Electromagnetic stimulation increases mitochondrial function in osteogenic cells and promotes bone fracture repair.

Bone fracture is a growing public health burden and there is a clinical need for non-invasive therapies to aid in the fracture healing process. Previous studies have demonstrated the utility of electromagnetic (EM) fields in promoting bone repair; however, its underlying mechanism of action is unclear. Interestingly, there is a growing body of literature describing positive effects of an EM field on mitochondria. In our own work, we have previously demonstrated that differentiation of osteoprogenitors into osteoblasts involves activation of mitochondrial oxidative phosphorylation (OxPhos). Therefore, it was reasonable to propose that EM field therapy exerts bone anabolic effects via stimulation of mitochondrial OxPhos. In this study, we show that application of a low intensity constant EM field source on osteogenic cells in vitro resulted in increased mitochondrial membrane potential and respiratory complex I activity and induced osteogenic differentiation. In the presence of mitochondrial inhibitor antimycin A, the osteoinductive effect was reversed, confirming that this effect was mediated via increased OxPhos activity. Using a mouse tibial bone fracture model in vivo, we show that application of a low intensity constant EM field source enhanced fracture repair via improved biomechanical properties and increased callus bone mineralization. Overall, this study provides supporting evidence that EM field therapy promotes bone fracture repair through mitochondrial OxPhos activation.

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.

Laser and LED photobiomodulation effects in osteogenic or regular medium on rat calvaria osteoblasts obtained by newly forming bone technique.

The purposes of this study are to evaluate the effects of photobiomodulation (PBM) with laser and LED on rat calvaria osteoblasts (rGO lineage), cultured in osteogenic (OST) or regular (REG) medium, after induction of a quiescent state and to test if PBM is capable of osteogenic induction and if there is a sum of effects when combining OST medium with PBM. Before irradiation, the cells were put in a quiescent state (1% FBS) 24 h, when red (AlGaInP-660 nm) and infrared laser (GaAlAs-808 nm) and LED (637 ± 15 nm) were applied. The groups were as follows: red laser (RL3-5 J/cm2, 3 s and RL5-8.3 J/cm2, 5 s, 1.66 W/cm2); infrared laser (IrL3-5 J/cm2, 3 s and IrL5-8.3 J/cm2, 5 s); LED (LED3-3 s and LED5-5 s, 0.02 J/cm2, 0.885 W/cm2); positive (C+, 10% FBS) and negative control (C-, 1% FBS). For alkaline phosphatase (ALP) and mineralization assays, the cells were cultured in REG (DMEM 10% FBS) and OST medium (DMEM 10% FBS, 50 µg/mL ascorbic acid, 10 mM ß-glycerophosphate). Statistical analysis was performed using ANOVA and Tukey's tests (p < 0.05). RL5 and LED5 increased proliferation, in vitro wound closure, ALP, and mineralization in rGO cells (p < 0.05). PBM with red laser and LED induced mineralization by itself, without osteogenic medium, not observed for infrared laser (p < 0.05). A sum of effects was observed in osteogenic medium and PBM by infrared, red laser, and LED (5 s). Red laser and LED increased proliferation, migration, and secretory phases in rGO cells in a dose-dependent manner. PBM with red laser and LED promotes osteogenic induction by itself. PBM with infrared laser and osteogenic medium potentializes mineralization.

Laser therapy increases the proliferation of preosteoblastic MC3T3-E1 cells cultured on poly(lactic acid) films.

