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Background: The differential responses of the myokine irisin, in combination with changes in markers and regulators of bone remodeling to high-intensity interval exercise of high and low impact, were examined in 18 young adult females (22.5 ± 2.7 years). Methods: Participants performed two high-intensity interval exercise trials in random order: running on a treadmill and cycling on a cycle ergometer. Trials consisted of eight 1 min running or cycling intervals at ≥ 90% of maximal heart rate, separated by 1 min passive recovery intervals. Blood samples were collected at rest (pre-exercise) and 5 min, 1 h, and 24 h following each exercise trial. Irisin, osteocalcin, sclerostin, osteoprotegerin (OPG), receptor activator nuclear factor kappa-β ligand (RANKL), and parathyroid hormone (PTH) were analyzed in serum, with post-exercise concentrations being corrected for exercise-induced changes in plasma volume. Results: Irisin was elevated 24 h post-exercise compared to its resting values in both trials (20%, p < 0.05) and was higher after cycling compared to running (exercise mode effect, p < 0.05) with no interaction. Osteocalcin, sclerostin, PTH, and RANKL increased from pre- to 5 min post-exercise (18%, 37%, 83%, and 33%, respectively, p < 0.05), returning to baseline levels in 1 h, with no trial or interaction effects. OPG showed a time effect (p < 0.05), reflecting an overall increase at 5 min and 1 h post-exercise, which was not significant after the Bonferroni adjustment. Conclusions: In young adult females, high-intensity interval exercise induced an immediate response in markers and regulators of bone remodeling and a later response in irisin concentrations, which was independent of the gravitational impact.

Fermented rice bran (FRB) is known to have numerous beneficial bioactivities, amongst which is its anti-inflammatory properties when used as a supplement. To determine its effects, we examined osteoclastogenesis and bone resorption caused by injections of lipopolysaccharide (LPS), using mice with and without FRB supplementation. The results were favorable: those that received FRB showed reduced osteoclast numbers and bone resorption compared to those with the control diet. Notably, receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-α (TNF-α) mRNA levels were shown to be lower in the LPS-treated animals with FRB supplementation. FRB’s inhibitory effect on RANKL- and TNF-α-induced osteoclastogenesis was further confirmed in vitro. In culture, macrophages exhibited decreased TNF-α mRNA levels when treated with FRB extract and LPS versus treatment with LPS alone, but there was no significant change in RANKL levels in osteoblasts. We can conclude that FRB supplementation dampens the effect of LPS-induced osteoclastogenesis and bone resorption by controlling TNF-α expression in macrophages and the direct inhibition of osteoclast formation.

Cardiomyopathy has become one of the leading causes of death in patients with Duchenne muscular dystrophy (DMD). We recently reported that the inhibition of the interaction between the receptor activator of nuclear factor κB ligand (RANKL) and receptor activator of nuclear factor κB (RANK) significantly improves muscle and bone functions in dystrophin-deficient mdx mice. RANKL and RANK are also expressed in cardiac muscle. Here, we investigate whether anti-RANKL treatment prevents cardiac hypertrophy and dysfunction in dystrophic mdx mice. Anti-RANKL treatment significantly reduced LV hypertrophy and heart mass, and maintained cardiac function in mdx mice. Anti-RANKL treatment also inhibited NFκB and PI3K, two mediators implicated in cardiac hypertrophy. Furthermore, anti-RANKL treatment increased SERCA activity and the expression of RyR, FKBP12, and SERCA2a, leading possibly to an improved Ca2+ homeostasis in dystrophic hearts. Interestingly, preliminary post hoc analyses suggest that denosumab, a human anti-RANKL, reduced left ventricular hypertrophy in two patients with DMD. Taken together, our results indicate that anti-RANKL treatment prevents the worsening of cardiac hypertrophy in mdx mice and could potentially maintain cardiac function in teenage or adult patients with DMD.

Mast cells may contribute to osteoporosis development, because patients with age-related or post-menopausal osteoporosis exhibit more mast cells in the bone marrow, and mastocytosis patients frequently suffer from osteopenia. We previously showed that mast cells crucially regulated osteoclastogenesis and bone loss in ovariectomized, estrogen-depleted mice in a preclinical model for post-menopausal osteoporosis and found that granular mast cell mediators were responsible for these estrogen-dependent effects. However, the role of the key regulator of osteoclastogenesis, namely, receptor activator of NFκB ligand (RANKL), which is secreted by mast cells, in osteoporosis development has, to date, not been defined. Here, we investigated whether mast-cell-derived RANKL participates in ovariectomy (OVX)-induced bone loss by using female mice with a conditional Rankl deletion. We found that this deletion in mast cells did not influence physiological bone turnover and failed to protect against OVX-induced bone resorption in vivo, although we demonstrated that RANKL secretion was significantly reduced in estrogen-treated mast cell cultures. Furthermore, Rankl deletion in mast cells did not influence the immune phenotype in non-ovariectomized or ovariectomized mice. Therefore, other osteoclastogenic factors released by mast cells might be responsible for the onset of OVX-induced bone loss.

Implant-related bone infections are a major complication in orthopedic surgery that lead to inflammation and bone destruction. Bacterial biofilm formation on the implant is discussed to polarize the immune response towards tolerance and to facilitate bacterial persistence. In addition to their role in the early immune response, macrophages are osteoclast precursor cells. Therefore, macrophages can link inflammation and RANKL-mediated osteoclastogenic bone destruction. We investigated the influence of Staphylococcus aureus (SA) and epidermidis (SE) biofilm formation on immune function and osteoclastogenesis using RAW264.7 cells and conditioned media (CM) of planktonic and biofilm cultures in the presence and absence of the osteoclastogenic transcription factor RANKL. Analysis of immune cell activation, metabolic activity and osteoclast formation revealed that a planktonic environment causes a pro-inflammatory response. This was also partially induced by biofilm CM. Simultaneous stimulation with CM and RANKL suppressed osteoclast formation in favor of a long-term immune activation. While the early macrophage response towards CM was dominated by glycolysis, the CM and RANKL approach shifted metabolism towards increased mitochondrial biomass and activity. This was most evident in biofilm CM. We further showed that planktonic CM effects are mediated through activation of TLR signaling and induction of IFN-β production. In biofilm CM, high lactate levels seem to significantly contribute to the modulation of macrophages. Our results can contribute to find targets for therapeutic intervention that restore an effective pro-inflammatory immune response, which could help to control implant-related bone infections. Importance The number of endoprosthetic and bone reconstructive surgeries is rising and accordingly incidences of implant-related bone infections increase. Biofilm formation on the implant surface protects bacteria against most antibiotics and the host’s immune response. Thus, novel therapeutic strategies for prevention or treatment of biofilm formation are needed. Furthermore, bone infections are associated with osteoclast-mediated bone destruction, which promotes infection progression and ultimately leads to implant loosening. We investigated the effect of staphylococci-mediated biofilm formation on macrophage immune activation and osteoclastogenic differentiation, respectively. In planktonic and biofilm environments, immune cell activation dominated over osteoclastogenesis. The effects of planktonic environments were mainly mediated through activation of TLR signaling and resulted in a pronounced pro-inflammatory and IFN-β macrophage immune response. In biofilm environments, high lactate levels impaired osteoclast formation but also prevented an effective long-term immune activation. Therefore, targeting biofilm-derived metabolites but also IFN-β signaling might be promising novel treatment strategies.

Udder traits, influencing udder health and function, are positively correlated with lactation performance. Among them, breast texture influences heritability and impacts on the milk yield of cattle; however, there is a lack of systematic research on its underlying mechanism in dairy goats in particular. Here, we showed the structure of firm udders with developed connective tissue and smaller acini per lobule during lactation and confirmed that there were lower serum levels of estradiol (E2) and progesterone (PROG), and higher mammary expression of estrogen nuclear receptor (ER) α and progesterone receptor (PR), in dairy goats with firm udders. The results of transcriptome sequencing of the mammary gland revealed that the downstream pathway of PR, the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) signal, participated in the formation of firm mammary glands. During the culture of goat mammary epithelial cells (GMECs), high RANKL level additions promote the Inhibitor kappaB (IκB)/p65/Cyclin D1 expression related to cell proliferation and decrease the phosphorylated signal transduction and transcription activator 5 (Stat5) expression related to milk-protein synthesis of GMECs, which is consistent with electron microscope results showing that there are fewer lactoprotein particles in the acinar cavity of a firm mammary. Furthermore, co-culturing with adipocyte-like cells for 7 d is beneficial for the acinar structure formation of GMECs, while there is a slightly negative effect of high RANKL level on it. In conclusion, the results of this study revealed the structure of firm udders structure and confirmed the serum hormone levels and their receptor expression in the mammary glands of dairy goats with firm udders. The underlying mechanism leading to firm udders and a decrease in milk yield were explored preliminarily, which provided an important foundation for the prevention and amelioration of firm udders and improving udder health and milk yield.

