phototherapy used, it can be concluded that phototherapy stimulated the viability of fibroblasts cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced.

Lasers Med Sci. 2011 Jan 28. [Epub ahead of print]

Low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells.

Alghamdi KM, Kumar A, Moussa NA.

Department of Dermatology, Vitiligo Research Chair, College of Medicine, King Saud University, PO Box 240997, Riyadh, 11322, Saudi Arabia, kmgderm@yahoo.com.

Abstract

The aim of this work is to review the available literature on the details of low-level laser therapy (LLLT) use for the enhancement of the proliferation of various cultured cell lines including stem cells. A cell culture is one of the most useful techniques in science, particularly in the production of viral vaccines and hybrid cell lines. However, the growth rate of some of the much-needed mammalian cells is slow. LLLT can enhance the proliferation rate of various cell lines. Literature review from 1923 to 2010. By investigating the outcome of LLLT on cell cultures, many articles report that it produces higher rates of ATP, RNA, and DNA synthesis in stem cells and other cell lines. Thus, LLLT improves the proliferation of the cells without causing any cytotoxic effects. Mainly, helium neon and gallium-aluminum-arsenide (Ga-Al-As) lasers are used for LLLT on cultured cells. The results of LLLT also vary according to the applied energy density and wavelengths to which the target cells are subjected. This review suggests that an energy density value of 0.5 to 4.0 J/cm(2) and a visible spectrum ranging from 600 to 700 nm of LLLT are very helpful in enhancing the proliferation rate of various cell lines. With the appropriate use of LLLT, the proliferation rate of cultured cells, including stem cells, can be increased, which would be very useful in tissue engineering and regenerative medicine.

Photomed Laser Surg. 2010 Aug;28(4):547-52.

Effect of LED phototherapy of three distinct wavelengths on fibroblasts on wound healing: a histological study in a rodent model.

de Sousa AP, Santos JN, Dos Reis JA Jr, Ramos TA, de Souza J, Cangussú MC, Pinheiro AL.

Laser Center, School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil.

Abstract

AIM: The aim of the present investigation was to evaluate histologically fibroblastic proliferation on dorsal cutaneous wounds in a rodent model treated or not with light-emitting diodes (LEDs) of three wavelengths.

BACKGROUND: Fibroblasts secrete substances essential for wound healing. There are few reports of LED phototherapy on fibroblast proliferation, mainly in vivo.

ANIMALS AND METHODS: Following approval by the Animal Experimentation Committee of the School of Dentistry of the Federal University of Bahia, we obtained 16 young adult male Wistar rats weighing between 200 and 250 g. Under general anesthesia, one excisional wound was created on the dorsum of each animal; they were then randomly distributed into four groups of four animals each: G0, untreated control; G1, red LED (700 +/- 20 nm, 15 mW, 10 J/cm(2)); G2, green LED (530 +/- 20 nm, 8 mW, 10 J/cm(2)); and G3, blue LED (460 +/- 20 nm, 22 mW, 10 J/cm(2)). The irradiation started immediately after surgery and was repeated every other day for 7 days. Animals were killed 8 days after surgery. The specimens were removed, routinely processed to wax, cut, and stained with hematoxylin/eosin (HE). Fibroblasts were scored by measuring the percentage of these cells occupying the area corresponding to wound healing on stained sections.

RESULTS: The quantitative results showed that red LED (700 +/- 20 nm) and green LED (530 +/- 20 nm) showed a significant increase in fibroblast numbers (p < 0.01 and p = 0.02) when compared with the control group.

CONCLUSION: The use of green and red LED light is effective in increasing fibroblastic proliferation on rodents.

J Periodontal Implant Sci. 2010 Jun;40(3):105-10. Epub 2010 Jun 25.

 

Biological effects of a semiconductor diode laser on human periodontal ligament fibroblasts.

 

Choi EJ, Yim JY, Koo KT, Seol YJ, Lee YM, Ku Y, Rhyu IC, Chung CP, Kim TI.

Department of Periodontology and Dental Research Institute, Seoul National University College of Dentistry, Seoul, Korea.

