Musculoskeletal disorders (MSDs) are associated with pain and lead to reduced mobility and quality of life for patients. Radon therapy is used as alternative or complementary to pharmaceutical treatments. According to previous reports, radon spa leads to analgesic and anti-inflammatory effects, but the cellular and molecular mechanisms are widely unknown. A previous study (RAD-ON01) revealed, that bone erosion markers like collagen fragments (C-terminal telopeptide, CTX) are reduced after radon spa treatment in serum of patients with degenerative MSDs. Within the scope of the prospective, placebo-controlled RAD-ON02 trial presented here, we analyzed the influence of radon and thermal spa treatment on osteoclastogenesis. From patient blood, we isolate monocytes, seeded them on bone slices and differentiated them in the presence of growth factors into mature osteoclasts (mOCs). Subsequent analysis showed a smaller fraction of mOCs after both treatments, which was even smaller after radon spa treatment. A significantly reduced resorbed area on bone slices reflects this result. Only after radon spa treatment, we detected in the serum of patients a significant decrease of receptor activator of NF-κB ligand (RANKL), which indicates reduced differentiation of OCs. However, other markers for bone resorption (CTX) and bone formation (OPG, OCN) were not altered after both treatments. Adipokines, such as visfatin and leptin that play a role in some MSD-types by affecting osteoclastogenesis, were not changed after both treatments. Further, also immune cells have an influence on osteoclastogenesis, by inhibiting and promoting terminal differentiation and activation of OCs, respectively. After radon treatment, the fraction of Treg cells was significantly increased, whereas Th17 cells were not altered. Overall, we observed that both treatments had an influence on osteoclastogenesis and bone resorption. Moreover, radon spa treatment affected the Treg cell population as well as the Th17/Treg ratio were affected, pointing toward a contribution of the immune system after radon spa. These data obtained from patients enrolled in the RAD-ON02 trial indicate that radon is not alone responsible for the effects on bone metabolism, even though they are more pronounced after radon compared to thermal spa treatment.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that exerts physiological effects via G protein-coupled receptor 120 (GPR120). In our previous studies, we figured out the inhibitory effects of DHA on TNF-α (Tumor necrosis factor-α)-induced osteoclastogenesis via GPR120 in vivo. Moreover, DHA directly suppressed RANKL expression in osteoblasts via GPR120 in vitro. In this study, we generated bone marrow chimeric mice using GPR120 deficient mice (GPR120-KO) to study the inhibitory effects of DHA on bone resorption and osteoclast formation. Bone marrow cells of wild-type (WT) or GPR120-KO mice were transplanted into irradiated recipient mice, which were WT or GPR120 deficient mice. The resulting chimeric mice contained stromal cells from the recipient and bone marrow cells, including osteoclast precursors, from the donor. These chimeric mice were used to perform a series of histological and microfocus computed tomography (micro-CT) analyses after TNF-α injection for induction of osteoclast formation with or without DHA. Osteoclast number and bone resorption were found to be significantly increased in chimeric mice, which did not express GPR120 in stromal cells, compared to chimeric mice, which expressed GPR120 in stromal cells. DHA was also found to suppress specific signaling pathways. We summarized that DHA suppressed TNF-α-induced stromal-dependent osteoclast formation and bone resorption via GPR120.

