Showing papers on "Tartrate-resistant acid phosphatase published in 2021"

A novel RANKL-targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species.

Abstract: Background and purpose Osteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (Rob) is a flavonoid glycoside that has shown anti-inflammatory and antioxidative effects in previous studies, but little is known about its effects on bone homeostasis. The purpose of our research was to investigate whether Rob could prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms. Methods The docking pose of Rob and RANKL was identified by protein-ligand molecular docking. Rob was added to bone marrow macrophages (BMMs) stimulated by nuclear factor-κB (NF-κB) ligand (RANKL). The effects of Rob on osteoclastic activity were evaluated by positive tartrate resistant acid phosphatase (TRAcP) staining kit and hydroxyapatite resorption assay. RANKL-induced ROS generation in osteoclasts was detected by H2 DCFDA and MitoSox Red staining. The classic molecular cascades triggered by RANKL, such as NF-κB, ROS, calcium oscillations, and NFATc1-mediated signaling pathways, were investigated using Fluo4 staining, western blot, and quantitative real-time polymerase chain reaction. In addition, an OVX mouse model mimicking estrogen-deficient osteoporosis was created to evaluate the therapeutic effects of Rob in vivo. Results Computational docking results showed that Rob could bind specifically to RANKL's predicted binding sites. In vitro, Rob inhibited RANKL-mediated osteoclastogenesis dose-dependently without obvious cytotoxicity at low concentrations. We also found that Rob attenuated RANKL-induced mitochondrial ROS production or enhanced activities of ROS-scavenging enzymes, and ultimately reduced intracellular ROS levels. Rob abrogated the RANKL-induced mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, and subsequently blocked NFATc1 signaling and TRAcP expression. In addition, Rob inhibited osteoclast proliferation by downregulating the expression of osteoclast target genes (Acp5, Cathepsin K, Atp6v0d2, Nfact1, c-Fos, and Mmp9) and reducing Ca2+ oscillations. Our in vivo results showed that Rob reduced bone resorption in OVX animal model by repressing osteoclast activity and function. Conclusions Rob inhibits the activation of osteoclasts by targeting RANKL and is therefore a potential osteoporosis drug.

Humulus lupulus L. Extract Prevents Ovariectomy-Induced Osteoporosis in Mice and Regulates Activities of Osteoblasts and Osteoclasts.

Abstract: To systematically evaluate the protective effects of Humulus lupulus L. extract (HLE) on osteoporosis mice. In vivo experiment, a total of 35 12-week-old female ICR mice were equally divided into 5 groups: the sham control group (sham); the ovariectomy with vehicle group (OVX); the OVX with estradiol valerate [EV, 0.2 mg/(kg•d)] the OVX with low- or high-dose HLE groups [HLE, 1 g/(kg•d) and 3 g/(kg•d)], 7 in each group. Treatment began 1 week after the ovariectomized surgery and lasted for 12 weeks. Bone mass and trabecular bone mircoarchitecture were evaluated by micro computed tomography, and bone turnover markers in serum were evaluated using enzyme-linked immunosorbent assay (ELISA) kits. In vitro experiment, osteoblasts and osteoclasts were treated with HLE at doses of 0, 4, 20 and 100 µg/mL. Biomarkers for bone formation in osteoblasts and bone resorption in osteoclasts were analyzed. Compared with the OVX group, HLE exerted bone protective effects by the increase of estradiol (P<0.05), the improvement of cancellous bone structure, bone mineral density (P<0.01) and the reduction of serum alkaline phosphatase (ALP), tartrate resistant acid phosphatase (TRAP), bone gla-protein, c-terminal telopeptides of type I collagen (CTX-I) and deoxypyridinoline levels (P<0.01 for all). In vitro experiment, compared with the control group, HLE at 20 µg/mL promoted the cell proliferation (P<0.01), and increased the expression of bone morphogenetic protein-2 and osteopontin levels in osteoblasts (both P<0.05). HLE at 100 µg/mL increased the osteoblastic ALP activities, and HLE at all dose enhanced the extracellular matrix mineralization (both P<0.01). Furthermore, compared with the control group, HLE at 20 µg/mL and 100 µg/mL inhibited osteoclastic TRAP activity (P<0.01), and reduced the expression of matrix metalloproteinase-9 and cathepsin K (both P<0.05). HLE may protect against bone loss, and have potentials in the treatment of osteoporosis.

