The Human Joint in Health and Disease [Reprint 2016 ed.] 9781512807004

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THE HUMAN JOINT IN HEALTH AND DISEASE

Contributors

Jonathan Black, Ph.D. Stanley M. K. Chung, M.D. Laura Fürst, B.A. Michael Harty, M.D., F.R.C.S. Wilson C. Hayes, Ph.D. David S. Howell, M.D. John J. Joyce, III, M.D. Joseph M. Lane, M.D. Paul A. Lotke, M.D. Charles J. Malemud, Ph.D. Henry J. Mankin, M.D. C. W. McCutchen, Ph.D. John R. Parsons, Ph.D. Eric L. Radin, M.D. Lawrence Rosenberg, M.D. Asher I. Sapolsky, Ph.D. H. Ralph Schumacher, Ph.D. William H. Simon, M.D. Leon Sokoloff, M.D. Marvin E. Steinberg, M.D. Charles Weiss, M.D. J. Frederick Woessner, Jr., Ph.D.

EDITED BY

William H. Simon,

M.D.

THE HUMAN JOINT IN HEALTH AND DISEASE ® UNIVERSITY 1978

OF PENNSYLVANIA

PRESS

Copyright © 1978 by the University of Pennsylvania Press, Inc. Chapter 12 copyright © 1976 by The International Academy of Pathology. Chapter 18 copyright © 1978 by Eric L. Radin, M.D. All rights reserved Manufactured in the United States of America

Library of Congress Cataloging in Publication Data Main entry under title: The Human joint in health and disease. Includes bibliographies. 1. Joints—Diseases. 2. Joints. I. William H. [DNLM: 1. Joints. 2. WE300H918] RC933.H85 616.7'2 77-20305 ISBN 0-8122-7738-4

Simon, Joint diseases.

Composition by Deputy Crown, Inc., Camden, N.J.

Contents

Introduction Section I. 1.

Human Joints in Health

Anatomy and Development of Joints

3

MICHAEL HARTY

2.

Light and Electron Microscopic Studies of Normal Articular Cartilage

9

CHARLES WEISS

3.

The Scanning Electron Microscopy of Articular Cartilage

21

JOHN R. PARSONS

4.

Structure of Cartilage Proteoglycans

26

LAWRENCE ROSENBERG

5.

Articular Cartilage Collagen in Health and Disease

31

JOSEPH M. LANE

6.

The Water of Articular Cartilage

37

HENRY J . MANKIN

7.

Cell Culture in the Study of Mammalian Chondrocyte Metabolism

43

CHARLES J . MALEMUD

8.

The Metabolism of Articular Cartilage

53

HENRY J . MANKIN

9.

Mechanical Properties and Wear of Articular Cartilage

71

WILLIAM H. SIMON

10.

Diffusion in Cartilage

76

PAUL A. LOTKE

11.

Cartilage Lubrication

81

C. W. MCCUTCHEN V

vi

Contents Section II.

12.

Human Joints in Disease

Osteoarthrosis

91

LEON SOKOLOFF

13.

Light and Electron Microscopic Studies of Osteoarthritic Articular Cartilage

112

CHARLES WEISS

14.

Experimental Degenerative Joint Disease: In Vivo Freezing of Rabbit Articular Cartilage

122

WILLIAM H. SIMON

15.

Enzymes in Degenerative Joint Disease and Antienzyme Therapy

128

DAVID S. HOWELL, J . FREDERICK WOESSNER, JR., AND ASHER I. SAPOLSKY

16.

The Role of Arthroscopy in the Diagnosis and Treatment of Joint Disorders

132

JOHN J . JOYCE, III

17.

Abnormal Joint Biomechanics

136

WILSON C. HAYES

18.

The Case for Biologic Healing in Osteoarthrosis: The Total Joint Replacement is Not for Everyone

142

ERIC L. RADIN

19.

Experimental Rheumatoid Arthritis

149

MARVIN E. STEINBERG

20.

Gout, Pseudogout, and Ochronosis

153

H. RALPH SCHUMACHER

21.

Joint Sepsis

160

STANLEY M. K. CHUNG

22.

The Response of Cartilage to Artificial Articular Surfaces

169

JONATHAN BLACK AND LAURA FÜRST

23.

Prosthetic Replacement Surgery PAUL A. LOTKE

175

Introduction

The mysteries of the human body are numerous. None is more fascinating than the function of the human joint. A biologic system acting as a bearing surface is able to move with almost frictionless precision under tremendous loads and impacts. There are no spare parts. Joints must last a lifetime. The amazing fact is not that they break down, but that they continue to function for years and years with no "pit stops" for repairs. Mechanical analogies alone are inadequate to describe joint function. In some miraculous fashion, nature has designed a biologic system that operates much more efficiently than any known mechanism. Understanding this unique biologic system is a goal of a multitude of scientists—the best and the brightest of whom are among the contributors to this symposium. As with the three blind men feeling the elephant, there are many ways to describe the human joint. Arthroscopy, light microscopy, and electron and scanning electron microscopy all present differing pictures. The biologist and the biomechanist work together on the problem. Whether it be protein synthesis, chondrocyte metabolism, the place of water, mucopolysaccharides, and collagen, or the effect of exogenous forces on the growth, development, and breakdown of the joint—all bases must be touched in the quest for the answer to how joints function. Disease processes such as degenerative arthritis, chondrocalcinosis, gout, and ochronosis all effect the human joint and are discussed by the authors within this volume. Curing diseases with unknown etiologies may be considered a hit-or-miss proposition. The following papers offer logical, if not the final, medical and surgical answers to the problem of diseases affecting human joints. Because research into the mysteries of joint function and disease is ongoing at this moment, the articles in this volume are necessarily out of date. This is as it should be. No apology is necessary. William H. Simon, M.D. vii

SECTION

Human

Joints in Health

MICHAEL HARTY, M.D., F . R . C . S .

Anatomy

and Development

of Joints

Arthrology is the study of joints or articulations that in the animal body are the connections between its denser and more rigid components. Etymologically, the term syndesmology refers to fibrous joints only. Long and learned discussions have been devoted to the classification of joints but the titles used have meant little, except to the student of classical Greek. Adjacent bones may be united by fibrous tissue, by cartilage, or by synovial joints. In fibrous joints, the opposed uneven and often interlocking bony margins are bound together by fibrous tissue that is continuous with the adjoining periosteum. The fibrous joints in the skull vault are immobile and represent growth lines where the bones continued to expand by surface accretion. The inferior tibiofibular articulation is the only noncranial fibrous joint; it does not ossify, enabling slight rotary and sliding motions. Cartilagenous joints are primary and secondary; they retain the fibroperiosteal connecting collar but also add a thin irregular cartilage plate. The primary cartilaginous joints have a layer of hyaline cartilage surrounded by the fibrous ring. They include the epiphyseal plates between the epiphyses and diaphyses, and a few local areas in the basal skull sutures. They allow no movement, and the epiphyseal plates are generally fused by the twenty-first birthday. Secondary cartilagenous joints still retain the fibrous annulus. The bone ends are covered by thin layers of hyaline cartilage united by a flat intervening layer of fibrocartilage. Practically all secondary cartilagenous joints are in the median plane, such as the intervertebral discs, symphysis, pubis, and manubriosternal junction. They allow only a slight range of motion (Fig. 1—1). 3

4

HUMA Ν JOINTS IN HE A LT Η FIBROUS

CARTILAGENOUS

JOINT PRIMARY

PERIOSTEUM

JOINTS

SECONDARY

ARTICULAR CARTILAGE

FIBROCARTILAGE

Figure 1-1. Fibrous and cartilagenous joints. Periosteum and fibrous capsule (thin solid lines). Articular cartilage (stippled) and fibrocartilage {parallel lines).

SYNOVIAL JOINTS Synovial joints retain the fibrous connecting ring ( n o w capsule) continuous with the adjoining periosteum. In most joints, the contiguous bone ends are covered by hyaline cartilage (articular cartilage) and are separated by a synovial cavity. This closed cavity is bounded by articular cartilage and synovial membrane; although it may communicate with local bursae, tendon sheaths or other joint cavities (Fig. 1 - 2 ) . Synovial joints are designed specifically to enable free movement and have the following common designations: plane joint (e.g., wrist), hinge joint (e.g., interphalangeal joint), condylar joint (two distinct articular surfaces, e.g., k n e e ) , ellipsoidal joint (e.g., radiocarpal or metacarpophalangeal joint), pivot joint (e.g., proximal radioulnar joint), saddle joint (e.g., carpometacarpal joint of t h u m b ) , and ball-and-socket or spheroidal joint (e.g., hip or shoulder).

