TRAP-positive Multinucleated Cells Research Articles

The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For invivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

Tmem119 deficiency delays bone repair in mice

Tmem119 was identified as a bone anabolic factor in osteoblasts, however the roles of Tmem119 on bone repair have remained unknown. Therefore, we herein investigated the roles of Tmem119 on bone repair by examining the bone repair process after a femoral bone defect using Tmem119-deficient mice. In Tmem119-deficient mice, bone repair after a femoral bone defect was significantly delayed 10 and 14 days after bone injury in female and male mice with 3-dimensional micro-computed tomography analyses, respectively. The number of alkaline phosphatase-positive cells at the damaged sites was significantly decreased 7 days after bone injury in Tmem119-deficient mice, although the number of Osterix-positive cells was not significantly different 4 days after bone injury. The number of tartrate-resistant acid phosphatase-positive multinucleated cells as well as the number and luminal area of CD31-positive vessels at the damaged sites were not significantly different between Tmem119-deficient and wild-type mice. The present study first showed that Tmem119 deficiency delayed bone repair partly through a decrease in the osteoblastic bone formation of differentiated osteoblasts.

14-3-3ζ suppresses RANKL signaling by destabilizing TRAF6

Macrophages are essential regulators of inflammation and bone loss. RANKL, a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages (BMDMs) obtained from wildtype (Wt) and YwhazKO animals, and RAW cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger TRAP-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

In vitro Assessment of the Effects of Host Modulatory Agents on Osteoclastogenesis.

Osteoclastogenesis, the formation of osteoclasts from precursor cells, plays a pivotal role in bone remodeling and associated pathologies like osteoporosis and rheumatoid arthritis. Host modulatory agents (HMAs) have emerged as potential therapeutic candidates for modulating osteoclastogenesis. However, their effects need comprehensive evaluation through in vitro studies. In this study, we conducted an in vitro assessment of the effects of a novel HMA on osteoclastogenesis. Primary murine bone marrow-derived macrophages were cultured with the receptor activator of nuclear factor kappa-B ligand to induce osteoclast differentiation. The HMA was administered at various concentrations, and osteoclastogenesis was evaluated through tartrate-resistant acid phosphatase (TRAP) staining, osteoclast size measurement, and gene expression analysis of osteoclast markers. Treatment with the HMA resulted in a dose-dependent inhibition of osteoclast formation. At the highest concentration (100 μM), osteoclastogenesis was significantly suppressed, with a reduction in the number of TRAP-positive multinucleated cells from 50 ± 5 to 10 ± 2 per field (P < 0.001). Moreover, the osteoclast size was markedly reduced, with an average diameter of 20 ± 3 μm compared to 35 ± 4 μm in the control group (P < 0.05). Gene expression analysis revealed downregulation of osteoclast-specific markers, including TRAP, Cathepsin K, and NFATc1, confirming the inhibitory effect of the HMA on osteoclastogenesis. Our findings demonstrate the potential of the investigated HMA as a modulator of osteoclastogenesis. By suppressing osteoclast formation and activity, this agent holds promise for the development of novel therapeutic strategies targeting bone resorption-associated disorders.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Effects of continuous and released compressive force on osteoclastogenesis invitro

ObjectiveCompressive force has been found to be catabolic to alveolar bone during orthodontic tooth movement. This study quantified the fusion of mononuclear RAW 264.7 cells (a murine osteoclastic-like cell line) into multinucleated osteoclasts under a hydrostatic pressure-generated mechanical compression-the new model of various magnitudes and durations. MethodsRAW 264.7 cells were subjected to 0.3, 0.6 or 0.9 g/cm2 of compressive force by an acrylic cylinder custom-made by laser cutting or no compressive force for 4 days during osteoclastogenic induction. TRAP-positive multinucleated cells were quantified. For the release from force experiment, osteoclastogenesis was induced by 0.6 g/cm2 mechanical stimuli for 0, 1, 2, 3 or 4 days. Cell viability, TRAP-positive multinucleated cells, DCSTAMP and Cathepsin K (CTSK) gene expression were evaluated 4 days after release from force. ResultsCompressive force at 0.6 and 0.9 g/cm2 significantly increase the number of TRAP-positive multinucleated cells (P < 0.05). Release from continuous mechanical compression after 4 days significantly elevated the number of TRAP-positive multinucleated cells and DCSTAMP and CTSK mRNA expression, with no adverse effects on cell viability (P < 0.05). ConclusionsContinuous stimulation with compressive force induced osteoclastogenesis in RAW 264.7 cells by enhancing DCSTAMP and CTSK expression, which provides new understanding of bone remodeling during orthodontic treatment.