Interleukin-4 repairs wear particle induced osteolysis by modulating macrophage polarization and bone turnover.

Abstract: Periprosthetic osteolysis remains as a major complication of total joint replacement surgery. Modulation of macrophage polarization with interleukin-4 (IL-4) has emerged as an effective means to limit wear particle-induced osteolysis. The aim of this study was to evaluate the efficacy of local IL-4 delivery in treating preexisting particle-induced osteolysis. To this end, recently established 8 week modification of murine continuous femoral intramedullary particle infusion model was utilized. Subcutaneous infusion pumps were used to deliver polyethylene (PE) particles into mouse distal femur for 4 weeks to induce osteolysis. IL-4 was then added to the particle infusion for another 4 weeks. This delayed IL-4 treatment (IL-4 Del) was compared to IL-4 delivered continuously (IL-4 Cont) with PE particles from the beginning and to the infusion of particles alone for 8 weeks. Both IL-4 treatments were highly effective in preventing and repairing preexisting particle-induced bone loss as assessed by μCT. Immunofluorescence indicated a significant reduction in the number of F4/80 + iNOS + M1 macrophages and increase in the number of F4/80 + CD206 + M2 macrophages with both IL-4 treatments. Reduction in the number of tartrate resistant acid phosphatase + osteoclasts and increase in the amount of alkaline phosphatase (ALP) + osteoblasts was also observed with both IL-4 treatments likely explaining the regeneration of bone in these samples. Interesting, slightly more bone formation and ALP + osteoblasts were seen in the IL-4 Del group than in the IL-4 Cont group although these differences were not statistically significant. The study is a proof of principle that osteolytic lesions can be repaired via modulation of macrophage polarization.

Microstructured titanium functionalized by naringin inserted multilayers for promoting osteogenesis and inhibiting osteoclastogenesis.

Abstract: Osteoporosis is the most common cause of fractures in middle-aged and elderly people. Fracture repair can be difficult due to the decreased bone volume in osteoporosis patients and implants are often required. In this study, a slow-release system for microstructured titanium (Micro-Ti) was designed to promote osteogenesis and inhibit osteoclastogenesis. Firstly, Micro-Ti was prepared on titanium surfaces by dual acid etching. Micro-Ti was covered with naringin (NA), chitosan (CHI) and gelatin (GEL) multilayers through layer by layer technique, which is denoted as LBL (NA) coated-Ti. Osteoblasts (ME3T3-E1) and macrophages (RAW 264.7) were cultured on untreated and treated titanium surfaces in vitro. Osteoblasts grown on LBL (NA) coated-Ti showed higher alkaline phosphatase (ALP) and mineralization, consistent with qRT-PCR analysis of osteoblast genes including runt-related transcription factor 2 (Runx2), ALP, collagen I (Col I), osteocalcin (OCN), osteopontin (OPN), and osteoprotegerin (OPG). In contrast, acid tartarate-resistant phosphatase activity and the expression of osteoclastic differentiation related genes comprising of cathepsin K (CTSK), nuclear factor of activated T cells (NFAT), tartrate resistant acid phosphatase (TRAP) and V-ATPase (VATP) in osteoclasts were significantly reduced on LBL (NA) coated-Ti surfaces compared with other groups. These results indicate that microstructured titanium functionalized by naringin inserted multilayers enhanced the differentiation of osteoblasts and inhibited osteoclast formation. The proposed approach in this research provides a novel way to modify titanium-based implants for fracture repair in osteoporosis patients.

