Photons Induce Vesicular Exocytotic Release of Glutamate in a Power-Dependent Way.

Increasing evidence indicates that photobiomodulation, based on tissue irradiation with photons in the red to near-infrared spectrum, may be an effective therapeutic approach to central nervous system disorders. Although nervous system functionality has been shown to be affected by photons in animal models, as well as in preliminary evidence in healthy subjects or in patients with neuropsychiatric disorders, the mechanisms involved in the photobiomodulation effects have not yet been clarified. We previously observed that photobiomodulation could stimulate glutamate release. Here, we investigate mechanisms potentially involved in the glutamate-releasing effect of photons from adult mouse cerebrocortical nerve terminals. We report evidence of photon ability to induce an exocytotic vesicular release of glutamate from the terminals of glutamatergic neurons in a power-dependent way. It can be hypothesized that photobiomodulation, depending on the potency, can release glutamate in a potentially neurotoxic or physiological range.

Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone.

The bone marrow-derived multipotent mesenchymal cells (MSCs) have captured scientific interest due to their multi-purpose features and clinical applications. The operational dimension of MSCs is not limited to the bone marrow reservoir, which exerts bone-building and niche anabolic tasks; they also meet the needs of quenching inflammation and restoring inflamed tissues. Thus, the range of MSC activities extends to conditions such as neurodegenerative diseases, immune disorders and various forms of osteopenia. Steering these cells towards becoming an effective therapeutic tool has become mandatory. Many laboratories have employed distinct strategies to improve the plasticity and secretome of MSCs. We aimed to present how photobiomodulation therapy (PBM-t) can manipulate MSCs to render them an extraordinary anti-inflammatory and osteogenic instrument. Moreover, we discuss the outcomes of different PBM-t protocols on MSCs, concluding with some perplexities and complexities of PBM-t in vivo but encouraging and feasible in vitro solutions.

A Narrative Review on Oral and Periodontal Bacteria Microbiota Photobiomodulation, through Visible and Near-Infrared Light: From the Origins to Modern Therapies.

Photobiomodulation (PBM) consists of a photon energy transfer to the cell, employing non-ionizing light sources belonging to the visible and infrared spectrum. PBM acts on some intrinsic properties of molecules, energizing them through specific light wavelengths. During the evolution of life, semiconducting minerals were energized by sun radiation. The molecules that followed became photoacceptors and were expressed into the first proto-cells and prokaryote membranes. Afterward, the components of the mitochondria electron transport chain influenced the eukaryotic cell physiology. Therefore, although many organisms have not utilized light as an energy source, many of the molecules involved in their physiology have retained their primordial photoacceptive properties. Thus, in this review, we discuss how PBM can affect the oral microbiota through photo-energization and the non-thermal effect of light on photoacceptors (i.e., cytochromes, flavins, and iron-proteins). Sometimes, the interaction of photons with pigments of an endogenous nature is followed by thermal or photodynamic-like effects. However, the preliminary data do not allow determining reliable therapies but stress the need for further knowledge on light-bacteria interactions and microbiota management in the health and illness of patients through PBM.

Near-Infrared 810 nm Light Affects Porifera Chondrosia reniformis (Nardo, 1847) Regeneration: Molecular Implications and Evolutionary Considerations of Photobiomodulation-Animal Cell Interaction.

Chemotrophic choice as a metabolic source of energy has characterised animal cell evolution. However, light interactions with animal cell photoacceptors that are able to increase energetic metabolism (photo-biomodulation (PBM)) have been previously described. In the present study, we cut three specimens of Chondrosia reniformis into four equal parts (12 fragments), and we irradiated the regenerating edge of six fragments with the previously characterised 810 nm near-infrared light, delivered at 1 W, 60 J/cm2, 1 W/cm2, and 60 J in a continuous-wave mode for 60 s through a flat-top hand-piece with a rounded spot-size area of 1 cm2. Six fragments were irradiated with 0 W for 60 s as the controls. We performed irradiation at the time 0 h and every 24 h for a total of five administrations. We monitored the regeneration process for five days (120 h) in aquaria by examining the macroscopic and histological changes. We analysed the gene expression profile of the inflammatory processes, apoptosis, heat stress, growth factors, and collagen production and determined oxidative stress enzyme activity and the total prokaryotic symbiont content. PBM sped up C. reniformis regeneration when compared to the controls. Particularly, transforming growth factor TGF3 and TGF6 upregulation during the early phase of regeneration and TGF5 upregulation 120 h postinjury in the irradiated samples supports the positive effect of PBM in sponge tissue recovery. Conversely, the expression of TGF4, a sponge fibroblast growth factor homologue, was not affected by irradiation, indicating that multiple, independent pathways regulate the TGF genes. The results are consistent with our previous data on a wide range of organisms and humans, suggesting that PBM interaction with primary and secondary cell targets has been conserved through the evolution of life forms.

