CR6-Interacting Factor-1 Promotes Osteoclastogenesis Through the NF-κB Signaling Pathway after Irradiation.

Radiation exposure arising from radiotherapy may induce rapid bone loss and an increase in the extent of bone resorption. Reactive oxygen species (ROS) caused by radiation exposure play a crucial role during the process of osteoclastogenesis. However, the pathological mechanisms underlying radiation-induced osteoclastogenesis have yet to be fully elucidated. CR6-interacting factor-1 (Crif1) as a multifunctional protein is involved in regulating multiple biological functions in cells. Here, we investigated the role of Crif1 in radiation-induced osteoclastogenesis and found that radiation exposure induced an increase in the expression level of Crif1 and enhanced osteoclastogenesis in osteoclast progenitors. Crif1 and NF-κB p65 co-localized in the cytoplasm after radiation exposure. Crif1 knockdown did not affect the phosphorylation and total protein levels of extracellular signal-regulated kinases (ERK), c-Jun amino (N)-terminal kinases (JNK), p38, and IκB-α before and after irradiation. However, Crif1 knockdown did lead to the reduced phosphorylation and nuclear translocation of NF-κB p65 after irradiation and resulted in a reduced level of osteoclastogenesis in RAW264.7 cells after irradiation. In vivo studies involving Lyz2Cre;Crif1fl/fl mice possessing the myeloid-specific deletion of Crif1 demonstrated the alleviation of bone loss after irradiation when compared with Crif1fl/fl mice. Our findings demonstrate that Crif1 mediated the phosphorylation and nuclear translocation of NF-κB p65 and promoted osteoclastogenesis via the NF-κB signaling pathway after radiation exposure. Thus, our analysis revealed a specific role for Crif1 in the mediation of radiation-induced bone loss and may provide new insight into potential therapeutic strategies for radiation-induced bone loss.

Ionizing Radiation Activates Mitochondrial Function in Osteoclasts and Causes Bone Loss in Young Adult Male Mice.

The damaging effects of ionizing radiation (IR) on bone mass are well-documented in mice and humans and are most likely due to increased osteoclast number and function. However, the mechanisms leading to inappropriate increases in osteoclastic bone resorption are only partially understood. Here, we show that exposure to multiple fractions of low-doses (10 fractions of 0.4 Gy total body irradiation [TBI]/week, i.e., fractionated exposure) and/or a single exposure to the same total dose of 4 Gy TBI causes a decrease in trabecular, but not cortical, bone mass in young adult male mice. This damaging effect was associated with highly activated bone resorption. Both osteoclast differentiation and maturation increased in cultures of bone marrow-derived macrophages from mice exposed to either fractionated or singular TBI. IR also increased the expression and enzymatic activity of mitochondrial deacetylase Sirtuin-3 (Sirt3)-an essential protein for osteoclast mitochondrial activity and bone resorption in the development of osteoporosis. Osteoclast progenitors lacking Sirt3 exposed to IR exhibited impaired resorptive activity. Taken together, targeting impairment of osteoclast mitochondrial activity could be a novel therapeutic strategy for IR-induced bone loss, and Sirt3 is likely a major mediator of this effect.

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.

Simulated Galactic Cosmic Rays Modify Mitochondrial Metabolism in Osteoclasts, Increase Osteoclastogenesis and Cause Trabecular Bone Loss in Mice.

Space is a high-stress environment. One major risk factor for the astronauts when they leave the Earth's magnetic field is exposure to ionizing radiation from galactic cosmic rays (GCR). Several adverse changes occur in mammalian anatomy and physiology in space, including bone loss. In this study, we assessed the effects of simplified GCR exposure on skeletal health in vivo. Three months following exposure to 0.5 Gy total body simulated GCR, blood, bone marrow and tissue were collected from 9 months old male mice. The key findings from our cell and tissue analysis are (1) GCR induced femoral trabecular bone loss in adult mice but had no effect on spinal trabecular bone. (2) GCR increased circulating osteoclast differentiation markers and osteoclast formation but did not alter new bone formation or osteoblast differentiation. (3) Steady-state levels of mitochondrial reactive oxygen species, mitochondrial and non-mitochondrial respiration were increased without any changes in mitochondrial mass in pre-osteoclasts after GCR exposure. (4) Alterations in substrate utilization following GCR exposure in pre-osteoclasts suggested a metabolic rewiring of mitochondria. Taken together, targeting radiation-mediated mitochondrial metabolic reprogramming of osteoclasts could be speculated as a viable therapeutic strategy for space travel induced bone loss.

