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.

Introduction and aims

Growth plate cartilage is responsible for bone growth in children. Injury to growth plate can often lead to faulty bony repair and bone growth deformities, which represents a significant clinical problem. This work aims to develop a biological treatment.

We have used a culture system of human peripheral blood mononuclear cells (PBMC)as a source of osteoclast (OC) precursors and murine stromal cells to define the cytokine environment in which human OC form, and to determine the separate contributions of the stromal and haemopoietic elements. We designed a panel of reverse transcription-polymerase chain reaction (RT-PCR) primers that specifically amplify the respective murine or human mRNA species that correspond to cytokines and their receptors previously shown to promote or inhibit OC formation. Murine ST-2 cells and human PBMC were cocultured for up to 21 days in the presence of 1,25(OH) 2vitD3, dexamethasone and human macrophage-colony stimulating factor (M-CSF). OC formation was monitored by the appearance of cells that were positive for tartrate resistant acid phosphatase and able to form resorption lacunae on slices of dentine. We found that the ST-2 cells in these cultures expressed mRNA encoding a repertoire of many of the reported osteoclastogenic factors, as well as the recently described OC differentiation factor (ODF/RANKL). The stromal cells also expressed mRNA encoding osteoprotegerin (OPG), a potent inhibitor of OC formation. We found that agonists and antagonists of OC formation were expressed by both the stromal cells and the PBMC. RANK, the receptor for ODF/RANKL, was expressed only by the PBMC as were IL-1R2 and c-FMS. We identified three features of the cytokine environment that may be a characteristic of normal OC formation. Firstly, the ratio of mouse ODF:OPG mRNA was found to increase during the cocultures, consistent with a key role for ODF in the promotion by stromal cells of OC formation. Secondly, we found that mRNA encoding IL-1 and IL-17, as well as IL-6 and sIL-6R, were coordinately expressed by the PBMC. Thirdly, analysis of the culture medium showed that the PBMC secreted IL-1, IL-6 and TNF-alpha protein only in coculture with ST-2 cells during the first few days of osteoclast development. Similarly, prostaglandin E2, shown to synergise with ODF during OC development, was secreted only in cocultures. Together, these data show OC develop in a complex cytokine environment and suggest that haemopoietic cells provide signals to stromal cells during OC development. Work is in progress to extend these studies to human PBMC interacting with normal human osteoblasts.