Loss of Grem1-lineage chondrogenic progenitor cells causes osteoarthritis.

Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.

Osteocytes respond to particles of clinically-relevant conventional and cross-linked polyethylene and metal alloys by up-regulation of resorptive and inflammatory pathways.

Periprosthetic osteolysis is a major cause of implant failure in total hip replacements. Aseptic loosening caused by osteolytic lesions is associated with the production of bioactive wear particles from the articulations of implants. Wear particles infiltrate the surrounding tissue of implants, promoting inflammation as well as bone resorption. Osteocytes have been shown to both regulate physiological osteoclastogenesis and directly remodel their perilacunar bone matrix by the process of osteocytic osteolysis. We hypothesise that osteocytes respond to wear debris of orthopaedic implant materials by adopting a pro-catabolic phenotype and thus contribute to periprosthetic osteolysis through the known pathways of bone loss. Osteocyte responses to particles derived from clinically relevant materials, ultra-high molecular weight polyethylene (UHMWPE), highly cross-linked polyethylene (XLPE) and metal alloys, Ti6Al4V and CoCrMo, were examined in vitro in human primary osteocyte-like cultures. Osteocyte-like cells exposed to both polyethylene and metal wear particle types showed upregulated expression of catabolic markers associated with osteocytic osteolysis, MMP13, carbonic anhydrase 2 (CA2) and cathepsin K (CTSK). In addition, pro-osteoclastogenesis markers RANKL and M-CSF were induced, as well as the expression of pro-inflammatory cytokines, IL-6 and TNFα, albeit with different kinetics. These findings suggest a previously unrecognised action of wear particles of multiple orthopaedic materials on osteocytes, and suggest a multifaceted role for osteocytes in periprosthetic osteolysis. STATEMENT OF SIGNIFICANCE: This study addresses periprosthetic osteolysis, a major clinical problem leading to aseptic loosening of orthopaedic implants. It is well accepted that wear particles of polyethylene and of other implant materials stimulate the activity of bone resorbing osteoclasts. Our recent work provided evidence that commercial particles of ultra-high molecular weight polyethylene (UHMWPE) stimulated osteocytes to adopt a bone catabolic state. In this study we demonstrate for the first time that particles derived from materials in clinical use, conventional UHMWPE, highly cross-linked polyethylene (XLPE), and Ti6Al4V and CoCrMo metal alloys, all stimulate human osteocyte activities of osteocyte-regulated osteoclastogenesis, osteocytic osteolysis, proinflammatory responses, osteocyte apoptosis, albeit to varying extents. This study provides further mechanistic insight into orthopaedic wear particle mediated bone disease in terms of the osteocyte, the most abundant and key controlling cell type in bone.

Yeast RNA extract suppresses human osteoclast resorption in vitro.

AIMS: The aim of this short study was to test the combinations of RNA extracts (both the connective tissue extracts-cartilage and synovia along with yeast extract) found in natural ribonucleotide extract Osteochondrin S (OST) on human osteoclast formation and activity in vitro. METHODS: In vitro human osteoclasts were treated with the RNA extracts (cartilage, synovia and yeast) at concentrations equivalent to those in OST starting from day 7 of the culture. A tartrate resistant acid phosphatase stain (TRAP) was used to indicate osteoclast formation and activity assessed by determining area of dentine resorption. RESULTS: The combination of all components as is found in OST suppressed both osteoclast formation and activity. The yeast extract suppressed osteoclast activity at similar levels to that observed with all components combined. CONCLUSIONS: Our findings indicate that yeast RNA extracts found in OST may be the key component responsible for suppression of osteoclast activity.

Evidence that osteocyte perilacunar remodelling contributes to polyethylene wear particle induced osteolysis.

