Histological identification of cells directly participating calcified tissue resorption and destruction

It is well established that multinucleated osteoclasts are the main cells to resorb calcified tissues. However, it still remains the possibility that the mononuclear cells other than osteoclasts directly resorb or destruct calcified tissues. In this study, we examined the ontogenic development of long bones in vivo and in vitro and the process of the bone destruction during the experimentally induced mouse arthritis model to detect the calcified tissue destruction by mononuclear cells. Materials and Methods Humeral bones from E16 Balb/c mice were dissected and processed for transmission electron microscopy (TEM). To inhibit resorption by osteoclasts, pregnant mice were treated with a daily administration of nitrogen‐containing bisphosphonate (alendronate) from E15–E17 before the formation of fetal humeral bone marrow cavities. Metatarsals at E16 were organ cultured for 7 days and processed for TEM. Ten‐weeks old male DBA/1 mice were treated with type II collagen to induced collagen‐induced arthritis. Alendronate was administered once a week before the onset of arthritis to inhibit osteoclastic bone resorption. At the indicated time, knee joints were dissected and processed for the histological and immunohistochemical analyses. Results During the development of the humeral primary bone marrow cavity formation, the density of type II collagen fibers in the cartilage decreased with the progress of the calcification. Finally, the calcified cartilage directly became fragmented and macrophages invading from the perichondrium phagocytosed the fragmented particles. The number of primary bone trabecula of new born mice increased with alendronate treatment. However, bone marrow was formed in humeral bones of alendronate –treated newborn mice where active hematopoiesis was observed. In the organ culture of mouse E15 metatarsals, the primary bone marrow was also formed where fibroblasts phagocytosed calcified cartilage particles. In the mouse collagen–induced arthritis model, alendronate induced more severe inflammation than the control, which leaded to the severe articular cartilage and bone destruction. Many osteoclasts actively resorbed bone in the control group whereas many neutrophils were accumulated on the bone surface in the alendronate‐treated group. Ultrastructural observation clearly showed the accumulation and rupture of neutrophils on the bone surface. No collagen fibers were detected in the bone matrix. Discussion Bone homeostasis is maintained by the functional coupling between osteoblasts and osteoclasts. It has been accepted that the aberration of these two cells lead to bone diseases. In this study, we strongly indicated the direct resorption and destruction of calcified tissues by mononuclear cells other than osteoclasts. The results may change the concept for bone diseases and help the way to the understanding and the development of new therapies for the disease mainly involving bone destruction.