Investigation of the optimal light parameters for photobiomodulation to induce osteogenic differentiation of the human bone marrow stem cell and human umbilical vein endothelial cell co-culture.

Bones have an important role in the human body with their complex nature. Mesenchymal stem cells and endothelial cells together support their unique and complex nature. Photobiomodulation (PBM) is a promising method that provides cell proliferation, osteogenic differentiation, and bone regeneration. However, there are still unknowns in the mechanism of osteogenic differentiation induced by PBM. The main aim of the study is to understand the molecular mechanism of PBM at 655 and 808 nm of wavelengths and identify the most effective energy densities of both wavelengths for osteogenic differentiation. The effect of PBM on osteogenic differentiation of Human Bone Marrow Stem Cell (hBMSC) and Human Umbilical Vein Endothelial Cell (HUVEC) co-culture was examined at 1, 3, and 5 J/cm2 energy densities of red and near-infrared light through different analysis such as cell viability, scratch assay, intracellular reactive oxygen species production, and ATP synthesis, nitric oxide release, temperature monitoring, and osteogenic differentiation analyses. Even though all PBM-treated groups exhibited better results compared to the control group, 5 J/cm2 energy density induced faster cell proliferation and migration at both wavelengths. The increases in ATP and NO levels as signaling molecules, and the increases in DNA, ALPase, and calcium contents as osteogenic markers were higher in the groups treated with 5 J/cm2 energy density at both wavelengths. Only a slight change was obtained in the level of intracellular ROS after any light applications. It can be concluded that NO release has a very important role together with ATP production in PBM therapy to trigger DNA synthesis, ALPase activity, and mineralization for osteogenic differentiation of the hBMSC and HUVEC co-culture at 655 and 808 nm of wavelengths.

Synergistic effects of integrin binding peptide (RGD) and photobiomodulation therapies on bone-like microtissues to enhance osteogenic differentiation.

Bone tissue engineering aims to diversify and enhance the strategies for bone regeneration to overcome bone-related health problems. Bone mimetic peptides such as Gly-Arg-Gly-Asp-Ser (RGD) are useful tools for osteogenic differentiation. Similarly, photobiomodulation (PBM) at 600-800 nm of wavelength range improves bone tissue healing via the production of intracellular reactive oxygen species (ROS), ATP synthesis, and nitric oxide (NO) release. Besides, traditional monolayer cell culture models have limited conditions to exhibit the details of a mechanism such as a peptide or PBM therapy. However, scaffold-free microtissues (SFMs) can mimic a tissue more properly and be an efficient way to understand the mechanism of therapy via cell-cell interaction. Thus, the synergistic effects of RGD peptide (1 mM) and PBM applications (1 J/cm2 energy density at 655 nm of wavelength and 5 J/cm2 energy density at 808 nm of wavelength) were evaluated on SFMs formed with the co-culture of Human Bone Marrow Stem Cells (hBMSC) and Human Umbilical Vein Endothelial Cells (HUVEC) for osteogenic differentiation. Cell viability assays, mechanistic analysis, and the evaluation of osteogenic differentiation markers were performed. Combined therapies of RGD and PBM were more successful to induce osteogenic differentiation than single therapies. Especially, RGD + PBM at 655 nm group exhibited a higher capability of osteogenic differentiation via ROS production, ATP synthesis, and NO release. It can be concluded that the concomitant use of RGD and PBM may enhance bone regeneration and become a promising therapeutic tool to heal bone-related problems in clinics.

Photobiomodulation therapy at red and near-infrared wavelengths for osteogenic differentiation in the scaffold-free microtissues.

