The effect of helium-neon laser irradiation on in vitro maturation and fertilization of immature bovine oocytes

A study was made of the effects of low power laser irradiation on in vitro oocyte maturation rates and subsequent fertilization of immature bovine oocytes. Immature bovine oocytes from cows sacrificed at a slaughterhouse were irradiated with Helium-Neon laser (HeNe laser) irradiation at an energy density of 0.4 and 2.0 J cm^–2. An oocyte group was left untreated, serving as control group. All oocytes were matured and fertilized in TCM-199 medium supplemented with 20% fetal calf serum (FCS). Maturation and fertilization rates obtained in the irradiated oocytes group were lower (p<0.001 andp<0.05, respectively) than those of the control group. Furthermore, the laser-treated oocytes showed important degenerative changes on both cytoplasm and chromosomes in comparison with untreated control oocytes which showed a homogenous cytoplasm and disperse chromosomes. It is concluded that the application of HeNe laser irradiation at 0.4 and 2.0 J cm^–2 energy densities has a detrimental effect on in vitro maturation and fertilization process of immature bovine oocytes.     

Low-level laser irradiation,cyclooxygenase-2 (COX-2) expression and necrosis of random skin flaps in rats

Skin flaps are still a matter of concern among surgeons, as failures can occur leading to flap necrosis. However, low-level laser irradiation has been reported as an effective tool to improve the viability of ischemic flaps, yet its mechanisms of action remain unclear. We investigated the effect of low-level laser irradiation on the viability of random skin flaps in rats and determined COX-2 expression in the flap pedicle. The study animals comprised 24 EPM-1 Wistar rats which were randomly allocated into three equal groups. A cranially based dorsal random skin flap measuring 10 × 4 cm was created in all the animals. In one group, laser irradiation was simulated (sham group), and in the other two groups the animals were irradiated at 12 points with 0.29 J at 20 mW (energy density 10.36 J/cm², irradiance 0.71 W/cm²), or with 7.3 J at 100 mW (energy density 260.7 J/cm², irradiance 3.57 W/cm²). These procedures were applied to the cranial half of the flap immediately after surgery and were repeated on days 2 and 5 after surgery. The percentage necrotic area was determined on day 7 after surgery by the paper template method. The immunohistochemical expression of COX-2 in the samples was given scores from 0 to 3. The necrotic area was smaller in group irradiated at 7.3 J compared to sham-treated group and to the group irradiated at 0.29 J (P < 0.05); there was no difference between the sham-treated group and group irradiated at 0.29 J. COX-2 expression was lower in the group irradiated at 7.3 J than in the sham-treated group and the group irradiated at 0.29 J (P < 0.001). Low-level laser therapy was effective in decreasing random skin flap necrosis in rats using a laser energy of 7.30 J per point. Laser irradiation also decreased the expression of COX-2 in the flap pedicle.     

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

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/cm² 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.     

Effect of Helium-Neon (He-Ne) Laser Irradiation on Dog Neoplasm Cells in Culture

The effects of laser light on the cellular proliferation have been extensively characterised. Low-power laser sources, such as the helium–neon (He-Ne) laser irradiation with a wavelength of 632.8 nm, have been found to produce photobiological and photodamaging effects with evidence of interference with cell proliferation functions. The present study has investigated the in vitro effect of He-Ne laser irradiation on the proliferative action of dog tumour cells in culture. Dose–response studies showed that repeated He-Ne irradiation (irradiance 12.8 mW/cm²) once a day for 4 consecutive days in a dose range between 0.13 and 2.08 J/cm² significantly increased with increasing energy density up to a laser dose of 0.26 J/cm², whereas at >1.04 J/cm², the cell proliferation decreased with increasing energy densities. It is concluded that the application of He-Ne laser irradiation at energy densities ranging from 0.13 J/cm² to 2.08 J/cm² produced different effects on cell proliferation in dog tumour cells in culture. Paper received for publication 27 June 1997; accepted following revision 6 February 1998.     