The goal of the current study was to examine the effects of prenatal exposure to fumonisins (FBs) on bone properties and metabolism in weaned rat offspring divided into groups intoxicated with FBs at either 0 (the 0 FB group), 60 (the 60 FB group), or 90 mg/kg b.w. 0 (the 90 FB group). Female and male offspring exposed to FBs at a dose of 60 mg/kg b.w. had heavier femora. Mechanical bone parameters changed in a sex and FBs dose-dependent manner. Growth hormone and osteoprotegerin decreased in both sexes, regardless of FBs dose. In males osteocalcin decreased, while receptor activator for nuclear factor kappa-Β ligand increased regardless of FBs dose; while in females changes were dose dependent. Leptin decreased in both male FBs-intoxicated groups, bone alkaline phosphatase decreased only in the 60 FB group. Matrix metalloproteinase-8 protein expression increased in both female FBs-intoxicated groups and decreased in male 90 FB group. Osteoprotegerin and tissue inhibitor of metalloproteinases 2 protein expression decreased in males, regardless of FBs dose, while nuclear factor kappa-Β ligand expression increased only in the 90 FB group. The disturbances in bone metabolic processes seemed to result from imbalances in the RANKL/RANK/OPG and the OC/leptin systems.

© 2021 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/), The effective osteointegration of orthopedic implants is a key factor for the success of orthopedic surgery. However, local metabolic imbalance around implants under osteoporosis condition could jeopardize the fixation effect. Inspired by the bone structure and the composition around implants under osteoporosis condition, alendronate (A) was grafted onto methacryloyl hyaluronic acid (H) by activating the carboxyl group of methacryloyl hyaluronic acid to be bonded to inorganic calcium phosphate on trabecular bone, which is then integrated with aminated bioactive glass (AB) modified by oxidized dextran (O) for further adhesion to organic collagen on the trabecular bone. The hybrid hydrogel could be solidified on cancellous bone in situ under UV irradiation and exhibits dual adhesion to organic collagen and inorganic apatite, promoting osteointegration of orthopedic implants, resulting in firm stabilization of the implants in cancellous bone areas. In vitro, the hydrogel was evidenced to promote osteogenic differentiation of embryonic mouse osteoblast precursor cells (MC3T3-E1) as well as inhibit the receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteoclast differentiation of macrophages, leading to the upregulation of osteogenic-related gene and protein expression. In a rat osteoporosis model, the bone-implant contact (BIC) of the hybrid hydrogel group increased by 2.77, which is directly linked to improved mechanical stability of the orthopedic implants. Overall, this organic-inorganic, dual-adhesive hydrogel could be a promising candidate for enhancing the stability of orthopedic implants under osteoporotic conditions., This work was supported by the National Key R&D Program of China (2020YFA0908200), National Natural Science Foundation of China (82120108017), Six talent peaks project in Jiangsu Province (WSW-018). This work was financed by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” UID/BIM/04293/2019.

Aseptic loosening of the prosthesis caused by wear-particle-induced osteolysis is a long-term complication and one of the most common reasons for the failure of joint implants. The primary cause of aseptic loosening of the prosthesis is overactive bone resorption caused by wear-particle-activated osteoclasts in both direct and indirect ways. Therefore, drugs that can inhibit differentiation and bone resorption of osteoclasts need investigation as a potential therapeutic strategy to prevent and treat peri-prosthetic osteolysis and thereby prolong the service life of the prosthesis. This study has verified the potential inhibitory effect of LY450139 on inflammatory osteolysis induced by titanium particles in a mice skull model. In addition, we found that LY450139 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, bone resorption, and podosomal actin belt formation in a dose-dependent manner without evidence of cytotoxicity in vitro. In addition, LY450139 significantly decreased the expression of osteoclast-specific markers, including TRAP, CTSK, V-ATPase d2, CTR, DC-STAMP, NFATc1, and the downstream target gene Hes1 in Notch signaling pathway. Further investigation of the molecular mechanism demonstrated that LY450139 inhibited the formation of osteoclasts via inhibition of the NF-κB and Notch signaling pathways. In summary, LY450139 inhibited the formation of RANKL-mediated osteoclasts via NF-κB and Notch signaling and inhibited Ti particle-induced inflammatory osteolysis in vivo. LY450139 is a potential targeted drug for the treatment of peri-prosthetic osteolysis and other osteolytic disease associated with overactive osteoclasts.

Osteoporosis is a reduction in skeletal mass due to the decrease of osteogenic ability and the activation of the osteoclastic function. Inhibiting bone resorption and accelerating the new bone formation is a promising strategy to repair the bone defect of osteoporosis. In this study, we first systematically investigated the roles of Chinese medicine Asperosaponin VI (ASP VI) on osteogenic mineralization of BMSCs and osteoclastogenesis of BMMs, and then explored the synergistic effect of ASP VI and BS (BMP-2 immobilized in 2-N, 6-O-sulfated chitosan) on bone formation. The result showed that ASP VI with the concentration lower than 10−4 M contributed to the expression of osteogenic gene and inhibited osteoclastic genes RANKL of BMSCs. Simultaneously, ASP VI significantly reduced the differentiation of mononuclear osteoclasts in the process of osteoclast formation induced by M-CSF and RANKL. Furthermore, by stimulating the SMADs, TGF-β1, VEGFA, and OPG/RANKL signaling pathways, ASBS (ASP VI and BS) substantially enhanced osteogenesis, greatly promoted angiogenesis, and suppressed osteoclastogenesis. The findings provide a new perspective on osteoporosis care and prevention., Graphical abstract Image 1, Highlights • Asperosaponin VI down-regulated the osteoclastic genes RANKL expression. • First combined Asperosaponin VI with rhBMP-2 and 2-N, 6-O-sulfated chitosan. • Show high osteogenic activity and excellent anti-osteoclastogenesis. • Explored mechanism on promoting osteogenesis and inhibiting osteoclastogenesis.

Postmenopausal osteoporosis is a systemic disease characterized by the loss of bone mass and increased bone fracture risk largely resulting from significantly reduced levels of the hormone estrogen after menopause. Besides the direct negative effects of estrogen-deficiency on bone, indirect effects of altered immune status in postmenopausal women might contribute to ongoing bone destruction, as postmenopausal women often display a chronic low-grade inflammatory phenotype with altered cytokine expression and immune cell profile. In this context, it was previously shown that various immune cells interact with osteoblasts and osteoclasts either via direct cell-cell contact, or more likely via paracrine mechanisms. For example, specific subtypes of T lymphocytes express TNFα, which was shown to increase osteoblast apoptosis and to indirectly stimulate osteoclastogenesis via B cell-produced receptor-activator of NF-κB ligand (RANKL), thereby triggering bone loss during postmenopausal osteoporosis. Th17 cells release interleukin-17 (IL-17), which directs mesenchymal stem cell differentiation towards the osteogenic lineage, but also indirectly increases osteoclast differentiation. B lymphocytes are a major regulator of osteoclast formation via granulocyte colony-stimulating factor secretion and the RANKL/osteoprotegerin system under estrogen-deficient conditions. Macrophages might act differently on bone cells dependent on their polarization profile and their secreted paracrine factors, which might have implications for the development of postmenopausal osteoporosis, because macrophage polarization is altered during disease progression. Likewise, neutrophils play an important role during bone homeostasis, but their over-activation under estrogen-deficient conditions contributes to osteoblast apoptosis via the release of reactive oxygen species and increased osteoclastogenesis via RANKL signaling. Furthermore, mast cells might be involved in the development of postmenopausal osteoporosis, because they store high levels of osteoclastic mediators, including IL-6 and RANKL, in their granules and their numbers are greatly increased in osteoporotic bone. Additionally, bone fracture healing is altered under estrogen-deficient conditions with the increased presence of pro-inflammatory cytokines, including IL-6 and Midkine, which might contribute to healing disturbances. Consequently, in addition to the direct negative influence of estrogen-deficiency on bone, immune cell alterations contribute to the pathogenesis of postmenopausal osteoporosis., publishedVersion

