Photosynthesis and calcification of the coccolithophore Emiliania huxleyi are more sensitive to changed levels of light and CO2 under nutrient limitation.

Photophysiological responses of phytoplankton to changing multiple environmental drivers are essential in understanding and predicting ecological consequences of ocean climate changes. In this study, we investigated the combined effects of two CO2 levels (410 and 925 µatm) and five light intensities (80 to 480 µmol photons m-2 s-1) on cellular pigments contents, photosynthesis and calcification of the coccolithophore Emiliania huxleyi grown under nutrient replete and limited conditions, respectively. Our results showed that high light intensity, high CO2 level and nitrate limitation acted synergistically to reduce cellular chlorophyll a and carotenoid contents. Nitrate limitation predominantly enhanced calcification rate; phosphate limitation predominantly reduced photosynthetic carbon fixation rate, with larger extent of the reduction under higher levels of CO2 and light. Reduced availability of both nitrate and phosphate under the elevated CO2 concentration decreased saturating light levels for the cells to achieve the maximal relative electron transport rate (rETRmax). Light-saturating levels for rETRmax were lower than that for photosynthetic and calcification rates under the nutrient limitation. Regardless of the culture conditions, rETR under growth light levels correlated linearly and positively with measured photosynthetic and calcification rates. Our findings imply that E. huxleyi cells acclimated to macro-nutrient limitation and elevated CO2 concentration decreased their light requirement to achieve the maximal electron transport, photosynthetic and calcification rates, indicating a photophysiological strategy to cope with CO2 rise/pH drop in shoaled upper mixing layer above the thermocline where the microalgal cells are exposed to increased levels of light and decreased levels of nutrients.

Growth, calcification, and photobiology of the threatened coral Acropora cervicornis in natural versus artificial light.

Land-based coral culture is of increasing interest for conservation and educational display. Shallow water corals generate most of their energy from photosynthesis, and light is a critical abiotic factor in their husbandry. We compared growth, calcification, and photobiology in the coral Acropora cervicornis between natural and artificial (light-emitting diode; LED) light to better understand the impact of light source on coral performance. One tank of a greenhouse recirculating system at The Florida Aquarium's Center for Conservation was used to culture replicate coral colonies. Half of the tank and corals were covered to block sunlight and illuminated with a commercial reef aquarium LED fixture, while the other half was exposed to natural sunlight. Treatments were matched in terms of maximum photosynthetically active radiation and spectral measurements characterized both light regimes. Coral growth and calcification were tracked over a period of 19 weeks by repeated measurements of total linear extension (TLE) and buoyant weight. For the first 5 weeks, photosynthetic yield was measured weekly using a pulse-amplitude-modulated fluorometer. Calcification was significantly higher under LED lighting relative to natural light, but TLE did not differ. Photobiology data suggest that corals in both treatments were acclimated to the same light level, but photosynthetic efficiency was ultimately greater in the natural light treatment. More consistent light delivery and different spectral composition under LED treatment conditions may explain the incongruity between calcification and photosynthetic efficiency. This experiment informs husbandry of shallow-water scleractinian corals maintained in both natural sunlight and enclosed structures.

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.

Irradiation by high-intensity red light-emitting diode enhances human bone marrow mesenchymal stem cells osteogenic differentiation and mineralization through Wnt/ß-catenin signaling pathway.

Photobiomodulation therapy (PBMT) using a light-emitting diode (LED) has been employed for various photomedicine studies. The aim of this study was to determine the effects of a high-intensity red LED on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) and the related mechanism. BMSCs were subjected to high-intensity red LED (LZ1-00R205 Deep Red LED) irradiations for 0 to 40 s with energy densities ranging from 0 to 8 J/cm2. The distance from the LED to the cell layer was 40 mm. The spot size on the target was 4 cm2. Cell proliferation was measured at 3, 24, 48, and 72 h. The effects of LED irradiation on osteogenic differentiation and mineralization were examined with a particular focus on the Wnt/ß-catenin signaling pathway. The high-intensity red LED irradiations did not alter BMSC proliferation after 72 h. LED exposure of 6 J/cm2 (30 s) led to significant enhancements of osteogenic differentiation and mineralization. Additionally, the high-intensity LED irradiation induced activation of Wnt/ß-catenin. The effects of the high-intensity LED irradiation on BMSC osteogenic differentiation and mineralization were suppressed by treatment with the Wnt/ß-catenin inhibitor XAV939. P < 0.05 was considered significant. The results indicate that high-intensity red LED irradiation increases BMSC osteogenic differentiation and mineralization via Wnt/ß-catenin activation. Therefore, short duration irradiation with a portable high-intensity LED may be used as a potential approach in hard tissue regeneration therapy.

