Effects of the invasive and non-invasive systemic photobiomodulation using low-level laser in experimental models: A systematic review.

Systemic photobiomodulation (PBM) of the blood or over blood vessels has been associated with bio-stimulating, vasodilating, and anti-inflammatory properties. This treatment modality has been used for modulating inflammatory processes, tissue repair, atherosclerosis, and systemic arterial hypertension, and is described more often in clinical studies than experimental models. Therefore, the aim of the present study was to conduct a literature review regarding the effect of systemic PBM involving the intravascular laser irradiation of blood (ILIB) or non-invasive vascular photobiomodulation (VPBM) using low-level laser (LLL) in experimental (animal) models. The PubMed/MEDLINE®, Scopus, SPIE Digital Library, and Web of Science databases were searched for articles on the use of VPBM with LLL in animal models. Nine original articles met the inclusion criteria and were critically evaluated. The variables of interest were the dosimetric laser parameters, different methods for delivering energy, and the main results. The use laser in the red spectrum was more prevalent and VPBM (non-invasive) predominated over ILIB (invasive). No standardization was found in the dosimetric parameters. However, the studies showed the positive effects of VPBM on arterial pressure and blood circulation, the positive effects of ILIB on blood composition and hematological markers, as well as positive effects of both forms of systemic PBM (ILIB and VPBM) on the tissue repair process. In conclusion, the studies evaluated in the present review showed that the use of systemic PBM with ILIB or non-invasive VPBM induced positive effects, modulating metabolic conditions and tissue repair. However, there is a need for standardization in the dosimetric parameters for the different conditions and processes evaluated using experimental models.

Optimizing Dacarbazine Therapy: Design of a Laser-Triggered Delivery System Based on ß-Cyclodextrin and Plasmonic Gold Nanoparticles.

Dacarbazine (DB) is an antineoplastic drug extensively used in cancer therapy. However, present limitations on its performance are related to its low solubility, instability, and non-specificity. To overcome these drawbacks, DB was included in ß-cyclodextrin (ßCD), which increased its aqueous solubility and stability. This new ßCD@DB complex has been associated with plasmonic gold nanoparticles (AuNPs), and polyethylene glycol (PEG) has been added in the process to increase the colloidal stability and biocompatibility. Different techniques revealed that DB allows for a dynamic inclusion into ßCD, with an association constant of 80 M-1 and a degree of solubilization of 0.023, where ßCD showed a loading capacity of 16%. The partial exposure of the NH2 group in the included DB allows its interaction with AuNPs, with a loading efficiency of 99%. The PEG-AuNPs-ßCD@DB nanosystem exhibits an optical plasmonic absorption at 525 nm, a surface charge of -29 mV, and an average size of 12 nm. Finally, laser irradiation assays showed that DB can be released from this platform in a controlled manner over time, reaching a concentration of 56 µg/mL (43% of the initially loaded amount), which, added to the previous data, validates its potential for drug delivery applications. Therefore, the novel nanosystem based on ßCD, AuNPs, and PEG is a promising candidate as a new nanocarrier for DB.

Synthesis of gold decorated silica nanoparticles and their photothermal properties.

Silica-Gold Nanostructures (SGNs), composed of a silica core decorated by gold nanoparticles, have the photothermal capacity to transform near-infrared (NIR) wavelengths into heat. This work presents a simple, efficient, and replicable method of synthesis of SGNs and their characterization by: (1) transmission electron microscopy to obtain micrographs of the particles and their corresponding diameter distribution; (2) diffraction patterns showing the amorphous atomic arraignment of the silica and the crystalline atomic arrangement of the gold nanoparticles; (3) zeta potential confirming the stability of the SGNs in a colloidal solution; and (4) thermal images displaying the capacity of SGNs to convert NIR irradiation into heat and their respective increment in temperature. SGNs were synthesized over silica cores with diameters of 63, 83, and 132 nm and decorated with a partial gold shell. They were heated with a coherent light intensity of 340 mW/cm2 with a wavelength of 852 nm. This wavelength is within the range of the optical window of the human body; therefore, SGNs may be used for the photothermal ablation of tumors with no damage to the tissue. The heating of different dimensions of SGNs took 6-8 min of NIR radiation, and their cooling, once the laser was turned off, was in the order of 2-3 min. It was found that SGNs, with a core diameter of 132 nm, have a notable photothermal capacity. That enables them to increase the temperature of their surroundings by 4.4 ºC. This increment in temperature is sufficient to induce cellular necrosis, which makes SGNs a good option for photothermal treatments.

Spatiotemporal Temperature Distribution of NIR Irradiated Polypyrrole Nanoparticles and Effects of pH.