Periprosthetic osteolysis (PO) leading to aseptic loosening, is the most common cause of failure of total hip replacement (THR) in the mid- to long-term. Polyethylene (PE) particulates from the wear of prosthesis liners are bioactive and are implicated in the initiation and or progression of osteolysis. Evidence exists that cells of the osteoblast/osteocyte lineage are affected by PE particles and contribute to the catabolic response by promoting osteoclastic bone resorption. In this study, we hypothesised that osteocytes contribute directly to PO by removing bone from their perilacunar matrix. Osteocyte responses to ultra-high molecular weight PE (UHMWPE) particles were examined in vitro in human primary osteocyte-like cultures, in vivo in the mouse calvarial osteolysis model, and in the acetabulum of patients undergoing revision total hip replacement (THR) surgery for PO. Osteocytes exposed to UHMWPE particles showed upregulated expression of catabolic markers, MMP-13, carbonic anhydrase 2 (CA2), cathepsin K (CTSK) and tartrate resistant acid phosphatase (TRAP), with no effect on cell viability, as assessed by Caspase 3 activity. Consistent with this catabolic activity causing perilacunar bone loss, histological analysis of calvarial sections from mice exposed to UHMWPE revealed a significant (p<0.001) increase in osteocyte lacunar area (Lac.Ar) compared to sham-operated animals. Furthermore, acetabular biopsies from patients with PO also showed significantly (p<0.001) increased osteocyte lacunar size in trabecular bone adjacent to PE particles, compared with osteocyte lacunar size in bone from primary THR patients. Together, these findings suggest a previously unrecognised action of UHMWPE wear particles on osteocytes, which directly results in a loss of osteocyte perilacunar bone. This action may exacerbate the indirect pro-osteoclastic action of UHMWPE-affected osteocytes, previously shown to contribute to aseptic loosening of orthopaedic implants. STATEMENT OF SIGNIFICANCE: This study addresses the clinical problem of periprosthetic osteolysis, bone loss in response to polyethylene wear particles derived from materials used in orthopaedic implants. Periprosthetic osteolysis has been thought to be due largely to wear particles stimulating the activity of bone resorbing osteoclasts. However, in this study we demonstrate for the first time that polyethylene particles stimulate another type of bone loss, mediated by the direct activity of bone mineral embedded osteocytes, termed osteocytic osteolysis or osteocyte perilacunar remodelling. This study provides new mechanistic insight into wear-particle mediated bone loss and represents a new paradigm for the way in which bone cells, namely osteocytes, the key controlling cell type in bone, react to biomaterials.

The X-Linked Inhibitor of Apoptosis Protein Inhibitor Embelin Suppresses Inflammation and Bone Erosion in Collagen Antibody Induced Arthritis Mice.

OBJECTIVE: To investigate the effect of Embelin, an inhibitor of X-Linked Inhibitor of Apoptosis Protein (XIAP), on inflammation and bone erosion in a collagen antibody induced arthritis (CAIA) in mice. METHODS: Four groups of mice (n = 6 per group) were allocated: CAIA untreated mice, CAIA treated with Prednisolone (10 mg/kg/day), CAIA treated with low dose Embelin (30 mg/kg/day), and CAIA treated with high dose Embelin (50 mg/kg/day). Joint inflammation was evaluated using clinical paw score and histological assessments. Bone erosion was assessed using micro-CT, tartrate resistant acid phosphatase (TRAP) staining, and serum carboxy-terminal collagen crosslinks (CTX-1) ELISA. Immunohistochemistry was used to detect XIAP protein. TUNEL was performed to identify apoptotic cells. RESULTS: Low dose, but not high dose Embelin, suppressed inflammation as reflected by lower paw scores (P < 0.05) and lower histological scores for inflammation. Low dose Embelin reduced serum CTX-1 (P < 0.05) and demonstrated lower histological score and TRAP counting, and slightly higher bone volume as compared to CAIA untreated mice. XIAP expression was not reduced but TUNEL positive cells were more abundant in Embelin treated CAIA mice. CONCLUSION: Low dose Embelin suppressed inflammation and serum CTX-1 in CAIA mice, indicating a potential use for Embelin to treat pathological bone loss.

Osteoimmunology: Major and Costimulatory Pathway Expression Associated with Chronic Inflammatory Induced Bone Loss.