Cucumber seed polypeptides regulate RANKL-induced osteoclastogenesis through OPG/RANKL/RANK and NF-κB

Postmenopausal osteoporosis (PMOP) is a common disease that endangers the health of elderly women. Cucumber seeds have shown excellent therapeutic effects on PMOP, but the mechanism of cucumber seed peptide (CSP) remains unclear. The expression levels of NF-κB and osteoclast-related genes were detected by RT-qPCR. The levels of apoptosis-related proteins were detected by Western blotting. Nuclear translocation of NF-κB p65 and osteoclast formation were detected by immunofluorescence and tartrate-resistant acid phosphatase (TRAP) staining, respectively. ELISA was used to detect the expression levels of OPG, M-CSF, and RANKL. Hematoxylin–eosin (H&E) and TRAP staining were used to observe the effects of CSP on bone formation. In RAW264.7 cells, CSP (0.4 mg/L, 4 mg/L, and 40 mg/L) effectively inhibited the expression of osteoclast-related genes (Cathepsin-K, MT1-MMP, MMP-9, and TRAP). TRAP-positive multinucleated giant cells gradually decreased. Furthermore, NF-κB pathway activation downstream of RANK was inhibited. In bone marrow stromal cells (BMSCs), the expression levels of M-CSF and RANKL gradually decreased, and OPG gradually increased with increasing CSP concentrations. Treatment of RAW264.7 cells with pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) prevented the formation of osteoclasts. Treatment with different concentrations of CSP effectively decreased the levels of RANKL and M-CSF in rat serum and increased the expression of OPG in the oophorectomy (OVX) rat model. Furthermore, different concentrations of CSP could ameliorate the loss of bone structure and inhibit the formation of osteoclasts in rats. CSP inhibits osteoclastogenesis by regulating the OPG/RANKL/RANK pathway and inhibiting the NF-kB pathway.

Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis.

Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.

Orientin suppresses osteoclastogenesis and ameliorates ovariectomy-induced osteoporosis via suppressing ROS production.

The aberrant differentiation of osteoclasts is a key feature of the pathogenesis of osteoporosis, which has a devastating impact on human health. While the effects of Orientin (Ori) on osteoporosis, particularly on RANKL-stimulated osteoclast production and activation, remain still unclear, Ori has been found to display several biological activities, including antioxidant and anti-inflammatory. In this work, we investigated the possible pathways through which Ori suppressed RANKL-induced osteoclast development and showed for the first time that it does so. The macrophages from the bone marrow (BMMs) were cultivated and then treated with Ori after being stimulated with RANKL. Then, TRAP-positive multinucleated cells were counted, and F-actin ring analysis was used to assess Ori's impact on mature osteoclast development. In addition, dihydroethidium (DHE) staining was used to evaluate the impact of Ori on RANKL-induced reactive oxygen species (ROS). In addition, we performed western blotting and quantitative RT-PCR analysis to investigate probable causes of these downregulation effects. We discovered that Ori inhibits the creation of osteoclasts, the gene and protein expressions unique to osteoclasts, and the ROS production. By activating Nrf2 and other ROS-scavenging enzymes, Ori reduces intracellular ROS levels. The expression of the main transcription factor of osteoclast development, c-Fos, was downregulated together with NFATc1, CTSK, and NFATc2, thanks to Ori's inhibition of RANKL-induced NF-κB. Consistent with its in vitro antiosteoclastogenic action, Ori therapy in the ovariectomized (OVX) rat model was also able to restore bone mass and improve microarchitecture in the distal femurs. Together, our results demonstrate that Ori is a flavonoid molecule with therapeutic promise for bone illnesses associated with osteoclasts, such as osteoporosis.