Introduction: Favipiravir and Vitamin C (Vit C) were used together in the treatment of the COVID-19 pandemic. However, the effects of favipiravir on the periodontium are still unknown. Therefore, the aim of this study was to investigate the effects of Favipiravir and Vit C treatment on alveolar bone metabolism. Experimental: Fifty healthy adult male Sprague-Dawley rats (2-3 months old) were randomly divided into five equal groups (n = 10): Control, Favi 20, Favi 100, Favi 20+Vit C, Favi 100+Vit C. Favipiravir (20 mg/kg and 100 mg/kg, i.m.) and Vit C (150 mg/kg/day, oral) were administered to the rats for 14 days. Alveolar bone loss (ABL) and histopathological changes were examined using a light microscope. Immunohistochemistry was used to determine levels of receptor activator of nuclear factor kappa-B ligand (RANKL), caspase-3, bone morphogenic protein 2 (BMP-2) and alkaline phosphaTaşe (ALP) in the bone tissues. Results: Favipiravir increased the levels of RANKL and caspase-3 expression but decreased BMP-2 and ALP levels in a dose-dependent manner. Favi 20+Vit C and Favi 100 +Vit C groups showed decreased RANKL and caspase-3 levels in addition to increased BMP-2 and ALP levels. Conclusion: Favipiravir can cause histopathological damage to the periodontium, but administration of favipiravir combined with Vit C can provide a protective effect against this damage.

Osteoclasts (OCs), the main cause of bone resorption irregularities, may ultimately cause various bone diseases, including osteoarthritis. The objective of this study was to investigate the effect of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) on OC formation induced by receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) and to further explore its underlying mechanism. IFIT1 expression in Raw264.7 cells treated with macrophage colony-stimulating factor (M-CSF) and RANKL was determined by qRT-PCR. OC formation was detected using tartrate-resistant acid phosphatase (TRAP) staining. The effect of IFIT1 on STAT3 activation was detected using Western blotting. Additionally, Western blotting was used to measure the change in the expression of OC-specific proteins. IFIT1 was highly expressed in Raw264.7 cells after stimulation with M-CSF and RANKL. IFIT1 overexpression accelerated the formation of OCs, as evidenced by the increased number and size of multinuclear cells, and the upregulation of OC-specific proteins, and activated the STAT3 pathway, by inducing phosphorylation of JAK1 and STAT3. However, silencing of IFIT1 inhibited the formation of OCs and a STAT3 inhibitor Stattic weakened the effects of IFIT1. In conclusion, IFIT1 accelerates the formation of OCs, which is caused by RANKL by STAT3 pathway regulation. This study provides a potential basis for further research and for development of drugs for treating bone resorption-related diseases.

Perillaldehyde (PAH), one of the active ingredients of the traditional Chinese medicine (TCM) plant Perilla frutescens, is widely used and exerts crucial anti-cancer activities. The aim of current study is to illustrate the potential mechanisms of PAH-mediated regulation of bone metastasis and osteoclastogenesis in prostate cancer (PCa) cell lines. Effects of PAH on proliferation, invasion and migration of PC-3 cells were assessed with the Cell Counting Kit-8 (CCK-8) assay and Transwell assays, respectively. Effects of PAH on stem cell characteristics of PC-3 cells were evaluated by cell-matrix adhesion assay, colony formation assay, spheroid formation assay, as well as western blot . The anti-metastasis and anti-osteoclastogenesis activity of PAH in RAW264.7 cells was examined by osteoclast differentiation assay and western blot. The protein levels of CD133 and CD44 in PC-3 cells and the activity of nuclear factor kappa B (NF-κB) signaling pathway in RAW264.7 cells were measured by western blot. PAH suppressed proliferation, invasion and migration of PC-3 cells, prevented stem cell characteristics including cell-matrix adhesion, colony formation, spheroid formation as well as CD133 and CD44 expression. PAH inhibited bone metastasis and osteoclastogenesis via repressing the activation of NF-κB pathway as well as (RANKL)- and cancer cell- induced osteoclastogenesis in PCa cells. These findings suggested the potential therapeutic effects of PAH on the metastasis of patients with PCa.

Cardiovascular calcifications (CVC) are frequently observed in chronic kidney disease (CKD) patients and contribute to their cardiovascular mortality. The aim of the present study was to investigate the impact of osteoprotegerin (OPG)/Receptor Activator of NF-κΒ (RANK)/RANK ligand (RANKL) pathway in the development and evolution of CVCs in hemodialysis patients. In total, 80 hemodialysis patients were assessed for the presence of vascular (abdominal aorta and muscular arteries) calcifications and results were correlated to serum OPG and RANKL levels and the OPG/RANKL ratio. Traditional cardiovascular risk factors and mineral bone disease parameters were also estimated. The presence of VCs was also evaluated 5 years after the initiation of the study, and results were correlated to the initial serum OPG levels. Age, diabetes mellitus, coronary artery disease and OPG levels (p < 0.001) were associated with VCs, whereas RANKL levels were not. Multivariate analysis though revealed that only OPG levels were significantly associated with abdominal aorta calcifications (p = 0.026), but they were not correlated with the progression of VCs. Serum OPG levels are positively and independently associated with VCs in HD patients, but not with their progression. RANKL levels did not show any associations, whereas further studies are needed to establish the significance of OPG/RANKL ratio.

Peficitinib, a pan-JAK inhibitor, is known to suppress the activation of fibroblast-like synoviocytes (FLSs) and thereby reduces joint inflammation associated with rheumatoid arthritis (RA). However, the effect on osteoporosis in RA remains to be elucidated. In this study, the effect of peficitinib or etanercept on joint inflammation, and consequently decreased bone mineral density (BMD) was evaluated in mice with collagen-induced arthritis (CIA). Additionally, the effect on RANKL production from osteoblasts differentiated from the mesenchymal stem cells of RA patients was evaluated. Administration of peficitinib for established CIA ameliorated arthritis and improved BMD in the femoral metaphysis, but not in the femoral diaphysis. Conversely, etanercept suppressed an increase in synovial inflammatory markers but did not improve arthritic conditions or the reduction of BMD in either region. All elevated bone formation and bone resorption markers were decreased with peficitinib but only partially decreased with etanercept. Furthermore, production of RANKL by human osteoblasts was suppressed by peficitinib but enhanced by etanercept. Unlike etanercept, peficitinib is thought to increase BMD by ameliorating the high bone turnover associated with RA states, resulting in improvement of bone fragility. Our data provide evidence that peficitinib would be expected to show efficacy for osteoporosis associated with RA.

Objective. To assess the effect of the combined treatment method including preoperative denosumab therapy on the results of treatment of patients with giant cell tumors of the spine.Material and Methods. A single-center retrospective-prospective study of a series of clinical cases included 15 patients with giant cell tumors of vertebrae. The average follow-up period was 56 months. A total of 11 patients received denosumab therapy according to the following scheme: 120 mg subcutaneously on the 1st, 8th, 15th and 28th days of the first month and then once every 28 days. Surgical options included marginal resection, segmental resection, or en-bloc resection with or without spinal reconstruction/stabilization. In the case of locally advanced and inoperable disease, long-term therapy with denosumab was carried out until the disease progressed or serious adverse events appeared.Results. Thoracic vertebrae were involved in 7 (46.6 %) of 15 cases, lumbar in 4 (26.7 %) and cervical in 4 (26.7 %). Local recurrence rate after surgery alone was 40 % (2/5), average time to recurrence onset was 4.5 months. No relapses were observed after combined treatment performed in four patients. Disease progression during long-term denosumab therapy for inoperable disease recurrence was not recorded (0/7). The average number of denosumab injections before surgery and during long-term therapy was 15 and 24 injections, respectively. Denosumab therapy allows reducing the duration of surgery and the volume of blood loss.Conclusion. Combined therapy of giant cell vertebral tumor allows to reduce the risk of recurrence of the disease, as well as to reduce surgery duration and blood loss. Long-term continuous therapy for inoperable cases allows achieving long-term stabilization of the effect. Due to the rarity of giant cell tumors of the spine, a further prospective recruitment of patients is required to study the efficacy and safety of combined therapies.

