The effects of low-level laser irradiation on cellular viability and proliferation of human skin fibroblasts cultured in high glucose mediums

Delayed wound healing is one of the most challenging complications of diabetes mellitus (DM) in clinical medicine. This study has aimed to evaluate the effects of low-level laser therapy (LLLT) on human skin fibroblasts (HSFs) cultured in a high glucose concentration. HSFs were cultured either in a concentration of physiologic glucose (5.5 mM/l) or high glucose media (11.1 and15?mM/l) for either 1 or 2 weeks after which they were subsequently cultured in either the physiologic glucose or high concentration glucose media during laser irradiation. LLLT was carried out with a helium–neon (He–Ne) laser unit at energy densities of 0.5, 1, and 2 J/cm², and power density of 0.66 mW/cm² on 3 consecutive days. HSFs’ viability and proliferation rate were evaluated with the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. The LLLT at densities of 0.5 and 1 J/cm² had stimulatory effects on the viability and proliferation rate of HSFs cultured in physiologic glucose (5.5 mM/l) medium compared to their control cultures (p?=?0.002 and p?=?0.046, respectively). All three doses of 0.5, 1, and 2 J/cm² had stimulatory effects on the proliferation rate of HSFs cultured in high glucose concentrations when compared to their control cultures (p?=?0.042, p?=?0.000, and p?=?0.000, respectively). This study showed that HSFs originally cultured for 2 weeks in high glucose concentration followed by culture in physiologic glucose during laser irradiation showed enhanced cell viability and proliferation. Thus, LLLT had a stimulatory effect on these HSFs.     

Effects of Nd:YAG laser irradiation on cultured human gingival fibroblasts

BACKGROUND AND OBJECTIVE: The Nd:YAG laser has been proposed to apply in minor soft tissue surgery, including various periodontal procedures. However, little information is available regarding the direct effect of Nd:YAG laser on gingival fibroblasts, which play an important role in the early healing processes of periodontal repair. STUDY DESIGN/MATERIALS AND METHODS: Nd:YAG laser irradiation was performed in pulsed mode on human gingival fibroblasts, which was derived from healthy human gingiva by an explant method. The size of laser diode was 400 microm in diameter. The parameters in laser delivery were pulse energy (50-150 mJ), power output (1.0-3.0 W), pulse rate (10-30 pps), and fixed duration of irradiation (10 seconds). The cell cultures were analysed by cytomorphologic examination under phase-contrast and scanning electron microscope. The vitality was also examined with the help of MTT staining. RESULTS: The area of laser damage on cell culture was circular in shape, with diameter beyond the size of laser diode. By scanning electron microscopy, we observed the cellular damage of cultured gingival fibroblasts induced by Nd:YAG laser irradiation, comparable with the progressive increased power settings. The cytomorphologic changes ranged from disappearance of cellular boundary, loss of identifiable cellular nucleus, and finally cell contraction and vacuolization. Significant decrease in cellular vitality (14% approximately 44%) after laser treatment with irradiation distance of nearly contact was noted. However, 2 mm defocusing irradiation with the same power settings did not significantly decrease cellular vitality. CONCLUSION: Our study demonstrated the cell damaging effects of Nd:YAG laser, ranging from degeneratively cytomorphologic change to cell death, on the cultured human gingival fibroblasts. It provided the dentist a chance to understand the potential hazard of laser application in periodontal treatment. If the energy output is enough for the clinical purposes, Nd:YAG laser with lower pulse energy and corresponding pulse rate should be selected to minimize the damage on adjacent soft tissue.     

Effects of high-frequency near-infrared diode laser irradiation on the proliferation and migration of mouse calvarial osteoblasts

