Studies on Cartilage: Electron Microscope Observations on Normal Rabbit Ear Cartilage*

BY HUNTINGTON SHELDON, M.D.~ AND ROBERT g. ROBINSON, M.D.

W I T H THE TECHNICAL ASSISTANCE OF ULLA F . AFZELIUS

(From The Johns Hopkins University School of Medicine, Department of Orthopaedic Surgery, Baltimore)

PLATES 213 TO 216

(Received for publ icat ion, Feb rua ry 3, 1958)

ABSTRACT

Normal rabbit ear cartilage studied with the light and electron microscope shows chondrocytes in which large lipide spherules, and an abundance of glycogen, a few small mitochondria, and relatively few elements of the endoplasmic reticulum can be identified. The chondrocytes contain, in addition, a material which stains strongly with acid fuchsin and appears in the electron microscope as a relatively dense JFelt-work. In electron micrographs, 1he matrix of normal rabbit ear cartilage consists of two components: a uniformly distributed moderately dense substance which appears as a fine meshwork without any particular pattern extending from cartilage cell border to cartilage cell border; and a three-dimensional anas- tomotic network of more dense material, which is best described as "felt-like" lying between the cells. The similarity between the felt-like material of the matrix and the elastic fibers described in previous electron microscope obser- vations is discussed.

Observat ions on the fine s t ructure of cartilage from the epiphyseal appara tus (1, 2) and from ar t icular surfaces (3) have been reported previ- ously. Since r abb i t ear cartilage ostensibly serves a different purpose, i t is of interest to determine whether this difference in funct ion is reflected in a difference in structure. The purpose of this com- munica t ion is to report a correlated l ight and electron microscope s tudy on r abb i t ear cartilage, pre l iminary to subsequent descriptions of experi- menta l ly induced changes in the cells and mat r ix of this type of cartilage. In addit ion, a mater ial was found within these cartilage cells which, to our knowledge, has no t been reported before in o ther cells studied wi th the electron microscope.

Materials and Methods

Ear cartilage was obtained by biopsy from young rabbits which weighed less than 1000 grams. For elec-

* This investigation was supported by research grant A-706-C from the United States Public Health Service, National Institute of Arthritis and Metabolic Diseases.

~: Instructor, Department of Pathology.

tron microscopy the cartilage was cut into 0.5 milli- meter square pieces and fixed for 45 minutes in 1 per cent buffered (pH 7.4) isotonic osmium tetroxide at approximately 0°C. (19). For light microscopy some specimens were fixed in osmium tetroxide, some in 30 per cent alcohol, and some in 10 per cent neutral for- malin. For vital staining, specimens were cut free hand and immersed for 30 seconds in 0.86 per cent saline which contained 0.1 per cent tolnidine blue. They were then rinsed and kept in Ringer's solution for periods up to 3 hours while under observation. Frozen sections for tat stains were cut from tissue fixed 1 hour in formalin.

The tissue for electron microscopy was routinely dehydrated in graded alcohols at room temperature and embedded in partially polymerized methacrylate. Thin sections for electron microscopy were cut with a glass knife, on a SjSstrand (4) microtome and examined in a RCA EMU 2B microscope. For light microscopy sections were cut from tissue that had been dehydrated in alcohols and embedded in paraffin.

A variety of staining techniques was used to eluci- date the light microscope image of the cells and matrix of this cartilage. These included hematoxylin and eosin, periodic acid-Schiff's reagent (5, 6), alcoholic and aque- ous solutions of toluidine blue (7), Verhoeff-Van Gieson (8), orcein (9), aldehyde fuchsin (10), and a modifica-

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402 STUDIES ON CARTILAGE

tion of Altmann's acid fuchsin (11). Acetic-carbol- Sudan I I I was used to stain for fat (12).

OBSERVATIONS

The electron and light microscope observations will be described in the order in which they were made; perhaps in that way, the correlative and complementary nature of the two techniques is most easily seen.

