Interferon-gamma down-regulates gene expression of cathepsin K in osteoclasts and inhibits osteoclast formation

The cytokine, IFN-gamma, has been shown in vitro to inhibit bone resorption, but the mechanisms responsible for this inhibition have not been clearly defined. Cathepsin K is a major protease responsible for bone resorption. IFN-gamma may inhibit bone resorption through down-regulation of osteoclast genes, including cathepsin K. To test the hypothesis, we investigated the effect of IFN-gamma on cathepsin K expression in the MOCP-5 and wild-type mouse bone marrow co-culture systems by Northern blot as well as osteoclast formation at different stages of differentiation. The results show that IFN-gamma down-regulates mRNA levels of cathepsin K in a time- and dose-dependent manner. Consequently, cathepsin K protein production is also reduced by IFN-gamma. Moreover, our results indicate that IFN-gamma inhibits osteoclast formation only early in osteoclast differentiation. IL-6 and TNFalpha did not significantly affect cathepsin K gene expression in osteoclasts. However, IL-1alpha stimulated gene expression. In conclusion, our data suggest that the actions of IFN-gamma on osteoclastic bone resorption may be mediated by its effects on both osteoclast formation at an early stage and osteoclast gene expression in mature osteoclasts.     

In situ hybridization for matrix metalloproteinase-1 and cathepsin K in rat root-resorbing tissue induced by tooth movement

The movement of teeth during orthodontic treatment occasionally induces undesirable root resorption. Although high collagenolytic activity has been detected in resorbing tissue of deciduous teeth, the cellular origin of collagenolytic enzymes in root-resorbing tissue caused by tooth movement has not been identified. Here, rats were subject to 7 days of experimental tooth movement to induce root resorption. In situ hybridization with digoxigenin-labelled RNA probes was performed on sections of the maxillary bone to detect the mRNAs that encode matrix metalloproteinase-1 (MMP-1) and cathepsin K in root-resorbing tissue. MMP-1 mRNA was detected in fibroblastic cells, cementoblasts and osteoblasts, but not in odontoclasts nor osteoclasts. Moreover, MMP-1 mRNA was highly expressed in some cementocytes located near odontoclasts and in many osteocytes. In contrast, cathepsin K mRNA was expressed only in odontoclasts and osteoclasts. These results suggest that MMP-1 and cathepsin K are important in root resorption during tooth movement in a mode similar to bone resorption.     

Phytochemicals That Stimulate Osteoblastic Differentiation and Bone Formation

Some bone diseases, such as osteoporosis and periodontitis, result from an imbalance in bone remodeling characterized by excessive bone resorption relative to bone formation. Two different pharmacological approaches can be used to treat such diseases: anti-resorptioe agents that inhibit osteoclastic bone resorption, and anabolic agents that stimulate osteoblastic bone formation. Anti-resorptioe agents, such as bisphosphonates and calcitonin, are currently available for the treatment of osteoporosis ; however, these anti-resorptioe agents are unlikely to have an anabolic effect, and the need for anabolic agents aimed at increasing bone mass has recently been a matter for concern. Natural compounds could provide the basis for developing anabolic agents. A wide variety of natural compounds has recently been found to stimulate osteoblastic bone formation. This review describes the effects of natural compounds on osteoblast proliferation, differentiation, and bone formation in vitro and in vivo.     

Histological and Histochemical Analyses of Cell-mediated Resorption of Anorganic Bovine Bone Matrix at the Site of Sinus Floor Augmentation in Humans