Abstract

PURPOSE: It has been reported that low-level semiconductor diode lasers could enhance the wound healing process. The periodontal ligament is crucial for maintaining the tooth and surrounding tissues in periodontal wound healing. While low-level semiconductor diode lasers have been used in low-level laser therapy, there have been few reports on their effects on periodontal ligament fibroblasts (PDLFs). We performed this study to investigate the biological effects of semiconductor diode lasers on human PDLFs.

METHODS: Human PDLFs were cultured and irradiated with a gallium-aluminum-arsenate (GaAlAs) semiconductor diode laser of which the wavelength was 810 nm. The power output was fixed at 500 mW in the continuous wave mode with various energy fluencies, which were 1.97, 3.94, and 5.91 J/cm(2). A culture of PDLFs without laser irradiation was regarded as a control. Then, cells were additionally incubated in 72 hours for MTS assay and an alkaline phosphatase (ALPase) activity test. At 48 hours post-laser irradiation, western blot analysis was performed to determine extracellular signal-regulated kinase (ERK) activity. ANOVA was used to assess the significance level of the differences among groups (P<0.05).

RESULTS: At all energy fluencies of laser irradiation, PDLFs proliferation gradually increased for 72 hours without any significant differences compared with the control over the entire period taken together. However, an increment of cell proliferation significantly greater than in the control occurred between 24 and 48 hours at laser irradiation settings of 1.97 and 3.94 J/cm(2) (P<0.05). The highest ALPase activity was found at 48 and 72 hours post-laser irradiation with 3.94 J/cm(2) energy fluency (P<0.05). The phosphorylated ERK level was more prominent at 3.94 J/cm(2) energy fluency than in the control.

CONCLUSIONS: The present study demonstrated that the GaAlAs semiconductor diode laser promoted proliferation and differentiation of human PDLFs.

Cell Tissue Bank. 2009 Nov;10(4):327-32. Epub 2009 Jul 11.

Effects of diode laser therapy on the acellular dermal matrix.

Soares LP, de Oliveira MG, de Almeida Reis SR.

Department of Oralmaxillofacial Surgery, PUCRS School of Dentistry, Porto Alegre, Rio Grande do Sul, Brazil. liviaps@ibest.com.br

Acellular dermal matrix (ADM) was subcutaneously implanted into calvarian skin of male Wistar rats (n = 40). Low-level laser (lambda 685 nm, 4 J/cm(2)) was locally applied in experimental group (n = 20) above the skin flap. Grafts were harvested at 1, 3, 7 and 14 days after surgery and underwent histological analyses. In treated animals, the extent of edema and the number of inflammatory cells were reduced (P < 0.05). The amount of collagen in graft treated with low-level laser were significantly higher than those of controls (P < 0.05) and were statistically more prominent on the 14th day after surgery. The mean count of fibroblasts was significantly higher in the low-laser therapy group within the 3rd day, showing a marked influx of fibroblasts into area. In conclusion, wound healing of the ADM appear to be positively affected by laser therapy.

Photomed Laser Surg. 2009 Sep 21. [Epub ahead of print]

Low-Level Laser Irradiation (InGaAlP-660 nm) Increases Fibroblast Cell Proliferation and Reduces Cell Death in a Dose-Dependent Manner.

Frigo L, Fávero GM, Campos Lima HJ, Maria DA, Bjordal JM, Joensen J, Iversen VV, Marcos RL, Parizzoto NA, Lopes-Martins RA.

1 Biological Sciences and Health Center, Cruzeiro do Sul University , São Paulo, Brazil .

Abstract Background and Objective: Impaired cell metabolism and increased cell death in fibroblast cells are physiological features of chronic tendinopathy. Although several studies have shown that low-level laser therapy (LLLT) at certain parameters has a biostimulatory effect on fibroblast cells, it remains uncertain if LLLT effects depend on the physiological state.

Study Design/Material and Methods: High-metabolic immortal cell culture and primary human keloid fibroblast cell culture were used in this study. Trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test were used to determine cell viability and proliferation. Propidium iodide stain was used for cell-cycle analysis by flow cytometry. Laser irradiation was performed daily on three consecutive days with a GaAlAs 660-nm laser (mean output: 50 mW, spot size 2 mm(2), power density =2.5 W/cm(2)) and a typical LLLT dose and a high LLLT dose (irradiation times: 60 or 420 s; fluences:150 or 1050 J/cm(2); energy delivered: 3 or 21 J).

