The fine structure of connective tissues

EXPERI\IENT:\L

AND

MOLECUL.JR

The

Fine

529-543 (1964)

3.

P.\TIIOLO(:Y

Structure IV.

of

The Intercellular

NEIL I?. P. FERNANDO,~ G. A. Department

of Pathology,

Connective

Bunting

VAN

Institzltc,

Tissues*

Elements

EKKEL, llrrir~ersitg

AND

HENRY Z. NOVAT

of Toronto,

Torotzfo.

Canada

INTRODUCTION , This paper deals with the fine structure of the intercellular elements of connective tissue. Earlier reports have described the cellular elements of connective tissue (Movat and Fernando, 1962a, b; Fernando and Movat, 1963b) and its terminal vascular bed (Movat and Fernando, 1963b, c; Fernando and Movat, 1964a, b). These studies were undertaken to give a baseline for investigations of certain pathological alterations which may occur in connective tissue (Fernando and Movat, 1963a; Movat ct al., 1963), particularly in normergic (Jlovat and Fernando, 1963d) and allergic inflammation (Movat and Fernando, 1963a; Fernando and Movat, 1963e: Criuhara and Movat, 1964). JIATERIALS

AND METHODS

To study ground substance,the frog web and the heart valves and synovial tissue of rabbits were examined. Basement membranes were studied in the frog web, the vesselsof the rabbit mesentery, the hamster cheek pouch, and in the glomeruli and tubules of the kidney in rabbits, rats, mice, and dogs. Elastic fibers and membraneswere examined in the ligamentum nuchae of cattle, the aorta of rabbits and in blood vesselsof the various tissueslisted above. Collagen fibrils were studied in dense (rabbit skin, rabbit tendon, hamster cheek pouch) and loose (frog web, rabbit heart valves, rabbit synovial tissue, rabbit mesentery) connective tissue. The only pathologically altered tissue used in this study was the cheek pouch of hamsters in which the reversedpassive Arthus reaction had beeninduced (Fernando and Movat, 1963e) and regenerating tendon of rabbits, (Fernando and Novat, 1963a). The tissueswere fixed in 1;: buffered (pH 7.4, Verona1acetate) osmium tetroxide containing sucrose (45 mg per milliliter) for l-15,; hours. Some tissue was postfixed for 1 hour in the same fixative, but with calcium chloride (0.2 mg per milliliter) instead of the sucrose.The latter procedure insured good cohesion of the tissue and prevented disruption of cell membranes. Following dehydration, the tissues were embedded either in Epon 612, Selectron, or, at the beginning of our studies, in methacrylate. Thick (0.5 1~) sections were stained with azure II alkalinized with borax and occasionally with periodic acid-silver methenamine. After identification of appropriate structures in the light microscope,the blocks were trimmed and ultrathin 1 Supported 2 Recipient

by grants from the Canadian of a Colomho Plan Fe!lowship.

Arthritis

Society-

530

NEILV.P.FERNANl)O,G.A.VA~

ERKEL,AND

HEXKT

Z.MOVAT

sections were cut for electron microscopy. These were stained with uranyl acetate, phosphotungstic acid, lead hydroxide, or Protargol. They were examined with an RCA-EMU-3E electron microscope.The negatives were enlarged photographically. OBSERVATIONS The intercellular elementsof connective tissue are generally divided into amorphous and formed components. The amorphous components are the ground and cementing substances,the term “cement substances” being applied to that part of the ground substancewhich is thought to bind fibrils together. The formed elementsinclude the collagen and reticular fibrils, the elastic fibrils, and the elastic membranes.The basement membrane may be considered intermediate between the amorphous ground substanceand the fibers. GROUND SUBSTANCE

