Structural and chemical characteristics and maturation of the calcium‐phosphate crystals formed during the calcification of the organic matrix synthesized by chicken osteoblasts in cell culture

The calcium‐phosphate (Ca‐P) crystals formed in the extracellular organic matrix synthesized by chicken osteoblasts in cell culture were examined after 30, 40, and 60 days of culture by a number of physical and chemical techniques including chemical analyses, X‐ray diffraction, transmission electron microscopy of isolated crystals, and resolution‐enhanced Fourier transform infrared (FTIR) spectroscopy. The data reveal that the solid inorganic calcium‐phosphate phase consists of a very poorly crystalline apatite, having a low carbonate content and containing acid phosphate groups. The chemical and structural characteristics are generally similar to the apatite crystals found in young newly synthesized bone but there were small but significant differences found. The major significant differences noted were the rate at which maturational changes occurred in the crystals formed in cell culture compared with those noted in vivo and in synthetic carbonate apatite crystals equilibrated with the same cell culture medium, and the persistence of labile groups, especially HPO4−2 ions during a relatively long period of incubation. Despite extensive chemical efforts to degrade the organic constituents and to disperse the individual crystals isolated from the organic matrix constituents, a large proportion of the crystals were found to be organized in both loosely and densely packed relatively large roughly spherical aggregates. A few of the aggregates were organized in the form of fibrils with the crystals oriented with their c‐axes roughly parallel to the long axes of the crystal aggregate. With briefer periods of chemical treatment, larger aggregates of crystals were occasionally observed in which there was a distinct axial periodicity of approximately 70 nm. In such collagen‐crystal fragments, the crystals were well‐oriented with their c‐axis roughly parallel to the long axes of the aggregate similar to the organization and relationships between crystals and collagen fibrils in native bone. Isolated crystals were in the shape of thin plates. At the end of 30 days of culture, many of the crystals were clearly larger than those observed in native chick bone, except for those in the very youngest (7‐ to 8‐day‐old) embryos. At the end of 40 and 60 days of culture, the crystal habit remained as thin plates but the crystals were predominantly smaller, similar to those found in older embryo and postnatal chicken bone. The marked tendency of the crystals to form relatively large aggregates that resist dispersion by techniques that readily disperse the crystals of bone, and the presence of a significant number of larger crystals has also been observed in studies of calcified cartilage. Resolution enhanced FTIR spectroscopy revealed the presence of a high concentration of labile phosphate groups, especially after 30 days of culture and just after the plateau of mineralization is reached. The concentration of the labile phosphate groups decreases very slowly with further increase in the time of culture consistent with a very modest process characteristic of the maturation of apatite crystals in vivo and of synthetic carbonate apatite crystals in vitro. Carbonate ions were found to be preferentially incorporated into phosphate sites as they do in native bone material (type B apatite). However, unlike the bone crystals formed in vivo, and the synthetic carbonate apatite crystals equilibrated in vitro, the immature apatite crystals deposited in this in vitro cell culture system are stabilized for a relatively long time in an “immature” form and mature only very slowly when compared with native bone mineral in vivo or small synthetic carbonate apatite crystals incubated in the same cell culture media.