Osteoporosis is a mineral bone disease arising from the predominance of osteoclastic bone resorption. Bisphosphonates which inhibit osteoclasts are commonly used in osteoporosis treatment, but are not without severe adverse effects like osteonecrosis of the jaw. The mechanisms behind the development of such phenomena is not well understood. Bone homeostasis is achieved through an intimate cross-talk between osteoclasts and osteoblasts. Thus, it is important to visualise activities of these cells simultaneously in situ. Currently, there are means to visualise osteoclast shape and numbers with tartrate-resistant alkaline phosphatase (TRAP) staining but no practical and accurate methods to quantify osteoclast activity in situ. This investigation aims to establish the use of ELF97, a substrate of TRAP, to visualise and quantify osteoclast activity. This provides vital clues to mechanisms of various bone disorders. TRAP dephosphorylation of ELF97 results in a detectable fluorescent product at areas of osteoclast activity. Osteoclastic activity was initiated in zebrafish by inducing crush injuries in tail fin rays. Colocalisation of ELF97 fluorescence with osteoclast-specific DsRed in transgenic zebrafish, visualised under confocal microscopy, is used to further establish the specificity of ELF97 to sites of osteoclastic activity. Quantification is established by comparing fluorescence between wild type, osteoclast-deficient mutants and bisphosphonate-treated zebrafish. The utility of ELF97 will also be investigated in terms of the stability of the florescent product. The investigation revealed that ELF97 and DsRed fluorescence were found commonly at crush sites with osteoclastic activity. Wild type zebrafish had greater fluorescence compared to osteoclast-deficient (p<0.0001) and bisphosphonate-treated zebrafish (p<0.0001) after 7 and 14 days post-crush, revealing that fluorescence from ELF97 corresponds to expected osteoclastic activity. Fluorescence of tail fins treated with ELF97 did not diminish over a period of 21 days of storage, demonstrating its stability. ELF97 is thus a useful means to visualise osteoclast activity, potentially crucial in more advanced investigations to understand bone disorders. It could be used in combination with other cellular markers in whole biological samples to study and experimentally manipulate bone remodelling

Aims. Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders. Methods. We examine the inhibitory effect of a high affinity, glycosyl-phosphatidylinositol-anchored TIMP variant named ‘T1. PrαTACE. ’ on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation. Results. Osteoclast progenitor cells transduced with T1. PrαTACE. failed to form tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts or exhibit bone-resorbing activity following treatment with RANKL. At the messenger RNA level, T1. PrαTACE. strongly attenuated expression of key osteoclast marker genes that included TRAP, cathepsin K, osteoclast stimulatory transmembrane protein (OC-STAMP), dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast-associated receptor (OSCAR), and ATPase H. +. -transporting V0 subunit d2 (ATP6V0D2) by blocking autoamplification of nuclear factor of activated T cells 1 (NFATc1), the osteoclastogenic transcription factor. T1. PrαTACE. selectively extended p44/42 mitogen-activated protein kinase activation, an action that may have interrupted terminal differentiation of osteoclasts. Inhibition studies with broad-spectrum hydroxamate inhibitors confirmed that the anti-resorptive activity of T1. PrαTACE. was not reliant on its metalloproteinase-inhibitory activity. Conclusion. T1. PrαTACE. disrupts the RANKL-NFATc1 signalling pathway, which leads to osteoclast dysfunction. As a novel candidate in the prevention of osteoclastogenesis, the TIMP could potentially be developed for the treatment of osteoclast-related disorders such as osteoporosis. Cite this article: Bone Joint Res 2022;11(11):763–776

Fractures and related complications are a common challenge in the field of skeletal tissue engineering. Vitamin D and calcium are the only broadly available medications for fracture healing, while zinc has been recognized as a nutritional supplement for healthy bones. Here, we aimed to use polaprezinc, an anti-ulcer drug and a chelate form of zinc and L-carnosine, as a supplement for fracture healing. Polaprezinc induced upregulation of osteogenesis-related genes and enhanced the osteogenic potential of human bone marrow-derived mesenchymal stem cells and osteoclast differentiation potential of mouse bone marrow-derived monocytes. In mouse experimental models with bone fractures, oral administration of polaprezinc accelerated fracture healing and maintained a high number of both osteoblasts and osteoclasts in the fracture areas. Collectively, polaprezinc promotes the fracture healing process efficiently by enhancing the activity of both osteoblasts and osteoclasts. Therefore, we suggest that drug repositioning of polaprezinc would be helpful for patients with fractures

