Semaphorin-3a, neuropilin-1 and plexin-A1 in prosthetic-particle induced bone loss.

Peri-prosthetic osteolysis (PPO) occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. Semaphorin-3a (SEM3A), neuropilin-1 (NRP1) and plexin-A1 (PLEXA1) are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This study investigated SEM3A, NRP1 and PLEXA1 protein and mRNA expression in human PPO tissue and polyethylene (PE) particle-stimulated human peripheral blood mononuclear cell (PBMC)-derived osteoclasts in vitro. In addition, the effects of tumour necrosis factor alpha (TNFα) on cultured osteoclasts was assessed. In PPO tissues, a granular staining pattern of SEM3A and NRP1 was observed within large multi-nucleated cells that contained prosthetic wear particles. Immunofluorescent staining confirmed the expression of SEM3A, NRP1 and PLEXA1 in large multi-nucleated human osteoclasts in vitro. Furthermore, SEM3A, NRP1 and PLEXA1 mRNA levels progressively increased throughout osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL), and the presence of PE particles further increased mRNA expression of all three molecules. Soluble SEM3A was detected in human osteoclast culture supernatant at days 7 and 17 of culture, as assessed by ELISA. TNFα treatment for 72h markedly decreased the mRNA expression of SEM3A, NRP1 and PLEXA1 by human osteoclasts in vitro. Our findings suggest that SEM3A, NRP1 and PLEXA1 may have important roles in PPO, and their interactions, alone or as a complex, may have a role in pathological bone loss progression. STATEMENT OF SIGNIFICANCE: Peri-prosthetic osteolysis occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. The rate of hip and knee arthroplasty is increasing by at least 5% per year. However, these joint replacements have a finite lifespan, with data from the National Joint Replacement Registry (Australia) showing that the major cause of failure of total hip replacements is aseptic loosening. In aseptic loosening, wear particles liberated from prostheses are phagocytosed by macrophages, leading to release of inflammatory cytokines and up-regulation of osteoclast formation and activity. Semaphorin-3a, neuropilin-1 and plexin-A1 are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This is the first report to show that these molecules may be involved in the implant failure.

Class I and II histone deacetylase expression in human chronic periodontitis gingival tissue.

BACKGROUND AND OBJECTIVE: Histone deacetylase inhibitors (HDACi) are being considered to treat chronic inflammatory diseases at low doses. Currently HDACi that are more specific are being developed to target particular HDACs; therefore, this study aimed to determine levels and distribution of class I and II HDAC in human gingival samples obtained from patients with chronic periodontitis. MATERIAL AND METHODS: Gingival biopsies were obtained from patients with and without (mild inflammation, no bone loss) periodontitis. Total RNA was isolated for real-time quantitative polymerase chain reaction to determine expression of HDACs 1-10. Immunohistochemistry was used to determine protein distribution of HDACs 1, 5, 8 and 9. Factor VIII, CD3 and tartrate resistant acid phosphatase (TRAP) were detected in serial sections to identify blood vessels, lymphocytes, pre-osteoclasts and osteoclasts cells respectively. Tumour necrosis factor α (TNF-α) expression was also assessed. RESULTS: mRNA for HDAC 1, 5, 8 and 9 were significantly upregulated in chronic periodontitis gingival tissues compared to non-periodontitis samples (p < 0.05). Significantly higher HDAC 1 protein expression was observed in chronic periodontitis samples (p < 0.05), and was associated with CD3, TRAP and TNF-α-positive cells. HDAC 1, 5, 8 and 9 were expressed strongly by the factor VIII-positive microvasculature in the chronic periodontitis gingival tissues. CONCLUSIONS: HDAC 1, 5, 8 and 9 expression was higher in gingival tissues from patients with chronic periodontitis compared to non-periodontitis samples. Results suggest that these HDACs could therefore be targeted with specific acting HDACi.

Caffeic acid phenethyl ester abrogates bone resorption in a murine calvarial model of polyethylene particle-induced osteolysis.

Particle-induced bone loss by osteoclasts is a common cause of aseptic loosening around implants. This study investigates whether caffeic acid phenethyl ester (CAPE), a potent and specific inhibitor of nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 and nuclear factor kappa B, at a low dose reduces bone resorption in a murine calvarial model of polyethylene (PE) particle-induced osteolysis. The effects of particles and CAPE treatment on gastrointestinal tract (GIT) histopathology were also evaluated. Mice were scanned using in vivo animal micro-computed tomography (µCT) as a baseline measurement. PE particles (2.82 × 10(9) particles/mL) were implanted over the calvariae on day 0. CAPE was administered subcutaneously (1 mg/kg/day) at days 0, 4, 7 and 10. Mice were killed at day 14 and serum was analysed for Type-1 carboxyterminal collagen crosslinks (CTX)-1 and osteoclast-associated receptor (OSCAR) levels. Ex vivo µCT scans were conducted to assess bone volume (BV) change and percentage area of calvarial surface resorbed. Calvarial and GIT tissue was processed for histopathology. By day 14, PE particles significantly induced calvarial bone loss compared with control animals as evidenced by resorption areas adjacent to the implanted PE in three-dimensional µCT images, an increase in percentage of resorbed area (p = 0.0022), reduction in BV (p = 0.0012) and increased Tartrate-resistant acid phosphatase positive cells. Serum CTX-1 (p = 0.0495) and OSCAR levels (p = 0.0006) significantly increased in the PE implant group. CAPE significantly inhibited PE particle-induced calvarial osteolysis, as evidenced by a significant reduction in surface bone resorption (p = 0.0012) and volumetric change (p = 0.0154) compared with PE only, but had no effect on systemic CTX-1. Neither particles nor CAPE had an effect on GIT histopathology.