Sterilized particles of anorganic bovine bone matrix (ABBM: Bio-Oss^®), widely used for sinus floor augmentation, are characterized by high bone conductivity and long persistence due to slow resorption. Here we report the histological and histochemical features of ABBM particles applied for bone augmentation in 5 adult patients with special reference to the mode of osteoclastic resorption of xenogenic material. Bone cores retrieved at 6 months after surgery contained densely packed ABBM particles fully or partially encapsulated by the newly induced bone and showed immunoreactivity for osteopontin (OPN) at the bone/ABBM boundary. Multinuclear cells showing enzymatic reactions for tartrate-resistant acid phosphatase (TRAP) were in contact with 48.26±9.43% of the ABBM surface but only 2.13±0.84% of that of induced bone. TRAP-positive multinuclear cells attached to the ABBM particles were enriched with mitochondria but, with a few exceptions, lacked a ruffled border and immunoreactivity for cathepsin K. These data indicate that a predominantly large proportion of TRAP-positive cells at the graft site are hypofunctional osteoclasts or osteoclastic cells at 6 months after surgery, and explain the long persistence of grafted ABBM particles despite the abundance of TRAP-positive multinuclear cells. Graft samples retrieved at 2 weeks from the extraction socket suggested the role of tiny ABBM fragments for the induction of TRAP-positive multinuclear cells in the early postoperative phase. Due to the limited availability of samples, how grafted xenogenic material is involved in bone remodeling in later time periods is yet to be determined.     

Effects of β-cryptoxanthin on bone-formation parameters in the distal femoral epiphysis of ovariectomized mice

Objective

To compare the effects of β-cryptoxanthin (β-cry) from citrus fruit on bone-formation parameters in the distal femoral epiphysis of ovariectomized (OVX) mice with the effects of osteoporosis therapeutic agents.

Methods

Ten-week-old OVX ICR mice were used. The mice were divided into five experimental groups: β-cry; bisphosphonate (treatment with the bone resorption inhibitor risedronate); vitamin K₂ [treatment with the bone formation stimulator and bone resorption inhibitor menaquinone 4 (MK4)]; OVX control; and sham (no OVX). Risedronate was used as the bisphosphonate (risedronate group), and menaquinone 4 was used as vitamin K₂ (MK4 group). Risedronate was administered orally, and β-cry and MK4 were mixed with the regular feed. The mice were fed the diets for 2 months. Femurs were resected, and blood samples were collected. Bone mineral density (BMD) was analyzed by peripheral quantitative computed tomography. Microcomputed tomography was also performed to evaluate the three-dimensional structure of the trabecular bone.

Results

In contrast to the risedronate and MK4 groups, there was no improvement in the trabecular BMD, cortical thickness, or periosteal perimeter in the β-cry group. Trabecular structural parameters including the morphometric indices, continuity index, and connectivity index were significantly improved in the β-cry group compared with the OVX control group. In particular, there was a marked improvement in morphometric indices.

Conclusion

β-Cry improved the trabecular structure. This suggests that β-cry might be useful as a dietary supplement to prevent bone degeneration and osteoporosis associated with aging.     

A concept for the treatment of various dental bone defects

Untreated dental bone defects usually lead to resorption of alveolar bone. Filling these defects with bone substitute material prevents resorption of bone, preserves the alveolar ridge, and provides sufficient bone for immediate or subsequent implant placement. A variety of bone substitutes is available. They differ in origin, consistency, particle size, porosity, and resorption characteristics. We have treated almost 1000 bony defect sites in 267 patients with the bone regeneration material Cerasorb. Being resorbed simultaneously with the formation of new bone, it is completely replaced by the patient's own vital bone within 6 to 12 months. The representative cases described in this paper demonstrate the successful use of the pure-phase beta-tricalcium phosphate ceramic in the treatment of all dental bone defects.     

The effect of two locally administered anti-resorptive agents on bone regeneration in a rat fibula model: Alendronate and 15-deoxy-Δ12,14-prostaglandin J2

Bisphosphonates are well-known drugs as inhibitors of bone resorption acting on inducing programmed cell death of osteoclasts. However, many in vitro studies report that optimal concentration of the bisphosphonate affects not only osteoclasts but also osteoblasts, that is, it induces the anabolic effects of osteoblasts. Recently reported 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is an endogenous ligand of peroxisome proliferator-activated receptor-gamma, with an inhibitory activity on bone loss. Researchers have also suggested that 15d-PGJ₂ has the ability to reduce bone destruction and as the possibility of regeneration of bone.The purpose of this study is to demonstrate the anabolic effect of two anti-resorptive materials, alendronate and 15d-PGJ₂, in a critical sized segmental defect model of rat fibula. The regenerated bone on the operative site was assessed through gross, radiographic (plain X-ray, and micro-computed tomography), histomorphologic evaluation, and statistical analysis. Consequently, the locally applied alendronate prevented resorption of grafted materials, and had a positive effect on bone regeneration with positive micro-architectural modification of the surrounding bone, although this study did not verify a significant capacity of bone regeneration of 15d-PGJ₂ and instead only shed a light on its possibility.     