Articular Cartilage T h e shape of the articular surface in the synovial joint depends on the functional requirements of that particular joint, and the range of motion is often indicated by the laxity or shortness of the articular capsule. T h e smooth articular cartilage is closely molded and firmly attached to the underlying bone, with minor variations. Although this cartilage can sustain a remarkable amount of weight, it still enables free slide or motion between the opposed surfaces. Its thickness ranges from 1 to 5 or 6 mm. Typically it is thickest at the central area of the convex surface and on the peripheral margins of the concave surface (Fig. 1 - 2 ) . Until recent years articular cartilage was considered one of the smoother body surfaces, but the higher magnifications of electron and scanning microscopy have shown irregular hills and hollows on cartilage surfaces. Adult cartilage is avascular, aneural, relatively acellular, and resilient, and it is not normally seen in radiograms. The central area of articular cartilage derives nutrition from the synovia and to a lesser extent f r o m the underlying bone. T h e peripheral margin of the cartilage gets a blood supply from the articular vascular circle of William Hunter, which lies immediately under the adjacent synovial mem-

Anatomy and Development

of Joints

5

Figure 1-2. Synovial joint. Subsynovial bursa {right) and intraarticular wedge (left). Periosteum and fibrous capsule ( thin solid line). Synovial membrane (broken line).

brane (Fig. 1 - 3 ) . Degenerative changes in the central area are characterized by cartilage ulceration and erosion, in contrast to the peripheral area, where excrescence is the more typical feature. Biological repairs and regrowth of articular cartilage are rarely if ever found.

Capsule and Ligaments The capsule and ligaments are composed of collagenous fibers and like the adjacent periosteum are extremely sensitive. This high sensitivity protects the joints, and the capsule and ligaments serve as special sense organs for static and dynamic feedback. All are aware of the nicety of finger-joint sensitivity during the adjustment of a high-power light microscope. Ligaments are often condensations of certain regions of the capsule—for example, the collateral ligaments of a hinge joint and the ligamentum patellae. Other ligaments may be slightly removed from the capsule or invaginated by synovial membrane into the joint cavity, such as the long biceps tendon in the shoulder. The ligaments play a major role in the stability

6

HUMAN

JOINTS

IN HE A LTH

Figure 1-3. Proximal femur to illustrate the pericapsular and subsynovial arterial anastomoses. of most joints. They reinforce specific areas and influence, direct, or restrict certain joint motions. Many muscles, their tendons, or expansions reinforce the joint capsule and ligaments. Capsular or ligamentous stretching often produces rapid, protective muscle contraction.

Synovial membrane This lines the joint cavity except on articular cartilage and on intraarticular fibrocartilagenous discs or menisci. The membrane may lie on and be attached to ligaments presenting a light gray appearance—for example, on the medial collateral ligament of knee or tendon of the popliteal muscle. It may be stretched loosely on the capsule (areolar), appearing pinkish gray; or it may cover extrasynovial fat, presenting a pale yellow hue. Arthroscopically, these colors are easily distinguished and aid in identification. Synovial membrane, unlike other connective tissues, produces a fluid ground substance rather than a gel. This fluid provides nutrition and lubrication for the articular cartilage.

A natomy and Development of Joints

7

Fat Pads The extrasynovial subcapsular collections of fat range in size from the large, very mobile, mercurial infrapatellar fat pad to the minute fingerlike villi typically seen at the junction of articular cartilage and synovial membrane. These irregular smaller pedunculated villi present diagnostic problems in arthrography and obstruct the visual field of the arthroscopist. The fat pads are highly sensitive, containing more naked (pain) nerve endings than any other intracapsular structure.

NUTRITION AND NERVE SUPPLY Joints derive their blood supply from their overlying blood vessels, which break up into a periarticular plexus at the bone capsule junction. From here vessels pierce the capsular attachment to form a finer vascular anastomosis in the subsynovium at the margins of the articular cartilage (Fig. 1 - 3 ) . Both these articular anastomoses provide a blood supply to the adjacent bones and muscles, to the capsule and to the synovium and its extrasynovial structures, such as fat pads. Hilton's statement (Law) of 1863 that "the same trunk of nerves whose branches supply the groups of muscles moving a joint, also furnish a distribution of nerves to the skin over the insertion of the same muscles and—what at this moment more especially merits our attention—the interior of the joint receives its nerves from the same source" is as germane today as it was a century ago. The major concentrations of sensory articular nerve endings are located in the capsule, the ligaments, and the fat pads. Freeman and Wyke found no nerve endings in synovial membrane but some in the synovial blood vessels.3 The nerve endings carry impulses of pain, static and dynamic joint position, motion, and reflex activity. The mechanoreceptors convey impulses of motion, its speed, intensity and duration. Sympathetic fibers influence the smooth muscle in the wall of the articular blood vessels.

DEVELOPMENT Inherited self-differentiating factors determine the species pattern of the future bones and joints; compression, distraction, growth, and muscle actions play but a minor part in the final configuration. About the fifth week of intrauterine life, the central mesenchyme of the limb bud display centers of chondrification, surrounded by a tube of perichondrium. A week later, expanded areas of the tube contain flattened cells—the interzone or future joint. The tube of mesenchyme, which was continuous with the perichondrium, is invaded by blood vessels and forms the capsule with its condensations of ligaments, synovial membrane, and discs. The undifferentiated mesenchyme of the interzone soon differentiates into three layers—two dense, covering the cartilagenous ends separated by a loose intermediate avascular zone. About the eighth week, ossification centers appear in the long bones; at approximately the tenth to twelfth week, small interzonal cavities appear that quickly coalesce to form joint cavities. Although the early formation of the joint cavity occurs independently of motion, later molding of the joint is related to active action—for example, the acetabulum is a shallow cavity at 16

8

HUMA Ν JOINTS

IN HE A LT Η

weeks, but only if occupied by the femoral head will it deepen to normal dimensions. Bursae close to joints develop about the same time as the joint cavity. A loosening of the local tissue is followed by liquefaction of the connective tissue spaces, which coalesce to form the bursae.

REFERENCES 1. Barnett, C. H., et al.: Synovial Joints: Their Structure and Mechanics. C. H. Thomas, 1961. 2. Dee, R.: Structure and function of hip joint innervation. Ann. Roy. Coll. Surg. Eng., 65:357, 1969. 3. Freeman, M. A. R., and Wyke, B.: The innervation of the knee joint. J. Anat., 101:3, 505, 1967. 4. Gardner, E., et al.: Anatomy. A regional study of human structure, 4th Ed. Philadelphia, W. B. Saunders, 1975. 5. Harty, M., and Joyce, J. J., III.: Surgical anatomy and exposures of the knee joint. AAOS Internat. Course Lect., 20:206, 1971. 6. Hilton, J.: Rest and Pain. Bell, London, 1863.

CHARLES WEISS, M.D.

Light and Electron Microscopic Studies of Normal Articular Cartilage

Under gross examination, the articular surfaces of normal joints appear to be smooth and regular; however, closer scrutiny with a hand lens under indirect light shows these surfaces to be irregular and undulating. These undulations depicted in the drawings of nineteenth century microscopists15 have recently been quantified by using incident light microscopy,14 Linnik interference photomicrography, 17 tallysurf tracing,49 reflected light interference microscopy,18 and scanning electron microscopy. 8 · 9 · 16 · 48 Three orders of joint irregularities have been described: the primary joint contours; secondary undulations (200 /xm.-500 /um. in diameter); and tertiary hollows (20 /¿m.-50 μηι. in diameter and 0.5 /un in depth in the young). With aging, there is an increased depth (up to 1.7 μηι.) and an apparent decrease in the frequency of these tertiary hollows. Light microscopy (Figs. 2-1 and 2 - 2 ) reveals a birefringent line on the articular surface (the lamina splendens) 23 that corresponds to a layer of fine fibrils (4-10 mm. in diameter) arranged in a random fashion and up to several microns in depth, seen with both the scanning10 and transmission electronmicroscope (Figs. 2-3 and 2-4). 5 3 The composition of this layer is thought to be hyaluronic acid2 or other adsorbed proteoglycan derived from the synovial fluid.36 With aging, gaps in this covering layer expose bundles of superficial collagen fibrils to the articular surface, accounting for increased irregularity.18 The surface of old articular cartilage is often covered with a thick accumulation of amorphous debris.51

