Influence of Different Photobiomodulation Parameters on Multi-Potent Adipose Tissue Mesenchymal Cells In Vitro.

Objective: Investigating the effect of different parameters of photobiomodulation (PBM) with low-power laser on multi-potent mesenchymal stem cells (MSCs) derived from adipose tissue in terms of proliferation and cell death. Methods: MSCs were submitted to PBM applications with combinations of the following physical parameters: control group (no intervention), wavelengths of 660 and 830 nm; energy of 0.5, 2, and 4 J; and power of 40 and 100 mW. MSC analysis was performed using MetaXpress® software at 24, 48, and 72 h. Results: Irradiation promoted a significant increase in cell proliferation (p < 0.05), with 830 nm laser, 100 mW, with energy of 0.5, 2, and 4 J in relation to the control group at all times. PBM with 660 nm, power of 40 mW, and energy of 0.5, 2, and 4 J produced greater cell death at 24 h compared with the control group. At the time of 72 h, there was no significant difference concerning cell death. Conclusions: According to the results found, we can conclude that both wavelengths were effective; however, the 830 nm laser was more effective in terms of cell proliferation compared with the 660 nm laser. The 660 nm wavelength showed a significant increase in cell death when compared with the 830 nm laser.

Effect of photobiomodulation therapy on the morphology, intracellular calcium concentration, free radical generation, apoptosis and necrosis of human mesenchymal stem cells-an in vitro study.

The aim of the study was to investigate the impact of multiwave locked system (MLS M1) emitting synchronized laser radiation at 2 wavelength simultaneous (λ = 808 nm, λ = 905 nm) on the mesenchymal stem cells (MSCs). Human MSCs were exposed to MLS M1 system laser radiation with the power density 195-318 mW/cm2 and doses of energy 3-20 J, in continuous wave emission (CW) or pulsed emission (PE). After irradiation exposure in doses of energy 3 J, 10 J (CW, ƒ = 1000 Hz), and 20 J (ƒ = 2000 Hz), increased proliferation of MSCs was observed. Significant reduction of Fluo-4 Direct™ Ca2+ indicator fluorescence over controls after CW and PE with 3 J, 10 J, and 20 J was noticed. A decrease in fluorescence intensity after the application of radiation with a frequency of 2000 Hz in doses of 3 J, 10 J, and 20 J was observed. In contrary, an increase in DCF fluorescence intensity after irradiation with laser radiation of 3 J, 10 J, and 20 J (CW, ƒ = 1000 Hz and ƒ = 2000 Hz) was also shown. Laser irradiation at a dose of 20 J, emitted at 1000 Hz and 2000 Hz, and 3 J emitted at a frequency of 2000 Hz caused a statistically significant loss of MSC viability. The applied photobiomodulation therapy induced a strong pro-apoptotic effect dependent on the laser irradiation exposure time, while the application of a sufficiently high-energy dose and frequency with a sufficiently long exposure time significantly increased intracellular calcium ion concentration and free radical production by MSCs.

Effects of 970 nm diode laser irradiation on morphology, proliferation, and differentiation of gingival mesenchymal stem cells / Efectos de la irradiación con láser de diodo de 970 m sobre la morfología, la proliferación y la diferenciación de las células madre mesenquimales gingivales

Objective: The objective of this study was to investigate the morphology, proliferation, and differentiation of gingival mesenchymal stem cells (GMSCs) irradiated with a 970 nm Diode Laser (LLLT). It is essential to validate the efficacy of treatment, optimize irradiation conditions and guarantee the safety and quality of stem cells for future use in dental applications. Materials and Methods: GMSCs were cultured in standard conditions and irradiated with a Diode laser (970 nm, 0.5W) with an energy density of 9J/cm2. Cell proliferation was assessed with the WST-1 proliferation kit. GMSCs were differentiated into chondrogenic and osteogenic lineages. Cell morphology was performed with Hematoxylin/eosin staining, and quantitative nuclear analysis was done. Cell viability was monitored with trypan blue testing. Results: GMSCs subjected to irradiation demonstrated a significant increase in proliferation at 72 hours compared to the non-irradiated controls (p=0.027). This indicates that the 970 nm diode laser has a stimulatory effect on the proliferation of GMSCs. LLLT-stimulated GMSCs exhibited the ability to differentiate into chondrogenic and osteogenic lineages. A substantial decrease in cell viability was observed 24 hours after irradiation (p=0.024). However, after 48 hours, the cell viability recovered without any significant differences. This indicates that there might be a temporary negative impact on cell viability immediately following irradiation, but the cells were able to recover and regain their viability over time. Conclusions: This study support that irradiation with a 970 nm diode laser could stimulate the proliferation of GMSCs, maintain their ability to differentiate into chondrogenic and osteogenic lineages, and has minimal impact on the mor- phological characteristics of the cells. These results support the potential use of NIR Lasers in combination with GMSCs as a promising strategy for dental treatments.

