Three-dimensional visualization of osteoclasts in embryonic mouse mandibles using SEM array tomography.

OBJECTIVE: Three-dimensional (3-D) images of osteoclasts in vivo have been elusive, due to their large size and intricate morphology. The present study was designed to reconstruct the 3-D morphology of whole osteoclasts in developing mouse mandibles using scanning electron microscopy (SEM) array tomography. METHODS: Mandibles of 16 days post coitum mouse embryos were fixed and embedded in epoxy resin after decalcification. Epoxy blocks were trimmed, and serial sections of 1 µm in thickness were cut with an ultramicrotome and mounted on glass microscope slides. Consecutive images of every fourth or fifth serial section were obtained by SEM after electron staining and platinum coating. Three dimensional reconstruction of osteoclasts was performed using these consecutive images. RESULTS: Multinucleated osteoclasts were observed to cluster around developing bone in the embryonic mouse mandible. The outlines of osteoclasts and their sealing zones were identified in the serial sections. The reconstructed 3-D image revealed whole osteoclast morphology with the sealing zone. Osteoclasts were adherent to bone with the anchoring structure between the osteoclast and the bone. CONCLUSIONS: SEM array tomography with our modification revealed 3-D imagery of a whole osteoclast and its sealing zone in vivo for the first time. This methodology could provide useful information on in vivo structures and dynamics of large cells, such as osteoclasts.

Degradation of extracellular matrices propagates calcification during development and healing in bones and teeth.

BACKGROUND: Bone, dentin, and enamel are tissues formed through calcification, a process involving deposition of calcium phosphate minerals on extracellular organic matrices. Calcification, the underlying mechanism of which is unknown, is initiated with mineral deposition followed by advancing of the deposit and subsequent maturation of the mineral crystal. HIGHLIGHT: We have reviewed the current knowledge of how calcification proceeds during bone development, bone healing, and enamel and dentin development, based on reported studies. Previous studies reported by us and by other authors have suggested that degradation of some extracellular matrix (ECM) proteins is involved in calcification during bone and dentin development and bone healing in a manner similar to that previously reported for enamel development. CONCLUSION: The ECM proteins may inhibit mineral deposition and calcification, similar to the role of amelogenin during enamel development. The candidates for the amelogenin equivalents in bone and dentin have not been identified. Further studies are required to elucidate the regulatory mechanisms of bone and dentin calcification in light of specific ECM proteins that prevent calcification and enzymes that degrade these ECM proteins.

Bone marrow PDGFRα+Sca-1+-enriched mesenchymal stem cells support survival of and antibody production by plasma cells in vitro through IL-6.

Plasma cells (PCs) acquiring long lifespans in the bone marrow (BM) play a pivotal role in the humoral arm of immunological memory. The PCs reside in a special BM niche and produce antibodies against past-encountered pathogens or vaccine components for a long time. In BM, cysteine-X-cysteine (CXC) chemokine receptor type 4 (CXCR4)-expressing PCs and myeloid cells such as dendritic cells are attracted to and held by CXC chemokine ligand 12 (CXCR12)-secreting stromal cells, where survival of the PCs is supported by soluble factors such as IL-6 and APRIL (a proliferation-inducing ligand) produced by neighboring myeloid cells. Although these stromal cells are also supposed to be involved in the support of the survival and antibody production, the full molecular mechanism has not been clarified yet. Here, we show that BM PDGFRα+Sca-1+-enriched mesenchymal stem cells (MSCs), which can contribute as stromal cells for hematopoietic stem cells, also support in vitro survival of and antibody production by BM PCs. IL-6 produced by MSCs was found to be involved in the support. Immunohistochemistry of BM sections suggested a co-localization of a minor population of PCs with PDGFRα+Sca-1+ MSCs in the BM. We also found that the sort-purified MSC preparation was composed of multiple cell groups with different gene expression profiles, as found on single-cell RNA sequencing, to which multiple roles in the in vitro PC support could be attributed.