Effect of bisphosphonates and gallium on dentin resorption in vitro

Abstract Replacement resorption may follow the replantation of an avulsed tooth. Currently there is no effective treatment for replacement resorption. The purpose of this study was to investigate the effect of bisphosphonates and gallium nitrate, which have been shown to reduce bone resorption, on cells which resorb dentin. Osteoclast-like cells were obtained by culturing cells from prenatal chick tibeas. These cells were seeded onto slices of human dentin which had been soaked in either saline (control), or solutions of 10^–5 M 1-hydroxyethylidene-1, 1-bisphosphonic acid (EHBP), 10^–6 M dichloromethylene bisphosphonic acid (Cl₂MBP), or 10^–6 M gallium nitrate. Resorption was measured by counting the number of resorptive lacunae produced by the cells. Results indicated that the experimental groups did not differ significantly from each other, but each exhibited significantly reduced resorption compared with saline controls (p<0.01). These results suggested that the experimental treatment reduced dentinal resorption by the osteoclast-like cells, and that these agents might be useful to prevent or at least postpone replacement resorption in avulsed teeth.     

RANKL and cathepsin K in diffuse sclerosing osteomyelitis of the mandible

BACKGROUND: Diffuse sclerosing osteomyelitis (DSO) of the mandible is characterized by mixed bone resorption and formation. METHODS: Immunohistopathology of DSO in the clinically acute and subacute phases was compared with healthy bone. RESULTS: Receptor activator of nuclear factor kappaB ligand (RANKL) was found in DSO lesions. When it was used in vitro to stimulate monocytes, cathepsin K expression was observed in mononuclear prefusion precursors and in multinuclear giant cells. Similarly, exacerbations of DSO were characterized by RANKL and induction of cathepsin K in mononuclear precursor cells, which subsequently seem to differentiate into osteoclasts or foreign body giant cells. The proportion of bone to soft tissue increased with the duration of disease. CONCLUSIONS: RANKL-driven osteoclastogenesis and acidic cysteine endoproteinase cathepsin K seem to play important roles in DSO as osteoclast-mediated bone resorption may represent the primary disease process later followed by new bone formation.     

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice

ObjectiveToll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2^−/−) mice to gain insight into TLR2 function in biological circumstances without microbial infection.MethodsEight-week-old male wild-type and TLR2^−/− mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis.ResultsTLR2^−/− tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2^−/− osteoclasts reduced cell fusion and bone resorption activity.ConclusionIt seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.     

iRoot BP Plus对小鼠炎性骨破坏作用的研究

目的:比较新型口腔生物陶瓷材料iRoot BP Plus和传统材料三氧化矿化聚合物(MTA)对LPS诱导小鼠炎症性骨破坏的影响。方法:24只BABL/C小鼠随机平分为4组,分别注射PBS、LPS(10 mg/kg)、iRoot BP Plus浸提液+LPS和MTA浸提液+LPS,1次/d。7 d后取颅盖骨,通过显微CT扫描、HE染色、酶组织化学和组织免疫荧光染色,观察骨破坏程度、破骨细胞以及组织蛋白酶K的表达。结果:iRoot BP Plus和MTA可有效减轻小鼠颅盖骨炎性骨破坏,抑制破骨细胞的形成和吸收能力,并明显降低与破骨功能相关的组织蛋白酶K的表达。 结论: iRoot BP Plus对小鼠炎性骨破坏有抑制作用。     