The ground substanceconsistsof acid mucopolysaccharidesand proteins. It fills the spacebetween the cellular and formed intercellular elementsof the connective tissue. In the light microscope it is difficult to recognize unless specifically stained. The intensity of staining with the commonly used dyes (Alcian blue; Hale’s colloidal iron ; metachromasia) is thought to depend on the concentration of acid mucopolysaccharides. By selecting tissues containing abundant ground substance, which stains with the above stains, the presence of ground substance in our electron microscopic preparations was insured. Electron micrographs of ordinary connective tissue often contain “empty” spaces between cells and formed intercellular elements.In tissuesin which cellular preservation is good it is often difficult to ascertain whether such spacesrepresent artefacts or whether the spacesare occupied by ground substance. Many electron microscopists believe that the ground substance of connective tissue cannot be identified in the electron microscope. In the frog web, there was some indication of a difference in density and LLtexture” between spacesthought to be occupied by ground substanceand the lumina of vessels (Fig. 1). The difference between the granular and slightly electron-dense ground substance of heart valves and the cardiac cavity was more apparent (Fig. 2). In synovial tissue likewise there was a difference in density between the ground substance, the lumina of vesselscontaining precipitated plasma, and the space beyond the edge of the sections, where the embedding medium only was present (Fig. 3). At high magnification a filamentous component could at times be identified in ground substance (Fig. 4). This has also been observed in cartilage (Godman and Porter, 1960; Sheldon and Robinson, 1960). BASEMENT

MEMBRANES

The nature and origin of basementmembrane is still controversial. Recent evidence suggeststhat somebut not all basement membranesare of epithelial origin (Pierce et al., 1962). The older view is that basement membranesare a condensation of ground substance (Gersh and Catchpole, 1949). Basement membranesstain moderately with aniline dyes, which stain collagen fibers, but stain intensely with the periodic acid-Schiff (PAS) and periodic acid-silver methenamine stains. Basement membranesare thought to be composedof mucoproteins (Pearse, 1954).

THE

FINE

STRUCTURE

OF CONNECTIVE

TISSUES

531

po~ver view of frog web. A venule is seen to the right, composed of endothelial FIG. 1. Low cells (ESD). basement membrane (BM), and perivascular cells (PER). The ground substance (GS) which fills the spaces between cells appears slightly darker and “clumpy” compared to the lumen of the vessel. RBC. Red blood cell; LEIK. leukocyte. Epon, Protargol, X11,200.

332

NEIL

V. P. FERNANDO,

G. A. VAN

EKKEL.

AND

HENRY

Z. MOVAT

FIG. 2. Mitral valve of rabbit. The ground substance (GS) between the and the collagen fibrils (COL) is granular to floccular and more electron ventricular cavity. EXD, Endothelium; RBC, red blood cell. Methacrylate, acid, X10,000. Fio. 3. Synovial connective tissue of rabbit knee joint. Sate the difference texture between ground substance (GS) and capillary lumen (CAP) as well as substance and the edge of the section (arrows), FB, Fibroblast ; END, endothelium; fibrils. Epon, Protarpol, X8500.

fibroblasts (FB) dense than the phosphotungstic in density and between ground COL, collagen

53.1

534

NEIL

FK. 4. Bovine (GS) is filamcntous

V. I’.

FERNANDO,

G. A.

VAN

ERKEL,

AND

ligamentum nuchae. The elastic fibers (EL) (FIL) and contains collagen fibrils (COL).

HENRY

Z. MOVAT

stain faintly. The ground substance Epon, uranyl acetate, x,i7,000.

THE

FINE

STRUCTURE

OF

CONNECTIVE

TISSL'ES

535

In vessels, basement membrane lies at the base of endothelial cells and also surrounds the perivascular cells or pericytes of capillaries (Fernando and Movat, 196413) (Fig. 5) and smooth muscle cells of arteries (Movat and Fernando, 196313) (Fig. 11). Such basement membranes may appear faintly fibrillar or filamentous (Fig. .5), but more often are homogeneous (Fig. 6). Apart from differences in thickness, epithelial and endothelial basement membranes look alike in the electron microscope (Fig. 7). n’hile usually regarded as a band or tube, the basement membrane may have irregular projections into the depth of the overlying cell (Fig. 8). Amphibian basement membrane is similar to that of mammals (Fig. 9 ). ELASTIC

