Observations With the Electron Microscope on the Solvation and Reconstitution of Collagen

Abstract

Electron microscope studies of collagen have dealt, for the most part, with the morphology of the mature or completely formed fibers.' It has been shown that fibers from a variety of sources have the same characteristic periodic structure which consists of regularly spaced bands of greater density to electrons, arranged crosswise to the long axis of the fibers. The combination of a dense (A band) and a light region (B band), which constitutes a single macroperiod, measures about 640 A (1, 2). Metal shadowing reveals that, in the dried fiber, the dense bands represent elevations in the contour and therefore regions containing greater amounts of non-volatile material, whereas the fight bands coincide with depressions (3). Studies at higher resolution reveal further complexity. The more opaque band is found to have two component striae and the more transparent band or depression, a single striation. The number of these intraperiod striations is usually greater in fibers "stained" with phosphotungstic acid but it may be that these are phases or regions within the organization that specifically fix the tungsten compound (4). In any case the over-all picture is one of extremely precise and complicated organization and this poses such questions of morphogenesis as (a) what is the form of the fundamental, possibly macromolecular, units which make up the fibers, and (b) how do these units combine to give the now familiar, striated fiber.Some information relative to these problems was obtained from a study of the natural formation of collagen as it occurs in cultures of collagen-containing tissues (5). It was observed that the smaller natural fibers start out with bands of more or less uniform size, spaced at 210 to 270 A, and that only as these fibers grow to greater diameters do their striae acquire the inequalities which account for the macroperiod of 640 A. However, the conditions of this study did not lend themselves to a demonstration of the means by which the fibers grew in size. While there was some indication that the narrower fibers fused to

Collagen Formation by Fibroblasts of the Chick Embryo Dermis

“…If collagen were present it should, on the basis of previous experience, precipitate out under these conditions (32), but again the results were negative.…”

“…In recent years, since attention was focused by the studies of Highberger et al (10) and of Vanamee and Porter (32) on fiber formation in the test tube from solutions of collagen (Nageotte (2l)), a 154 COLLAGEN FORMATION BY FIBROBLASTS strong inclination has become evident to relegate control of in vivo fiber production to the inanimate, physicochemical conditions of the intercellular milieu. Thus Gross el aI.…”

The Formation of Fibrils From Collagen Solutions

“…It has been suggested that these filaments aggregate to form the striated fibrils (7,12). Although a constant feature of all the DNA experiments, they were quite different from the linear aggregates resembling branching "filaments" found in the air-dried sample of DNA alone.…”

“…Narrow fibrils and visualization of the tapered fibril ends characterize young skin or actively growing tissue (4)(5)(6). The presence of acute bends is evidence of flaccidity of the fibrils as the suspension settles on the grid during drying (7). As all the replicated precipitates described here were satisfactory, uncontaminated preparations consisting almost entirely of collagen bearing 640 A striations, with hardly any debris and no elastin-like structures, amorphous material or buffer salts, these characteristics at each time interval could be easily established.…”

The Behavior of Collagen Units as a Model in Morphogenesis

“…An exciting development was the revelation by electron microscopy that the reconstituted fibrils had the same precisely ordered morphology as did the native fibrils, namely the repeating axial period of about 640 A plus the detailed fine structure (1,21,38,42,51,52). The two aspects of the problem which have engaged the attention of most investigators in this area are the characterization of the molecule and elucidation of the mechanism of in vitro fibrogenesis with the hope of relating it to the in vivo physiological process.…”