Objetivo: El objetivo de este estudio fue investigar la morfología, proliferación y diferenciación de las células madre mesenquimatosas (GMSC) irradiadas con un láser de diodo de 970 nm (LLLT). Es fundamental validar la eficacia del tratamiento, optimizar las condiciones de irradiación y garantizar la seguridad y calidad de las células madre para su uso futuro en aplicaciones dentales.Materiales y Métodos: Las GMSC se cultivaron en condiciones estándar y se irradiaron con un láser de diodo (970 nm, 0,5 W) con una densidad de energía de 9 J/cm2. La proliferación celular se evaluó con el kit de proliferación WST-1. Las GMSC se diferenciaron en linajes condrogénicos y osteogénicos. La morfología celular se realizó con tinción de hematoxilina/eosina y se realizó un análisis nuclear cuantitativo. La viabilidad celular se controló con prueba de azul de tripano. Resultados: Las GMSC sometidas a irradiación demostraron un aumento significativo en la proliferación a las 72 horas en comparación con los controles no irradiados (p=0,027). Esto indica que el láser de diodo de 970 nm tiene un efecto estimulante sobre la proliferación de GMSC. Las GMSC estimuladas con LLLT exhibieron la capacidad de diferenciarse en linajes condrogénicos y osteogénicos. Se observó una disminución sustancial de la viabilidad celular 24 horas después de la irradiación (p=0,024). Sin embargo, después de 48 horas, la viabilidad celular se recuperó sin diferencias significativas. Esto indica que podría haber un impacto negativo temporal en la viabilidad de las células inmediatamente después de la irradiación, pero las células pudieron recuperarse y recuperar su viabilidad con el tiempo. Conclusión: En conclusión, este estudio respalda que la irradiación con un láser de diodo de 970 nm podría estimular la proliferación de GMSC, mantener su capacidad para diferenciarse en linajes condrogénicos y osteogénicos y tiene un impacto mínimo en las características morfológicas de las células. Estos resultados respaldan el uso potencial de láseres NIR en combinación con GMSC como una estrategia prometedora para tratamientos dentales.

Low-level laser therapy with different irradiation methods modulated the response of bone marrow mesenchymal stem cells in vitro.

Low-level laser therapy (LLLT) also known as photobiomodulation is a treatment to change cellular biological activity. The exact effects of LLLT remain unclear due to the different irradiation protocols. The purpose of this study was to investigate the effects of LLLT by three different irradiation methods on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs were inoculated in 24-well plates and then irradiated or not (control) with a laser using three different irradiation methods. The irradiation methods were spot irradiation, covering irradiation, and scanning irradiation according to different spot areas (0.07 cm2 or 1.96 cm2) and irradiation areas (0.35 cm2 or 1.96 cm2), respectively. The laser was applied three times at energy densities of 4 J/cm2. The cell proliferation by CCK-8. ALP activity assay, alizarin red, and quantitative real-time polymerase chain reaction (RT-PCR) were performed to assess osteogenic differentiation and mineralization. Increases in cell proliferation was obvious following irradiation, especially for covering irradiation. The ALP activity was significantly increased in irradiated groups compared with non-irradiated control. The level of mineralization was obviously improved following irradiation, particularly for covering irradiation. RT-PCR detected significantly higher expression of ALP, OPN, OCN, and RUNX-2 in the group covering than in the others, and control is the lowest. The presented results indicate that the biostimulative effects of LLLT on BMSCs was influenced by t he irradiation method, and the covering irradiation is more favorable method to promote the proliferation and osteogenic differentiation of BMSCs.