Bone marrow coagulated and low-level laser therapy accelerate bone healing by enhancing angiogenesis, cell proliferation, osteoblast differentiation, and mineralization.

The present study evaluated bone marrow aspirate (BMA) and low-level laser therapy (LLLT) on bone healing. It was created critical-size defects (CSD) of 5 mm diameter in rat calvaria of 64 rats. Animals were randomly divided into four groups: Control (blood clot), BMA (coagulated BMA), LLLT (laser irradiation and blood clot), and BMA/LLLT (laser irradiation and coagulated BMA). Euthanasia was performed at 15 or 30 days postoperative. Immunohistochemical reactions were performed to identify vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). The markers were quantified, and data were statistically analyzed. Groups BMA/LLLT and LLLT presented significantly higher VEGF expression than group control. Group BMA/LLLT presented a significantly higher expression of PCNA than all experimental groups. Groups BMA and BMA/LLLT presented significantly higher expression of BMP-2 than all experimental groups. Groups LLLT and BMA/LLLT presented significantly higher expression of OPN than groups control and BMA. Groups LLLT, BMA, and BMA/LLLT presented a significantly higher expression of OCN than group control. It can be concluded that the association of BMA and LLLT enhanced bone healing by improving expression of VEGF, PCNA, Runx2, BMP-2, OPN, and OCN.

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.

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.

Environmental influence on calcification of the bivalve Chamelea gallina along a latitudinal gradient in the Adriatic Sea.

Environmental factors are encoded in shells of marine bivalves in the form of geochemical properties, shell microstructure and shell growth rate. Few studies have investigated how shell growth is affected by habitat conditions in natural populations of the commercial clam Chamelea gallina. Here, skeletal parameters (micro-density and apparent porosity) and growth parameters (bulk density, linear extension and net calcification rates) were investigated in relation to shell sizes and environmental parameters along a latitudinal gradient in the Adriatic Sea (400 km). Net calcification rates increased with increasing solar radiation, sea surface temperature and salinity and decreasing Chlorophyll concentration in immature and mature shells. In immature shells, which are generally more porous than mature shells, enhanced calcification was due to an increase in bulk density, while in mature shells was due to an increase in linear extension rates. The presence of the Po river in the Northern Adriatic Sea was likely the main driver of the fluctuations observed in environmental parameters, especially salinity and Chlorophyll concentration, and seemed to negatively affect the growth of C. gallina.

Irradiation by blue light-emitting diode enhances osteogenic differentiation in gingival mesenchymal stem cells in vitro.

The aim of this study was to investigate the effects of blue light irradiation on the process of osteogenic differentiation in stem cells. The cells used in this study were derived from human gingival mesenchymal stem cells (hGMSCs), and were treated with 0 (control group), 1, 2, 4 or 6 J/cm2 blue light using blue light-emitting diodes. Cell growth was assessed by the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-Diphenyl-2H-tetrazolium bromide (MTT) cell proliferation assay and osteogenic differentiation was evaluated by monitoring alkaline phosphatase (ALP) activity, alizarin red staining and real-time PCR (RT-PCR). The results of the MTT assay indicated that blue light inhibited hGMSC proliferation, and the ALP and alizarin red results showed that blue light promoted osteogenesis. The expression levels of the osteogenic genes runt-related transcription factor2 (Runx2), collagen type I (Col1) and osteocalcin (OCN) increased significantly (P < 0.05) when cells were irradiated with 2 or 4 J/cm2 of blue light. In conclusion, irradiation with blue light inhibits the proliferation of hGMSC and promotes osteogenic differentiation.

Vulnerability of juvenile hermit crabs to reduced seawater pH and shading.