Osteoporosis is a common problem in postmenopausal women and the elderly. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional enzyme that primarily activates glucocorticoids (GCs) in vivo, which is a considerable potential target as treatment for osteoporosis. Previous studies have demonstrated its effect on osteogenesis, and our study aimed to demonstrate its effect on osteoclast activation. In vivo, we used 11β-HSD1 knock-off (KO) and C57BL6/J mice to undergo the ovariectomy-induced osteoporosis (OVX). In vitro, In vivo, We used 11β-HSD1 knockoff (KO) and C57BL6/J mice to undergo the ovariectomy-induced osteoporosis (OVX). In vitro, bone marrow-derived macrophages (BMM) and bone marrow mesenchymal stem cell (BMSC) of KO and C57BL6/J mice were extracted to test their osteogenic and osteoclastic abilities. We then created osteoclastic 11β-HSD1 elimination mice (Ctsk::11β-HSD1fl/fl) and treated them with OVX. Micro-CT analysis, H&E, immunofluorescence staining, and qPCR were performed. Finally, we conducted the high-throughput sequencing to find out 11β-HSD1 and osteoclast activation related genes. We collected 6w samples after modeling. We found that KO mice were resistant to loss of bone trabeculae. The same effect was observed in osteoclastic 11β-HSD1 elimination mice. Meanwhile, BVT-2733, a classic inhibitor of 11β-HSD1, inhibited the osteoclast effect of cells without affecting osteogenic effect in vitro. High-throughput sequencing suggested that glucocorticoid receptor (GR) may play a key role in the activation of osteoclasts, which was verified by immunofluorescence staining and WB in vivo and in vitro. In the process of osteoporosis, 11β-HSD1 expression of osteoclasts is abnormally increased, which may be a new target for inhibiting osteoclast activation and treating osteoporosis

To date there is no medical treatment alternative to surgery for osteolysis after THA. In this proof-of-concept clinical trial we examined the effect of a human monoclonal antibody against osteoclasts versus placebo on osteolytic lesion activity in patients undergoing revision surgery. Patients scheduled for revision for symptomatic osteolysis were randomised (1:1) to receive either denosumab 60mg or placebo subcutaneously eight weeks prior to operation. At surgery, biopsies from the osteolytic membrane-bone interface were taken for histomorphometric analysis of osteoclast number. Secondary outcome measures included systemic bone turnover markers. 22 subjects completed the study (10 denosumab). The denosumab group had 83% (−63 to −97), P=0.011 fewer osteoclasts at osteolytic lesion sites, 87% lower osteoclast surface (−65 to −95, P=0.009), and 72% lower eroded surface (−35 to −93, P=0.020) versus the placebo group. At surgery, serum CTX-I, TRAP5b and PINP were 80% (−65 to −95, p<0.001), 57% (−40 to −90, p<0.001), and 44% (−41 to −65, p<0.001) lower in the denosumab versus placebo groups, respectively. The rate of adverse events (denosumab 6, placebo 7) were similar between groups (P>0.05). These data provide a biological basis for a definitive clinical trial using pain, function and prosthesis survival as the study endpoints. As osteolysis/ aseptic loosening is the leading cause of prosthesis failure world-wide, the establishment of a non-surgical solution would reduce patient suffering and dramatically reducing the cost to healthcare economies

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development. Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining. Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production. Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA

Abstract. Objective. In this phase 2 clinical trial (EudraCT 2011-000541-20) we examined the effect of denosumab versus placebo on osteolytic lesion activity in patients undergoing revision surgery after THA. Methods. Men and women ≥ 30 years old scheduled for revision surgery for symptomatic, radiologically-confirmed osteolysis were randomised (1:1) to receive either denosumab 60mg or placebo subcutaneously eight weeks prior to operation. At surgery, biopsies from the osteolytic membrane-bone interface were taken for histomorphometric analysis of osteoclast number, the primary outcome measure. Secondary outcome measures included other static histomorphometric indices and systemic bone turnover markers. Adverse events and patient-reported clinical outcome scores were recorded as safety endpoints. Results. Of the 24 subjects enrolled, 22 completed the study (10 denosumab) and comprise the per-protocol analysis. There were no differences in baseline characteristics and bone turnover markers between groups (p>0.05). The denosumab group had 78% fewer osteoclasts at osteolytic lesion sites (95% CI −61 to −95, P=0.011), 81% lower osteoclast surface (−70 to −95, P=0.009), and 73% lower eroded surface (−54 to −92, P=0.020) compared to the placebo group. Number of osteoblasts and osteoblast surface were also reduced by 81% (−62 to −100, p=0.021) and 82% (−64 to −101, p=0.017), respectively. Immunocytochemistry for cell proliferation (Ki67) and apoptosis (Caspase 3) identified no differences between the groups (p>0.05). At surgery, serum CTX-I in the denosumab group was 80% lower (−65 to −95, p<0.001), TRAP5b −65% (−40 to −90, p<0.001), PINP −53% (−41 to −65, p<0.001). Patient-reported outcome measures and the rate of adverse events (denosumab 6, placebo 7) were similar between groups (P>0.05). Conclusion. A single dose of denosumab reduced osteoclast activity within osteolytic lesions and was safe to administer. These data provide a biological basis for a phase 3 trial using clinical outcomes of pain, function and prosthesis survival as the study endpoints. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