Osteoporosis is a systemic metabolic bone disease with an increasing incidence rate. Chinese medicinal herbs have a long history of treating bone diseases. Polysaccharides are an important category of phytochemicals in Chinese medicinal herbs, and their health benefits have increased the interest of the public. Numerous studies have indicated that polysaccharides exhibit anti-osteoporosis effects by balancing bone resorption and bone formation, but the detailed effects and mechanism have not been systematically summarized. We performed a comprehensive review of the literature to consolidate studies for the period 2000–2021 by conducting electronic searches on the PubMed, CNKI, VIP, and Wanfang databases. In total, polysaccharides from 19 kinds of Chinese medicinal herbs in 54 studies have shown bone homeostasis protective properties. In vivo and in vitro experiments have demonstrated that polysaccharides present properties in the treatment of postmenopausal osteoporosis, senile osteoporosis, and glucocorticoid-induced secondary osteoporosis, especially postmenopausal osteoporosis. Moreover, a number of signalling pathways, such as the Wnt/β-catenin signalling pathway, BMP/SMAD/RUNX2 signalling pathway, OPG/RANKL/RANK signalling pathway, apoptosis pathway, and transcription factors, are regulated by polysaccharides and participate in improving bone homeostasis. This review will provide a better understanding of the anti-osteoporotic effects of polysaccharides and the concomitant modulations of signalling pathways.

Mechanical strain regulated osteoclastic differentiation and angiogenesis are crucial for bone modeling and remodeling, and previous data indicate that high-magnitude strain within physiological load regulates osteoclastic differentiation. However, the underlying mechanisms are still not fully understood. In the present study, the RAW264.7 mouse monocyte/macrophage was used as an osteoclast precursor, and the bone marrow-derived macrophages (BMMs) were isolated and cultured in vitro. The above cells were subjected to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) for the induction of osteoclast differentiation. Subsequently, the above cells were stretched by differential strain magnitudes to simulate the mechanical stimuli in the physiological conditions, and we found that low-magnitude strain (100 με) increased the expression levels of Acp5, Clcn7, MMP9 and Ctsk to promote osteoclastogenesis, while high-magnitude strain (3000 με) had opposite effects. In addition, we noticed that high-magnitude strain upregulated PTEN to inactivate the PI3K/Akt signaling pathway, and silencing of PTEN abrogated the suppressing effects of high-magnitude strain on osteoclastic differentiation. Next, we screened out that high-magnitude strain downregulated miR-21 to promote PTEN expressions in a competing endogenous RNA (ceRNA)-dependent manner. Finally, upregulation of miR-21 recovered osteoclastic differentiation in RAW264.7 and BMMs cells stimulated with high-magnitude strain. Collectively, our findings suggested that high-magnitude mechanical strain affected osteoclastic differentiation through modulating the miR-21/PTEN/PI3K/Akt signaling cascade, which provided potential strategies for the treatment of bone-related diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00507-x.

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and bone quality and increased bone porosity, which increase the risk of bone fracture. Inflammation, one of the important mechanisms related to aging, is associated with osteoporosis. Treatment with anti-inflammatory agents is effective for alleviating senile osteoporosis. Alginate oligosaccharide (AOS) can prevent and treat diseases related to inflammation, oxidative stress, and immunity. This study evaluates the effect of AOS on osteoporosis and investigates the underlying mechanism. Osteoporosis model was induced by D-galactose (D-gal) (200 mg kg-1 day-1 ) for eight weeks. Three groups were administered via AOS (50, 100, and 150 mg kg-1 day-1 ) for four weeks, while a control group received sterile water (5 ml kg-1 day-1 ) for 8 weeks. The results showed that AOS improved bone density and bone microstructure in D-gal-induced osteoporosis mice. AOS inhibited osteoclast proliferation, probably through the suppression of receptor activator of nuclear factor-kappa B ligand (RANKL)-associated nuclear factor kappa B (NF-κB) and c-Fos signaling pathway. AOS also increased osteoprotegerin (OPG) expression and competitively inhibited the binding between RANK and RANKL in senile osteoporosis. Further, AOS decreased the secretion of serum osteocalcin and reduced bone conversion. Together, these results demonstrate the anti-osteoporosis activity of AOS in mice with osteoporosis.

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen-activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/β-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/β-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SNP rs3755955 (major/minor allele: G/A) located in Iduronidase-Alpha-L- (IDUA) gene was reported to be significant for human bone mineral density (BMD). This follow-up study was to uncover the underlying association mechanism through molecular and cellular functional assays relevant to bone. We tested the effects of single nucleotide polymorphisms (SNP) rs3755955 (defined allele G as wild-type and allele A as variant-type) on osteoblastic and osteoclastic functions, as well as protein phosphorylation in stably transfected human fetal osteoblast (hFOB) cell and mononuclear-macrophage (RAW264.7) cell. In hFOB cells, transfection with variant-type IDUA significantly decreased osteoblastic gene expression (OPN, COL1A1 and RANKL) (p

Vertebral osteomyelitis (VO) is an infection of the spine mainly caused by bacterial pathogens. The pathogenesis leading to destruction of intervertebral discs (IVDs) and adjacent vertebral bodies (VBs) is poorly described. The present study aimed at investigating the connection between infection and bone/disc metabolism in VO patients. 14 patients with VO (infection group) and 14 patients with burst fractures of the spine (fracture group; control) were included prospectively. Tissue biopsies from affected IVDs and adjacent VBs were analysed by RT-qPCR for mRNA-expression levels of 18 target genes including chemokines, adipokines and genes involved in bone metabolism. Most importantly, the receptor activator of NF-κB/osteoprotegerin (RANK/OPG) expression ratio was drastically elevated in both VBs and IVDs of the infection group. In parallel, expression of genes of the prostaglandin-E2-dependent prostanoid system was induced. Such genes regulate tissue degradation processes via the triad OPG/RANK/RANKL as well as via the chemokines IL-8 and CCL-20, whose expression was also found to be increased upon infection. The gene expression of the adipokine leptin, which promotes inflammatory tissue degradation, was higher in IVD tissue of the infection group, whereas the transcription of omentin and resistin genes, whose functions are largely unknown in the context of infectious diseases, was lower in infected VBs. In summary, similar expression patterns of pro-inflammatory cytokines and pro-osteoclastogenic factors were identified in VBs and IVDs of patients suffering from VO. This suggests that common immuno-metabolic pathways are involved in the mechanisms leading to tissue degradation in VBs and IVDs during VO.

Osteoporosis is caused by an osteoclast activation mechanism. People suffering from osteoporosis are prone to bone defects. Increasing evidence indicates that scavenging reactive oxygen species (ROS) can inhibit receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and suppress ovariectomy-induced osteoporosis. It is critical to develop biomaterials with antioxidant properties to modulate osteoclast activity for treating osteoporotic bone defects. Previous studies have shown that manganese (Mn) can improve bone regeneration, and Mn supplementation may treat osteoporosis. However, the effect of Mn on osteoclasts and the role of Mn in osteoporotic bone defects remain unclear. In present research, a model bioceramic, Mn-contained β-tricalcium phosphate (Mn-TCP) was prepared by introducing Mn into β-TCP. The introduction of Mn into β-TCP significantly improved the scavenging of oxygen radicals and nitrogen radicals, demonstrating that Mn-TCP bioceramics might have antioxidant properties. The in vitro and in vivo findings revealed that Mn2+ ions released from Mn-TCP bioceramics could distinctly inhibit the formation and function of osteoclasts, promote the differentiation of osteoblasts, and accelerate bone regeneration under osteoporotic conditions in vivo. Mechanistically, Mn-TCP bioceramics inhibited osteoclastogenesis and promoted the regeneration of osteoporotic bone defects by scavenging ROS via Nrf2 activation. These results suggest that Mn-containing bioceramics with osteoconductivity, ROS scavenging and bone resorption inhibition abilities may be an ideal biomaterial for the treatment of osteoporotic bone defect., Graphical abstract A schematic diagram showing the function of Mn-TCP bioceramics in osteoporotic bone defect regeneration. Compared to TCP bioceramics, Mn-TCP bioceramics significantly inhibit osteoclastic differentiation and promote osteoporotic bone regeneration by scavenging ROS through Nrf2 activation.Image 1, Highlights • Mn-containing bioceramics with osteoconductivity, ROS scavenging and bone resorption inhibition abilities were prepared. • Mn-containing bioceramics inhibited osteoclastogenesis by scavenging ROS via Nrf2 activation in vitro. • Mn-containing bioceramics acted as antioxidant biomaterials accelerated bone defect regeneration in osteoporotic rats. • Mn-containing bioceramics can be further applied as a biomaterial for treating osteoporotic bone defects.

