The effect of continuous long-term illumination with visible light in different spectral ranges on mammalian cells.

One of the biggest challenges in tissue engineering and regenerative medicine is to ensure oxygen supply of cells in the (temporary) absence of vasculature. With the vision to exploit photosynthetic oxygen production by microalgae, co-cultivated in close vicinity to oxygen-consuming mammalian cells, we are searching for culture conditions that are compatible for both sides. Herein, we investigated the impact of long-term illumination on mammalian cells which is essential to enable photosynthesis by microalgae: four different cell types-primary human fibroblasts, dental pulp stem cells, and osteoblasts as well as the murine beta-cell line INS-1-were continuously exposed to warm white light, red or blue light over seven days. We observed that illumination with red light has no adverse effects on viability, metabolic activity and growth of the cells whereas exposure to white light has deleterious effects that can be attributed to its blue light portion. Quantification of intracellular glutathione did not reveal a clear correlation of this effect with an enhanced production of reactive oxygen species. Finally, our data indicate that the cytotoxic effect of short-wavelength light is predominantly a direct effect of cell illumination; photo-induced changes in the cell culture media play only a minor role.

A Numerical Study on Mechanical Effects of Low-Intensity Pulsed Ultrasound on Trabecular Bone and Osteoblasts.

The lack of sufficient mechanical stimulation to the human bone, results in disuse osteoporosis. Low-intensity pulsed ultrasound (LIPUS) promotes fracture healing and the treatment of disuse osteoporosis, but its biomechanical mechanism remains unknown. Simulative research on the mechanical effects of LIPUS on disuse trabecular bone and osteoblasts have been performed. The von Mises stress of disuse trabecular bone and osteoblasts obviously increased under LIPUS irradiation. The average von Mises stress of osteoblasts were two orders of magnitude higher under the irradiation of simulant LIPUS than that without LIPUS irradiation, and the von Mises stress of osteoblasts was positively correlated with the amplitude of sound pressure excitation. The results showed that LIPUS irradiation could obviously improve the mechanical micro-environment of trabecular bone and osteoblasts to alleviate the lack of mechanical stimulation. The results of the research can reveal the biomechanical mechanism of LIPUS in the treatment of disuse osteoporosis to some extent and provide theoretical guidance for clinical treatment of disuse osteoporosis through physical methods.

Systematic Review on Multilevel Analysis of Radiation Effects on Bone Microarchitecture.

Introduction: Modern radiation therapy has become an effective method to treat and monitor tumour growth in cancer patients. It has proved to be a successful way to minimise mortality rates. However, the adverse effects of radiation have been historical evidence in the clinical environment involving diminishing the quality and density of bone and causing fragility fracture to the bone in the long run. This systematic review was aimed at identifying and evaluating the effects of irradiation on morphology and mechanical properties of murine model bone in previous publications. Methods: A systematic literature review was undertaken following the Preferred Reporting Items for Systemic Reviews and Meta-analysis (PRISMA) guidelines. A comprehensive literature search was performed using Scopus, Web of Science, and Science Direct databases (English only studies published between 2015 and 2020). The selected studies were evaluated according to three criteria: (1) criteria for study sample selection; (2) criteria for methodological procedures; and (3) criteria for detection and evaluation. Results: The initial search strategy identified 1408 related studies, 8 of were included based on inclusion and exclusion criteria. This review revealed an association between bone destruction and the magnitude of time and dose postirradiation. We agreed that the effect of radiation on bone morphology and strength primarily is a later stage event but noticeable in both low (1 Gy) and high dose (30 Gy) radiation. Trabecular and cortical bone microstructures were significantly altered at irradiation and contralateral sites. Besides, the mechanical strength was significantly impacted in both shorter and longer periods. Conclusion: Overall, the radiotherapy altered bone microstructures and substantially decreases bone mechanical properties. The alteration was related to quantity and the activity of the osteoblast and osteoclast. Early detection of those most at risk for radiation-induced bone alterations could lead to better prophylactic intervention decisions.