Azithromycin suppresses P. gingivalis LPS-induced pro-inflammatory cytokine and chemokine production by human gingival fibroblasts in vitro.

OBJECTIVE: Azithromycin is a macrolide antibiotic that appears to have both antibacterial and anti-inflammatory properties. This study aimed to investigate the effect of azithromycin on the production of pro-inflammatory cytokines and chemokines by human gingival fibroblasts (HGF) in vitro. MATERIALS AND METHODS: The effects of azithromycin (0.1 to 10 µg/mL) on the production of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), and growth-regulated oncogene (GRO) by human gingival fibroblasts cultured in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (LPS) was studied. Cytokine and chemokine protein levels in the culture supernatant were assessed using a Luminex® multiplex immunoassay. RESULTS: P. gingivalis LPS induced cytokine/chemokine (IL-6, IL-8, MCP-1, and GRO) protein production in HGFs, and this effect was suppressed by azithromycin at all concentrations tested. CONCLUSIONS: This study demonstrates that azithromycin suppresses P. gingivalis LPS-induced cytokine/chemokine protein production in HGF, which may explain some of the clinical benefits observed with the adjunctive use of azithromycin in the treatment of periodontitis. CLINICAL RELEVANCE: The current study examines the anti-inflammatory properties of azithromycin which may make it useful as an adjunct treatment to periodontitis. Specifically, we used azithromycin to modulate the production of pro-inflammatory cytokines by gingival fibroblasts known to be important in periodontal inflammation.

Quantifying not only bone loss, but also soft tissue swelling, in a murine inflammatory arthritis model using micro-computed tomography.

In rodent models of inflammatory arthritis, bone erosion has been non-invasively assessed by micro-computed tomography (micro-CT). However, non-invasive assessments of paw swelling (oedema) are still based on clinical grading by visual evaluation, or measurements by callipers, not always reliable for the tiny mouse paws. The aim of this work was to demonstrate a novel straightforward 3D micro-CT analysis protocol capable of quantifying not only joint bone erosion, but also soft tissue swelling, from the same scans, in a rodent inflammatory arthritis model. Balb/c mice were divided into two groups: collagen antibody-induced arthritis (CAIA) and CAIA treated with prednisolone, the latter reflecting an established treatment in human rheumatoid arthritis. Clinical paw scores were recorded. On day 10, front paws were assessed by micro-CT and histology. Micro-CT measurements included paw volume (bone and soft tissue together) and bone volume at the radiocarpal joint, and bone volume from the radiocarpal to the metacarpophalangeal joint. Micro-CT analysis revealed significantly lower paw volume (-36%, P < 0.01) and higher bone volume (+17%, P < 0.05) in prednisolone-treated CAIA mice compared with untreated CAIA mice. Paw volume and bone volume assessed by micro-CT correlated significantly with clinical and histological scores (|r| > 0.5, P < 0.01). Untreated CAIA mice showed significantly higher clinical scores, higher inflammation levels histologically, cartilage and bone degradation, and pannus formation, compared with treated mice (P < 0.01). The presented novel micro-CT analysis protocol enables 3D-quantification of paw swelling at the micrometre level, along with the typically assessed bone erosion, using the same images/scans, without altering the scanning procedure or using contrast agents.

Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation.

Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin (ß3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p<0.05) reduced the expression of NFATc1, CathK, OSCAR, FcRγ, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p<0.05) decreased CathK, OSCAR, FcRγ, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the calcineurin-NFAT signalling cascade to suppress key mediators of the ITAM pathway during late stage osteoclast differentiation and this is associated with a reduction in both osteoclast differentiation and activity.

Inhibition of apoptosis in periodontitis.

This study investigated whether the prolonged survival of inflammatory cells in periodontal disease could be due to the inhibition of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptors and inhibition of the terminal stages of apoptosis signaling by inhibitor of apoptosis (IAP) family members. Gingival tissue samples were taken from healthy individuals and those with chronic periodontitis. The expression of TRAIL, TRAIL receptors, TUNEL, cleaved caspase-3, xIAP, and survivin was determined immunohistologically and at the level of mRNA expression. Higher levels of TRAIL and the TRAIL decoy receptor, TRAIL R4, were expressed in the diseased periodontal tissues (p < 0.005). Statistically (p < 0.05) higher levels of cleaved caspase-3 and the cleaved caspase-3 inhibitors, xIAP and survivin, were seen. Similar changes were seen at the level of mRNA. The results indicate that apoptosis in periodontitis may be inhibited by elevated expression of TRAIL decoy receptors and cleaved caspase-3 inhibitors.