Notopterol Attenuates Estrogen Deficiency-Induced Osteoporosis via Repressing RANKL Signaling and Reactive Oxygen Species

Abstract: Integrity of the skeleton is sustained through the balanced activities of osteoblasts and osteoclasts in bone remodeling unit. The balance can be disrupted by excessive osteoclasts activation commonly seen in osteoporosis. Notopterol (NOT) is a main component of Notopterygium incisum which exerts a wide spectrum effect on biomedical pharmacology. In our study, we found NOT serves as an inhibitor in regulating RANKL-activated osteoclasts formation and bone resorption function by calculating tartrate resistant acid phosphatase (TRAcP) staining and hydroxyapatite resorption assays. Furthermore, RANKL-mediated signaling pathways including MAPK, NF-κB and calcium ossification were hampered, whereas ROS scavenging enzymes in Nrf2/Keap1/ARE signaling pathways were promoted by NOT. In addition, the activation of the essential transcription factor NFATc1 in RANKL-mediated osteoclastogenesis was almost totally suppressed by NOT. What is more, NOT diminished the loss of bone mass in preclinical model of OVX mice by blocking osteoclastogenesis determined by bone histomorphometry, TRAcP staining and H&E staining. Conclusively, our findings demonstrated that NOT could arrest osteoclastogenesis and bone resorptive activity by attenuating RANKL-mediated MAPK, NF-κB, calcium and NFATc1 signaling transduction pathways and enhancing ROS scavenging enzymes in Nrf2/Keap1/ARE pathways in vitro, and prohibit bone loss induced by OVX in vivo. Taken together, NOT may be identified to be a natural and novel treatment for osteolytic diseases.

Inhibition of Sirtuin 3 prevents titanium particle-induced bone resorption and osteoclastsogenesis via suppressing ERK and JNK signaling.

Abstract: Implant-derived wear particles can be phagocytosed by local macrophages, triggering an inflammatory cascade that can drive the activation and recruitment of osteoclasts, thereby inducing peri-prosthetic osteolysis. Efforts to suppress pro-inflammatory cytokine release and osteoclastsogenesis thus represent primary approaches to treating and preventing such osteolysis. Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylases that control diverse metabolic processes. However, whether SIRT3 could mitigate wear debris-induced osteolysis has not been reported. Herein we explored the impact of the SIRT3 on titanium particle-induced osteolysis. Tartrate resistant acid phosphatase (TRAP) staining revealed that the inhibition of SIRT3 suppressed nuclear factor-κB ligand (RANKL)-mediated osteoclasts activation in a dose-dependent fashion. Notably, inhibition of SIRT3 also suppressed matrix metallopeptidase 9 (MMP9) and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) expression at the mRNA and protein levels, while also inhibiting the mRNA expression of dendritic cell-specific transmembrane protein (DC-STAMP), ATPase H+ Transporting V0 Subunit D2 (Atp6v0d2), TRAP and Cathepsin K (CTSK) . In addition, inhibition of SIRT3 suppressed titanium particle-induced tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) expression and prevented titanium particle-induced osteolysis and bone loss in vivo. This inhibition of osteoclasts differentiation was found to be linked to the downregulation and reduced phosphorylation of JNK and ERK. Taken together, inhibition of SIRT3 may be a potential target for titanium particle-induced bone loss.

Overexpression of miRNA-22-3p attenuates osteoporosis by targeting MAPK14.

Abstract: Osteoporosis (OP) results from an imbalance between bone formation, which is regulated by osteoblasts, and bone resorption, which is mediated by osteoclasts. MicroRNA-22-3p (miR-22-3p) expression is decreased during the process of osteoclast differentiation and p38α mitogen-activated protein kinase (MAPK)14 promotes the proliferation and differentiation of osteoclast progenitors. However, whether miR-22-3p could target MAPK14 to regulate the progression of OP remains unknown, which was the aim of the present study. CD14+ PBMCs were used for the establishment of osteoclastic differentiation in vitro. In the present study, reverse transcription quantitative PCR was used to determine the mRNA expression of MAPK14, tartrate resistant acid phosphatase (TRAP), nuclear factor of activated T-cells (NFATC1) and cathepsin K (CTSK). Western blotting was applied to determine the protein expression of MAPK14, TRAP, NFATC1, CTSK, p-p65 and p65. Dual luciferase reporter assay was applied to confirm the relation between miR-22-3p and MAPK14. Cell Counting Kit-8 assay and flow cytometry assays were used to determine the cell proliferation and cell apoptosis, respectively. The results demonstrated that miR-22-3p expression was lower while MAPK14 expression was higher in the serum from patients with OP compared with healthy volunteers. Furthermore, miR-22-3p expression was negatively correlated with MAPK14 expression in patients with OP. In addition, miR-22-3p expression was decreased and MAPK14 expression was increased during the progression of CD14+peripheral blood mononuclear cells (PBMCs) osteoclastic differentiation in a time-dependent manner. Furthermore, miR-22-3p inhibited the proliferation and differentiation and promoted the apoptosis of CD14+PBMCs by targeting MAPK14. In summary, the findings from the present study suggested that miR-22-3p may serve a potential therapeutic role in patients with OP.