Evaluation of osteoblastic cell behavior upon culture on titanium substrates photo-functionalized by vacuum ultra-violet treatment.

Photo-functionalization of titanium orthopedic/prosthetic implants using ultraviolet illumination is known to improve osteogenesis. Therefore, in this study, we aimed to examine the influence of vacuum ultraviolet (VUV)-treated titanium surfaces on osteoblast cell adhesion, activity, and differentiation. Osteoblastic cells were cultured on titanium substrates treated with various VUV treatment conditions (0, 6.2, 18.7, and 37.4 J/cm2) and their behavior was evaluated. The results revealed that cell adhesion was increased whereas cell activity and differentiation ability were decreased upon cell culture on VUV-treated substrates. In particular, cell activity and differentiation ability were dramatically suppressed with 18.7 J/cm2 VUV irradiation. Within the limitations of this cell-based experiment, we clarified the VUV treatment conditions in which cell adhesion was improved but cell activity and differentiation ability were suppressed. These results indicate that VUV-treatment can be used to influence cell growth properties and can be used to accelerate or suppress cell differentiation on implant substrates.

635 nm LED irradiation may prevent endoplasmic reticulum stress in MC3T3-E1 cells.

Although endoplasmic reticulum (ER) stress is thought to be involved in various diseases such as cancer, metabolic, and inflammatory disorders, the relationship between ER stress and bone diseases, are remains unclear. Tunicamycin-treated MC3T3-E1 osteoblasts were used as the ER stress model in this study. 635 nm light-emitting diode irradiation (635 nm-IR) was carried out for 1 h before and after inducing ER stress. To investigate the effects of 635 nm-IR on ER stress-induced MC3T3-E1 osteoblasts and the underlying mechanism, western blot, reverse transcription polymerase chain reaction, alkaline phosphatase and Alizarin red staining, 2',7'-dichlorodyhydrofluorescein diacetate assay, Fluo-3AM and immunocytochemistry were performed. Pretreatment with 635 nm-IR effectively prevented intracellular reactive oxygen species production and alleviated ER stress through the pancreatic ER kinase (PERK)-eukaryotic initiation factor 2 (eIF2)-activating transcription factor 4 (ATF4)-nuclear factor-like 2 (Nrf2) signaling pathway. Hence, 635 nm-IR may serve a protective role in the treatment of ER stress-related bone diseases.

Effect of High Static Magnetic Fields on Biological Activities and Iron Metabolism in MLO-Y4 Osteocyte-like Cells.

There are numerous studies that investigate the effects of static magnetic fields (SMFs) on osteoblasts and osteoclasts. However, although osteocytes are the most abundant cell type in bone tissue, there are few studies on the biological effects of osteocytes under magnetic fields. Iron is a necessary microelement that is involved in numerous life activities in cells. Studies have shown that high static magnetic fields (HiSMF) can regulate cellular iron metabolism. To illustrate the effect of HiSMF on activities of osteocytes, and whether iron is involved in this process, HiSMF of 16 tesla (T) was used, and the changes in cellular morphology, cytoskeleton, function-related protein expression, secretion of various cytokines, and iron metabolism in osteocytes under HiSMF were studied. In addition, the biological effects of HiSMF combined with iron preparation and iron chelator on osteocytes were also investigated. The results showed that HiSMF promoted cellular viability, decreased apoptosis, increased the fractal dimension of the cytoskeleton, altered the secretion of cytokines, and increased iron levels in osteocytes. Moreover, it was found that the biological effects of osteocytes under HiSMF are attenuated or enhanced by treatment with a certain concentration of iron. These data suggest that HiSMF-regulated cellular iron metabolism may be involved in altering the biological effects of osteocytes under HiSMF exposure.

Pilot investigation on the dose-dependent impact of irradiation on primary human alveolar osteoblasts in vitro.

Radiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

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.

The small protein MafG plays a critical role in MC3T3-E1 cell apoptosis induced by simulated microgravity and radiation.

