Boundary mode lubrication of articular cartilage by recombinant human lubricin

Lubrication of cartilage involves a variety of physical and chemical factors, including lubricin, a synovial glycoprotein that has been shown to be a boundary lubricant. It is unclear how lubricin boundary lubricates a wide range of bearings from tissue to artificial surfaces, and if the mechanism is the same for both soluble and bound lubricin. In the current study, experiments were conducted to investigate the hypothesis that recombinant human lubricin (rh-lubricin) lubricates cartilage in a dose-dependent manner and that soluble and bound fractions of rh-lubricin both contribute to the lubrication process. An rh-lubricin dose response was observed with maximal lubrication achieved at concentrations of rh-lubricin greater than 50 µg/mL. A concentration–response variable-slope model was fit to the data, and indicated that rh-lubricin binding to cartilage was not first order. The pattern of decrease in equilibrium friction coefficient indicated that aggregation of rh-lubricin or steric arrangement may regulate boundary lubrication. rh-lubricin localized at the cartilage surface was found to lubricate a cartilage–glass interface in boundary mode, as did soluble rh-lubricin at high concentrations (150 µg/mL); however, the most effective lubrication occurred when both soluble and bound rh-lubricin were present at the interface. These findings point to two distinct mechanisms by which rh-lubricin lubricates, one mechanism involving lubricin bound to the tissue surface and the other involving lubricin in solution. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 771–777, 2009     

Binding and localization of recombinant lubricin to articular cartilage surfaces.

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.     

Genetic polymorphisms in IL‐2, IL‐10, TGF‐β1, and IL‐2RB and acute rejection in renal transplant patients

Acute rejection (AR) remains a concern for kidney transplantation. Cytokines are key mediators in the induction and effector phases of all immune and inflammatory responses. Single nucleotide polymorphisms (SNPs) in cytokines and their receptors may relate to AR. We investigated the relation between AR and SNPs in the genes encoding for IL‐2(–330G>T), IL‐10(?592C>A and ?1082G>A), TGF‐β1(915G>C), and IL‐2RB(rs228942 C>A and rs228953 C>T) in 325 renal transplant patients during the first year after transplantation. The overall incidence of AR was 15.4%. In multivariate analysis, only the use of induction therapy was correlated with AR (odds ratio 1.9; 95% confidence interval 1.1–3.7; p = 0.04). No statistically significant associations between the SNPs studied and AR were observed. SNPs in the investigated cytokines and their receptors were not associated with the risk of AR. Genotyping patients for these SNPs is unlikely to aid the clinician in adjusting the immunosuppressive therapy for individual patients.     

Association of TNF‐α, TGF‐β1, IL‐10, IL‐6, and IFN‐γ gene polymorphism with acute rejection and infection in lung transplant recipients

Infection and rejection are common complications faced by lung transplant recipients (LTRs) and have become major impediments to long‐term survival. Cytokines may play an important role in the development of these complications. In this study, we explored the correlation between TNF‐α (?308 A/G), TGF‐β1 (+869 T/C, +915 G/C), IL‐10 (?592 C/A, ?819 T/C, ?1082 G/A), IL‐6 (?174 G/C), and IFN‐γ (+874 T/A) gene polymorphisms and the incidence of acute rejection and infection. Transplant outcomes were reviewed in a retrospective cohort of 113 LTRs from a single center between December 2004 and November 2012. Cytokine polymorphisms were measured using sequence‐specific primer‐based PCR. HLA typing was performed for the donors and recipients. We found that the LTRs with the IL‐10 ?819 CC and ?592 CC genotypes had a significantly decreased risk of infection (p = 0.017, OR = 0.177, 95% CI = 0.04–0.85). However, we found no significant association between cytokine polymorphisms and acute rejection. Furthermore, the data revealed that the occurrence of acute rejection was strongly associated with infection episodes (χ² = 8.5256, p < 0.01). These results suggest that LTRs possessing the IL‐10 ?819 CC and ?592 CC genotype may be protected from the occurrence of infection. Our results demonstrated that infection is an important cause of acute rejection for LTRs.     

Increased accumulation of superficial zone protein (SZP) in articular cartilage in response to bone morphogenetic protein-7 and growth factors.