Laser irradiation activates a range of cellular processes and can promote tissue repair. Here, we examined the effects of high-frequency near-infrared (NIR) diode laser irradiation on the proliferation and migration of mouse calvarial osteoblastic cells (MC3T3-E1). MC3T3-E1 cells were cultured and exposed to high-frequency (30 kHz) 910-nm diode laser irradiation at a dose of 0, 1.42, 2.85, 5.7, or 17.1 J/cm². Cell proliferation was evaluated with BrdU and ATP concentration assays. Cell migration was analyzed by quantitative assessment of wound healing using the Incucyt^® ZOOM system. In addition, phosphorylation of mitogen-activated protein kinase (MAPK) family members including p38 mitogen-activated protein kinase (p38), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), and extracellular signal-regulated protein kinase (ERK)1/2) after laser irradiation was examined with western blotting. Compared to the control, cell proliferation was significantly increased by laser irradiation at a dose of 2.85, 5.7, or 17.1 J/cm². Laser irradiation at a dose of 2.85 J/cm² induced MC3T3-E1 cells to migrate more rapidly than non-irradiated control cells. Irradiation with the high-frequency 910-nm diode laser at a dose of 2.85 J/cm² induced phosphorylation of MAPK/ERK1/2 15 and 30 min later. However, phosphorylation of p38 MAPK and SAPK/JNK was not changed by NIR diode laser irradiation at a dose of 2.85 J/cm². Irradiation with a high-frequency NIR diode laser increased cell division and migration of MT3T3-E1 cells, possibly via MAPK/ERK signaling. These observations may be important for enhancing proliferation and migration of osteoblasts to improve regeneration of bone tissues.     

Effect of low-level laser irradiation and epidermal growth factor on adult human adipose-derived stem cells

The study investigated the effects of low-level laser radiation and epidermal growth factor (EGF) on adult adipose-derived stem cells (ADSCs) isolated from human adipose tissue. Isolated cells were cultured to semi-confluence, and the monolayers of ADSCs were exposed to low-level laser at 5 J/cm² using 636 nm diode laser. Cell viability and proliferation were monitored using adenosine triphosphate (ATP) luminescence and optical density at 0 h, 24 h and 48 h after irradiation. Application of low-level laser irradiation at 5 J/cm² on human ADSCs cultured with EGF increased the viability and proliferation of these cells. The results indicate that low-level laser irradiation in combination with EGF enhances the proliferation and maintenance of ADSCs in vitro.     

Effects of increased low-level diode laser irradiation time on extraction socket healing in rats

Lasers in Medical Science - In our previous studies, we confirmed that low-level laser therapy (LLLT) with a 980-nm gallium–aluminum–arsenide diode laser was beneficial for the healing...     

Effect of low-level laser irradiation on cytotoxicity of benzene in human normal fibroblast cells

Lasers in Medical Science - Benzene is volatile organic hydrocarbon which is widely used in a wide range of industries. Studies have shown that exposure to benzene consequences serious health risks...     

Vitamin E inhibits proliferation of primary cultured human mesangial and endothelial cells

BACKGROUND: Vitamin E (VE) has been used as an antioxidant and has been suggested to inhibit the proliferation of mesangial cells in rat and vascular endothelial cells. The direct effect of VE on primary cultures of mesangial cells (MC) and endothelial cells (EC) from the human glomerulus was studied. METHODS: (1) MC (in 17 or 2.5% FCS DMEM) or EC (in 10 or 5% FCS CSC) at 5,000 cells/well was incubated with serial concentrations of VE from 0.05 to 50 microg/ml (0.06 to 60 IU/l). (2) MC was cocultured with 160, 80, 40 or 20 microg/ml of low-density lipoprotein (LDL) or oxidized LDL (ox-LDL) in 17 or 2.5% FCS DMEM with or without VE. After 3 days of incubation at 37 degrees C in 5% CO(2), cell proliferation was measured by the Premix WST-1 Assay System. RESULTS: The concentration of VE that significantly inhibited the proliferation of MC cultured in 17 or 2.5% FCS DMEM was 50 or 2.5 microg/ml (60 or 3.0 IU/l), respectively, and that of EC in 10 or 5% FCS medium was 50 or 25 microg/ml (60 or 30 IU/l). VE at 25 microg/ml (30 IU/l) inhibited the LDL proliferative effect on MC cultured in 2.5 FCS DMEM by 21.79-93.21% in a LDL concentration-dependent manner. There was little difference between the effects of LDL and ox-LDL on the VE inhibitory effect on MC under our experimental conditions. CONCLUSION: VE at low concentrations had no effect on the proliferation of both MC and EC, but at high concentrations, it showed an inhibitory effect on both cells.     