Microgravity and radiation exposure-induced bone damage is one of the most significant alterations in astronauts after long-term spaceflight. However, the underlying mechanism is still largely unknown. Recent ground-based simulation studies have suggested that this impairment is likely mediated by increased production of reactive oxygen species (ROS) during spaceflight. The small Maf protein MafG is a basic-region leucine zipper-type transcription factor, and it globally contributes to regulation of antioxidant and metabolic networks. Our research investigated the role of MafG in the process of apoptosis induced by simulated microgravity and radiation in MC3T3-E1 cells. We found that simulated microgravity or radiation alone decreased MafG expression and elevated apoptosis in MC3T3-E1 cells, and combined simulated microgravity and radiation treatment aggravated apoptosis. Meanwhile, under normal conditions, increased ROS levels facilitated apoptosis and downregulated the expression of MafG in MC3T3-E1 cells. Overexpression of MafG decreased apoptosis induced by simulated microgravity and radiation. These findings provide new insight into the mechanism of bone damage induced by microgravity and radiation during space flight.

0.5­Gy X­ray irradiation induces reorganization of cytoskeleton and differentiation of osteoblasts.

Osteoblasts are sensitive to ionizing radiation. The small GTPase RhoA and its effector Rho­associated protein kinase (ROCK) are critical to several cellular functions, including cytoskeleton reorganization, cell survival, and cell differentiation. However, whether the RhoA/ROCK signaling pathway is involved in the regulation of osteoblast cytoskeleton reorganization and differentiation induced by low­dose X­ray irradiation remains to be determined. The aim of the present study was to investigate the role of the RhoA/ROCK signaling pathway in mediating differentiation of osteoblasts and reorganization of the cytoskeleton under low­dose X­ray irradiation. Osteoblasts were pretreated with the ROCK kinase­specific inhibitor (Y­27632) before exposure to low­dose X­ray irradiation. The changes of F­actin in MC3T3 cells were observed at different time points following X­ray irradiation. Cell Counting Kit­8 assay, alkaline phosphatase activity, Alizarin red staining and western blotting were used to detect the proliferation and differentiation of osteoblasts after 0.5­Gy X­ray irradiation. In the present study, low­dose X­ray irradiation promoted the expression of genes associated with the cytoskeleton reorganization. Indeed, the results showed that, 0.5­Gy X­ray irradiation can induce reorganization of cytoskeleton and promote differentiation of osteoblasts through the RhoA/ROCK signaling pathway. Additionally, inhibiting ROCK activity blocked low­dose X­ray irradiation­induced LIMK2 phosphorylation, stress fiber formation and cell differentiation. Thus, these results demonstrated the excitatory effects of low­dose X­ray irradiation on MC3T3­E1 cells, including reorganization of the cytoskeleton and differentiation of osteoblasts.

Pulsed electromagnetic field (PEMF) transiently stimulates the rate of mineralization in a 3-dimensional ring culture model of osteogenesis.

Pulsed Electromagnetic Field (PEMF) has shown efficacy in bone repair and yet the optimum characteristics of this modality and its molecular mechanism remain unclear. To determine the effects of timing of PEMF treatment, we present a novel three-dimensional culture model of osteogenesis that demonstrates strong de novo generation of collagen and mineral matrix and exhibits stimulation by PEMF in multiple stages over 62 days of culture. Mouse postnatal day 2 calvarial pre-osteoblasts were cast within and around Teflon rings by polymerization of fibrinogen and cultured suspended without contact with tissue culture plastic. Ring constructs were exposed to PEMF for 4h/day for the entire culture (Daily), or just during Day1-Day10, Day11-Day 27, or Day28-Day63 and cultured without PEMF for the preceding or remaining days, and compared to no-PEMF controls. PEMF was conducted as HF Physio, 40.85 kHz frequency with a 67 ms burst period and an amplitude of 1.19 mT. Osteogenesis was kinetically monitored by repeated fluorescence measurements of continuously present Alizarin Red S (ARS) and periodically confirmed by micro-CT. PEMF treatment induced early-onset and statistically significant transient stimulation (~4-fold) of the mineralization rate when PEMF was applied Daily, or during D1-D10 and D11-D27. Stimulation was apparent but not significant between D28-D63 by ARS but was significant at D63 by micro-CT. PEMF also shifted the micro-CT density profiles to higher densities in each PEMF treatment group. Ring culture generated tissue with a mineral:matrix ratio of 2.0 by thermogravimetric analysis (80% of the calvaria control), and the deposited crystal structure was 50% hydroxyapatite by X-ray diffraction (63% of the calvaria and femur controls), independent of PEMF. These results were consistent with backscatter, secondary electron, and elemental analysis by scanning electron microscopy. Thus, in a defined, strong osteogenic environment, PEMF applied at different times was capable of further stimulation of osteogenesis with the potential to enhance bone repair.