Biological Effect of Helium-Neon (He-Ne) Laser Irradiation on Mouse Myeloma (Sp2-Ag14) Cell Line In Vitro

We examined the effect of helium-neon (He-Ne) laser irradiation with a wavelength of 632.8 nm on cell cycle synchronisation of monolayer growing mouse myeloma (Sp2-Ag14) cell line. The monolayer cultures were exposed to repeated doses of different energy densities (4–64 J/cm²). The nuclear DNA content has been studied by flow cytometry to obtain the cell percentage in each cell cycle phase. Results showed that the He-Ne laser irradiation at energy densities of 8–64 J/cm² produced a significative and different effect on the G0–G1 and S phases of cell cycle over control. In contrast, no significant effect in G2–M phase was produced by He-Ne laser irradiation at any energy density compared with non-treated control. These results support previous observations suggesting that He-Ne laser irradiation of low energy density interferes with cell cycling and may inhibit cell proliferation when irradiation is performed at doses of 8 J/cm² or more. Received for publication 22 July 1997; accepted following revision 6 February 1998.     

Effect of diode laser on enzymatic activity of parotid glands of diabetic rats

The aim of this study was to evaluate the effect of laser irradiation (LI) on enzymatic activities of amylase, catalase and peroxidase in the parotid glands (PG) of diabetic and non-diabetic rats. Ninety-six female rats were divided into eight groups: D0; D5; D10; D20 and C0; C5; C10; C20, respectively. Diabetes was induced by administration of streptozotocin and confirmed later by the glycemia results. Twenty-nine (29) days after the induction, the PGs of groups D5 and C5; D10 and C10; D20 and C20, were irradiated with 5 J/cm², 10 J/cm² and 20 J/cm² of laser diode (660 nm/100 mW) respectively. On the following day, the rats were euthanized and the enzymatic activity in the PGs was measured. Diabetic rats that had not been irradiated (group D0) showed higher catalase activity (P < 0.05) than those in group C0 (0.14 ± 0.02 U/mg protein and 0.10 ± 0.03 U/mg protein, respectively). However, laser irradiation of 5 J/cm² and 20 J/cm² decreased the catalase activity of the diabetic groups (D5 and D20) to non-diabetic values (P > 0.05). Based on the results of this study, LI decreased catalase activity in the PGs of diabetic rats.     

Microtensile bond strength of resin composite to dentin treated with Er:YAG laser of bleached teeth

The objective of this study was to evaluate the influence of Er:YAG laser (λ = 2.94 μm) on microtensile bond strength (μTBS) and superficial morphology of bovine dentin bleached with 16% carbamide peroxide. Forty bovine teeth blocks (7 × 3 × 3 mm³) were randomly assigned to four groups: G1- bleaching and Er:YAG irradiation with energy density of 25.56 J/cm² (focused mode); G2 - bleaching; G3 - no-bleaching and Er:YAG irradiation (25.56 J/cm²); G4 - control, non-treated. G1 and G2 were bleached with 16% carbamide peroxide for 6 h during 21 days. Afterwards, all blocks were abraded with 320 to 600-grit abrasive papers to obtain flat standardized dentin surfaces. G1 and G3 were Er:YAG irradiated. Blocks were immediately restored with 4-mm-high composite resin (Adper Single Bond 2, Z-250-3 M/ESPE). After 24 h, the restored blocks (n = 9) were serially sectioned and trimmed to an hour-glass shape of approximately 1 mm² at the bonded interface area, and tested in tension in a universal testing machine (1 mm/ min). Failure mode was determined at a magnification of 100× using a stereomicroscope. One block of each group was selected for scanning electron microscope (SEM) analysis. μTBS data was analyzed by two-way ANOVA and Tukey test (α = 0.05). Mean bond strengths (SD) in MPa were: G1- 32.7 (5.9)^A; G2- 31.1 (6.3)^A; G3- 25.2 (8.3)^B; G4- 36.7 (9.9).^A Groups with different uppercase letters were significantly different from each other (p < .05). Enamel bleaching procedure did not affect μTBS values for dentin adhesion. Er:YAG laser irradiation with 25.56 J/cm² prior to adhesive procedure of bleached teeth did not affect μTBS at dentin and promoted a dentin surface with no smear layer and opened dentin tubules observed under SEM. On the other hand, Er:YAG laser irradiation prior to adhesive procedure of non-bleached surface impaired μTBS compared to the control group.     

The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts—an in vitro study

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm² for BMSCs being induced to neurons, and 2 or 4 J/cm² for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm² in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm² dramatically facilitated the differentiation of BMSCs into neurons (p < 0.001). Also, ALP activity was significantly enhanced in irradiated BMSCs differentiated to osteoblast on the second, fifth, seventh, and tenth day of differentiation (p < 0.001 except for the second day). Using LLLI at 810 nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2–6 J/cm², and at the same time increases BMSCs proliferation (except for 6 J/cm²). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation.     