Multiple simultaneous stressors induced by anthropogenic activities may amplify their impacts on marine organisms. The effects of ocean acidification, in combination with other anthropogenic impacts (apart from temperature) are poorly understood, especially in coastal regions. In these areas, shading caused by infrastructure development, such as harbor construction, may potentially interact with CO2-induced pH reduction and affect invertebrate populations. Here, we evaluated the effects of reduced pH (7.6) and shading (24h in darkness) on mortality, growth, calcification and displacement behavior to live predator (danger signal) and dead gastropod (resource availability signal) odors using juveniles of the hermit crab Pagurus criniticornis collected in Araçá Bay (São Paulo state, Southeastern Brazil). After a 98 day experimental period, both stressors had a significant interaction effect on mortality, and an additive effect on total growth. No difference in calcification was recorded among treatments, indicating that individuals were able to maintain calcification under reduced pH conditions. When exposed to odor of live predators, crab responses were only affected by shading. However, an interactive effect between both stressors was observed in response to gastropod odor, leading to reduced displacement behavior. This study shows how local disturbance impacts may enhance the effects of global environmental change on intertidal crustacean populations.

Red (635 nm), Near-Infrared (808 nm) and Violet-Blue (405 nm) Photobiomodulation Potentiality on Human Osteoblasts and Mesenchymal Stromal Cells: A Morphological and Molecular In Vitro Study.

Photobiomodulation (PBM) has been used for bone regenerative purposes in different fields of medicine and dentistry, but contradictory results demand a skeptical look for its potential benefits. This in vitro study compared PBM potentiality by red (635 ± 5 nm) or near-infrared (NIR, 808 ± 10 nm) diode lasers and violet-blue (405 ± 5 nm) light-emitting diode operating in a continuous wave with a 0.4 J/cm² energy density, on human osteoblast and mesenchymal stromal cell (hMSC) viability, proliferation, adhesion and osteogenic differentiation. PBM treatments did not alter viability (PI/Syto16 and MTS assays). Confocal immunofluorescence and RT-PCR analyses indicated that red PBM (i) on both cell types increased vinculin-rich clusters, osteogenic markers expression (Runx-2, alkaline phosphatase, osteopontin) and mineralized bone-like nodule structure deposition and (ii) on hMSCs induced stress fiber formation and upregulated the expression of proliferation marker Ki67. Interestingly, osteoblast responses to red light were mediated by Akt signaling activation, which seems to positively modulate reactive oxygen species levels. Violet-blue light-irradiated cells behaved essentially as untreated ones and NIR irradiated ones displayed modifications of cytoskeleton assembly, Runx-2 expression and mineralization pattern. Although within the limitations of an in vitro experimentation, this study may suggest PBM with 635 nm laser as potential effective option for promoting/improving bone regeneration.

Single vs. multiple fraction regimens for palliative radiotherapy treatment of multiple myeloma : A prospective randomised study.

PURPOSE: To compare the impact of a single fraction (8 Gy × 1 fraction) and multifraction (3 Gy × 10 fractions) radiotherapy regimens on pain relief, recalcification and the quality of life (QoL) in patients with bone destructions due to multiple myeloma (MM). PATIENTS AND METHODS: In all, 101 patients were included in a randomised prospective clinical trial: 58 patients were included in the control arm (3 Gy × 10 fractions) and 43 patients into the experimental arm (8 Gy × 1 fraction). The response rate was defined according to the International Consensus on Palliative Radiotherapy criteria. Recalcification was evaluated with radiographs. QoL questionnaires were completed before and 4 weeks after treatment. RESULTS: Pain relief was obtained in 81/101 patients (80.2%): complete response in 56 (69%) and partial in 25 patients (30.9%). No significant differences were observed in analgesic response between the groups. Significant factors for pain relief were female gender, age under 65, IgG MM type, presence of recalcification at the irradiated site. Recalcification was found in 32/101 patients (33.7%): complete in 17 (53.2%) and partial in 15 (46.2%). No significant differences were observed in recalcification between the groups. Significant factors for recalcification were Karnofsky index ≥ 60%, haemoglobin level ≤ 80 g/dl, MM stage II and analgesic response at the irradiated site. The QoL after radiotherapy was improved in the control group. CONCLUSION: The same analgesic and recalcification response was observed using two different radiotherapy regimens. Higher doses should be used to achieve a better QoL.

Differences in responses to X-ray exposure between osteoclast and osteoblast cells.