Results: Primary fibroblast cell culture from human keloids irradiated with 3 J showed significant proliferation by the trypan blue exclusion test (p < 0.05), whereas the 3T3 cell culture showed no difference using this method. Propidium iodide staining flow cytometry data showed a significant decrease in the percentage of cells being in proliferative phases of the cell cycle (S/g(2)/M) when irradiated with 21 J in both cell types (hypodiploid cells increased). Conclusions: Our data support the hypothesis that the physiological state of the cells affects the LLLT results, and that high-metabolic rate and short- cell-cycle 3T3 cells are not responsive to LLLT.

In conclusion, LLLT with a dose of 3 J reduced cell death significantly, but did not stimulate cell cycle. A LLLT dose of 21 J had negative effects on the cells, as it increased cell death and inhibited cell proliferation.

Photomed Laser Surg. 2009 Jun;27(3):461-6.

Assessment of cytoskeleton and endoplasmic reticulum of fibroblast cells subjected to low-level laser therapy and low-intensity pulsed ultrasound.

Oliveira DA, De Oliveira RF, Magini M, Zangaro RA, Soares CP.

Laboratório de Dinâmica de Compartimento Celular, Instituto de Pesquisa e Desenvolvimento (IP&D), UNIVAP, São José dos Campos, São Paulo, Brazil.

OBJECTIVE: The aim of the present study was to compare the effect of low-level laser therapy (LLLT) and low-intensity pulsed ultrasound (LIPUS) on the cytoskeleton and endoplasmic reticulum of L929 cells. Thermal and non-thermal physical mechanisms such as LLLT and LIPUS induce clinically significant responses in cells, tissues, and organs.

MATERIALS AND METHODS: L929 fibroblast cell cultures were irradiated with LLLT and subjected to LIPUS. Cultures irradiated with the laser (904 nm) were divided into three groups: group I, control (no irradiation); group II, irradiated at 6 J/cm(2); and group III, irradiated at 50 mJ/cm(2). Cultures subjected to ultrasound were divided into five groups: group I, control (no LIPUS); group II, LIPUS at 0.2 W/cm(2) in pulsed mode at 10% (1:9 duty cycle); group III, LIPUS at 0.6 W/cm(2) in pulsed mode at 10% (1:9 duty cycle); group IV, LIPUS at 0.2 W/cm(2) in pulsed mode at 20% (2:8 duty cycle); and group V, LIPUS at 0.6 W/cm(2) in pulsed mode at 20% (2:8 duty cycle). Each group was irradiated at 24-h intervals, with the following post-treatment incubation times: 24, 48, and 72 h. The effects of LLLT and LIPUS on the cytoskeleton and endoplasmic reticulum was evaluated by the use of fluorescent probes and with fluorescence microscopy analysis.

RESULTS: The results following LLLT and LIPUS demonstrate that ultrasound was more effective than laser on fibroblast cell cultures when the endoplasmic reticulum was assessed, whereas there was a better distribution of the filaments of the cytoskeleton in the cells subjected to laser irradiation.

CONCLUSION: The study demonstrated that both LLLT and LIPUS promote changes on the cellular level. However, LIPUS was more effective than LLLT at the doses used here, as assessed by fluorescence microscopy, which revealed increased reticulum activity and increased protein synthesis. However, when the organization of actin filaments was assessed, LLLT achieved a better result.

Lasers Med Sci. 2009 Nov;24(6):885-91. Epub 2008 Jul 4.

 

Effect of laser phototherapy on the release of fibroblast growth factors by human gingival fibroblasts.

 

Damante CA, De Micheli G, Miyagi SP, Feist IS, Marques MM.

Departamento de Periodontia, Faculdade de Odontologia, Universidade de São Paulo, São Paulo, Brazil.

The effects of laser phototherapy on the release of growth factors by human gingival fibroblasts were studied in vitro. Cells from a primary culture were irradiated twice (6 h interval), with continuous diode laser [gallium-aluminum-arsenium (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP),_660 nm] in punctual and contact mode, 40 mW, spot size 0.042 cm(2), 3 J/cm(2) and 5 J/cm(2) (3 s and 5 s, respectively). Positive [10% fetal bovine serum (FBS)] and negative (1%FBS) controls were not irradiated. Production of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) was quantified by enzyme-linked immunosorbent assay (ELISA). The data were statistically compared by analysis of variance (ANOVA) followed by Tukey’s test (P </= 0.05). The characterization of the cell line indicated a mesenchymal nature. KGF release was similar in all groups, while that of bFGF was significantly greater (1.49-times) in groups treated with infra-red laser. It was concluded that increased production of bFGF could be one of the mechanisms by which infra-red laser stimulates wound healing.