The spatiotemporal temperature distributions of NIR irradiated polypyrrole nanoparticles (PPN) were evaluated by varying PPN concentrations and the pH of suspensions. The PPN were synthesized by oxidative chemical polymerization, resulting in a hydrodynamic diameter of 98 ± 2 nm, which is maintained in the pH range of 4.2-10; while the zeta potential is significantly affected, decreasing from 20 ± 2 mV to -5 ± 1 mV at the same pH range. The temperature profiles of PPN suspensions were obtained using a NIR laser beam (1.5 W centered at 808 nm). These results were analyzed with a three-dimensional predictive unsteady-state heat transfer model that considers heat conduction, photothermal heating from laser irradiation, and heat generation due to the water absorption. The temperature profiles of PPN under laser irradiation are concentration-dependent, while the pH increase only induces a slight reduction in the temperature profiles. The model predicts a value of photothermal transduction efficiency (η) of 0.68 for the PPN. Furthermore, a linear dependency was found for the overall heat transfer coefficient (U) and η with the suspension temperature and pH, respectively. Finally, the model developed in this work could help identify the exposure time and concentration doses for different tissues and cells (pH-dependent) in photothermal applications.

In vitro evaluation of EDTA combined with photodynamic therapy to reduce Streptococcus mutans in carious dentin.

BACKGROUND: This in vitro study aimed to evaluate the use of EDTA combined with photodynamic therapy to reduce Streptococcus mutans in carious dentin. METHODS: Sixty third molars were sectioned to obtain flat dentin surfaces. All specimens were waterproofed, except for the coronal dentin, and subjected to cariogenic challenge in brain-heart infusion (BHI) broth supplemented with 0.5% yeast extract, 1% glucose, 1% sucrose, and standard strain of S. mutans (ATCC 25175). The specimens were divided into 6 groups (n = 10 each): (1) control - caries collection; (2) EDTA - 17% EDTA was actively applied with a microbrush for 1 min; (3) aPDT - antimicrobial photodynamic therapy with 0.01% methylene blue photosensitizer (wavelength of 660 nm, energy of 4 J, power of 100 mW, spot size of 0.028 cm2, energy density of 142 J/cm2 for 40 s); (4) EDTA+aPDT - 17% EDTA actively applied for 1 min plus aPDT; (5) (EDTA+PT) + L - application of EDTA compounded with photosensitizer plus laser irradiation; and (6) PT - photosensitizer alone. Collection of caries was performed after the different cavity disinfection protocols. Aliquots from each dilution were seeded for colony-forming unit (CFU) counts. The results were log10-transformed and analyzed by the Kruskal-Wallis test (Student-Newman-Keuls). RESULTS: There was a significant reduction in S. mutans after aPDT (p<0.05), EDTA+aPDT (p<0.001), and (EDTA+PT) + L (p<0.001). The percentage of microbial reduction in ascending order was as follows: EDTA: 1.65%; PT: 15.51%; aPDT: 38.28%; EDTA+aPDT: 75.24%; and (EDTA+PT) + L: 97.35%. CONCLUSION: Application of 17% EDTA prior to photosensitization or compounded with a photosensitizer increased the antimicrobial effect of aPDT on S. mutans in carious dentin.

Post-resistance exercise photobiomodulation therapy has a more effective antioxidant effect than pre-application on muscle oxidative stress.

This study evaluated the effect of photobiomodulation therapy (PBMt) before or after a high-intensity resistance exercise (RE) session on muscle oxidative stress. Female Wistar rats were assigned to one of the following groups: Sham (non-exercised, undergoing placebo-PBMt); NLRE (exercised, undergoing placebo-PBMt); PBMt + RE (pre-exercise PBMt); RE + PBMt (post-exercise PBMt). The RE comprised four climbs bearing the maximum load with a 2 min rest between each climb. An 830-nm aluminum gallium arsenide diode laser (100 mW; 0.028 cm2; 3.57 mW/cm2; 142.8 J/cm2; 4 J; Photon Laser III, DMC, São Paulo, Brazil) was applied 60 s before or after RE in gastrocnemius muscles. Analyses were performed at 24 h after RE: lipoperoxidation using malondialdehyde (MDA) and protein oxidation (OP) on Western blot. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity were spectrophotometrically assessed. Nitric oxide (NO) level was determined by the Griess reaction. The MDA and OP levels were significantly higher in the NLRE group. Increased OP was prevented in all PBMt groups; however, increased MDA was prevented only in the RE + PBMT group. The RE + PBMt group had higher SOD activity compared to all other groups. A higher GPx activity was observed only in the PBMT + RE compared to Sham group, and CAT activity was reduced by RE, without PBMt effect. NO levels were unchanged with RE or PBMt. Therefore, PBMt application after a RE section has a more potent antioxidant effect than previous PBMt. Rats submitted to post-RE PBMt illustrated prevention of increased lipoperoxidation and protein oxidation as well as increased SOD activity. The photobiomodulation can attenuate oxidative stress induced by resistance exercise. A more evident benefit shows to be obtained with the application after exercise, in which it has increased the activity of superoxide dismustase.

Effect of cetrimide 2% with and without photodynamic therapy to reduce Streptococcus mutans burden in dentinal carious lesions.