Radiation-induced bone loss is a potential health concern for cancer patients undergoing radiotherapy. Enhanced bone resorption by osteoclasts and decreased bone formation by osteoblasts were thought to be the main reasons. In this study, we showed that both pre-differentiating and differentiating osteoclasts were relatively sensitive to X-rays compared with osteoblasts. X-rays decreased cell viability to a greater degree in RAW264.7 cells and in differentiating cells than than in osteoblastic MC3T3-E1 cells. X-rays at up to 8 Gy had little effects on osteoblast mineralization. In contrast, X-rays at 1 Gy induced enhanced osteoclastogenesis by enhanced cell fusion, but had no effects on bone resorption. A higher dose of X-rays at 8 Gy, however, had an inhibitory effect on bone resorption. In addition, actin ring formation was disrupted by 8 Gy of X-rays and reorganized into clusters. An increased activity of Caspase 3 was found after X-ray exposure. Actin disorganization and increased apoptosis may be the potential effects of X-rays at high doses, by inhibiting osteoclast differentiation. Taken together, our data indicate high radiosensitivity of osteoclasts. X-ray irradiation at relatively low doses can activate osteoclastogenesis, but not osteogenic differentiation. The radiosensitive osteoclasts are the potentially responsive cells for X-ray-induced bone loss.

Indirect effects of X-irradiation on proliferation and osteogenic potential of bone marrow mesenchymal stem cells in a local irradiated rat model.

Cancer survivors after radiotherapy may suffer a variety of bone­related adverse side effects, including radioactive osteoporosis and fractures. Localized irradiation is a common treatment modality for malignancies. Recently, a series of reactions and injuries called indirect effects (remote changes in bone when other parts of the body are irradiated) have been reported on the indirect irradiated area of bone tissue after radiotherapy. To address this issue, we developed a rat localized irradiation model. Rats were irradiated with a single dose of X-rays to the left hind limbs, and bone marrow mesenchymal stem cells (BMMSCs) were isolated from bone marrow of the left (direct irradiated) and right (indirect irradiated) hind limbs 3, 7 and 14 days after irradiation, and assayed for the proliferation ability and osteogenic potential by alkaline phosphatase (ALP) activity, mineralization assay, RT­PCR and western blot analysis. The results showed that there were significant morphology changes in the BMMSCs from direct and indirect irradiated bone tissue with bigger cell bodies and increased granules. The proliferation of BMMSCs decreased both in the direct irradiated and non­irradiated bone tissue. The ALP expression and activities of BMMSCs from direct irradiated bone was consistently defected following a transient enhancement, the mRNA levels of RUNX2 and OCN, the protein expression of RUNX2, and the mineralization ability also showed the same trend. Simultaneously, in indirect irradiated group, the osteogenic potential indicators of BMMSCs decreased in the early stage of post­irradiation and were still impaired 14 days after irradiation. Our data demonstrate that localized irradiation may have both direct and indirect adverse effects on BMMSCs' proliferation and osteogenic potential into osteoblast, which may be the mechanism of radiation-induced abscopal impairment to the skeleton in the cancer radiotherapy-induced bone loss.

Biostimulation with diode laser positively regulates cementoblast functions, in vitro.

The aim of this study was to evaluate the effects of diode laser biostimulation on cementoblasts (OCCM.30). A total of 40 root plates were obtained from healthy third molar teeth and assigned to the following two groups: (1) control group and (2) laser-treated group. Root plates were placed into the cell culture inserts, and OCCM.30 cells were seeded onto root plates. Cells were irradiated with a low level of diode laser (power: 0.3 W in continuous wave, 60 s/cm2). Proliferation and mineralized tissue-associated gene's and BMP's messenger RNA (mRNA) expressions of cementoblasts were evaluated. Total RNAs were isolated on day 3 and integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap), Type I collagen (Col1a1), osteoblastic transcription factor, runt-related transcription factor (Runx2), and Bone Morphogenetic Protein (BMP)-2, 3, 4, 6, and 7 mRNA expressions were determined using quantitative RT-PCR. von Kossa staining was performed to evaluate biomineralization of OCCM.30 cells. In the proliferation experiment, while there was no significant difference until 96 h, laser irradiation retarded the decrease in cell proliferation trend after 96 h compared to the untreated control group. Statistically significant increase in Ibsp, Bglap, and BMP-2,3,6,7 mRNA expressions were noted in the laser groups when compared to the untreated control group (p < 0.05). Laser irradiation induced mineralized nodule formation of cementoblasts. The results of this study reveal that the biostimulation setting of diode laser modulates the behavior of cementoblasts inducing mineralized tissue-associated gene's mRNA expressions and mineralization. Therefore, biostimulation can be used during regenerative periodontal therapies to trigger cells with periodontal attachment apparatus.