ELEMENTS

Elastic fibers may form a network in loose connective tissue, may form thick ligaments as in the ligamentum nuchae, or may form membranes as commonly encountered in vessels, particularly in elastic arteries. Elastic tissue stains specifically with certain dyes, such as resorcin fuchsin and orcein. Little is known about the chemical structure of elastic tissue. Hall (1957) has suggested that there is no single entity of “elastin” but a series of elastins differing in their amino acid composition. He suggested that the elastic fiber consists of a central protein core surrounded by a protein mucopolysaccharide complex. Pease and Molinari (1960) had proposed that elastin is formed in a mucopolysaccharide matrix by addition of a second material which is composed of gIobuIar units which fuse into a continuum. M’ith the electron microscope, no fibrils or periodicity had been reported in elastic fibers or membranes. The fibers are generally said to be electron translucent. In our material, elastic membranes were always electron translucent in unstained sections. Thick elastic membranes and fibers appeared white or stained faintly with uranyl acetate and lead (Fig. 4). Thin membranes stained intensely, particularly with phosphotungstic acid (Fig. 11). (When stained with resorcin-fuchsin, thick membranes are reddish-purple, while thin ones are dark purple to black.) From our electron micrographs, we gained the impression that a filamentous material surrounding the dense homogeneous core could be demonstrated around the elastica (Fig. 12). Phosphotungstic acid often showed a sharply demarcated elastic membrane surrounded by a faintly stained area (Fig. 13). Our observations made with the electron microscope can be integrated we]] with light microscopic observations. In the light microscope one can see in sections stained simultaneously with resorcin-fuchsin and Alcian blue (hlovat, 1955) that elastic membranes are surrounded by Alcian blue-positive material. The latter is probably acid mucopolysaccharide-rich ground substance and corresponds to the filamentous material in the electron microscope. COLLAGEP;

FIBERS

AND

FIBRILS

With light microscopy collagen fibers are unbranching and stain typically with aniline dyes, such as aniline blue or fast green in the Masson or Mallory staining procedures. The oldest collagen stain is the Van Gieson picrofuchsin procedure. When impregnated with silver, collagen fibers are brown to purple, in contrast to wellimpregnated reticular fibers, which are black. While no difference between collagen

536

NEIL

V. P. PERNANDO,

G. A. VAN

ERKEL,

AND

HENRY

Z. MOVAT

5. Portion of capillary of frog web. The endothelium (END) rests on basement membrane The latter is cut tangentially and is faintly fibrilar. LUM, Lumen; JUN, junction; collagen fibrils. Epon, many1 acetate, X55,000. FIG. 6. Portion of capillary of hamster cheek pouch. The basement membrane (BM) is seen as a homogeneous band, limited to the right by collagen fibrils (COL), but with no definite limitation in the left half of the photograph. LUM, Lumen; END, endothelium; PER, perivascular cell. Epon, phosphotungstic acid (block staining), X19,300. FIG. 7. Base of proximal convoluted tubule and peritubular capillary of rat. Note the difference in width between the tubular basement membrane (TBM) and the capillary basement membrane (CBM). The pores in the endothelium (END) are bridged by a thin membrane. LUM, Capillary lumen; PCT, proximal convoluted tubule; COL, collagen fibrils. Selectron, uranyl acetate. X52,000. FIG.

(BM). COL,

THE

FINE

STRUCTURE

OF

CONNECTIVE

TISSUES

537

FIG. 8. Base of proximal convoluted tubule (PCT) of mouse. The tubular basement membrane (TBM), which is cut tangentially, is irregular toward the tubular cell. LUM, Lumen of peritubular capillary; END, end&helium; CBM, capillary basement membrane. Selectron-methacrylate; lead hydroxide, ~30,000. FIG. 9. Base of a mucous gland of frog skin. The epithelial cell (EP) of the gland rest on basement membrane (BM), which is faintly fillamentous. A finely floccular material, presumably ground substance, is seen between the collagen fibrils (COL), which are cut in part across and in part longitudinally. Epon. uranyl acetate, ~48,000. 53s

THE

FIG. center. fihrils,

FINE

STRUCTURE

OF

CONNECTIVE

TISSUES

10. Bo\-ine ligamentum nuchae. A moderately electron dense elastic fiber It is connected with filaments (FIL), n,hich mcrpe \vith the ground substance. phosphotunpstic acid, Epon, ~79,500.