808-nm Photobiomodulation Affects the Viability of a Head and Neck Squamous Carcinoma Cellular Model, Acting on Energy Metabolism and Oxidative Stress Production.

Photobiomodulation (PBM) is a form of low-dose light therapy that acts through energy delivery from non-ionizing sources. During the recent two decades, there has been tremendous progress with PBM acceptance in medicine. However, PBM effects on potential stimulation of existing malignant or pre-malignant cells remain unknown. Thus, the primary endpoint was to assess the safety of PBM treatment parameters on head and neck squamous cell carcinoma (HNSCC) proliferation or survival. The secondary endpoint was to assess any putative anti-cancer effects of PBM treatments. Cell viability, energy metabolism, oxidative stress, and pro- and anti-apoptotic markers expression were investigated on a Human Head and Neck Squamous Cell Carcinoma cellular model (OHSU-974 FAcorr cell line). PBM therapy was administered through the 810 nm diode laser (GaAlAs) device (Garda Laser, 7024 Negrar, Verona, Italy) at the powers of 0, 0.25, 0.50, 0.75, 1.00, or 1.25 W in continuous wave (CW) mode for an exposure time of 60 s with a spot-size of 1 cm2 and with a distance of 1.86 cm from the cells. Results showed that 810-nm PBM affected oxidative phosphorylation in OHSU-971 FAcorr, causing a metabolic switch to anaerobic glycolysis. In addition, PBM reduced the catalase activity, determining an unbalance between oxidative stress production and the antioxidant defenses, which could stimulate the pro-apoptotic cellular pathways. Our data, at the parameters investigated, suggest the safeness of PBM as a supportive cancer therapy. Pre-clinical and clinical studies are necessary to confirm the in vitro evidence.

Improving Consistency of Photobiomodulation Therapy: A Novel Flat-Top Beam Hand-Piece versus Standard Gaussian Probes on Mitochondrial Activity.

The tremendous therapeutic potential of photobiomodulation therapy in different branches of medicine has been described in the literature. One of the molecular mechanisms for this treatment implicates the mitochondrial enzyme, cytochrome C oxidase. However, the efficacy and consistency of clinical outcomes with photobiomodulation treatments has been fiercely debated. This work was motivated by this need to improve photobiomodulation devices and delivery approaches. We designed a novel hand-piece with a flat-top beam profile of irradiation. We compared the beam profile versus a standard hand-piece and a fibre probe. We utilized isolated mitochondria and performed treatments at various spots within the beam, namely, the centre, left and right edge. We examined mitochondrial activity by assessing ATP synthesis with the luciferin/luciferase chemiluminescent method as a primary endpoint, while mitochondrial damage was assessed as the secondary endpoint. We observed a uniform distribution of the power density with the flat-top prototype compared to a wide Gaussian beam profile with the standard fibre and standard hand-piece. We noted increased production of ATP in the centre of all three beams with respect to the non-treated controls (p < 0.05). Both the fibre and standard hand-piece demonstrated less increase in ATP synthesis at the edges than the centre (p < 0.05). In contrast, ATP synthesis was increased homogenously in the flat-top handpiece, both in the centre and the edges of the beam. Fibre, standard hand-piece and the flat-top hand-piece prototype have discrete beam distribution characteristics. This significantly affected the mitochondrial activity with respect to their position within the treated areas. Flat-top hand-piece enhances the uniformity of photobiomodulation treatments and can improve the rigour and reproducibility of PBM clinical outcomes.

Photobiomodulation by Near-Infrared 980-nm Wavelengths Regulates Pre-Osteoblast Proliferation and Viability through the PI3K/Akt/Bcl-2 Pathway.

