Engineering Osteoclasts to Prevent Diseases Caused by Osteoclast Deficiency

Abstract

Osteoclasts are bone-resorbing cells that bind to mineralized surfaces and resorb calcification via formation of resorption lacunae. However, their ability to prevent calcification via elaboration of anticalcific factors has not been investigated. To test this, RAW264.7 murine monocytic cells were engineered with an inducible receptor activator of nuclear factor kappa-B (iRANK) construct to induce differentiation into osteoclasts under the control of a chemical inducer of dimerization (CID). iRANK cells treated with CID formed TRAP-positive multinucleated osteoclasts that were capable of mineral resorption. We demonstrated that iRANK osteoclasts could inhibit mineralization of C2C12 myoprogenitor cells and bovine aortic valve interstitial cells in a co-culture system. Analyses of candidate anticalcific proteins identified osteopontin (OPN) in the culture media of CID-treated iRANK cells at significantly higher levels compared to untreated cells. Immunodepletion of OPN from the media conditioned by CID-treated iRANK cells significantly reduced its ability to inhibit C2C12 calcification, as well as progressive mineralization of human heterotopic ossification samples in vitro. These data suggest that engineered osteoclasts may be useful for preventing ectopic calcification through elaboration of potent anticalcific factors. Both anticalcific and bone resorptive functions of osteoclasts could thus prove useful as therapeutic tools for the treatment of heterotopic ossification and other ectopic calcification disorders. One such disorder, medication-related osteonecrosis of the jaw or MRONJ, stems from a serious side effect of treatment with antiresorptive medications (such as denosumab) used in patients with cancer and metastatic bone disease. It often occurs after dental-related interventions such as tooth extraction, and is thought to occur due to inhibition of osteoclastic bone resorption. In this study, we showed that iRANK cells are resistant to inhibition by anti-mouse RANKL antibody, a mouse analog of denosumab. Thus, we proposed to utilize the engineered cells to elucidate the role of osteoclasts in MRONJ development and subsequently use these cells to prevent this disease. In this study, we developed a mouse model of MRONJ in nude mice and validated cell delivery method and osteoclast induction in vivo. These techniques will be used in future studies to test the hypothesis that MRONJ occurs as a result of impaired osteoclasts due to the inhibition by antiresorptive drugs such as denosumab. Thus, delivering engineered osteoclasts, resistant to this inhibition, may prevent the development of MRONJ after dental-related trauma.