Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Histochemical assessment for biological interplay of endothelial cells and perivascular cell in endochondral ossification (#346)

Erika Tsuchiya 1 , Tomoka Hasegawa 1 , Hiromi Hongo 1 , Tomomaya Yamamoto 1 , Norio Amizuka 1
  1. Graduate School of Dental Medicine, Hokkaido University, Sapporo, HOKKAIDO, Japan

Purpose: Endochondral ossification is essential for not just ossification but also fracture healing. Chondro-osseous junction is the site of vascular invation in the process of endochondral ossification, which replaces epiphyseal cartilage with bone. Osteoclasts, bone-resorbing cells, aren’t intrinsic for endochondral ossification, because the long bones of osteoclast-less mice can grow longitudinally. In this study, we have histologically examined vascular cell and the cell exist around it at the chondro-osseous junction.

Materials and Methods: ICR mice at six week of age were perfused with 4% paraformaldehyde solution or 1/2Karnovskyl solution from the left ventricle, and then, tibiae were extracted and immersed in the samefixatives. The speciments were decalcified with 5% EDTA and embedded into paraffin or epoxy resin. Paraffin sections were employed for histochemistry of CD31, MMP-9, F4/80 and DBA lectin. Ultrathin sentions were staind with uranyl acetate and lead citrate prior to TEM observation.

Result and discussion: DBA-positive perivascular cells were localized in the close proximity of the invading vascular endothelial cells, but didn’t precede vascular invasion. MMP-9 was shown to be positive in CD31-positive endothelial cells and osteoclasts. Cytoplasmic processes of vascular endothelial cells were shown to extend into the transverse partitions of cellular columns of hypertrophic chondrocytes. Hypertrophic chondrocytes adjacent to such vascular invasion seemed to be often intact, featuring normal cell organelles and enlarged cell bodies. But, some cell debris was observed in blood vessels close to the junction. Moreover perivascular cell exsist in c-fos-/- mice, but not in RANKL-/- mice. Thus, DBA-positive perivascular cells may have relation to expression of RANKL. Some vascular endothelial cells made cell-to-cell contact with osteoblasts. EphrinB2 is shown to be positive in endothelial cells. The cellular interplay between the endothelial cells and the surrounding cells seems to be essential for vascular invasion during endochondral ossification.