ProjectInvestigation of soft tissue toxicity in anti-osteoclastic drug-related osteonecrosis of the jaw (ARONJ)

Investigation of soft tissue toxicity in anti-osteoclastic drug-related osteonecrosis of the jaw (ARONJ)

10/31/2016 to 2/28/2017

Although the first anti-osteoclastic drug-related osteonecrosis of the jaw (ARONJ) case was reported over a decade ago, the pathogenesis of the disease remains unclear. Consequently there is no unified recommended prevention or treatment protocol. One of the proposed mechanisms of medication-related osteonecrosis of the jaw (MRONJ) involves the toxicity of the surrounding mucosa. However, the role of soft tissue in the pathogenesis of MRONJ is not well defined, and may play a key role in bone exposure and impaired bone healing.

This project will attempt to clarify the pathogenesis of MRONJ due to the medication-related damage of the surrounding soft tissue by investigating the role of human gingival fibroblasts (HGF) to induce or inhibit apoptosis and inflammation that may influence angiogenesis and wound healing in response to bisphosphonates, denosumab, and combination therapies. We will attempt to investigate if this effect is medication dose-dependent, and whether it could be augmented by bacterial lipopolysaccharide (LPS).

Zolendronate, denosumab, and combination therapy with other medications reported in association with MRONJ (sunitinib, sorafenib, bevacizumab, and sirolimus) will be tested on an in vitro HGF culture model in the absence and presence of LPS from P.gingivalis. The readout will be cell adhesion, proliferation, cell death of HGF at various dosages via the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences) system, a non-invasive approach to assess cellular activity via electrical impedance measurements in real-time. LIVE/DEAD fluorescent staining will confirm these findings.

Quantitative PCR assay will be performed for pro-inflammatory (IL-1, TNF-α, IFN-γ, IL-6) and angiogenic (IL-8, VEGF) gene expression. A further co-culture of peripheral blood mononuclear cells (THP-1 cells) with HGFs aims to explore additional immunological reactions triggered by these medications. ELISA of the co-culture media in the presence and absence of various anti-osteoclastic medications will be performed for the same inflammatory and angiogenic mediators with additional osteogenic/osteoclastogenic markers (RANKL, Osteoprotegerin).