530

is seen in the COL. collagen

540

NEIL

V. P. FERNANDO,

G. A. VAN

ERKEL,

AND

HENRY

2.. MOVAT

and reticular fibrils can be distinguished by electron microscopy, chemical analysis showed a greater carbohydrate content in the reticular fibrils (Glegg et al., 1953; Windrum et al., 1955). This is thought by some to be responsible for their intense staining, although the difference may be purely physical. Collagen and reticulin have the same amino acid composition, characterized by a high concentration of prolin, hydroxyprolin, and glycine (Jackson, 1959). The fiber seen with the light microscope can be resolved into fibrils by electron microscopy. The fibers have a diameter of the order of microns, but the width of the fibrils in only several hundred or thousand ‘4 units. Figure 14 shows an example of dense connective tissue, composed of bundles of collagen fibrils and cells. The collagen fibrils shown in Fig. 15 would correspond to argentophilic or reticular fibers by light microscopy. The bundle of iibrils seen in Fig. 16 would correspond to a collagen fiber in the light microscope.

FIG. 11. Small mesenteric artery of rabbit. The elastic elements (EL) of the internal lamina are stained black. The space between endothelium (END) and smooth muscle cells not occupied by elastic tissue, is probably ground substance. BM, Basement membrane; collagen fibrils. Selectron, phosphotungstic acid, X 16,300.

elastic (SM), COL,

Collagen fibrils can be stained by a variety of heavy metal salts for electron microscopy, the most intense staining being obtained with phosphotungstic acid (Figs. 15 and 16). In all these preparations, a typical periodicity is seen (Figs. 17-19). The periodicity of native collagen is 640 pi. However, in plastic-embedded and dehydrated tissue there is some shrinkage, which varies with the embedding medium used. Schmidt and co-workers (1955) assumed that the axial periodicity of native collagen fibrils as seen in small X-ray diagrams and in the electron microscope represents parallel arrangements of tropocollagen macromolecules, displaced in an axial direction by one quarter of a length in adjacent macromolecules.

Fm. 12. Internal elastic lamina of small renal artery a; r-t. The e!astic membrane (EL) is homogeneous and moderately electron dense. It is surrounded by a filamentous material, which is more electron dense and probably represents acid mucopolysaccharide-rich ground substance. Between the arrows the elastic membrane is interrupted and the fenestra is filled by the dense filamentous substance. SM, Smooth muscle cell. Selectron, many1 acetate, X56,700. FIG. 13. Same artcry as in Fig. 12 at different level. The elastic membrane (EL) is moderately dense and well demarcated. The filamentous ground substance seen in Fig. 12 stains very faintly. In addition to ground substance (GS) the space between the elastic membrane and the smooth muscle cell (SM) also contains cross-sectioned, electron-dense collagen fibrils (CO). END, Endothelial cell; SM. smooth muscle cell; CM, cell membrane. Selectron. phosphotungstic acid, X69,000. 541

542

NEIL

V. P. FERNANDO,

G. A. VAN

ERKEL,

AND

HENRY

Z. MOVAT

Fro. 14. Dense collagcnous connective tissue of hamster cheek pouch. The bundles of collagen fibrils (BC) run in various directions. Note the good coherence of the tissue. MON, Mononuclear cell; FB, fibroblast; STR, edge of striated muscle. Epon, uranyl acctatc, x7000.