This study aimed to verify the efficacy of low-level laser irradiation (LLLI) on the proliferation of MC3T3-E1 preosteoblasts cultured on poly(lactic acid) (PLA) films. The produced films were characterized by contact angle tests, scanning electron microscopy (SEM), atomic force microscopy, differential scanning calorimetry, and X-ray diffraction. The MC3T3-E1 cells were cultured as three different groups: Control-cultured on polystyrene plastic surfaces; PLA-cultured on PLA films; and PLA + Laser-cultured on PLA films and submitted to laser irradiation (660 nm; 30 mW; 4 J/cm2 ). Cell proliferation was analyzed by Trypan blue and Alamar blue assays at 24, 48, and 72 h after irradiation. Cell viability was assessed by Live/Dead assay, apoptosis-related events were evaluated by Annexin V/propidium iodide (PI) expression, and cell cycle events were analyzed by flow cytometry. Cell morphology on the surface of films was assessed by SEM. Cell counting and biochemical assay results indicate that the PLA + Laser group exhibited higher proliferation (p < 0.01) when compared with the Control and PLA groups. The Live/Dead and Annexin/PI assays indicate increased cell viability in the PLA + Laser group that also presented a higher percentage of cells in the proliferative cell cycle phases (S and G2/M). These findings were also confirmed by the higher cell density observed in the irradiated group through SEM images. The evidence from this study supports the idea that LLLI increases the proliferation of MC3T3-E1 cells on PLA surfaces, suggesting that it can be potentially applied in bone tissue engineering.

The Effects of Photobiomodulation on MC3T3-E1 Cells via 630 nm and 810 nm Light-Emitting Diode.

BACKGROUND Photobiomodulation (PBM) has been explored as a promising therapeutic strategy to regulate bone cell growth; however, the effects of PBM on osteoblast cell lines remains poorly understood. In addition, as a light source of PBM, the light uniformity of light-emitting diode (LED) devices has not been given enough attention. MATERIAL AND METHODS Here, we sought to investigate the effects of PBM on MC3T3-E1 cells via 630 nm and 810 nm light from a newly designed LED with high uniformity of light. Cell proliferation, flow cytometric analysis, alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red S staining, and quantitative real-time polymerase chain reaction (qRT-PCR) were carried out to assess treatment response. MC3T3-E1 cells were irradiated with LED devices (630±5 nm and 810±10 nm, continuous wave) for 200 seconds at a power density of 5 mW/cm² once daily. RESULTS Increases in cell proliferation and decreases in cell apoptosis were evident following irradiation. ALP staining intensity and activity were also significantly increased following irradiation. Level of mineralization was obviously enhanced in irradiated groups compared with non-irradiated controls. qRT-PCR also showed significant increases in mRNA expression of osteocalcin (OCN) and osteoprotegerin (OPG) in the irradiated groups. CONCLUSIONS Our results showed that LED PBM could promote the proliferation, ALP staining intensity and activity, level of mineralization, gene expression of OCN and OPG of MC3T3-E1 cells, with no significant difference between the 630 nm- and 810 nm-irradiated groups.

Effect of low power laser in biomodulation of cultured osteoblastic cells of Wistar rats1.

PURPOSE: To analyze aspects of the biomodulating effect of light in biological tissues, bone cells from surgical explants of the femur of rats were irradiated with low intensity laser. METHODS: Bone cells were cultured and irradiated with LASER light (GaAlAs). Growth, cell viability, mineralized matrix formation, total protein dosage, immunostimulatory properties, cytochemical analysis, gene expression of bone proteins were examined using live cell imaging and cell counting by colorimetric assay. The gene expression of: alkaline phosphatase (ALP), type 1 collagen, osteocalcin and osteopontin through the real-time polymerase chain reaction. RESULTS: At 8 days, the viability of the irradiated culture was 82.3% and 72.4% in non-irradiated cells. At 18 days, the cellular viability (with laser) was 77.42% and 47.62% without laser. At 8 days, the total protein concentration was 21.622 mg / mol in the irradiated group and 16, 604 mg / mol in the non-irradiated group and at 18 days the concentration was 37.25 mg / mol in the irradiated group and 24, 95 mg / mol in the non-irradiated group. CONCLUSION: The laser interfered in the histochemical reaction, cell viability, matrix mineralization, and maintained the cellular expression of proteins.

Optimal currents for electrical stimulation of bone fracture repair: A computational analysis including variations in frequency, tissue properties, and fracture morphology.