Intracellular Signaling Responses Induced by Radiation within an In Vitro Bone Metastasis Model after Pre-Treatment with an Estrone Analogue.

2-Ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) is an in silico-designed estradiol analogue which has improved the parent compound's efficacy in anti-cancer studies. In this proof-of-concept study, the potential radiosensitizing effects of ESE-16 were investigated in an in vitro deconstructed bone metastasis model. Prostate (DU 145) and breast (MDA-MB-231) tumor cells, osteoblastic (MC3T3-E1) and osteoclastic (RAW 264.7) bone cells and human umbilical vein endothelial cells (HUVECs) were representative components of such a lesion. Cells were exposed to a low-dose ESE-16 for 24 hours prior to radiation at non-lethal doses to determine early signaling and molecular responses of this combination treatment. Tartrate-resistant acid phosphatase activity and actin ring formation were investigated in osteoclasts, while cell cycle progression, reactive oxygen species generation and angiogenic protein expression were investigated in HUVECs. Increased cytotoxicity was evident in tumor and endothelial cells while bone cells appeared to be spared. Increased mitotic indices were calculated, and evidence of increased deoxyribonucleic acid damage with retarded repair, together with reduced metastatic signaling was observed in tumor cells. RAW 264.7 macrophages retained their ability to differentiate into osteoclasts. Anti-angiogenic effects were observed in HUVECs, and expression of hypoxia-inducible factor 1-α was decreased. Through preferentially inducing tumor cell death and potentially inhibiting neovascularization whilst preserving bone physiology, this low-dose combination regimen warrants further investigation for its promising therapeutic application in bone metastases management, with the additional potential of limited treatment side effects.

The Influence of Radiation on Bone and Bone Cells-Differential Effects on Osteoclasts and Osteoblasts.

The bone is a complex organ that is dependent on a tight regulation between bone formation by osteoblasts (OBs) and bone resorption by osteoclasts (OCs). These processes can be influenced by environmental factors such as ionizing radiation (IR). In cancer therapy, IR is applied in high doses, leading to detrimental effects on bone, whereas radiation therapy with low doses of IR is applied for chronic degenerative and inflammatory diseases, with a positive impact especially on bone homeostasis. Moreover, the effects of IR are of particular interest in space travel, as astronauts suffer from bone loss due to space radiation and microgravity. This review summarizes the current state of knowledge on the effects of IR on bone with a special focus on the influence on OCs and OBs, as these cells are essential in bone remodeling. In addition, the influence of IR on the bone microenvironment is discussed. In summary, the effects of IR on bone and bone remodeling cells strongly depend on the applied radiation dose, as differential results are provided from in vivo as well as in vitro studies with varying doses of IR. Furthermore, the isolated effects of IR on a single cell type are difficult to determine, as the bone cells and bone microenvironment are building a tightly regulated network, influencing on one another. Therefore, future research is necessary in order to elucidate the influence of different bone cells on the overall radiation-induced effects on bone.

Radiation alters osteoclastogenesis by regulating the cytoskeleton and lytic enzymes in RAW 264.7 cells and mouse bone marrow-derived macrophages.