The purpose of this study was to investigate the role of bone morphogenetic proteins (BMPs), such as BMP-7, growth factors, and cytokines, in the accumulation of superficial zone protein (SZP) in bovine articular cartilage. Calf superficial articular cartilage discs and chondrocytes were obtained for explant and monolayer culture systems, respectively. Dose- and time-dependent actions of BMP-7 on SZP accumulation were investigated in both explant and monolayer culture systems. In addition, actions of various morphogens and growth factors [BMP-2, BMP-4, fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-beta1)], and cytokines [interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor (TNF-alpha)] alone, and in combination with BMP-7, on SZP accumulation were investigated in monolayer culture systems. SZP accumulation was quantified in both the cartilage and the medium using SDS-PAGE and subsequent immunoblotting. In both explant and monolayer cultures, BMP-7 increased SZP accumulation in a dose- and time-dependent fashion (p < 0.05). Furthermore, SZP accumulation was significantly increased in monolayer cultures by FGF-2, IGF-1, PDGF, and TGF-beta1 (p < 0.05). Both IL-1alpha and TNF-alpha significantly reduced SZP accumulation (p < 0.05). The inhibition of SZP accumulation by TNF-alpha was partially alleviated by concurrent treatment with BMP-7. The results of this investigation provide novel insights into the role of morphogens, especially BMP-7, growth factors, and cytokines in the accumulation of SZP in articular cartilage. This information has clinical implications because stimulation of SZP may ameliorate the pathology of joint function in arthritis. Furthermore, tissue engineering approaches to articular cartilage may depend on the optimal synthesis and assembly of SZP in the superficial zone to ensure functional tissue architecture.     

Induction of chondrogenesis and superficial zone protein accumulation in synovial side population cells by BMP‐7 and TGF‐β1

Synovial cells are known to contain a sub‐population of cells with multipotent differentiating capacity including chondrocytes. However, the stem/progenitor cells in synovial cells have not been well characterized. Stem/progenitor cells can exclude Hoechst 33342 dye, and the cell fraction with this property is called “side population (SP).” SP cells are present in many adult tissues. The aim of this investigation was to identify, isolate, and characterize SP cells from bovine synovium. Hoechst dye efflux and fluorescence activated cell sorting showed that synovial cells contained 0.60% SP cells. In the presence of verapamil, an inhibitor of ABC transporters critical for the dye efflux property, the SP cell fraction was not observed, indicating the critical role of ABC transporters. Isoforms of ABC transporters (ABCG2 and ABCB1 mRNA) were highly expressed in SP cells derived from the synovial cells by real‐time RT‐PCR analysis. Bone morphogenetic protein‐7 (BMP‐7) induced type II collagen mRNA expression characteristic of chondrogenesis in articular cartilage with both SP and non‐SP cells. In addition, expression of SZP mRNA, a marker of the surface layer of articular cartilage, was significantly up‐regulated by BMP‐7, and the protein accumulation of SZP was stimulated by both BMP‐7 and TGF‐β1. Thus, synovial cells contain ABC transporter‐dependent SP cells. These findings demonstrate that side population cells of synovium differentiate toward an articular chondrocyte phenotype of the surface layer and have direct implications for tissue engineering and regeneration of articular cartilage. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:485–492, 2008     

B cell–derived IL‐1β and IL‐6 drive T cell reconstitution following lymphoablation

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti‐thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell–derived soluble factors. B cell production of interleukin (IL)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, IL‐6, IL‐1R, MyD88, or IL‐6R impair CD4⁺ and CD8⁺ T cell recovery and significantly enhance the graft‐prolonging efficacy of lymphoablation. Adoptive co‐transfer experiments demonstrate a direct effect of IL‐6 but not IL‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β or IL‐6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell–derived IL‐1β and IL‐6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.     

Influence of the growth factors PDGF‐BB,TGF‐β1 and bFGF on the replicative aging of human articular chondrocytes during in vitro expansion

Decreasing replicative potential and dedifferentiation of articular chondrocytes during expansion in cell culture are essential limitations for tissue engineering and cell therapy approaches. Telomeres and telomerase play a key role in cell development, aging, and tumorigenesis. There is evidence that growth factors are involved in regulating telomerase activity. Therefore, the objective was to evaluate the effect of selected growth factors on telomere biology of serially passaged chondrocytes. Human articular chondrocytes were isolated from cartilage of three patients undergoing total knee arthroplasty. The chondrocytes were cultured in monolayer with the growth factors PDGF‐BB, TGF‐β1, and bFGF. Telomere length was measured by telomere restriction fragment length assay, and telomerase activity was determined by quantifying the gene expression of its catalytic subunit hTERT by rtPCR. Chondrocytes cultured with PDGF‐BB and TGF‐β1 showed a significantly higher proliferation rate than control cells. None of the growth factor cultures revealed an accelerated rate of telomere shortening. Telomerase was not expressed in significant amounts in any of the chondrocyte cultures. Growth factor treatment of chondrocyte cell cultures for cell therapy purposes can be regarded as safe in terms of telomere biology. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:354–360, 2010     

Autologous solution protects bovine cartilage explants from IL‐1α‐ and TNFα‐induced cartilage degradation