Fracture healing happens naturally in most bone break cases. Occasionally prolongation of restoration period or non-union of the fracture may occur, where electrical stimulation has been shown to facilitate bone restoration by stimulating osteoblasts. Despite clinical use, a comprehensive computational model linking the applied currents to the stimulating field in the fracture has been missing. In this paper, we investigate the input current needed to stimulate osteoblasts in a fracture in the human forearm. Optimal current is computed for various fracture configurations, and sensitivity to frequency and inter/intrapersonal variance in dielectric properties are analyzed. Stimulation thresholds at the fracture site are based on detailed review of experimental studies. Our results show that for a 1 mm thick 30° fracture with a 15 Hz sinusoidal field, the input current amounts to a maximum of 3.77 µA. Minimum and maximum required current levels are plotted versus fracture parameters, all of which comply with the ICNIRP standard. Simulation results are supported by several experimental reports. Our model is useful for understanding the effects of various geometrical and electrical factors on clinical outcome, and serves as a theoretical aid in the design of more efficient systems. Bioelectromagnetics. 40:128-135, 2019. © 2019 Bioelectromagnetics Society.

Effects of Nd:YAG low-level laser irradiation on cultured human osteoblasts migration and ATP production: in vitro study.

Low-level laser therapy has become one of the fastest growing fields of medicine in recent years. Many in vivo and in vitro studies have shown that laser irradiation activates a range of cellular processes in a variety of cell types and can promote tissue repair. However, few in vitro experiments have evaluated the effects of laser irradiation on cells in real time. The purpose of this study was to examine the effects of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on the migration of cultured human osteoblasts. A dedicated 96-well plate was used, and confluent cultures of the human osteoblast-like cell line, Saos-2, were injured with a wound maker. The wounded cells were then exposed to the Nd:YAG laser (wavelength of 1064 nm) for 60 s at 0.3 W (10 pps, 30 mJ). The total energy density was about 10.34 J/cm2. Images of the wounds were automatically acquired inside the CO2 incubator by the IncuCyte ZOOM™ software. In addition, after laser irradiation, the production of adenosine triphosphate (ATP) was measured using the CellTiter-Glo™ Luminescent Cell Viability Assay. Migration of cells from the border of the original scratch zone was accelerated by laser irradiation. In addition, compared with the control group, significant enhancement of ATP production was observed in the irradiated group. The present study showed that Nd:YAG laser irradiation (wavelength of 1064 nm, 0.3 W, 10 pps, 30 mJ, 10.34 J/cm2, irradiation time 60 s) may contribute to the regeneration of bone tissues owing to enhanced osteoblast cell migration.

The effects of photobiomodulation therapy on mouse pre-osteoblast cell line MC3T3-E1 proliferation and apoptosis via miR-503/Wnt3a pathway.

Photobiomodulation therapy (PBMT) has been demonstrated as regulating osteoblast proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in osteoblast, but the role of miRNAs in the PBMT-based promotion of osteoblast proliferation remains unclear. This study aimed to investigate the effects of PBMT treatment (3.75 J/cm2) on mouse pre-osteoblast cell line MC3T3-E1 proliferation and apoptosis via the miR-503/Wnt3a pathway; meanwhile, detect the expressions of miR-503 and Wnt3a after PBMT treatment and the role of miR-503 in regulating Wnt signaling molecules Wnt3a, ß-catenin, Runx2, apoptotic proteins caspase-3, and Bcl-2 in vitro. The PBMT parameters were as follows: 808 nm continuous wavelength, 0.401 W output power, 0.042 W/cm2 power density, 9.6 cm2 spot size, 36 J energy, 3.75 J/cm2 energy density, 90 s irradiation for three times per 12 h, 14.5 cm distance of the laser source and the angle of divergence of the laser beam was 7°. In our present study, the target relationship was predicted and verified by bioinformatics analysis and luciferase reporter assays. Gene mRNA and protein expressions were examined by qPCR and western blot analysis. The MTT method was used to evaluate the effect of miR-503 on MC3T3-E1 cells proliferation. And cell apoptosis was examined by flow cytometry. The results showed that PBMT treatment reduced the expression of miR-503 and increased the level of Wnt3a (p < 0.01). Bioinformatics analysis and luciferase reporter assays revealed that Wnt3a was a target of miR-503, and Wnt3a was regulated by miR-503. Furthermore, miR-503 was found to functionally inhibit proliferation and promote apoptosis (p < 0.01). And during this process, Wnt3a, ß-catenin, Runx2, and Bcl-2 expressions were significantly inhibited (p < 0.01); however, caspase-3 level was upregulated (p < 0.01). These results suggest that miR-503 plays a role in osteoblast proliferation and apoptosis in response to PBMT, which is potentially amenable to therapeutic manipulation for clinical application.