We have developed precision-engineered strontium eluting nanopatterned surfaces. Nanotopography has been shown to increase osteoblast differentiation, and strontium is an element similar to calcium, which has been proven to increase new bone formation and mineralization. This combination has great potential merit in fusion surgery and arthroplasty, as well as potential to reduce osteoporosis. However, osteoclast mediated osteolysis is responsible for the aseptic failure of implanted biomaterials, and there is a paucity of literature regarding osteoclast response to nanoscale surfaces. Furthermore, imbalance in osteoclast/osteoblast resorption is responsible for osteoporosis, a major healthcare burden. We aimed to assess the affect of strontium elution nanopatterned surfaces on osteoblast and osteoclast differentiation. We developed a novel human osteoblast/osteoclast co-culture system without extraneous supplementation to closely represent the in vivo environment. We assessed the surfaces using electron microscopy (SEM), protein expression using immunofluorescence and histochemical staining and gene expression using polymerase chain reaction (PCR). In complex co-culture significantly increased osteoblast differentiation and bone formation was noted on the strontium eluting, nanopatterned and nanopatterned strontium eluting surfaces, suggesting improved osteointegration. There was a reduction in macrophage attachment on these surfaces as well, suggesting specific anti-osteoclastogenic properties of this surface. Our results show that osteoblast and osteoclast differentiation can be controlled through use of nanopatterned and strontium eluting surface features, with significant bone formation seen on these uniquely designed surfaces

Summary Statement. Obovatol inhibits receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and prevents inflammatory bone loss in mice. Introduction. Adult skeletal mass and integrity are maintained by balancing osteoclast-mediated bone resorption and osteoblast-induced bone formation during bone remodeling. Abnormal increases in osteoclastic bone resorption can lead to excessive bone destruction as observed in osteoporosis, rheumatoid arthritis, and metastatic cancers Therefore, Modulation of osteoclast formation and function is a promising strategy for the treatment of bone-destructive diseases. To search for compounds that inhibit osteoclast formation, we tested the effect of obovatol, a natural product isolated from the medicinal plant Magnolia obovata, on osteoclastogenesis and inflammatory bone loss. Methods. Osteoclastogenesis was assessed using bone marrow-derived macrophages. RANKL signaling was assessed by immunoblotting and apoptosis by cell death ELISA assay. Actin ring staining and resorption pit assay was performed. Bone morphometric parameters were determined using a microcomputed tomography system. Results. We found that obovatol strongly inhibited osteoclast formation from bone marrow-derived macrophages in a dose-dependent manner without cytotoxicity. Obovatol significantly suppressed RANKL-induced activation of NF-κB, JNK, and ERK signaling pathways. Obovatol also inhibited RANKL-induced expression of the genes c-Fos and nuclear factor of activated T cells c1, which are transcription factors important for osteoclastogenesis. In addition to osteoclast differentiation, obovatol blocked cytoskeletal organization and abrogated the bone resorbing activity of mature osteoclast. Obovatol also accelerated osteoclast apoptosis through the induction of caspase-3 activation. Consistent with its in vitro anti-resorptive effect, obovatol prevented bone loss induced by lipopolysaccharide (LPS) in vivo. Conclusion. Our findings demonstrate that obovatol, a natural compound isolated from Magnolia obovata, suppresses the differentiation, function, and survival of osteoclasts. Furthermore, obovatol protected against LPS-induced bone loss in vivo. Therefore, we suggest that obovatol may have therapeutic potential for the treatment of bone-destructive diseases characterised by increased osteoclast number and/or activity