Context: Osteoporosis occurs not only in the lumbar bone, femur, and radius but also occurs in the mandibular bone. Therefore, osteoporosis is a disease that required attention from the dentists. Aims: To determine the ratio of Receptor Activator of Nuclear Factor kB-Ligand:Osteoprotegerin (RANKL:OPG) expression and the ratio of osteoclasts:osteoblasts number in the mandibular bone of the osteoporosis rat models. Methods: The research subjects used were 20 Rattus novergicus, which were randomly divided into two groups, namely ovariectomy (OVX) and sham surgery (SHS). Twelve weeks after surgery, the animals were terminated and mandibular bone specimens were taken for histological examination of osteoclasts, osteoblasts, RANKL, and OPG expression. An examination of estrogen levels in the blood of the research subjects was also carried out. All data obtained were tested statistically using a t-test. Results: There was a decrease in the amount of estrogen in the OVX group rather than SHS (p = 0.005). In the SHS group, the RANKL:OPG expression value ratio was 0.51 ± 0.15, while in the OVX group was 0.86 ± 0.22 (p = 0.001). In the SHS group, the ratio of osteoclasts:osteoblasts number was 0.012 ± 0.004, while in the OVX group was 0.061 ± 0.023 (p = 0.001). Conclusions: A decrease in estrogen significantly induces the ratio of RANKL:OPG and osteoclast:osteoblasts in the mandibular bone.

Periodontal disease is an inflammatory disease caused by periodontopathogenic bacteria, which eventually leads to bone tissue (alveolar bone) destruction as inflammation persists. Periodontal tissues have an immune system against the invasion of these bacteria, however, due to the persistent infection by periodontopathogenic bacteria, the host innate and acquired immunity is impaired, and tissue destruction, including bone tissue destruction, occurs. Osteoclasts are essential for bone destruction. Osteoclast progenitor cells derived from hematopoietic stem cells differentiate into osteoclasts. In addition, bone loss occurs when bone resorption by osteoclasts exceeds bone formation by osteoblasts. In inflammatory bone disease, inflammatory cytokines act on osteoblasts and receptor activator of nuclear factor-κB ligand (RANKL)-producing cells, resulting in osteoclast differentiation and activation. In addition to this mechanism, pathogenic factors of periodontal bacteria and mechanical stress activate osteoclasts and destruct alveolar bone in periodontitis. In this review, we focused on the mechanism of osteoclast activation in periodontitis and provide an overview based on the latest findings.

Jun-ming Huang, Chen-zhong Wang, Shun-yi Lu, Zhe Wang, Zuo-qin Yan Department of Orthopaedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of ChinaCorrespondence: Zuo-qin Yan 180 Feng Lin Road, Xuhui District, Shanghai, 200032, People’s Republic of ChinaEmail yan.zuoqin@zs-hospital.sh.cnBackground: Osteoclasts are the major players in bone resorption and have always been studied in the prevention and treatment of osteoporosis. Previous studies have confirmed that a variety of flavonoids inhibit osteoporosis and improve bone health mainly through inhibiting osteoclastogenesis. Oroxin B (OB) is a flavonoid compound extracted from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent, exerts potent antitumor and anti-inflammation effect, but its effect on osteoclastogensis remains unknown.Methods: We comprehensively evaluated the effect of OB on the formation and function of osteoclasts and the underling mechanism by bone marrow-derived macrophage in vitro. In vivo, we used mice ovariectomized model to verify the protective effect of OB.Results: OB was found to inhibit osteoclast formation and bone resorption function in vitro, in a dose-dependent manner and the increased osteoclastic-related genes induced by RANKL (NFATc1, c-fos, cathepsin K, RANK, MMP9 and TRAP) were also attenuated following OB treatment. Mechanistical investigation showed OB abrogated the increased phosphorylation level of MAPK and NF-κB pathway, and diminished the expression of the vital transcription factors for osteoclastogenesis. OB also prevented ovariectomy (OVX)-induced bone loss by inhibiting osteoclast formation and activity in mice.Conclusion: Our study demonstrated that OB may act as an anti-osteoporosis agent by inhibiting osteoclast maturation and attenuating bone resorption.Keywords: osteoclast, Oroxin B, MAPK, NF-κB, RANKL, osteoporosis

BACKGROUND: In fibrous dysplasia/McCune-Albright syndrome (FD/MAS), mosaic mutations in the GNAS gene lead to locally abnormal bone turnover. Additionally, patients with FD/MAS, particularly with thoracic lesions, have an increased risk for breast cancer. Development and progression of breast cancer has been associated with expression of Receptor Activator of NF-κB ligand (RANKL) in mammary tissue, and due to the GNAS mutation, RANKL is systemically increased in patients with FD/MAS. Yet it is unknown whether breast cancer in FD/MAS is also dependent on RANKL. We hypothesized that the GNAS mutation might induce RANKL overproduction and an oncogenic niche in mammary tissue, and examined RANKL expression in breast cancer tissue of patients with FD/MAS compared to controls.METHODS: Nine patients with FD/MAS and breast cancer were included and clinical data were retrieved. Patients were matched to controls with breast cancer without FD/MAS based on age and tumor type. Three pregnant breast cancer patients were included as positive controls. Immunohistochemical detection of RANKL was performed on formalin-fixed paraffin-embedded breast cancer specimens. Staining intensity was classified as weak, moderate or intense. The area of positive RANKL staining divided by the total ductal-lobular area was assessed (positive area percentage, PAP). Number of patients with RANKL expression was compared between FD/MAS and control group by chi-square (χ 2) test, the PAP by Mann-Whitney U test (MWU). RESULTS: RANKL expression was observed in 3 patients with FD/MAS (38 %), mainly in healthy tissue, and none of the control patients (χ 2p = 0.055). The FD/MAS group demonstrated considerably more intense staining than the control group, comparable to positive controls. The median PAP was 0.64 % (range 0.14-2.04 %) in the 3 FD/MAS patients with RANKL expression, 0.01 % (Q1-Q3: 0.0003-0.514 %) in the entire FD/MAS group, 0.006 % (Q1-Q3: 0.001-0.012 %) in the control group (MWU = 0.574), and 0.19 % (0.08-0.32 %) in the pregnant patients. All patients with FD/MAS and RANKL expression had thoracic bone lesions, but no correlation was observed between RANKL expression and presence of the GNAS mutation or FD disease burden. CONCLUSIONS: The triad of a higher number of patients, higher positive area percentage and stronger intensity in the FD/MAS compared to the control group indicates that RANKL may be upregulated in mammary tissue in a subset of patients with FD/MAS, which may explain the increased risk for breast cancer, although the clinical significance remains unclear. Further research is needed to establish risk profiles for the development of RANKL-positive breast cancer and to improve early screening and treatment.

Intestinal microfold (M) cells are critical for sampling antigen in the gut and initiating the intestinal mucosal immune response. In this study, we found that the differentiation efficiency of M cells was closely related to the colitis severity. The expression levels of M cells differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines expression originated from dextran sulfate sodium (DSS) induction and Salmonella infection. Compared with wild-type (WT) mice, MyD88-/- mice exhibited significantly lower expression levels of M cells differentiation-related genes. However, DSS could induce colitis in MyD88-/- mice but failed to promote M cells differentiation. Furthermore, the receptor activator of the Nuclear Factor-κB ligand (RANKL) induced M cells differentiation in murine intestinal organoids prepared from both WT and MyD88-/- mice. However, less M cells differentiation were found in MyD88-/- mice as compared with WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related M cells differentiation.

