The inflammatory cascade associated with prosthetic implant wear debris, in addition to diseases such as rheumatoid arthritis and periodontitis, it is shown to drastically influence bone turnover in the local environment. Ultimately, this leads to enhanced osteoclastic resorption and the suppression of bone formation by osteoblasts causing implant failure, joint failure, and tooth loosening in the respective conditions if untreated. Regulation of this pathogenic bone metabolism can enhance bone integrity and the treatment bone loss. The current study used novel compounds that target a group of enzymes involved with the epigenetic regulation of gene expression and protein function, histone deacetylases (HDAC), to reduce the catabolism and improve the anabolism of bone material in vitro.

Human osteoclasts were differentiated from peripheral blood monocytes and cultured over a 17 day period. In separate experiments, human osteoblasts were differentiated from human mesenchymal stem cells isolated from bone chips collected during bone marrow donations, and cultured over 21 days. In these assays, cells were exposed to the key inflammatory cytokine involved with the cascade of the abovementioned conditions, tumour necrosis factor-α (TNFα), to represent an inflammatory environment in vitro. Cells were then treated with HDAC inhibitors (HDACi) that target the individual isoforms previously shown to be altered in pathological bone loss conditions, HDAC-1, −2, −5 and −7. Analysis of bone turnover through dentine resorptive measurements and bone mineral deposition analyses were used to quantify the activity of bone cells. Immunohistochemistry of tartrate resistant acid phosphatase (TRAP), WST-assay and automated cell counting was used to assess cell formation, viability and proliferation rates. Real-time quantitative PCR was conducted to identify alterations in the expression of anti- and pro-inflammatory chemokines and cytokines, osteoclastic and osteoblastic factors, in addition to multiplex assays for the quantification of cytokine/chemokine release in cell supernatant in response to HDACi treatments in the presence or absence of TNFα.

TNFα stimulated robust production of pro-inflammatory cytokines and chemokines by PBMCs (IL-1β, TNFα, MCP1 and MIP-1α) both at the mRNA and protein level (p < 0 .05). HDACi that target the isoforms HDAC-1 and −2 in combination significantly suppressed the expression or production of these inflammatory factors with greater efficacy than targeting these HDAC isoforms individually. Suppression of HDAC-5 and −7 had no effect on the inflammatory cascade induced by TNFα in monocytes. During osteoclastic differentiation, TNFα stimulated the size and number of active cells, increasing the bone destruction observed on dentine slices (p < 0 .05). Targeting HDAC-1 and −2 significantly reduced bone resorption through modulation of the expression of RANKL signalling factors (NFATc1, TRAF6, CatK, TRAP, and CTR) and fusion factors (DC-STAMP and β3-integerin). Conversely, the anabolic activity of osteoblasts was preserved with HDACi targeting HDAC-5 and −7, significantly increasing their mineralising capacity in the presence of TNFαthrough enhanced RUNX2, OCN and Coll-1a expression.

These results identify the therapeutic potential of HDACi through epigenetic regulation of cell activity, critical to the processes of inflammatory bone destruction.

Osteoclast Associated Receptor (OSCAR) is a novel member of leucocyte receptor complex (LCR)-encoded family expressed by pre-osteoclasts and mature osteoclasts (OC). Blocking of OSCAR binding to its putative ligand has been shown to inhibit osteoclast formation. To date there is no data available regarding the expression of OSCAR in tissues associated with osteolysis and the objective of this study is to determine if OSCAR is expressed adjacent to focal bone osteolysis near failed implants. A total of 22 samples (10 Peri-implant tissue and 12 OA) were studied. OSCAR antibodies were a gift from R& D Systems Inc. (Minneapolis, MN, USA). The tissues were analysed semi-qualitatively using semi-quantitative scoring (SQA) independently by two observers. Non-parametric Mann Whitney-U test was used to test statistical significance. Dual labelling for OSCAR and CD68 expression was also carried out. Strong expression of OSCAR was seen in the majority of multinucleated cells in peri-implant tissues while OA tissues showed very low levels of OSCAR expression. Dual labelling studies revealed that the cells expressing OSCAR also expressed CD68. There was a significant difference in the expression of OSCAR between peri-implant tissue and OA synovial tissue (p< 0.003). This study shows that OSCAR is expressed at high levels by the numerous CD68 multinucleated cells present is these tissues in peri-implant tissues. These findings and recent reports on the role OSCAR may play in OC formation indicate that OSCAR could be an important mediator of peri-implant osteolysis

There is growing evidence that RANKL (also known as osteoclast differentiation factor), its receptor RANK and its natural inhibitor osteoprotegerin (OPG) are involved in bone loss in a number of pathologies. The aim of this study was to determine if these factors are expressed in a number of bone loss pathologies and what cell types were producing these factors in the tissues using reverse transcription polymerase chain reaction (RT-PCR), in situ hybridisation and immunostaining techniques. Periarticular tissue was obtained from 15 patients undergoing revision of aseptic loose implants. Rheumatoid joint tissue was obtained from the pannus region of 12 patients diagnosed with rheumatoid arthritis undergoing joint replacement or joint fusion. Inflamed gingival tissue from sites near bone erosion were obtained from 11 patients with periodontal disease. 6 normal periodontal and periarticular tissue from 6 osteoarthritic patients was used as controls.

RANK, RANKL, OPG and M-CSF mRNA were expressed in tissues obtained from all the pathologies. Higher ratio’s of RANKL to OPG were observed in the pathological tissues compared to their respective controls. In revision tissues many multinucleated giant cell containing particles expressed RANK mRNA. The pattern of staining of RANK mRNA was markedly different in the rheumatoid and periodontal tissues. Differences were also seen in the pattern of expression for RANKL using both in situ and immunostaining. Overall our results indicate that although similar osteoclastogenic factors are fundamentally involved in these bone loss pathologies, different cell types may be producing and/or responding to these factors. Identifying fundamental mechanisms such as these may indicate that similar treatments, such as using OPG or related compounds, may be used for a diverse range of bone loss diseases.

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.