PURPOSE: The aim of the present study was to investigate the duality of irradiation effect on osteoclastogenesis, particularly on the cytoskeleton and expression of lytic enzymes in osteoclast precursors. Therefore, the present study may serve as a useful reference for the prevention and treatment of radiation-induced bone loss in the clinic. MATERIALS AND METHODS: Two typical osteoclast precursors, murine RAW 264.7 macrophage cells and mouse bone marrow-derived macrophages (BMMs), were exposed to radiation in the order of 0.25-8 Gy, and the effects on cell viability, TRAP activity and bone resorption were subsequently investigated. Furthermore, changes in the cytoskeleton, cell apoptosis, and expression of lytic enzymes in osteoclasts were examined to elucidate the molecular mechanism of the duality of irradiation on osteoclastogenesis. RESULTS: Morphological changes and impaired viability were observed in RAW 264.7 cells and BMMs treated with 1-8 Gy irradiation with or without RANKL. However, the cell fusion tendency of osteoclasts was enhanced after 2 Gy irradiation, and an increased number of fused giant osteoclasts and enhanced F-actin ring formation were observed. Consistently, the bone resorption activity and the enzyme expression of TRAP, cathepsin K, matrix metalloproteinase 9, activator protein 1, and Caspase 9 were increased following irradiation with 2 Gy. Furthermore, intracellular ROS production and apoptosis of osteoclast precursors were increased. CONCLUSIONS: Irradiation with 2 Gy inhibited the viability of osteoclast precursors, but increased osteoclastogenesis by enhancing cell fusion and increasing the secretion of lytic enzymes, which may be an important mechanism of radiation-induced bone loss.

Radioprotection and cross-linking of allograft bone in the presence of vitamin E.

Bone allografts are the preferred method for bone augmentation in over 500,000 orthopedic surgical procedures in the US. Sterilization by ionizing radiation is the most effective method of minimizing the bioburden of bone allografts; however, radiation causes chain scission of collagen, resulting in the reduction of the allografts' mechanical strength. In this study, we doped bone allografts with vitamin E as radioprotectant using a novel two-step process to protect the collagen architecture against radiation damage and to preserve the mechanical strength of the construct. In addition, combining the radioprotectant with a cross-linking agent further minimized collagen degradation and further preserved the mechanical strength of the allografts. Both vitamin E and combined vitamin E/genipin-treated allograft were less cytotoxic to both osteoblasts and osteoclasts when compared to irradiated-only allografts. Host bone-allograft unionization was faster in a rat calvaria defect model with vitamin E-treated and combined vitamin E and genipin-treated allograft when compare to irradiated-only allografts. This method can enable the efficient and uniform radioprotective treatment of bone allograft of desired shapes for sterilization with improved mechanical strength and biointegration.

Amifostine inhibited the differentiation of RAW264.7 cells into osteoclasts by reducing the production of ROS under 2 Gy radiation.

Patients with malignant tumors receive radiotherapy, and radiation could harm the skeletal system, leading to radiation-induced osteoporosis. A major cause of this phenomenon is the activation of osteoclasts by radiotherapy. In this study, we studied whether amifostine (AMI) could affect the differentiation of osteoclast precursor cells (RAW264.7 cells) into osteoclasts under 2 gray (Gy) radiation. Four groups were used in the experiment: (a) 0 Gy (no radiation); (b) 0 Gy + AMI; (c) 2 Gy radiation; and (d) 2 Gy radiation + AMI. After radiation, a proliferation assay, a reactive oxygen species (ROS) assay, a comet assay, Trap staining, reverse transcription polymerase chain reaction, and an animal study to test the effect of AMI on osteoclast precursor cells under 2 Gy radiation were conducted. Cell proliferation was significantly inhibited by AMI (P < .05). In addition, 2 Gy radiation led to longer "comet tails", high level of ROS, and more Trap-positive cells in vivo and in vitro (P < .05). Radiation improved the expression of CSTK, NFAT, and Rankl/OPG gene (P < .05), as well as Trap-5b levels in the serum, and decreased bone mineral density. AMI inhibited the differentiation of RAW264.7 cells, shortened the tail moment length of comets, and decreased the level of ROS induced by radiation. The expression of NFAT, CTSK, and Rankl/OPG was decreased by AMI at the detection time point in radiation groups (P < .05). AMI inhibits the maturation and differentiation of osteoclasts under radiation conditions by reducing DNA damage and ROS induced by radiation, thereby reducing the adverse effects of radiation in the skeletal system, indicating that AMI might be used to treat osteoradionecrosis.