Cells:

The chondroeytes in hematoxylin and eosin- stained sections appear in the light microscope image as ellipsoids of various sizes in which a moder- ately densely stained nucleus is eccentrically placed (Fig. 1). Large vacuoles are prominent within the chondrocyte cytoplasm and little cytoplasmic de- tail is apparent. Interlacing, anastomosing "fibers" lie between the cells, yet each cell appears to be immediately surrounded by material which does not contain these fibers. This region in the matrix in which the chondrocyte lies stains only lightly, and many cells are seen to be separated from the tightly stained matrix by a small space which does not stain at all. I t is this appearance of the areas immediately around the chondrocytes that has provided the excuse for the introduction of an abundance of descriptive terms such as capsule, lacuna, and interterritorial substance (13). The picture of osmium tetroxide--fixed specimens em- bedded, sectioned, and stained in an identical way differed in two respects from the formalin- or alcohol-fixed specimens. After osmium fixation the small clear space between the chondrocyte and the pale matrix is seldom seen, and instead of large, empty intracytoplasmic vacuoles, there are one or more black dense spherules within the chondro- cytes.

Electron microscopy of thin sections of osmium tetroxide-fixed material showed that the large dense spherules occupy a prominent place in the cytoplasm (Fig. 5). This prompted us to attempt to characterize the spherules by means of polarized light and fat stains. Their appearance in living cells is depicted in Fig. 2. Polarized light observations on free hand sections immersed in 0.86 per cent saline failed to show any birefringent quality to the spherules, but the matrix revealed an intensely anisotropic network. The Sudan stain on frozen sections shows that the spherules have a marked sudanophilia and so are identified as lipide, (Fig. 3). Observed at higher magnifications, the periphery of each lipide spherule is found to possess

a rim of very small dense particles which seem to be embedded in the cytoplasmic border of the spherule (Fig. 6). The bulk of the dense material of the vacuole appears separated from this rim by a clear space that is presumed to be a shrinkage artifact.

A second component of these chondrocytes ap- pears in the form of a cloudy, moderately dense, amorphous material, dispersed randomly through- out the cytoplasm. This is most clearly seen in the chondrocyte in Fig. 5 in which, apart from the nucleus and the lipide spherule, there is little else to be seen. I t closely corresponds to Fawcett 's description (14) of glycogen within the liver cells. In the light microscope the PAS technique applied to osmium tetroxide-, or alcohol-, or formalin- fixed tissue shows an abundance of PAS material randomly distributed within the cells (Fig. 4). The intense PAS staining of the chondrocytes disappeared after treatment with salivary amylase, leaving only a small amount of intracellular PAS- positive materialJ

A third recognizable component within the chondrocyte cytoplasm is the endoplasmic reticu- lum, a few elements of which are scattered about in the cytoplasm and do not appear preferentially oriented as in the cytoplasm of pancreatic exocrine cells (15, 16) or osteoblasts (17). Small mitochon- dria with characteristic double limiting membranes and transverse membranes or cristae are recogniz- able (Fig. 7), but are not so abundant as in kidney tubule cells (18) or intestinal epithelia (19). The nucleus is, of course, easily recognized, and many cells are observed to have two nuclei within the cytoplasm.

In addition, there is another component within the cartilage cell cytoplasm which appears in the electron microscope as much more dense than the cloudy, amorphous material, and looks somewhat like a felt-work of fine fibrils. However, with the present resolution it could not be determined whether this material is truly fibrillar in nature. This element of the cytoplasm appears different from any hitherto described in the literature and may be peculiar to so called "elastic" material in

1 The PAS staining of the chondrocyte cytoplasm, the disappearance of this staining quality following amylase treatment, and the appearance in the elec- tron micrographs of the cloudy moderately dense amorphous material without particular orientation all indicate that these cells contain a large amount of gly- cogen.