Osteoclasts are cells that resorb bone. They derive from haemopoietic precursors in the presence of Macrophage-Colony Stimulating Factor (M-CSF) and the osteoclast growth factor, Receptor Activator of Nuclear Factor–kB Ligand (RANKL). Tumour Necrosis Factor-a (TNF-a) and M-CSF has been shown to form mature osteoclastic bone resorption in vitro murine cultures in the absence of RANKL. The aim of this study was to investigate the mechanism of action of the pro-inflammatory cytokine Tumour Necrosis Factor-a (TNF-a) with respect to osteoclastic bone resorption. Development of osteoclasts was performed using an in vitro assay of healthy human peripheral blood mononuclear culture (PBMNC) in the presence of M-CSF and RANKL. In the same cultures RANKL was replaced by TNF-a over a wide range of concentrations. Osteoclasts were generated in the presence of M-CSF, TNF-a and RANKL from human PBMNC. However, in the same experiments M-CSF and TNF-a in the absence of RANKL failed to support human osteoclast formation. Aseptic loosening and osteolysis are considered the main long-term complications of hip arthroplasty. Pathogenesis of peri-prosthetic osteolysis is multifactorial and both biological and mechanical factors are important. TNF-a is thought to be involved in orthopaedic implant oste-olysis induced by prosthesis-derived wear particles. The final osteolytic step is undertaken mainly by osteoclasts. This is the first report showing that TNF-a and M-CSF in the absence of RANKL in human PBMNC is not capable of inducing osteoclast formation. TNF-a therefore may increase peri-prosthetic loosening by enhancing the activity of the mature osteoclast

Recent studies have shown that random disorder nanotopography increases osteoblast differentiation and bone formation. This has great potential merit in producing surfaces where osteointegration is required such as spinal fusion surgery and arthroplasty. However, the long-term failure of orthopaedic implants is often related to osteoclast mediated osteolysis and loosening. It is vitally important that we understand the effect of nanotopography on osteoclast formation and bone remodeling. We developed an unique osteoblast/osteoclast co-culture system derived from human mesenchymal and haematopoetic stem cells. This was co-cultured on both nanopatterned and unpatterned polycarbonate substrates. We assessed the co-culture using electron microscopy (SEM), protein expression using immunofluorescence and histochemical staining and gene expression using polymerase chain reaction (PCR). Co-culture of both osteoclasts and osteoblasts was confirmed with mature bone nodules and resorption pits identified on both surfaces. Significantly increased osteoblast differentiation and bone formation was noted on disordered nanotopography. Antagonistic genes controlling osteoclast activity were both upregulated with no significant difference in osteoclast marker gene expression. Our results confirm successful co-culture of osteoblasts and osteoclasts using an unique method closely resembling the in vivo environment encountered by orthopaedic implants. Nanotopography increases osteoblast differentiation and bone formation as previously identified, with possible subsequent increase in osteoclast mediated bone turnover

Cellular mechanisms that account for tumour osteolysis associated with Ewing’s sarcoma are uncertain. Osteoclasts are marrow-derived multinucleated cells that effect tumour osteolysis. Osteoclasts are known to form from macrophages by both receptor activator for nuclear factor κB ligand (RANKL)-dependent and RANKL-independent mechanisms. In this study our aim has been to determine whether tumour-associated macrophages (TAMs) isolated from Ewing’s sarcoma are capable of differentiating into osteoclasts and to characterise the cellular and humoral mechanisms whereby this occurs. TAMs were isolated from two Ewing’s sarcomas and cultured on both coverslips and dentine slices for up to 21 days with soluble RANKL and human macrophage colony stimulating factor (M-CSF). Osteoclast formation from TAMs (CD14+) was evidenced by the formation of tartrate–resistant acid phosphatase and vitronectin receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. This osteoclast formation and resorption was inhibited by the addition of the bisphosphonate, zoledronate. Osteoclast formation was also seen when Ewing’s sarcoma-derived TAMs were cultured with TNF α in the presence of M-CSF. We also found that TC71 Ewing’s sarcoma cells were capable of independently stimulating osteoclast formation through the release of a soluble factor. These results indicate that TAMs in Ewing’s sarcoma are capable of osteoclast differentiation by both RANKL-dependent and RANKL-independent mechanisms and that Ewing’s sarcoma cells produce an osteoclastogenic factor. The role bisphosphonates may play in inhibiting osteoclast formation and osteolysis in Ewing’s sarcoma merits further investigation