BACKGROUND: bone tissue regeneration remains a current challenge. A growing body of evidence shows that mitochondrial dysfunction impairs osteogenesis and that this organelle may be the target for new therapeutic options. Current literature illustrates that red and near-infrared light can affect the key cellular pathways of all life forms through interactions with photoacceptors within the cells' mitochondria. The current study aims to provide an understanding of the mechanisms by which photobiomodulation (PBM) by 900-nm wavelengths can induce in vitro molecular changes in pre-osteoblasts. METHODS: The PubMed, Scopus, Cochrane, and Scholar databases were used. The manuscripts included in the narrative review were selected according to inclusion and exclusion criteria. The new experimental set-up was based on irradiation with a 980-nm laser and a hand-piece with a standard Gaussian and flat-top beam profile. MC3T3-E1 pre-osteoblasts were irradiated at 0.75, 0.45, and 0.20 W in continuous-wave emission mode for 60 s (spot-size 1 cm2) and allowed to generate a power density of 0.75, 0.45, and 0.20 W/cm2 and a fluence of 45, 27, and 12 J/cm2, respectively. The frequency of irradiation was once, three times (alternate days), or five times (every day) per week for two consecutive weeks. Differentiation, proliferation, and cell viability and their markers were investigated by immunoblotting, immunolabelling, fluorescein-FragELTM-DNA, Hoechst staining, and metabolic activity assays. RESULTS AND CONCLUSIONS: The 980-nm wavelength can photobiomodulate the pre-osteoblasts, regulating their metabolic schedule. The cellular signal activated by 45 J/cm2, 0.75 W and 0.75 W/cm2 consist of the PI3K/Akt/Bcl-2 pathway; differentiation markers were not affected, nor do other parameters seem to stimulate the cells. Our previous and present data consistently support the window effect of 980 nm, which has also been described in extracted mitochondria, through activation of signalling PI3K/Akt/Bcl-2 and cyclin family, while the Wnt and Smads 2/3-ß-catenin pathway was induced by 55 J/cm2, 0.9 W and 0.9 W/cm2.

Electromagnetic Dosimetry for Isolated Mitochondria Exposed to Near-Infrared Continuous-Wave Illumination in Photobiomodulation Experiments.

This paper presents results on the electromagnetic field computed inside isolated mitochondria when they are exposed to near-infrared illuminations with reference to photobiomodulation experiments. The accurate calculation of the electromagnetic dose is considered to be important for a better understanding of the mechanism of interaction of light with these organelles and to improve the reliability and repeatability of the experiments. To get such results, we introduce several models. Even though they refer to a well-defined experimental setup, different models are necessary to take into account the possible different dispositions of the mitochondria, and of the differences in their dimensions and in their constitutive parameters. Different wavelengths and polarizations are considered as well. The effects of all parameters on the electromagnetic field inside mitochondria are discussed. © 2021 Bioelectromagnetics Society.

Experimental and Clinical Applications of Red and Near-Infrared Photobiomodulation on Endothelial Dysfunction: A Review.

BACKGROUND: Under physiological conditions, endothelial cells are the main regulator of arterial tone homeostasis and vascular growth, sensing and transducing signals between tissue and blood. Disease risk factors can lead to their unbalanced homeostasis, known as endothelial dysfunction. Red and near-infrared light can interact with animal cells and modulate their metabolism upon interaction with mitochondria's cytochromes, which leads to increased oxygen consumption, ATP production and ROS, as well as to regulate NO release and intracellular Ca2+ concentration. This medical subject is known as photobiomodulation (PBM). We present a review of the literature on the in vitro and in vivo effects of PBM on endothelial dysfunction. METHODS: A search strategy was developed consistent with the PRISMA statement. The PubMed, Scopus, Cochrane, and Scholar electronic databases were consulted to search for in vitro and in vivo studies. RESULTS: Fifty out of >12,000 articles were selected. CONCLUSIONS: The PBM can modulate endothelial dysfunction, improving inflammation, angiogenesis, and vasodilatation. Among the studies, 808 nm and 18 J (0.2 W, 2.05 cm2) intracoronary irradiation can prevent restenosis as well as 645 nm and 20 J (0.25 W, 2 cm2) can stimulate angiogenesis. PBM can also support hypertension cure. However, more extensive randomised controlled trials are necessary.