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro‐ and anti‐inflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL‐1α‐ or TNFα‐challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL‐1α and TNFα. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL‐1ra, sTNF‐RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin‐O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL‐1α‐induced collagen release over a 21‐day culture period. APS treatment reduced the degree of Safranin‐O staining loss when cartilage explants were cultured with IL‐1α or TNFα. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1929–1935, 2013     

Phospho‐Smad1 modulation by nedd4 e3 ligase in BMP/TGF‐β signaling

A considerable number of studies have focused on the regulation of mothers against decapentaplegic homologue (Smad)–dependent or –independent pathways in the signaling by each transforming growth factor β (TGF‐β) superfamily member in diverse biologic contexts. The sophisticated regulation of the actions of these molecules and the underlying molecular mechanisms still remain elusive. Here we show new mechanisms of ambilateral R (receptor‐regulated)–Smad regulation of bone morphogenetic protein 2 (BMP‐2)/TGF‐β1 signals. In a specific context, both signals regulate the nonclassic Smads pathway reciprocally, BMP‐2 to Smad2/3 and TGF‐β1 to Smad1/5/8, as well as their own classic linear Smad pathway. Interestingly, in this study, we found that C‐terminal phosphorylated forms of each pathway Smad degraded rapidly 3 hours after stimulation of nonclassic signals but are dramatically restored by treatment with via proteasomal inhibition. Furthermore, an E3 ligase, neural precursor cell expressed, developmentally down‐regulated 4 (Nedd4), also was found as one of the important modulators of the p‐Smad1 in both BMP‐2 and TGF‐β1 action. Overexpressed Nedd4 suppressed the BMP‐induced osteoblast transdifferentiation process of premyoblast C2C12 cells or alkaline phosphatase (ALP) level of human osteosarcoma cells and promoted TGF‐β1‐induced degradation of p‐Smad1 via physical interaction and polyubiquitination. Conversely, siNedd4 potentiated BMP signals through upregulation of p‐Smad1 and ALP activity, the effect of which led to an increased the rate of P_i‐induced calcification of human vascular smooth muscle cells. These new insights about proteasomal degradation–mediated phosphorylated nonclassic Smad regulation of BMP‐2/TGF‐β1 could, in part, help to unravel the complex mechanisms of abnormal nonosseous calcification by the aberrant activity of BMP/TGF‐β/Smads. © 2011 American Society for Bone and Mineral Research.     

Synoviocytes are more sensitive than cartilage to the effects of minocycline and doxycycline on IL‐1α and MMP‐13‐induced catabolic gene responses

The objective of this study was to determine the primary articular tissue target of doxycycline and minocycline. Synoviocytes—cartilage cocultures (n = 4) were treated with MMP‐13 (25 ng/mL medium) or IL‐1 (1.0 ng/mL medium) for 24 h. Doxycycline (4.3, 0.43, 0.043 µM) or minocycline (10, 1.0 or 0.1 µM) were then added and cultures were continued for 96 h. Cartilage and media were analyzed for GAG content. Quantitative PCR was used to measure cartilage MMP‐3, MMP‐13, aggrecan, COL2A1, ADAMTS‐4, and ADAMTS‐5 expression, and synoviocyte MMP‐3, MMP‐13, ADAMTS‐4, and ADMATS‐5 expression. Total and active MMP‐3, MMP‐13, and ADAMTS 4/5 enzymes were measured in culture medium. All concentrations of doxycycline and minocycline diminished GAG accumulation in the media. All concentrations of minocycline, but only the highest concentration of doxycycline decreased MMP‐3 and MMP‐13 expression in synoviocytes but not cartilage, and basal ADAMTS‐5 mRNA levels in both synoviocytes and cartilage. Only minocycline decreased active MMP‐13 protein in synoviocytes. In summary, the protective effects of tetracycline compounds are more pronounced in synoviocytes than cartilage, and following minocycline compared to doxycycline. Studies to determine the molecular mechanism of action of the tetracyclines in synoviocytes might lead to the design of targeted therapeutics for the treatment of OA or RA. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:522–528, 2010     

Autologous protein solution inhibits MMP‐13 production by IL‐1β and TNFα‐stimulated human articular chondrocytes

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet‐rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF‐β1, TGF‐β2, EGF, IGF‐1, PDGF‐AB, PDGF‐BB, and VEGF) and anti‐inflammatory (IL‐1ra, sTNF‐RI, sTNF‐RII, IL‐4, IL‐10, IL‐13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL‐1α, IL‐1β, TNFα, IL‐6, IL‐8, IL‐17, and IL‐18). Human articular chondrocytes were pre‐incubated with the antagonists IL‐1ra, sTNF‐RI, or APS prior to the addition of recombinant human IL‐1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP‐13 in the culture medium were evaluated by ELISA. MMP‐13 production stimulated in chondrocytes by IL‐1β or TNFα was reduced by rhIL‐1ra and sTNF‐RI to near basal levels. APS was also capable of inhibiting the production of MMP‐13 induced by both IL‐1β and TNFα. The combination of anabolic and anti‐inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1320–1326, 2011     