9

10

HUMA Ν JOINTS

IN HE A LT Η

Figure 2-1. Photomicrograph of normal adult articular cartilage. The birefringent line on the articular surface (LS) is the lamina splendens. Cells of the tangential zone (TN) are arranged with their long axes parallel to the articular surface. Rounded cells of the transitional zone (TR) are randomly arranged. Cells of the radial zone (R) are in short columns. Cells of the calcified zone (C) appear to be degenerated. The tidemark (T) separates the radial and calcified zones. Blood vessels do not penetrate the tidemark. (H&E; X200)

Figure 2-2. Photomicrograph of articular cartilage from an immature animal. The tangential zone (TN) consists of flattened cells. The underlying cartilage contains two zones of proliferating cells. The more superficial zone (1) accounts for growth of the articular cartilage and the deeper zone (2) acts as a microepiphyseal plate for the underlying epiphysis. Large vessels (V) penetrate the basilar portion of the cartilage (H&E; X 100)

THE MATRIX The matrix of articular cartilage is composed primarily of collagen fibrils imbedded in a gel-like substance composed of polyanionic proteoglycans and water. The arrangement of collagen fibers has fascinated anatomists since the 18th century: Hunter, in 1743, suggested that the articular cartilage was covered by a "skin," beneath which the fibers were arranged in a radial fashion. 2 1 Analyzing the way in which cartilage could be split, Benninghoff in 1925 concluded that individual fibers were anchored in the calcified zone, ran vertically in the radial zone, turned obliquely in the transitional zone, ran parallel to the articular surface in the tangential zone, and then returned to the calcified zone, reversing the order just

Light and Electron

Microscopic

Studies of Normal Articular

Cartilage

11

4

described. This gave rise to the " a r c a d e " concept of collagen fiber organization. The arcade concept remained unchallenged until X-ray diffraction studies in 1958 revealed that collagen fibrils in the tangential zone were arranged tangential to the articular surface, while those of the deeper zones appeared arranged in a random fashion except in the calcified zone, where they were perpendicular to the articular surface. 1 ' 2 Transmission and scanning electron microscopic studies of many species, 3 · I3 · 41· * 2 · 4 3 , 56 including m a n , 7 · 1 0 · 3 2 , 39· 53 have shown that collagen fibrils of the tangential zone in normal, young articular cartilage are arranged in tight bundles, which are frequently at right angles to each other and are oriented parallel to the articular surface (Figs. 2 - 3 and 2 - 4 ) . This tangential zone is thinnest at the -

L S

-

TAN

w§m TRANS

RAD

-

CAL

Figure 2-3. Diagramatic representation of the fibrous architecture of normal, young adult human articular cartilage. The lamina splendens (LS) is a layer of fine fibrils (4 nm.-10 nm. in diameter) several microns thick that covers the articular surface. The tangential zone ( T A N ) consists of tightly packed bundles of collagen fibrils (30 nm.-32 nm. in diameter) arranged parallel to the articular surface and often at right angles to each other. The transitional zone ( T R A N S ) and radial zone (RAD) consist of randomly arranged collagen fibrils (40 nm.-100 nm. in diameter) and significant amounts of proteoglycans. The predominant organization of collagen fibrils in the basilar portion of the radial zone is perpendicular to the joint surface. There is an increased concentration of fine fibers and filamentous fibrils (4 nm.-10 nm. in diameter) in the lacunae (L) and in the interterritorial regions of the transitional and radial zones. Mature collagen fibrils appear to encapsulate (C) the lacunae. Collagen fibrils of the calcified zone (CAL) are usually greater than 100 nm. in diameter and are arranged perpendicular to the joint surface. (From Lane, J. M., and Weiss, C.: Arth. Rheum., 18:553, 1975.)

12

HUMA Ν JOINTS

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Figure 2-4. Electronmicrograph of the tangential zone of normal, young adult human articular cartilage. The lamina splendens consists of a layer of fine fibers (/) and filamentous fibrils i f f ) covering the articular surface (insert A). Collagen fibrils (C) are arranged in large bundles that run parallel to the articular surface and at right angles to each other (CB1, CB2). Elongated fibrocyte-like cells of the tangential zone have hyperchromic nuclei (Ν), endoplasmic reticulum (E), dilated to form cisternae (CS), Golgi apparatus (G), vacuoles (V), and cytoplasmic processes (CP), ( x 16,000; insert, x 30,000) (From Weiss, C., et al.: J. Bone Joint Surg., 50/4:663, 1968.)

apex of the joint ( 2 0 0 μ in d e p t h at the summit of the m e d i a l f e m o r a l c o n d y l e ) and thickest at peripheral margins of the joint ( m o r e than 6 0 0 μ in d e p t h ) . 5 1 Developmentally, this zone o r skin a p p e a r s to be c o n t i n u o u s with m e m b r a n o u s structures peripheral to the articular cartilage such as the p e r i c h o n d r i u m , periosteum, joint

Light and Electron Microscopic

Studies of Normal Articular Cartilage

13

Figure 2-5. Electronmicrograph from transitional zone of normal-appearing cartilage in a 67-year-old. Collagen fibrils (F) in the territory sweep in capsular fashion about the lacunae. Fine fibers and filamentous fibrils form a halo (H) about the chondrocytes (insert A). Collagen fibrils in the interterritorial regions are randomly arranged and vary in diameter from 30 to 120 nm. (arrow heads) (insert C). Osmophilic bodies consistent with matrix vesicles ( M V ) and extracellular lipids are present in the matrix (insert B). Portions of two viable chondrocytes are present and contain normal cell membranes (CM), rough-surfaced endoplasmic reticulum (R), mitochondria (M), Golgi apparatus (G), lipid droplets (L), a complex body (Cx), and some lysosomal-like structures. (X 73,000; inserts x 12,500)

capsule, and synovial membrane. 5 4 Collagen fibrils of the transitional and radial zones appear randomly arranged except in the immediate vicinity of cells, where collagen fibrils sweep in capsular fashion about the lacunae (Figs. 2 - 5 and 2—6) 1 0 · 1 3 · 5 1 - 5 3 In the deepest calcified portions of the radial zone and in the calcified zone, the predominant orientation of collagen fibrils is perpendicular to the joint surface. The diameter of individual collagen fibrils is increased with increased distance f r o m the articular surface (Figs. 2 - 3 , 2 - 4 , and 2 - 5 ) . 1 0 · 3 4 · 39· 53 Collagen fibrils in the tightly packed bundles of the tangential zone are 30 mm. to 32 mm. in diameter

14

HUMAN

JOINTS IN

HEALTH

Figure 2-6. Electronmicrograph of a chondrocyte in the transitional zone of normal, young adult human articular cartilage. The cell is rounded and surrounded by a pericellular halo (H) of fine fibers and filamentous fibrils. Mature collagen fibrils (C) are arranged concentrically about the cell. The cell membrane has a scalloped appearance. There are an extensive rough-surfaced endoplasmic reticulum (R), a large Golgi apparatus (G), and many large vacuoles (V) containing material similar in density to that of the extracellular matrix. Intracytoplasmic filaments (F) and glycogen deposits ( G Y ) are present. (X8000) Figure 2-7. Electronmicrograph of chondrocytes from the transitional zone of normalappearing cartilage in an 86-year-old. A degenerating chondrocyte (D) lies adjacent to a hypertrophied cell and contains extensive rough-surfaced endoplasmic reticulum (R), an extensive Golgi apparatus (G), many enlarged mitochondria (M), lipid droplets (L), lysosomal-like structures (LY), and a complex body (Cx). (x 10,000) (From Weiss, C.: Fed. Proc., 32:1459, 1973.)

with a periodicity of 64 nm. (Fig. 2 - 4 ) . 5 3 Individual fibrils in the transitional and radial zones are of larger diameter (40 n m . - 1 0 0 nm.) and more loosely arranged with greater interfibrillar distances (Fig. 2 - 3 ) . Collagen fibrils of the calcified zone are larger than those found within the radial zone, many being more than 100 nm. in diameter. Hydroxyapatite crystals are found both in and between collagen fibrils in a manner similar to that seen in bone. However, the fibrils of this zone are not continuous with those of the underlying bony endplate. Between the radial and calcified zones a wavy blue line can be seen on hematoxylin stain (Fig. 2 - 1 ) . This structure was termed the "tidemark" by Collins, 11 and ultrastructural observations suggest that it functions as an anchoring, stress-relieving mechanism between the fibrils of the radial and calcified zones. 37 The periodicity of collagen fibrils in all zones is partially obscured by adherent proteoglycan. This same coating accounts for the thicker appearance of collagen fibrils observed with the scanning electron microscope. 10, 37 The increased concentration of fine fibers and filamentous fibrils (4 n m . - 1 0 nm. in diameter) in areas of high metachromasia, such as in the lacunae (the cell "territory"), in the areas between cells of the transitional and radial zones (the "interterritorial" areas), and