To evaluate the use of cetrimide alone and combined with photodynamic therapy to reduce S. mutans burden in carious lesions. Sixty permanent third molars were sectioned and the coronal dentin exposed. A cariogenic challenge was performed using brain-heart infusion (BHI) medium supplemented and S. mutans ATCC 25175. Specimens were incubated in anaerobic jars at 37 °C for 15 days, with BHI renewed every 24 h. After 15 days, specimens were randomly divided into six groups (n = 10): C, control (no treatment); CHX, application of chlorhexidine 2%; CT, application of cetrimide 2%; CT+aPDT, application of cetrimide 2% followed by methylene blue dye and aPDT (antimicrobial photodynamic therapy: wavelength 660 nm, energy 4J, power 100 mW, spot size 0.0028 cm2, energy density 142 J/cm2 for 40 s); ES+aPDT, application of experimental solution (methylene blue dye with cetrimide) and aPDT; and aPDT alone. Carious tissue from each specimen was collected before and after the applications. Five decimal dilutions were performed, and the resulting solution was seeded in mitis-salivarius-bacitracin agar. Plates were incubated in anaerobic jars at 37 °C for 48 h. Analysis of variance (ANOVA) with post hoc Tukey's test was used to compare total S. mutans counts. Significant reductions in S. mutans were observed after application of CT+aPDT (0.30 (0.97), p < 0.0001) and ES+aPDT (0.52 (1.13), p < 0.0001). Cetrimide 2% with methylene blue dye, applied consecutively or as a mixture, can be used as a photosensitizing agent for aPDT to reduce S. mutans burden in dentinal caries.

mILIB: a new concept in laser therapy / mILIB: un nuevo concepto en laserterapia

mILIB (Intravascular Laser Irradiation in the Modified Bloodstream) presents itself as one more photobiomodulation technique, among the many therapeutic possibilities we know. The advantage in this case is the possibility of making application in a systemic way in which the organism as a whole is benefited. The use of the red therapeutic laser in a transcutaneous way, has been a great option as an added therapeutic value to the treatment of several pathologies, providing systemic stimuli, similar to hemotherapy. Thus, mILIB presents itself as an effective and increasingly widespread technique for professionals working with photobiomodulation

mILIB (Irradiación láser intravascular en el torrente sanguíneo modificado) se presenta como una técnica de fotobiomodulación más, entre las muchas posibilidades terapéuticas que conocemos. La ventaja en este caso es la posibilidad de realizar una aplicación de forma sistémica en la que se beneficia al organismo en su conjunto.El uso del láser terapéutico rojo de forma transcutánea, ha sido una gran opción como valor terapéutico añadido al tratamiento. de varias patologías, proporcionando estímulos sistémicos, similar a la hemoterapia. Así, mILIB se presenta como una técnica eficaz y cada vez más extendida para los profesionales que trabajan con fotobiomodulación.

A simple synthesis of ZnO:Co2O3 nanocomposites by pulsed laser irradiation in liquid.

Nanocomposite materials are emerging in popularity due to their enhanced performance over the constituent materials. In this work, we report the fabrication of zinc oxide: cobalt oxide nanocomposites in a simple, fast and room temperature synthesis with good productivity. The nanocomposites synthesized were characterized by SEM, XPS and UV-Visible spectroscopy to analyze their morphology, composition, chemical states, optical absorption, band gap etc. The nanocolloids of the composite were drop casted to form thin films for photocatalytic studies. In SEM analysis, the morphological transformation of the material is observed where it transformed from agglomerated spherical particles to petals shaped and then to partially spherical forms due to pulsed laser irradiation. XPS analysis showed a gradual change in oxygen high resolution spectra in the samples with respect to the concentration difference of cobalt oxide. The optical studies show an enhanced absorption in visible region for the nanocomposite and the energy band gap reduced to 2.4 eV. All the thin films of nanocomposite showed photocatalytic decay of methylene blue dye under visible light irradiation. The results of this study support the effective use of laser irradiation in liquid to obtain nanocomposites of metal oxides for photocatalytic applications.

Effect of 12 Weeks of Endurance Training Combined with Creatine Supplement, Photobiomodulation Therapy, or Both on Performance and Muscle Damage in Rats.

Background: Photobiomodulation therapy (PBMT) and creatine (Cr) intake have been used in conjunction with heavy training, but little is known about their possible effects during a long-term training program. Objective: We assessed long-term use of PBMT and Cr in an exercise training program. Methods: Twenty-five male Wistar rats weighing ∼300 g were randomly allocated to one of five groups: a nontraining control group, a training group, a training group receiving Cr, a training group receiving PBMT, and a training group receiving both PBMT and Cr. The training program consisted of 12 weeks of daily swimming training. PBMT was delivered in six points with a laser device (808 nm, 100 mW, 30 sec per point of irradiation, 3 J, 75 J/cm2). Results: All training groups showed significantly higher peak force and longer time to 50% decay of force, and lower creatine kinase (CK) levels than the nontraining control group, thus confirming the benefit of the training program. In all outcomes related to muscle performance, the groups receiving PBMT with or without Cr supplement performed significantly better (p < 0.05) peak force and time of force decay during an electrical stimulation protocol than all the other groups. In addition, CK levels were also significantly lower for the PBMT groups than for the other groups. Conclusions: We conclude that PBMT alone or in conjunction with Cr supplement during a 12-week training program resulted in significantly better muscle performance and lower levels of CK, a biochemical marker of muscle damage.