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HUNTINGTON SHELDON AND ROBERT A. ROBINSON 403

the chondrocyte. This felt-like material is most often seen in close topographic relationship to the lipide spherules, and in some areas (Fig. 8) the smallest recognizable particles of the lipide appear to lie within the felt-like material.

Matrix:

The chondrocyte-matrix relationship as de- picted by the electron microscope appears different from what has been described in the light micro- scope studies, a difference due to the better re- solving power of the former rather than to any difference in preparatory techniques. The major proportion of the matrix appears in the electron micrographs as a moderately dense substance of which one component has the form of delicate, fine meshwork without any particular pattern. This appearance (Figs. 5 to 8) of the major part of the matrix is somewhat similar to earlier electron microscope descriptions of cartilaginous matrices (1 to 3). The meshwork may represent an arti- ficial condensation of a portion of the substance which in the living state is homogeneously distrib- uted. I t is important to remark that at the achieved resolutions no periodicity is observed in this material, nor have any recognizable collagen fibrils ~ been seen by us in this matrix. This matrix material with its delicate meshwork extends right up to the cell membrane of the chondrocytes (Figures 5 to 7), and there is no suggestion of an empty space between the cell and the matrix except in specimens which have obviously under- gone severe changes in preparation for microscopy.

In addition to this predominant and more or less homogenous material, there is a more dense component in the matrix of the normal cartilage of the rabbit ear. I t appears to lie approximately midway between the chondrocytes and forms what can best be described as a three-dimensional web or latticework within which the chondrocytes are situated. However, this web or latticework is not in direct continuity With the chondrocytes so far as can be seen. The distribution of this more dense material is seen at the arrows in Figs. 5 and 8, and its appearance at higher magnification is shown in Fig. 7. This material has no obvious structure at this resolution, suggesting that its structural sub- units remain to be resolved. The similarity of the

Collagen fibrils are defined as fibrils of indeter- minate length which show a characteristic periodicity within which sub-bandings are seen (20).

density and general appearance of this felt-like material in the matrix and the felt-like material within the chondrocytes mentioned above is striking.

An examination of routine hematoxylin and eosin stain preparations with the light microscope shows a network of large refractile fibers in the matrix which are distributed between the chondrocytes, but always keep their distance from the chondro- cytes in such a manner that they create the illusion of a capsule around the cells (Figures 1, 3, 4, and 9). As these large fibers are the only component seen in the extracellular matrix with the light microscope, they must correspond to the felt-like network of dense matter which appears to lie in the matrix between the chondrocytes in the elec- tron micrographs. I t remains to identify the nature of the substances which constitute the bulk of the matrix, and the felt-like material of the matrix, and to determine the relationship between this dense felt-like material of the matrix and the similar appearing material Within the chondrocyte.

For many years auricular cartilage has been considered "elastic" cartilage (13). Various stains for elastic tissue were used in this study, but such staining techniques only serve to beg the question of the real nature of the substances which take a positive elastic stain, since there is little informa- tion as to the mode of action of these stains. Hence, substances which stain positively are called elastic fibers. In the present studies Verhoeff-Van Gieson (8), orcein (9), aldehyde fuchsin staining methods (10) were all used, and light microscopy showed that the large matrix fibers stain with these tech- niques. However, none of the structures within the cells could be seen to have taken up these stains. Largely through chance, an acid-fuchsin stain (11) was used. This reveals an intracellular material which is usually distributed in the cell about the periphery of the lipide spherule (Fig. 9) in a manner similar to the felt-like material of the chondrocytes shown in the electron micrographs (Fig. 7). With this acid-fuchsin technique the in- tracellular material but not the lipide spherule is stained magenta (Fig. 9). At the same time the fibers of the matrix appear to take on a similar though fainter purple color.