Mitochondrial Bioenergetic, Photobiomodulation and Trigeminal Branches Nerve Damage, What's the Connection? A Review.

BACKGROUND: Injury of the trigeminal nerve in oral and maxillofacial surgery can occur. Schwann cell mitochondria are regulators in the development, maintenance and regeneration of peripheral nerve axons. Evidence shows that after the nerve injury, mitochondrial bioenergetic dysfunction occurs and is associated with pain, neuropathy and nerve regeneration deficit. A challenge for research is to individuate new therapies able to normalise mitochondrial and energetic metabolism to aid nerve recovery after damage. Photobiomodulation therapy can be an interesting candidate, because it is a technique involving cell manipulation through the photonic energy of a non-ionising light source (visible and NIR light), which produces a nonthermal therapeutic effect on the stressed tissue. METHODS: The review was based on the following questions: (1) Can photo-biomodulation by red and NIR light affect mitochondrial bioenergetics? (2) Can photobiomodulation support damage to the trigeminal nerve branches? (preclinical and clinical studies), and, if yes, (3) What is the best photobiomodulatory therapy for the recovery of the trigeminal nerve branches? The papers were searched using the PubMed, Scopus and Cochrane databases. This review followed the ARRIVE-2.0, PRISMA and Cochrane RoB-2 guidelines. RESULTS AND CONCLUSIONS: The reliability of photobiomodulatory event strongly bases on biological and physical-chemical evidence. Its principal player is the mitochondrion, whether its cytochromes are directly involved as a photoacceptor or indirectly through a vibrational and energetic variation of bound water: water as the photoacceptor. The 808-nm and 100 J/cm2 (0.07 W; 2.5 W/cm2; pulsed 50 Hz; 27 J per point; 80 s) on rats and 800-nm and 0.2 W/cm2 (0.2 W; 12 J/cm2; 12 J per point; 60 s, CW) on humans resulted as trustworthy therapies, which could be supported by extensive studies.

Photobiomodulation and Oxidative Stress: 980 nm Diode Laser Light Regulates Mitochondrial Activity and Reactive Oxygen Species Production.

Photobiomodulation with 808 nm laser light electively stimulates Complexes III and IV of the mitochondrial respiratory chain, while Complexes I and II are not affected. At the wavelength of 1064 nm, Complexes I, III, and IV are excited, while Complex II and some mitochondrial matrix enzymes seem to be not receptive to photons at that wavelength. Complex IV was also activated by 633 nm. The mechanism of action of wavelengths in the range 900-1000 nm on mitochondria is less understood or not described. Oxidative stress from reactive oxygen species (ROS) generated by mitochondrial activity is an inescapable consequence of aerobic metabolism. The antioxidant enzyme system for ROS scavenging can keep them under control. However, alterations in mitochondrial activity can cause an increment of ROS production. ROS and ATP can play a role in cell death, cell proliferation, and cell cycle arrest. In our work, bovine liver isolated mitochondria were irradiated for 60 sec, in continuous wave mode with 980 nm and powers from 0.1 to 1.4 W (0.1 W increment at every step) to generate energies from 6 to 84 J, fluences from 7.7 to 107.7 J/cm2, power densities from 0.13 to 1.79 W/cm2, and spot size 0.78 cm2. The control was equal to 0 W. The activity of the mitochondria's complexes, Krebs cycle enzymes, ATP production, oxygen consumption, generation of ROS, and oxidative stress were detected. Lower powers (0.1-0.2 W) showed an inhibitory effect; those that were intermediate (0.3-0.7 W) did not display an effect, and the higher powers (0.8-1.1 W) induced an increment of ATP synthesis. Increasing the power (1.2-1.4 W) recovered the ATP production to the control level. The interaction occurred on Complexes III and IV, as well as ATP production and oxygen consumption. Results showed that 0.1 W uncoupled the respiratory chain and induced higher oxidative stress and drastic inhibition of ATP production. Conversely, 0.8 W kept mitochondria coupled and induced an increase of ATP production by increments of Complex III and IV activities. An augmentation of oxidative stress was also observed, probably as a consequence of the increased oxygen consumption and mitochondrial isolation experimental conditions. No effect was observed using 0.5 W, and no effect was observed on the enzymes of the Krebs cycle.

Effects of photobiomodulation on bone defects grafted with bone substitutes: A systematic review of in vivo animal studies.