Avaliação de protocolo para terapia com laser de baixa intensidade no tratamento da síndrome da ardência bucal: um estudo clínico cego e randomizado / Evaluation of a protocol for laser therapy of low-intensity treatment of the syndrome of burning mouth: a blinded, randomized clinical trial

Até o momento, não existe consenso quanto ao tratamento para a síndrome da ardência bucal (SAB), definida como queimação idiopática que afeta a mucosa bucal e a região perioral clinicamente normais. A terapia a laser de baixa intensidade (TLBI) tem se mostrado método não invasivo favorável, entretanto, a metodologia utilizada na literatura é bastante variada, gerando resultados discrepantes. O presente trabalho objetivou desenvolver um protocolo de TLBI para a SAB e avaliar sua eficácia em atenuar e/ou eliminar os sintomas. O estudo consistiu em um ensaio clínico, cego, randomizado, com controle por placebo. Valeu-se de uma amostra de 10 voluntários com SAB, separados em dois grupos por alocação oculta: 5 do Grupo Intervenção (GI) e 5 do Grupo Controle (GC). A randomização foi feita em blocos. Os voluntários compareceram a 8 consultas, em 5 semanas, e foram submetidos à TLBI ou placebo a partir da segunda consulta, até a penúltima. Além da laserterapia, foram aplicados os questionários de dor Douleur Neuropathique in 4 questions (DN4) e McGill (MPQ) e o Brief Pain Inventory (BPI), utilizados para avaliação clínica da dor. Indivíduos do GI apresentaram redução estatisticamente significativa nos escores do DN4 (p = 0.03), o que não ocorreu com o GC (p > 0.05). Também houve diferença entre os grupos para o MPQ, nos tempos (T) 1 (p = 0.02), 5 (p = 0.04) e 8 (p = 0.03), com redução significativamente distinta entre GI e GC. Tiveram significância estatística no BPI os critérios "habilidade para apreciar a vida" e "percentual de melhora com a laserterapia". No primeiro, houve diferença na comparação entre T1 e os demais tempos (T2-T8) para o GI (p < 0.05), e não para o GC (p > 0.05). Para o segundo critério BPI, no GI, o escore foi reduzido a zero no T2, mantendo-se assim até o T8. O GC, todavia, não expressou resultados estatisticamente significativos na comparação entre os tempos (p > 0.05). Os achados observados neste estudo indicam que a TLBI representa um potencial tratamento para atenuar os sintomas em pacientes com SAB. (au)

Until the present moment there is no consensus regarding to the treatment of the burning mouth syndrome (BMS), which is defined as an idiopathic burning symptom that affects clinically normal oral mucosa and perioral region. Low-level laser therapy (LLLT) has been proved favorable non-invasive method; however, the protocols in the literature is quite varied achieving different results. The present study aimed to develop a LLLT protocol for BMS and evaluate its effectiveness in mitigating and/or eliminating symptoms. The study consisted of a randomized, blinded, placebocontrolled clinical trial. A sample of ten volunteers with BMS was recruited divided into two groups by hidden allocation: five in the Intervention Group (IG) and five in the Control Group (CG). Randomization was performed in sets. The volunteers came to eight appointments, in five weeks, and were submitted to LLLT or placebo from the second appointment, until the day before the last appointment. In addition to laser therapy, the Douler Neuropathique in 4 questions (DN4) and McGill (MPQ) pain questionnaires and a brief pain inventory (BPI), for clinical pain assessment, were applied. IG showed a statistically significant improvement in the DN4 scores (p = 0.03), which did not occur in the CG (p > 0.05). There was also a difference between the groups for the MPQ, at day (T) 1 (p = 0.02), 5 (p = 0.04) and 8 (p = 0.03), with a significantly difference improvement when compare IG with CG. The criteria "ability to enjoy life" and "percentage of improvement with laser therapy" were statistically significant at BPI. In the first, there was a difference in the comparison between T1 and the other days (T2-T8) for the IG (p < 0.05), and not for the CG (p > 0.05). For the second BPI assessment the score for IG was reduced to zero in T2, thus maintaining it until T8. The CG, however, did not express statistically significant results when compared the times (p > 0.05). The findings observed in this study indicate that LLLT represents a potential treatment to improve symptoms in patients with BMS. (AU)

Four-Session Protocol Effectiveness in Reducing Cervical Dentin Hypersensitivity: A 24-Week Randomized Clinical Trial.