Introduction: Osseous metastases from melanoma are relatively common (7% of cases), and occur most often in the axial skeleton. Bone destruction in skeletal metastases of solid tumours is due to stimulation of osteoclast formation and bone resorption. Osteoclasts are formed by the fusion of marrow-derived mononuclear phagocyte precursors which express RANK (receptor activator of nuclear factor κB) which interacts with RANKL expressed by osteoblasts/bone stromal cells in the presence of macrophage colony-stimulating factor (M-CSF). Osteoclast formation by a RANKL-independent, tumour necrosis factor α (TNFα)-induced mechanism has also been reported. Tumour-associated macrophages (TAMs) are present in both primary and secondary tumours and TAMs are known to be capable of osteoclast differentiation. Our aim in this study was to determine the role of TAMs and the humoral mechanisms of osteolysis associated with melanoma metastases. Materials and Method: In this study we isolated TAMs from extraskeletal primary melanomas and lymph node metastases. TAMs were cultured for up to 21 days in the presence and absence of M-CSF and RANKL or TNF. In a separate experiment, conditioned medium was extracted from the melanoma cell line, SK-Mel-29, and cultured with human peripheral blood mononuclear cells in the presence of M-CSF. Results: TAM-osteoclast differentiation, as evidenced by the expression of tartrate-resistant acid phosphatase, vitronectin receptor and lacunar resorption pit formation, occurred by both RANKL-dependent and RANKL-independent mechanisms. Osteoclast formation induced by RANKL-independent mechanism was not abolished by the addition of osteoprotegerin or RANK:Fc, decoy receptors for RANK. Conditioned medium from SK-Mel-29 could support osteoclast differentiation in the absence of RANKL. This effect was not abolished by antibodies to RANKL, TNFα, TGFβ, IL-8 or gp130. Discussion: These results indicate that melanoma TAMs are capable of differentiation into osteoclasts and that both RANKL-dependent and RANKL-independent (TNFα) mechanisms are involved. Melanoma tumour cells also secrete a soluble factor that supports osteoclastogenesis. Conclusion: Inhibitors of osteoclast formation targeting TAM-osteoclast differentiation and osteoclast activity and identification of the osteoclastogenic factor produced by melanoma cells may have a therapeutic potential in controlling tumour osteolysis

In rheumatoid arthritis (RA) and other arthritic disorders e.g. gout, there is destruction of articular cartilage and juxta-articular bone. Osteoclasts are specialised multinucleated cells (MNCs) that carry out bone resorption. It has previously been shown that circulating monocytes and synovial macrophages in RA can be stimulated to differentiate into functional osteoclasts in the presence of RANKL and M-CSF. The aim of this study was to determine whether the mononuclear cells present in synovial fluid of RA patients are capable of differentiating into functional osteoclasts in the presence of osteogenic factors. Mononuclear cells were isolated from the synovial fluid obtained from patients with Ra, osteoarthritis (OA) gout and joint trauma. The cells were seeded onto dentine slices and coverslips and cultured for up to 21 days in the presence/absence of RANKL (30ng/ml) and M-CSF (25ng/ml). Cells cultured on coverslips for 24h, 14 and 21 days were assessed for the expression of the monocyte-macrophage antigen CD14 that is known to be expressed by osteoclasts, and the osteoclast associated markers; tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR). After 21 days, dentine slices were assessed for evidence of osteoclastic lacunar resorption. After 24 h culture on coverslips mononuclear cells isolated from the synovial fluid of all the above joint conditions were largely CD14+, and entirely negative for TRAP and VNR. After 14 days culture, in the presence of RANKL and M-CSF these synovial fluid macrophages were stimulated to form multinucleated osteoclasts which were TRAP+ and VNR+ and capable of forming resorption pits on dentine slices. In the absence of either RANKL or M-CSF osteoclast formation did not occur. The osteogenic factors RANKL and M-CSF have been shown to be present in the synovial fluid of patients with RA, OA, gout and joint trauma. Results from this study demonstrate that CD14+ mononuclear cells (macrophages) in the synovial fluid of patients with the above conditions have the capacity to differentiate into functional multinucleated osteoclasts in the presence of RANKL and M-CSF. These findings show that one cellular mechanisms whereby bone erosions many occur in arthritic disorders is through increased osteoclast formation of synovial fluid macrophages; this process requires RANKL and m-CSF, both of which are produced by inflammatory cells e.g. T Cells found in the synovial fluid and the arthritic synovial membrane