To recapitulate, this study demonstrates, by correlated light and electron microscopy, that there is an hitherto undescribed substance within the chondrocytes of rabbit ear cartilage. With ap- propriate staining techniques for light and electron

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404 STUDIES ON CARTILAGE

microscopy, this intracellular material appears similar to the fibers seen in the matrix between the chondrocytes. This matrix contains no structures with any obvious periodicity such as are seen in classic descriptions of collagen. Furthermore, there are no "lacunae" about the chondrocytes in rabbit ear cartilage if shrinkage artifacts during prepara- tion for microscopy are assiduously avoided. The illusion of a "capsule" is attributed to the fact that the dense fibers of the matrix are arranged at some distance from the cells, while in the immediate vicinity of the cells there is only a much less dense component of the matrix. Finally, the electron microscopic appearance of many previously recog- nized cellular components (mitochondria, lipide droplets, and glycogen) are described.

DISCUSSION

The observations ~above amplify and extend earlier microscopic observations on this particular type of cartilage. A brief review of some previous microscope studies on cartilage will serve to put the present findings in perspective.

Clark and Clark (21) observed cartilage forma- tion in vivo by means of the ear chamber technique. They remarked on both the presence of lipides 3 within the living cells and on the mode of matrix formation. They stated that under these in vivo conditions cartilage formed only in areas which had been vascularized for some time and had reached a stable condition. The rate of cartilage growth was slow, but the cells, which contained small and larger refractile granules, soon appeared to be separated from one another by an homog- enous matrix. The intracellular granules coalesced until, in the mature state, there might be only a single 15 to 20 micron lipide granule. Some time after the first appearance of the homogenous matrix the vascular supply to the developing cartilage disappeared. The mechanism for this was believed to be the squeezing off of capillaries by the matrix between the chondrocytes. At this time the car- tilage cells appeared to be surrounded by bands 3 to 5 microns in diameter which were referred to as "capsules." No mention of any matrix fibers was made in these light microscope studies.

In the present communication it was dearly evident that the matrix fibers are more difficult to visualize in the living tissue (Fig. 2) than in the

a The first recognition of the presence of lipides in chondrocytes is attributed to Leydig in 1857 (22).

fixed and stained preparation (Figs. 1, 4, and 9). I t might be remarked that this same matrix in freshly cut free hand sections exhibits a very strong birefringent quality under polarized light.

The demonstration of substantial amounts of glycogen in addition to lipide within these cartilage cells confirms earlier observations on the presence of these two substances within chondrocytes. Harris (23) showed that glycogen deposition pre- cedes the appearance of lipide in embryonic chon- drocytes, and Fawcett (24) observed the same sequential relationship between glycogen and fat in developing adipose tissue. In studies of enzy- matic activities in auricular cartilage cells, Mon- tagna (25) demonstrated a high lipase activity, as well as a strong reaction for alkaline phosphatase. Follis and Berthrong (26), using epiphyseal car- tilage, showed little cytochrome oxidase, but some citric acid and succinic dehydrogenase activity in the chondrocytes. I t is difficult to integrate these incomplete histochemical observations on different types of cartilage, and it would be unwise to try to relate them at this time to the morphology of the tissue. Chondrocytes from different types of cartilage may well have different metabolic pat- terns just as they may serve different functions. But the coincidence of large amounts of glycogen and lipide in one cell suggests this particular cell might be a suitable one in which to look for a structural basis for intermediary metabolism at the macromolecular level. For example, we wonder whether the very small dense particles at the cytoplasmic rim of the lipide spherule in Fig. 6 may represent the end product of an intracellular synthesis of lipide. Such very small particles could then coalesce to form the larger lipide spherule which is visible in the light microscope.