One of the novel strategies for bone tissue regeneration is photobiomodulation (PBM) which depends on the red and near-infrared light absorption by mitochondria and may trigger bone tissue regeneration via the production of intracellular ROS and ATP, NO release, etc. It is also important to identify the changes in those signal molecule levels in an in vivo mimicking platform such as 3-Dimensional (3D) Scaffold Free Microtissues (SFMs) that may serve more natural osteogenic differentiation responses to PBM. Herein, we aimed to increase the osteogenic differentiation capability of the co-culture of Human Bone Marrow Stem Cells (hBMSC) and Human Umbilical Vein Endothelial Cells (HUVECs) on 3D SFMs by triple light treatment at 655 and 808-nm of wavelengths with the energy densities of 1, 3, and 5 J/cm2. We performed the analysis of cell viability, diameter measurements of SFMs, intracellular ROS production, NO release, ATP activity, temperature measurements, DNA content, ALPase activity, calcium content, and relative gene expressions of ALP, Collagen, and Osteopontin by qRT-PCR. It was found that both wavelengths were effective in terms of the viability of SFMs. 1 and 5 J/cm2 energy densities of both wavelengths increased the SFM diameter with significant changes in intracellular ROS, ATP, and NO levels compared to the control group. We concluded that PBM therapy was successful to induce osteogenesis. 1 J/cm2 at 655 nm of wavelength and 5 J/cm2 at 808 nm of wavelength were the most effective energy densities for osteogenic differentiation on SFMs with triple light treatment.

The antibacterial activity of photodynamic agents against multidrug resistant bacteria causing wound infection.

Antimicrobial photodynamic inactivation (aPDI) of multidrug-resistant (MDR) wound pathogens was evaluated with cationic porphyrin derivatives (CPDs). MDR bacterial strains including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae were used. The CPDs named PM, PE, PN, and PL were synthesized as a photosensitizer (PS). A diode laser with a wavelength of 655 nm was used as a light source. aPDI of the combinations formed with different energy densities (50, 100, and 150 J/cm²) and PS concentrations (ranging from 3.125 to 600 µM) were evaluated on each bacterial strain. Dark toxicity, cytotoxicity, and phototoxicity were determined on fibroblast cells. In the aPDI groups, survival reductions of up to 5.80 log10 for E. coli, 5.90 log10 for P. aeruginosa, 6.11 log10 for K. pneumoniae, and 6.78 log10 for A. baumannii were obtained. The cytotoxic effect of PL and PM on fibroblast cells was very limited. PN was the type of CPD with the highest dark toxicity on fibroblast cells. In terms of providing broad-spectrum aPDI without or with very limited cytotoxic effect, the best result was observed in aPDI application with PL. The other CPDs need some modifications to show bacterial selectivity for use at 50 µM and above.

Mechanistic approaches to the light-induced neural cell differentiation: Photobiomodulation vs Low-Dose Photodynamic Therapy.

BACKGROUND: Neurodegenerative diseases are the result of irreversible damage in the neuronal cells by effecting vital functions temporarily or even permanently. The use of light for the treatment of these diseases is an emerging promising innovative method. Photobiomodulation (PBM) and Photodynamic Therapy (PDT) are the modalities that have a wide range of use in medicine and have opposite purposes, biostimulation and cell death respectively. METHODS: In this study, we aimed to compare these two modalities (PDT and PBM) at low-level intensities and create a stimulatory effect on the differentiation of PC12 cells. Three different energy densities (1, 3, and 5 J/cm2) were used in PBM and Chlorin e6-mediated PDT applications upon irradiation with 655-nm laser light. The light-induced differentiation profile of PC12 cells was analyzed by morphological examinations, qRT-PCR, cell viability assay, and some mechanistic approaches such as; the analysis of intracellular ROS production, NO release, and mitochondrial membrane potential change. RESULTS: It has been observed that both of these modalities were successful at neural cell differentiation. PBM at 1 J/cm2 and low-dose PDT at 3 J/cm2 energy densities provided the best differentiation profiles which were proved by the over-expressions of SYN-1 and GAP43 genes. It was also observed that intracellular ROS production and NO release had pivotal roles in these mechanisms with more cell differentiation obtained especially in low-dose PDT application. CONCLUSION: It can be concluded that light-induced mechanisms with properly optimized light parameters have the capacity for neural cell regeneration and thus, can be a successful treatment for incurable neurodegenerative diseases.