Light and Electron Microscopic Studies of Normal Articular Cartilage

15

Figure 2-8. Electronmicrograph from the radial zone of articular cartilage from a 67year-old. Collagen fibrils (F) up to 250 nm. in diameter with 64 nm. periodicity are adjacent to a degenerating cell (D). (x 10,000) (From Weiss, C.: Fed. Proc., 52:1459, 1973.) Figure 2-9. Electronmicrograph of a microscar in the radial zone of articular cartilage from an 80-year-old. Remnants of a degenerated cell (D) are present within the lacuna space. These remnants are consistent with matrix vesicles. The pericellular halo is filled with collagen fibrils of up to 450 nm. in diameter and containing nuclei of calcification (arrows and insert). (X3100; insert X8000) (From Weiss, C.: Fed. Proc., 32:1459, 1973.)

in intracellular secretory vacuoles, provides indirect evidence that they are proteoglycan molecules (Figs. 2 - 5 and 2 - 6 ) . 4 0 The histologic characteristics of the proteoglycan molecules have recently come under intense investigation. A number of dyes have been shown to react with these polyanions as both metachromatic stains (for example, alcian blue) or orthochromatic stains (for example, safranin-O). Safranin-O has been shown by Rosenberg 3 8 to react in a quantitative fashion with the glycosaminoglycans, and although this is a useful procedure, it is not totally specific because of its affinity for R N A . Alcian blue does react specifically in cartilage with the sulfated polyanions, chondroitin sulfate and keratan sulfate. Dye displacement techniques (varying the salt concentration) have confirmed the increase of keratan sulfate with aging, the localized increases in chondroitin sulfate surrounding cells (compared to the interterritorial regions where there is an increase in keratan sulfate), the increased keratan sulfate with increased depth of cartilage, and the relative absence of glycosaminoglycan from the tangential zone. 4 0 · 4 5 With aging, the diameter of individual collagen fibrils increases significantly; however, the overall arrangement remains generally similar (Fig. 2 - 5 ) . 4 3 · 5 0 ' 5 1 Although individual collagen fibrils of the tangential zone remain aligned tangential to the articular surface, they are no longer arranged in tight bundles and often appear fragmented. 5 1 Collagen fibrils of exceedingly large diameter (more than 450 n m . ) , often containing nuclei of calcification, are commonly found in the vicinity of degenerating cells, matrix vesicles, and lipid accumulations (Figs. 2 - 8 and 2 - 9 ) . 1 · 6 · 7 · 5 0 · 5 1

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THE CELLS Immature articular cartilage contains two morphologically and metabolically distinct cell populations (Fig. 2 - 2 ) . At the surface, cells are elongated and tightly packed with their long axes parallel to the joint surface. These cells appear continuous with the perichondrium. There is an average of 1.5 times as many cells in the tangential or superficial zone per unit volume of tissue than is present in the underlying articular cartilage, and these cells incorporate one-third as much as proline and glycine, one-fifth as much sulfate and only one-tenth as much glucosamine as do the cells of the underlying articular cartilage. 54 The underlying cartilage cells are widely dispersed and rounded. Autoradiographic techniques have demonstrated two zones of cellular proliferation within this deeper layer.- 4 One of these zones is present immediately beneath the superficial or tangential zone, presumably accounting for growth of the articular cartilage. A second zone deep to this acts as a microepiphyseal plate for endochondral ossification of the underlying epiphysis (Fig. 2—2).24 The basilar portion of the articular cartilage is perforated by a rich vasculature similar to that seen in the epiphyseal plate. At the end of the epiphyseal growth, mitotic activity in normal cartilage ceases. 26 · 27 During maturation, the cell density of articular cartilage falls to about 14,500 cells/cjt. mm. in human femoral condylar cartilage 47 and appears to remain fairly constant. 29 · 44 At maturation, the cells in normal adult articular cartilage can be divided into four distinct zones (Fig. 2 - 1 ) . The zone closest to the articular surface—the tangential zone (the "skin" of cartilage)—contains several layers of flattened, discoid cells whose long axes are parallel to the articular surface. Deep to the tangential zone lies the transitional zone, which contains larger, rounded, and apparently randomly arranged cells. Below this region lie rounded cells, often in short irregular columns—the radial zone. Beneath the radial zone, cells in short columns are surrounded by a calcified matrix—the calcified zone. These cells often have pyknotic nuclei and do not incorporate 3H-cytidine.2r> In normal articular cartilage, vessels present at the basilar portion of the calcified zone do not penetrate the tidemark. In recent years, extensive ultrastructural studies have been performed on both human and other mammalian articular cartilage. These studies have further shown the consistent zonal differences in cellular morphology. Most of the discoid cells of the tangential zone appear quite similar to fibrocytes (Fig. 2 - 4 ) . 1 3 · 4 3 · 5 3 The nuclei are elongated and irregularly shaped, and contain a dense nucleoplasm with chromatin clumping. The cytoplasm is moderately dense and contains short cisternae of rough endoplasmic reticulum, a small Golgi apparatus, and small and rounded mitochondria. 13 · 35 · 41 · 53 Large lipid accumulations, glycogen deposits, and matrix containing vacuoles are rare. The cell membrane is fairly smooth on the superficial surface; however, the deep surface contains many short cytoplasmic processes. Pinocytotic vesicles are common. In the transitional zone (Figs. 2 - 5 and 2 - 6 ) , cells are rounded and are surrounded by a dense accumulation of fine fibers and filamentous fibrils ( 4 - 1 0 mm. in diameter). 5 3 These cells have many elongated cytoplasmic processes extending from the cell membrane. The nucleus is rounded, eccentric, and finely granular, and it often contains more than one nucleolus. The nuclear membranes have regularly

Light and Electron

Microscopic

Studies of Normal

Articular

Cartilage

17

35

spaced nucleopores. The ground plasm is of moderately low electron density and contains aggregates of glycogen particles and intracytoplasmic filaments (7 mm.— 10 mm. in diameter). 53 There are an extensive rough-surfaced endoplasmic reticulum, a large Golgi apparatus, and secretory vacuoles.3· "· 3 5 · 4 1 · 53 The roughsurfaced endoplasmic reticulum, Golgi apparatus, and secretory vacuoles contain fibrillar material similar in electron density and size to that found in the extracellular matrix.53 The juxtanuclear Golgi apparatus is especially well-developed, containing many lamellae of closely packed agranular membranes with small vesicles and larger vacuoles up to 2.5 μ in diameter. 13 · 39 · 41 ~ 43 · 53 The cell surface has a scalloped appearance, owing to the fusion of secretory vesicles with the external cell membrane and the discharge of intracellular matrix components to the external matrix.20 In the radial zone, cells have a less well-developed rough-surfaced endoplasmic reticulum, a sparse Golgi apparatus, small dense mitochondria, and an increased number of intracellular filaments (7 mm.-10 mm. in diameter). 53 Lipid and glycogen deposits are more common in the cells of the transitional and radial zone than in cells of the tangential zone. Cells within the calcified zone are in advanced stages of degeneration, with hyperchromic nuclei, disruption of cellular and nuclear membranes, and loss of organellar substructure. With aging, a number of significant changes can be observed in the cells of all zones. Viable cells appear greatly hypertrophied with extensive development of the rough surfaced endoplasmic reticulum, Golgi apparatus, and many mitochondria (Fig. 2-7). 5 0 · 5 1 Accumulations of glycogen and lipid droplets are larger and more frequent in older chondrocytes.3· 5 · 2 8 · 3 0 · 39· 45>50 There is an increased number of complex bodies found in many cells, and it has been suggested that these are derived from lysosomal degradative enzyme activity. An increase in the number of intracellular filaments (70 nm.-10 nm.) is also found (Fig. 2-5). 3 1 · 5 0 · 5 1 These intracellular filaments were thought to represent degenerative changes; however, recent studies have shown that despite the presence of extensive intracellular filaments, the synthetic capacity of the cells was not significantly altered if cell and nuclear membranes remained intact.55 Both intracellular and extracellular lipids increase with aging, most prominently in the superficial and transitional zones. 3 · 5 ·, 12 · 19 · 60 The extracellular lipids, by virtue of their distribution and composition, are thought to be of cellular origin, consisting of triglycerides, cholesterol, and phospholipids.5 The matrix of older articular cartilage contains many vesicles, which are often associated with matrix calcification and are probably secondary to fragmentation and degeneration of chondrocytes in aging articular cartilage (Figs. 2-5, 2-8, and 2 - 9 ) .