Osteomyelitis commonly causes bone destruction and is most frequently due to infection by Staphylococcus aureus. S. aureus is known to secrete a number of surface-associated proteins which are extremely potent stimulators of bone resorption in the mouse calvarial assay system. The precise cellular and humoral mechanisms whereby this stimulatory effect is mediated, in particular whether osteoclast formation or activity is directly promoted by these factors, have not been determined by this study. Surface-associated material (SAM)(0.001ug/ml)obtained from 24 hour cultures of S. aureus was added to cultures of mouse and human osteoclast precursors (RAW 264.7 cells and human peripheral blood mononuclear cells respectively). These cultures were incubated in the presence and absence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). It was found that independent of RANKL, SAM was capable of inducing osteoclast formation in cultures of RAW cells and human monocytes. This was evidenced by the generation of tartrate-resistant acid phosphatase-positive multinucleated cells, which formed lacunar resorption pits when these cells were cultured on dentine slices. In cultures where M-CSF, RANKL and SAM were added, osteoclast formation was increased, but did not exceed the osteoclast formation in cultures with M-CSF and RANKL. These findings indicate that S. aureus produces a soluble factor which can promote osteoclast formation. Identification of this factor may help to develop therapeutic strategies for treating bone destruction due to Staphylococcal osteomyelitis

The aseptic loss of bone after hip replacement is a serious problem leading to implant instability. Hydroxyapatite coating of joint replacement components produces a bond with bone and helps to reduce loosening. However, over time bone remodeling at the implant interface leads to loss of hydroxyapatite. One possible solution would be to develop a coating that reduces hydroxyapatite and bone loss. Hydroxyapatite can be chemically modified through the substitution of ions to alter the biological response. Zinc is an essential trace element that has been found to inhibit osteoclast-like cell formation and decrease bone resorption. It was hoped that by substituting zinc into the hydroxyapatite lattice, the resultant zinc-substituted hydroxyapatite (ZnHA) would inhibit ceramic resorption and the resorption of bone. The aim of this work was to investigate the effect of ZnHA on the number and activity of osteoclasts. Discs of phase pure hydroxyapatite (PPHA), 0.37wt% ZnHA and 0.58wt% ZnHA were produced, sintered at 1100 degrees Celsius and ground with 1200 grit silicon carbide paper. They were cultured in medium containing macrophage colony stimulating factor and receptor activator of nuclear factor kappa B ligand (RANKL) for 11 and 21 days. A control disc of PPHA cultured in medium containing no RANKL was also used. On the required dates the discs were removed and the cells stained for actin with phalloidin-TRITC and the cell nuclei with 4',6-Diamidino-2-phenylindole dihydrochloride. Cells with 3 or more nuclei were classed as osteoclasts and counted using ImageJ. On day 21 after the cells had been counted, the cells were removed and the discs coated in platinum before viewing with a scanning electron microscope. Resorption areas were then measured using ImageJ. The addition of zinc was observed to significantly decrease the number of differentiated osteoclasts after 21 days (p<0.005 for 0.58wt% ZnHA compared to PPHA and p<0.01 for 0.37wt% ZnHA compared to PPHA). The area of resorption was also significantly decreased with the addition of zinc (p<0.005 for the comparison of 0.58wt% ZnHA with PPHA). The work found that zinc substituted hydroxyapatite reduced the number and subsequent activity of osteoclasts