Effect of 650-nm low-level laser irradiation on c-Jun, c-Fos, ICAM-1, and CCL2 expression in experimental periodontitis.

This study was designed to investigate the effect of 650-nm low-level laser irradiation (LLLI) as an adjunctive treatment of experimental periodontitis. To investigate possible LLLI-mediated anti-inflammatory effects, we utilized an experimental periodontitis (EP) rat model and analyzed c-Jun, c-Fos, ICAM-1, and CCL2 gene expressions on PB leukocytes and in the gingival tissue. Total RNA was isolated from the gingivae and peripheral blood (PB) leukocytes of normal, EP, scaling, and root planing (SRP)-treated EP and LLLI + SRP-treated EP rats, and gene expressions were analyzed by real-time PCR. The productions of c-Jun, c-Fos, ICAM-1, and CCL2 in gingivae were analyzed immunohistochemically. Tartrate-resistant acid phosphatase (TRAP) staining was used to determine osteoclast activity in alveolar bone. The c-Jun and ICAM-1 messenger RNA (mRNA) levels were significantly decreased in the EP rat gingival tissue treated by SRP + LLLI than by SRP, the c-Jun, ICAM-1, and c-Fos mRNA levels on PB leukocytes reduced after LLLI treatment but did not show any significant differences in both groups. There was no significant difference in CCL2 mRNA levels on PB leukocytes and in gingivae between the SRP + LLLI and the SRP groups. The c-Fos mRNA levels in gingivae did not show significant difference in both groups. Immunohistochemistry showed that the CCL2, ICAM-1, c-Jun, and c-Fos productions were significantly reduced in rats of the SRP + LLLI group compared with the only SRP group. LLLI significantly decreased the number of osteoclasts as demonstrated by TRAP staining. The 650-nm LLLI might be a useful treatment modality for periodontitis.

Influence of radiation exposure pattern on the bone injury and osteoclastogenesis in a rat model.

Radiotherapy, one of the clinical treatments of cancer, is accompanied by a high risk of damage to healthy tissues, such as bone loss and increased risk of fractures. The aim of the present study was to establish a rat model of local and systemic bone injury by focal irradiation, in order to study the etiological mechanism and intervention. The proximal metaphyseal region of the left hindlimb of male Sprague­Dawley rats were exposed to a single 2 Gy or three 8 Gy doses delivered on days 1, 3 and 5 using a small animal irradiator, the changes in bone volume and microarchitecture were evaluated, and the mineral apposition rate (MAR) was assessed. Furthermore, bone marrow­derived macrophages (BMMs) were isolated and induced to osteoclasts. It has been demonstrated that a single dose of 2 Gy may result in a significant loss of lumbar bone density at 3 days post­irradiation, however this is restored at 30 days post­irradiation. In the 3x8 Gy irradiation rat model, there was a rapid decrease in the aBMD of lumbar spine at 3 days and at 7 days post­irradiation, and the aBMD decline persisted even at 60 days post­irradiation. In addition, microCT analysis revealed a persistent decline in bone volume and damage in microarchitecture in the 3x8 Gy irradiation model, accompanied by a decrease in MAR, index of the decline in bone­forming ability. In the cellular mechanism, a single 2 Gy local irradiation mainly manifested as an enhancement of the BMMs osteoclastogenesis potential, which was different from the osteoclastogenesis inhibition after high­dose focal irradiation (3x8 Gy). In summary, the irradiation with simulated clinical focal fractionated radiotherapy exerts short­ and long­term systemic injury on bone tissue, characterized by different osteoclastogenesis potential between the high dose mode and a single 2 Gy focal irradiation. Physicians must consider the irreversibility of bone damage in clinical radiotherapy.