The effect of 805 nm near-infrared photobiomodulation on proliferation and differentiation of bone marrow stem cells in murine rats.

OBJECTIVE: To evaluate the effect of near infra-red gallium-aluminium-arsenide (GaAlAs) diode laser (805 nm) irradiation on proliferation and differentiation of rat femoral bone marrow-derived mesenchymal stem cells (BMSCs) cultured in osteogenic medium. MATERIALS AND METHODS: BMSCs were obtained from femurs of 60 Sprague Dawley rats (200 gm). The control group comprised isolated BMSCs supplemented with an osteogenic differentiation medium. On the other hand, in the experimental group, the BMSCs were irradiated with a near-infrared laser in addition to an osteogenic differentiation medium. The experimental group was irradiated with a soft tissue laser comprising of allium-aluminium-arsenic (Ga-Al-Ar) Diode at a near-infrared wavelength of 805 nm in continuous mode. The different output powers applied were 0.5 W, 1.0 W, 1.5 W and 2.0 W respectively. Various energy levels of 1, 4, 7 and 10 J were used for irradiation. Alkaline phosphatase (ALP) assay and Alizarin staining were performed to confirm osteogenic differentiation. Statistical analysis was done using a one-way ANOVA and a p-value of <0.05 was considered significant. RESULTS: According to our findings, 1.27 J/cm2 was the optimal energy density value that significantly increased the BMSC proliferation at the output of 1.5 W with the power density of 1.27 W/cm2. On 1.27 J/cm2, there was a significant difference compared to the control group on the first day, and the osteogenic differentiation increased significantly on the 4th day compared to the 1st day. CONCLUSIONS: According to our findings, 1.27 J/cm2 was the optimal energy density value that significantly increased the BMSC proliferation at the output of 1.5 W with the power density of 1.27 W/cm2.

Photobiomodulation has rejuvenating effects on aged bone marrow mesenchymal stem cells.

The plasticity and proliferative capacity of stem cells decrease with aging, compromising their tissue regenerative potential and therapeutic applications. This decline is directly linked to mitochondrial dysfunction. Here, we present an effective strategy to reverse aging of mouse bone marrow mesenchymal stem cells (BM-MSCs) by restoring their mitochondrial functionality using photobiomodulation (PBM) therapy. Following the characterization of young and aged MSCs, our results show that a near-infrared PBM treatment delivering 3 J/cm2 is the most effective modality for improving mitochondrial functionality and aging markers. Furthermore, our results unveil that young and aged MSCs respond differently to the same modality of PBM: whereas the beneficial effect of a single PBM treatment dissipates within 7 h in aged stem cells, it is lasting in young ones. Nevertheless, by applying three consecutive treatments at 24-h intervals, we were able to obtain a lasting rejuvenating effect on aged MSCs. Our findings are of particular significance for improving autologous stem cell transplantation in older individuals who need such therapies most.

In Vitro Wound Healing Potential of Photobiomodulation Is Possibly Mediated by Its Stimulatory Effect on AKT Expression in Adipose-Derived Stem Cells.

Increasing evidence suggests that adipose-derived stem cells (ADSCs) serve as a therapeutic approach for wound healing. The aim of this study was to determine the effect of photobiomodulation (PBM) on antioxidant enzymes in ADSCs. Four ADSC cell models, namely, normal, wounded, diabetic, and diabetic wounded, were irradiated with 660 nm (fluence of 5 J/cm2 and power density of 11.2 mW/cm2) or 830 nm (fluence of 5 J/cm2 and power density of 10.3 mW/cm2). Nonirradiated cells served as controls. Cell morphology and wound migration were determined using light microscopy. Cell viability was determined by the trypan blue exclusion assay. The enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of antioxidants (superoxide dismutase (SOD), catalase (CAT), and heme oxygenase (HMOX1)). AKT activation and FOXO1 levels were determined by immunofluorescence and western blotting. The gaps (wound) in PBM-treated wounded and diabetic wounded cell models closed faster than the controls. PBM treatment significantly increased antioxidant levels in all cell models. This reflects that oxidative stress is reduced on the counterpart of increased antioxidant levels. This might be due to the activation of the AKT signaling pathway as evidenced by the increased AKT signals via western blotting and immunofluorescence. This data suggests that PBM promotes wound healing by increasing antioxidant levels by activating AKT signaling.