FIG. 15. The collagen fibrils (COL) in the wall of this wnule (rabbit mesentery) correspond to argentophilic or reticular libers by light microscopy. LtrM, Lumen; E-ND, endothelium: PER, perivascular cell. Selectron-methacrylate, phosphotungstic acid, X10,000. FIG. 16. Rabbit skin; bundle of collagen fibrils !BC) corresponding to a collagen fiber b) light microscopy. FB, Fibroblast. methacrylate, phosphotungstic acid, X 12,000. FIGS. 17-19. High magnification of collagen fibrils. Figures 17 and 18: regenerating tendon of rabbit; Fig. 19 from frog web. Note the periodicity in all fibrils and the floccular material between the fibrils in Fig. 17. Sections are stained with uranyl acetate. Figure 17: methacrylate, X51.600 ; Fig. 18: Epon. X 160,000; Fig. 18: Selectron, X 130,000.

544

NEIL

V. P. FERNANDO,

G. A. VAN

ERKEL,

AND

HENRY

Z. MOVAT

Little is known about the formation of the intercellular elements of connective tissue. It is now fairly well established that collagen fibrils form extracellularly, but that the building material is manufactured by the fibroblasts (Fernando and Movat, 1963a). Some evidence has been presented (Godman and Porter, 1960) that ground substance of cartilage is produced by the chondrocytes. In contrast, the fibroblasts in the combs of newly hatched chicks stimulated with androgen showed no changes which could be interpreted as the secretion of ground substance or of mucopolysaccharide (Movat and Fernando, unpublished observations). SUMMARY

The fine structure of the extracellular elements of connective tissue is described. Ground substance, which because of its high concentration of acid mucopolysaccharide, stains intensely with certain stains, can be visualized in the electron microscope as a faintly electrondense substance. At high magnification, ground substance often looks filamentous. Basement membranes which usually appear homogenous are at times faintly filamentous. Elastic fibers and membranes seem to consist of a central homogeneous core surrounded by a filamentous layer. The latter is probably acid mucopolysaccharide-rich ground substance. Collagen and reticular fibrils cannot be identified as separate entities by electron microscopy. They show the well-known periodicity. -4CKNOWLEDGMENTS The authors wish to thank Dr. A. C. Ritchie for the valuable help and criticism in preparation of this manuscript. They also wish to express their gratitude to Mr. David Macmorine, Mrs. Ottie Freitag, Mrs. Anneliesse Carre, and Miss Charlote Turnbull for the skillful preparation of the numerous sections examined in this study. Grateful acknowledgment is made to Mr. Giinter Thomas for his invaluable assistance in the operation of the electron microscope. REFERENCES N. V. P., and MOVAT, H. Z. (1963a). Fibrillogenesis in the regenerating tendon. Lab. Invest. 12, 214-229. FERNANDO, N. V. P., and MOVAT, H. Z. (1963b). The fine structure of connective tissue. III. The mast cell. Enptl. Mol. Pathol. 2, 450-463. FERNANDO, N. V. P., and Mo~:!T, H. Z. (1964a). The fine structure of the terminal vascular bed. II. The smallest arterial vessels: terminal arterioles and metarterioles. Erptl. Mol. Pathol. 3, l-9. FERNANDO, N. V. P., and MOVAT, H. Z. (1964b). The fine structure of the terminal vascular bed. III. The capillaries. Exptl. M 01. Pathol. 3, 87-97. Allergic inflammation. II. Identification of FERNANDO, N. V. P., and MOVAT, H. Z. (1963c). antigen-antibody complexes in the electron microscope during the early phase of allergic inflammation. Am. J. Pathol. 43, 381-390. GERSH, I., and CATCIIPOLE, H. R. (1949). The organization of ground substance and basement membrane and its significance in tissue injury, disease and growth. Am. J. Anat. 85, 457-521. GLEGG, R. E., EIDINGER, D., and LEBLOND, C. P. (1953). Some carbohydrate components of reticular fibers. Science 118, 614-616. GODMAN, G. C., and PORTER, K. R. (1960). Chondrogenesis, studied with the electron microscope. J. Biophys. Biochem. Cytol. 8, 719-760. HALL, D. A. (1957). Chemical and enzymatic studies on elastin. In “Connective Tissue” (R. E. Tunbridge, ed.), pp. 238-253. Blackwell, Oxford. JACKSON, D. S. (1959). Chemistry of the fibrous elements of connective tissue. In “Connective Tissue, Thrombosis and Arteriosclerosis” (J. H. Page. ed.), pp. 67-76. Academic Press, New York. MOVAT, H. Z. (1955). The demonstration of all connective tissue elements in a single section. Pentachrome stains. Arch. Pathol. 60, 289-295. MOVAT, H. Z., and FERNANDO, N. V. P. (1962a). The fine structure of connective tissue. I. The fibroblast. Exptl. Mol. Pathol. 1, 509-534.