Periodontal Fibroblasts—Macrophage Crosstalk in External Inflammatory Root Resorption

IntroductionThis study aimed to understand the influence of periodontal fibroblasts (PDLFs) on clastic differentiation of macrophages (Mφ) in different resorptive environments. MethodsPDLF-Mφ direct coculture (juxtacrine) was seeded on dentin, cementum, and polystyrene with/without lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand for 7 and 14 days and stained for tartrate-resistant acid phosphatase (TRAP) activity. PDLF-Mφ cocultured on polystyrene were immunostained for CD80, CD206, NFATc1, STAT6, and periostin, and cell culture supernatants were assessed for cytokines on days 2 and 7. Mφ grown in conditioned media of PDLFs (paracrine) and Mφ monoculture were used as controls. Data was analyzed using Student t test and one-way analysis of variance with the Tukey multiple comparisons test (P < .05). ResultsPDLF-Mφ coculture showed a higher number of TRAP-positive multinucleated cells than Mφ monoculture on dentin and polystyrene. No TRAP-positive multinucleated cells were observed in paracrine and cementum. The expression of CD80 and CD206 in PDLF-Mφ was similar at day 2, whereas CD206 was greater than CD80 at day 7. The expression of STAT6 was greater than NFATc1 at both days 2 and 7 (P < .05). Periostin expression in the presence of the lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand combination was down-regulated in PDLF monoculture, whereas it was up-regulated in PDLF-Mφ coculture. The cytokine profile of PDLF-Mφ on day 2 was predominated by interleukin (IL)-1β, tumor necrosis factor alpha, and MMP9 and MMP2 on day 7. IL-6 and IL-8 showed steady expression at both days 2 and 7. ConclusionsThe study highlights the juxtacrine effect of PDLFs on the clastic differentiation of Mφ with a difference in clastic activity between dentin and cementum. The study also emphasizes the temporal effect of tumor necrosis factor alpha, MMP2, MMP9, and IL-1β on intercellular crosstalk in resorptive environments.

Novel Tuning of PMMA Orthopedic Bone Cement Using TBB Initiator: Effect of Bone Cement Extracts on Bioactivity of Osteoblasts and Osteoclasts.

Bone cement containing benzoyl peroxide (BPO) as a polymerization initiator are commonly used to fix orthopedic metal implants. However, toxic complications caused by bone cement are a clinically significant problem. Poly (methyl methacrylate) tri-n-butylborane (PMMA-TBB), a newly developed material containing TBB as a polymerization initiator, was found to be more biocompatible than conventional PMMA-BPO bone cements due to reduced free radical generation during polymerization. However, free radicals might not be the only determinant of cytotoxicity. Here, we evaluated the response and functional phenotypes of cells exposed to extracts derived from different bone cements. Bone cement extracts were prepared from two commercial PMMA-BPO cements and an experimental PMMA-TBB. Rat bone marrow-derived osteoblasts and osteoclasts were cultured in a medium supplemented with bone cement extracts. More osteoblasts survived and attached to the culture dish with PMMA-TBB extract than in the culture with PMMA-BPO extracts. Osteoblast proliferation and differentiation were higher in the culture with PMMA-TBB extract. The number of TRAP-positive multinucleated cells was significantly lower in the culture with PMMA-TBB extract. There was no difference in osteoclast-related gene expression in response to different bone cement extracts. In conclusion, PMMA-TBB extract was less toxic to osteoblasts than PMMA-BPO extracts. Although extracts from the different cement types did not affect osteoclast function, PMMA-TBB extract seemed to reduce osteoclastogenesis, a possible further advantage of PMMA-TBB cement. These implied that the reduced radical generation during polymerization is not the only determinant for the improved biocompatibility of PMMA-TBB and that the post-polymerization chemical elution may also be important.

Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κβ ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.