Induced photo-cytotoxicity on prostate cancer cells with the photodynamic action of toluidine Blue ortho.

BACKGROUND: Photodynamic therapy (PDT) has become an advantageous therapeutic approach for the treatment of select cancers and microbial infections. PDT generates toxic reactive oxygen species as an end product of the interaction between the photosensitizer and light with an appropriate wavelength. Toluidine blue ortho is a photosensitizer that is commonly used in the photodynamic treatment of bacterial infection and a promising photosensitizer for cancer treatment. This study aims to evaluate the potential photo-cytotoxicity of toluidine blue ortho-mediated photodynamic therapy on PC-3 prostate cancer cells. METHODS: In this study toluidine blue ortho-mediated photodynamic therapy was assessed on PC-3 cancer cells with various photosensitizer concentrations and light energy densities of the 655-nm diode laser. MTT analysis was used for the determination of the cytotoxicity on the cells and viability/cytotoxicity assay was used for live/dead cell staining after the applications. The mechanism of this application was further analyzed with the determination of intracellular reactive oxygen species and nitric oxide release. RESULTS: The light applications and the photosensitizer alone did not inhibit the cell viability of PC-3 cells. 20 J/cm2 laser energy density together with 100 µM photosensitizer concentration resulted in maximum cancer cell death with a rate of approximately 89 %. The level of intracellular reactive oxygen species increased with the increasing parameters of the applications that resulted in more cell death. CONCLUSION: This study showed the successful anticancer activity of toluidine blue ortho upon irradiation with 655 nm of laser light against PC-3 cancer cells and it was mediated with the production of reactive oxygen species.

Comparative analysis of the light parameters of red and near-infrared diode lasers to induce photobiomodulation on fibroblasts and keratinocytes: An in vitro study.

BACKGROUND: Photobiomodulation (PBM) depends on the use of non-ionizing light energy to trigger photochemical changes, particularly in light-sensitive mitochondrial structures. It triggers proliferation and the metabolic activity of the cells, primarily by utilizing the energy from the near-infrared to the red wavelength of the light. PURPOSE: This in vitro study has analyzed comparatively the most appropriate energy doses and wavelengths to induce PBM on keratinocytes and fibroblasts for the accelerated wound healing process. METHODS: 1, 3, and 5 J/cm2 energy densities of 655 and 808-nm diode lasers were used to promote cell proliferation and wound healing process. Scratch assay and MTT analysis were performed on keratinocytes and fibroblasts for wound closure and cell proliferation after the triple light applications, respectively. RESULTS: 655-nm of wavelength was more successful on keratinocytes to induce wound healing and cell proliferation, whereas 808-nm of wavelength was so effective on fibroblasts to heal the wounds totally and it induced cell proliferation almost 3 times compared to the untreated control group. CONCLUSION: This study revealed that PBM with 655 and 808 nm of wavelengths was effective to speed up the wound healing process at specific energy densities. In general 808-nm of wavelength was more successful. However, the proper wavelength and the energy density may differ according to the cell type. Thus, every light parameter should be chosen properly to obtain better outcomes during PBM applications.

The effect of indocyanine green-based photodynamic therapy on healthy fibroblast and keratinocyte cells.

BACKGROUND: Photodynamic therapy is a promising invention to treat infections and cancer where conventional treatments are insufficient and have many side effects. Photodynamic therapy is mainly emphasized as having minimal side effects on healthy cells during local applications, even so photosensitizer can accumulate in any cell and unwanted deaths may occur upon irradiation. This study focused on the degree of photodynamic action with indocyanine green against healthy cells, when it has phototoxic effects on pathogens. METHODS: Healthy mouse skin fibroblast and human skin keratinocyte cells were exposed to energy densities of 84 and 252 J/cm2 with 4, 10, 25, 50,100, 125 and 150 µg/mL indocyanine green which have efficiently killed gram-positive and gram-negative pathogens. Cell Viability, Lipid Peroxidation and Live/Dead Cell Staining analysis were performed to assess the phototoxicity with defined parameters on the healthy cells. RESULTS: 84 J/cm2 energy density was quite safe for keratinocytes with indocyanine green concentrations ranging from 4 to 125 µg/mL. When 252 J/cm2 energy density was used, most of the keratinocytes were damaged with any photosensitizer concentration. Fibroblasts only tolerate these energy densities together with 4 and 10 µg/mL indocyanine green. Increasing photosensitizer concentrations resulted in high phototoxic effect on them. CONCLUSION: Photodynamic therapy applications, which destroy pathogens, may also kill healthy eukaryotic cells. While some energy densities are safe, but others cause serious mortality rate on fibroblasts and keratinocytes. Therefore, harm to healthy cells related to photodynamic therapy parameters should be minimized by the optimization of energy densities and photosensitizer concentration properly.