The field of osteoimmunology has emerged in response to the range of evidences demonstrating the close interrelationship between the immune system and bone metabolism. This is pertinent to immune-mediated diseases, such as rheumatoid arthritis and periodontal disease, where there are chronic inflammation and local bone erosion. Periprosthetic osteolysis is another example of chronic inflammation with associated osteolysis. This may also involve immune mediation when occurring in a patient with rheumatoid arthritis (RA). Similarities in the regulation and mechanisms of bone loss are likely to be related to the inflammatory cytokines expressed in these diseases. This review highlights the role of immune-related factors influencing bone loss particularly in diseases of chronic inflammation where there is associated localized bone loss. The importance of the balance of the RANKL-RANK-OPG axis is discussed as well as the more recently appreciated role that receptors and adaptor proteins involved in the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway play. Although animal models are briefly discussed, the focus of this review is on the expression of ITAM associated molecules in relation to inflammation induced localized bone loss in RA, chronic periodontitis, and periprosthetic osteolysis, with an emphasis on the soluble and membrane bound factor osteoclast-associated receptor (OSCAR).

Parthenolide reduces empty lacunae and osteoclastic bone surface resorption induced by polyethylene particles in a murine calvarial model of peri-implant osteolysis.

The study aimed to determine the effects of parthenolide (PAR) on bone volume (BV) and bone surface resorption as assessed by live-animal microcomputed tomography (µCT) and possible osteocyte death as indicated by empty lacunae histologically in polyethylene (PE) particle-induced calvarial osteolysis in mice. Baseline µCT scans were conducted 7 days preimplantation of 2 × 10(8) PE particles/mL over the calvariae (day 0). PAR at 1 mg/kg/day was subcutaneously injected on days 0, 4, 7, and 10. At day 14, BV and surface resorption was analyzed with µCT. Calvarial tissue was processed for histomorphometric osteocyte evaluation. Serum was analyzed for type-1 carboxy-terminal collagen crosslinks (CTX-1) and osteoclast associated receptor (OSCAR) levels by ELISA. PE significantly decreased BV (p = 0.0368), increased surface bone resorption area (p = 0.0022), and increased the percentage of empty lacunae (p = 0.0043). Interestingly, PAR significantly reduced the resorption surface area (p = 0.0022) and the percentage of empty osteocyte lacunae (p = 0.0087) in the PE-calvariae, but it did not affect BV, serum CTX-1 or OSCAR levels. The ability of PAR to inhibit PE-induced surface bone erosion may better reflect the in vivo situation, where bone resorption occurs on the surface at the bone-implant interface and may also be related to the role of osteocytes in this pathology.

Inhibiting histone deacetylase 1 suppresses both inflammation and bone loss in arthritis.

OBJECTIVE: Histone deacetylase 1 (HDAC1) is highly expressed in the synovium of RA patients. Thus we aimed to investigate a novel HDAC inhibitor (HDACi), NW-21, designed to target HDAC1. The effect of NW-21 on osteoclast formation and activity, cytokine and chemokine expression in vitro and arthritis in mice was assessed. METHODS: The effects on human osteoclast formation and activity derived from human blood monocytes stimulated with receptor activator of nuclear factor κB ligand (RANKL) and M-CSF were assessed. The anti-inflammatory activity of NW-21 was assessed using human monocytes stimulated with either TNF-α or lipopolysaccharide for 24 h. mRNA expression of monocyte chemotactic protein 1 (MCP-1), TNF-α, macrophage inflammatory protein 1α (MIP-1α), IL-1 and RANTES (regulated on activation, normal T cell expressed and secreted) was assessed. The effect of NW-21 in the collagen antibody-induced arthritis model was assessed following daily oral administration at 5 mg/kg/day. The HDAC1 inhibitors NW-21 and MS-275 were compared with a broad-acting HDACi, 1179.4b. Effects on inflammation and bone were assessed using paw inflammation scoring, histology and live animal micro-CT. RESULTS: NW-21 suppressed osteoclast formation and activity as well as significantly reducing mRNA expression of MCP-1 and MIP-1α in monocytes stimulated by lipopolysaccharide or TNF-α (P < 0.05) in vitro. Only inhibitors that targeted HDAC1 (NW-21 and MS-275) reduced inflammation and bone loss in the arthritis model. CONCLUSION: The results indicate that inhibitors targeting HDAC1, such as NW-21 and MS-275, may be useful for treating RA, as such drugs can simultaneously target both inflammation and bone resorption.

