Avian medullary bone in organ culture: Effects of vitamin D metabolites on collagen synthesis

Summary A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of³H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10–15% of the total protein labeled. The addition of 1,25-dihydroxycholecalciferol (1,25 (OH)₂D₃) resulted in a dose-dependent inhibition of³H-proline incorporation into CDP at doses from 10⁻¹⁰M to 10⁻⁷M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since³H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)₂D₃-induced decrease in the³H-hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)₂D₃ is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaiored incorporation of collagen into bone matrix, or bone resorption Medullary bone collagen synthesis was not affected by 24,25(OH)₂D₃, either alone or in combination with 1,25(OH)₂D₃. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)₂D₃ and 1,24,25(OH)₃D₃ were the most potent metabolites tested, followed by 1-alpha(OH)D₃. 25(OH)D₃ and 24,25(OH)₂D₃ had no effect at concentrations as high as 10⁻⁷M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as anin vitro model for the study of bone formation