Photomed Laser Surg.. [Epub ahead of print]

Effect of LED Phototherapy of Three Distinct Wavelengths on Fibroblasts on Wound Healing: A Histological Study in a Rodent Model.

de Sousa AP, Santos JN, Dos Reis JA, Ramos TA, de Souza J, Cangussú MC, Pinheiro AL.

1 Laser Center, School of Dentistry, Federal University of Bahia , Salvador, Bahia, Brazil.

Abstract Aim: The aim of the present investigation was to evaluate histologically fibroblastic proliferation on dorsal cutaneous wounds in a rodent model treated or not with light-emitting diodes (LEDs) of three wavelengths. Background: Fibroblasts secrete substances essential for wound healing. There are few reports of LED phototherapy on fibroblast proliferation, mainly in vivo.

Animals and Methods: Following approval by the Animal Experimentation Committee of the School of Dentistry of the Federal University of Bahia, we obtained 16 young adult male Wistar rats weighing between 200 and 250 g. Under general anesthesia, one excisional wound was created on the dorsum of each animal; they were then randomly distributed into four groups of four animals each: G0, untreated control; G1, red LED (700 nm +/- 20 nm, 15 mW, 10 J/cm(2)); G2, green LED (530 nm +/- 20 nm, 8 mW, 10 J/cm(2)); and G3, blue LED (460 nm +/- 20 nm, 22 mW, 10 J/cm(2)). The irradiation started immediately after surgery and was repeated every other day for 7 days. Animals were killed 8 days after surgery. The specimens were removed, routinely processed to wax, cut, and stained with hematoxylin/eosin (HE). Fibroblasts were scored by measuring the percentage of these cells occupying the area corresponding to wound healing on stained sections.

Results: The quantitative results showed that red LED (700 +/- 20 nm) and green LED (530 +/- 20 nm) showed a significant increase in fibroblast numbers (p < 0.01 and p = 0.02) when compared with the control group.

Conclusion: The use of green and red LED light is effective in increasing fibroblastic proliferation on rodents.

Photomed Laser Surg. 2009 Oct 1. [Epub ahead of print]

 

Effects of 780-nm Low-level Laser Therapy with a Pulsed Gallium Aluminum Arsenide Laser on the Healing of a Surgically Open Skin Wound in Rat.

Bayat M, Azari A, Golmohammadi MG.

Physical Therapy Research Group, Academic Center for Education, Culture, and Research, Iran Medical Science Branch University , Vanak, Tehran, Iran .

Abstract Objective: The aim of the present investigation is to evaluate the effects of a 780-nm low-level laser on open skin wound healing. Background Data: Optimal parameters of low-level laser therapy (LLLT) for wound healing are discussed.

Methods: One full-thickness skin wound was surgically induced in the dorsum skin of 30 rats. The rats were divided into two groups. Rats in the experimental group were daily treated with a gallium aluminum arsenide (GaAlAs) laser (2 J/cm(2), lambda = 780 nm, pulse frequency of 2336 Hz). Rats in the sham-exposed group received LLLT with switched off equipment. After 4, 7, and 15 days, wounds were checked by histological and biomechanical methods. Data were analyzed by the Mann-Whitney U-test.

Results: Fibroblasts, endothelium of blood vessels, blood vessel sections, and maximum stress were significantly increased, whereas macrophages were significantly decreased, compared with those of the sham-exposed group.

Conclusion: Pulsed LLLT with a 780-nm GaAlAs laser significantly accelerates the process of healing of surgically induced, full-thickness skin wounds in rat.

J Biomed Opt. 2009 May-Jun;14(3):034002.

Effect of low-level laser treatment of tissue-engineered skin substitutes: contraction of collagen lattices.

Ho G, Barbenel J, Grant MH.