Periprosthetic osteolysis after total hip replacement: molecular pathology and clinical management.

Periprosthetic osteolysis is a serious complication of total hip replacement (THR) in the medium to long term. Although often asymptomatic, osteolysis can lead to prosthesis loosening and periprosthetic fracture. These complications cause significant morbidity and require complex revision surgery. Here, we review advances in our understanding of the cell and tissue response to particles produced by wear of the articular and non-articular surfaces of prostheses. We discuss the molecular and cellular regulators of osteoclast formation and bone resorptive activity, a better understanding of which may lead to pharmacological treatments for periprosthetic osteolysis. We describe the development of imaging techniques for the detection and measurement of osteolysis around THR prostheses, which enable improved clinical management of patients, provide a means of evaluating outcomes of non-surgical treatments for periprosthetic osteolysis, and assist in pre-operative planning for revision surgery. Finally, there have been advances in the materials used for bearing surfaces to minimise wear, and we review the literature regarding the performance of these new materials to date.

Effects of Osteochondrin S and select connective tissue ribonucleinate components on human osteoclasts in vitro.

OBJECTIVES: Osteochondrin S, a natural product derived from connective tissues and yeast, is used to treat osteoarthritis. The aim of this study was to determine the effect of Osteochondrin S on human osteoclast activity in vitro. METHODS: Osteoclasts were derived from human peripheral blood mononuclear cells stimulated with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B (RANK) ligand. Cells were treated with 23.5-587.2 ng/ml Osteochondrin S or 0.2-5 mg/ml of RNA components (synovia, placenta, intervertebral disc or cartilage). The effects on osteoclast formation and resorptive activity were assessed. Real-time polymerase chain reaction was conducted to assess the expression of key osteoclast genes. KEY FINDINGS: Osteochondrin S and the individual RNA extracts resulted in a concentration-dependent inhibition of human osteoclast activity. Osteochondrin S did not affect RANK, nuclear factor of activated T cells (NFATc1), osteoclast-associated receptor or cathepsin K expression. However, there was a significant (P < 0.05) reduction in mRNA expression of calcitonin receptor. Osteochondrin S treatment also significantly increased the expression of osteoclast inhibitory factor interferon-ß and, interestingly, increased the expression of tumour necrosis-α-like weak inducer of apoptosis (TWEAK). CONCLUSIONS: Osteochondrin S inhibited the resorptive ability of osteoclasts. These actions are likely to occur at a late stage during osteoclast formation, downstream of NFATc1. Overall, the findings show that Osteochondrin S inhibition of osteoclast activity may be responsible for its beneficial effects on diseases such as osteoarthritis.

Epigenetic regulation of inflammation: progressing from broad acting histone deacetylase (HDAC) inhibitors to targeting specific HDACs.

Inhibition of histone deacetylases (HDAC) is emerging as a novel approach to treat a variety of diseases. Recently, broad acting inhibitors of HDAC have been shown to have anti-inflammatory effects both in vitro and in vivo. It is significant that these anti-inflammatory effects are observed at 10-100 fold lower concentrations than their anti-cancer effects. The broad action of these compounds makes it difficult to determine which HDAC enzymes are important in inflammation. Although showing promise it is unlikely that these drugs will progress to the clinic for treating inflammatory diseases due to number of HDACs they affect and the widespread activity of the enzymes throughout the body. Accordingly, research is now progressing to targeting specific HDAC enzymes to improve efficacy of treatment as well as reduce the risk of any unwanted side effects. Understanding the role specific HDACs play in inflammatory disease will help us to identify novel anti-inflammatory treatments. This manuscript is designed to review our limited knowledge in this field.