We have the impression from examining both acid-fuchsin-stained light microscope preparations and electron micrographs that the dense felt-like material inside the cell is seen most often in juxtaposition to the lipide spherules. In some elec- tron micrographs the smallest visible accumula- tions of lipide appear in the middle of the felt-like material (Fig. 8). The significance of this relation- ship is not understood. We also have the impression that with better resolution a fibrillar structure could be demonstrated in the cytoplasmic felt-like material, while the felt-like material of the matrix appears more homogenous, which suggests the intra- and extracellular materials may represent a structural variation of a basically similar sub-

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HUNTINGTON SHELDON AND ROBERT A. ROBINSON 405

stance. This impression is substantiated by the difference in affinity the intracellular and matrix material show for acid fuchsin. The intracellular material stains somewhat more vividly than the matrix fibers.

The elastic stains for light microscopy suggest that the felt-like matrix material seen in the elec- tron micrographs is elastin, a component of con- nective tissue which is different from collagen and reticulin. Elastic fibers are said to branch and anastomose while collagen fibrils do not. Moreover, there is a significant difference in chemical struc- ture; there is very little hydroxyproline in elastin and elastin has only 7 per cent polar side chains in contrast to 34 per cent polar side chains in col- lagen (27).

Previous electron microscope studies on elastic tissue (largely from beef l igamentum nuchae) re- port substantial ly uniform findings in that they fail to demonstrate a definite fine structure for elastin. Wolpers (28), Gross (29, 30), Bahr (31), and Franchi and De Robertis (32) describe elastin as long branching fibers without any well defined dimension or periodicity. A more recent report on thin sections of the aorta (33) also shows an amorphous material as the predominent component of elastin. The similarity between these previous studies on tissue known to contain elastin and the present observations on the felt-like matrix mate- rial is apparent. I t is conceivable, however, that further studies with improved techniques, might resolve the elastin of the auricular cartilage matrix into its component parts.

In conclusion we would like to suggest that there is considerable circumstantial evidence that in rabbit ear cartilage the felt-like material which is arranged in the matrix as a three-dimensional network between the chondrocytes is elastin. The origin of this material is suggested by the presence of a material with similar density and appearance within the chondrocytes.

REFERENCES

1. Robinson, R. A., and Cameron, D. A., Electron microscopy of cartilage and bone matrix at the distal epiphyseal line of the femur in the new- born infant, J. Biophysic. and Biochem. Cytol., 1956, 2, No. 4, suppl., 253.

2. Scott, B. L., and Pease, D. C., Electron microscopy of the epiphyseal apparatus, Anat. Rec., 1956, 126, 465.

3. Cameron, D. A., and Robinson, R. A., Electron microscopy of epiphyseal and articular cartilage

matrix in the femur of the newborn infant, J. Bone and Joint Surg., 1958, 40, 163.

4. Sjsstrand, F. S., A new microtome for ultrathin sectioning for high resolution electron micros- copy, Experientia, 1953, 9, 114.

5. Hotchkiss, R. D., A microchemical reaction result- ing in the staining of polysaccharide structures in fixed tissue preparations, Arch. Biochem., 1948, 16, 131.

6. McManus, J. F. A., Histological and histochemical uses of periodic acid, Stain Technol., 1948, 23, 99.

7. Glick, D., Techniques of histo- and cytochemistry, New York, Interscience Publishers, 1949, 47.

8. Verhoeff, F. H., Some new staining methods of wide applicability including a rapid differential stain for elastic tissue, J. Am. Med. Assn., 1908, 50, 876.

9. Unna, P. G., Elastin und Elacin, Z. wissensch. Mikr., 1895, 12, 240.

10. Gomori, G., Aldehyde fuchsin: a new stain for elastic tissue, Am. J. Clin. Path., 1950, 20, 665.

11. Bensley, R. R., Studies on the pancreas of the guinea pig, Am. J. Anat., 1911, 12, 297.

12. Jackson, C., An improved method of staining lipids: acetic-carbol-Sudan, Onderstepoort J. Vet. Sc. Animal Ind., 1944, 19, 169.

