UCLA

Osteonecrosis of jaw, is a bone disorder affecting the jaw bone. Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a condition found in patients who have received intravenous and oral forms of bisphosphonate therapy for various bone-related conditions. Bisphosphonates (BPs) are a class of drugs used to treat osteoporosis and malignant bone metastasis. Osteoclast's are the bone cells capable of resorbing bone, are the potential target of BP therapy. The objective of this study is to demonstrate the role of osteoclasts in ONJ, effect of BP on the differentiation and function of osteoclasts, inflammatory mediators secreted by osteoclasts involved in ONJ

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model, interaction of osteoclasts with other immune cells especially NK cells, as well as role of

IL-10 in osteoclasts and BP interaction. This study demonstrate that, i.v nitrogen-containing

bisphosphonates Zolendronic Acid and oral nitrogen-containing bisphosphonates Alendronate and oral non-nitrogen containing Etidronate modulate the pro-inflammatory functions of osteoclasts. Nitrogen containing Bisphosphonates mediated significant dose dependent release of pro-inflammatory cytokines IL-6, TNF-α, IL-1b and they inhibited anti-inflammatory IL-10 secretion by osteoclasts. Zolendronic Acid, Alendronate and Etidronate each had a different effect on the secretion of cytokines, with Zolendronic Acid having the highest and Etidronate the lowest ability to up-regulate the cytokine secretion. Zolendronic Acid, Alendronate and Etidronate each had a different effect on cell viability, Zolendronic Acid caused highest reduction in osteoclasts numbers, followed by Alendronate and Etidronate almost cause no cell death. The surface expression of CD14, CD33, CD54, CD44, CD11b, MHC class I and II and B7H1 were significantly increased when osteoclasts were treated with Zolendronic Acid, effect was higher at 500nM conc. and down-modulated at higher concentration of Zolendronic Acid, but still higher than untreated OC. All three bisphosphonates were capable of decreasing pit formation by osteoclasts. Osteoclasts act as key immune effectors capable of modulating the function of Natural Killer (NK) cells. Treatment of osteoclasts with Zolendronic Acid and much less with Alendronate was capable of inhibiting NK cell cytotoxicity whereas it induced significant secretion of cytokines, IFN-γ, IL-6, IL-10 and IL-18 and chemokines in the cultures of NK cells with osteoclasts. Resistance to NK cell cytotoxicity was higher at low concentration of Zolendronic Acid, comparative to higher concentration. NK cells were able to lyse osteoclasts much more than their precursor cell monocytes and this correlated with the decreased

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expression of MHC class I and CD54 expression on osteoclasts. These results suggest that Zolendronic Acid treated osteoclasts may remain viable in the microenvironment for a prolonged period of time during interaction with NK cells providing continuous secretion of pro-inflammatory cytokines and chemokines in the absence of anti-inflammatory cytokine IL-10 resulting in the chronicity of inflammation.

Zolendronic Acid injections could similarly increase IFN-g and IL-6 secretion and Cytotoxicity against the tumor cells, in the bone marrows but decrease the secretion of IFN-g in gingival, pancreatic and adipose tissues. These observations are of significant value since they may provide potential mechanisms for the pathogenesis seen in Osteonecrosis of the Jaw (ONJ). It is possible that an increase in activation of Bone Marrow derived cells by Zolendronic Acid result in the eventual loss or inhibition of IFN-g secreting cells when they reach to the tissues. Since gingival derived immune cells are of activated phenotype when compared to bone marrow derived immune cells in healthy subjects, prior activation of bone marrow derived cells when mobilized to the gingival tissues and are exposed to additional activation signals from antigens in gingiva may undergo activation induced cell death, or simply become exhausted, and result in the lower secretion of IFN-g by the immune cells as seen in our in vivo model system. These possibilities are under investigation in our laboratory. The phenotype and nature of immune cells, including NK cells, one of the main contributors of IFN-g secretion in bone marrow and tissues are currently being investigated in our laboratory.