Pulsed electromagnetic fields promote bone formation by activating the sAC-cAMP-PKA-CREB signaling pathway.

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly.

Effect of low power laser in biomodulation of cultured osteoblastic cells of Wistar rats

Abstract Purpose: To analyze aspects of the biomodulating effect of light in biological tissues, bone cells from surgical explants of the femur of rats were irradiated with low intensity laser. Methods: Bone cells were cultured and irradiated with LASER light (GaAlAs). Growth, cell viability, mineralized matrix formation, total protein dosage, immunostimulatory properties, cytochemical analysis, gene expression of bone proteins were examined using live cell imaging and cell counting by colorimetric assay. The gene expression of: alkaline phosphatase (ALP), type 1 collagen, osteocalcin and osteopontin through the real-time polymerase chain reaction. Results: At 8 days, the viability of the irradiated culture was 82.3% and 72.4% in non-irradiated cells. At 18 days, the cellular viability (with laser) was 77.42% and 47.62% without laser. At 8 days, the total protein concentration was 21.622 mg / mol in the irradiated group and 16, 604 mg / mol in the non-irradiated group and at 18 days the concentration was 37.25 mg / mol in the irradiated group and 24, 95 mg / mol in the non-irradiated group. Conclusion: The laser interfered in the histochemical reaction, cell viability, matrix mineralization, and maintained the cellular expression of proteins

Pulsed electromagnetic fields prevented the decrease of bone formation in hindlimb-suspended rats by activating sAC/cAMP/PKA/CREB signaling pathway.

Microgravity is one of the main threats to the health of astronauts. Pulsed electromagnetic fields (PEMFs) have been considered as one of the potential countermeasures for bone loss induced by space flight. However, the optimal therapeutic parameters of PEMFs have not been obtained and the action mechanism is still largely unknown. In this study, a set of optimal therapeutic parameters for PEMFs (50 Hz, 0.6 mT 50% duty cycle and 90 min/day) selected based on high-throughput screening with cultured osteoblasts was used to prevent bone loss in rats induced by hindlimb suspension, a commonly accepted animal model to simulate the space environment. It was found that hindlimb suspension for 4 weeks led to significant decreases in femoral and vertebral bone mineral density (BMD) and their maximal loads, severe deterioration in bone micro-structure, and decreases in levels of bone formation markers and increases in bone resorption markers. PEMF treatment prevented about 50% of the decreased BMD and maximal loads, preserved the microstructure of cancellous bone and thickness of cortical bone, and inhibited decreases in bone formation markers. Histological analyses revealed that PEMFs significantly alleviated the reduction in osteoblast number and inhibited the increase in adipocyte number in the bone marrow. PEMFs also blocked decreases in serum levels of parathyroid hormone and its downstream signal molecule cAMP, and maintained the phosphorylation levels of protein kinase A (PKA) and cAMP response element-binding protein (CREB). The expression level of soluble adenylyl cyclases (sAC) was also maintained. It therefore can be concluded that PEMFs partially prevented the bone loss induced by weightless environment by maintaining bone formation through signaling of the sAC/cAMP/PKA/CREB pathway. Bioelectromagnetics. 39:569-584, 2018. © 2018 Wiley Periodicals, Inc.

Low-Dose Radiotherapy Ameliorates Advanced Arthritis in hTNF-α tg Mice by Particularly Positively Impacting on Bone Metabolism.