Electroless Palladium-Coated Polymer Scaffolds for Electrical Stimulation of Osteoblast-Like Saos-2 Cells.

Three-dimensional porous scaffolds offer some advantages over conventional treatments for bone tissue engineering. Amongst all non-bioresorbable scaffolds, biocompatible metallic scaffolds are preferred over ceramic and polymeric scaffolds, as they can be used as electrodes with different electric field intensities (or voltages) for electric stimulation (ES). In the present work we have used a palladium-coated polymeric scaffold, generated by electroless deposition, as a bipolar electrode to electrically stimulate human osteoblast-like Saos-2 cells. Cells grown on palladium-coated polyurethane foams under ES presented higher proliferation than cells grown on foams without ES for up to 14 days. In addition, cells grown in both conditions were well adhered, with a flat appearance and a typical actin cytoskeleton distribution. However, after 28 days in culture, cells without ES were filling the entire structure, while cells under ES appeared rounded and not well adhered, a sign of cell death onset. Regarding osteoblast differentiation, ES seems to enhance the expression of early expressed genes. The results suggest that palladium-coated polyurethane foams may be good candidates for osteoblast scaffolds and demonstrate that ES enhances osteoblast proliferation up to 14 days and upregulate expression genes related to extracellular matrix formation.

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.

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.

First radiobiological characterization of the McCune-Albright syndrome: influence of the ATM protein and effect of statins + bisphosphonates treatment.

PURPOSE: MacCune-Albright syndrome (MAS) is a rare autosomal dominant osteo-hormonal disorder. MAS is characterized by a severe form of polyostotic fibrous dysplasia, 'café-au-lait' pigmentation of the skin and multiple endocrinopathies. MAS was shown to be caused by mosaic missense somatic mutations in the GNAS gene coding for the alpha-subunit of the stimulatory G-protein. MAS is also associated with radiation-induced malignant tumors, like osteosarcoma, fibrosarcoma and chondrosarcoma but their origin remains misunderstood. In parallel, bisphosphonates treatment was shown to improve the MAS patients' outcome, notably by increasing bone density but, again, the molecular mechanisms supporting these observations remain misunderstood. MATERIALS AND METHODS: Here, by using fibroblast and osteoblast cell lines derived from 2 MAS patients, the major radiobiological features of MAS were investigated. Notably, the clonogenic cell survival, the micronuclei and the γH2AX, pATM and MRE11 immunofluorescence assays were applied to MAS cells. RESULTS: It appears that cells from the 2 MAS patients are associated with a moderate but significant radiosensitivity, a delayed radiation-induced nucleoshuttling of the ATM kinase likely caused by its sequestration in cytoplasm, suggesting impaired DNA double-strand breaks (DSB) repair and signaling in both fibroblasts and osteoblasts. Such delay may be partially corrected by using bisphosphonates combined with statins, which renders cells more radioresistant. CONCLUSIONS: Our findings represent the first radiobiological characterization of fibroblasts and osteoblasts providing from MAS patients. Although the number of studied cases is reduced, our findings suggest that the MAS cells tested belong to the group of syndromes associated with moderate but significant radiosensitivity. Further investigations are however required to secure the clinical transfer of the combination of bisphosphonates and statins, to reduce the disease progression and to better evaluate the potential risks linked to radiation exposure.