Low­frequency pulsed electromagnetic field inhibits RANKL­induced osteoclastic differentiation in RAW264.7 cells by scavenging reactive oxygen species.

Bone homeostasis is a dynamic balance maintained by bone formation and resorption. An increase in the number and activity of osteoclasts leads to excessive bone resorption, which in turn results in bone disease, including osteoporosis. Therefore, inhibiting the differentiation and activity of osteoclasts is important for maintaining bone mass. Several studies have revealed that the use of a low­frequency pulsed electromagnetic field (PEMF) is an effective method to treat osteoporosis. However, its exact mechanism remains to be fully clarified. Therefore, the present study was designed to examine the effects that PEMF exerts on receptor activator of nuclear factor­κB ligand (RANKL)­induced osteoclastogenesis and intracellular reactive oxygen species (ROS) production in RAW264.7 cells. The viability of cells was determined using a Cell Counting Kit­8 assay, and gene and protein expression were investigated via reverse transcription­quantitative polymerase chain reaction and western blot analyses. Furthermore, microscopy was performed to detect the levels of intracellular ROS and tartrate­resistant acid phosphatase (TRAP). Following the culture of RAW264.7 cells with RANKL (50 ng/ml) for 4 days (3 h/day) under PEMF (75 Hz, 1 mt) exposure, it was observed that PEMF had an inhibitory effect on RANKL­induced osteoclastic differentiation. Multinucleated osteoclast formation, the activity of TRAP and the expression of osteoclastogenesis­associated genes, including cathepsin K, nuclear factor of activated T cells cytoplasmic 1 and TRAP, were significantly reduced by PEMF. Furthermore, PEMF effectively decreased the generation of intracellular ROS during osteoclastic differentiation. In addition, the results demonstrated that ROS are the key factor in osteoclast differentiation and formation. Reducing intracellular ROS with diphenylene­iodonium chloride significantly inhibited RANKL­induced osteoclast differentiation. Taken together, the results of the present study demonstrated that PEMF may inhibit RANKL­induced osteoclastogenesis by scavenging intracellular ROS. These results may provide the groundwork for future PEMF clinical applications in osteoclast­associated bone disease.

Pulsed electromagnetic fields regulate osteocyte apoptosis, RANKL/OPG expression, and its control of osteoclastogenesis depending on the presence of primary cilia.

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).

Potential role of senescence in radiation-induced damage of the aged skeleton.

Human aging-related changes are exacerbated in cases of disease and cancer, and conversely aging is a catalyst for the occurrence of disease and multimorbidity. For example, old age is the most significant risk factor for cancer and among people who suffer from cancer, >60% are above the age of 65. Oxidative stress and DNA damage, leading to genomic instability and telomere dysfunction, are prevalent in aging and radiation-induced damage and are major cellular events that lead to senescence. Human exposures from nuclear fallout, cosmic radiation and clinical radiotherapy (RT) are some common sources of irradiation that affect bone tissue. RT has been used to treat malignant tumors for over a century, but the effects of radiation damage on tumor-adjacent normal tissue has largely been overlooked. There is an increase in the percent survivorship among patients post-RT, and it is in older survivors where the deleterious synergy between aging and radiation exposure conspires to promote tissue deterioration and dysfunction which then negatively impacts their quality of life. Thus, an aging skeleton is already pre-disposed to architectural deterioration, which is further worsened by radiation-induced bone damage. Effects of senescence and the senescence associated secretory phenotype (SASP) have been implicated in age-associated bone loss, but their roles in radiation-associated bone damage are still elusive. RT is used in treatment for a variety of cancers and in different anatomical locations, the sequelae of which include long-term morbidity and lifelong discomfort. Therefore, consideration of the growing evidence that implicates the role of senescence in radiation-induced bone damage argues in favor of exploiting current senotherapeutic approaches as a possible prevention or treatment.

Lowering iron level protects against bone loss in focally irradiated and contralateral femurs through distinct mechanisms.