REFERENCES 1. Ali, S. Y., Bayliss, M. T.: Enzymic changes in human osteoarthritic cartilage. In Normal and Osteoarthritic Articular Cartilage, (Ali, S. Y., Elves, M. W., and Leaback, D. H., eds.). Instut. Ortho., London, 1974, p. 189. 2. Balazs, Ε. Α., Bloom, G. D., and Swann, D. Α.: Fine structure and glycosaminoglycan content of the surface layer of articular cartilage. Fed. Proc., 25:1813, 1966.

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3. Barnett, C. H., Cochrane, W., and Palfrey, A. J.: Age changes in articular cartilage of rabbits. Ann. Rheum. Dis., 22:389, 1963. 4. Benninghoff, Α.: Form and Bau der Gelenk-Knorpel in ihren Beziehungen zur Funktion. S. Anat. Entwicklungsgesch., 76:43, 1925. 5. Bonner, W. M., Jonsson, H., Malanos, C., and Bryant, M.: Changes in the lipids of human articular cartilage with age. Arth. Rheum. 18:461, 1975. 6. Bonucci, E., and Dearden, L. C.: Matrix vesicles in aging cartilage. Fed. Proc., 55:163, 1976. 7. Cameron, D. Α., and Robinson, R. Α.: Electromicroscopy of epiphyseal and articular cartilage matrix in the femur of the newborn infant. J. Bone Joint Surg., 40A: 163, 1958. 8. Clark, 1. C.: Surface characteristics of human articular cartilage: A scanning electron microscope study. J. Anat., 108:23, 1971. 9. Clark, I. C.: H u m a n articular cartilage surface contours and related surface depression frequency studies. Ann. Rheum. Dis., 50:15, 1971. 10. Clark, I. C.: Articular cartilage: A review and scanning electron microscope study. J. Bone Joint Surg., 5S:732, 1971. 11. Collins, D. H.: The Pathology of Articular and Spinal Disease. London, E. Arnold, 1949. 12. Collins, D. H., Ghadially, F. N., and Meachim, G.: Intracellular lipids of cartilage. Ann. Rheum. Dis., 24:123, 1965. 13. Davies, D. V., Barnett, C. H., Cochran, W., and Palfrey, A. J.: Electron microscopy of articular cartilage in the young adult rabbit. Ann. Rheum. Dis., 27:11, 1962. 14. Gardner, D. L., and McGillivray, D. C.: Living cartilage is not smooth. Ann. Rheum. Dis., 50:3, 1971. 15. Gardner, D. L., and McGillivray, D. C.: Surface structure of articular cartilage: Historical review. Ann. Rheum. Dis., 50:10, 1971. 16. Gardner, D. L., and Woodward, D.: Scanning electron microscopy and replica studies of articular surfaces of guinea pig synovial joints. Ann. Rheum. Dis., 28:319, 1969. 17. Gardner, D. L.: The influence of microscopic technology on knowledge of cartilage surface structure. Ann. Rheum. Dis., 57:235, 1972. 18. Gardner, D. L., and Longmore, R. B.: Age related studies of human articular cartilage. In Normal and Osteoarthritic Articular Cartilage (Ali, S. Y., Elves, M. W„ and Leaback, D. H., eds.). Instut. Ortho., London., 747:152, 1974. 19. Ghadially, F. N., Meachim, G., and Collins, D. H.: Extracellular lipid in the matrix of human articular cartilage. Ann. Rheum. Dis., 24:136, 1965. 20. Godman, G. C., and Lane, N.: On the site of sulfation in the chondrocyte. J. Cell Biol., 27:353, 1964. 21. Hunter, W.: On the structure and diseases of articulating cartilage. Phil. Trans., 42:514, 1743. 22. Little, K., Pimm, L. H., and Trueta, J.: Osteoarthritis of the hip: An electron micrographie study. J. Bone Joint Surg., 405:123, 1958. 23. McConnaill, Μ. Α.: The movements of bone and joints. IV. The mechanical structure of articulating cartilage. J. Bone Joint Surg., 33B:251, 1951. 24. Mankin, H. J.: Localization of tritiated thymidine in articular cartilage of rabbits. I. Growth and immature cartilage. J. Bone Joint Surg., 44A:682, 1962. 25. Mankin, H. J.: Localization of tritiated cytidine in articular cartilage of immature rabbits after intraarticular injection. Lab. Invest., 72:543, 1963. 26 Mankin, H. J.: Localization of tritiated thymidine in articular cartilage of rabbits. III. Mature articular cartilage. J. Bone Joint Surg., 45/1:529, 1963.

Light and Electron Microscopic

Studies oj Normal Articular Cartilage

19

27. Mankin, H. J.: The articular cartilages: A review. AAOS Instruct. Course Lect., 79:204, 1970. 28. Meachim, G. Age changes in articular cartilage. Clin. Orthop. 64:33, 1969. 29. Meachim, G., and Collins, D. H.: Cell counts of normal and osteoarthritic cartilage in relation to the uptake of sulfate ( 3r, S0 4 ) in vitro. Am. Rheum. Dis., 27:45, 1962. 30. Meachim, G., Ghadially, F. N., and Collins, D. H.: Regressive changes in the superficial layer of human articular cartilage. Ann. Rheum. Dis., 24:23, 1965. 31. Meachim, G., and Roy, S.: Intracytoplasmic filaments in the cells of adult human articular cartilage. Ann. Rheum. Dis., 26:50, 1967. 32. Meachim, G., and Roy, S.: Surface ultrastructure of mature adult human articular cartilage. J. Bone Joint Surg., 51B:529, 1969. 33. Miller, E. J., and Matukas, V. J.: Chick cartilage collagen: A new type of a l chain not present in bone or skin of the species. Proc. Nat. Acad. Sci. USA., 64:1264, 1969. 34. Muir, H., BuUough, P., and Maroudas, Α.: The distribution of collagen in human articular cartilage with some of its physiological implications. J. Bone Joint Surg., 52B:554-563, 1970. 35. Palfrey, A. J., and Davies, D. V.: The fine structure of chondrocytes. J. Anat., 100:213, 1966. 36. Radin, E. L., Swann, D., and Weisser, P. Α.: Separation of a hyaluronate-free lubricating fraction from synovial fluid. Nature, 228:337, 1970. 37. Redler, I., Mow, V. C., Zimny, M. L., and Mansell, J.: The ultrastructure and biomechanical significance of the tidemark of articular cartilage. Clin. Orthop. Rei. Res., 112:357, 1975. 38. Rosenberg, L.: Chemical basis for the histological use of safranin-O in the study of articular cartilage. J. Bone Joint Surg., 53A:69, 1971. 39. Rutner, J. R., and Spycher, Μ. Α.: Electron microscopic investigations on aging and osteoarthritic human cartilage. Pathol. Microbiol. 31:14, 1968. 40. Scott, S. E.: The histochemistry of cartilage proteoglycans in light and electron microscopes. In Normal and Osteoarthritic Articular Cartilage (Ali, S. Y., Elves, M. W., and Leaback, D. H., eds.) Instut. Ortho., London., 1974, p. 19. 41. Silberberg, R.: Ultrastructure of articular cartilage in health and disease. Clin. Orthop., 57:233, 1968. 42. Silberberg, R., Silberberg, M., and Feir, D.: Life cycle of articular cartilage cells: An electronmicroscopic study of the hip joint of the mouse. Am. J. Anat., 114: 17, 1964. 43. Silberberg, R., Silberberg, M., Vogel, Α., and Wettstein, W.: Ultrastructure of articular cartilage of mice of various ages. Am. J. Anat., 709:251, 1961. 44. Stockwell, R. Α.: The cell density of human articular and costal cartilage. J. Anat., 707:753, 1967. 45. Stockwell, R. Α.: The lipid and glycogen content of rabbit articular hyaline cartilage. J. Anat., 702:1, 1967. 46. Stockwell, R. Α., and Meachim, G.: Adult Articular Cartilage (M.A.R. Freeman, ed.). London, Pitman Medical, 1973, p. 55. 47. Stockwell, R. Α., and Scott, J. E.: Distribution of acid glycosaminoglycans in human articular cartilage. Nature, 275:1376, 1967. 48. Walker, P. S., Dowson, D., Longfield, M. D., and Wright, V.: Boosted lubrication in synovial joints by fluid enlargement and enrichment. Ann. Rheum. Dis., 27: 512, 1968. 49. Walker, R. S., et al.: Behavior of synovial fluid on surfaces of articular cartilage. Ann. Rheum. Dis., 25:1, 1969.