Inflammation and bone erosion are central in rheumatoid arthritis (RA). Even though effective medications for control and treatment of RA are available, remission is only seen in a subset of patients. Treatment with low-dose radiotherapy (LD-RT) which has been already successfully used for amelioration of symptoms in benign diseases should be a promising approach to reduce pain, inflammation, and particularly bone erosion in patients with RA. Even though anti-inflammatory effects of LD-RT are already described with non-linear dose response relationships, and pain-reducing effects have been clinically observed, the underlying mechanisms are widely unknown. Besides immune cells many other cell types, such as fibroblast-like synoviocytes (FLS), osteoclasts, and osteoblast are present in the affected joint and might be modulated by LD-RT. For this study, these cell types were obtained from human tumor necrosis factor-α transgenic (hTNF-α tg) mice and were consecutively exposed to different doses of ionizing radiation (0.1, 0.5, 1.0, and 2.0 Gy, respectively) in vitro. In order to study the in vivo effects of LD-RT within the arthritic joint, hind paws of arthritic hTNF-α tg mice were locally irradiated with 0.5 Gy, a single dose per fraction that is known for good clinical responses. Starting at a dose of 0.5 Gy, proliferation of FLS was reduced and apoptosis significantly enhanced with no changes in necrosis. Further, expression of RANK-L was slightly reduced following irradiation with particularly 0.5 Gy. Starting from 0.5 Gy, the numbers of differentiated osteoclasts were significantly reduced, and a lower bone resorbing activity of treated osteoclasts was also observed, as monitored via pit formation and Cross Laps presence. LD-RT had further a positive effect on osteoblast-induced mineralization in a discontinuous dose response relationship with 0.5 Gy being most efficient. An increase of the gene expression ratio of OPG/RANK-L at 0.1 and 0.5 Gy and of production of OPG at 0.5 and 1.0 Gy was observed. In vivo, LD-RT resulted in less severe arthritis in arthritic hTNF-α tg mice and in significant reduction of inflammatory and erosive area with reduced osteoclasts and neutrophils. Locally applied LD-RT can, therefore, induce a beneficial micro-environment within arthritic joints by predominantly positively impacting on bone metabolism.

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.

Dose analysis of photobiomodulation therapy on osteoblast, osteoclast, and osteocyte.

The objective of this study was to evaluate the effects of varying light doses on the viability and cellular activity of osteoblasts, osteocytes, and osteoclasts. A light application device was developed to apply 940-nm wavelength light from light-emitting diodes on three cultured cells, MC3T3-E1, MLO-A5, and RANKL-treated RAW264.7 cells. The doses (energy density) on cells were 0, 1, 5, and 7.5 J / cm2. The corresponding light power densities at the cell site were 0, 1.67, 8.33, and 12.5 mW / cm2, respectively, and the duration was 10 min. The results showed that the three cell types respond differently to light and their responses were dose dependent. Low-dose treatment (1 J / cm2) enhanced osteoblast proliferation, osteoclast differentiation, and osteoclastic bone resorption activity. Osteocyte proliferation was not affected by both low- and high-dose (5 J / cm2) treatments. While 1 J / cm2 did not affect viability of all three cell types, 5 J / cm2 significantly decreased viability of osteocytes and osteoclasts. Osteoblast viability was negatively impacted by the higher dose (7.5 J / cm2). The findings suggest that optimal doses exist for osteoblast and osteoclast, which can stimulate cell activities, and there is a safe dose range for each type of cell tested.

Low-level laser irradiation promotes the differentiation of bone marrow stromal cells into osteoblasts through the APN/Wnt/ß-catenin pathway.