Lithium chloride prevents interleukin‐1β induced cartilage degradation and loss of mechanical properties

Osteoarthritis is a chronic degenerative disease that affects the articular cartilage. Recent studies have demonstrated that lithium chloride exhibits significant efficacy as a chondroprotective agent, blocking cartilage degradation in response to inflammatory cytokines. However, conflicting literature suggests lithium may affect the physicochemical properties of articular cartilage and thus long‐term exposure may negatively affect the mechanical functionality of this tissue. This study aims to investigate the effect of lithium chloride on the biomechanical properties of healthy and interleukin‐1β treated cartilage in vitro and examines the consequences of long‐term exposure to lithium on cartilage health in vivo. Bovine cartilage explants were treated with lithium chloride for 12 days. Chondrocyte viability, matrix catabolism and the biomechanical properties of bovine cartilage explants were not significantly altered following treatment. Consistent with these findings, long term‐exposure (9 months) to dietary lithium did not induce osteoarthritis in rats, as determined by histological staining. Moreover, lithium chloride did not induce the expression of catabolic enzymes in human articular chondrocytes. In an inflammatory model of cartilage destruction, lithium chloride blocked interleukin‐1β signaling in the form of nitric oxide and prostaglandin E2 release and prevented matrix catabolism such that the loss of mechanical integrity observed with interleukin‐1β alone was inhibited. This study provides further support for lithium chloride as a novel compound for the treatment of osteoarthritis. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. J Orthop Res 33:1552–1559, 2015.     

Improved outcomes of islet autotransplant after total pancreatectomy by combined blockade of IL‐1β and TNFα

The efficacy of islet transplant is compromised by a significant loss of islet mass posttransplant due to an innate inflammatory reaction. We report the use of a combination of etanercept and anakinra (ANA+ETA) to block inflammatory islet damage in 100 patients undergoing total pancreatectomy with islet autotransplant. The patients were divided into 3 groups: no treatment (control [CTL]), etanercept alone (ETA), or a combination of etanercept and anakinra (ANA+ETA). Peritransplant serum samples were analyzed for protein markers of islet damage and for inflammatory cytokines. Graft function was assessed by fasting blood glucose, basal C‐peptide, secretory unit of islet transplant objects (SUITO) index, and hemoglobin A_1c. Administration of both antiinflammatory drugs was well tolerated without any major adverse events. Reductions in interleukin‐6, interleukin‐8, and monocyte chemoattractant protein 1 were observed in patients receiving ANA+ETA compared with the CTL group, while also showing a modest improvement in islet function as assessed by basal C‐peptide, glucose, hemoglobin A_1c, and SUITO index but without differences in insulin dose. These results suggest that double cytokine blockade (ANA+ETA) reduces peritransplant islet damage due to nonspecific inflammation and may represent a promising strategy to improve islet engraftment, leading to better transplant outcomes.     

Role of IL‐17 and TGF‐β in peritoneal adhesion formation after surgical trauma

Peritoneal adhesions are fibrous tissues formed after surgery. Both cytokines and transforming growth factors (TGFs) are involved in this process. The objective of this study was to investigate the cross talk between these entities. Peritoneal drainage fluid after surgery from patients and rodent models was examined by enzyme‐linked immunosorbent assay and fluorescence‐activated cell sorter. Data showed that the concentrations of interferon (IFN)‐γ and interleukin (IL)‐17 reached their peaks 6–12 hours after surgery, whereas TGF‐β1 concentrations showed two postoperative peak time points at 2 and 72–96 hours. By neutralizing IFN‐γ, IL‐17 6–12 hours, and TGF‐β1 72–96 hours after surgery, the degree of adhesion reduced significantly. However, neutralizing TGF‐β1 2 hours after surgery did not affect adhesion formation. Furthermore, in vitro studies showed that compared with the fibroblasts that were directly stimulated with TGF‐β1, the prestimulation of IL‐17 promoted plasminogen activator inhibitor‐1 production while inhibiting tissue‐type plasminogen activator production. Moreover, additional stimulation with IFN‐γ enhanced this effect. Together, these data indicate that IL‐17 may promote adhesion formation by increasing the reaction of fibroblasts against TGF‐β1. Blocking IL‐17 might have a therapeutic potential in preventing adhesion formation after surgery.     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.