Dose-related effects of 1,25-dihydroxyvitamin D3 on growth, modeling, and morphology of fetal mouse metatarsals cultured in serum-free medium.

Abstract

A serum-free, fetal bone organ culture model that permits the simultaneous determination of modeling and growth parameters was used to examine the effects of a near physiologic and a pharmacologic dose of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. The fetuses of pregnant mice were removed on day 17 of gestation, and three medial metatarsal rudiments were cleaned and after preculturing were cultured as pair-matched groups for 4 days in MEM supplemented with 0.2% BSA. 1,25-(OH)2D3 was added to the cultures at concentrations of 10(-12) or 10(-6) M. Cultures treated with the carrier and devitalized bones served as controls. For resorption studies, pregnant mice were given 45Ca on day 17 of pregnancy and fetal metatarsals harvested 24 h later. Resorption was determined by the amount of 45Ca released into the media. DNA synthesis was estimated by determining the incorporation of [3H]thymidine, collagen synthesis by measuring the incorporation of [3H]proline, mineralization by the incorporation of 45Ca, and proteoglycan synthesis by the incorporation of 35S. The amount of radiolabel was detected in media, as well as in noncultured, dead, and cultured rudiments. The total length of the rudiments and length of the calcified diaphyses were measured daily. In addition, rudiments from all experimental groups were prepared for light and electron microscopy. The high dose (10(-6) M) of 1,25-(OH)2D3 suppressed total rudiment growth but not the growth of the calcified diaphysis, 1,25-(OH)2D3 also decreased DNA, collagen, and proteoglycan synthesis, reduced calcification, and increased bone resorption in a dose-related manner. There were morphologic and ultrastructural changes in the osseous tissues and cells, particularly with the high dose of vitamin D, that supported the biochemical findings of suppressed activity of the osteogenic and chondrogenic cells. However, the suppression of collagen production and bone cell proliferation observed with the pharmacologic dose of vitamin D may be partially attributable to the decrease in bone mass (from increased resorption), thus resulting in less osseous tissue surface for these events to occur as endochondral osteogenesis progressed. The lower dose of vitamin D, however, had effects on 35S and 45Ca incorporation that could not be attributed to a decreased osseous tissue mass. This study emphasizes the importance of measuring specialized activities of the various cell populations in bone rudiment culture models to more fully understand the changes in tissue metabolism that result in changes in rudiment growth and modeling.