A present, photobiomodulation therapy (PBMT) effectiveness in enhancing bone regeneration in bone defects grafted with or without biomaterials is unclear. This systematic review (PROSPERO, ref. CRD 42019148959) aimed to critically appraise animal in vivo published data and present the efficacy of PBMT and its potential synergistic effects on grafted bone defects. MEDLINE, CCCT, Scopus, Science Direct, Google Scholar, EMBASE, EBSCO were searched, utilizing the following keywords: bone repair; low-level laser therapy; LLLT; light emitting diode; LEDs; photobiomodulation therapy; in vivo animal studies, bone substitutes, to identify studies between 1994 and 2019. After applying the eligibility criteria, 38 papers included where the results reported according to "PRISMA." The results revealed insufficient and incomplete PBM parameters, however, the outcomes with or without biomaterials have positive effects on bone healing. In conclusion, in vivo animal studies with a standardized protocol to elucidate the effects of PBMT on biomaterials are required initially prior to clinical studies.

Efficacy of Flat-Top Hand-Piece Using 980 nm Diode Laser Photobiomodulation on Socket Healing after Extraction: Split-Mouth Experimental Model in Dogs.

The objective was to evaluate the effect of photobiomodulation (PBM) using 980 nm diode laser therapy (0.60 W, 0.77 W cm-2 , 36 J, 46 J cm-2 , 60 s) irradiated in continuous wave mode by flat-top hand-piece on socket healing in the maxilla and mandible. A split-mouth experimental design was performed on 6 dogs. The 3rd premolar tooth was extracted from the maxilla and mandibles for both sides. The right-sided sockets were irradiated (PBM group), and the left-sided sockets were kept as control. Irradiation was done after extraction and at 48-h interval for 14 days. Both the buccal and lingual sides were irradiated to reach a total irradiation time of 120 s. Bone density was evaluated at 3, 4 and 5 weeks using cone beam computed tomography. We showed that maxillary sockets in the PBM group had higher bone density compared to control one at 3, 4, 5 weeks (P = 0.029, <0.001, <0.001), respectively. Mandibular sockets revealed no significant difference between PBM and control at 3 weeks (P = 0.347), while at 4 and 5 weeks PBM group showed higher bone density (P = 0.004, <0.001). In both groups, there was a significant increase (P < 0.001) in bone density by time which was higher in the PBM group. We concluded that PBM using a flat-top hand-piece of 980-nm improved the bone density of extraction sockets.

Interaction between Laser Light and Osteoblasts: Photobiomodulation as a Trend in the Management of Socket Bone Preservation-A Review.

Bone defects are the main reason for aesthetic and functional disability, which negatively affect patient's quality of life. Particularly, after tooth extraction, the bone of the alveolar process resorbs, limiting the optimal prosthetic implant placement. One of the major pathophysiological events in slowly- or non-healing tissues is a blood supply deficiency, followed by a significant decrease in cellular energy amount. The literature shows that photons at the red and infrared wavelengths can interact with specific photoacceptors located within the cell. Through this mechanism, photobiomodulation (PBM) can modify cellular metabolism, by increasing mitochondrial ATP production. Here, we present a review of the literature on the effect of PBM on bone healing, for the management of socket preservation. A search strategy was developed in line with the PRISMA statement. The PubMed and Scholar electronic databases were consulted to search for in vivo studies, with restrictions on the year (<50 years-old), language (English), bone socket preservation, and PBM. Following the search strategy, we identified 269 records, which became 14, after duplicates were removed and titles, abstract and inclusion-, exclusion-criteria were screened. Additional articles identified were 3. Therefore, 17 articles were included in the synthesis. We highlight the osteoblast-light interaction, and the in vivo therapeutic tool of PBM is discussed.

Photobiomodulation Therapy in Oral Mucositis and Potentially Malignant Oral Lesions: A Therapy Towards the Future.