Mineral trioxide aggregate inhibits osteoclastic bone resorption

Mineral trioxide aggregate (MTA), a commonly used endodontic repair material, is useful for both basic and clinical research, and the effect of MTA on osteoblast differentiation has been well-defined. However, the effects of MTA on osteoclastic bone resorption are not fully understood. Hence, the aim of this study is to examine the effect of MTA solution in the regulation of osteoclast bone-resorbing activity using osteoclasts formed in co-cultures of primary osteoblasts and bone marrow cells. MTA solution dose-dependently reduced the total area of pits formed by osteoclasts. The reduction of resorption induced by 20% MTA treatment was due to inhibition of osteoclastic bone-resorbing activity and had no effect on osteoclast number. A 20% MTA solution disrupted actin ring formation, a marker of osteoclastic bone resorption, by reducing phosphorylation and kinase activity of c-Src, and mRNA expressions of cathepsin K and mmp-9. A high concentration of MTA solution (50%) induced apoptosis of osteoclasts by increasing the expression of Bim, a member of the BH3-only (Bcl-2 homology) family of pro-apoptotic proteins. Taken together, our results suggest that MTA is a useful retrofilling material for several clinical situations because it both stimulates osteoblast differentiation and inhibits bone resorption.     

Expression of cathepsin K mRNA during experimental tooth movement in rat as revealed by in situ hybridization

The expression of cathepsin K. a novel collagenolytic enzyme specifically expressed in osteoclasts, was investigated in the rat maxillary dentoalveolar unit during experimental tooth movement by in situ hybridization histochemistry with a non-radioisotopic cRNA probe for rat cathepsin K. Orthodontic elastics were inserted into the interproximal space between the maxillary first and second molars of 7-week-old male SD rats according to Waldo's method and sections prepared from tissues obtained at 12 hr, 1, 2, 3, 4, 7, and 12 days after orthodontic force application. Cathepsin K mRNA expression was detected in the mono- and multinuclear osteoclasts on the pressure side of the alveolar bone at 12 hr after force application, and the distribution and number of cathepsin K mRNA-positive osteoclasts increased time-dependently on the pressure side. At 3-4 days, a marked increase in cathepsin K mRNA-positive osteoclasts was found not only on the pressure side but also on the tension side of the alveolar bone in response to tooth movement. At 7-12 days, the cathepsin K mRNA-positive osteoclasts on both sides had disappeared. These findings suggest that the recruitment of osteoclasts on the pressure side begins during the initial stage of orthodontic tooth movement and the site-specific early induction of cathepsin K mRNA may cause an imbalance in the relative resorption activities on the pressure and tension side incident to such movement.     

Immediately loaded bar-connected implants with an anodized surface inserted in the anterior mandible in a patient treated with diphosphonates for osteoporosis: a case report with a 12-month follow-up

Background: It has been suggested that tooth loss is greater in the osteoporotic patient population. Only a few cases have been reported in the literature about the use of dental implants in patients with osteoporosis. Diphosphonates are stable analogs of pyrophosphate, a physiologic regulator of calcification and bone resorption. Multiple implant failures have been reported in a patient undergoing treatment with diphosphonates. Recently, several clinical and experimental reports have shown that immediate loading of dental implants is possible in selected situations. Purpose: The aim of this case report was to present the clinical outcome of immediate loading of implants in a patient undergoing diphosphonate treatment for osteoporosis. Materials and Methods: In a 65‐year‐old patient undergoing diphosphonate treatment for osteoporosis, four implants were inserted in the anterior mandible. The implants were connected with a bar supporting an overdenture and were then loaded the same day. Results: No problems occurred in the postoperative period. At 1‐year follow‐up, all four of the implants appeared to be clinically osseointegrated, and no mobility was present. Minimal bone resorption was present around all implants. Conclusions: Our case report points to the fact that, contrary to what has been reported in the literature, it is possible to successfully insert and load immediately after surgery dental implants in a patient undergoing diphosphonate treatment for osteoporosis     

Slow Resorption of Anorganic Bovine Bone by Osteoclasts in Maxillary Sinus Augmentation