UNASSIGNED: Osteoclastogenesis, the process of osteoclast differentiation, plays a critical role in bone homeostasis. Overexpression of osteoclastogenesis can lead to pathological conditions, such as osteoporosis and osteolysis. This study aims to investigate the role of Engelitin in the process of RAW264.7 cell differentiation into osteoclasts induced by RANKL, as well as in a mouse model of bone loss following ovariectomy.
UNASSIGNED: We used RANKL-stimulated RAW264.7 cells as an in vitro osteoclast differentiation model. The effects of Eng on morphological changes during osteoclast differentiation were evaluated using TRAP and F-actin staining. The effects of Eng on the molecular level of osteoclast differentiation were evaluated using Western blot and q-PCR. The level of reactive oxygen species was evaluated using the DCFH-DA staining method. We then used ovariectomized mice as a bone loss animal model. The effects of Eng on changes in bone loss in vivo were evaluated using micro-CT and histological analysis staining.
UNASSIGNED: In the in vitro experiments, Eng exhibited dose-dependent inhibition of osteoclast formation and F-actin formation. At the molecular level, Eng dose-dependently suppressed the expression of specific RNAs (NFATc1, c-Fos, TRAP, Cathepsin K, MMP-9) involved in osteoclast differentiation, and inhibited the phosphorylation of proteins such as IκBα, P65, ERK, JNK, and P38. Additionally, Eng dose-dependently suppressed ROS levels and promoted the expression of antioxidant enzymes such as Nrf2, HO-1, and NQO1. In the in vivo experiments, Eng improved bone loss in ovariectomized mice.
UNASSIGNED: Our study found that Eng inhibited RANKL-induced osteoclast differentiation through multiple signaling pathways, including MAPKs, NF-κB, and ROS aggregation. Furthermore, Eng improved bone loss in ovariectomized mice.

BACKGROUND: Monocyte-macrophage lineage cells are committed towards osteoclast differentiation in vitro by the downregulation of microphthalmia-induced transcription factor (MITF) by miRNA-155. Therefore, we aimed to evaluate miRNA-155 expression and explore the regulation of MITF by miRNA-155 during osteoclastogenesis in periodontitis.
METHODS: Ninety-eight subjects were recruited and categorized into the following: group I (cases)-systemically healthy with localized stage III/IV periodontitis (N = 49) and group II (controls)-systemically and periodontally healthy (N = 49). Gingival tissue samples were procured and qRT-PCR analysis was carried out for relative gene expression.
RESULTS: The mean ΔCT of miRNA-155 expression was -1.04 ± 2.26 and -0.01 ± 1.4 respectively for groups I and II. There was a statistically significant difference in the miRNA-155 expression (P ≤ 0.01) between the groups. The mean ΔCT of MITF expression for groups I and II was 4.15± 2.16 and 3.51± 1.57 respectively with no significant difference (P > 0.01) between the groups. In the periodontitis group, miRNA-155 expression increased by fivefolds (P ≤ 0.01) whereas MITF expression showed no significant difference in the fold change between the groups (P > 0.01). The site-specific clinical parameters showed a statistically significant strong negative and positive correlation with the ΔCT and fold change values of miRNA-155 respectively in the total 98 samples (P < 0.01). miRNA-155 was able to discriminate between periodontal health and disease with a diagnostic accuracy of 96.9% (95%CI: 91.38-98.95) and the AUC was 0.98 (95%CI: 0.97-1.0, SE = 0.008, P < 0.001) in ROC analysis with a sensitivity of 93.8% (95%CI: 83.48-97.9) and specificity of 100% (95%CI: 92.73-100).
CONCLUSIONS: miRNA-155 was dysregulated and upregulated by fivefolds in periodontal disease. It can be used as a potential biomarker to discriminate between periodontal health and disease. No difference in the MITF gene expression was demonstrated between periodontal health and disease. The result suggested that miRNA-155 does not affect the expression of MITF gene in the process of osteoclastogenesis in localized stage III/IV periodontitis within this study design and limitations.

With the introduction of a new interdisciplinary field, osteoimmunology, today, it is well acknowledged that biomaterial-induced inflammation is modulated by immune cells, primarily macrophages, and can be controlled by nanotopographical cues. Recent studies have investigated the effect of surface properties in modulating the immune reaction, and literature data indicate that various surface cues can dictate both the immune response and bone tissue repair. In this context, the purpose of the present study was to investigate the effects of titanium dioxide nanotube (TNT) interspacing on the response of the macrophage-like cell line RAW 264.7. The cells were maintained in contact with the surfaces of flat titanium (Ti) and anodic TNTs with an intertube spacing of 20 nm (TNT20) and 80 nm (TNT80), under standard or pro-inflammatory conditions. The results revealed that nanotube interspacing can influence macrophage response in terms of cell survival and proliferation, cellular morphology and polarization, cytokine/chemokine expression, and foreign body reaction. While the nanostructured topography did not tune the macrophages\' differentiation into osteoclasts, this behavior was significantly reduced as compared to flat Ti surface. Overall, this study provides a new insight into how nanotubes\' morphological features, particularly intertube spacing, could affect macrophage behavior.