Oat Seedlings Extract Inhibits RANKL-Induced c-Fos/NFATc1 Transcription Factors in the Early Stage of Osteoclast Differentiation

Osteoporosis is a common disease that increases the risk of fractures due to decreased bone density and weakens the bone microstructure. Preventing and diagnosing osteoporosis using the available drugs can be a costly affair with possible side effects. Therefore, natural product-derived therapeutics are promising alternatives. Our study demonstrated that the oat seedlings' extract (OSE) inhibited the receptor activator of the nuclear factor κB ligand (RANKL)-induced osteoclastogenesis from the bone marrow-derived macrophages (BMMs). The OSE treatment significantly attenuated the RANKL-mediated induction of the tartrate-resistant acid phosphatase (TRAP) activity as well as the number of TRAP-positive (TRAP+) multinucleated cells (MNCs) counted through the TRAP staining in a dose-dependent manner. It was also confirmed that the OSE suppressed the formation of the TRAP + MNCs in the early stage of differentiation and not in the middle and late stages. The results of the real-time quantitative polymerase chain reaction (qPCR) and the western blotting showed that the OSE dramatically inhibited the mRNA and protein expressions of the osteoclastogenesis-mediated transcription factors such as the c-Fos and the nuclear factor-activated T cells c1 (NFATc1). In addition, the OSE strongly attenuated the mRNA induction of the c-Fos/NFATc1-dependent molecules such as the TRAP, the osteoclast-associatedimmunoglobulin-like receptor (OSCAR), the dendritic cell-specific transmembrane protein (DC-STAMP), and the cathepsin K. These results suggest that the naturally derived OSE may be useful for preventing bone diseases.

Caffeic Acid Inhibits Swelling, Bone Loss, and Osteoclastogenesis in Adjuvant-induced Arthritis Rats

BACKGROUND: Increase in inflammatory cytokine levels promotes pathological osteoclast differentiation. Caffeic acid has anti-inflammatory properties and can inhibit osteoclast bone resorption. In vitro studies have reported the ability of caffeic acid in inhibiting osteoclastogenesis pathways, however the in vivo study is rarely conducted. The aim of this study is to examine the role of caffeic acid in reducing inflammation and inhibiting osteoclastogenesis in Adjuvant-Induced Arthritis (AIA) rats.METHODS: Rats were injected with Freund’s Complete Adjuvant (CFA) and mineral oil. One day after injection, various concentration (0, 5, 25, 125 mg) of caffeic acid were given gastro-intestinally. Swelling degree in rats’ ankle joints was determined by measuring height and width of each ankle joint. Bone loss level was examined with soft X-ray, and then bone density was calculated. To examine osteoclastogenesis, ankle joints were stained with Tartrate-Resistant Acid Phosphatase (TRAP) and evaluated microscopically. RESULTS: Ankle joints of AIA rats had severe swelling before treated, yet the swelling was reduced based on concentration-dependent after receiving caffeic acid. Severe bone loss in AIA rats’ ankle joints were also observed, however the treatment of 125 mg caffeic acid showed remarkable inhibition effect toward rats’ bone loss. Osteoclastogenesis in AIA rats’ ankle joints were higher than the normal ones, as indicated with high TRAP-positive Multinucleated Cells (MNCs). But low number of TRAP-positive MNCs was observed in ankle joint of AIA rats that received 125 mg caffeic acid.CONCLUSION: Administration of caffeic acid can reduce the degree of swallowing, inhibit bone loss, and inhibit osteoclastogenesis in ankle joint of arthritis-induced rats.KEYWORDS: caffeic acid, osteoclastogenesis, bone loss, swelling, inflammation, RANKL, TNF-α

Curcumin reduces inflammation in rat apical periodontitis.