(1) Background: Osteoporosis is a disease in which bones are weakened and fractured easily because of various factors. It is mainly observed in elderly and postmenopausal women, and it continues to carry high economic costs in aging societies. Normal bone maintains a healthy state through a balanced process of osteoclast suppression and osteoblast activation; (2) Methods: In this study, osteoclast inhibition was induced by inhibiting osteoclast differentiation using ginseng protopanaxadiol-enriched rice (PPD-rice) seed extract. To analyze the effect of PPD-rice extract on the inhibition of osteoclast differentiation, bone marrow macrophages extracted from mice were treated with PPD-rice and Dongjin seed (non-transformed rice) extracts and analyzed for the inhibition of osteoclast differentiation; (3) Results: The results illustrated that PPD-rice extract reduced the transcription and translation of NFATc1, a modulator of osteoclast formation, decreased the mRNA expression of various osteoclast differentiation marker genes, and reduced osteoclast activity. Moreover, the bone resorptive activity of osteoclasts was diminished by PPD-rice extract on Osteo Assay plates; (4) Conclusions: Based on these results, PPD-rice extract is a useful candidate therapeutic agent for suppressing osteoclasts, an important component of osteoporosis, and it could be used as an ingredient in health supplements.

The present study aimed to investigate any biochemical and histological changes of the rat condyle and mandible in animals that had sustained mandibular growth restriction. Seventy-two male Wistar rats were divided into two equal groups, experimental and control. Each group consisted of three equal subgroups. The animals were sacrificed 30, 60, and 90 days after the start of the experiment. Blood samples were collected from the eye, and the osteoprotegerin (OPG), Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), and Macrophage Colony-Stimulating factor (MCSF)concentrations were measured by using enzyme-linked immunosorbent assay (ELISA) kits. A histological analysis was performed on the mandibular condyles. The blood serum values of OPG, RANKL, and MCSF did not exhibit any statistically significant difference between groups or subgroups. However, significant histological changes became evident after a histomorphometric condylar examination was performed. The Bone Surface/Total Surface ratio appeared reduced in the anterior and posterior regions of the condyle. In addition, the Posterior Condylar Cartilage Thickness was measured and determined to be significantly diminished. The present intervention that employed orthodontic/orthopedic devices did not prove to have any significant effect on the circulating proteins under study. Posterior displacement of the mandible may culminate only in local histological alterations in condylar cartilage thickness and its osseous microarchitecture.

The possible mechanisms underlying the quantitative and qualitative effects of cinacalcet on bone were explored in a chronic kidney disease-mineral and bone disorder (CKD-MBD) mouse model in relation to the influence of the interactions among the osteoclast (OC) endoplasmic reticulum (ER) stress, autophagy and apoptosis pathways on OC differentiation. Body weight and biochemical parameters improved significantly in the CKD + cinacalcet groups compared to the CKD group. Micro-computed tomography (μCT) revealed both cortical and trabecular parameters deteriorated significantly in the CKD group and were reversed by cinacalcet in a dose-dependent manner. Nanoindentation analysis of bone quality proved that both cortical hardness and elastic modulus improved significantly with high dose cinacalcet treatment. In vitro studies revealed that cinacalcet inhibited receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK)-induced OC differentiation in a concentration-dependent manner through a close interaction between activation of caspase-related apoptosis, reversal of OC autophagy through the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) pathways, and attenuation of the OC ER stress/CREBH/NFATc1 signaling pathway. Cinacalcet improves both bone quantity and bone quality in CKD mouse model and inhibits OC differentiation through regulation of the interactions among the apoptosis, ER stress, and autophagy pathways within OCs. © 2021 American Society for Bone and Mineral Research (ASBMR).

As part of our continuous program to identify new potential candidates for controlling osteolytic bone diseases from natural products, the alkaloid fraction of barley (Hordeum vulgare var. hexastichon) grass (HVA) significantly inhibited RANKL-induced osteoclast formation and protected mice from LPS-induced bone loss. A phytochemical investigation of HVA afforded nine indole alkaloids, including one new compound [hordeumin A (1)] and eight known analogues (2-9). Of them, four (1, 2, 4, and 5) were anti-osteoclastogenic compounds. Of these four, compound 5 significantly suppressed RANKL-induced osteoclast formation, actin ring formation, and bone resorption in a concentration-dependent manner. It also suppressed the RANKL-induced NF-κB and MAPK signaling pathways and the activation of c-Fos and NFATc1. Compound 5 also reduced the expression levels of osteoclast-specific marker genes, including TRAP, CtsK, DC-STAMP, OSCAR, and MMP9. Our findings suggest that HVA and its alkaloid constituents could be valuable candidates for the prevention and treatment of osteolytic bone diseases.

Cadmium (Cd) contamination in the environment is a major public health concern since it has been linked to osteoporosis and other bone deformities. Linarin is a flavonoid glycoside, and it can promote osteoblastogenesis. This research aimed to investigate the potential role of linarin against Cd-exposed bone deformations in mice model. In our research, male mice were randomly allocated into four groups: control, Cd-exposed, and Cd + linarin (20 and 40mg/kg/bw, respectively). Linarin prevented body weight loss, increased serum calcium (Ca) and phosphorus (P), and bone alkaline phosphatase (BAP) levels in Cd-exposed groups. Furthermore, linarin treatment at 20 and 40mg/kg/bw significantly decreased RANK and OPG, resulting in an increase in RANKL mRNA levels and protein distribution in the bone of Cd-exposed mice. In addition, the bone of Cd-exposed mice administered with linarin showed higher TRAP, NFATc1, MMP9, and RUNX2 mRNA levels and protein distribution. Linarin significantly decreased oxidative stress in Cd-exposed mice bone by decreasing MDA, a lipid peroxidation product. Moreover, linarin protects Cd-exposed mice antioxidant enzymes by increasing bone SOD, CAT, and GPx levels. Besides, linarin suppresses alterations in the inflammatory system, i.e., NF-κB p65/IKKβ, by reducing NF-κB p65, IKKβ, IL-6, and TNF-α in the bone of Cd-exposed animals. This study concluded that linarin has potential to cure osteoporosis in Cd-exposed mice by reducing oxidative stress and inflammation and modulating the RANK/RANKL/OPG pathway.

Objective Receptor activator of NF-κβ ligand (RANKL) is crucial for the development of hepatic insulin resistance and poor glucose uptake; therefore, inhibiting RANKL with Denosumab could improve fasting plasma glucose (FPG) and insulin (FPI). Methods A systematic review was conducted to evaluate the effects of Denosumab on glycemic parameters. PubMed, SCOPUS, EBSCO, and LILACS databases were searched for studies that investigated the effect of Denosumab on FPG, glycated hemoglobin (HbA1c), FPI, and Homeostatic Model Assessment for Insulin Resistance (HOMA1-IR). The pooled standard difference in means (SDM) and 95% confidence intervals (95%CI) were calculated. The results were stratified into (1) Normal Glucose Tolerance (NGT) and (2) Impaired Glucose Tolerance (IGT). Results Six publications (1203 participants) were included. There was a significant association between Denosumab and FPG (SDM = −0.388, 95%CI: −0.705 to −0.070, p = .017) and with HOMA1-IR (SDM = −0.223, 95%CI: −0.388 to −0.058, p = .008), but not for HbA1c and FPI. When stratified by glucose tolerance, the association between Denosumab and FPG, HbA1c, and HOMA1-IR was present for the IGT group. Lastly, Denosumab had a time-dependent effect on HbA1c (slope = −0.037, 95%CI: −0.059 to −0.015, p

Type 2 Diabetes Mellitus is a chronic metabolic disease that causes endothelial damage and is an important risk factor for atherosclerosis. In the present study vitamin D3 supplementation in rats was used to determine the role of Osteoprotegerin (OPG)/Receptor activator kB ligand (RANKL) signalling in endothelial damage and changes in the expression levels of genes involved in this pathway. We hypothesized that vitamin D3 supplementation affects OPG and RANKL activity in the aorta. Diabetes was induced in rats via injections of 40 mg/kg of streptozotocin followed by a high fructose (10%) diet. Group 2 (healthy) and 4 (diabetic) received 170 IU/kg of vitamin D3 weekly for 5 weeks, while Group 1 (healthy) and 2 (diabetic) received sterile saline. The aortas of each group were collected to analyse mRNA expression using the real-time PCR method and also to evaluate magnesium and calcium levels using inductively coupled plasma mass spectrometry. Opg and Il-1b expression levels were significantly associated with both diabetes and vitamin D3 supplementation in the aortas of the study groups (p ≤ 0.05). Opg mRNA expression was also found to correlate with both Icam-1 and Nos3 mRNA expression levels (r = 0.699, p = 0.001 and r = 0.622, p = 0.003, respectively). In addition, when mineral levels in the aortic tissues were compared among all groups, it was found that the interaction of diabetes and vitamin D3 supplementation significantly affected Mg levels and Mg/Ca ratios. It is concluded that vitamin D3 supplementation has a modulatory effect on OPG/RANKL activity in the vessel wall by ameliorating endothelial damage in diabetes. This effect may contribute to the regulation of cytokine-mediated vascular homeostasis and mineral deposition in the aorta; therefore, further comprehensive studies are proposed to demonstrate this relationship.