Objective: A single-blind randomized clinical trial was conducted to evaluate the effectiveness of desensitizing agents with different action mechanisms in reducing cervical dentin hypersensitivity (CDH) after four application sessions, with 24-week follow-up. Materials and methods: Sixty patients with CDH were selected in the study and were allocated in three groups of treatment: Desensibilize KF 2%, Clinpro XT Varnish, and Photon Lase III (100 mW, 4 J/cm2-1 J/cm2 each point, 10 sec per point with wavelength of 808 nm). There were four application sessions performed, with a 48-h interval between each one. The evaporative stimuli and visual analog scale were used to evaluate the CDH level at baseline, immediately after treatment, and at 2, 4, 8, and 24 weeks after the application. Mixed-model effects test was used for comparison (α = 0.05).Results: All three groups showed significant reduction in CDH from baseline to each all-subsequent follow-up. All the groups maintained the CDH reduction, and presented no statistical differences between each other after treatment (p = 0.885), 2 (p = 0.857), 4 (p = 0.928), 8 (p = 0.206), and 24 weeks (p = 0.073) of follow-up.Conclusions: The four-session protocol was an effective approach in reduction of CDH (even after 24 weeks), regardless of desensitization mechanism.

Tailoring the Bactericidal Activity of Ag Nanoparticles/α-Ag2WO4 Composite Induced by Electron Beam and Femtosecond Laser Irradiation: Integration of Experiment and Computational Modeling.

In nanotechnology research, significant effort is devoted to fabricating patterns of metallic nanoparticles on the surfaces of different semiconductors to find innovative materials with favorable characteristics, such as antimicrobial and photocatalytic properties, for novel applications. We present experimental and computational progress, involving a combined approach, on the antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) of as-synthesized α-Ag2WO4 samples and Ag nanoparticle composites (Ag NPs)/α-Ag2WO4. The former included two morphologies: hexagonal rod-like (α-Ag2WO4-R) and cuboid-like (α-Ag2WO4-C), and the latter included composites formed under electron beam, Ag NPs/α-Ag2WO4-RE and Ag NPs/α-Ag2WO4-CE, and femtosecond (fs) laser irradiation, Ag NPs/α-Ag2WO4-RL and Ag NPs/α-Ag2WO4-CL. Direct observations of the arrangement of Ag NPs on the Ag NPs/α-Ag2WO4 composites irradiated with an electron beam and laser, through transmission electron microscopy (TEM), high-resolution TEM, energy-dispersive X-ray spectroscopy, and field-emission scanning electron microscopy, allow us to investigate the surface morphology, chemical composition, homogeneity, and crystallinity. Therefore, these experimental factors, and in particular, the facet-dependent response of Ag NPs/α-Ag2WO4 composites were discussed and analyzed from the perspective provided by the results obtained by first-principles calculations. On this basis, α-Ag2WO4-R material proved to be a better bactericidal agent than α-Ag2WO4-C with minimum bactericidal concentration (MBC) values of 128 and 256 µg/mL, respectively. However, the Ag NPs/α-Ag2WO4-CL composite is the most efficient bactericidal agent of all tested samples (MBC = 4 µg/mL). This superior performance can be attributed to the cooperative effects of crystal facets and defect engineering. These results on the synthesis and stability of the Ag NPs/α-Ag2WO4 composites can be used for the development of highly efficient bactericidal agents for use in environmental remediation and the potential extension of methods to produce materials with catalytic applications.

Dental acid etchant as a sensitizing agent in photodynamic therapy to reduce S. mutans in dentinal carious lesions.

The study aims to assess the utility of dental acid etchant containing 37% phosphoric acid and methylene blue dye (DAE) as a sensitizing agent for photodynamic therapy (PDT) to reduce Streptococci mutans in dentinal caries. Forty-five permanent third molars were sectioned and the coronal dentin exposed. A cariogenic challenge was performed using brain-heart infusion (BHI) supplemented with 0.5% yeast extract, 1% glucose, 1% sucrose, and S. mutans ATCC 25175 standardized to 0.5 McFarland turbidity. Specimens were incubated in anaerobic jars at 37 °C for 15 days. During this period, BHI broth was renewed every 24 h. After 15 days, specimens were randomly divided into three groups (n = 15): DAE, application of dental acid etchant containing 37% phosphoric acid and methylene blue dye for 15 s; LLL, application of low-level laser (wavelength 660 nm, energy 4 J/cm2, power 5 W) for 15 s; and PDT, application of DAE for 15 s followed by LLL irradiation (660 nm, 4 J/cm2, 5 W). Carious tissue from each specimen was collected before and after the applications. Five decimal dilutions were performed and the resulting solution was seeded in mitis-salivarius-bacitracin agar. Plates were incubated in anaerobic jars at 37 °C for 48 h. Analysis of variance (ANOVA) with post hoc Tukey's test was used to compare total S. mutans counts. Significant reductions in S. mutans were observed after DAE application (40.70%, p < 0.0001), LLL (12.35%, p = 0.0036), and PDT (55.22%, p < 0.0001). Dental acid etchant containing 37% phosphoric acid and methylene blue dye can be used as a photosensitizing agent for PDT to reduce S. mutans burden in dentinal caries.