Photodynamic antimicrobial activity of new porphyrin derivatives against methicillin resistant Staphylococcus aureus.

Methicillin resistant Staphylococcus aureus (MRSA) with multiple drug resistance patterns is frequently isolated from skin and soft tissue infections that are involved in chronic wounds. Today, difficulties in the treatment of MRSA associated infections have led to the development of alternative approaches such as antimicrobial photodynamic therapy. This study aimed to investigate photoinactivation with cationic porphyrin derivative compounds against MRSA in in-vitro conditions. In the study, MRSA clinical isolates with different antibiotic resistance profiles were used. The newly synthesized cationic porphyrin derivatives (PM, PE, PPN, and PPL) were used as photosensitizer, and 655 nm diode laser was used as light source. Photoinactivation experiments were performed by optimizing energy doses and photosensitizer concentrations. In photoinactivation experiments with different energy densities and photosensitizer concentrations, more than 99% reduction was achieved in bacterial cell viability. No decrease in bacterial survival was observed in control groups. It was determined that there was an increase in photoinactivation efficiency by increasing the energy dose. At the energy dose of 150 J/cm2 a survival reduction of over 6.33 log10 was observed in each photosensitizer type. While 200 µM PM concentration was required for this photoinactivation, 12.50 µM was sufficient for PE, PPN, and PPL. In our study, antimicrobial photodynamic therapy performed with cationic porphyrin derivatives was found to have potent antimicrobial efficacy against multidrug resistant S. aureus which is frequently isolated from wound infections.

Antibacterial photodynamic therapy with 808-nm laser and indocyanine green on abrasion wound models.

Infections with pathogens could cause serious health problems, such as septicemia and subsequent death. Some of these deaths are caused by nosocomial, chronic, or burn-related wound infections. Photodynamic therapy (PDT) can be useful for the treatment of these infections. Our aim was to investigate the antibacterial effect of indocyanine green (ICG) and 808-nm laser on a rat abrasion wound model infected with the multidrug resistant Staphylococcus aureus strain. Abrasion wounds were infected with a multidrug resistant clinical isolate of S. aureus. ICG concentrations of 500, 1000, and 2000 µg∕ml were applied with a 450 J∕cm2 energy dose. Temperature change was monitored by a thermocouple system. The remaining bacterial burden was determined by the serial dilution method after each application. Wounds were observed for 11 days posttreatment. The recovery process was assessed macroscopically. Tissue samples were also examined histologically by hematoxylin­eosin staining. Around a 90% reduction in bacterial burden was observed after applications. In positive control groups (ICG-only and laser-only groups), there was no significant reduction. The applied energy dose did not cause any thermal damage to the target tissue or host environment. Results showed that ICG together with a 808-nm laser might be a promising antibacterial method to eliminate infections in animals and accelerate the wound-healing process.

Antimicrobial photodynamic therapy of resistant bacterial strains by indocyanine green and 809-nm diode laser.