Photobiomodulation therapy (PBMT) is an effective treatment modality, which has the significant advantage of enhancing a patient's quality of life (QoL) by minimising the side effects of oral cancer treatments, as well as assisting in the management of potentially cancerous lesions. It is important to note that the major evidence-based documentation neither considers, nor tackles, the issues related to the impact of PBMT on tumour progression and on the downregulation of cellular proliferation improvement, by identifying the dose- and time-dependency. Moreover, little is known about the risk of this therapy and its safety when it is applied to the tumour, or the impact on the factor of QoL. The review aimed to address the benefits and limitations of PBMT in premalignant oral lesions, as well as the conflicting evidence concerning the relationship between tumour cell proliferation and the applied dose of photonic energy (fluence) in treating oral mucositis induced by head and neck cancer (H&N) treatments. The objective was to appraise the current concept of PBMT safety in the long-term, along with its latent impact on tumour reaction. This review highlighted the gap in the literature and broaden the knowledge of the current clinical evidence-based practice, and effectiveness, of PBMT in H&N oncology patients. As a result, the authors concluded that PBMT is a promising treatment modality. However, due to the heterogeneity of our data, it needs to undergo further testing in well-designed, long-term and randomised controlled trial studies, to evaluate it with diligent and impartial outcomes, and ensure laser irradiation's safety at the tumour site.

Evaluation of the outcome of various laser therapy applications in root canal disinfection: A systematic review.

BACKGROUND: Any successful endodontic therapy requires elimination of the endodontic biofilms through meticulous root canal disinfection methods. Sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) are the most common effective irrigants, in removing smear-layer from the coronal and middle thirds of the dental canals but reduced considerably towards the apical one third. In recent years, newly alternative treatment modalities have been proposed, including high-power lasers and antimicrobial photodynamic therapy (aPDT). Our work was conducted to evalaute the outcome of root canal disinfection in relation to the efficacy of various treatment modalities. Furthermore, every effort was made to present an overview of the aPDT outcomes, as a model for this application, and to propose laser parameters protocol with positive results. METHODS: The electronic databases PubMed was searched from January 2013- January 2019. Our inclusive criteria based on laser therapy applications, as a model for root canal disinfection. The search terms utilised various combinations as follows: photodynamic therapy or antimicrobial photodynamic therapy or photoactivated disinfection or light activated disinfection or laser activated disinfection or laser therapy, and endodontic. RESULTS: The results of this systematic review concluded that the effectiveness of aPDT and various laser wavelengths protocols, in removing endodontic biofilms from infected root canals, remains unattainable. CONCLUSIONS: Study concluded that the combination of aPDT with antimicrobial irrigants could provide a synergetic effect. However, due to the heterogeneity of the selected studies and their limitations, in terms of lack of standardised protocol or discrepancy in the methodology, authors suggest further validated approaches to achieve optimal outcomes.

The 808 nm and 980 nm infrared laser irradiation affects spore germination and stored calcium homeostasis: A comparative study using delivery hand-pieces with standard (Gaussian) or flat-top profile.

Photobiomodulation relies on the transfer of energy from incident photons to a cell photoacceptor. For many years the concept of photobiomodulation and its outcome has been based upon a belief that the sole receptor within the cell was the mitochondrion. Recently, it has become apparent that there are other photoacceptors operating in different regions of the electromagnetic spectrum. Alternative photoacceptors would appear to be water and mechanisms regulating calcium homeostasis, despite a direct effect of laser photonic energy on intracellular calcium concentration outwith mitochondrial activity or influence, have not been clearly demonstrated. Therefore, to increase the knowledge of intracellular­calcium and laser photon interaction, as well as to demonstrate differences in irradiation profiles with modern hand-pieces, we tested and compared the photobiomodulatory effect of 808 nm and 980 nm diode laser light by low- and higher-energy (60s, 100 mW/cm2, 100 mW/cm2, 500 mW/cm2, 1000 mW/cm2, 1500 mW/cm2, 2000 mW/cm2) irradiated with a "standard" (Gaussian fluence distribution) hand-piece or with a "flat-top" (uniform fluence) hand-piece. For this purpose, we used the eukaryote unicellular-model Dictyostelium discoideum. The 808 nm and 980 nm infrared laser light, at the energy tested directly affect the stored Ca2+ homeostasis, independent of the mitochondrial respiratory chain activities. From an organism perspective, the effect on Ca2+-dependent signal transduction as the regulator of spore germination in Dictyostelium, demonstrates how a cell can respond quickly to the correct laser photonic stimulus through a different cellular pathway than the known light-chromophore(mitochondria) interaction. Additionally, both hand-piece designs tested were able to photobiomodulate the D. discoideum cell; however, the hand-piece with a flat-top profile, through uniform fluence levels allows more effective and reproducible effects.