Radiation therapy leads to increased risk of late-onset fragility and bone fracture due to the loss of bone mass. On the other hand, iron overloading causes osteoporosis by enhancing bone resorption. It has been shown that total body irradiation increases iron level, but whether the systemic bone loss is related to the changes in iron level and hepcidin regulation following bone irradiation remains unknown. To investigate the potential link between them, we first created an animal model of radiation-induced systemic bone loss by targeting the mid-shaft femur with a single 2 Gy dose of X-rays. We found that mid-shaft femur focal irradiation led to structural deterioration in the distal region of the trabecular bone with increased osteoclasts surface and expressions of bone resorption markers in both irradiated and contralateral femurs relative to non-irradiated controls. Following irradiation, reduced hepcidin activity of the liver contributed to elevated iron levels in the serum and liver. By injecting hepcidin or deferoxamine (an iron chelator) to reduce iron level, deterioration of trabecular bone microarchitecture in irradiated mice was abrogated. The ability of iron chelation to inhibit radiation-induced osteoclast differentiation was observed in vitro as well. We further showed that ionizing radiation (IR) directly stimulated osteoclast differentiation and bone resorption in bone marrow cells isolated not from contralateral femurs but from directly irradiated femurs. These results suggest that increased iron levels after focal radiation is at least one of the main reasons for systemic bone loss. Furthermore, bone loss in directly irradiated bones is not only due to the elevated iron level, but also from increased osteoclast differentiation. In contrast, the bone loss in the contralateral femurs is mainly due to the elevated iron level induced by IR alone. These novel findings provide proof-of-principle evidence for the use of iron chelation or hepcidin as therapeutic treatments for IR-induced osteoporosis.

Effects of the photobiomodulation using different energy densities on the periodontal tissues under orthodontic force in rats with type 2 diabetes mellitus.

To evaluate the impact of the GaAlAs diode laser with energy densities of 160 J/cm2, 320 J/cm2, and 640 J/cm2 on the periodontal tissues under continuous orthodontic force application and on the rate of orthodontic tooth movement in rats with type-2 diabetes mellitus. The intensity of primary alveolar bone formation was also investigated through the immune-positive osteocytes for OPN antibody. Forty adult male Wistar rats were divided into eight groups of 5 rats: normoglycemic (N), 160 J-laser-normoglycemic (160 J-LN), 320 J-laser-normoglycemic (320 J-LN), 640 J-laser-normoglycemic (640 J-LN), diabetic (D), 160 J-laser-diabetic (160 J-LD), 320 J-laser-diabetic (320 J-LD), and 640 J-laser-diabetic (640 J-LD) rats. Diabetes mellitus was induced by a single intravenous injection of 40 mg/kg monohydrated-alloxan. An orthodontic force magnitude of 20cN was applied. The laser parameters were continuous emission of 780-nm wavelength, output power of 20mW, and fiber probe with a spot size of 0.04 cm in diameter. Radiographic, histomorphological, and immunohistochemical analysis were performed after a period of 21 days. The photobiomodulation using the energy density of 640 J/cm2 strongly stimulated the alveolar bone formation and contributed the reorganization of the soft periodontal tissues, followed by the 320 J/cm2. Extensive alveolar bone loss, intense infiltration of inflammatory cells, and degradation of the PDJ tissue were mainly found in the D and 160 J-LD groups. The rate of orthodontic tooth movement was represented by the interdental distance between the cementoenamel junctions of the right mandibular first and second molars . This distance was larger in the diabetic groups (D: 39.98±1.97, 160 J-LD: 34.84±6.01, 320 J-LD: 29.82±1.73, and 640 J-LD: 35.47±4.56) than in the normoglycemic groups (N: 21.13±1.19; 160 J-LN: 22.69±0.72, 320 J-LN: 22.28±0.78, and 640 J-LN: 24.56±2.11). The number of osteopontin-positive osteocytes was significantly greater in the 640 J-LD (14.72 ± 0.82; p < 0.01) and 640 J-LN (13.62 ± 1.33; p < 0.05) groups than with D (9.82 ± 1.17) and 160 J-LD (9.77 ± 1.10) groups. Therefore, the energy density of 640 J/cm2 provided the best maintenance and integrity of the periodontal tissue microarchitecture under continuous orthodontic force when compared with the other dosages, mainly in the uncontrolled diabetic rats. The interdental distance was greater in the D and 160 J-LD groups due to presence of severe periodontitis caused by diabetes plus the mechanical stress generated by continuous orthodontic forces, implying, thus, an insufficient biostimulatory effect for the dosage of 160 J/cm2.