Evaluation of Dexamethasone-Induced Osteoporosis In Vivo Using Zebrafish Scales.

Abstract: Glucocorticoid-induced osteoporosis (GIOP) is a major cause of secondary osteoporosis, and the pathogenic mechanisms of GIOP remain to be elucidated. Here, we show a rapid dexamethasone-induced osteoporosis animal model using zebrafish scales. Intraperitoneal injection of dexamethasone over a 5-day period suppressed the regeneration of scales. Furthermore, the circularity of the newly formed regenerated scales was also slightly reduced compared to that of the control group on day 5. The changes in bone-related enzymes, such as cathepsin K, tartrate-resistant acid phosphatase (TRAP) for bone resorption, and alkaline phosphatase (ALP) for bone formation, provide insight into the progression of bone diseases; therefore, we further developed a method to measure the activities of cathepsin K, TRAP, and ALP using zebrafish scales. We found that a lysis buffer with detergent at neutral pH under sonication efficiently helped extract these three enzymes with high activity levels. Interestingly, treatment with a dexamethasone injection produced considerably higher levels of cathepsin K activity and a lower Ca/P ratio than those in the control group, suggesting that dexamethasone increased osteoclast activity, with no significant changes in the activities of TRAP and ALP. Our GIOP model and enzyme assay method could help to design better treatments for GIOP.

Expression of tartrate-resistant acid phosphatase and cathepsin K during osteoclast differentiation in developing mouse mandibles

Abstract: The present study was designed to test the hypothesis that osteoclasts appear after or at the same time as the initiation of bone mineralization in developing intramembranous bones. We examined mineral deposition via Von Kossa staining to determine when bone mineralization begins, tartrate-resistant acid phosphatase (TRAP) activity and cathepsin K immunoreactivity to identify the presence of osteoclasts, and their mRNA expression levels to assess osteoclastic differentiation in the embryonic mouse mandible. Cathepsin K-immunopositive cells were detected around the same time as the onset of bone mineralization, whereas TRAP-positive cells appeared prior to bone mineralization. Cathepsin K protein was expressed only in multinucleated osteoclasts, whereas TRAP activity was identified in both mono- and multinucleated cells. During bone development, TRAP-positive cells altered their morphology, which was related to the number of their nuclei. The elevated mRNA levels of TRAP and cathepsin K were consistent with the increased percentage of multinucleated osteoclasts and the progression of bone development. Our study revealed that TRAP-positive cells appear prior to bone mineralization, and TRAP- and cathepsin K-positive multinucleated osteoclasts appear at the same time as the initiation of bone mineralization in embryonic mouse mandibles, suggesting that osteoclasts contribute to bone matrix maturation during intramembranous ossification.

The W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis.

Abstract: The W9 peptide has been shown to act as a receptor activator for nuclear factor-κB ligand (RANKL) antagonist and tumor necrosis factor (TNF)-α antagonist, which can promote bone formation and inhibit bone resorption. Studies on the W9 peptide at the cellular level have mainly focused on osteoblasts, and little research on the mechanism by which the W9 peptide regulates osteoclasts has been reported, which was the aim of this work. In this study, a rat mandibular defect model was established in vivo and implanted with hydrogel containing the W9 peptide for 2 weeks and 4 weeks, and histochemical staining was used to evaluate the formation of new bone and the changes in osteoclasts. RAW264.7 cells were cultured in vitro for osteoclast induction, and different concentrations of W9 peptide were added. Tartrate resistant acid phosphatase staining, monodansylcadaverine staining, TdT-mediated dUTP Nick-End Labeling assay, real-time PCR and Western blot were used to detect osteoclast differentiation, autophagy and apoptosis. Our results showed that the W9 peptide could reduce osteoclastogenesis and osteoclast activity induced by RANKL, and these effects were partly due to the inhibition of osteoclast autophagy. On the other hand, the W9 peptide could promote mature osteoclast apoptosis, in which autophagy might play an antagonistic role. Taken together, these results suggest that the W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis. Our results will benefit the development and application of new small molecule peptides for the treatment of bone resorption diseases.