Efeito do laser de baixa intensidade na atividade biológica de células-tronco do ligamento periodontal humano cultivadas sobre arcabouços de quitosana / Effect of low-intensity laser on the biological activity of human periodontal ligament stem cells cultured on chitosan scaffolds

O laser de baixa intensidade (LBI) é capaz de estimular a proliferação de diferentes tipos celulares, porém pouco se sabe sobre sua eficácia na proliferação de células cultivadas na superfície dos biomateriais. O objetivo deste estudo foi avaliar a influência do LBI na proliferação e viabilidade de células-tronco do ligamento periodontal humano (PDLSCs) cultivadas em arcabouços de quitosana. A quitosana foi submetida a testes para identificação do teor real de massa e grau de desacetilação. Membranas de quitosana foram preparadas pela técnica de evaporação de solvente e submetidas a caracterização de morfologia e de superfície. PDLSCs previamente isoladas e caracterizadas foram cultivadas sobre quatro superfícies: (P) plástico da placa de cultivo, não irradiado, como controle positivo de crescimento celular; (Q) quitosana, não irradiado; (L1) quitosana, irradiado com dose de 1 J/cm²; e (L4) quitosana, irradiado com 4 J/cm². As irradiações foram realizadas com laser diodo InGaAlP, com comprimento de onda de 660 nm, potência de 30 Mw, diâmetro da ponta de 0.01cm² e modo de ação contínuo, em dose única. Os dados mostraram que a quitosana apresentou um teor real de massa de 88,08% e grau de desacetilação de 91,37±3,77%. A análise das membranas por MEV mostrou superfície uniforme e homogênea, com espessura média de 68,71 µm. A análise por microscopia de força atômica revelou uma rugosidade média de 285 nm. O peso das membranas variou de 0,03 a 0,04 g, indicando a sua uniformidade, e o pH de superfície exibiu média de 6,9±0,25, valor próximo ao pH da saliva. A viabilidade e a proliferação celular foram avaliadas através dos ensaios de Alamar Blue, Live/Dead, Annexin V/PI e Ki67, além da análise do ciclo celular, e a morfologia celular foi avaliada por microscopia eletrônica de varredura (MEV). O ensaio do Alamar Blue mostrou diferenças significativas na atividade mitocondrial entre os grupos nos intervalos de 24 h (L1>Q, p= 0,0118) e em 48 h (L1>Q, p= 0,0022; L4>Q, p=0,0002; L4>L1, p= 0,0022). O ensaio Live/Dead mostrou maior densidade de células vivas nos grupos irradiados (L1 e L4) em relação ao grupo sem irradiação (Q), o que foi comprovado pelo ensaio da Annexin V/PI, que mostrou um maior percentual de células viáveis em L4 (89,5%) e L1 (87,0%) em comparação com Q (78,4%) em 72 h. A imunoexpressão da proteína Ki67 foi maior em L4 e L1 e estes dois grupos apresentaram também um maior percentual de células nas fases proliferativas do ciclo celular (S e G2/M). A análise por MEV mostrou no grupo Q células com morfologia mais arredondada e com poucas projeções, além de debris celulares, enquanto nos grupos irradiados as células exibiram um arranjo mais plano, com projeções mais distribuídas e pontos de adesão focal, especialmente em L4. Em conjunto, os resultados do presente trabalho permitem concluir que a laserterapia nos padrões estudados, especialmente na dose de 4 J/cm², influencia positivamente a viabilidade e a proliferação de PDLSCs em membranas de quitosana, permitindo assim que as células superem eventuais efeitos adversos do microambiente do arcabouço (AU).

Low-level laser irradiation (LLLI) is able to stimulate the proliferation of various cell types, but little is known about its effectiveness on the proliferation of cells cultured on biomaterial surfaces. The aim of this study was to evaluate the influence of LLLI on the proliferation and viability of human periodontal ligament stem cells (PDLSCs) cultured on chitosan scaffolds. Chitosan was submitted to tests to identify the real mass content and degree of deacetylation. Chitosan membranes were prepared by solvent evaporation technique and submitted to morphology and surface characterization. PDLSCs previously isolated and characterized were grown on the surfaces of four groups: (P) culture plate plastic, non-irradiated, as a positive control of cell growth; (C) chitosan, non-irradiated; (L1) chitosan irradiated with a dose of 1 J/cm²; and (L4) chitosan, irradiated with 4 J/cm². The irradiations were performed with InGaAlP diode laser with wavelength of 660 nm, power 30 mW, tip diameter of 0.01cm², and continuous action mode in a single dose. Cell viability and proliferation were evaluated by Alamar Blue, Live/Dead, Annexin V/PI and Ki67 assays, as well as cell cycle analysis, whereas cell morphology was evaluated by MEV. The data showed that the chitosan presented a real mass content of 88.08% and degree of deacetylation of 91.37 ± 3.77%. SEM analysis showed membranes with uniform and homogeneous surface, with a mean thickness of 68.71 µm. Analysis by AFM revealed roughness around 285 nm. The weight of the membranes ranged from 0.03 to 0.04 g, indicating their uniformity, and the surface pH exhibited a mean of 6.9 ± 0.25, a value close to the pH of the saliva. The Alamar Blue assay showed significant differences in mitochondrial activity between groups at 24 h (L1> C, p = 0.0118) and at 48 h (L1> C, p = 0.0022; L4> C, p = 0.0002; L4>L1, p = 0.0022). The Live/Dead assay showed higher density of live cells in irradiated groups (L1 and L4) compared to the group without irradiation (C), which was confirmed by assay of Annexin V/PI, which showed a greater percentage of viable cells in L4 (89.5%) and L1 (87.0%) compared to C (78.4%) at 72 h. The Ki67 immunoexpression was higher in L4 and L1 and these two groups also showed a higher percentage of cells in the proliferative phases of the cell cycle (S and G2/M). The SEM analysis showed in group C cells with more rounded morphology and with few projections, as well as cell debris, whereas in the irradiated groups the cells exhibited a more flat arrangement, more distributed projections and focal adhesion points, especially in L4. Taken together, the results of the present study shown that laser therapy in the studied patterns, especially at the dose of 4 J/cm², has a positive effect on viability and proliferation of PDLSCs on chitosan membranes, thereby allowing the cells to overcome any adverse effects of the scaffold microenvironment (AU).