The aim of the study was to evaluate the effect of systemic curcumin administration on the severity of apical periodontitis (AP). Forty male Wistar rats weighing 250-280 g each, age 2.5months, were distributed into four groups (n=10): control untreated rats (C), control rats treated with curcumin (CUR), rats with pulp exposure-induced apical periodontitis (AP) and rats with pulp exposure-induced apical periodontitis treated with curcumin (AP-CUR). Curcumin treatment was administered orally once daily for 15 days before pulp exposure and continued for 30 days after pulp exposure. The rats were sacrificed at 30 days, and the jaws were collected and reconstructed in a programme specific for micro-CT. The jaws were processed for analysis of the inflammatory process using haematoxylin and eosin staining and immunohistochemical assays for interleukin tumour necrosis factor alpha (TNF-α), interleukin (Il)-6 and Il-1β. Tartrate-resistant acid phosphatase (TRAP) and osteocalcin (OCN) staining were used to analyse the resorptive process on the bone surface of periapical area. Kruskal-Wallis with Dunn's test was performed for nonparametric data and anova with Tukey's test for parametric data, p< .05. Micro-CT revealed no statistically significant differences in bone resorption between the AP and AP-CUR groups (p > .05). The levels of inflammatory cell infiltration and immunoreactivity for the proinflammatory cytokines TNF-α, Il-6 and Il-1β were significantly higher in the periapical lesions of the AP group than in the AP-CUR group (p < .05). The number of TRAP-positive multinucleated cells was higher in the AP group than in the AP-CUR group (p < .05). In OCN-positive cells, no differences were observed between the AP and AP-CUR groups (p > .05). Oral supplementation with curcumin had a significant effect on the AP severity in rats, suggesting an anti-inflammatory effect of curcumin on AP development.

Calcium phosphate-adsorbable and acid-degradable carboxylated polyrotaxane consisting of β-cyclodextrins suppresses osteoclast resorptive activity.

Recently, the potential of β-cyclodextrin-thread acid-degradable polyrotaxane (AdPRX) has been emphasized as a therapeutic agent for cholesterol-related metabolic disorders. In this study, we investigated whether carboxymethyl carbamate-modified AdPRX (CMC-AdPRX) can be used for adsorption to calcium phosphate to treat bone diseases. We first synthesized CMC-AdPRX and used it to coat the calcium phosphate plate. RAW264.7 cells were then differentiated into osteoclasts via a receptor activator of nuclear factor-κB ligand, and the number of osteoclasts and the area of absorption lacunae were determined. The number of tartrate-resistant acid phosphatase-positive multinucleated cells was reduced on the CMC-AdPRX-coated plate. The area of the absorption lacunae was smaller with CMC-AdPRX than with AdPRX, which was not carboxy-modified. Our results suggest that CMC-AdPRX can adsorb to calcium phosphate and act on differentiated osteoclasts to suppress their functional expression.

Systemic Dietary Hesperidin Modulation of Osteoclastogenesis, Bone Homeostasis and Periodontal Disease in Mice.

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (μCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using μCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.

Protection against Osteoporosis by Fermented Mulberry Vinegar Supplementation via Inhibiting Osteoclastic Activity in Ovariectomized Rats and Osteoclastic Cells

Menopause increases the osteoporosis risk, to which phytoestrogen intake can be beneficial. This study hypothesized that mulberry vinegar had a preventive effect on osteoporosis by decreasing osteoclastic activity. The hypothesis was tested in ovariectomized (OVX) rats and RANKL-differentiated osteoclast cells. OVX rats were given 0(OVX-CON), 0.5(OVX-MVL), 1(OVX-MVM), and 2(OVX-MVH) fermented mulberry vinegar (MV) mL/kg body weight (BW) daily for 12 weeks. Sham-operated rats had no MV supplementation (Normal-CON). The osteoporosis-related biomarkers were measured, and Micro-CT determined the bone mass of the femur. RANKL-differentiated Raw 264.7 cells were treated with MV (0–100 μg/mL). The cell viability, osteoporosis-related mRNA expression, and protein contents were measured. MV contained Acetobacter pasteurianus (7.31 log CFU/mL), citric acid (106 mg/mL), lactic acid (19.2 mg/mL), acetic acid (15.0 mg/mL), and rutin (0.36 mg/mL). OVX-MVM elevated the serum 17β-estradiol concentration similar to the Normal-CON group, but it did not prevent the decrease in uterine weight. OVX-MVM prevented the increase in osteoclastic-related parameters, including cathepsin K(CtsK), receptor activator of NF-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) in the circulation. OVX-MVH also lowered C-telopeptide of type Ⅰ collagen as much as the Normal-CON group (p &lt; 0.05). By contrast, OVX-MVH increased the serum osteoprotegerin concentration, an inhibitor of osteoclasts, better than the Normal-CON group (p &lt; 0.05). These changes were integrated to alter the bone mineral density (BMD) in Micro-CT analysis: OVX-MVM and OVX-MVH prevented BMD decrease after OVX as much as the Normal-CON. In RANKL-differentiated osteoclast cells, the MV treatment for 24 and 48 h decreased RANKL-induced differentiation in osteoclast cells dose-dependently up to 100 µg/mL. Its decrease was related to inhibiting the TRAP activity and reducing TRAP-positive multinucleated cells during the five-day administration of RANKL. MV treatments also decreased mRNA expression of osteoclast-related genes (TRAP, Ctsk, OSCAR, and NFATc1). MV suppressed the protein contents of NFATc1 and c-FOS-related osteoclast. In conclusion, MV intake (1 mg/kg bw) protected against BMD loss mainly by inhibiting the osteoclastic activity (RANKL/RANK/TRAP) in OVX rats. MV may develop as a functional food for anti-osteoporosis in menopausal women.