Investigation of photobiomodulation potentiality by 635 and 809 nm lasers on human osteoblasts.

Photobiomodulation (PBM) describes light-induced photochemical reactions achieved by the application of red or near infrared lasers/LED light with low energy densities. This noninvasive and painless method has been used in some clinical areas but controversial outcomes demand a skeptical look for its promising and potential effects. In this detailed in vitro study, the osteoblast cells were irradiated with 635 and 809 nm diode lasers at energy densities of 0.5, 1, and 2 J/cm2. Cell viability, proliferation, bone formation, and osteoblast differentiation were evaluated by methylthiazole tetrazolium (MTT) assay, Alamar Blue assay, acridine orange/propidium iodide staining, alkaline phosphatase (ALP) activity, Alizarin red staining, and reverse-transcription polymerase chain reaction (RT-PCR) to test the expression of collagen type I, ALPL, and osteocalcin. The results indicate that studied energy doses have a transient effect (48 h after laser irradiation) on the osteoblast viability and proliferation. Similarly, laser irradiation did not appear to have any effect on ALP activity. These results were confirmed by RT-PCR analysis of osteoblast markers. This study suggests that several irradiation parameters and variations in the methods should be clearly established in the laboratory before laser treatment becomes a postulated application for bone tissue regeneration in clinical level.

Photothermal effects of laser-activated surface plasmonic gold nanoparticles on the apoptosis and osteogenesis of osteoblast-like cells.

The specific properties of gold nanoparticles (AuNPs) make them a novel class of photothermal agents that can induce cancer cell damage and even death through the conversion of optical energy to thermal energy. Most relevant studies have focused on increasing the precision of cell targeting, improving the efficacy of energy transfer, and exploring additional functions. Nevertheless, most cells can uptake nanosized particles through nonspecific endocytosis; therefore, before hyperthermia via AuNPs can be applied for clinical use, it is important to understand the adverse optical-thermal effects of AuNPs on nontargeted cells. However, few studies have investigated the thermal effects induced by pulsed laser-activated AuNPs on nearby healthy cells due to nonspecific treatment. The aim of this study is to evaluate the photothermal effects induced by AuNPs plus a pulsed laser on MG63, an osteoblast-like cell line, specifically examining the effects on cell morphology, viability, death program, and differentiation. The cells were treated with media containing 50 nm AuNPs at a concentration of 5 ppm for 1 hour. Cultured cells were then exposed to irradiation at 60 mW/cm(2) and 80 mW/cm(2) by a Nd:YAG laser (532 nm wavelength). We observed that the cytoskeletons of MG63 cells treated with bare AuNPs followed by pulsed laser irradiation were damaged, and these cells had few bubbles on the cell membrane compared with those that were not treated (control) or were treated with AuNPs or the laser alone. There were no significant differences between the AuNPs plus laser treatment group and the other groups in terms of cell viability, death program analysis results, or alkaline phosphatase and calcium accumulation during culture for up to 21 days. However, the calcium deposit areas in the cells treated with AuNPs plus laser were larger than those in other groups during the early culture period.

Low intensity lasers differently induce primary human osteoblast proliferation and differentiation.