Purpose: Different biomaterials have been suggested for guided bone regeneration (GBR). These might show the ideal properties to let a new bone formation in the grafted area. Among these ideal features, it is essential their controlled resorption in order to be replaced for new vital bone. Bovine bone has been used widely as a good biomaterial for GBR, however there is still an interesting controversy about its resorbable capacity. In this sense, the objective of this study was to examine the behavior of anorganic bovine bone (ABB) in long‐term maxillary sinus graft healing and study its relationship with morphological and morphometrical variables. Materials and Methods: Seventeen maxillary sinus augmentation procedures were performed in patients. Bone cores were obtained from implant receptor sites at 6 months, 3 years, and 7 years of implant placement for histological, morphometric, and immunohistochemical (tartrate resistant acid phosphatase [TRAP]/cathepsin K/CD68) studies. Results: The percentages of bone, ABB particles, connective tissue, osteocytes, and osteoblasts in maxillary sinus grafts were similar at 6 months, 3 years, and 7 years. A progressive and significant decrease was detected in osteoclasts (p = .05, Kruskal‐Wallis test), TRAP and cathepsin K expression (p = .014 and p = .021, respectively), and osteoid lines (p = .038). Conclusion: According to these data, a decrease in osteoclasts over time may, partially, explain the ABB persistence observed in core biopsies. Further studies with more cases and different graft maturation times are required to elucidate the resorption rates and cell events underlying these phenomena.     

BioAggregate和MTA对体内破骨细胞分化和功能的影响

目的研究口腔生物陶瓷材料Bio Aggregate及MTA对体内LPS诱导的炎性骨吸收的影响。方法 6周龄C57/BL6雄性小鼠随机分为四组:PBS组、LPS组、Bio Aggregate+LPS组和MTA+LPS组,每组6只。LPS干预小鼠7 d后,取出颅盖骨进行显微-CT扫描、HE染色、酶组织化学染色和组织免疫荧光染色,观察骨破坏面积、破骨细胞的形成以及组织蛋白酶K(cathepsin K)的表达。结果 Bio Aggregate和MTA浸提液明显减少LPS诱导的破骨细胞形成和小鼠颅盖骨炎性骨破坏。与破骨细胞功能密切相关的组织蛋白酶K的表达在Bio Aggregate和MTA浸提液的作用下被显著下调。结论新型口腔生物陶瓷材料Bio Aggregate和传统材料MTA能够抑制体内炎性骨吸收。     

Micro-structured calcium phosphate ceramic for donor site repair after harvesting chin bone for grafting alveolar clefts in children

ObjectivesThe purpose of this study was to evaluate the use of synthetic bone graft material as a filling material at the mandibular symphysis donor site of autologous bone in children.Materials and methodsA blinded patient group comprised 20 patients with unilateral (UCLP) or bilateral (BCLP) cleft of lip and palate, all with an indication for alveolar cleft repair. The study took the form of a prospective randomized clinical trial. We used lateral cephalograms for the measurement of the symphyseal donor area defect both peroperatively and at 12 months postoperatively. The data obtained were digitalized and the treatment outcome expressed in numbers. Comparisons with a previous study were made. Histology of biopsies and CT scans were used for visualising bone formation.ResultsThis study demonstrates that the micro-structured, resorbable calcium phosphate ceramic provides good regeneration properties for the repair of a critical size bony defect in children. One year postoperatively, the measurements taken from lateral cephalograms show that there is scarcely any visible residual defect. Histological investigations of the bone biopsies show solid, induced bone formation and almost complete resorption of the micro-structured calcium phosphate.ConclusionsThe findings of this study (novel in children) indicate that micro-structured resorbable calcium phosphate is an excellent alternative to autologous bone. The digital findings showed a restored donor site defect significantly indicating the efficacy (i.e. osteoconductivity and resorbability) of this bone substitute. The biopsy histology demonstrated the overall presence of newly formed vital bone and the resorption of the bone substitute. Its use for grafting the alveolar cleft is currently researched and it may become the new standard.Clinical relevanceAs co-morbidity and prolonged operation time at the donor operation site are inherent to the alveolar cleft repair procedure, the use of the described bone substitute is winning progress.     