Azithromycin suppresses human osteoclast formation and activity in vitro.

Azithromycin is an antibiotic with anti-inflammatory properties used as an adjunct to treat periodontitis, a common inflammatory mediated condition featuring pathologic alveolar bone resorption. This study aimed to determine the effect of azithromycin on human osteoclast formation and resorptive activity in vitro. Osteoclasts were generated from peripheral blood mononuclear cells stimulated with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B (RANK) ligand. The effects of azithromycin at concentrations ranging from 0.5 to 40 µg/ml were tested. Osteoclast formation and activity, acidification, actin ring formation and expression of mRNA, and protein encoding for key osteoclast genes were assessed. The results demonstrated that azithromycin reduced osteoclast resorptive activity at all concentrations tested with osteoclast formation being significantly reduced at the higher concentrations (20 and 40 µg/ml). mRNA and protein expression of key osteoclast transcription factor Nuclear Factor of Activated T cells (NFATc1) was significantly reduced by azithromycin at later stages of osteoclast development (day 17). Azithromycin also reduced tumor necrosis factor receptor associated factor-6 (TRAF6) mRNA expression at day 14, and cathepsin K mRNA expression at days 14 and 17. Integrin ß3 and MMP-9 mRNA expression was reduced by azithromycin at day 17 in osteoclasts cultured on dentine. The osteoclast proton pump did not appear to be affected by azithromycin, however formation of the actin ring cytoskeleton was inhibited. This study demonstrates that azithromycin inhibits human osteoclast function in vitro, which may account for at least some of the beneficial clinical effects observed with azithromycin treatment in periodontitis.

Effect of Porphyromonas gingivalis on citrullination of proteins by macrophages in vitro.

BACKGROUND: Citrullination of proteins within inflamed periodontal tissues may provide an important link between periodontitis and rheumatoid arthritis. The aim of this study is to determine whether the presence of Porphyromonas gingivalis peptidylarginine deiminase (PPAD) can influence citrullination of proteins by either increasing the amount of local citrullinated protein or influencing the peptidylarginine deiminase (PAD) enzymes found in the monocyte/macrophage population. METHODS: Human peripheral blood monocytes and macrophages were incubated in the presence of live or heat-killed P. gingivalis. Expression of PAD2 and PAD4, PPAD, and citrullinated proteins were assessed by either a combination of real-time polymerase chain reaction, Western blotting, or a colorimetric assay. RESULTS: PPAD was detected only in mononuclear cells incubated in the presence of live P. gingivalis and resulted in increased extracellular citrullination. Endogenous PAD (mRNA and protein) expression was detected in monocytes and macrophages but was not affected by P. gingivalis. CONCLUSION: Although P. gingivalis produces a PAD that can citrullinate extracellular proteins and may contribute to the citrullinated protein load in gingival tissues, it does not appear to affect PAD expression or citrullination by host monocytes or macrophages.

The immunoreceptor tyrosine-based activation motif (ITAM) -related factors are increased in synovial tissue and vasculature of rheumatoid arthritic joints.