In vitro evaluation of erbium, chromium:yttrium-scandium-gallium-garnet laser-treated enamel demineralization

This study evaluated the effect of different parameters of erbium, chromium:yttrium–scandium–gallium–garnet (Er,Cr:YSGG) laser irradiation on enamel mineral loss in a simulated caries model. Forty-five enamel samples obtained from third molar teeth (3 mm× 3 mm) were randomly divided into five groups (n = 9): G1—Er,Cr:YSGG laser at 0.25 W, 20 Hz, 2.8 J/cm²; G2—Er,Cr:YSGG laser at 0.50 W, 20 Hz, 5.7 J/cm²; G3—Er,Cr:YSGG laser at 0.75 W, 20 Hz, 8.5 J/cm²; G4—sodium fluoride (NaF) dentifrice (positive control); G5—no treatment (negative control). After irradiation, the samples were submitted to 2 weeks of pH cycling. After the acid challenge, the samples were assessed by cross-sectional microhardness at different depths from the enamel surface. Analysis of variance (ANOVA) and Student–Newman–Keuls tests were performed (α = 5%). The percentage of lesion inhibition for each group was: G1 37%; G2 38%; G3 64%, and G4 50.5%. Regarding the relative mineral loss values (micrometers × volume percent), groups G1 (1,392 ± 522) and G2 (1,292 ± 657) did not differ significantly from each other, but both had higher values than group G3 (753 ± 287); the groups irradiated with Er,Cr:YSGG laser did not differ from group G4. Although the findings of the study revealed that Er,Cr:YSGG laser irradiation at 8.5 J/cm² can be an alternative for the enhancement of the enamel’s resistance to acid, lower energy densities also produced a cariostatic potential comparable to the use of fluoride dentifrice.     

Metrical and histological investigation of the effects of low-level laser therapy on orthodontic tooth movement

The aim of this study was to evaluate the effects of 820-nm diode laser on osteoclastic and osteoblastic cell proliferation-activity and RANKL/OPG release during orthodontic tooth movement. Thirty-eight albino Wistar rats were used for this experiment. Maxillary incisors of the subjects were moved orthodontically by a helical spring with force of 20 g. An 820-nm Ga-Al-As diode laser with an output power of 100 mW and a fiber probe with spot size of 2 mm in diameter were used for laser treatment and irradiations were performed on 5 points at the distal side of the tooth root on the first, second, and 3rd days of the experiment. Total laser energy of 54 J (100 mW, 3.18 W/cm², 1717.2 J/cm²) was applied to group II and a total of 15 J (100 mW, 3.18 W/cm², 477 J/cm²) to group III. The experiment lasted for 8 days. The number of osteoclasts, osteoblasts, inflammatory cells and capillaries, and new bone formation were evaluated histologically. Besides immunohistochemical staining of PCNA, RANKL and OPG were also performed. No statistical difference was found for the amount of tooth movement in between the control and study groups (p > 0.05). The number of osteoclasts, osteoblasts, inflammatory cells, capillary vascularization, and new bone formation were found to be increased significantly in group II (p < 0.05). Immunohistochemical staining findings showed that RANKL immunoreactivity was stronger in group II than in the other groups. As to OPG immunoreactivity, no difference was found between the groups. Immunohistochemical parameters were higher in group III than in group I, while both were lower than group II. On the basis of these findings, low-level laser irradiation accelerates the bone remodeling process by stimulating osteoblastic and osteoclastic cell proliferation and function during orthodontic tooth movement.     

Phototherapy promotes cell migration in the presence of hydroxyurea

Phototherapy has been shown to cause an increase in cell proliferation and migration. This study focused on viability (trypan blue), proliferation [sodium 3′-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro)-benzene sulphonic acid hydrate (XTT) and adenosine triphosphate (ATP)] and migration of WS1 cells following irradiation in the presence of hydroxyurea (HU), which is an inhibitor of proliferation. Wounded cells were irradiated on days 1 and 4 with a fluence of 5 J/cm² with a helium–neon (He-Ne) laser at 632.8 nm. After a repair time of 24 h, cellular responses were assessed. Wounded irradiated cells without HU showed an increase in cell viability and proliferation, which was confirmed by complete wound closure by day 4. Although wounded irradiated cells treated with 5 mM HU showed incomplete wound closure, these cells showed increased migration compared with that of control cells. This study showed that laser irradiation using an He-Ne laser with a fluence of 5 J/cm² stimulates cell viability. The HU results confirmed that laser irradiation promotes cell migration and proliferation.     

