Collagen Formation by Fibroblasts of the Chick Embryo Dermis

Abstract

This investigation has sought to determine the relation between collagen fiber and fibroblast during fibrogenesis. Toward this end the surfaces of chick fibroblasts grown under in vitro conditions have been examined with the electron microscope after fixation in Os04. Supplementary information has been obtained from thin sections of fibroblasts fixed in situ during phases of fiber production.The evidence provided by these studies and by various conditions of the experiments indicates that the unit fibrils of collagen form in close association with the cell surface. They were never observed within the cell. When these unit fibrils form in bundles it appears as though templates of scme nature, possibly coinciding with stress fibers within the cell cortex, influence the polymerization of the fibrils out of material available at the cell surface. From here the fibrils and bundles of them are shed into the intercellular spaces and there grow to limited diameters by accretion of materials from the general mi]ieu. I N T R O D U C T I O NAlthough the origin and formation of collagen fibers has been studied and debated for over a century, reports of observations and conclusions, even up to the present time, are contradictory and inconclusive. As recently as 1954 Klemperer (15), drawing on years of observation and study in this field, concludes that "the relationship between cell and intercellular substance of the connective tissue...is just as controversial (a question) as it was" in the middle of the 19th century.To review again the history of investigations into this controversial problem and its several ramifications seems unnecessary for the purposes of this report. Besides the brief but excellent treatment of the historical aspects in Klemperer's article, there is a summary in Cameron's Pathology of the Cell (5) and literature reviews in papers by M. Lewis (17), M. Stearns (29, 30), F. K. Studnicka (31), and F. Wassermann (33), to mention only a few. The broad problem is one of defining * Supported during the period of this investigation by a grant from the Eli Lilly Research Laboratories, Indianapolis.:~ Present address: Anatomy Department, College of Physicians and Surgeons, Columbia University, New York. the role of the fibroblast in collagen production. A subdivision of this, which is more especially the concern of experiments reported here, is the site of initial fiber formation. Do the fibers form apart from the cell in the ground substance of the intercellular spaces as proposed by Maximow (19), Nageotte (21), Doljanski and Roulet (7); within the cell to be later shed, as M. Lewis (17) proposed years ago; or are they formed at the cell surface, which is to say at the interphase between cell cortex or ectoplasm and the extracellular ground substance (Studnicka (31))?Interest in the question derives from a number . of sources. We are, primarily, interested in the mechanisms involved in the biogenesis of extracellular coverings and matrices, of which collagen plus ground substance constitute an important...

Morphological and Chemical Studies of Collagen Formation

“…The fixative was 1 per cent ice-cold osmium tetroxide solution, veronal acetate-buffered at pH 7.4, the osmolar concentration being adjusted to isotonicity by the addition of NaCI. A small concentration of calcium ions was present in the fixing solution (Palade,16;Porter and Pappas,19); the calcium, in the form of chloride, was added to the fixative to give a final concentration of 0.02 per cent. Fixation was carried out for 1 hour.…”

“…The mechanism in the living tissue is unlikely to be as simple as this. The observations of Porter and Pappas (19) on collagen formation in tissue-cultured fibroblasts indicate that the cell not only is involved in producing the fibril, but is to some extent responsible for its initial orientation. These authors suggest that the unit fibrils of collagen form in close association with the cell surface and, from here, are shed by the cells into the extracellular phase.…”

Fixation of Neural Tissues for Electron Microscopy by Perfusion With Solutions of Osmium Tetroxide

“…In 1956 Pappas (31) noted that 0.01 per cent CaCI~ in the fixative improved the preservation of Amoeba proteus for electron microscopy. Porter and Pappas (33) reported that 0.001 per cent CaC12 was necessary for successful fixation of chick embryo skin (previously disclosed in Palade's review of fixing procedures (26)). Off and on since 1953 the senior author has added calcium chloride to solutions of osmium tetroxide without noticing any significant difference in the preservation of membranes at the electron microscopic level.…”

Fine Structure of the Reticular Cells in the Rat Spleen, with Special Reference to Their Fibro-Muscular Features

“…Either hypothesis lacks direct proof, but it is agreed that those cells which have a large amout of granular endoplasmic reticulum and a prominent Golgi complex represent the main source of fibrous proteins (PORTER and PAPPAS, 1959;KARRER, 1960;GOLDBERG and GREEN, 1964;ROSS andBENDITT, 1964, 1965).…”