Role of enhancer of zeste homolog 2 in osteoclast formation and periodontitis development by downregulating microRNA-101-regulated VCAM-1.

Abstract: The enhancer of zeste homolog 2 (EZH2) represents a potential target for periodontitis treatment; however, its role in the development of periodontitis remains unclear. The current study aimed to elucidate the role of EZH2 in osteoclasts (OCs) growth as well as the mechanism underpinning the related process. The potential interaction among EZH2, microRNA-101 (miR-101), and vascular cell adhesion molecule 1 (VCAM-1) was evaluated using chromatin immunoprecipitation and dual-luciferase reporter gene assay. The expressions of EZH2 and miR-101 in OCs were examined by Western blot analysis and reverse transcription squantitative polymerase chain reaction. Loss- and gain-function assays were then performed to determine the role of EZH2/miR-101/VCAM-1 in periodontitis and OCs proliferation, followed by OC growth and proliferation detected using tartrate resistant acid phosphatase (TRAP) and 5-ethynyl-2'-deoxyuridine staining. Enzyme-linked immunoassay was conducted to determine the expression of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α). A periodontitis rat model was established to investigate the effect of EZH2 and VCAM-1 in vivo. EZH2 was overexpressed, while miR-101 was downregulated in the OCs of periodontitis. Silencing of EZH2, VCAM-1 repression, or miR-101 elevation suppressed the growth and proliferation of OC while acting to encumber the release of IL-1β and TNF-α. EZH2 negatively targeted miR-101, while miR-101 negatively targeted VCAM-1. Moreover, silencing of EZH2 or VCAM-1 was observed to attenuate periodontitis which was evidenced by an increase in BMD, BV/TV, and BS/BV as well as reduction in TRAP and cathepsin K in vivo. Taken together, the key findings of the current study demonstrate that EZH2 knockdown inhibited OC formation by elevating the expression of miR-101 via suppression of VCAM-1, ultimately attenuating periodontitis.

Osteoclasts May Affect Glucose Uptake-Related Insulin Resistance by Secreting Resistin.

Abstract: Objectives Bone may play a role in the modulation of insulin sensitivity. Insulin resistance can be caused by increased resistin. However, whether osteoclasts affect the insulin resistance via resistin remains unclear. In the present study, we show the expression of resistin in osteoclasts and the possible underlying role of resistin on glucose uptake-related insulin resistance in vitro. Methods Conditioned mediums (CM) were collected from Raw264.7 cells treated without (CCM) or with RANKL (CM3, treated with RANKL for 3 days; CM5, treated with RANKL for 5 days) and transfected with control or resistin siRNA (CMsiRNA). The osteoclast formation was examined by tartrate resistant acid phosphatase (TRAP) staining. C2C12 myoblasts were cultured with the CM or CMsiRNA. Glucose uptake was evaluated by 2-NBDG fluorescence intensity. Resistin expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay. Statistical analysis was performed by an independent two sample t-test or one-way ANOVA. Results The 2-NBDG fluorescence intensity was higher in C2C12 cells treated with CCM compared to those that received CM3 and CM5 (p < 0.05). Resistin mRNA and protein expressions were both increased in RAW264.7 cells treated with RANKL for 3 days and 5 days compared with those cells without RANKL administration. The 2-NBDG fluorescence intensities in C2C12 cells treated with CMsiRNA and CM5+Anti-resistin antibody were significantly higher than those cultured with CM5 (p < 0.05). Conclusion Osteoclasts may promote glucose uptake-related insulin resistance by secreting resistin.