13. Schaffer, J., Pas Knorpelgewebe, in Handbuch der mikroskopischen Anatomie des Menschen, (W. von M611endorff, editor), Berlin, Verlag yon Julius Springer, 1930, 2, part 2, 210.

14. Fawcett, D. W., Observations on the cytology and electron microscopy of hepatic cells, J. Nat. Can- cer Inst., 1955, 18, suppl. 5, 1475.

15. SjSstrand, F. S., and Hanzon, V., Membrane structures of cytoplasm and mitochondria in exocrine cells of mouse pancreas as revealed by high resolution electron microscopy, Exp. Cell Research, 1954, 7, 393.

16. Palade, G. E., The endoplasmic reticulum, J. Bio- physic, and Biochem. Cytol.. 1956, 2, No. 4, suppl., 85.

17. Sheldon, H., and Robinson, R. A., Electron micro- scope studies of crystal-collagen relationships in bone. IV. The occurrence of crystals within col- lagen fibrils, J. Biophysic. and Biochem. C)tol., 1957, 3, 1011.

18. Rhodin, J., Correlation of ultrastructural organiza- tion and function in normal and experimentally changed proximal convoluted tubule cells of the mouse kidney, Karolinska Institutet, Stockholm, Aktieholaget Godvil, 1954.

19. Zetterqvist, H., The ultrastructural organization of the columnar absorbing cells oi the mouse je- junum, Karolinska Institutet, Stockholm, Aktie- bolaget Godvil, 1956.

20. Bear, R. S., The structure of collagen fibrils, Ad- vances Protein Chem., 1952, 1, 69.

21. Clark, E. R., and Clark, E. L., Microscopic obser-

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vations on new formation of cartilage and bone in the living animal, Am. J. Anat., 1942, 70, 167.

22. Leydig, F., Lehrbuch der Histologie des Menschen und der Thiere, Frankfurt a. M., Verlag yon Meidinger Sohn und Co., 1857.

23. Harris, H. A., Glycogen in cartilage, Nature, 1932, 180, 996.

24. Fawcett, D. W., Histological observations on the relation of insulin to the deposits of glycogen in adipose tissue, Endocrinology, 1948, 42, 454.

25. Montagna, W., Glycogen and lipids in human cartilage with some cytochemical observations on the cartilage of the dog, cat and rabbit, Anat. Rec., 1949, 103, 77.

26. Follis, R. H., and Berthrong, M., Histochemical studies on cartilage and bone. I. The normal pattern, Bull. Johns Hopkins Hosp., 1949, 85, 281.

27. Kendrew, J. C., Structure proteins, in The Pro-

teins, (H. Neurath, and K. Bailey, editors), New York, Academic Press, Inc., 1954, 2, Part B, 946.

28. Wolpers, C., Klin. Woch., 1944, 23, 169, (cited in reference 27).

29. Gross, J., The structure of elastic tissue as studied with the electron microscope, J. Exp. Med., 1949, 89, 699.

30. Gross, J., Fiber formation in trypsinogen solutions: An electron optical study, Proc. Soc. Exp. and Biol. Med., 1951, 78, 241.

31. Bahr, G. F., l~ber die Feinstruktur elastischer Fasern., Z. Anat. u. Entwcklngsgesch., 1951, 116, 134.

32. Franchi, C. M., and De Robertis, E., Electron microscope observations on elastic fibers, Proc. Soc. Exp. Biol. and Med., 1951, 76, 515.

33. Karrer, H. E., Fine structure of the tunica propria in bronchioles of young adult mice [Abstract], 15th Annual Meeting Electron Microscope So- ciety of America, J. Appl. Physics, 1957, 28, 1368.

EXPLANATION OF PLATES

PLATE 213

FIC. 1. Light micrograph of formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained section from normal young rabbit ear cartilage. The chondrocytes contain what appear to be empty vacuoles (arrow) in addition to an eccentrically placed nucleus. The matrix shows anastomosing refractile fibers. Magnification, 300.