The Mechanistic Basis for Photobiomodulation Therapy of Neuropathic Pain by Near Infrared Laser Light

Background and Objective Various irradiances have been reported to be beneficial for the treatment of neuropathic pain with near infrared light. However, the mechanistic basis for the beneficial outcomes may vary based on the level of irradiance or fluence rate used. Using in vivo and in vitro experimentalmodels, this study determined the mechanistic basis of photobiomodulation therapy (PBMT) for the treatment of neuropathic pain using a high irradiance. Study Design/Materials and Methods ln vitro experiments: Cultured, rat DRG were randomly assigned to control or laser treatment (L T) groups with different irradiation times (2, 5, 30, 60 or 120s). The laser parameters were: output power » 960 mW, irradiance » 300mW/cm2, 808 nm wavelength and spot size » 3cm diameter/ area » 7.07cm2, with different fluences according to irradiation times. Mitochondrial metabolic activity was measured with the MTS assay. The DRG neurons were immunostained using a primary antibody to ß-Tubulin III. ln vivo experiments: spared nerve injury surgery (SNI), an animal model of persistent peripheral neuropathic pain, was used. The injured rats were randomly divided into three groups (n » 5). 1) Control: SNI without LT, 2) Short term: SNI with LT on day 7 and euthanized on day 7, 3) Long term: SNI with LT on day 7 and euthanized on day 22. An 808 nm wavelength laser was used for all treatment groups. Treatment was performed once on Day 7 post-surgery. The transcutaneous treatment parameters were: output power: 10 W, fluence rate: 270 mW/cm2, treatment time: 120s. The laser probe was moved along the course of the sciatic/sural nerve during the treatment. Within 1 hour of irradiation, behavior tests were performed to assess its immediate effect on sensory allodynia and hyperalgesia caused by SNI. Results ln vitro experiments: Mitochondrial metabolism was significantly lower compared with controls for all LT groups. Varicosities and undulations formed in neurites of DRG neurons with a cell body diameter 30µm or less. ln neurites of DRG neurons with a cell body diameter of greater than 30µm, varicosities formed only in the 120s group. ln vivo experiments: For heat hyperalgesia, there was a statistically significant reduction in sensitivity to the heat stimulus compared with the measurements done on day 7 prior to LT. A decrease in the sensitivity to the heat stimulus was found in the LT groups compared with the control group on day 15 and 21. For cold allodynia and mechanical hyperalgesia, a significant decrease in sensitivity to cold and pin prick was found within 1 hour after L T. Sensitivity to these stimuli returned to the control levels after 5 days post-L T. No significant difference was found in mechanical allodynia between control and L T groups for all time points examined. Conclusion These in vitro and in vivo studies indicate that treatment with an irradiance/fluence rate at 270 m W/cm2 or higher at the level of the nerve can rapidly block pain transmission. A combination therapy is proposed to treat neuropathic pain with initial high irradiance/fluence rates for fast pain relief, followed by low irradiance/ fluence rates for prolonged pain relief by altering chronic inflammation.

Photobiomodulation therapy improves multilineage differentiation of dental pulp stem cells in three-dimensional culture model.

Photobiomodulation therapy (PBM) has shown positive effects on stem cell differentiation in monolayer cell culture model, but little is known about its effect on three-dimensional (3-D) agarose gel culture. This study evaluated the PBM effect of human dental pulp stem cells (hDPSCs) differentiation and phosphatase alkaline activity (ALP) using an agarose 3-D model under different nutritional conditions. hDPSCs were characterized and seeded on a 0.3% agarose gel layer with different media (osteogenic, adipogenic, chondrogenic) and were assigned into four groups: control 10% fetal bovine serum (FBS), control 5% FBS, PBM 10% FBS, and PBM 5% FBS. Irradiation was performed with continuous-wave InGaAlP laser, 660 nm, 100 mW, 3,3 J / cm2, spot size 0.3 cm2, 10 s of exposure time, and 1 J of energy per point with 6-h interval between sessions. All groups were evaluated at 7 and 14 days. ALP assay was performed to analyze the deposition of mineralized tissue. At 7 days, PBM 5% FBS group presented better stimulation in osteogenic and adipogenic differentiation compared with control. After 14 days, hDPSCs cultured in 3-D exhibited osteogenic, adipogenic, and chondrogenic differentiation; furthermore, compared to control, PBM significantly stimulated all differentiation processes (p < 0.05). It can be concluded that hDPSCs cultured in 3-D agarose associated to PBM could be a promising tool for tissue engineering applications.

