S T U D I E S ON C A R T I L A G E II. Electron Microscope Observations on Rabbit Ear Cartilage following the Administration of Papain

Electron microscope observations on rabbit ear cartilage following the administration of papain show that both the elastic component of the matrix and the amorphous material disappear leaving a matrix which consists of delicate fibrils which are presumed to be collagen. This unmasking of fibrils coincides with the appearance of an abnormal component in the electrophoretic pattern of the rabbit 's serum. The chondrocytes show vacuoles in their cytoplasm which appear at the same time that the cells appear crenated in the light microscope. A ruffly appearance of the cell surface membrane coincides with this vacuolization, and vacuoles often appear open and in continuity with the extracellular space. The resurgence of the rabbit ear is accompanied by a reconstitution of both the amorphous material and the elastic component of the matrix. During this period numerous dilated cisternae of the endoplasmic reticulum which contain a moderately dense material are present in the chondrocyte cytoplasm. We have been unable to demonstrate a direct relationship between the elastic component of the matrix and a particular component of the chondrocyte cytoplasm, but it is clear that changes occur in the cartilage cell cytoplasm during both the depletion and reconstitution of the matrix. Previous studies on the effect of papain on elastic tissue are noted and the possible relationships between changes in the cells and matrix of this elastic cartilage are discussed. The phenomenon of collapse of the ears of young rabbits after papain is administered intravenously was observed by Thomas (1) who reported the reversibility of this phenomenon. He described the appearance of auricular and other cartilage in the light microscope during the phase of collapse, and commented on a loss of basophilia in the cartilage matrix. Further studies on this phenomenon have been reported by Spicer and Bryant (2, 3), McCluskey and Thomas (4), Tsahas (5), and Bryant, Leder, and Stetten (6), who describe the appearance of an abnormal acid component in the electrophoretic pattern of serum after the administration of papain. The present authors thought that this phenomenon of collapse and recovery might prove suitable for an electron microscope This investigation was supported by research grant A-706-C from the United States Public Health Service, National Institute of Arthritis and Metabolic Diseases. Dr. Sheldon is a Markle Scholar in Medical Science, Department of Pathology, McGill University School of Medicine, Montreal, Canada. Received for publication, December 29, 1959. 151 on O cber 0, 2017 jcb.rress.org D ow nladed fom study of the relationship between the matrix and the cellular constituents of cartilage. This communication reports electron microscope observations on rabbit ear cartilage taken at intervals from 30 minutes to 48 hours after the administration of papain. An electron microscope study of the ear cartilage of normal rabbits has been reported previously (7). M A T E R I A L S A N D M E T I t O D S One cc. of 1 per cent solution of crude papain 1 was administered intravenously to 12 young rabbits each of which weighed less than 1000 grams. Specimens of ear cartilage were removed 30 minutes, 2, 4, 8, 12, 18, 24, and 48 hours later. To avoid the effects of trauma no more than 2 interval biopsies were performed on any one ear. The tissue was examined with the light and electron microscope. Tissue for light microscopy was fixed in 10 per cent neutral formalin. Tissue for electron microscopy was prepared in the same manner as in the study on normal rabbit ear cartilage (7). Thin sections were cut either on a Sj6strand or Porter-Blum nficrotome with a glass knife and were examined in either an RCA EMU-2B or 3C electron microscope. O B S E R V A T I O N S In the electron microscope study of the ear cartilage of normal rabbi t the matrix is described as consisting of two components: a uniformly distributed moderately dense substance which appears as a fine meshwork without any particular pat tern extending from cartilage cell border to cartilage cell border; and a second component, a three-dimensional network of more dense material, which is best described as "felt-like," lying midway t Papain obtained from Delta Chemical Works, Inc., New York. between the cells. Since the characteristic difference between this "elastic" cartilage and cartilage from the epiphyseal plate (8) is the presence of this "felt-like" material, for the sake of brevity, this material will be referred to subsequently as the elastic component. The normal chondrocyte (vtoplasm shows, in addition to mitochondria, lipid spherules, glycogen, and components of the endoplasmic reticulum, a material which had not been described previously in light microscope studies and which appears as a relatively dense felt-work (7). The relationship in normal elastic cartilage between this cytoplasmic material and the elaslic component of the matrix is not clear. The observations in the present communicat ion are correlated with light microscope observations and with the electron microscope study of the ear cartilage of normal rabbits. Thirty minutes after papain is injected, before any changes in the normal posture of the rabbit ear are apparent , there is an evident increase in the eosinophilia of the cartilage matrix when sections are stained with hematoxylin and eosin. At this early time, in areas where the extracellular matrix normally shows an amorphous line meshwork, the electron microscope now reveals abundan t delicate fibrils (Fig. 1). In areas where the elastic component normally lies, one sees this material, but each large fibril of it appears surrounded by a clear halo (Fig. 1). The chondrocytes showno significant changes in the appearance of their cell surface membrane, cytoplasm, or nucleus. Two hours after papain has been given, the matrix continues to show fibrils, and we have the impression that the elastic component may have diminished in amount. The cytoplasm of many chondrocytes shows numerous vacuoles (Fig. 3) which are not present in normal cartilage cells. In sections from cells which contain many vacuoles