Developing an Inducible Osteoclast System as a Cell Therapy for Ectopic Calcification

Abstract

Ectopic calcification (EC) is a complex disease in which mineral develops in abnormal locations. It can develop in a variety of locations throughout the body, including the vasculature, heart valves, and in orthopedic settings where it is more commonly referred to as heterotopic ossification (HO). Current therapies for these conditions are limited and new treatment modalities would benefit a wide variety of patients. Osteoclasts resorb mineral in vivo and we hypothesized that a cell therapy based on inducible osteoclast differentiation could be used as a cell therapy to treat EC and HO. The RANK/RANKL pathway is a critical step in osteoclast differentiation and we created an inducible RANK (iRANK) fusion protein which could be activated by a small molecule chemical inducer of dimerization (CID). This fusion protein was introduced into a macrophage cell line, and the resulting RAW264.7+iRANK cells were characterized using in vitro assays. In the presence of CID, the cells formed large, multinucleated, tartrate-resistant acid phosphatase (TRAP)-positive cells capable of resorbing two-dimensional mineral substrates. We then developed three-dimensional assays that were used to test the capabilities of our cell line, including mineralized fibrin scaffolds, murine calvarial discs, and samples of human HO. These cells were capable of resorbing existing mineral, but also of preventing further mineralization of the human HO samples in a contact-independent manner. Finally, we characterized the behavior of RAW264.7+iRANK cells in vivo. We labeled and tracked the cells using bioluminescence and developed a collagen cell delivery vehicle to retain cells at the injection site. The cells were introduced into a murine model of HO where bone morphogenic protein 2 (BMP-2) is injected into the calf muscle to induce formation of an HO lesion. Using microcomputed tomography to quantify the volume of the HO over time, we found that in mice treated with RAW264.7+iRANK cells, there was a significant decrease in HO volume compared to baseline over a two week period, whereas in mice which did not receive cells there was no significant change over this period. Histological examination allowed us to locate the HO lesion and GFP-positive cells in close proximity. This research represents a novel approach to the treatment of EC and the in vitro study of osteoclastogenesis using the RAW264.7+iRANK cell line, and also the three-dimensional mineral assays.