Photobiomodulation: A review of the molecular evidence for low level light therapy.

Light energy is harnessed for therapeutic use in a number of ways, most recently by way of photobiomodulation (PBM). This phenomenon is a cascade of physiological events induced by the nonthermal exposure of tissue to light at the near infrared end of the visible spectrum. Therapeutic PBM has become a highly commercialized interest, marketed for everything from facial rejuvenation to fat loss, and diode-based devices are popular in both the clinic setting and for use at home. The lack of regulatory standards makes it difficult to draw clear conclusions about efficacy and safety but it is crucial that we understand the theoretical basis for PBM, so that we can engage in an honest dialogue with our patients and design better clinical studies to put claims of efficacy to the test. This article presents a summary of the science of PBM and examines the differences between laser light, on which much of the preclinical evidence is based and light from diodes, which are typically used in a clinical setting.

Effects of green light photobiomodulation on Dental Pulp Stem Cells: enhanced proliferation and improved wound healing by cytoskeleton reorganization and cell softening.

Photobiomodulation (PBM) has been shown to improve cell proliferation and cell migration. Many cell types have been investigated, with most studies using deep penetrating red light irradiation. Considering the interest of surface biostimulation of oral mesenchymal cells after surgical wound, the present study aimed to assess green light irradiation effects on Dental Pulp Stem Cells' (DPSC) proliferation and migration. To understand the mechanisms underlying these effects, we investigated cytoskeleton organization and subsequent cell shape and stiffness. A 532-nm wavelength Nd:YAG laser (30 mW) was applied between 30 and 600 s on DPSC in vitro. Cell proliferation was analyzed at 24, 48, and 72 h after irradiation, by cell counting and enzymatic activity quantification (paranitrophenylphosphate phosphatase (pNPP) test). A wound healing assay was used to study cell migration after irradiation. Effects of PBM on cytoskeleton organization and cell shape were assessed by actin filaments staining. Elasticity changes after irradiation were quantified in terms of Young's modulus measured using Atomic Force Microscopy (AFM) force spectroscopy. Green light significantly improved DPSC proliferation with a maximal effect obtained after 300-s irradiation (energy fluence 5 J/cm2). This irradiation had a significant impact on cell migration, improving wound healing after 24 h. These results were concomitant with a decrease of cells' Young's modulus after irradiation. This cell softening was explained by actin cytoskeleton reorganization, with diminution of cell circularity and more abundant pseudopodia. This study highlights the interest of green laser PMB for the proliferation and migration of mesenchymal stem cells, with encouraging results for clinical application, especially for surgical wound healing procedures.

Magnetic Stimulation Drives Macrophage Polarization in Cell to-Cell Communication with IL-1ß Primed Tendon Cells.

Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1ß. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1ß-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.

Photobiomodulation therapy compensate the impairments of diabetic bone marrow mesenchymal stem cells.

Pathophysiologic conditions associated with diabetes mellitus affect mesenchymal stem cells (MSCs), and this phenomenon may lead to some diabetic secondary complications. The present study was conducted to evaluate the impact of photobiomodulation (PBM) on rat diabetic MSC (DMSC) behavior in vitro. For the purpose of PBM, we used helium-neon laser with a wavelength of 632.8 nm at three different energy densities (0.5, 1, 2 J/cm2) and radiation periodicity of once, twice, and thrice. The survival, proliferation, and apoptosis in the normal MSCs (NMSCs), DMSCs, and diabetic MSCs, which were laser irradiated (DMSCs+L), were assessed using MTT assay, Ki67 immunofluorescence staining, and TUNEL assay, respectively. Our results demonstrated that DMSCs have significantly lower survival (P < 0.05) and proliferation rates (P < 0.001), and dramatically higher population doubling time (PDT, P < 0.001) and apoptosis rates (P < 0.001) as compared to NMSCs. Moreover, PBM with energy density of 1 J/cm2 and the periodicity of 1 or 2 times could improve diabetic MSC capabilities in the term of survival, proliferation, and apoptosis. Considering these findings, it is suggested that PBM could improve the ability of diabetic MSCs in vitro prior to transplantation or may rise their capabilities in their native niche in vivo.