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50. Weiss, C.: An ultrastructural study of aging human articular cartilage (abstract). J. Bone Joint Surg., 53Λ:803, 1971. 51. Weiss, C.: Ultrastructural characteristics of osteoarthritis. Fed. Proc., 32:1459, 1973. 52. Weiss, C.: The structure and composition of articular cartilage. In Disorders of the Knee (A. J. Helfet, ed.). Philadelphia, Lippincott, 1974. 53. Weiss, C., Rosenberg, L., and Helfet, A. J.: An ultrastructural study of normal young adult human articular cartilage. J. Bone Joint Surg., 50A:663, 1968. 54. Weiss, C., Shapiro, F., Trahan, C., and Altmann, K.: The tangential zone of articular cartilage. J. Bone Joint Surg., 57/1:584, 1975. 55. Weiss, C., Trahan, C., Lippiello, L., and Altmann, K.: Intracellular filaments of articular cartilage: An ultrastructural and autoradiographic study. J. Bone Joint Surg., 57A:584, 1975. 56. Zelander, R.: Ultrastructure of articular cartilage. Z. Zellforsch. Mikrosk. Anat., 49:720, 1959.

JOHN R. PARSONS, Ph.D.

The Scanning

Electron

of Articular

Microscopy

Cartilage

The transmission electron microscope (TEM) revolutionized many aspects of biological research. A more recent development—the scanning electron microscope (SEM)—has a similar potential. For the most part, this potential has not been realized because of the fundamental differences between the two instruments. The TEM is similar in many respects to the optical microscope. Illumination, in this case produced by electrons, is collimated, passes through a thin specimen, and is focused on a phosphor screen. The phosphors are excited, giving rise to an image. In biologic specimens, contrast arises principally from variations in specimen density. Electron-dense areas appear dark; less dense areas are lighter. Contrast can be enhanced by using specific electron-dense stains. Particular specimen morphologies long recognized in the optical microscope are visible in the TEM, but in much greater detail. Furthermore, substructures, too small to be resolved optically, are available for study in the TEM. The principles of the SEM are quite different. In the SEM, an electrically conductive material* is scanned in a regular array by a small, intense beam of electrons. Backscattered or secondary electrons are emitted from the specimen surface as a result of this electron bombardment. These electrons are collected and processed. The net result is a three-dimensional picture of the specimen displayed on a television screen. It is possible to see only the surface of the bulk specimen with the SEM. The surface can be seen in great detail, however. Extreme depth of field and large range of magnification ( X 20 to X 100,000) make possible study of the roughest, most undulating surface. Unfortunately, morphologies associated with thin sections, as observed with the optical microscope or the TEM, are not easily recognized in the SEM. Specific optical or electron-dense strains have no application in the SEM. Additionally, * Biologic specimens must be dehydrated since the SEM operates at high vacuum. Additionally, the specimen must be made electrically conductive by applying a thin metallic layer, using evaporation techniques.

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artifacts associated with specimen preparation can be produced, tissue shrinkage during dehydration being the primary source. Even careful drying procedures involving critical-point or freeze-drying techniques produce notable shrinkage. For these reasons, the SEM has remained little more than a curiosity in many areas of biological research. The study of articular cartilage is an exception, however.

ARTICULAR CARTILAGE The function of articular cartilage is mechanical in nature, and is reflected in its physical structure. McCalP recognized this and was the first to use the S E M to study the surface of articular cartilage. He demonstrated differences in the surface appearance of normal and pathologic cartilage. Gardner and Woodward 7 pointed out that the three-dimensional structure of cartilage varied f r o m specimen to specimen, and even within areas of the same joint specimen. Shortly thereafter, they studied in detail the articular surfaces of guinea pig joints. 8 Walker et al. 14 described the behavior of synovial fluid on the surface of cartilage. In another study, Redler and Zimny 1 - used the SEM to examine the surface of normal and abnormal (rheumatoid and osteoarthritic) articular cartilage and synovium. This early work clearly demonstrated that in the dehydrated state, as prepared for the SEM, the surface of articular cartilage is not smooth. It consists of a dense fibrillar network. The fibrils (0.1 μ - 0 . 2 μ in diameter) appear to lay parallel to the surface. Parallel arrays of ridges 2 μ to 6 μ in width are noted in selected areas. Round or oval, mound-like undulations 10 μ to 4 0 μ in diameter are prominent features. Pathologic tissue, even that which appears grossly normal, has a severely disrupted surface at the ultrastructural level. Normal surface structures and contours are, in general, obliterated. Are these observations artifact? Do they exist in vivo in the hydrated state? What physical significance, if any, can be attributed to these features? These are the logical questions posed by this early work. Gardner and McGilliwray"'· 6 using optical microscopy techniques, examined fresh (in vivo), fully hydrated cartilage in a number of species, including human. R o u n d undulations of 10 μ to 4 0 μ were visible. Clark 1 observed similar 10 μ to 4 0 μ undulations in another SEM study. Using scanning and optical techniques, Clark 2 , * subsequently demonstrated a correspondence in size, shape, and distribution between those round protuberences and subsurface chondrocytes. Presumably, these undulations are formed by a surface layer that closely conforms to the contours of underlying lacunar structures. These contours are present in both hydrated and dehydrated states. More recently, Mow et al. 10 examined the parallel ridge patterns observed in some areas of the surface of articular cartilage. These patterns were first observed by McCall, 9 Walker et a l . , " and Clarke. 1 Clarke 1 3 thought ridge patterns to be artifactual, occurring near the edge of specimens as the result of tissue shrinkage. Mow et al. 10 suggested that the parallel ridge patterns are due to instability of the articular surface. This instability results from the dynamic interaction of synovial fluid and articular cartilage during sliding motion of the joint. T h e ridge patterns occur in vivo and are presumable preserved by rapid-fixation and dehydration techniques.

The Scanning Electron Microscopy of Articular Cartilage

23

In addition to surface structure, the structure of deeper layers, as seen in cross section, is of interest. Clark 3 reviewed the literature and presented an SEM study concerned primarily with the structure and organization of the fibrillar architecture of articular cartilage. Both surface and deeper layers were examined. Later, Redler 11 described the fibrillar structure of cartilage. Both authors are in general agreement. These works suggest zones having distinct ultrastructural characteristics. The superficial tangential zone of cartilage (50 μ-200 μ thick) consists of sheets composed of a network of fine collagen fibrils. In the midzone, between the superficial, tangential zone and the calcified zone, fibrils vary in diameter from approximately 0.2 μ in the upper portion to 1 μ in lower levels. Also fibrils in the upper portion of the midzone are tightly compacted and randomly oriented. In the lower portion of the midzone, fibrils are less tightly packed and are more nearly perpendicular to the surface. Many fibrils are continuous with those in the calcified zone. The fibrillar structure of the midzone, and particularly superficial tangential zone, is severely disrupted in osteoarthritic tissue. The relationship of chondrocytes to this fibrillar structure was investigated by Zimmy and Redler. 15 The cells appeared round to oval. The cell surface shared intimate contact with fibrous elements of the matrix. Matrix fibrils were seen to form a nest about the cell. In osteoarthritic tissue, many chondrocytes were devoid of this well-defined lacunar structure. Clumps and rows of cells were often seen." Similar clumps of cells were found in an experimentally induced degenerative condition. 13 This increased cellularity suggests chondrocyte stimulation in an attempt to repair or maintain cartilage integrity.

SUMMARY Based on the literature reviewed, and on personal observation, 13 articular cartilage as viewed in the SEM appears as follows: 1. Articular surface : This surface is composed of a dense network of fibrils 0.1 μ to 0.2 μ in diameter. Parallel ridges 2 μ to 6 μ in width exist in selected areas. Round or oval undulations 10 μ to 40 μ in diameter are frequently observed. These structures often appear in pairs. These undulations correspond to subjacent condrocytes. Surface structure is severely disrupted in pathologic tissue. 2. Fibrillar Architecture: The fibrillar structure of the superficial tangential zone is that of thin sheets composed of networks of fine collagen fibrils. The midzone is composed of fibrils ranging in diameter from 0.2 μ in the upper portion to 1 μ in the lower portion. The fibrils in the upper portion of the midzone are closely packed and randomly oriented. In the lower portion of the midzone, the fibrils are less closely packed and are more nearly perpendicular to the articular surface. Many of these fibrils continue into the calcified zone. Fibrillar structure, particularly in the superficial tangential zone, is obliterated in pathologic tissue. 3. Chondrocytes: The cells of articular cartilage appear round or oval. The cell surface seems to share an intimate contact with the fibrous elements of the matrix. These elements form a cell nest. These fibrous structures are disrupted in osteoarthritic tissue. Clumps and rows of cells are often seen in disrupted tissue. Many of these general features are seen in the following series of micrographs (Fig. 3 - 1 ) .

