Synchrotron radiation x-ray scattering in the early stages of in vitro collagen fibril formation.

The time course of in vitro collagen fibril formation was monitored by synchrotron radiation x-ray scattering. Collagen polymerization was induced by a temperature jump from 4 degrees C to 32 degrees C and the solution scattering pattern was recorded continuously with a time resolution of a few seconds. The scattered intensity increased as soon as the final temperature was attained, without discernible lag phase, when the collagen concentration was about 0.56 mg/ml, whereas turbidimetric measurements revealed a lag phase of ca. 2.5 min at 1.05 mg/ml. A direct correlation was found between the temperature and both the rate of formation and the total amount of early aggregates. The formation of these aggregates was only partially reversed by lowering the temperature, except when the collagen had been prevented from forming Schiff base-mediated intermolecular crosslinks by sodium borohydride reduction. In this case, formation of aggregates was completely reversible. We conclude that the aggregates that appear to correspond to the subfibrils proposed on the basis of independent methods are formed and simultaneously crosslinked in the early phases of in vitro collagen self-assembly.