OBJECTIVE: This research aimed to investigate the bactericidal effect of indocyanine green (ICG) with 809-diode laser on wild type and resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa in vitro. BACKGROUND DATA: ICG and 809 nm combination can be a powerful tool for the treatment of wound-infecting, antibiotic-resistant bacteria. METHODS: The effect of ICG and 809 nm laser light on wild type and resistant strains of S. aureus and P. aeruginosa was examined in vitro. ICG concentrations and laser doses were initially optimized for wild type S. aureus and P. aeruginosa. After determining the most effective ICG concentrations with specified light dose, they were applied on resistant strains. Viable bacterial cells were counted by serial dilution method. RESULTS: Photodynamic therapy (PDT) with ICG was totally efficient to kill all of these bacterial strains, and light/ICG alone did not cause any lethal effect on any of the strains. Optimum ICG laser doses varied with respect to the bacteria type: 84 J/cm(2) of light dose with 6 µg/mL of ICG concentration caused more than 95% killing of wild type S. aureus strains. The same bactericidal effect was achieved with a lower amount of ICG (4 µg/mL) on resistant strain S. aureus. Optimum parameters for 99% killing of wild type P. aeruginosa were 125 µg/mL ICG and 252 J/cm(2) of light dose. Similarly, their bactericidal effect was stronger on resistant strain; 100 µg/mL ICG with 252 J/cm(2) was enough to cause a 99% decrease in viable cells. CONCLUSIONS: The combination of ICG and 809 nm laser light was found as an effective antibacterial method to destroy antibiotic-resistant strains of gram-positive and gram-negative bacteria.

Modulated and continuous-wave operations of low-power thulium (Tm:YAP) laser in tissue welding.

Our aim is to explore the welding capabilities of a thulium (Tm:YAP) laser in modulated and continuous-wave (CW) modes of operation. The Tm:YAP laser system developed for this study includes a Tm:YAP laser resonator, diode laser driver, water chiller, modulation controller unit, and acquisition/control software. Full-thickness incisions on Wistar rat skin were welded by the Tm:YAP laser system at 100 mW and 5 s in both modulated and CW modes of operation (34.66 Wcm(2)). The skin samples were examined during a 21-day healing period by histology and tensile tests. The results were compared with the samples closed by conventional suture technique. For the laser groups, immediate closure at the surface layers of the incisions was observed. Full closures were observed for both modulated and CW modes of operation at day 4. The tensile forces for both modulated and CW modes of operation were found to be significantly higher than the values found by conventional suture technique. The 1980-nm Tm:YAP laser system operating in both modulated and CW modes maximizes the therapeutic effect while minimizing undesired side effects of laser tissue welding. Hence, it is a potentially important alternative tool to the conventional suturing technique.

The effect of irradiance level in 980-nm diode laser skin welding.

OBJECTIVE: Current research aimed to investigate the role of irradiance in skin laser welding. BACKGROUND DATA: Optical and thermal responses of tissue to infrared irradiation are highly dependent on both wavelength and tissue type. The desired effect on tissue is created by proper selection of laser power, application time, and spot size. METHODS: Full-thickness skin incisions on Wistar rat dorsum were welded with 980-nm diode laser application. Two irradiance levels (200 and 16.6 W/cm(2)) were applied with high (6 W, 400 ms) and low (0.5 W, 5 s) powers of laser with the same spot size (0.03 cm(2)). Subjects were monitored throughout a 21-day recovery period; incisions were sampled for histology and mechanical tests on particular control days (1, 4, 7, 14, and 21). Closure index, thermally altered areas, epidermal thickness, and granulation areas of H&E (eosin) stained samples were calculated. The breaking point during a mechanical tensile test that ran at 5-mm/min crosshead speed was recorded. RESULTS: In the suture group, there was no closure 24 h postoperation. For laser groups, immediate closure at the surface layers of the incisions was observed: Almost half-thickness (from surface to deep dermis) welding was achieved. Granulation tissue level and epidermal thickness level for all groups were similar on postoperative day 21. CONCLUSION: The laser welding technique was found reliable in terms of immediate and mechanically strong closure compared with suture. Low irradiance of a 980-nm laser (16.6 W/cm(2)) yielded noticeably stronger bonds at the end of 21 days of recovery, as well as minimal thermal damage.