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Figure 3-1. Articular surface of femoral condyle of a rabbit containing fissure of undetermined origin. A, Note textured appearance of surface, ( x 100) B, 20 μ. undulations ira the surface are visible. (X300) C, Detail of fissure, showing a subsurface matrix and chondrocyte (arrow), ( x 1000) D, Chondrocyte and lacunar structure, ( x 10,000)

REFERENCES 1. Clarke, I. C.: Surface characteristics of human articular cartilage—A scanning electron microscopic study. J. Anal., 108:23, 1971. 2. Clarke, I. C.: Human articular surface contours and related surface depression frequency studies. Ann. Rheum. Dis., 30:15, 1971.

The Scanning Electron Microscopy

of A rticular Cartilage

25

3. Clarke, I. C.: Articular cartilage: A review and scanning electron microscope study. J. Bone Joint Surg., 53B:732, 1971. 4. Clarke, I. C. T h e Microevaluation of articular surface contours. Ann. Eng., 7:31, 1972.

Biomed.

5. Gardner, D. L., and McGilliwray, D. C.: Living articular cartilage is not smooth. Ann. Rheum. Dis., 30:3, 1971. 6. Gardner, D. L., and McGilliwray, D. C.: Surface structure of articular cartilage: A historical revue. Ann. Rheum. Dis., 30:10, 1971. 7. Gardner, D. L., and Woodward, D. H.: Scanning electron microscopy of articular surfaces. Lancet, 2:1246, 1968. 8. Gardner, D. L., and Woodward, D. H.: Scanning electron microscopy and replica studies of articular surfaces of guinea-pig synovial joints. Ann. Rheum. Dis., 28:379, 1969. 9. McCall, J. G.: Scanning electron microscopy of articular surfaces. Lancet, 2:1194, 1968. 10. Mow, V. C., Lai, W. M., and Redler, I.: Some surface characteristics of articular cartilage. I. A scanning electron microscopy study and a theoretical model for the dynamic interaction of synovial fluid and articular cartilage. Π. On the stability of articular surface and a possible biomechanical factor in etiology of chondrodegeneration. J. Biomechan., 7:449, 1974. 11. Redler, I.: A scanning electron microscopic study of human normal and osteoarthritic articular cartilage. Clin. Orthop., 103:262, 1974. 12. Redler, I., and Zimny, M. L.: Scanning electron microscopy of normal and abnormal articular cartilage and synovium. J. Bone Joint Surg., 52A: 1395, 1970. 13. Simon, W. H., Richardson, S., Herman, W., Parsons, J. R., and Lane, J.: Longterm effects of chondrocyte death on rabbit articular cartilage in vivo. J. Bone Joint Surg., S8A:517, 1976. 14. Walker, R. S., et al.: Behavior of synovial fluid on surfaces of articular cartilage. Ann. Rheum. Dis., 25:1,1969. 15. Zimny, M. L., and Redler, I.: Scanning electron microscopy of chondrocytes. Acta Anat., 55:398, 1972.

L

±f

LAWRENCE ROSENBERG, M.D.

Structure

of Cartilage

Proteoglycans

Articular cartilage is a highly specialized connective tissue whose mechanical properties are essential to the function of the musculoskeletal system. Articular cartilage is a hard yet elastic tissue that provides a smooth gliding surface for diarthrodial joints. Articular cartilage transmits load, absorbs impact, and resists shearing forces, yet it resists wear to a surprising degree. The remarkable mechanical properties of articular cartilage are directly related to the structure of the tissue. Articular cartilage consists of relatively few cells distributed throughout an abundant extracellular matrix. The extracellular matrix is composed mainly of collagen, proteoglycans, and water. Collagen is an insoluble fibrous protein with tensile strength. Proteoglycans are elastic molecules that tend to expand in solution, and that resist compression into a smaller volume of solution. The mechanical properties of cartilage appear to result from the tensile properties of collagen, the elastic properties of proteoglycans, and the properties of the fibrous composite formed when proteoglycans at high concentration are entangled in a dense network of collagen fibers. In the formation of both collagen and proteoglycans, a basic structural unit of relatively low molecular weight is first formed. Many basic structural units then associate with one another to form an aggregate of much higher molecular weight that possesses the characteristic properties of the biochemical species. T h e basic structural unit of cartilage ground substance is the proteoglycan subunit ( P G S ) . Figure 1 - 4 shows a tentative diagrammatic model of the structure of the PGS. The PGS consists of a protein core to which are attached chondroitin sulfate and keratan sulfate side chains. Chondroitin sulfate is covalently attached to the hydroxyl groups of serine residues of the protein core via the neutral sugar trisaccharide Gal—Gal—Xyl—Ser. Keratan sulfate is attached to serine and threonine residues via ^-acetylgalactosamine, to which residues of galactose and sialic acid are also attached. Chondroitin sulfate and keratan sulfate are two mem26

Structure of Cartilage Proteoglycans

II 1 1 1 1 1 1 J," 1 III "1 "1 "1 "1 1

27

KERATAN SULFATE

J,, 1 T \

CORE PROTEIN LINKAGE REGION CHONDROITIN SULFATE

Figure 4-1.

The proteoglycan subunit (PGS).

bers of a group of linear polymers composed of sugar residues called glycosaminoglycans. Glycosaminoglycans are composed of two different sugar residues that alternate regularly in the polymer chain. These alternating sugar residues form disaccharide repeating units in terms of which the structure of a particular glycosaminoglycan is described. In chondroitin sulfate, ^-acetylgalactosamine alternates with glucuronic acid to form the disaccharide repeating unit of the polymer. In keratan sulfate, the disaccharide repeating unit consists of /V-acetylglucosamine alternating with galactose. The amino sugars of both chondroitin sulfate and keratan sulfate carry ester sulfate groups bound to carbon number four or six. The galactose residue of keratan sulfate may also be variably sulfated. Chondroitin sulfate and keratan sulfate therefore carry negatively charged sulfate and carboxylate groups as closely spaced as 5 A intervals along the polymer chain. The repelling forces of these closely spaced negatively charged groups cause the glycosaminoglycan chains to assume a stiffly extended conformation and to stick out from the protein core of the proteoglycan subunit like teeth on a comb or bristles on a brush. Because of the repelling forces of these closely spaced, negatively charged groups, the PGS molecule resists compression into a smaller volume of solution.

STRUCTURE OF PROTEOGLYCAN AGGREGATES In the ground substance of native cartilage, most of the proteoglycan exists as aggregates of high molecular weight, formed by the noncovalent association of PGS molecules with hyaluronic acid and link protein. Figure 4 - 2 shows a twodimensional model of the structure of the proteoglycan aggregate. Hyaluronic acid forms the filamentous backbone of the proteoglycan aggregate, to which many proteoglycan subunit molecules are noncovalently bound at fairly regular intervals. 1-9 PGS core protein contains a globular region devoid of glycosaminoglycan chains at one terminus, which contains the binding site for hyaluronic acid. This globular region has been called the hyaluronic acid-binding region of the PGS core protein. 7-9 The other terminus of the PGS core protein appears to have an extended conformation and contains the serine and threonine residues to which chondroitin

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sulfate and keratan sulfate chains are attached. This region has been called the polysaccharide-attachment region of the PGS core protein. Between the hyaluronic acid-binding region and the polysaccharide-attachment region, the PGS core protein contains a relatively small region of peptide containing keratan sulfate but essentially no chondroitin sulfate, called the keratan sulfate-rich region. The elastic properties of cartilage depend in part on the presence in ground substance of proteoglycan aggregates. The formation of aggregates depends in turn on the formation of a noncovalent bond between the hyaluronic acid-binding region of the PGS core protein and hyaluronate. The nature and properties of this bond, specifically the residues and functional groups involved in its formation, and its stability under various conditions are of special current interest. The noncovalent bond between the PGS and hyaluronate is broken in concentrated solutions of guanidine hydrochloride, calcium chloride, and magnesium chloride, leading to a reversible dissociation of the proteoglycan aggregate. This effect is the basis for the use of these agents to dissociatively extract PGSs, link protein, and hyaluronate from cartilage. The bond is also broken at pH 4, at which the carboxyl groups of hyaluronate are protonated, suggesting that these groups may be involved in the binding of the PGSs to hyaluronate.