Human Umbilical Cord Mesenchymal Stem Cell-induced Osterix, Bone Morphogenetic Protein-2, and Tartrate-resistant Acid Phosphatase Expression in Osteoporotic Mandibular Bone

Abstract: Objectives The aim of this study was to prove that human umbilical cord mesenchymal stem cell (hUCMSC) therapy conducted according to the mandibular osteoporotic model will increase Osterix (Osx) and bone morphogenetic protein-2 (BMP-2) expression, while reducing tartrate-resistant acid phosphatase (TRAP) expression. PKH26 labeling proves that mandibular bone regeneration is produced by hUCMSCs induction. Materials and Methods This study incorporated a true posttest only control group design. Twenty-five female Wistar rats were randomly divided into five groups consisting of the sham surgery (N) group, osteoporotic groups injected with gelatin for 4 weeks (G4) and 8 weeks (G8), and osteoporotic groups injected with hUCMSC-gelatin for 4weeks (SC4) and 8 weeks (SC8). All subjects were provided for BMP-2, Osx, and TRAP on immunohistochemistry examination and PKH-26 labeling. Statistical Analysis All data were analyzed using ANOVA and Tukey HSD tests with p Results Compared with other groups, the highest level of BMP-2 and Osx occurred in the sham surgery (N) and osteoporotic groups injected with hUCMSCs-gelatin (SC), while the lowest level of TRAP was found in SC4. During 4- and 8-week observation periods, the PKH 26 appeared green (fluorescent). Conclusions hUCMSC demonstrates high-osteogenic activity and increased osteoporotic mandibular bone regeneration, as shown by increased expression of Osx and BMP-2 and decreased TRAP expression. From the labeling, PKH-26 proved that viable hUCMSCs in gelatin solvent can be present in the mandibular bone and be capable of promoting osteogenic differentiation and increasing mineralization and bone formation in the osteoporotic mandibular bone.

4‑Hexylresorcinol inhibits osteoclastogenesis by suppressing the NF‑κB signaling pathway and reverses bone loss in ovariectomized mice

Abstract: 4-Hexylresorcinol (4HR) is a small organic compound that is widely used as an antiseptic and antioxidant. In the present study, its role in osteoclastogenesis was investigated. Bone marrow-derived macrophages from mice were used to examine the role of 4HR in osteogenesis. An ovariectomy (OVX) mouse model was constructed to examine the effect of 4HR in vivo, followed by hematoxylin and eosin and tartrate resistant acid phosphatase staining. In the present study, 4HR effectively suppressed receptor activator of NF-κB ligand-induced osteoclastogenesis in a dose-dependent manner. 4HR was also found to significantly suppress the expression of osteoclast (OC)-specific markers, including tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cell cytoplasmic 1 and c-Fos in the presence of RANKL in BMMs. Furthermore, 4HR inhibited osteoclastogenesis by inhibiting the activation of the NF-κB signaling pathway in BMMs. Consistent with the in vitro results, 4HR effectively ameliorated OVX-induced bone loss and markedly reduced OC number in the proximal tibia in vivo. In conclusion, the present results suggested that 4HR inhibited osteoclastogenesis in vitro and rescued bone loss in vivo, suggesting that 4HR may serve as a novel therapeutic agent for osteoporosis treatment.

Role of osteoclast differentiation in the occurrence of osteoarthritis of temporomandibular joint.

Abstract: Objectives This study aimed to explore the role of osteoclast differentiation in the occurrence of temporomandibular joint osteoarthritis (TMJOA). Methods A mouse TMJOA model was constructed. Micro-CT was used to observe the changes in condylar bone during the development of TMJOA. Hematoxylin-eosin (HE) staining was used to observe the histological structure changes of the condyle of TMJOA mice. Tartrate resistant acid phosphatase (TRAP) staining was used to observe the presence of osteoclasts in TMJOA joint tissue. The synovial fluid of patients with TMJ-OA was collected to determine the effect on osteoclast differentiation. Results Micro-CT revealed that the condyle of the TMJOA group had the most obvious damage in the second and third weeks, and the shape of the condyles also changed in a beak-like manner. HE staining showed that the condyle cartilage and subchondral bone structure of TMJOA mice were disordered in the second week. TRAP tissue staining showed that the number of osteoclasts of the TMJOA group obviously increased in the second week. Results of cell experiments showed that the number of osteoclast differentiation significantly increased after stimulation of synovial fluid from TMJOA patients, and the cell volume increased. Conclusions TMJOA animal models and TMJOA patient synovial cell experiments could induce osteoclast differentiation, indicating that osteoclast differentiation plays an important role in TMJOA occurrence.