The role of Piezo1 mechanosensor in osteoclastogenesis from monocytes

Abstract Objectives Interrelations between bone and the immune system are involved in both homeostatic and pathogenic bone remodeling processes. Although emerging evidence supports that mechanosensitive Ca2+-channel Piezo1 plays a pivotal role in osteoblast-mediated bone regeneration, possible engagement of Piezo1 in the differentiation of osteoclasts from monocytes and resulting bone resorption is largely unknown. In the present study, we sought to establish the role of Piezo1 in RANKL-stimulated monocytes for their differentiation to osteoclasts in vitro. Methods Mouse RAW 264.7 monocyte cell line or bone marrow derived mononuclear cells (BMMCs) were stimulated with M-CSF and/or RANKL, in the presence or absence of Yoda1 (Piezo1 activator) or with or without hydrostatic pressure (HP). Ca2+ influx assay was employed to confirm Piezo1 channel activity. To evaluate the level of osteoclastogenesis and bone-resorptive function by mature osteoclasts, TRAP staining and pit formation assays were performed. To monitor the expressions of mRNAs for Piezo1 as well as OC-STAMP, TRAP (ACP5) and MMP9, qPCR were employed. Results Yoda1-mediated Ca2+ influx was significantly elevated in RANKL BMMCs and RAW264.7 cells, while significantly suppressing the expressions of osteoclastogenesis-related genes, OC-STAMP, MMP9 and ACP5. Moreover, the number and size of multi-nucleated TRAP-positive cells as well as formation of resorption pits were significantly down-regulated by the treatment with Yoda1 as well as HP. Conclusions The present study suggests that mechanosensitive Piezo1 channel expressed on monocytes may play a gatekeeper role in down-modulating over-activation of osteoclastogenesis and function to resorb bone. Supported by NIH NIDCR grants, DE-027851, DE-028715 and DE-331851

OC_Finder: Osteoclast segmentation, counting, and classification using watershed and deep learning.

Osteoclasts are multinucleated cells that exclusively resorb bone matrix proteins and minerals on the bone surface. They differentiate from monocyte/macrophage lineage cells in the presence of osteoclastogenic cytokines such as the receptor activator of nuclear factor-κB ligand (RANKL) and are stained positive for tartrate-resistant acid phosphatase (TRAP). In vitro osteoclast formation assays are commonly used to assess the capacity of osteoclast precursor cells for differentiating into osteoclasts wherein the number of TRAP-positive multinucleated cells is counted as osteoclasts. Osteoclasts are manually identified on cell culture dishes by human eyes, which is a labor-intensive process. Moreover, the manual procedure is not objective and results in lack of reproducibility. To accelerate the process and reduce the workload for counting the number of osteoclasts, we developed OC_Finder, a fully automated system for identifying osteoclasts in microscopic images. OC_Finder consists of cell image segmentation with a watershed algorithm and cell classification using deep learning. OC_Finder detected osteoclasts differentiated from wild-type and Sh3bp2 KI/+ precursor cells at a 99.4% accuracy for segmentation and at a 98.1% accuracy for classification. The number of osteoclasts classified by OC_Finder was at the same accuracy level with manual counting by a human expert. OC_Finder also showed consistent performance on additional datasets collected with different microscopes with different settings by different operators. Together, successful development of OC_Finder suggests that deep learning is a useful tool to perform prompt and accurate unbiased classification and detection of specific cell types in microscopic images.