The success of biomaterials lies in the direct interaction with the host tissue. Calcium phosphates (CaP) stand as an alternative graft material for bone regeneration due to their similar composition to natural bone. Few studies have focused on the early stages of bone-like material remodeling by osteoclasts (OC), though the CaP fate is to be resorbed and then replaced by new bone. Instead, to understand how osteoclasts modify the CaP surface and initiate resorption, so as to influence subsequent osteoblast activities and bone formation, is mandatory. Sintered hydroxyapatite (s-HA) and biomimetic hydroxyapatite with two different microstructures (b-HA-C, coarse and b-HA-F, fine) discs (1500×250 µm. 2. ) were produced from the same reagents [1]. Tissue culture polystyrene (TCPS) was used as control. Precursor human OC from buffy coats were seeded on ceramic substrates [6·10. 6. cells/cm. 2. ] and supplemented with RANKL-containing osteoblast supernatant as differentiation medium over 21 days. Cell interaction with the biomaterials was investigated in terms of OC adhesion and differentiation, with gene expression, tartrate-resistant acid phosphatase (TRAP) and Hoechst staining for OC maturation. Cell culture supernatants were analyzed for ionic exchange, namely Ca and P, due to biomaterials or cells. Osteoclasts morphology was evaluated using SEM at 21 days. Innovatively, focused ion beam (FIB) was used to evaluate biomaterial structure beneath the OC to further investigate the resorption effects. To this aim, selected OC were cut cross-sectioned using a Gallium ion beam at an acceleration of 30KV, followed by a coarse milling at 10nA and a deposition of platinum to achieve a fine milling at 500pA. Clear differences in cellular behavior were noted relative to the different substrate microstructures. Control TCPS and s-HA showed similar TRAP-positive staining and gene expression for mature OC. Several resorption pits with partial dissolution of the equiaxial grains of s-HA were noticed. b-HA substrates also showed attached and differentiated TRAP-positive OC, but gene expression resulted lower than control and s-HA. However, morphological evaluation with SEM-FIB interestingly showed early stages of osteoclast-mediated degradation on b-HA-F, i.e.an increased surface roughness in the substrate underlying cells. B-HA-C also showed attached and mature OC with a scarce degradation activity. FIB technique has been applied to cell-seeded CaP and shown as a viable method to investigate OC morphology and resorption. Though gene expression showed similarities for both biomimetic substrates, substrate morphology observed underneath OC was significantly different. b-HA-F showed early stages of OC mediated degradation underneath well spread cells similar to those seen on s-HA. No resorptive activity was found on b-HA-C even though gene expression values were similar to b-HA-F: both the acute ion exchange and the surface tortuosity on b-HA-C could explain the difficulty with the resorptive process by OC. In conclusion focused ion beam technique complements SEM imaging and may disclose changes in the inner structure of materials due to cell/material interactions

Wear particles are thought to be a major factor causing osteolysis that leads to aseptic loosening. The aim of this study was to investigate the role of primary regulators of osteoclast development, RANKL (also known as osteoclast differentiation factor), its receptor RANK and natural inhibitor osteoprotegerin (OPG) in aseptic loosening. Cells were isolated from periprosthetic tissues taken at revision from more than 30 patients and the expression of these mediators in vivo was assessed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). These cells were also cultured on dentine to determine their ability to become mature osteoclasts. In situ hybridisation using DIG labelled riboprobes specific for RANK mRNA was used to identify cells likely to become osteoclasts. We were able to compare revison tissues containing several different types of prosthetic wear particles. RANKL, RANK and OPG mRNA were found in samples of periprosthetic revision tissues. Cells derived from this tissue developed into mature osteoclasts capable of resorbing dentine. Cells that rapidly formed osteoblasts expressed a fifteen fold higher ratio of RANKL:OPG mRNA. In situ hybridisation showed RANK expression by macrophages and giant cells, many of which contained wear particles. Significantly, cells from tissues containing silastic wear particles expressed higher levels of RANKL relative to OPG and more produced large numbers of osteoclasts in vitro. This study shows that different bio materials in a particulate form may differ in their ability to form osteoclasts and that the relative levels of RANKL and OPG are likely to be important in determining if osteolysis will occur. In the future molecules that inhibit RANKL binding, such as OPG, may be considered for therapy of periprosthetic osteolysis