Nucleotides modulate synoviocyte proliferation and osteoclast differentiation in macrophages with potential implications for rheumatoid arthritis

Abstract: P2 receptors are nucleotide-activated receptors involved in inflammation, cell proliferation osteoblastogenesis, osteoclastogenesis and their function. They can be potential role players in the pathophysiology of rheumatoid arthritis (RA). Our analysis of gene expression datasets of synovial tissue biopsy from the GEO database shows changes in the expression levels of P2 receptors. HIG-82, a synovial fibroblast cell line and RAW 264.7, a macrophage cell line are good in vitro models to study RA. Nucleotide addition experiments showed UDP Glucose significantly increased the proliferation of synovial fibroblasts (HIG-82). Similarly, nucleotides such as Adenosine tri-phosphate (ATP), Adenosine di-phosphate (ADP), Uridine tri-phosphate (UTP), Uridine di-phosphate (UDP) and Uridine diphosphoglucose (UDPG) induced elevated reactive oxygen species (ROS) and tartrate Resistant Acid Phosphatase (TRAP) activity in RAW264.7 cells. The ADP-induced TRAP could be inhibited by clopidogrel a P2Y12 inhibitor. ATP, ADP, UTP, UDP and UDPG also induced osteoclastogenesis as evident from fused multinucleate cells and expression of osteoclast markers (TRAP, Cathepsin K [CTSK]) as determined by Q-PCR. Apyrase (APY) a nucleotidase and an enzyme that is used to modulate extracellular nucleotide concentration is sufficient to induce osteoclastogenesis. Taken together our results show that nucleotides modulate synoviocyte proliferation and macrophage differentiation into osteoclast and play an important role in RA. Nucleotide receptors might be potential therapeutic targets in RA.

The function of the calcium channel Orai1 in osteoclast development.

Abstract: To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1fl/fl -LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.

TLR-4 targeting contributes to the recovery of osteoimmunology in periodontitis.

Abstract: Objective The aim of this study was to determine the potential role of TLR-4 in the osteoimmunological imbalance of periodontitis. Background Although current evidence supports that TLR-4 plays an important role in the inflammatory response of periodontal tissues triggered by microorganisms, little information is available regarding the function of TLR-4 in the osteoimmune regulation of homeostasis in periodontitis. Methods Human gingival epithelial cells (HGEC) were isolated from the gingival tissues of 3 healthy volunteers and the expression of osteoclastogenic cytokines was evaluated by ELISA and real time RT-PCR. In addition, 30 C57BL/6 mice were used and randomly divided into three groups: control group, periodontitis group (CP) and periodontitis+TAK-242 (a specific inhibitor of TLR-4) group (TAK-242) and the expression of osteoclastogenic cytokines and the osteoclast density in the periodontal tissue were evaluated by immunohistochemical staining and tartrate resistant acid phosphatase staining. Moreover, micro-computed tomography (Micro-CT) was used to assess bone resorption. Results The in vitro results showed that TAK-242 blocked the overproduction of IL-1, IL-6, TNF-α and RANKL in HGEC treated with LPS. The in vivo results revealed that TAK-242 also effectively decreased these osteoclastogenic cytokines in periodontal tissue of mice with periodontitis. More importantly, Micro-CT analysis showed a significant reduction of the alveolar bone loss in the TAK-242 group compared with the CP group. Furthermore, the TRAP staining showed a significant lower density of osteoclasts in the alveolar bone area of the TAK-242 group. Conclusion TLR-4 inhibition decreased the differentiation of osteoclast through the inhibition of the overproduction of osteoclastogenic cytokines and the prevention of the alveolar bone absorption in mouse periodontitis models. Therefore, the use of TAK-242 might contribute to the recovery of the osteoimmunological homeostasis and might provide a potential strategy to treat periodontal diseases.

Schistosoma japonicum cystatin suppresses osteoclastogenesis via manipulating the NF‑κB signaling pathway.