1064 nm Nd:YAG laser light affects transmembrane mitochondria respiratory chain complexes.

Photobiomodulation (PBM) is a non-plant-cell manipulation through a transfer of energy by means of light sources at the non-ablative or thermal intensity. Authors showed that cytochrome-c-oxidase (complex IV) is the specific chromophore's target of PBM at the red (600-700 nm) and NIR (760-900 nm) wavelength regions. Recently, it was suggested that the infrared region of the spectrum could influence other chromospheres, despite the interaction by wavelengths higher than 900 nm with mitochondrial chromophores was not clearly demonstrated. We characterized the interaction between mitochondria respiratory chain, malate dehydrogenase, a key enzyme of Krebs cycle, and 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in the ß-oxidation (two mitochondrial matrix enzymes) with the 1064 nm Nd:YAG (100mps and 10 Hz frequency mode) irradiated at the average power density of 0.50, 0.75, 1.00, 1.25 and 1.50 W/cm2 to generate the respective fluences of 30, 45, 60, 75 and 90 J/cm2 . Our results show the effect of laser light on the transmembrane mitochondrial complexes I, III, IV and V (adenosine triphosphate synthase) (window effects), but not on the extrinsic mitochondrial membrane complex II and mitochondria matrix enzymes. The effect is not due to macroscopical thermal change. An interaction of this wavelength with the Fe-S proteins and Cu-centers of respiratory complexes and with the water molecules could be supposed.

A Comparative Study Between the Effectiveness of 980 nm Photobiomodulation Delivered by Hand-Piece With Gaussian vs. Flat-Top Profiles on Osteoblasts Maturation.

Photobiomodulation (PBM) is a clinically accepted tool in regenerative medicine and dentistry to improve tissue healing and repair and to restore the functional disability. The current in vitro study aimed to investigate the photobiomodulatory effects of 980 nm wavelength (the real energy at the target: ~0.9 W, ~0.9 W/cm2, 60 s, ~55 J/cm2 and a single energy ~55 J in CW) on MC3T3-E1 pre-osteoblast, delivered with flattop profile in comparison to the standard profile. The laser groupings and their associated energies were: Group 1 - once per week (total energy 110 J); Group 2 - three times per week (alternate day) (total energy 330 J); Group 3 - five times per week (total energy 550 J). The metabolic activity and the osteoblasts maturation were analyzed by alkaline phosphatase assay, alizarin red S histological staining, immunoblot and/or double immunolabeling analysis for Bcl2, Bax, Runx-2, Osx, Dlx5, osteocalcin, and collagen Type 1. Our data, for the first time, prove that laser irradiation of 980 nm wavelength with flat-top beam profile delivery system, compared to standard-Gaussian profile, has improved photobiomodulatory efficacy on pre-osteoblastic cells differentiation. Mechanistically, the irradiation enhances the pre-osteoblast differentiation through activation of Wnt signaling and activation of Smads 2/3-ßcatenin pathway.

Photobiomodulation Affects Key Cellular Pathways of all Life-Forms: Considerations on Old and New Laser Light Targets and the Calcium Issue.

After 50 years of studies on photobiomodulation (PBM), there is still so much to investigate to understand the laser light-nonplant cells interactions. The current scientific knowledge allows to say that the phenomena induced by PBM are based on cellular pathways that are the key points of cell life. The mitochondria chromophores, also present on the bacterial membrane, the calcium channels, ion that regulates the life-and-death cellular processes, as well as the TRP family, whose genes have been found in protozoa and suggest that its basic mechanism evolved long before the appearance of animals, seem to be elective targets in photobiomodulatory events by wavelengths from 600 up to 980 nm. The ambiguous resulting cellular communication way, mediated by ATP, ROS and/or calcium, leads to cell manipulation, which modifies its metabolism and whose response connects all life-forms from bacteria to vertebrates. Because of the Giano-Bifronte features of ROS and calcium, as well as the fine balance of energetic mitochondrial processes, whose alteration is responsible for several diseases, the PBM can show unpredictable results and it requires scrupulous approach to avoid cellular damages. However, when carefully applied, PBM is able to improve nonhealthy cell's responses and represents a reliable support in human and veterinary medicine.