Engineered Osteoclasts Prevent Cell-Mediated Calcification Through Elaboration of Anticalcific Factors

Objectives: Osteoclasts are bone-resorbing cells that bind to mineralized surfaces and resorb calcification via formation of resorption lacunae. However, their ability to prevent further calcification via elaboration of soluble, anticalcific factors has not been investigated. In this study, we hypothesized that osteoclasts can prevent calcification through elaboration of soluble calcification inhibitors.
Methods: RAW264.7 murine monocytic cells were engineered with an inducible receptor activator of nuclear factor kappa-B (iRANK) construct to enforce differentiation into osteoclasts under the control of a chemical inducer of dimerization (CID). iRANK cells were co-cultured with C2C12 myoprogenitor cells in media containing high inorganic phosphate using Transwell inserts. ELISA was used to detect a candidate anticalcific molecule, osteopontin (OPN), in culture supernatants. OPN in conditioned media was immunodepleted using Dynabead and OP-199 antibody to further confirm the role of OPN in inhibiting calcification.
Results: iRANK cells treated with CID formed TRAP positive multi-nucleated osteoclasts in a dose-dependent manner and could functionally resorb two-dimensional mineralized substrates in vitro, similar to RANKL-induced osteoclasts. In contrast to RANKL-induced osteoclasts, engineered osteoclast differentiation was independent of RANKL and M-CSF, and resistant to inhibition by OPG. Using a co-culture system, iRANK cells induced with CID inhibited matrix calcification of osteogenic precursors in the absence of cell contact. Candidate anticalcific protein analysis showed that OPN levels in culture supernatant of CID-treated iRANK cells were ~25 fold higher than in untreated cells (p<0.05). Immunodepletion of OPN in conditioned media from CID-treated iRANK cells significantly reduced its ability to inhibit calcification of C2C12 cells.
Conclusions: To the best of our knowledge, this is the first study to demonstrate the contact-independent ability of osteoclasts to inhibit cell-mediated calcification in vitro. Our findings suggest that osteoclasts function not only to resorb mineralized matrix through physical contact, but also to prevent cell-mediated calcification by elaborating OPN, a potent inhibitor for mineral deposition.