Improvement in viability and mineralization of osteoporotic bone marrow mesenchymal stem cell through combined application of photobiomodulation therapy and oxytocin.

The probable positive effects of photobiomodulation therapy (PBMT) and oxytocin (OT) treatments together or alone were evaluated on cell viability along with the changes in the gene expression of Osteocalcin (OC), Osteoprotegerin (OPG), and Runt-related transcription factor 2 (Runx2) levels of sham (healthy)-Bone marrow mesenchymal stem cell(BMMSC) and ovariectomy-induced osteoporosis (OVX)-BMMSC. BMMSC was harvested from healthy and OVX rats and was cultured in osteogenic induction medium (OIM). There were five groups of BMMSCs: (1) sham -BMMSCs; (2) control -OVX-BMMSCs; (3) OT-treated-OVX-BMMSCs; (4) PBMT-treated-OVX-BMMSCs, and (5) OT + PBMT-OVX-BMMSCs. In all 5 groups, BMMSC viability and proliferation as well as gene expression of OC, OPG, and RUNX2 were evaluated. PBMT and PBMT + OT treatments showed a promising effect on the increased viability of OVX-BMMSC (ANOVA test; LSD test, p = 0.01, p = 0.002). The results of gene expression analysis revealed that the sham- BMMSCs responded optimally to OT treatment. It was also found that OVX-BMMSCs responded optimally to PBMT + OT and PBMT treatments at early and middle stages of osteogenic induction process. Nevertheless, they responded optimally to PBMT + OT and OT especially at the late stage of osteogenic induction process. PBMT and PBMT + OT treatments significantly increased viability of OVX-BMMSC in OIM in vitro. Both PBMT and PBMT + OT treatments could promote mineralization of OVX-BMMSC in the culture medium at early and middle stages of osteogenic induction process. Both OT and PBMT + OT treatments could promote mineralization of OVX-BMMSC in vitro at late stages of osteogenic induction process.

Photobiomodulation with 630 plus 810 nm wavelengths induce more in vitro cell viability of human adipose stem cells than human bone marrow-derived stem cells.

The goal of the current experiment is to explore the influence of combined and/or single applications of red and near infrared (NIR) photobiomodulation (PBM) at different wavelengths, energy densities and times on cell viability, population doubling time (PDT), and apoptosis of in vitro cultures of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and h adipose-derived stem cells (hASCs). Both in vitro hBM-MSCs and hASCs were irradiated with 36 protocols using two different laser types (helium­neon [He-Ne] and diodes), four different laser wavelengths (HeNe laser, 630 nm, 810 nm, 630 + 810 nm); three different energy densities (0.6 J/cm2, 1.2 J/cm2, 2.4 J/cm2); and three different PBM times (1, 2, and 3). One-way ANOVA analysis showed that PBM with the 630 nm red laser significantly stimulated cellular viability of both hBM-MSCs and hASCs. The 630 nm red laser significantly decreased PDT of hBM-MSCs. One-way ANOVA demonstrated that the 630 + 810 laser significantly stimulated cellular viability, and significantly decreased PDT and apoptosis of hBM-MSCs and hASCs. Two-way ANOVA analysis showed that PBM with the 630 nm red laser and 630 + 810 nm laser significantly stimulated cellular viability of hASCs compared to the control hASCs, and experimental and control hBM-MSCs. Our study demonstrated that PBM with the combined 630 + 810 nm lasers significantly stimulated cell viability, and significantly decreased PDT and apoptosis of hBM-MSCs and hASCs in vitro. We reported new in vitro evidence where PBM administered at 630 nm (one and two times, 0.6 and 1.2 J/cm2) and 630 + 810 nm (three times, 2.4 J/cm2) significantly increased hASC cell viability compared to its control and the PBM-treated hBM-MSC groups.

Effect of Low-Level Laser Irradiation on Proliferative Activity of Wharton's Jelly Mesenchymal Stromal Cells.