INTRODUCTION: The immunoreceptor tyrosine-based activation motif (ITAM) pathway provides osteoclast co-stimulatory signals and regulates proliferation, survival and differentiation of effector immune cells. In the osteoclast, the receptors Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Osteoclast Associated Receptor (OSCAR) and their respective adaptor proteins, DAP12 and FcRγ mediate ITAM signals and induce calcium signaling and the crucial transcription factor, NFATc1. In rheumatoid arthritis (RA), OSCAR expression by monocytes is inversely correlated with disease activity. Additionally, serum levels of OSCAR are reduced in RA patients versus healthy controls suggesting that expression and secretion or cleavage of soluble (s) OSCAR is immune modulated. Recent data suggest that endothelial cells may also be a source of OSCAR. METHODS: ITAM receptors, their adaptor proteins, and NFATc1 and cathepsin K were detected in human synovial tissues by immunohistochemistry. Synovial tissues from patients with active RA were compared with tissue from patients in remission, osteoarthritis (OA) patients and healthy individuals. OSCAR was measured by immunoassay in synovial fluids recovered from active RA and OA patients. Endothelial cells were cultured with or without 5 ng/mL TNF-α or IL-1ß over 72 hours. Temporal expression of OSCAR mRNA was assessed by qRT PCR and OSCAR protein in the supernatant was measured by ELISA. RESULTS: Significantly higher (P < 0.05) NFATc1-positive inflammatory cell aggregates were found in active RA tissues than in healthy synovial tissue. Similarly, the percentage of OSCAR, FcRγ, DAP12 and TREM2 positive cells was significantly higher in active RA tissues compared to the healthy synovial tissue. Notably, OSCAR was strongly expressed in the microvasculature of the active RA tissues (9/9), inactive RA (8/9) weakly in OA (4/9) but only in the lumen of healthy synovial tissue (0/8). OSCAR levels were detected in synovial fluids from both RA (47 to 152 ng/mL) and OA (112 to 145 ng/mL) patients. Moreover, OSCAR mRNA expression and soluble OSCAR release was stimulated by TNF-α and IL1-ß in cultured endothelial cells. CONCLUSIONS: Increased levels of ITAM related factors were present in synovial tissue from active RA joints compared to OA and healthy joints. OSCAR was strongly expressed by the vasculature of active RA patients and membrane bound and soluble OSCAR was stimulated by inflammatory mediators in endothelial cells in vitro.

Histone deacetylase inhibitors as suppressors of bone destruction in inflammatory diseases.

OBJECTIVES: Despite progress in developing many new anti-inflammatory treatments in the last decade, there has been little progress in finding treatments for bone loss associated with inflammatory diseases, such as rheumatoid arthritis and periodontitis. For instance, treatment of rheumatic diseases with anti-tumour necrosis factor-alpha agents has been largely successful in reducing inflammation, but there have been varying reports regarding its effectiveness at inhibiting bone loss. In addition, there is often a delay in finding the appropriate anti-inflammatory therapy for individual patients, and some therapies, such as disease modifying drugs, take time to have an effect. In order to protect the bone, adjunct therapies targeting bone resorption are being developed. This review focuses on new treatments based on using histone deacetylase inhibitors (HDACi) to suppress bone loss in these chronic inflammatory diseases. KEY FINDINGS: A number of selected HDACi have been shown to suppress bone resorption by osteoclasts in vitro and in animal models of chronic inflammatory diseases. Recent reports indicate that these small molecules, which can be administered orally, could protect the bone and might be used in combination with current anti-inflammatory treatments. SUMMARY: HDACi do have potential to suppress bone destruction in chronic inflammatory diseases including periodontitis and rheumatoid arthritis.

Osteoclast resorption of thermal spray hydoxyapatite coatings is influenced by surface topography.

Coating characteristics such as composition, crystallite features and topography collectively impact the cell response. The influence from splats has not yet been assessed for hydroxyapatite (HAp) thermal spray coatings. The objective of this work is to (a) survey the topography on commercial implants, (b) ascertain topography formation from single splats, and (c) determine the osteoclast resorption pattern on a topographically refined coating compared to dentine. Coatings on dental implants, an orthopedic screw, a femoral stem and a knee implant were studied for reference. The effects of substrate pre-heat, roughness, spray distance and particle size on the coating roughness and topography were studied. Human-derived osteoclasts were placed on a coating with refined topography and compared to dentine, a polished coating and polished sintered HAp. A pre-heat of at least 200°C on titanium was required to form rounded splats. The greatest influence on coating roughness and topography arose from particle size. A 2-fold increase in the mean particle size from 30 to 72 µm produced a significant difference (P<0.001) in roughness from 4.8 and 9.7 µm. A model is shown to illustrate topography formation, nanostructure evolution on single splats, and the topography as seen in commercial implants. Osteoclasts showed a clear preference for activity on coatings with refined topography. A one-way ANOVA test revealed a significantly greater pit depth (P=0.022) for dentine (14 µm) compared to the as-sprayed and polished coating (5 µm). Coatings with topography display a similar number of resorption pits with dentine, but a 10-fold greater number than polished coatings, emphasizing the importance of flattened droplet topography on implant surfaces.