Osteoporosis is an age‐related disease characterized by low mineral density, compromised bone strength and increased risk of fragility fracture. Most agents for treating osteoporosis focus primarily on anti‐resorption by inhibiting osteoclast activity. Bisphosphonate (BP) is a potent anti‐resorptive agent that has been used clinically for decades and is proven to be effective. However, BP has a variety of side effects and is far from being an ideal anti‐osteoporosis agent. BP selectively binds to calcium crystals, which are subsequently taken up or released by osteoclasts. Based on the action of BP, we previously demonstrated the inhibitory effect of a novel bone‐targeting BP derivative, bisphosphonate‐enoxacin (BE). In the current study, we used bone marrow‐derived osteoclast cultures to further assess the inhibitory effect of BE on osteoclastogenesis and employed reverse transcription PCR and real‐time PCR to examine expression of osteoclast‐specific genes. Additionally, we used bone resorption and F‐actin immunofluorescence assays to evaluate the effect of BE on osteoclast function and investigated the potential mechanisms affecting osteoclast differentiation and function in vitro. Furthermore, an ovariectomized (OVX) rat model was established to evaluate the therapeutic effects of BE on preventing bone loss. Results showed that BE exerted potent inhibitory effects on osteoclast formation and bone resorption by specifically abrogating RANKL‐induced JNK signalling, and that it preserved OVX rat bone mass in vivo without any notable side effects. Collectively, these results indicated that the BP derivative BE may have significant potential as a treatment for osteoporosis and other osteolytic diseases.

Osteosarcoma is a major form of malignant bone tumor that typically occurs in young adults and children. The combination of aggressive surgical strategies and chemotherapy has led to improvements in survival time, although individuals with recurrent or metastatic conditions still have an extremely poor prognosis. This disappointing situation strongly indicates that testing novel, targeted therapeutic agents is imperative to prevent the progression of osteosarcoma and enhance patient survival time. Curcumin, a naturally occurring phenolic compound found in Curcuma longa, has been shown to have a wide variety of anti-tumor, anti-oxidant, and anti-inflammatory activities in many types of cancers including osteosarcoma. Curcumin is a highly pleiotropic molecule that can modulate intracellular signaling pathways to regulate cell proliferation, inflammation, and apoptosis. These signaling pathways include RANK/RANKL, Notch, Wnt/β-catenin, apoptosis, autophagy, JAK/STAT, and HIF-1 pathways. Additionally, curcumin can regulate the expression of various types of microRNAs that are involved in osteosarcoma. Therefore, curcumin may be a potential candidate for the prevention and treatment of osteosarcoma. This comprehensive review not only covers the use of curcumin in the treatment of osteosarcoma and its anti-cancer molecular mechanisms but also reveals the novel delivery strategies and combination therapies with the aim to improve the therapeutic effect of curcumin.

Fracture healing is a complex event with the involvement of many cell systems, cytokines, as well as mRNAs. Herein, we report the interactions among long noncoding RNA X-inactive specific transcript (XIST)/microRNA-135 (miR-135)/cAMP response element-binding protein 1 (CREB1) axis during fracture healing. We observed increased expression of XIST in patients with long-term unhealed fracture by microarray analysis. Subsequently, a mouse model with tibial fracture and a cell model using osteoblast-like MC3T3-E1 cells were generated. The XIST overexpression during fracture healing decreased proliferation and differentiation of MC3T3-E1 cells, while silencing of XIST facilitated MC3T3-E1 cell growth. Furthermore, miR-135 targeted CREB1 and negatively regulated its expression. XIST acted as a sponge for miR-135, thereby upregulating CREB1 and promoting the activity of the TNF-α/RANKL pathway. Transfection of miR-135 inhibitor or CREB1 overexpression blocked the stimulating effects of XIST knockdown on MC3T3-E1 cell growth. Besides, specific inhibitors of the TNF-α/RANKL pathway reversed the repressive role of XIST in cell osteogenic differentiation. All in all, these findings suggest that XIST knockdown induces the differentiation of osteoblast-like cells via regulation of the miR-135/CREB1/TNF-α/RANKL axis. XIST, as a consequence, represents an attractive therapeutic strategy to accelerate fracture healing.

A simple methylenedioxy dibromoindole alkaloid, amakusamine (1), was isolated from a marine sponge of the genus Psammocinia, and its structure was determined from spectroscopic data, time-dependent density-functional theory calculations, and synthesis. Compound 1 inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinuclear osteoclasts with an IC50 value of 10.5 μM in RAW264 cells. The structure-activity relationship of 1 was also investigated with synthetic derivatives.

Objective MicroRNA-146a (miR-146a) is a regulator of inflammatory response. Periodontitis is a disease with immune pathophysiology of the periodontium in which the inflammation results in the destruction of the soft tissues and alveolar bone. Therefore, the aim of this study was to investigate the expressions of miR-146a, OPG, and RANKL in diseased and healthy periodontal tissues to understand whether miR-146a expression level may associate with OPG and RANKL mRNA levels and OPG/RANKL ratio after non-surgical periodontal treatment. Methods The levels of miR-146a, RANKL, and OPG in gingival tissues from patients with generalized periodontitis stages II and III and grades A and B (n = 15, group A), patients with generalized periodontitis stages III and IV and grade C (n = 15, group B), and healthy individuals (n = 10) were determined by real-time PCR. The associations of miR-146a expression with OPG and RANKL levels were evaluated. Results The levels of miR-146a in two subgroups within periodontitis patients were significantly higher than healthy subjects (P P P Conclusions The results of this study failed to show the associations of miR-146a level with OPG and RANKL levels and OPG/RANKL ratio in periodontitis after non-surgical periodontal treatment.

Background/objectives Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule expressed on T cells. CTLA-4 also binds to the surfaces of monocytes and macrophages, precursors of osteoclasts. Research on rheumatoid arthritis demonstrated that CTLA-4 suppresses inflammation and bone resorption. However, its effects on alveolar bone have yet to be understood. The purpose of this study was to investigate the role and potential mechanism of CTLA-4 in bone resorption in periodontitis. Materials and methods In vivo, the effects of systemic administration of CTLA-4 immunoglobulin fusion protein (CTLA-4-Ig) on alveolar bone resorption were investigated using a periodontitis mouse model. A total of 20 C57BL/6J mice were randomly assigned to two groups according to the administration modes. Periodontitis was induced by placing a ligature around the left maxillary second molar. The contralateral tooth was left un-ligated. In the CTLA-4-Ig (+) group, CTLA-4-Ig was administered by intraperitoneal injection at 1 and 3 days after ligature placement. Animals in the CTLA-4-Ig (-) group were given only phosphate-buffered saline each time. At 5 days after ligature placement, bone resorption was assessed by micro-computed tomography and histological examination, and the prevalence of osteoclast-like cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining. In vitro, the effects of CTLA-4-Ig on osteoclasts were evaluated. Viability of RAW 264.7 cells treated with receptor activator of nuclear factor-κB ligand (RANKL) and CTLA-4-Ig was tested by WST-1 assay. Osteoclast-like cells were enumerated by TRAP staining, and osteoclast activity was evaluated by resorption pit assay. Gene expression levels of osteoclast differentiation markers (macrophage-colony stimulating factor receptor, carbonic anhydrase II, cathepsin K, and Trap) and protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, were assessed by quantitative real-time polymerase chain reaction. The effect of CTLA-4-Ig on the nuclear factor-κB (NF-κB) activation was assessed by enzyme-linked immunosorbent assay. Results In vivo, ligature-induced bone resorption and the numbers of osteoclast-like cells were significantly decreased by the administration of CTLA-4-Ig. In vitro, treatment with RANKL and CTLA-4-Ig had no significant effect on cell viability. CTLA-4-Ig significantly reduced the prevalence and activation of osteoclast-like cells and decreased the expressions of osteoclast differentiation markers, compared with the RANKL-treated control. CTLA-4-Ig significantly suppressed RANKL-induced phosphorylation of NF-κB p65 but increased PP2A expression. Conclusion These results suggest that CTLA-4-Ig abrogates bone resorption in induced periodontitis, possibly via inhibition of osteoclast differentiation and activation. The regulation of the NF-κB pathway and PP2A expression may be one mechanism by which CTLA-4-Ig suppresses osteoclast behavior.