Low-level laser irradiation (LLLI) promotes proliferation of mesenchymal and cardiac stem cells in culture

BACKGROUND AND OBJECTIVES: Low-level laser irradiation (LLLI) was found to promote the proliferation of various types of cells in vitro. Stem cells in general are of significance for implantation in regenerative medicine. The aim of the present study was to investigate the effect of LLLI on the proliferation of mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs). STUDY DESIGN/MATERIALS AND METHODS: Isolation of MSCs and CSCs was performed. The cells were cultured and laser irradiation was applied at energy densities of 1 and 3 J/cm2. RESULTS: The number of MSCs and CSCs up to 2 and 4 weeks respectively, post-LLLI demonstrated a significant increase in the laser-treated cultures as compared to the control. CONCLUSION: The present study clearly demonstrates the ability of LLLI to promote proliferation of MSCs and CSCs in vitro. These results may have an important impact on regenerative medicine.     

Effects of low-level laser irradiation on human blood lymphocytes in vitro

Low-level laser irradiation (LLLI) has various effects on cultured human lymphocytes in vitro, but little is known about such effects in whole blood. This study investigated whether LLLI affected lymphocyte count in human whole blood in vitro. A total number of 130 blood samples were collected from apparently healthy adult patients through venipuncture into tubes containing EDTA. Each sample was divided into two equal aliquots to be used as a non-irradiated control sample and an irradiated sample. The irradiated aliquot was subjected to laser wavelengths of 405, 589, and 780 nm with different fluences of 36, 54, 72, and 90 J/cm², at a fixed irradiance of 30 mW/cm². A paired student t test was used to compare between non-irradiated and irradiated samples. The lymphocyte counts were measured using a computerized hematology analyzer and showed a significant (P?<?0.02) maximum increase (1.6%) at a fluence of 72 J/cm² when compared with non-irradiated samples. This increase in lymphocyte count upon irradiation was confirmed by flow cytometry. At a wavelength of 589 nm and fluence of 72 J/cm², irradiation of whole blood samples showed a significant increase in CD45 lymphocytes and natural killer (NK) (CD16, CD56) cells, but no significant changes in CD3 T lymphocytes, T-suppressor (CD3, CD8) cells, T-helper (CD3, CD4) cells, and CD19 B lymphocytes when compared with their non-irradiated counterparts. Our results clearly demonstrate that NK cell count is altered by irradiation, which ultimately affects the whole lymphocyte count significantly.     

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.     

Effect of diode laser irradiation on the survival rate of gingival fibroblast cell cultures.

BACKGROUND AND OBJECTIVE: The present study is part of a basic research program investigating the cellular effects of the GaAlAs-diode laser with a wavelength of 810 nm on human periodontal tissues. The aim of the investigation was to evaluate the effects of the laser treatment on the survival rate of human gingival fibroblasts (HGF) in monolayer cell culture at different power settings and durations. STUDY DESIGN/MATERIALS AND METHODS: HGF were obtained from a human gingival connective tissue explant. Cells were cultured on 24-well tissue culture plates. One hundred and thirty multi well cell cultures were laser treated. The variable parameters were power output (0.5-2.5 W) and exposure time (60-240 seconds per well). The cultures were analyzed by means of trypan blue staining, and the cell numbers counted under a light microscope. Photographs before and after irradiation were taken and evaluated. RESULTS: Depending on different settings, the laser irradiation caused significant (P < 0.05, t-test) reductions of cell numbers. Exposure time was more relevant to this phenomenon than the power output. Linear regression analysis revealed no unambiguous correlation between power output and cell death, when exposure time was kept constant. CONCLUSIONS: When used for periodontal pocket decontamination, the laser beam may cause damage to collateral periodontal tissues, if the power setting and the duration of the treatment are not adequate. Further investigation, especially with regard to the effects on hard tissue and microorganisms, are needed to give a secure recommendation for irradiation parameters at pocket decontamination.     

Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation

Background and Objective: As Light Emitting Diode (LED) devices are commercially introduced as an alternative for Low Level Laser (LLL) Therapy, the ability of LED in influencing wound healing processes at cellular level was examined. Study Design/Materials and Methods: Cultured fibroblasts were treated in a controlled, randomized manner, during three consecutive days, either with an infrared LLL or with a LED light source emitting several wavelengths (950 nm, 660 nm and 570 nm) and respective power outputs. Treatment duration varied in relation to varying surface energy densities (radiant exposures). Results: Statistical analysis revealed a higher rate of proliferation (p 0.001) in all irradiated cultures in comparison with the controls. Green light yielded a significantly higher number of cells, than red (p 0.001) and infrared LED light (p 0.001) and than the cultures irradiated with the LLL (p 0.001); the red probe provided a higher increase (p 0.001) than the infrared LED probe and than the LLL source. Conclusion: LED and LLL irradiation resulted in an increased fibroblast proliferation in vitro. This study therefore postulates possible stimulatory effects on wound healing in vivo at the applied dosimetric parameters.     