Exploit Technologies, Biomedical Sciences Division, Agency of Science and Technology (A STAR), 30 Biopolis Street, Singapore 138671, Singapore.

Fibroblast-populated collagen lattices (FPCL) are widely used in tissue-engineered artificial skin substitutes, but their main drawback is that interaction of fibroblasts and matrix causes contraction of the lattice, reducing it to about 20% of its original area. The effect of low-level laser treatment (LLLT) on the behavior of 3T3 fibroblasts seeded in collagen lattices containing 20% chondroitin-6-sulphate was investigated to determine whether LLLT could control the contraction of FPCL. A He-Ne laser was used at 632.8 nm to deliver a 5-mW continuous wave with fluences from 1 to 4 J/cm(2). Laser treatment at 3 J/cm(2) increased contraction of collagen lattices in the absence of cells but decreased contraction of cell seeded lattices over a 7-day period. The effect was energy dependent and was not observed at 1, 2, or 4 J/cm(2). There was no alteration in fibroblast viability, morphology, or mitochondrial membrane potential after any laser treatments, but the distribution of actin fibers within the cells and collagen fibers in the matrices was disturbed at 3 J/cm(2). These effects contribute to the decrease in contraction observed. LLLT may offer a means to control contraction of FPCL used as artificial skin substitutes.

Lasers Med Sci. 2008 Apr;23(2):211-5. Epub 2007 Jul 10.

Effects of laser irradiation on the release of basic fibroblast growth factor (bFGF), insulin like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3) from gingival fibroblasts.

 

Saygun I, Karacay S, Serdar M, Ural AU, Sencimen M, Kurtis B.

Department of Periodontology, Gulhane Military Medical Academy, Etlik, Ankara, 06018 Turkey. saygunisil@yahoo.com

Various studies have shown biostimulation effects of laser irradiation by producing metabolic changes within the cells. Little is known about the biological effect of laser irradiation on the oral tissues. Among the many physiological effects, it is important to recognize that low-level laser therapy (LLLT) may affect release of growth factors from fibroblasts. Therefore, the aim of the present study was to determine whether the laser irradiation can enhance the release of basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3) from human gingival fibroblasts (HGF). The number of all samples in the study were 30, and the samples were randomly divided into three equal groups; In the first group (single dose group), HGF were irradiated with laser energy of 685 nm, for 140 s, 2 J/cm(2) for one time, and in the second group, energy at the same dose was applied for two consecutive days (double dose group). The third group served as nonirradiated control group. Proliferation, viability, and bFGF, IGF-1, IGFBP3 analysis of control and irradiated cultures were compared with each other. Both of the irradiated groups revealed higher proliferation and viability in comparison to the control group. Comparison of the single-dose group with the control group revealed statistically significant increases in bFGF (p < 0.01) and IGF-1 (p < 0.01), but IGFBP3 increased insignificantly (p > 0.05). When the double dose group was compared with the control group, significant increases were determined in all of the parameters (p < 0.01). In the comparison of the differences between the two irradiated groups (one dose and two doses), none of the parameters displayed any statistically significant difference (p > 0.05). In both of the laser groups, LLLT increased the cell proliferation and cell viability. The results of this study showed that LLLT increased the proliferation of HGF cells and release of bFGF, IGF-1, and IGFBP3 from these cells. LLLT may play an important role in periodontal wound healing and regeneration by enhancing the production of the growth factors.

Photomed Laser Surg. 2007 Apr;25(2):78-84.

In vitro exposure of wounded diabetic fibroblast cells to a helium-neon laser at 5 and 16 J/cm2.

Houreld N, Abrahamse H.

Faculty of Health Sciences, Laser Research Unit, University of Johannesburg, Doornfontein, South Africa.

OBJECTIVE: The aim of the present investigation was to assess morphological, cellular, and molecular effects of exposing wounded diabetic fibroblast cells to He-Ne (632.8 nm) laser irradiation at two different doses.

BACKGROUND DATA: An alternative treatment modality for diabetic wound healing includes low-level laser therapy (LLLT). Although it’s used in many countries and for many medical conditions, too many health care workers are unaware of this therapy, and there is still controversy surrounding its effectiveness.