ODONTOLOGICAL EFFECTS OF IONIZING RADIATION (review). / ODONTOLOGIChNI EFEKTY IONIZUIuChOGO VYPROMINIuVANNIa (ogliad).

BACKGROUND: Odontological effects of ionizing radiation (IR) as a result of radiotherapy, the consequences of accidents at nuclear power plants and industry, individual occupational exposure, etc. deserve significant attention interns of radiation medicine and radiation safety. OBJECTIVE: to analyze and summarize clinical and experimental data on the odontological radiation effects. OBJECT: the pathological changes in the hard tissues of teeth, pulp, periodontium, mucousmembranes of the mouth and jaws due to exposure to IR. METHOD: search in the PubMed / MEDLINE, Google Scholarabstract medical and biological databases, scientific libraries of the relevant sources of scientific information. RESULTS: Radiobiological effects of IR due to its direct and indirect action are manifested throughout the period ofodontogenesis and formation of the facial skeleton. Experimental and clinical data (in children and adults) indicatethe increased risk of dental caries, reduction of pain threshold and vascularization of tooth pulp along with its fibrosis and atrophy, periodontal dysfunction, which predispose to a high probability of tooth loss. Abnormalities in theactivity of osteoblasts and cementoblasts of dental periosteum and osteoblasts of alveolar process in combinationwith circulatory disorders due to endothelial cell death, hyalinization, thrombosis and vascular obliteration increasethe risk of jaw osteoradionecrosis. Children who have undergone a prenatal exposure to IR as a result of theChornobyl NPP accident have a premature change of teeth. Deterioration of periodontal tissues and early development of acute and complicated dental caries are typical for children and adults affected by the Chornobyl disaster. CONCLUSIONS: Summarized data on the effects of radiation exposure under different conditions on teeth primordia(i.e. immature teeth), their formation and eruption in experimental and clinical settings, as well as on the odontological radiation effects in adults are summarized. Condition of the teeth in the Chornobyl NPP accident survivorsis described. Understanding and taking into account the radiobiological odontological effects is necessary in thelight of planning, preparing, and conducting local radiation therapy and developing the standards of radiation safety and measures to protect professionals and the public in the event of possible radiation accidents at the nuclearpower plants and industry facilities.

Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells.

Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.

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.

Effect of autophagy on irradiation­induced damage in osteoblast­like MC3T3­E1 cells.

Autophagy is activated under radiation stress, which serves an important role in maintaining bone homeostasis. However, the underlying mechanisms of irradiation­induced autophagy in bone homeostasis is not well understood. The present study aimed to determine the effects of radiation­activated autophagy on pre­osteoblastic MC3T3­E1 cells. X­ray irradiation activated autophagy in a dose­dependent manner, with an increased fluorescence intensity of monodansylcadaverine staining, increased ratio of microtubule­associated protein 1 light chain 3ß (LC3)­II/LC3­I, decreased p62 expression, and increased ATG5 and beclin­1 expression levels in MC3T3­E1 cells 72 h after irradiation compared with those in non­irradiated MC3T3­E1 cells. Irradiation reduced colony formation and mineralization in a dose­dependent manner in MC3T3­E1 cells at 2 and 3 weeks after irradiation, respectively. Decreased levels of alkaline phosphatase activity and runt­related transcription factor 2 expression were observed at 72 h post­irradiation. In addition, irradiation­induced apoptosis was accompanied by a decreased ratio of Bcl­2/BAX protein and increased the activity of caspase­3. By contrast, doxycycline (DOX)­inhibited autophagy attenuated the decreased colony formation and mineralization, and aggravated the increased cell apoptosis in irradiated MC3T3­E1 cells. Furthermore, the ratio of phosphorylated P38/P38 was observed to be higher following DOX treatment within 1 week of irradiation, which was reversed 2 weeks post­irradiation. In conclusion, DOX­inhibited autophagy aggravated X­ray irradiation­induced apoptosis at an early stage, but maintained cell proliferation and mineralization at a late stage in irradiated MC3T3­E1 cells.