The regenerative potential of bone grafts is tightly linked to the interaction of the biomaterial with the host tissue environment. Hence, strategies to confer artificial extracellular matrix (aECM) cues on the material surface are becoming a powerful tool to trigger the healing cascade and to stimulate bone regeneration. The use of glycosaminoglycans (GAGs), such as heparin, as aECM components has gained interest in the last years as a strategy to improve biological response. Calcium phosphates (CaP) are extensively used as bone grafts, however no studies have investigated the effect of GAG functionalisation on their surface. Some authors have focused on the effects of GAGs on osteoblastic cells, however, little work has been performed on the interaction with osteoclasts (OC), and still the reported effects are controversial [1]. The aim of this study was to investigate the effect of heparin on osteoclastic fate in terms of adhesion and differentiation. Sintered CaP (β-TCP) and biomimetic CaP (calcium-deficient hydroxyapatite, CDHA) discs were synthesized at 1100 ºC and at 37ºC, respectively. Heparinisation was achieved though silane coupling (APTES) followed by amidation in the presence of EDC/NHS to covalently link heparin. The osteoclast response of heparinised (H) vsnon-heparinised substrates was studied using human monocytes as OC precursors. Tissue culture plastic (TCPS) was used as a control sample. Cell densities were 6·10. 6. and 3·10. 6. cells/cm. 2. for biomaterials and TCPS, respectively. Cell cultures were supplemented every 3 days with 25% supernatant of osteoblast-like cell line as a source of RANKL, as well as other stimulating factors [2]. Tartrate-resistant acid phosphatase and Hoechst staining were used to evaluate OC adhesion, differentiation and morphology at different time points from seeding on the surfaces (14–21–28 days). OC precursors showed adhesion on all substrates. β-TCP and β-TCP-H hosted higher number of OC precursors which might be related to the smoother sintered surface of the materials. Oppositely, the high roughness of CDHA and CDHA-H hamper the adhesion of OC, hence a lower number of cells was observed on heparin-coated and uncoated biomimetic apatites. However, the maturation of OC precursors was found to take place at earlier times (14days) on biomimetic substrates compared to sintered ones. TCPS, CDHA, CDHA-H and β-TCP-H showed clearly differentiated OC at 14 days, as revealed by TRAP positivity and multinuclearity. Interestingly, CDHA-H and β-TCP-H induced the highest multinuclearity among all differentiated OC. Both heparinised substrates point at an enhancing effect of heparin on OC maturation. OC precursors are able to differentiate on β-TCP and CDHA substrates, a process enhanced when heparin functionalisation is performed on the materials surface. In our hands heparinisation is promoting OC differentiation at early time points, similarly to TCPS control. Interestingly, heparin substrates induced larger TRAP positive-OC and higher multinuclearity in the mature OC than TCPS control. As pointed out by Irie et al., heparin might interact through the RANKL/OPG ratio [3], thus inhibiting OPG activity and enhancing RANKL which triggers OC maturation

Introduction. Both the RANK/RANKL system and the endocannabinoid system have roles in bone remodelling. Activation of CB1 receptors on sympathetic nerve terminals in trabecular bone modulates bone remodelling by attenuating adrenergic inhibition over bone formation. CB2 receptors are involved in the local control of bone cell differentiation and function. Osteoblastic CB2 receptor activation negatively regulates RANKL mRNA expression indicating an interaction between the two systems and that efficient bone remodelling requires a balance between these two systems. The aim of the study was to establish the presence of the different components of the endocannabinoid system and the RANK/RANKL signalling pathway in human bone and osteoclast culture. Methods. Levels of endocannabinoids (AEA, 2-AG) and their related compounds (OEA, PEA) in human trabecular bone, obtained from patients undergoing elective orthopaedic surgery, were measured using Liquid Chromatography Mass Spectrometry (LC-MS-MS). mRNA for the endocannabinoid synthetic and catabolic enzymes (NAPE-PLD, DAGLa, FAAH, MAGL), cannabinoid-activated receptors (CB1, CB2, PPARs, TRPV1), and RANK, RANKL and NFkB were determined using Taqman Real-Time PCR. Osteoclasts were differentiated from U-937 cells (Human leukaemic monocyte lymphoma cell line), following the sequential treatment using TPA (0.1μg/ml) followed by either TNF-a (3ng/ml) or calcitriol (10. −8. M), cultured for up to 30 days. Osteoclasts were identified by positive staining with tartrate resistant acid phosphatase (TRAP), multinucleation and the ability to form resorption pits on calcium phosphate coated discs. Taqman Real-Time PCR was performed to detect the expression of the osteoc!. last marker genes TRAP and cathepsin K, together with genes of the endocannabinoid and RANK/RANKL signalling pathways. Results. AEA (5.1±0.7pmol/g), 2-AG (527.0±78.6 pmol/g), PEA (122.2±5.1pmol/g) and OEA (122.8±4.3pmol/g) were present in human trabecular bone. All components of the endocannabinoid system and RANK/RANKL pathways were present at the mRNA level in human trabecular bone. TRAP positive, multinucleated, calcium phosphate resorbing osteoclasts were observed from day 8 to day 23 of culture. mRNA expression of the osteoclast specific markers TRAP and cathepsin K and components of the endocannabinoid and the RANK/RANKL systems (with the exception of CB1) were up-regulated with osteoclast maturation with highest levels of expression on day 14. Conclusion. The detection of both synthetic and catabolic enzymes of the endocannabinoids in human trabecular bone and osteoclast culture indicates local skeletal production and regulation of endocannabinoids. The co-expression of all components of the endocannabinoid and the RANK/RANKL systems in human trabecular bone and osteoclast culture suggest possible interactions between the 2 systems in maintaining balanced bone remodelling, which may impact upon bone resorption seen in many bone diseases