ABNORMAL SOFTENING IN ARTICULAR CARTILAGE

Transcription

1 1209 ABNORMAL SOFTENING IN ARTICULAR CARTILAGE Its Relationship to the Collagen Framework NEIL D. BROOM Abnormal softening in articular cartilage is related to the presence of collagen fibers strongly aligned in a radial direction. In this paper, a morphologic model is proposed that provides a mechanism by which these radial arrays can develop passively from the more random, 3-dimensional network of fibers observed in normal articular cartilage. The ability of articular cartilage to sustain repeatedly applied compressive stresses over a lifetime of normal joint function is attributed to the arrangement of collagen fibers that form a captive interlocking meshwork for the hydrophilic matrix proteoglycan molecules (1,2). As the cartilage is compressed, the collapse of the collagen network is countered by the resistance offered by the proteoglycan gel. The precise nature of the forces exerted through the network will be highly complex and related both to the mechanical entrapment and chemical bonding effects between these fibers and the proteoglycans. Very little is known about the nature of these interaction effects, largely because it is impossible to examine articular cartilage ultrastructurally while simultaneously applying deforming loads to the tissue in its normal functional condition. Any significant change in the configuration of the collagen network will in turn influence the way in which the proteoglycan aggregates are immobi- From the Department of Mechanical Engineering, School of Engineering, University of Auckland, New Zealand. Neil D. Broom, PhD: Medical Research Council Senior Fellow, School of Engineering, University of Auckland. Address reprint requests to Neil D. Broom, PhD, Department of Mechanical Engineering, School of Engineering, University of Auckland, Private Bag, Auckland, New Zealand. Submitted for publication July 21, 1981; accepted in revised form March 4, lized within the cartilage, thus altering its functional behavior. In considering the general problem of abnormal softening in articular cartilage, a thorough understanding of the relationship between structure and functional response of normal articular cartilage is important. Only then can comparative studies of the 2 conditions provide some insight into the origin of softening in articular cartilage. Recently, a preliminary study of the morphologic/mechanical response of normal articular cartilage maintained in a nonviable physiologic condition was made (3-5). With the use of simultaneous micromechanical testing and interference light microscopy, combined with transmission electron microscopy, it has been possible to image the fibrous components of articular cartilage while simultaneously subjecting it to loading conditions in its wet functional state. These studies have helped explain how the collagenous structures, in their primary task of load bearing, are integrated from the articulating surface through to the calcified cartilage. The following is a study of abnormally soft articular cartilage. It clarifies the relationship between abnormal mechanical function and changes in the morphology of the collagen framework. MATERIALS AND METHODS Tissue sources. Fresh samples of softened articular cartilage were obtained from the knee joint tibia1 surfaces of 16 mature bovine animals. The particular region of the joint surface sampled was that corresponding to the central area, which is not normally in direct contact with the semilunar menisci. Tissue from these central areas, in contrast to the surrounding tissue, appeared less shiny and manifested a distinct softening when indented with a blunt probe. These Arthritis and Rheumatism, Vol. 25, No. 10 (October 1982)

2 1210 BROOM NE E ol g 100 LU E v) W h v) v) W g 5c I s patellar groove/ / COMPRESSIVE STRAIN pct Figure 1. Compressive stresdstrain curves for normal and softened bovine articular cartilage. characteristics are consistent with those observed in previous studies of tissue from similar regions of canine knee joints exhibiting early stages of either natural or surgically induced osteoarthritis (6,7). Tissue testing and observation. The procedure used for obtaining fresh specimens suitable for combined optical and deformation studies was similar to that described by Broom and Myers (4). The system of Nomarski interference optical microscopy and simultaneous micromechanical testing was also described in that paper. Basically, this technique makes it possible to view microscopically the structural response of cartilage under conditions of compressive loading that approximate the physiologic in vivo response of the tissue. When load is applied to articular cartilage, there is a time-dependent expression of fluid from the matrix when the highly polyanionic proteoglycans release hydrated water. This process is sensitive to the rate of application of load. To eliminate variations in stresshtrain response caused by different loading rates, all tissue specimens were deformed at a predetermined strain rate of 6 x sec-'. When applied to the study of fresh, unstained articular cartilage, the optical technique cannot make the distinction between dead and living cells that is normally indicated through loss of nuclear staining (8). Nor can it indicate depletion of matrix proteoglycans. Several samples were therefore stained in their fresh condition with toluidine blue A Figure 2. Response of slice of normal bovine articular cartilage to compression. C = Compression anvil; B = subchondral bone. A, Zero load. B, 200 gm/mm2. (Original magnification ~33.) B