We have previously indicated that a single injection of alendronate, one of the nitrogen-containing bisphosphonates (NBPs), affects murine hematopoietic processes, such as the shift of erythropoiesis from bone marrow (BM) to spleen, disappearance of BM-resident macrophages, the increase of granulopoiesis in BM and an increase in the number of osteoclasts. NBPs induce apoptosis and the formation of giant osteoclasts in vitro and/or in patients undergoing long-term NBP treatment. Therefore, the time-kinetic effect of NBPs on osteoclasts needs to be clarified. In this study, we examined the effect of alendronate on mouse osteoclasts and osteoclastogenesis. One day after the treatment, osteoclasts lost the clear zone and ruffled borders, and the cell size decreased. After 2 days, the cytoplasm of osteoclasts became electron dense and the nuclei became pyknotic. Some of the cells had fragmented nuclei. After 4 days, osteoclasts had euchromatic nuclei attached to the bone surface. Osteoclasts had no clear zones or ruffled borders. After 7 days, osteoclasts formed giant osteoclasts via the fusion of multinuclear and mononuclear osteoclasts. These results indicate that NBPs affect osteoclasts and osteoclastogenesis via two different mechanisms.

OBJECTIVE: To evaluate the effects of alcohol and nicotine, when used alone or simultaneously, in the development of apical periodontitis induced in rats, using a correlative analytic approach with micro-CT, histological and immunohistochemical analysis.
METHODS: Twenty-eight male Wistar rats were arranged into four groups: Control, Nicotine, Alcohol and Alcohol + Nicotine. The alcohol groups were exposed to self-administration of a 25% alcohol solution, whilst the other groups drunk only filtered water. The nicotine groups received daily intraperitoneal injections of a solution with 0.19 μL of nicotine per mL, whilst the other groups received saline solution. The pulps of the left mandibular first molars were exposed for 28 days to induce periapical lesions. Throughout the experiment, drug administration was maintained, and the animals had their weight and solid and liquid consumption measured. After euthanasia, the mandibles were removed and the area, volume and major diameter of the periapical lesions were measured using micro-computed tomography images. The samples were submitted to histopathological evaluation and immunohistochemistry for RANKL and PTHrP. Statistical analysis was undertaken with a significance level of 5%. Nonparametric data were analysed using the Kruskal-Wallis test followed by Dunn\'s test, whilst one-way anova followed by Tukey\'s test was performed for parametric data.
RESULTS: The alcohol groups had lower solid and liquid consumption and gained less weight when compared to the nonalcohol groups (P < 0.05). The Alcohol + Nicotine group had lesions with significantly larger volume and area when compared to the other groups (P < 0.05), whilst the Alcohol or Nicotine groups had significantly larger lesions than the control group (P < 0.05). There was no significant difference in the largest diameter of the lesions amongst groups (P > 0.05). The experimental groups had greater inflammatory response scores than the control group (P < 0.05), and the representative samples had more pronounced immunoreaction against RANKL and PTHrP antibodies.
CONCLUSIONS: Alcohol and nicotine consumption exacerbated the inflammatory response and the development of periradicular lesions in rats. The association of both substances enhanced their harmful effects.

