Endochondral Ossification of Chick Embryonic Femora in vitro and on Chorioallantoic Membrane

We observed angiogenesis and endochondral ossification of the femora of chick embryos in vitro, on chorioallantoic membrane (CAM), and in vivo to clarify the cellular processes of avian endochondral ossification. We found that the inside of the in vitro femora was still filled with chondrocytes, despite being cultured for 10 days, while calcification of the diaphysis was not observed. As well, only the cartilage tissue of the epiphysis was enlarged. By contrast, blood vessels invaded into the diaphysis and the marrow cavity was formed in CAM-cultured femora. Cartilage canals extended from the marrow cavity and reached to the resting chondrocyte zone, with normal endochondral ossification occurring as in vivo. This study demonstrates that endochondral ossification occurs in femora in CAM culture similar to that in in vivo femora, but not in in vitro femora. The ossification is dependent on vascular invasion into the embryonic femora. In conclusion, for the endochondral ossification of long bones it is essential to supply the embryo with blood vessels. CAM culture system was found to be a superior endochondral ossification model of the embryonic femur

Osteoclast-Like Cell Formation in Medullary Bone Marrow Cell Culture of Laying Hens

Marrow cells were isolated from medullary bone of hens at two opposing phases, the bone formative and resorptive phases, of the egg-laying cycle. These cells were cultured for up to 14 days on cover slips. After culture, the osteoclast-like cells which were multinuclear, positive for tartrate-resistant acid phosphatase (TRAP) and formed bone-resorption pit, were observed on the cover slip. The number of them increased by 8 days of culture and decreased thereafter. Also, the osteoclast-like cell formation was prominent in culture of medullary bone marrow cells at the bone resorptive phase, compared with those at the bone formative phase. Additionally, before forming osteoclast-like cells, TRAP-positive mononuclear cells were adherent to the cover slip and later aggregated as clusters. In the clusters, the TRAP-positive mononuclear cells contacted each other and partially became multinucleated. These results show that medullary bone marrow cells contain osteoclast progenitors or precursor, and these cells differentiate into TRAP-positive precursors and terminally fuse each other to form mature and functional osteoclast-like cells. Also, the medullary bone marrow cells at the bone resorptive phase contain many osteoclast progenitors or precursors, suggesting that medullary bone marrow cells at the bone resorptive phase have high potential to form osteoclasts