Structure of Cartilage Proteoglycans

29

Link protein is a low-molecular-weight protein (40,000 to 50,000 daltons) that is a component of proteoglycan aggregates from every cartilage so far examined. In the absence of link protein, the noncovalent bond between the PGS core protein and hyaluronate is relatively weak. This bond is strong enough to be maintained under some conditions, such as experiments involving chromatography, but it is weak enough to be broken under other conditions, such as experiments in the analytical ultracentrifuge. Therefore, although the PGS and hyaluronate associate in the absence of link proteins to form aggregates demonstrable on gel chromatography, these aggregates dissociate on analytical ultracentrifugation. When large PGSs are mixed with hyaluronate and the mixture is examined in the analytical ultracentrifuge, no aggregates are demonstrable in the absence of link protein. When link protein is added to the mixture, typical proteoglycan aggregates are formed that are stable during analytical ultracentrifugation. Link protein appears to strengthen and stabilize the binding of the PGS to hyaluronate.

POLYDISPERSITY OF PROTEOGLYCAN SUBUNITS (PGSs) Closer examination of Figure 4-2 indicates that PGSs are polydisperse in length, molecular weight, and composition. The PGSs are a polydisperse population of molecules in which the molecular weights of individual members increase in proportion to their chondroitin sulfate content and their chondroitin sulfate-to-protein ratio. One question of current interest is the structural basis for the observed polydispersity of PGSs. To answer this question, polydisperse PGSs from bovine articular cartilage have been separated into a series of monodisperse fractions that have been characterized in terms of chemical composition and physical properties.® The chondroitin sulfate content of each fraction increased from 27.2% to 56.3% as the sedimentation coefficient of the PGS species increased from 5.7 S to 14.3 S. The protein content of the fractions increased from 9.9% to 30.7% as the size of the PGS species decreased from 14.3 S to 5.7 S. As the PGS species decreased in molecular weight, a larger proportion of the molecule appeared to consist of protein devoid of glycosaminoglycan. In addition, the amino acid composition of the PGS changed characteristically with decreasing molecular weight. The amino acid composition of PGS molecules of the lowest molecular weight was relatively high in aspartic acid, cysteine, and methionine and closely resembled that of the hyaluronic acid-binding region isolated by Heinegard and Hascall.7 As the size of the PGS species increased from 5.7 S to 14.3 S, there was a progressive increase in serine and glycine residues found in the attachment sites of chondroitin sulfate chains within the polysaccharide attachment region. The pattern of polydispersity observed supports the concept that PGS core protein contains a hyaluronic acid-binding region of constant size and composition, rich in cysteine, methionine, and aspartic acid, located at one terminus of the molecule, and a polysaccharide-attachment region of variable length composed of variable numbers of Ser—Gly— containing peptides providing attachment sites for chondroitin sulfate chains, extending toward the other terminus of the molecule. The polydispersity of PGS appears to be determined largely by the variable length of the polysaccharide attachment region of PGS core protein. 7 · 8

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REFERENCES 1. Hardingham, Τ. E., and Muir, H.: The specific interaction of hyaluronic acid with cartilage proteoglycan. Biochim. Biophys. Acta, 279:401, 1972. 2. Hardingham, T. E., and Muir, H.: Hyaluronic acid in cartilage. Biochem. Soc. Trans., 1:282, 1973. 3. Hardingham, T. E., and Muir, H.: Binding of Oligosaccharides of Hyaluronic Acid to Proteoglycans. Biochem. J., 135:905, 1973. 4. Hardingham, T. E., and Muir, H.: Hyaluronic acid in cartilage and proteoglycan aggregate. Biochem. J., 139:565, 1974. 5. Hascall, V. C., and Heinegard, D.: Aggregation of cartilage proteoglycans. I. The role of hyaluronic acid. J. Biol. Chem., 249:4232, 1974. 6. Hascall, V. C., and Heinegard, D.: Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors of the proteoglycan-hyaluronic acid interaction. J. Biol. Chem., 249:4242, 1974. 7. Heinegard, D., and Hascall, V. C.: Aggregation of cartilage proteoglycans. III. Characteristics of the proteins isolated from trypsin digests of aggregates. J. Biol. Chem., 249:4250, 1974. 8. Rosenberg, L., Hellmann, W., and Kleinschmidt, A. K.: Electron microscopic studies of the proteoglycan aggregates from bovine articular cartilage. J. Biol. Chem., 250:1877, 1975. 9. Rosenberg, L., Wolfenstein-Todel, C., Margolis, R., Pal, S. and Strider, W.: Proteoglycans from bovine proximal humeral articular cartilage. Structural basis for the polydispersity of proteoglycan subunit. J. Biol. Chem., 257:6439, 1976.

JOSEPH M. LANE, M D.

Articular

Cartilage

in Health

and

Collagen Disease

Technical advances in the study of mammalian collagen have enabled the detailed investigation of this major structural protein of articular cartilage. The difficulty of collagen extraction from cartilage and bone has prevented its characterization in hard connective tissues until recently. Since the initial isolation of a separate collagen from cartilage by Miller and Matukas in 1969, there have been many new advances in detailing cartilage collagen metabolism and structure in normal and disordered cartilage. Articular cartilage is a highly specialized connective tissue consisting of a relatively small number of cells distributed throughout abundant extracellular matrix. The unique composition of the extracellular matrix accounts for the specialized properties of articular cartilage, particularly in its role as a bearing in locomotion. Collagen constitutes approximately 10% of the wet weight and 50% of the dry weight of cartilage. The bulk of the noncollagenous mass of articular cartilage is proteoglycans. The biomaterial properties of cartilage are a consequence of the interaction of the proteoglycans and the collagen in the extracellular matrix. Whether the interaction is a spatial mechanical intertwining of the collagen and the proteoglycans or a chemical relationship, the collagen effectively serves as a membrane to resist the flow of proteoglycans while preserving the proteoglycan colloidal properties. The collagen provides a taut structure to restrain the proteoglycan gel as it sustains the compressive loads of the articular cartilage. The collagen fibrils anchor the ground substance to the subchondral bone by being directly embedded in the calcified cartilage matrix. Alterations in collagen metabolism, structure, or content would be expected to have significant effects on the biomaterial properties of articular cartilage. Under electron microscopy, collagen microfibrils demonstrate a typical banded pattern which results from the quarter stagger layering of the individual collagen molecules. The single tropocollagen molecular unit of the microfibrils has the dimensions of a rod 3000 A long and 15 A in diameter. Each molecule consists of 31

32

HUMAN

JOINTS

IN

HEALTH Table 5-1

Amino Acid Composition of Interstitial and Cartilage Collagen Alpha Chains from Chick Bone [«1 (I)]2a- and Cartilage [α1(Π)]3 Collagen Chick Cartilage [«1 (Π)] 3

Chick Bone [αϊ (I)] 2 a2 a2

a l (1) (residues/1000

Amino Acid 3 -Hydroxyproline 4-HydroxyproIine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine

0.9 102.0 42.0 19.0 29.0 78.0 118.0 330.0 129.0 14.0 8.6 6.1 20.0 2.1 14.0 5.5 29.0 2.8 51.0

a l (Π) total residues)

0.8 100.0 48.0 19.0 29.0 67.0 118.0 329.0 102.0 28.0 6.0 18.0 32.0 2.0 14.0 8.0 24.0 7.1 49.0

2.2 103.0 42.0 26.0 26.0 87.0 115.0 329.0 104.0 16.0 11.0 7.8 26.0 2.2 15.0 23.0 13.0 2.0 50.0

three polypeptide chains coiled in a unique rigid helical structure. The triple helix imparts a specific X-ray diffraction pattern to the collagen molecule. Most body collagens consist of two alpha-1 chains and a single alpha-2 chain—designated («1) 2 α2. The alpha-1 and alpha-2 chains are similar in polar groupings, but their amino acid compositions differ sufficiently for the chains to be separable by SDS electrophoresis and ion-exchange chromatography. Each chain contains about 1000 amino acids, with a total molecular weight of 95,000 daltons. The properties of the polypeptide chains are in part due to the rigid amino acid repeating triplets of G l y — X — Y , in which X is frequently proline and Y is hydroxyproline. Hydroxylysine, an amino acid essentially unique to collagen, and lysine provide the cross-linkage between collagen molecules by forming aldol condensation products and Schiff bases with the epsilon amino group and the oxidative deaminated aldehyde forms. Involved cross-link forms have been identified, including a Afunctional cross-link involving an aldol condensation product and a histidine residue. Some of the hydroxylysine residues are glycosylated and may function as determinants of fibril diameter. Miller, Nimni, and Trelstad have recently shown that cartilage contains a genetically distinct type of collagen. Unlike the interstitial collagen, cartilage col-

Articular

Cartilage Collagen in Health and Disease

33

Table 5-2 Amino Acid Composition of Homologous Cyanogen Bromide Peptides from Interstitial «1 (I) and Cartilage a l (II) from Chick Bones «1 (I)-CB2 and Cartilage