Regulation of bone remodeling by vitamin K2

All living tissues require essential nutrients such as amino acids, fatty acids, carbohydrates, minerals, vitamins, and water. The skeleton requires nutrients for development, maintaining bone mass and density. If the skeletal nutritional requirements are not met, the consequences can be quite severe. In recent years, there has been growing interest in promotion of bone health and inhibition of vascular calcification by vitamin K2. This vitamin regulates bone remodeling, an important process necessary to maintain adult bone. Bone remodeling involves removal of old or damaged bone by osteoclasts and its replacement by new bone formed by osteoblasts. The remodeling process is tightly regulated, when the balance between bone resorption and bone formation shifts to a net bone loss results in the development of osteoporosis in both men and women. In this review, we focus on our current understanding of the effects of vitamin K2 on bone cells and its role in prevention and treatment of osteoporosis.     

Osteoporosis and oral bone resorption: a review

This paper is a summary of the published studies on the possible association between osteoporosis and alveolar bone loss. Osteoporosis and low bone mass are considered as a major public health problem. The mandible like other bones of the body has a series of anatomical landmarks that can serve as radiographic indicators. Using these indicators it is possible to evaluate changes in bone with respect to its quantity or quality by different methods of taking images. Higher bone resorption was detected in women with a higher number of pregnancies. Also, the higher educated the patient, the less bone resorption. Women with a background of backaches had more bone resorption to those who did not have this backache background. Finally, it was recognized that it would be possible to clear the quality dimension of the process of mandibular bone resorption. If we can identify the osteoporotic process using a basic panoramic radiography measurement technique, then it is possible to intercept the progress of the disease through early warning and treatment. From the results of this study, it can be concluded the thickness of the mandibular angular cortex can be used as an index for bone resorption. A healthy lifestyle has multiple benefits for the mouth and throughout the body. Dental professionals can play a role in preventing osteoporosis by reinforcing this message.     

Td92, an outer membrane protein of Treponema denticola,induces osteoclastogenesis via prostaglandin E2‐mediated RANKL/osteoprotegerin regulation

Kim M, Jun H‐K, Choi B‐K, Cha J‐H, Yoo Y‐J. Td92, an outer membrane protein of Treponema denticola, induces osteoclastogenesis via prostaglandin E₂‐mediated RANKL/osteoprotegerin regulation. J Periodont Res 2010; 45: 772–779. © 2010 John Wiley & Sons A/S Background and Objective: Periodontitis is a chronic inflammatory disease of the periodontium that causes significant alveolar bone loss. Osteoclasts are bone‐resorbing multinucleated cells. Osteoblasts regulate osteoclast differentiation by expression of RANKL and osteoprotegerin (OPG). Td92 is a surface‐exposed outer membrane protein of Treponema denticola, a periodontopathogen. Although it has been demonstrated that Td92 acts as a stimulator of various proinflammatory mediators, the role of Td92 in alveolar bone resorption remains unclear. Therefore, in this study, we investigated the role of Td92 in bone resorption. Material and Methods: Mouse bone marrow cells were co‐cultured with calvariae‐derived osteoblasts in the presence or absence of Td92. Osteoclast formation was assessed by TRAP staining. Expressions of RANKL, osteoprotegerin (OPG) and prostaglandin E₂ (PGE₂) in osteoblasts were estimated by ELISA. Results: Td92 induced osteoclast formation in the co‐cultures. In the osteoblasts, RANKL and PGE₂ expressions were up‐regulated, whereas OPG expression was down‐regulated by Td92. The addition of OPG inhibited Td92‐induced osteoclast formation. The prostaglandin synthesis inhibitors NS398 and indomethacin were also shown to inhibit Td92‐induced osteoclast formation. The effects of Td92 on the expressions of RANKL, OPG and PGE₂ in osteoblasts were blocked by NS398 or indomethacin. Conclusion: These results suggest that Td92 promotes osteoclast formation through the regulation of RANKL and OPG production via a PGE₂‐dependent mechanism.