Post-translational control of collagen fibrillogenesis in mineralizing cultures of chick osteoblasts

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Abstract
Cultured osteoblasts from chick embryo calvaria were used as a model system to investigate the post-translational extracellular mechanisms controlling the macroassembly of collagen fibrils. The results of these studies demonstrated that cultured osteoblasts secreted a collagenous extracellular matrix that assembled and mineralized in a defined temporal and spatial sequence. The assembly of collagen occurred in a polarized fashion, such that successive orthogonal arrays of fibrils formed between successive cell layers proceeding from the culture surface toward the media. Mineralization followed in the same manner, being observed first in the deepest and oldest fibril layers. Collagen fibrillogenesis, the kinetics of cross-link formation, and collagen stability in the extracellular matrix of the cultures were examined over a 30 day culture period. Between days 8 and 12 in culture, collagen fibril diameters increased from < 30 nm to an average of 30–45 nm. Thereafter, diameters ranged in size from 20 to 200 nm. Quantitation of the collagen cross-linking residues, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), showed that these mature cross-links increased from undetectable levels to concentrations found in normal chick bone. Analysis of the kinetics of their formation by pulse-chase labeling the cultures with [3H]lysine showed a doubling time of ˜ 5 days. The relationships between cross-link formation, fibrillogenesis, and collagen stability were examined in cultures treated with β-aminopropionitrile (β-APN), a potent inhibitor of lysyl oxidase and cross-link formation. In β-APN-treated cultures, total collagen synthesis was increased twofold, with no change in mRNA levels for type I collagen, whereas the amount of collagen accumulated in the cell layer was decreased by 50% and mineral deposition was reduced. The rate of collagen retention in the matrix was assessed by pulse-chase analysis of [3H]proline over a 16 day period in control and β-APN-treated cultures. In control cultures, about 20% of the labeled collagen was lost from the cell layers over a 16 day period compared with > 80% in the presence of β-APN. The β-APN-treated cultures also showed a wider diversity of fibril diameters with a median in the > 45–60 nm range. In summary, these data suggest that cross-linking and assembly of collagen fibrils secreted by osteoblasts in vitro occur in a fashion similar to that found in vivo. The rate of cross-link formation is relatively constant and may be correlated with increasing collagen mass. With progressive cross-linking, collagen fibrils become more stable and less susceptible to loss from the cell layer. Thus, collagen deposition is probably controlled at several post-translational stages during fibrillogenesis, including cross-link formation, lateral fibril aggregation, and increasing fibril stability.
Funding Information
  • NIH (HD22400, AR34078, AR34081, AR-41452)
  • NASA (NAG 2-538)