Among various compounds used in research and clinic for degenerative bone diseases, low level laser therapy (LLLT), comprising low level lasers (LLL) and light emitting diodes (LEDs), has been investigated regarding its effects on bone metabolism. They have specific wavelengths but in general act as a cellular biomodulator, and as a therapeutic agent, rebalancing and normalizing their activity. However, they are not standardized yet, since their parameters of use are relevant for the effects and mechanisms of action. Therefore, the aim of this study was to compare the influence of two spectrums of LLL and LED phototherapy, at the same energy densities (10 and 50J/cm(2)), on human osteoblasts proliferation and differentiation. The involvement of ERK signaling on proliferation was also investigated by evaluating its activation during proliferation under different phototherapies by western blotting and CFSE-based osteoblast proliferation was measured in a presence or absence of the ERK-specific inhibitor. Osteogenic differentiation was evaluated through in vitro mineralization and gene expression of type I collagen (COL1A1) and osteonectin (SPARC) by Real Time- PCR. Increases in viable cells and proliferation were obtained after irradiation, regardless of LLLT type. However, only red at 10J/cm(2) and infrared at both doses, but not LED, induced ERK1/2 activation. In the presence of ERK inhibitor, the LLL-induced proliferation was prevented. In addition, while COL1A1 gene expression was upregulated by red laser, SPARC does so by infrared stimulation. However, LED, at both doses, increased both COL1A1 and SPARC expression. All LLLT increased mineralization, dependent on the dose and time. Thus, LLL and LED differently modulated the metabolism of human osteoblasts, increasing proliferation by mechanism dependent or not of ERK signaling activation and osteogenic differentiation markers.

Comparison between one-session root canal treatment with aPDT and two-session treatment with calcium hydroxide-based antibacterial dressing, in dog's teeth with apical periodontitis.

To evaluate one-session endodontic treatment with aPDT and two-session treatment with calcium hydroxide (CH)-based dressing in dog's teeth with apical periodontitis. After experimental induction of apical periodontitis, 48 teeth were randomly assigned to the following groups: groups OS/aPDT120d and OS/aPDT180d (one-session treatment with aPDT) and groups TS/CH120d and TS/CH180d (two-session treatment with CH-based dressing-control groups). The animals were euthanized after 120 and 180 days. After histotechnical processing, microscopic and radiographic analyses were performed. Data were analyzed by Kruskal-Wallis and Fisher's exact tests (α = 0.05). Groups TS/CHs presented repaired resorbed cemental areas, with collagen bundles and few inflammatory cells. In groups OS/aPDTs, the areas of cemental resorption were not repaired with reduced presence of cells and fibers. In the analysis of the apical closure, fluorescence microscopy and percentage of radiographic reduction of lesions, there was significant difference between groups TS/CH120d and OS/aPDT120d and between TS/CH180d and OS/aPDT180d (p < 0.05). Groups TS/CHs had weak RANKL expression and positive immunostaining for RANK and OPG. In OS/aPDT120d, there was positive immunostaining for RANKL. In OS/aPDT180d, the three osteoclastogenesis markers were expressed. The results using aPDT were worse than those obtained with two-session endodontic treatment using a CH-based dressing in teeth with apical periodontitis.

Parathyroid hormone attenuates radiation-induced increases in collagen crosslink ratio at periosteal surfaces of mouse tibia.

As part of our ongoing efforts to understand underlying mechanisms contributing to radiation-associated bone fragility and to identify possible treatments, we evaluated the longitudinal effects of parathyroid hormone (PTH) treatment on bone quality in a murine model of limited field irradiation. We hypothesized PTH would mitigate radiation-induced changes in the chemical composition and structure of bone, as measured by microscope-based Raman spectroscopy. We further hypothesized that collagen crosslinking would be especially responsive to PTH treatment. Raman spectroscopy was performed on retrieved tibiae (6-7/group/time point) to quantify metrics associated with bone quality, including: mineral-to-matrix ratio, carbonate-to-phosphate ratio, mineral crystallinity, collagen crosslink (trivalent:divalent) ratio, and the mineral and matrix depolarization ratios. Irradiation disrupted the molecular structure and orientation of bone collagen, as evidenced by a higher collagen crosslink ratio and lower matrix depolarization ratio (vs. non-irradiated control bones), persisting until 12weeks post-irradiation. Radiation transiently affected the mineral phase, as evidenced by increased mineral crystallinity and mineral-to-matrix ratio at 4weeks compared to controls. Radiation decreased bone mineral depolarization ratios through 12weeks, indicating increased mineral alignment. PTH treatment partially attenuated radiation-induced increases in collagen crosslink ratio, but did not restore collagen or mineral alignment. These post-radiation matrix changes are consistent with our previous studies of radiation damage to bone, and suggest that the initial radiation damage to bone matrix has extensive effects on the quality of tissue deposited thereafter. In addition to maintaining bone quality, preventing initial radiation damage to the bone matrix (i.e. crosslink ratio, matrix orientation) may be critical to preventing late-onset fragility fractures.