Abstract: Abnormal osteoclastic activation and secretion of cysteine proteinases result in excessive bone resorption, which is one of the primary factors in the development of bone metabolic disorders, such as rheumatoid arthritis and osteoporosis. Mammalian cystatins have been demonstrated to restrain osteoclastic bone resorption and to alleviate severe osteolytic destruction via blocking the activity of cysteine proteinases. However, the specific effects of parasite cystatins on the formation and function of osteoclasts remain unclear. The purpose of the current study was to explore the effects of cystatins from Schistosoma japonicum (Sj‑Cys) on macrophage colony‑stimulating factor (M‑CSF) and receptor activator of NF‑κB ligand (RANKL)‑induced osteoclast differentiation, as well as the underlying molecular mechanisms. Recombinant Sj‑Cys (rSj‑Cys) dose‑dependently restrained osteoclast formation, with a half‑maximal inhibitory concentration (IC50) value of 0.3 µM, and suppressed osteoclastic bone resorptive capability in vitro. The findings were based on tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays, respectively. However, the cell viability assay showed that the repression of rSj‑Cys on osteoclast formation did not depend on effects on cell viability or apoptosis. Based on the results of reverse transcription‑quantitative PCR and western blot analysis, it was found that rSj‑Cys downregulated the expression levels of osteoclastogenesis‑related genes and proteins, by interfering with M‑CSF and RANKL‑induced NF‑κB signaling and downstream transcription factors during early‑phase osteoclastogenesis. Overall, the results of the present study revealed that rSj‑Cys exerted an inhibitory role in osteoclast differentiation and could be a prospective biotherapeutic candidate for the treatment and prevention of bone metabolic disorders.

The study of gp130/the inflammatory factors regulating osteoclast differentiation in rheumatoid arthritis.

Abstract: Rheumatoid arthritis (RA) is a chronic immune disease characterized by synovitis and bone destruction. The osteoclasts play a critical role in pathologic bone loss during inflammatory arthritis. In this paper, we report that Interleukin (IL)-6, IL-6Rα/gp130, IL-11, IL-27, and Matrix Metallo Proteinases (MMP)-9 expression results in serum of the RA group were significantly higher than that of the control group. The gp130 positive cells in peripheral blood mononuclear cell (PBMC) and osteoclast-like cells (OLC) which had been induced with receptor activator of nuclear factor κB ligand (RANKL) in RA group were also higher than that in the control group. In addition, after OLC in RA group is cultured with anti-gp130 Monoclonal antibody (McAb), the IL-6 and MMP-9 expression in osteoclast supernatant insignificantly decreased. Meanwhile, the expression results of Tartrate Resistant Acid Phosphatase (TRAP)-positive cells and osteoclasts were also decreased significantly. Our study suggests that regulating gp130 receptor can be used to control the differentiation and formation of osteoclasts, which provides a new clinical strategy for RA patients in the future.

Effect of zoledronic acid on bone nanocomposites organization and prevention of bone mineral density loss in ovariectomized rats.

Abstract: OBJECTIVES Osteoporosis often occurs in individuals of different age groups, frequently during menopause and after ovariectomy. It increases the risk of pathological fractures almost twice. The aim of our research was to assess bone metabolism, nanocomposite structure of the tibia under conditions of ovariectomy and zoledronic acid treatment. METHODS X-ray diffraction has been performed for nanostructure analysis of mineral crystallites and crystal lattice of hydroxyapatite in the tibia samples of ovariectomized rats with additional application of bisphosphonate zoledronic acid (0.025 mg/kg). Markers of remodeling - osteocalcin, alkaline phosphatase, tartrate resistant acid phosphatase 5b - were determined. Quantitative amount of calcium in the bones was detected by atomic absorption method. RESULTS Zoledronic acid prevented loss of mineral mass after ovariectomy. Rats after ovariectomy, treated with zoledronic acid, showed statistically higher (р<0.05) values of crystalline phase and calcium content compared with the SHAM-surgery and ovariectomy groups (р<0.05). Zoledronic acid inhibited bone remodeling, which is proved by tartrate resistant acid phosphatase 5b reduction and inhibition of osteoclasts during the experiment. CONCLUSIONS These results enable to suggest that zoledronic acid can improve mineral mass of the bone during menopause in individuals of different age groups.