3 ABNORMAL SOFTENING IN CARTILAGE A B Figure 3. Response of slice of softened bovine articular cartilage to compression. C = Compression anvil; B = subchondral bone. A, Zero load. B, 50 gm/mm*. (Original magnification ~33.) (Michaelis-veronal acetate hydrogen chloride buffer at ph 4.5) to provide some qualitative indication of proteoglycan depletion. Comparison of staining patterns was made only between different regions of the same slice exposed uniformly to the histologic dye. Figure 4. Low-magnification view of a section of articular cartilage spanning the normal (N) and softened (S) regions of the joint surface. (Original magnification ~7.) RESULTS Stresslstrain and flow-pattern behavior. Compressive stress/strain curves for both normal and softened bovine tibia1 plateau articular cartilage were obtained (Figure 1). Also included for comparison were data from normal patellar and femoral condyle surfaces. Although responses varied considerably within both conditions, the softened tissue exhibited only minimal resistance to extensive deformation as compared with the normal articular cartilage. Representative examples of the difference in the pattern of compressive deformation between the 2 conditions of articular cartilage are shown in Figures 2 and 3. Whereas the tissue approximately 42%, with a sustained compression of 200 gm/ mm2 (Figure 2), the softened cartilage deformed to 71%

4 1212 BROOM A B C Figure 5. A, Middle zone of normal articular cartilage exhibiting a fine, relatively amorphous matrix texture. Some radial alignment is visible in the pencellular regions (X). Arrow indicates radial direction. B, Middle zone of transition region between normal and softened articular cartilage. Arrow indicates radial direction. C, Relatively coarse, radial arrays of fibers in middle zone of softened articular cartilage; uncompressed tissue. D, Same region as C, compressed in radial direction (arrows) to demonstrate collapse of the radial structure. (Original magnification x 525.) D

5 ABNORMAL SOFTENING IN CARTILAGE 1213 strain, with a sustained stress of less than SO gm/mm2 (Figure 3). At this low magnification, the chondrocytes image as small dots and conveniently act as markers delineating the pattern of flow as the tissue is compressed. The pattern of flow was quite different in the 2 conditions. The softened tissue exhibited a visible horizontal flow line, indicating that considerable sideways or lateral flow had occurred right up to the calcified cartilage substrate. In contrast, the normal tissue exhibited only minimal lateral flow. Microstructure and response to compression. Some aspects of the structural response of normal articular cartilage have been described in earlier studies (4). Figure 4 shows a low-magnification view of an articular cartilage section. After its release from the calcified substrate, the section curled up toward the articular surface. Note the gross change in appearance from the normal to the softened sites and also the greater thickness of the softened tissue perpendicular to the joint surface. Proceeding across the joint surface from the normal to the softened region revealed a striking change in matrix texture. The matrix of the normal tissue was extremely fine and, except for some local regions of radial texture (i.e., in surface-to-bone direction), difficult to resolve optically (Figure SA). Any waveform was very fine, and mostly confused, possessing only a limited phase relationship with adjacent fibers. The extent and prominence of this radial structure progressively increased as the softened region was approached (Figure SB), until, in the softened tissue proper, a relatively coarse collagenous structure, strongly aligned in the radial direction and frequently possessing a waveform or crimp, dominated the middle and deep zones of the tissue (Figure SC). In response to direct compression, these radial arrays of fiber bundles collapsed in sympathy into an acute waveform with little increase in load (Figure SD). In some parts of the softened cartilage, these prominent radial arrays had degenerated into a loose teased-out network of fiber bundles (Figure 6). Staining behavior of the middle and deep zones. The normal tissue stained metachromatically with toluidine blue. The capsular or territorial regions of Figure 6. Middle zone of softened articular cartilage exhibiting a very loosened radial structure of collagen bundles. (Original magnification x 525.) Figure 7. Intact surface (arrow) of normal bovine articular cartilage. (Original magnification ~210.)