OBJECTIVE: The relationship between adiponectin (APN) pathway and Wnt pathway was explored through BMSCs, and the effect of low-level laser irradiation (LLLI) on bone marrow stromal cells (BMSCs) and its mechanism were further studied. MATERIALS AND METHODS: 3-week-old Sprague-Dawley (SD) rats were selected, and mesenchymal stem cells were separately cultured and purified. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze cell proliferation. After osteogenic and adipogenic induction, cultures were conducted, respectively, cells were stained with alizarin red and oil red O. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expressions of osteogenesis-related genes, runt-related transcription factor 2 (RUNX2), and osteocalcin (OC) and those of adipogenesis-related genes, peroxisome proliferator-activated receptor-gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (c/EBPα). Western blotting was used to detect the expressions of ß-catenin in the cytoplasm and nucleus. The lentiviral expression vector of adiponectin receptors (APN-R) was constructed, and the expression of APN receptor genes was silenced. The expressions of ß-catenin in APN receptors and the nucleus within cells were detected. RESULTS: LLLI promoted the bone formation by inducing the differentiation direction of mesenchymal stem cells, increasing the number of osteoblasts in the bone marrow and inhibiting the reduction of the number of adipocytes. LLLI regulates the Wnt pathway, promotes the entry of ß-catenin into the nucleus, activates the osteogenic effect of the Wnt pathway so as to promote the bone formation of osteoblasts and inhibit bone resorption of osteoclasts. LLLI promotes the entry of ß-catenin into the nucleus and the osteogenic differentiation of BMSCs through the APN pathway. CONCLUSIONS: In summary, LLLI can promote osteogenesis and inhibit adipocytes formation, thus attenuating bone resorption of osteoclasts. The mechanism of LLLI is that it promotes the entry of ß-catenin into the nucleus and regulates the Wnt pathway and the differentiation direction of mesenchymal stem cells through the APN signal pathway, thus promoting bone formation.

The impact of photobiomodulation on osteoblast-like cell: a review.

In this study, we present a review of the literature on the impact of photobiomodulation on osteoblast-like cell culture. Searches were performed in the PubMed/MEDLINE (Medical Literature Analysis and Retrieval System Online), SCOPUS, and SPIE digital library databases for original articles regarding the effects of LLLT on osteoblast-like cells in experimental models using LLLT published in English from the last 20 years. The search identified 1439 studies. After the analysis of the abstracts, 1409 studies were excluded and 30 studies were then selected for the full-text analysis, 8 of which were excluded. Thus, 22 studies were included for a critical evaluation of the impact of photobiomodulation on osteoblast-like cell culture. The cell lineages studied were primary rat, primary human, saos-2, Osteo-1, MC3T3, MG63, and OFCOL II. Moreover, a wide variety of experimental models were used to experimentally analyze the impact of photobiomodulation, the most common of which were alkaline phosphatase, MTT, and cell count. This review suggests that osteoblastic-like cells are susceptible to photobiomodulation but that most of the light parameters varied by different authors have little to no influence on proliferation but very high levels of irradiance have demonstrated deleterious effects on proliferation, highlighting the bi-phasic effect of photobiomodulation.

Determination of Optimum Operation Parameters for Low-Intensity Pulsed Ultrasound and Low-Level Laser Based Treatment to Induce Proliferation of Osteoblast and Fibroblast Cells.

OBJECTIVE: The aim of this study was to determine the optimum operating parameters (pulse duration, energy levels, and application time) to promote induction of osteoblast and fibroblast cell proliferation and to maintain cell viability treated with low-intensity pulsed ultrasound (LIPUS) and low-level laser therapy (LLLT). BACKGROUND DATA: The positive effects of LIPUS and LLLT on cellular activity have been reported in recent years. Comparisons between experimental parameters of previous studies are difficult because scientific studies reported frequencies and the duty cycles of LIPUS and wavelengths and doses of LLLT in a wide range of parameters. However, optimum amount of energy and optimum time exposure must be determined to induce bone and tissue cell proliferation for effective healing process and to avoid cell damage. MATERIAL AND METHODS: Fibroblast and osteoblast cell cultures were irradiated with LIPUS (10-50% pulse and continuous mode at 1 and 3 MHz for 1, 3, and 5 min) and LLLT (4, 8, and 16 J at 50, 100, 200, 300, 400, and 500 mW). Cell cultures were analyzed using XTT assay. RESULTS: For both cell types, LIPUS treatment with 10% pulse (1:9 duty cycle), 3 MHz, and for 1 min and LLLT treatment over 100 mV for 4, 8, and 16 J modalities contributed to the growth, and may help bone repair and tissue healing process optimally. CONCLUSIONS: Bio-stimulating effects of LLLT irradiation promote proliferation and maintain cell viability better than LIPUS treatment without causing thermal response for both cell types, and the therapeutic modality above 200 mV has maximum effectiveness.