Stimulatory effect of low-level laser therapy on root development of rat molars: a preliminary study

Several studies suggest a biomodulatory influence of low-level laser irradiation in the inflammatory and reparative processes of dental tissues. The aim of this study was to investigate the stimulatory effect of 808-nm laser irradiation on root development of rat molars and also to evaluate the histological reaction of pulp and periapical tissues. Twenty-four 30-day-old Wistar male rats were randomly assigned to three-time and five-time laser therapy groups. After initial x-ray, using mammography equipment, laser energy was applied at a wavelength of 808 nm (2 J/cm², 100 mW, 20 s) to the midroot area of the lower molars of one side of mouth at repeated intervals of the 48 h. The animals were killed 1 day after the final treatment, and root length development of the experimental samples was compared to contra-lateral non-irradiated molars using mammography. The histological reaction of the pulp and periapical tissue was evaluated under light microscopy. Root development was more advanced in irradiated groups than in the non-irradiated controls (p < 0.001). No significant differences, however, could be found between the root development changes in the three-time and five-time laser therapy groups (p > 0.05). Histological findings showed that the occurrence of secondary cement formation was significantly higher in the irradiation groups compared to the controls (p = 0.003). However, there were no statistically significant differences for the frequencies of pulp hyperemia, periodontal ligament fiber organization, or lamina dura remodeling between the groups (p > 0.05). Under the conditions used in this study, 808-nm low-level laser accelerates the rat molar root development in the presence of favorable histological reactions.     

Histomorphometric analysis of inflammatory response and necrosis in re-implanted central incisor of rats treated with low-level laser therapy

Low-level laser therapy is a tool employed in the management of post-operative inflammation process and in the enhancement of reparative process. The aim of the study was to perform histological evaluation of dental and periodontal ligament of rats central upper-left incisor teeth re-implanted and irradiated with low-level laser (InGaAl, 685 nm, 50 J/cm²) 15, 30, and 60 days after re-implantation. Seventy-two male rats had the central upper left incisor removed and kept for 15 min on dry gauze before replantation. Laser was irradiated over the root surface and empty alveolus prior replantation and over surrounding mucosa after the re-implantation. After histological procedures, all slices were analyzed regarding external resorption area and histological aspects. We observed an increase of root resorption (p < 0.05) in the control group compared to the laser group at 15, 30, and 60 days. These results showed that the laser groups developed less root resorption areas than the control group in all experimental periods. Additionally, histological analysis revealed less inflammatory cells and necrotic areas in laser groups.     

Photodynamic Inactivation of the Single Crayfish Nerve Cell: Dynamics of Electrophysiological Responses and Comparison of Photosensitisers

. The study of single neuron response to photodynamic effect provides a means for the study of the dynamics of cytotoxic events leading to cell death and allows comparison of the phototoxicity of different photosensitisers. Isolated crayfish stretch receptor neurons were photosensitised for 30 min, then irradiated with a He-Ne laser (632.8 nm; 0.3 W/cm²) until irreversible firing cessation. The dynamics of neuron firing frequency were continuously recorded throughout. The following photosensitisers were studied: methylene blue, janus green B, protoporphyrin IX, chlorins e ₆ and p ₆, haematoporphyrin derivative (Photoheme) and sulphonated aluminium phthalocyanine (Photosens). Nerve cells were found to be insensitive to either He-Ne laser irradiation or photosensitisation alone, but very vulnerable to the photodynamic effect: neurons changed firing rate and died at nanomolar concentrations of photosensitisers. The dynamics of neuron responses was found to depend on photosensitiser type and concentration. The current approach provides a means of evaluation of initial threshold cell membrane alteration and cytotoxic events leading to cell death. The dependence of firing acceleration rate and neuron lifetime on photosensitiser concentration additionally allowed comparison of efficiencies of different photosensitisers. Photosens, Photoheme and chlorin p ₆ were found to be the most potent photosensitisers: neurons responded to their photodynamic effects at concentrations as low as 1–5 nM. Paper received 23 February 1998; accepted following revision 7 December 1998.     

