Incorporation of In Vitro Formed 4D-Staggered Linear Polymers of Collagen into D-Periodic Fibrils

Abstract

We have previously demonstrated in vitro formation of acid-labile, unidirectional, and 4D-staggered linear polymers of collagen molecules at low ionic strength, low temperature, and pH 4.5. The possibility was investigated in this paper whether the 4D-staggered linear polymers thus formed could be incorporated into native-type, D-periodic collagen fibrils. For this purpose, acid-labile, 4D-staggered linear polymers of collagen were formed in vitro by dialysing monomeric collagen solution against acetate buffer, pH 4.5, and the 4D-staggered association was chemically fixed with glutaraldehyde. The 4D-staggered linear polymers gave fibrous, or end-overlapped segment-long-spacing (SLS) forms when dialysed against ATP/acetic acid solution. 4D-Periodic SLS forms were dialysed against 0.14 M NaCl-30 mM phosphate buffer (pH 7.4) at 37°C. Samples were taken at intervals and examined under an electron microscope. After 4D-periodic SLS being loosened, D-periodic fine fibril structure appeared within 4 hours of dialysis. After longer incubation, the preference of 4D-periodicity completely disappeared and D-periodic polar fibrils of collagen were formed with negligible fine filaments in the background.It is concluded that in vitro formed 4D-staggered linear polymers can be incorporated into D-periodic collagen fibrils under physiological conditions.