The development of fetal mice long bones in vitro: An assay of bone modeling

This study was carried out to develop an in vitro system for the analysis of bone modeling (coincidentally occurring bone growth, formation, mineralization, and resorption) as is seen during bone development. The fetuses of pregnant mice previously labeled with ⁴⁵Ca were removed on Days 15,16, and 17 of gestation. The radii and ulnae were dissected free and cultured for up to 6 days in a chemically defined medium (BGJ) supplemented with fetal calf serum or human serum albumin and 150 μg/ml vitamin C. The change in bone length over the culture period was measured as were the changes in calcium and phosporus content, the hydroxyproline: protein ratio, and the percent ⁴⁵Ca released into the medium. The effect of insulin and parathyroid extract on the system was also examined. The results indicate that cultures of 16-dayold fetal bones provided the most suitable model. During culture there was a continuous increase in bone length as well as calcium and phosphorus content in the ratio of 2:1, a significant increase in the hydroxyproline content, and a continuous release of ⁴⁵Ca into the medium. Parathyroid extract caused a dose-dependent inhibition of both growth in diaphyseal length and calcium and phosphorus uptake with an increase in ⁴⁵Ca release into the medium. Insulin at 10^-9M and 10^-10M resulted in a significant increase in diaphyseal length and calcium and phosphorus uptake without affecting ⁴⁵Ca release. These results indicate that the assay described is suitable for the study of bone modeling, providing a means to measure bone growth, formation, calcification, and resorption. The direct effect of various factors on bone modeling can also be measured.