We studied the effect of low-level laser irradiation on proliferative activity of cultured human Wharton's jelly mesenchymal stromal sells. Cells were irradiated with a solid-state laser emitting at 650 nm; irradiation doses were 0.04, 0.4, or 4 J/cm2. Laser irradiation was performed once at the start of the cell proliferation experiment or daily throughout the experiment. Cells were cultured for 7 days. The number of viable cells was assessed using the MTT test. An increase in cell proliferative activity was detected after daily laser irradiations; the maximum stimulating effect was achieved at a dose of 0.04 J/cm2. These results substantiate medical use of lasers for expansion of cells intended for transplantation.

Effect of Photobiomodulation Therapy on the Increase of Viability and Proliferation of Human Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: We have investigated how low intensity laser irradiation emitted by a multiwave-locked system (MLS M1) affects the viability and proliferation of human bone marrow mesenchymal stem cells (MSCs) depending on the parameters of the irradiation. STUDY DESIGN/MATERIALS AND METHODS: Cells isolated surgically from the femoral bone during surgery were identified by flow cytometry and cell differentiation assays. For irradiation, two wavelengths (808 and 905 nm) with the following parameters were used: power density 195, 230, and 318 mW/cm 2 , doses of energy 3, 10, and 20 J (energy density 0.93-6.27 J/cm 2 ), and in continuous (CW) or pulsed emission (PE) (frequencies 1,000 and 2,000 Hz). RESULTS: There were statistically significant increases of cell viability and proliferation after irradiation at 3 J (CW; 1,000 Hz), 10 J (1,000 Hz), and 20 J (2,000 Hz). CONCLUSIONS: Irradiation with the MLS M1 system can be used in vitro to modulate MSCs in preparation for therapeutic applications. This will assist in designing further studies to optimize the radiation parameters and elucidate the molecular mechanisms of action of the radiation. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Astragalus Polysaccharide Eases G1 Phase-Correlative Bystander Effects through Mediation of TGF- ß R/MAPK/ROS Signal Pathway After Carbon Ion Irradiation in BMSCs.

Although Astragalus polysaccharide (APS) has been shown to have various pharmacological effects, there have been no studies concerning the inhibitory effects of APS on the radiation-induced bystander effects (RIBE). The aim of this study was to investigate whether APS could suppress RIBE damage by inhibiting cell growth, micronucleus (MN) formation and 53BP1 foci number increased in bone marrow mesenchymal stem cells (BMSCs), named bystander cells, as well as to explore its mechanism. In this study, APS decreased proliferation and colony rate of bystander cells by inducing cell cycle arrest at G1 phase via extrinsic and intrinsic DNA damage. Regarding mechanism, APS inhibited mitogen-activated protein kinase (MAPK) signal pathway by down-regulating the expression of the key proteins, phosphorylated JNK (p-JNK), phosphorylated ERK (p-ERK) but not phosphorylated P38 (p-P38), and down-regulating their downstream function protein and molecule, cyclooxygenase-2 (COX-2) and reactive oxygen species (ROS). Moreover, in bystander cells, APS inhibits expression of transforming growth factor ß receptor II (TGF- ß R II), a cell membrane receptor, resulting in lower ROS production and secretion via TGF- ß R-JNK/ERK-COX-2/ROS not P38 signaling. They gave a hint that the decreased RIBE damage induced by APS treatment involved TGF- ß R-JNK/ERK-COX-2/ROS down-regulation.

Low-level laser and adipose-derived stem cells altered remodelling genes expression and improved collagen reorganization during tendon repair.

OBJECTIVES: The cellular therapy using adipose-derived mesenchymal stem cells (ASCs) aims to improve tendon healing, considering that repaired tendons often result in a less resistant tissue. Our objective was to evaluate the effects of the ASCs combination with a low-level laser (LLL), an effective photobiostimulation for the healing processes. MATERIALS AND METHODS: Rats calcaneal tendons were divided into five groups: normal (NT), transected (T), transected and ASCs (SC) or LLL (L), or with ASCs and LLL (SCL). RESULTS: All treated groups presented higher expression of Dcn and greater organization of collagen fibres. In comparison with T, LLL also up-regulated Gdf5 gene expression, ASCs up-regulated the expression of Tnmd, and the association of LLL and ASCs down-regulated the expression of Scx. No differences were observed for the expression of Il1b, Timp2, Tgfb1, Lox, Mmp2, Mmp8 and Mmp9, neither in the quantification of hydroxyproline, TNF-α, PCNA and in the protein level of Tnmd. A higher amount of IL-10 was detected in SC, L and SCL compared to T, and higher amount of collagen I and III was observed in SC compared to SCL. CONCLUSIONS: Transplanted ASCs migrated to the transected region, and all treatments altered the remodelling genes expression. The LLL was the most effective in the collagen reorganization, followed by its combination with ASCs. Further investigations are needed to elucidate the molecular mechanisms involved in the LLL and ASCs combination during initial phases of tendon repair.