Effects of low-level laser therapy (685 nm) at different doses in osteogenic cell cultures

The present in vitro study evaluated parameters of osteogenesis under the influence of low-level laser therapy (LLLT) at different doses. Osteogenic cells originated from rat calvaria were cultivated in polystyrene plates and exposed to a laser irradiation using an indium-gallium-aluminum phosphide therapeutic laser (InGaAIP), at wavelength of 685 nm, power of 35 mW, 600-μm-diameter optical fiber, and continuous wave. In the attempt of observing the existence of a dose response and its effects, laser irradiation was performed at 25, 77, and 130 J/cm² (7, 22, and 37 s, respectively). The following parameters were assessed: growth curve (4, 7, and 11 days), cell viability (24 h), and nodular formation of mineralized matrix (14 days). The results did not show significant differences related to the growth curve (4, 7, and 11 days) and cell viability (24 h). Within 14 days, osteogenic cultures showed nodular areas with well-defined calcified matrix. The total area stained with Alizarin Red did not show any differences between doses of 25 and 130 J/cm². However, the percentage of stained area was significantly higher in the 25 J/cm² group when compared to the group of 77 J/cm² (Kruskal-Wallis test, p < 0.05). It was possible to conclude that the 685-nm laser irradiation (at 25, 77, and 130 J/cm²) did not influence cell growth and proliferation, although the extracellular mineralization process may have its pattern altered by the LLLT on osteogenic cell cultures.     

Histological and clinical responses of articular cartilage to low-level laser therapy: Experimental study

This study was carried out to evaluate the effects of low-level laser irradiation on experimental lesions of articular cartilage. A standard lesion was practised on the femoral trochlea of both hindlimbs of 20 clinically normal Californian rabbits. These animals were divided into two groups of 10 individuals each, depending on the laser equipment used for treatment. Onc group was treated with He-Ne laser (8 J cm^-2, 632.8 nm wavelength) and the other with infra-red (IR) laser (8 J cm^-2, 904 nm wavelength). In both groups, five points of irradiation to the right limb alone were irradiated per session for a total of 13 sessions, applied with an interval of 24 h between sessions. These points were the following: left and right femoral epicondyles, left and right tibial condyles and the centre of articulation. The distance between these points was approximately 1 cm. The untreated left limb was left as a control. During treatment, extension angle and periarticular thickness were considered. At the end of the treatment, samples were collected for histopathologi-cal study and stained with: Haematoxylin-Eosin, PAS and Done. The results show a statistically higher anti-inflammatory capacity of the IR laser (p < 0.0001). The functional recovery was statistically similar for both treatments (p < 0.176). Histological study showed, at the end of the treatment, hyaline cartilage in the IR group, fibrocartilage in the He-Ne group and granulation tissue in the control limbs. Clinical and histological results indicated that this laser treatment had a clear anti-inflammatory effect that provided a fast recuperation and regeneration of the articular cartilage.     

Laser and light-emitting diode effects on pre-osteoblast growth and differentiation

The acceleration of bone regeneration by low-intensity laser irradiation may hold potential benefits in clinical therapy in orthopedics and dentistry. The purpose of this study is to compare the effects of light-emitting diode (LED) and laser on pre-osteoblast MC3T3 proliferation and differentiation. Cells were irradiated with red, infrared, and LED (3 and 5 J/cm²). Lasers had a power density of 1 W/cm² and irradiation time of 2 and 5 s. LED had a power density of 60 mW/cm² and irradiation time of 50 and 83 s. Control group did not receive irradiation. Cell growth was assessed by a colorimetric test (MTT) (24, 48, 72, and 96 h), and cell differentiation was evaluated by alkaline phosphatase (ALP) quantification after growth in osteogenic medium (72 and 96 h and 7 and 14 days). At 24 h, the cell growth was enhanced 3.6 times by LED (5 J/cm²), 6.8 times by red laser (3 J/cm²), and 10.1 times by red laser (5 J/cm²) in relation to control group (p?<?0.05). At the other periods, there was no influence of irradiation on cell growth (p?>?0.05). The production of ALP was not influenced by irradiation at any period of time (p?>?0.05). Low-intensity laser and LED have similar effects on stimulation of cell growth, but no effect on cell differentiation.