Accumulating evidence during the last two decades has addressed the potential anti‐inflammatory properties of berberine (BBR), a bioactive alkaloid compound isolated from Coptidis rhizoma, in controlling or treating several inflammatory diseases. Periodontitis is one of the most common chronic and serious inflammatory diseases, in which uncontrolled and unabated host immune responses against periodontopathic pathogens play critical and crucial roles in the disease pathogenesis. Hence, regulating inflammatory responses in periodontitis has a valuable approach and holds promise in treating periodontitis. For the first time, this paper reviews the evidence from in vitro and in vivo experimental models to explore the anti‐inflammatory effects of BBR in periodontitis and exhibits that BBR has the high potency to exert anti‐inflammatory effects by reducing expression and secretion of pro‐inflammatory mediators including TNF‐α, IL‐1β, IL‐17, RANKL, MMP‐2, MMP‐9 and MCP‐1. The BBR‐mediated anti‐inflammatory actions could translate into the inhibition of the periodontal tissues and alveolar bone destruction and the control of the disease in vivo. As the second aim of this paper, we also paid attention to the therapeutic potential of BBR in treating human diseases regarding its anti‐inflammatory properties.

Background/purpose Orthodontic tooth movement (OTM) is facilitated by two events; bone resorption on the compression side and bone formation on the tension side simultaneously termed bone remodeling. Osteocytes play a critical role in bone remodeling during OTM, as they have been described as the critical source of nuclear factor-κB ligand (RANKL) necessary for bone remodeling during OTM. Tumor necrosis factor (TNF)-α is a cytokine that acts by amplifying RANKL expression in osteocytes. In this study, we evaluated the effects of TNF-α on RANKL expression in osteocyte during OTM. Materials and methods We assessed whether TNF-α influenced RANKL expression in osteocyte during orthodontic tooth movement by using wild-type (WT) and TNF receptor I and II deficient (TNFRsKO) mice. A Nickel-titanium closed coil spring was attached to the maxillary alveolar bone near the incisors and the upper left first molar, and the first molars were moved mesially in WT and TNFRsKO mice. After OTM, the number of RANKL-positive osteocytes in the alveolar bone was evaluated by immunohistochemistry. Results The number of RANKL-positive osteocyte in the alveolar bone significantly increased in WT mice than in TNFRsKO mice after OTM. Conclusion The results indicate that TNF-α induces the expression of RANKL in osteocyte during OTM.

Background/purpose: Local anesthesia (LA) application is a routine dental work in clinic. The aim of present study was to evaluate the extent of biologic effects of LA on periodontal ligaments (PDL) or bone cells (MG63). Materials and methods: Local anesthetics (LAs) at different concentrations were added to PDL and MG 63 cells. The viability of the cells was analyzed using an MTT assay. The inflammatory markers, COX-2, IL-1, IL-6 and TNF-A, of PDL and MG63 cells treated with LAs were analyzed with a Western blot assay. The extract medium of the LA-treated PDL cells was added to the MG63 cells for subsequent culture and to examine the RANKL, ALP, and OPG expression. The data were statistically analyzed with p 0.05). The RANKL expression in the MG63 cells increased as the Septanest and Scandonest concentrations were increased in the PDL extract medium (p

The main metabolic function of bone is the homeostasis of calcium and phosphate. This is achieved through a balance of bone resorption and formation. To gain an understanding of bone turnover it is important to appreciate the different cells, regulatory steroids and hormones involved and how these have their actions. This article summarizes bone cells, normal bone metabolism, bone turnover, regulatory mechanisms and the clinical applications of these. The receptor activator of nuclear factor-kB, receptor activator of nuclear factor-kB ligand and osteoprotegerin (RANK/RANKL/OPG) cell signalling pathway is discussed in detail. Understanding these topics enhances knowledge of the pathological processes and diseases that result from failures of bone metabolism.

OBJECTIVES Notch signaling pathway, known to influence bone resorption in several oral diseases, has not been analyzed in peri-implantitis yet. Therefore, the aims of the present study were to determine the levels of Notch cascade, bone remodeling mediators, and pro-inflammatory cytokines, in conjunction with clinical parameters, in subjects with peri-implant mucositis and peri-implantitis. MATERIAL AND METHODS Clinical parameters: peri-implant probing depth, bleeding on probing, suppuration on probing, and plaque index (PI) were recorded. Samples were collected from 130 participants, divided into peri-implantitis (PI), peri-implant mucositis (PM), and healthy implants (HI) group. Relative expression levels (REL) of Notch 1, Notch 2, Jagged 1, Hes 1, Hey 1, TNF-α, IL-17, IL-1β, IL-6, RANKL, and OPG mRNA were determined by reverse transcriptase-real-time polymerase chain reaction. Quantitation of Notch 1, Il-17, and IL-6 proteins was performed using ELISA assays. RESULTS All clinical parameters were significantly higher in PI compared to HI. Significant decrease of Notch 1, and higher REL of Hey 1, IL-1β, IL-6, and RANKL were found in PI compared to HI. PM showed significant increase of IL-1β REL in comparison with HI. In PI versus PM, significantly higher REL was found for Hey 1, TNF-α, IL-17, IL-1β, IL-6, and RANKL. Additionally, higher protein concentrations of IL-6 and IL-17 were detected in PI versus PM and versus HI group. CONCLUSION The combined effect of Notch 1 down-regulation and elevated expression of some key inflammation modulators might result in osteoclast activity increase and subsequent osteolysis in peri-implantitis.

Purpose Emerging evidence suggests that the progesterone-mediated receptor activator of nuclear factor κB (RANK)/soluble RANK ligand (sRANKL)/osteoprotegerin (OPG) pathway plays an important role in mammary carcinogenesis and is hyperactivated in germline (g)BRCA1/2 mutation carriers. We analyzed the effects of a 3-month intensive lifestyle intervention within the LIBRE-1 study on the serum levels of OPG and sRANKL and hypothesized that the intervention program provides a beneficial impact on the biomarkers by increasing OPG and reducing sRANKL serum concentrations. Methods Serum levels of OPG and sRANKL of 49 gBRCA1/2 mutation carriers were quantified using enzyme-linked immunosorbent assays. We used previously collected blood samples from participants of the prospective LIBRE-1 study, who were randomized into an intervention group (IG), increasing physical activity and adherence to the Mediterranean diet (MedD) through supervised sessions from study entry to the first study visit after 3 months and a usual-care control group (CG). Differences in biomarker levels before and after the 3-month intervention were tested within and between study groups. Results The lifestyle intervention resulted in a significant increase in OPG for participants in both the IG (q = 0.022) and CG (q = 0.002). sRANKL decreased significantly in the IG (q = 0.0464) and seemed to decrease in the CG (q = 0.5584). An increase in the intake of Omega-3 polyunsaturated fatty acids was significantly associated with an increase in OPG (r = 0.579, q = 0.045). Baseline serum levels of sRANKL were a strong predictor for the change of sRANKL in the course of the intervention (ß-estimate = − 0.70; q = 0.0018). Baseline physical fitness (assessed as VO2peak) might predict the change of OPG in the course of the intervention program (ß-estimate = 0.133 pg/ml/ml/min/kg; p = 0.0319; q = 0.2871). Conclusion Findings from this pilot study seem to confirm our hypothesis by showing an increase in OPG and decrease in sRANKL over a 3-month lifestyle intervention and suggest that increased physical activity and adherence to the MedD are potent modulators of the biomarkers OPG and potentially sRANKL.