FERNANDO,

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CONNECTIVE

TISSUES

545

H. Z., and FERTLANIIO, N. V. P. (1962b). The line structure of connective tissue. II. The plasma cell. Exptl. Mol. Pathol. 1, 535-553. MOVAT, H. Z., and FERNANDO, N. V. P. (1963a). Allergic Inflammation. I. The earliest fine structural changes at the blood-tissue barrier during antigen-antibody interaction. .4m. J. Pathol. 42, 41-59. MOVAT, H. Z., and FERNANDO, N. V. P. (1963b3. The fme structure of the terminal vascular bed. I. Small arteries with an internal elastic lamina. Esptl. Mol. Pathol. 2, 549-563. MOVAT, H. Z., and FERNAKDO, N. V. P. (1963~). The fine structure of the terminal vascular bed. IV. The venules and their perivascular cells (pcricytes, adventitial cells). Exptl. Mol. Pathol. 3, 98-114. MOTAT, H. Z., and FERKANDO, N. \:. P. (1963d). Acute inflammation. The earliest changes at The blood-tissue barrier. Lab. Invest. 12, 895-910. MOUNT, H. Z., FERNAKCO, S. \‘. P., URIUIIARA, T., and WEISEK, R. J, (1963). Allergic Inflammation. III. The fine structure of collagen fibrils at sites of antigen-antibody interaction in Arthustype lesions. J. ExP. Med. 118, 557-564. PEI\RSL, r\. G. E. (1954). “Histochemistry, Theoretical and Applied,” Churchill, London. PEASE, D. C., and MOLIS~RI, S. (1960). Elertron microscopy of muscular arteries; pial vessels of the cat and monkey. J. Ultrastluct. Res. 3. 437-465. PIERCE, G. B., JR., MIDGLEY, A. R., JR., SRI RAIN, J., and FELD~I~S, J. D. (1962 1. Parietal yolk sac carcinoma: clue to the histogenesis of Reichcrt’s membrane of the mouse embryo. Am. J. Pathol. 41, 549-566. SHELDON, H., and ROBINSOX, R. .4. (1960). Studies on cartilage. II. Electron microscope observations on rabbit ear cartilage following the administration of papain. J. Biophys. Biochrnz. Cxtol. 8, 151-163. SCHMITT, F. O., GROSS, J., and HIGIIBERGER, J, H. (1955). Sgnzp. Sot. Esptl. Biol. 9, 148. Quoted by SCIXVIITT (1959a, b). SCHMITT, F. 0. (1959a). The macromolecular basis of collagen structure. In “Connective Tissue, Thrombosis, and .4rteriosclerosis” (J. H. Page, ed.), pp. 43-66. Academic Press, New York. SCHMITT, F. 0. (1959b). Interaction properties of elongate protein macromolecules with particular reference to collagen (tropocollagen). lit “Biophysical Science-A Study Program” (J. L. Oncelay, ed.), pp. 349-358. Wiley, New York. URIUIIARA, T.. and Mo\~T, H. Z. (1964). Allergic inflammation. IV. The vascular changes during the development and progression of the direct active and passive .4rthus reactions. Lab. Inwest. 13, 1057-1079. \VINDRU~~, G. M., KU-T, P. \V., and EATOX., J. E. (195.;). The constitution of human renal reticulum. Brit. J. Esptl. Pathol. 36, 49-59.

MOVAT,