Fro. 2. Light micrograph of unfixed free hand section from same animal. This tissue had been briefly immersed in 1 per cent aqueous toluidine blue, and the small dark cytoplasmic bodies represent supravitally stained elements. The lipide spherule appears as an unstained globule. The matrix fibers are barely visible. Magnification, 600.

FIG. 3. Light micrograph of formalin-fixed section from same animal, stained with acetic-carbol-Sudan. The lipide spherule appears at s. The cell surface is resolved as a fine membrane with microvilli which is surrounded by the homogeneous appearance of the "lacuna." The refractile matrix fibers are visible at some distance from the cell. Magnification, 600.

FIG. 4. Light micrograph of alcohol-fixed, para~n-embedded, periodic acid-Schiff-stained section from same animal. The chondrocyte cytoplasm shows abundant PAS-positive material which is not distributed in any spe- cific way. Compare the distribution of this material with the appearance of the acid-fuchsin stained material (Fig. 9) which appears around the lipide spherule. Magnification, 600.

The light micrographs were taken by Chester Reather.

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FIG. 5. Electron micrograph of a section from normal rabbit ear cartilage, fixed with osmium tetroxide. The chondrocyte in the middle of the field has a cell membrane with microvilli, and a small portion of another chon- drocyte can be seen in the upper left corner. This chondrocyte contains abundant cloudy amorphous material (c) which apparently has no specific topographical relation to any cytoplasmic organelles. The abundance of this material in the electron micrographs and the appearance of abundant PAS-positive material in the light microscope suggests that the cloudy amorphous material represents glycogen. The nucleus at the upper pole shows a small nucleolus, while a lipide spherule (s) is seen at the lower pole of the cell. At this magnification the structural de- tails of mitochondria (m) and the endoplasmic reticulum are not resolved. The cell is surrounded by a moderately dense matrix meshwork without any well defined pattern. A more dense matrix material is distributed approxi- mately midway between the chondrocytes (between arrows). Magnification, 7,300.

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Flo. 6. Electron micrograph of a portion of a chondrocyte which shows a segment of a lipide spherule (s) sepa- rated by a less dense area (presumed to be a shrinkage artifact) from the cytoplasmic rim. Around the cytoplasmic rim of the lipide spherule are very small dense particles (arrow). Within the cytoplasm the felt-like material (f) can be seen near the lipide spherule. The extracellular matrix is in the upper left corner. Magnification, 24,000.

FIG. 7. Electron micrograph of a portion of a chondrocyte, showing mitochondria (m) and membranes with attached granules (e) which have been described as a component of the endoplasmic reticulum. The appearance of the chondrocyte cell membrane arranged in microvilli is clearly apparent. The felt-like material of the matrix (/) is seen at some distance from the cell. Compare the appearance of this felt-like matrix material with the appearance of the cytoplasmic felt-like material in Fig. 6. Magnification, 28,000.

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FIO. 8. A survey electron micrograph of osmium tetroxide-fixed rabbit ear cartilage, showing a chondrocyte within which a large lipide spherule (s) may be seen at one pole and a very small portion of another lipide spherule (the dense material seen five millimeters to the left of (f)) at the opposite pole. Distributed in a figure eight pattern within the cell and surrounding both lipide spherules is the felt-like (f) material of the cytoplasm. The dense felt- like material of the matrix is distributed in bundles at some distance from the cell (between arrows). Magnification, 5,300.

FIo. 9. A light micrograph of osmium tetroxide-fixed rabbit ear cartilage embedded in paraffin and stained with acid fuehsin. The lipide spherules (s) are black. At the arrow is the cytoplasmic material which stains a bril- liant magenta. This material also is seen in the two chondrocytes to the left of the arrow. The fibers of the cartilage matrix are also fuchsinophilic, and their disposition at some distance from the cell surface can be seen in this light micrograph. Magnification, 600.

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