Low-level laser irradiation modulates the proliferation and the osteogenic differentiation of bone marrow mesenchymal stem cells under healthy and inflammatory condition.

The aim of this in vitro study was to evaluate the effects of low-level laser therapy (LLLT) at different energy intensities on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under healthy and inflammatory microenvironments. Human BMSCs and BMSCs from inflammatory conditions (i-BMSCs, BMSCs treated with tumor necrosis factor α; TNF-α) were subject to LLLT (Nd:YAG;1064 nm) at different intensities. We designed one control group (without irradiation) and four testing groups (irradiation at 2, 4, 8, and 16 J/cm2) for both BMSCs and i-BMSCs. Cell proliferation was measured using colony-forming unit fibroblast assay and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic capacity of cells was determined by alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin Red S staining and the mRNA transcript levels of genes runt-related transcription factor 2 (Runx2), ALP, and osteocalcin. Moreover, the effects of LLLT on secretion of TNF-α in BMSCs and i-BMSCs were measured by enzyme-linked immunosorbent assay. Our results demonstrated LLLT could significantly promote BMSC proliferation and osteogenesis at densities of 2 and 4 J/cm2. LLLT at density of 8 J/cm2 could promote the proliferation and osteogenesis of i-BMSCs. However, LLLT at 16 J/cm2 significantly suppressed the proliferation and osteogenesis of BMSCs both in healthy and in inflammatory microenvironment. Moreover, we also found that the expression of TNF-α was obviously inhibited by LLLT at 4, 8, and 16 J/cm2, in an inflammatory microenvironment. Considering these findings, LLLT could improve current in vitro methods of differentiating BMSCs under healthy and inflammatory microenvironments prior to transplantation.

Photobiomodulation effect on the proliferation of adipose tissue mesenchymal stem cells.

The use of mesenchymal stem cells (MSCs) in tissue engineering has been extensively investigated. The greater the proliferation of this cellular group, the greater the regenerative and healing capacity of the tissue to which they belong. In this context, photobiomodulation (PBM) is an efficient technique in proliferation of distinct cell types. However, its parameters and mode of action are still unclear and require further investigation. This study aimed to evaluate the PBM action with different energies in MSCs of adipose tissue (hASCs). We used hASCs, seeded in 24-well plates, with 3 × 104 cells per well, in culture media. We used a total of four experimental groups, one with hASCs and simulated PBM and three other groups, which received PBM irradiation at 24, 48, and 72 h, with a 660-nm laser and power of 40 mW and energy of 0.56, 1.96, and 5.04 J. We performed analyses of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor) and trypan blue to evaluate cell proliferation and viability, 1 h after PBM irradiation. Software Graph PadPrism 7.0 was used. Intergroup comparisons were performed with ANOVA two-way and we used the Tukey post hoc test. Mitochondrial activity evaluated by MTT revealed the statistical difference in the first 24 h for group with more high energy when compared to control group; and in the 72 h for two irradiated groups when compared to the control group. The trypan blue test showed significant differences at the end of the experiment for two irradiated groups LG1 (4.52 × 104 ± 0.2) and LG2 (4.85 × 104 ± 0.8), when compared to the control group (1.87 × 104 ± 0.7). Both tests failed to be statistically different at the end of the experiment for groups LG1 and LG2 and observed a reduction in cellular mitochondrial growth and activity for group LG3. We conclude that PBM with energy close to 0.56 and 1.96 J promote proliferation of hASCs, and higher energy, such as 5.04 J, can be harmful.