Effects of low-level laser irradiation on mesenchymal stem cell proliferation: a microarray analysis

Increased proliferation after low-level laser irradiation (LLLI) has been well demonstrated in many cell types including mesenchymal stem cells (MSCs), but the exact molecular mechanisms involved remain poorly understood. The aim of this study was to investigate the change in mRNA expression in rat MSCs after LLLI and to reveal the associated molecular mechanisms. MSCs were exposed to a diode laser (635 nm) as the irradiated group. Cells undergoing the same procedure without LLLI served as the control group. Proliferation was evaluated using the MTS assay. Differences in the gene expression profiles between irradiated and control MSCs at 4 days after LLLI were analyzed using a cDNA microarray. Gene ontology and pathway analysis were used to find the key regulating genes followed by real-time PCR to validate seven representative genes from the microarray assays. This procedure identified 119 differentially expressed genes. Real-time PCR confirmed that the expression levels of v-akt murine thymoma viral oncogene homolog 1 (Akt1), the cyclin D1 gene (Ccnd1) and the phosphatidylinositol 3-kinase, catalytic alpha polypeptide gene (Pik3ca) were upregulated after LLLI, whereas those of protein tyrosine phosphatase non-receptor type 6 (Ptpn6) and serine/threonine kinase 17b (Stk17b) were downregulated. cDNA microarray analysis revealed that after LLLI the expression levels of various genes involved in cell proliferation, apoptosis and the cell cycle were affected. Five genes, including Akt1, Ptpn6, Stk17b, Ccnd1 and Pik3ca, were confirmed and the PI3K/Akt/mTOR/eIF4E pathway was identified as possibly playing an important role in mediating the effects of LLLI on the proliferation of MSCs.     

Effects of low-level laser irradiation on proliferation and osteoblastic differentiation of human mesenchymal stem cells seeded on a three-dimensional biomatrix: in vitro pilot study

Mesenchymal stem cells (MSCs) from bone marrow are a recent source for tissue engineering. Several studies have shown that low-level laser irradiation has numerous biostimulating effects. The purpose of this trial was to evaluate the effects of Nd:Yag laser irradiation on proliferation and differentiation of MSCs induced into the osteoblastic lineage. MSCs were collected from adult human bone marrow, isolated, and cultured in complete medium (α-MEM). Subsequently, they were treated with osteogenic medium, seeded in three-dimensional collagen scaffolds, and incubated. We used six scaffolds, equally divided into three groups: two of these were irradiated with Nd:Yag laser at different power levels (15 Hz, 100 mJ, 1.5 W, and one with a power level of 15 Hz, 150 mJ, 2.25 W), and one was left untreated (control group). Evaluations with specific staining were performed at 7 and 14 days. After 7 days, proliferation was significantly increased in scaffolds treated with laser, compared with the control scaffold. After 14 days, however, laser irradiation did not appear to have any further effect on cell proliferation. As concerns differentiation, an exponential increase was observed after 14 days of laser irradiation, with respect to the control group. However, this was a pilot study with very limited sample size, we conclude, that low-level laser irradiation might lead to a reduction in healing times and potentially reduces risks of failure.     

Management of gingival hyperpigmentation by semiconductor diode laser

Gingival hyperpigmentation is caused by excessive deposition of melanin in the basal and suprabasal cell layers of the epithelium. Although melanin pigmentation of the gingiva is completely benign, cosmetic concerns are common, particularly in patients having a very high smile line (gummy smile). Various depigmentation techniques have been employed, such as scalpel surgery, gingivectomy, gingivectomy with free gingival autografting, cryosurgery, electrosurgery, chemical agents such as 90% phenol and 95% alcohol, abrasion with diamond burs, Nd:YAG laser, semiconductor diode laser, and CO(2) laser. The present case report describes simple and effective depigmentation technique using semiconductor diode laser surgery - for gingival depigmentation, which have produced good results with patient satisfaction.