Abstract. A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels forpro al(I) and pro ~t2 collagen and total collagen synthesis increased 1.2-and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to ~t chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times.The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase.These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.T HE processes by which mineralization is initiated and regulated in bone and other vertebrate calcifying tissues are incompletely understood. It is known, however, that a prerequisite for mineralization is the synthesis and assembly of an extracellular matrix into which mineral may be deposited. Collagen type I has been shown to be the major extracellular matrix protein of bone. It comprises between 60-70% of its organic components and between 20-30% of its total dry mass (20). Physiologically, type I collagen provides the protein basis for the architecture of bone and the scaffold into which mineral is accumulated (5,20,41). The importance of collagen type I in maintaining structural integrity and proper mineralization of bone has been demonstrated for one form of inherited osteogenesis imperfecta. A specific frame shift m...

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Chondrocytes, derived from a tissue that remains as permanent hyaline cartilage in vivo (embryonic chicken caudal sterna) were treated with 10(-8) to 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. These nonadherent rounded chondrocytes acquired an adherent, polygonal morphology in a dose-dependent fashion with 1,25(OH)2D3 treatment. During the first 4 days of 1,25(OH)2D3 treatment cell flattening was associated with a 10-fold increase in beta-actin and fibronectin and their corresponding messenger RNAs (mRNAs). After adherence over the 12 days of continuous hormone treatment, a 2- to 4-fold increase in DNA synthesis and DNA accumulation were observed for the highest hormone dose (10(-8) M). Over the same time course total collagen synthesis decreased 35-50% primarily due to decreased type II collagen synthesis, which accompanied comparable decreases in its mRNA. In contrast, both alpha 1(I) and alpha 2(I) showed a continuous 5- to 10-fold increase; however, type I collagen protein synthesis remained undetectable, indicating translational control of the type I collagen synthesis. alpha 1(X) mRNAs showed a 2- 3-fold increase after 12 days of hormone treatment, and its polypeptide was clearly detected by sodium dodecyl sulfate polyacrylamide gel analysis. Type IX collagen synthesis showed a 2-fold increase in synthesis and its mRNA levels during the first 4 days of 1,25(OH)2D3 treatment but thereafter had levels comparable to control cultures. Analysis of proteoglycan synthesis and core protein mRNA levels showed there was a 2-fold increase in core protein mRNAs while proteoglycan synthesis, as assessed by 35S incorporation, showed only a 10-20% increase. Direct hormone effects vs. those secondary to altered cellular morphology were determined by blocking cell adherence by growth of the 1,25(OH)2D3-treated cultures on bacteriological petri dishes. All of the observed effects on cytoskeletal and collagen mRNAs were blocked except the elevations observed in proteoglycan core protein and alpha 1(IX) mRNAs. DNA contents in hormone-treated cultures also remained elevated. These results suggest that 1,25(OH)2D3 both activates and suppresses specific genes, promoting chondrocyte maturation toward a more hypertrophic phenotype. However, prevention of the initial morphological alterations that are induced by 1,25(OH)2D3 blocks many of the subsequent changes in connective tissue expression.