OBJECTIVE: To establish the in vitro study model of osteoclasts induced by RANKL, to elaborate the effect of formononetin (FO) , an effective component of Caulis Spatholobi, on the differentiation and function of bone marrow mononuclear macrophages (BMMs) into osteoclasts, and to explore the molecular mechanism of its inhibition.
METHODS: The BMMs in femur and tibia were isolated from 20 clean C57/BL6 mice of 4 to 6 weeks old, 10 males and 10 females, each weighing (20± 2) g. The BMMs in femur and tibia were cultured and proliferated in vitro with α-MEM medium. BMMs were cultured with MCSF and different concentrations of anthocyanin (5 to 50 μm) respectively for 4 days, and CCK8 of cell proliferation and toxicity was detected. BMMs in good growth condition were added to M-CSF and RANKL to induce osteoclast differentiation in turn. There was no special treatment in the control group. DMSO was added to the control group with DMSO solvent. Each observation group was added with different concentrations of awnasin (1 to 20 μm) . After 6 days of culture, the osteoclasts were counted and statistically analyzed. The expression of NFATc1, c-Fos and ERK, the key transcription factors in osteoclast differentiation, were detected by Western blot, RNA was extracted at 4 days, and the activity of ctsk, trap, MMP9 and Car2 were detected by real time PCR.
RESULTS: CCK8 test results showed that awnstein could inhibit the activity of BMMs in a dose-dependent manner, and had no significant toxic effect on the growth of bmms within the safe concentration range of ≤20 μM (P= 0.278>0.05) . The results of trap staining showed that awnstein could inhibit osteoclast production in a dose-dependent manner in the concentration range of (1 to 20 μM) , especially in 10 μM (P=0.000<0.05) . Western blot showed that 10 μ m could significantly inhibit the expression of NFATc1 and c-Fos, but not the expression of ERK. In terms of osteoclast function, the expression of ctsk (P=0.000<0.05) , trap (P=0.000<0.05) , MMP9 (P=0.000<0.05) and Car2 (P=0.000<0.05) related to osteoclast function were detected by real time PCR.
CONCLUSIONS: The effective component of Caulis Spatholobi can inhibit the proliferation and differentiation of primary mononuclear macrophages into osteoclasts, and down regulate the expression of osteoclast bone resorption related proteins and genes, which may be one of the mechanisms of its prevention and treatment of bone destruction and collapse in osteonecrosis of femoral head.

Although studies have established the role of integrins in bone homeostasis, especially in osteoclastogenesis, and these molecules are novel and promising therapeutic drug targets for bone loss diseases, such as: osteolysis, the cellular mechanism still elusive. Bone homeostasis takes place through the interaction of bone cells (osteoblasts and osteoclast) via the activation of intercellular adhesion molecule-1 (ICAM-1), which is a critical submolecule of integrin. In the present study, we reviewed several novel studies on integrins and their submolecule, ICAM-1, in bone homeostasis. In order to demonstrated that ICAM-1 might exert dual effects on osteoclastogenesis by directly affecting the adhesive ability of mature osteoclasts and indirectly participating in RANKL/RANK induced osteoclastic precursors differentiation. Although these results still need to be verified in the future, the extending study about the role of ICAM-1 in osteoclastogenesis will cerntainly provide a promising therapeutic target for the treatment of bone loss diseases.

Despite the response to the receptor activator of nuclear factor κ-Β ligand (RANKL), a study has reported that lipopolysaccharide (LPS) could induce RAW264.7 linage osteoclastic differentiation. However, on the contrary, another study recently showed that the LPS-induced multinuclear cells from RAW264.7 did not express osteoclastic functions. Interestingly, in our previous study, we found that RAW264.7 cells pretreated with 10 ng LPS plus macrophage-colony stimulating factor did not show any effects for enhancing RANKL-induced osteoclastic cell differentiation. Therefore, in our current study, we aim to investigate the oteoclastogesis induction ability and efficacy of LPS in the RAW264.7 cell line and relevant molecular signaling. The osteoclastogenic activity of LPS-treated RAW264.7 linage was studied by bone resorption pits and fibrous actin study. Besides that, through polymerase chain reaction and western blot, we showed that the transcriptional factor c-Fos and Nfatc1 might be associated with LPS-induced osteoclastogenesis. Overall, the results of our current study showed positive proof for osteoclast generation from LPS-independent treatment, as well as established an optimal and efficient method for this process.