The expression of RANKL and OPG in the various grades of osteoarthritic cartilage.

The objective of the study was to determine whether cartilage expression of the bone regulating molecules receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) varies between the different grades of osteoarthritis (OA). Cartilage samples were obtained from 30 patients undergoing total hip/knee replacement surgery. Tissue sections were stained with Safranin O and graded. Immunohistochemical staining was then performed, and levels of RANKL and OPG expression were assessed using a semi-quantitative scoring system. In addition, levels of mRNA encoding for RANKL and OPG were determined by a relative real-time reverse transcription-polymerase chain reaction technique. We found that expression of RANKL protein, mRNA expression, and the ratio of RANKL: OPG mRNA was greater in grade 2 cartilage in comparison with grade 0 cartilage (P < 0.05). Increased RANKL staining in the grade 2 cartilage was predominantly in the peri-cellular region of the middle and deep zones as well as in the matrix of the superficial zone. OPG mRNA expression was greater in grade 3 cartilage in comparison with grade 0 cartilage (P < 0.05). Cartilage and subchondral bone are in close proximity and soluble proteins produced in the cartilage are likely to move from one compartment to the other. Our finding of increased expression of RANKL in grade 2 OA cartilage might explain the increase in bone turnover reported in the subchondral bone of OA patients. The changes seen in the different grades of tissue may also indicate that this effect occurs during the early stages of OA development.

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis.

INTRODUCTION: TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro. METHODS: TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry. RESULTS: TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts. CONCLUSIONS: The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.

Pre-existing periodontitis exacerbates experimental arthritis in a mouse model.

AIMS: Previous studies have shown a higher incidence of alveolar bone loss in patients with rheumatoid arthritis (RA) and that patients with periodontitis are at a greater risk of developing RA. The aim of this study was to develop an animal model to assess the relationship between pre-existing periodontitis and experimental arthritis (EA). METHODS: Periodontitis was first induced in mice by oral gavage with Porphyromonas gingivalis followed by EA using the collagen antibody-induced arthritis model. These animals were compared with animals with periodontitis alone, EA alone and no disease (controls). Visual changes in paw swelling were assessed to determine clinical development of EA. Alveolar bone and joint changes were assessed using micro-CT, histological analyses and immunohistochemistry. Serum levels of C-reactive protein were used to monitor systemic inflammation. RESULTS: Mice with pre-existing periodontitis developed more severe arthritis, which developed at a faster rate. Mice with periodontitis only also showed evidence of loss of bone within the radiocarpal joint. There was also evidence of alveolar bone loss in mice with EA alone. CONCLUSIONS: The results of this study indicate that pre-existing periodontitis exacerbated experimental arthritis in a mouse model.

Role of polyethylene particles in peri-prosthetic osteolysis: A review.

There is convincing evidence that particles produced by the wear of joint prostheses are causal in the peri-prosthetic loss of bone, or osteolysis, which, if it progresses, leads to the phenomenon of aseptic loosening. It is important to fully understand the biology of this bone loss because it threatens prosthesis survival, and loosened implants can result in peri-prosthetic fracture, which is disastrous for the patient and presents a difficult surgical scenario. The focus of this review is the bioactivity of polyethylene (PE) particles, since there is evidence that these are major players in the development and progression of osteolysis around prostheses which use PE as the bearing surface. The review describes the biological consequences of interaction of PE particles with macrophages, osteoclasts and cells of the osteoblast lineage, including osteocytes. It explores the possible cellular mechanisms of action of PE and seeks to use the findings to date to propose potential non-surgical treatments for osteolysis. In particular, a non-surgical approach is likely to be applicable to implants containing newer, highly cross-linked PEs (HXLPEs), for which osteolysis seems to occur with much reduced PE wear compared with conventional PEs. The caveat here is that we know little as yet about the bioactivity of HXLPE particles and addressing this constitutes our next challenge.