Beneficial Role of Low-Intensity Laser Irradiation on Neural ß-tubulin III Protein Expression in Human Bone Marrow Multipotent Mesenchymal Stromal Cells.

The purpose of the present study was to evaluate the neural protein expression pattern of human multipotent mesenchymal stromal cells (hMSCs) treated with forskolin (free-form/FF). The study investigated forskolin's capacity to enhance intracellular levels of cyclic adenosine monophosphate (cAMP) by activating adenylate cyclase and probably by inducing neuron-like cells in vitro. In addition, because nanotechnology is a growing field of tissue engineering, we also assessed the action of a new system called the nanostructured-forskolin (NF) to examine the improvement of drug delivery. Afterwards, the cells were submitted to low-level laser irradiation to evaluate possible photobiostimulatory effects. Investigations using the immunofluorescence by confocal microscopy and Western blot methods revealed the expression of the neuronal marker ß-tubulin III. Fluorescence intensity quantification analysis using INCell Analyzer System for ß-tubulin III was used to examine significant differences. The results showed that after low-level laser irradiation exposure, there was a tendency to increase the ß-tubulin III expression in all groups, as expected in the photobiostimulation process. Notably, this process induced for irradiation was more pronounced in irradiated nanoforskolin cells (INF) compared to non-irradiated free-forskolin control cells (NFFC). However, there was also an increase in ß-tubulin III protein expression in the groups: irradiated nanocontrol cells (INC) compared to non-irradiated free-forskolin control cells (NFF) and after treatment with non-irradiated free-forskolin (NFF) and non-irradiated nanoforskolin (NNFC). We concluded that the methods using low-level laser irradiation and/or nanoparticles showed an up-regulation of neural-protein expression in hMSCs that could be used to facilitate cellular therapy protocols in the near future.

Low-Power Laser Irradiation (LPLI): A Clinical Point of View on a Promising Strategy to Improve Liver Regeneration.

The capacity of the liver to regenerate is an important clinical issue after major hepatectomies and makes the difference between life and death in some cases of post-operative malfunction when the liver remnant is too small or has an impaired regenerative capacity. Several approaches have been tested to stimulate hepatic regeneration after post-operative hepatic failure syndrome; however, they have produced controversial results. A quick, simple, and harmless method that can be used intraoperatively and capable of promoting an increased regenerative capacity of the remaining liver would be very welcome. Thus, based on the data in the literature, we presented low-power laser irradiation (LPLI) as a quick, simple, and harmless method to improve liver regeneration after major hepatectomies. This article highlights the current evidence about the effects of LPLI on liver regeneration, and also suggests laser therapy as an important tool for regenerative stimulation in clinical practice.

Effects of photobiomodulation therapy on Bothrops moojeni snake-envenomed gastrocnemius of mice using enzymatic biomarkers.

Bothropic venom contains a range of biologically active substances capable of causing severe local and systemic envenoming symptomatology within its victims. The snake anti-venom is effective against systemic effects but has no neutralizing effect against the fast developing local effects. Herein, mice gastrocnemius injected with Bothrops moojeni venom (40 µg/kg) or saline solution were irradiated with HeNe (632.8 nm) and GaAs (904 nm) lasers (daily energy density of 4 J/cm2; 0.03/0.21 power density; 0.07/0.16 spot size; 1.2/0.04 total energy, 1 cm off contact, for HeNe and GaAs lasers, respectively) and euthanized in periods ranging from 3 h to 21 days. Blood biochemistry for creatine kinase (CK), alkaline phosphatase (ALP), acid phosphatase (AP), lactate dehydrogenase (LDH), aspartate transaminase (AST), and myoglobin and histopathological analysis, for assessing the degree of myonecrosis and regeneration of gastrocnemius, were done at every time interval. GaAs laser promoted faster photobiomodulation therapy (PBMT) effects, and the GaAs group exhibited a better clinical outcome than the HeNe group. Within the GaAs group, the serum levels of CK, LDH, AP, AST, and myoglobin, which were increased by the physiological effects of the venom, were reduced to initial baseline before snake envenomation in less time than those irradiated by the HeNe laser. However, the group receiving irradiation from the HeNe laser returned the levels of ALP activity to baseline faster than those of the GaAs group. Histopathological analysis revealed enhanced muscle regeneration in mice groups treated with both lasers. PBM promoted by GaAs and HeNe showed well-developed centrally nucleate regenerating cells and an increased number of newly formed blood vessels when compared to unirradiated muscle. We therefore suggest that GaAs had the best outcomes likely derived from a deeper penetrating longer wavelength. We conclude that PMBT is a promising, non-invasive approach to be further tested in pre-clinical studies with a goal to further its clinical use in skeletal muscle recovery in snakebite victims.