METHODS: Normal human skin fibroblast cells (WS1) were used to simulate a wounded diabetic model. The effect of LLLT (632.8 nm, 5 and 16 J/cm(2) once a day on two non-consecutive days) was determined by analysis of cell morphology, cytotoxicity, apoptosis, and DNA damage.

RESULTS: Cells exposed to 5 J/cm(2) showed a higher rate of migration than cells exposed to 16 J/cm(2), and there was complete wound closure by day 4. Exposure of WS1 cells to 5 J/cm(2) on two non-consecutive days did not induce additional cytotoxicity or genetic damage, whereas exposure to 16 J/cm(2) did. There was a significant increase in apoptosis in exposed cells as compared to unexposed cells.

CONCLUSION: Based on cellular morphology, exposure to 5 J/cm(2) was stimulatory to cellular migration, whereas exposure to 16 J/cm(2) was inhibitory. Exposure to 16 J/cm(2) induced genetic damage on WS1 cells when exposed to a He-Ne laser in vitro, whereas exposure to 5 J/cm(2) did not induce any additional damage.

Lasers Med Sci. 2007 Mar;22(1):1-3. Epub 2006 Nov 25.

A reasonable mechanism for visible light-induced skin rejuvenation.

Lubart R, Friedmann H, Lavie R, Longo L, Jacobi J, Baruchin O, Baruchin AM.

Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900, Israel.

In recent years, much research has been done in the field of non-ablative skin rejuvenation. This comes as a response to the continuous demand for a simple method of treating rhytides, UV exposure, and acne scars. Numerous researches involve visible light-pulsed systems (20-30 J/cm(2)). The mechanism of action is believed to be a selective heat-induced denaturalization of dermal collagen that leads to subsequent reactive synthesis (Bitter Jr., Dermatol. Surg., 26:836-843, 2000; Fitzpatrick et al., Arch. Dermatol., 132:395-402, 1996; Kauvar and Geronemus, Dermatol. Clin., 15:459-467, 1997; Negishi et al., Lasers Surg. Med., 30:298-305, 2002; Goldberg and Cutler, Lasers Surg. Med., 26:196-200, 2000; Hernandez-Perez and Ibeitt, Dermatol. Surg., 28:651-655, 2002). In this study, we suggest a different mechanism for photorejuvenation based on light-induced reactive oxygen species (ROS) formation. We irradiated collagen in vitro with a broadband of visible light (400-800 nm, 24-72 J/cm(2)) and used the spin trapping coupled with electron paramagnetic resonance spectroscopy to detect ROS. Irradiated collagen resulted in hydroxyl radicals formation. We propose, as a new concept, that visible light at the energy doses used for skin rejuvenation (20-30 J/cm(2)) produces high amounts of ROS, which destroy old collagen fibers, encouraging the formation of new ones. On the other hand, at inner depths of the skin, where the light intensity is much weaker, low amounts of ROS are formed, which are well known to stimulate fibroblast proliferation.

Photomed Laser Surg. 2005 Apr;23(2):167-71.

Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level.

Vinck EM, Cagnie BJ, Cornelissen MJ, Declercq HA, Cambier DC.

Department of Rehabilitation Sciences and Physiotherapy, Ghent University, 9000 Ghent, Belgium. elke.vinck@UGent.be

BACKGROUND AND OBJECTIVE: The chronic metabolic disorder diabetes mellitus is an important cause of morbidity and mortality due to a series of common secondary metabolic complications, such as the development of severe, often slow healing skin lesions. In view of promoting the wound-healing process in diabetic patients, this preliminary in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on fibroblast proliferation and viability under hyperglycemic circumstances.

MATERIALS AND METHODS: To achieve hyperglycemic circumstances, embryonic chicken fibroblasts were cultured in Hanks’ culture medium supplemented with 30 g/L glucose. LED irradiation was performed on 3 consecutive days with a probe emitting green light (570 nm) and a power output of 10 mW. Each treatment lasted 3 min, resulting in a radiation exposure of 0.1 J/cm2.

RESULTS: A Mann-Whitney U test revealed a higher proliferation rate (p = 0.001) in all irradiated cultures in comparison with the controls.

CONCLUSION: According to these results, the effectiveness of green LED irradiation on fibroblasts in hyperglycemic circumstances is established. Future in vivo investigation would be worthwhile to investigate whether there are equivalent positive results in diabetic patients.