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.

Low-Dose Radiotherapy Has No Harmful Effects on Key Cells of Healthy Non-Inflamed Joints.

Low-dose radiotherapy (LD-RT) for benign inflammatory and/or bone destructive diseases has been used long. Therefore, mechanistic investigations on cells being present in joints are mostly made in an inflammatory setting. This raises the question whether similar effects of LD-RT are also seen in healthy tissue and thus might cause possible harmful effects. We performed examinations on the functionality and phenotype of key cells within the joint, namely on fibroblast-like synoviocytes (FLS), osteoclasts and osteoblasts, as well as on immune cells. Low doses of ionizing radiation showed only a minor impact on cytokine release by healthy FLS as well as on molecules involved in cartilage and bone destruction and had no significant impact on cell death and migration properties. The bone resorbing abilities of healthy osteoclasts was slightly reduced following LD-RT and a positive impact on bone formation of healthy osteoblasts was observed after in particular exposure to 0.5 Gray (Gy). Cell death rates of bone-marrow cells were only marginally increased and immune cell composition of the bone marrow showed a slight shift from CD8⁺ to CD4⁺ T cell subsets. Taken together, our results indicate that LD-RT with particularly a single dose of 0.5 Gy has no harmful effects on cells of healthy joints.

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.

Sema3a inhibits the differentiation of Raw264.7 cells to osteoclasts under 2Gy radiation by reducing inflammation.

Astronauts and cancer patients receive different types of radiation, and radiation decreases bone strength and leads to radiation-induced osteoporosis. This effect is attributed to the activation of osteoclasts. Our aim was to study the effect of Sema3a on the differentiation of the murine macrophage cell line Raw264.7 into osteoclasts upon irradiation. Raw264.7 cells were divided into four groups: A, receiving no radiation; B, receiving no radiation + 50ngng/ml Sema3a; C, receiving 2Gy radiation; and D, receiving 2Gy radiation +50ngng/ml Sema3a. After treatment, cells were subjected to a proliferation assay, migration assay, live and apoptosis assay, and an ROS assay, along with analyses of bone resorption activity, TRAP staining and RT-PCR to assess the effect of Sema3a on Raw264.7 cells under 2Gy radiation. Sema3a inhibited the proliferation of Raw264.7 cells and showed statistical significance at a concentration of 100ngng/ml (P<0.05). Under 2Gy radiation, cell migration was reduced (P<0.05). In addition, 2Gy radiation resulted in more apoptotic cells, a higher level of ROS, larger bone resorption lacunae and more Trap-positive cells (p<0.05), and radiation increased CSTK, NFAT, TRAP-5b, Rankl/OPG, IL-1, IL-6, TNFa and P53 gene expression (P<0.05). Sema3a had an inhibitory effect on the differentiation of Raw264.7 cells and the migration and activity of osteoclasts upon irradiation but did not affect ROS. Sema3a also decreased the expression of CSTK, NFAT, TRAP-5b, Rankl/OPG, IL-1, IL-6 and TNFa on the 3rd and 7th days after irradiation (p<0.05), whereas P53 expression was increased (P<0.05). Sema3a reduced the inflammation induced by radiation and negatively regulated osteoclast differentiation. Sema3a promoted Raw264.7 cell apoptosis after irradiation, indicating that Sema3a could be a potential therapeutic target for radiation-induced osteoporosis.