Exposure Duration Is a Determinant of the Effect of Sinusoidal Electromagnetic Fields on Peak Bone Mass of Young Rats.

We proposed a three-step strategy to obtain the optimal therapeutic parameters, which is composed of large-scale screening at cellular level, verification in animal experiments, and confirmation by a clinical trial. The objective of the current study was to test the feasibility of our strategy. Newborn rat calvarial osteoblasts were treated by 50 Hz 1.8 mT sinusoidal electromagnetic fields (SEMFs) with 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h/days, respectively. The osteogenic differentiation and maturation of the osteoblast were assayed and compared to obtain the optimal duration. One-month-old growing rats were then treated by the same SEMFs with 0.5, 1.5, and 2.5 h/days, respectively, and the peak bone mass was analyzed after 2 months. It was found that the optimal exposure duration to promote the osteogenic differentiation and maturation of osteoblasts was 1.5 h/days, judging by the increasing degrees of ALP activity, calcified nodules formed, the gene and protein expression levels of Runx-2, BMP-2, and Col-I, as well as the expression levels of signaling proteins of the BMP-2/Smad1/5/8 pathway. The highest increase of peak bone mass after 2 months was also obtained by 1.5 h/days, judging by the results of X-ray dual-energy absorptiometry, mechanical property analysis, micro-CT scanning, and serum bone turnover marker examinations. The above results indicated that exposure duration is a determinant for the therapeutic effect of EMFs, and the optimal therapeutic effects only can be obtained by the optimal exposure duration.

Low-level ultrahigh-frequency and ultrashort-pulse blue laser irradiation enhances osteoblast extracellular calcification by upregulating proliferation and differentiation via transient receptor potential vanilloid 1.

BACKGROUND AND OBJECTIVE: Low-level laser irradiation (LLLI) exerts various biostimulative effects, including promotion of wound healing and bone formation; however, few studies have examined biostimulation using blue lasers. The purpose of this study was to investigate the effects of low-level ultrahigh-frequency (UHF) and ultrashort-pulse (USP) blue laser irradiation on osteoblasts. STUDY DESIGN/ MATERIALS AND METHODS: The MC3T3-E1 osteoblast cell line was used in this study. Following LLLI with a 405 nm newly developed UHF-USP blue laser (80 MHz, 100 fs), osteoblast proliferation, and alkaline phosphatase (ALP) activity were assessed. In addition, mRNA levels of the osteoblast differentiation markers, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), and osteopontin (Opn) was evaluated, and extracellular calcification was quantified. To clarify the involvement of transient receptor potential (TRP) channels in LLLI-induced biostimulation, cells were treated prior to LLLI with capsazepine (CPZ), a selective inhibitor of TRP vanilloid 1 (TRPV1), and subsequent proliferation and ALP activity were measured. RESULTS: LLLI with the 405 nm UHF-USP blue laser significantly enhanced cell proliferation and ALP activity, compared with the non-irradiated control and LLLI using continuous-wave mode, without significant temperature elevation. LLLI promoted osteoblast proliferation in a dose-dependent manner up to 9.4 J/cm2 and significantly accelerated cell proliferation in in vitro wound healing assay. ALP activity was significantly enhanced at doses up to 5.6 J/cm2 , and expression of Osx and Alp mRNAs was significantly increased compared to that of the control on days 3 and 7 following LLLI at 5.6 J/cm2 . The extent of extracellular calcification was also significantly higher as a result of LLLI 3 weeks after the treatment. Measurement of TRPV1 protein expression on 0, 3, and 7 days post-irradiation revealed no differences between the LLLI and control groups; however, promotion of cell proliferation and ALP activity by LLLI was significantly inhibited by CPZ. CONCLUSION: LLLI with a 405 nm UHF-USP blue laser enhances extracellular calcification of osteoblasts by upregulating proliferation and differentiation via TRPV1. Lasers Surg. Med. 50:340-352, 2018. © 2017 Wiley Periodicals, Inc.