6 1214 BROOM Figure 8. Radial arrays of fibers exposed at the articular surface (arrow). (Original magnification ~210.) the chondrocytes exhibited the usual, more intense staining pattern, reflecting the higher acid mucopolysaccharide concentrations in the vicinity of the chondrocytes (9). The softened tissue also stained metachromatically but at a reduced intensity compared with the normal tissue. There was a marked loss or even absence of staining in those regions of the softened tissue where the fiber bundles had become loosened into an open structure (Figure 6). Surface morphologies. The surface of the normal tissue was generally intact (Figure 7), whereas the softened tissue exhibited varying degrees of disruption. This disruption took a variety of forms: 1. Minor irregularities in the surface 2. Tuft formation, resulting from partial delamination of the tangential surface structure 3. Surface clefts, which frequently extended into the underlying, radially orientated fibers 4. A complete removal of the superficial layers that exposed the underlying radial structure to the articulating surface (Figure 8). Erosion along the direction of fiber alignment in these exposed radial arrays produced a teasing-out of individual fiber bundles. DISCUSSION The single most distinctive feature in this study was the gradual change in the appearance of the collagenous structure from the normal to the softened regions of the joint surface. The fibrous structures observed in the normal regions were consistent with both the earlier studies of the present author (3,4) as well as those of several other workers. These studies have described the fibrous architecture in the middle zones of articular cartilage as a largely random network of fibers with some increased radial alignment in the deep zone to facilitate the tethering of the compliant tissue to the subchondral bone (l,lo,ll). It is generally agreed that the fine, 3-dimensional network produced by this largely random distribution of fibers provides the most effective structure for containing the hydrated proteoglycan gel under direct compressive loading. In the present study, a well-defined radial structure progressively developed as the soft-tissue region was approached. In this fully softened condition, the collagen fibers in the middle and deep zones were almost entirely grouped into radial arrays easily imaged in the optical microscope. Such a fibrous structure is no longer able to offer a 3-dimensional captive environment for the proteoglycan gel and will readily collapse under compressive loading (Figure 5). How then has this dramatic change in the fibrous structure taken place? If it were the result of a major rebuilding of the collagen framework, it would require a complete turnover of the matrix collagen, with the original network being replaced by this new radial structure. Not only is there little evidence for significant turnover in mature cartilage (12,13), but also it is difficult to envisage physically a strategy of morphologic change in which an active remodeling process could produce such distinct radial arrays without forming disruptive discontinuities between the existing, old framework and the new. A morphologic model. What is required, then, is a model that can satisfactorily account for the observed gradual increase in the number of radial fiber bundles as the softened tissue is approached. The model must comply with the following conditions: 1. It should involve little or no metabolic turnover of collagen and yet provide a way of changing from an apparently random 3-dimensional network to a radial structure without any gross repositioning of whole lengths of fibers. 2. It should account for the observed continuity of structure from normal to softened regions, i.e., it

7 ABNORMAL SOFTENING IN CARTILAGE 1215 a I b Figure 9. A schematic representation of the morphologic model proposed by the author. a, Normal tissue, with radial configuration of fibers; b, ultrastructural level of each fiber; c, ultrastructural view of array of adjacent fibers. should account for the radial structure s gradual development from the original, more random network of fibers. 3. It should explain the diverse range of morphologies of the collagenous structure in articular cartilage reported in the literature (10,14). 4. It should be consistent with the observed large difference in compressive compliance between normal and softened tissue. 5. It should account for the increase in thickness of the softened tissue. A schematic of the proposed model is shown in Figure 9. In the normal tissue the overall configuration of fibers beneath the superficial layers is radial (Figure 9a). Imposed upon this at an ultrastructural level, however, is a repeated, short-range change in direction along the length of each fiber (Figure 9b). Therefore, when viewed ultrastructurally, an array of adjacent fibers will present as a more random, 3- dimensional network (Figure 9c). The mechanical integrity of this interlocking network would presumably result from the strong collagen-proteoglycan interactions believed to occur in articular cartilage (12,15). It is likely, in fact, that the short-range changes in direction in the collagen fibers are a direct result of the complex-constraining influences of these interactions. The interlocking network would therefore represent an equilibrium config- C uration of high stored energy or internal stress. If there were a loss of such matrix interactions in any region, this configuration would revert to a more aligned, local configuration of adjacent fibers. Aggregates of such fibers would then be easily viewed in the light microscope. A more extensive breakdown of the matrix interactions would ultimately lead to the re-formation of the strong radial configurations (Figure 9). This structure, having lost its 3-dimensional character, would have only limited ability to contain the swelling proteoglycan aggregates remaining in the tissue, and would therefore reflect a state of softening in articular cartilage. This model satisfies all of the conditions outlined earlier. No metabolic turnover of collagen is required, nor need there be any gross movement or repositioning of whole lengths of fibers. The random, 3-dimensional network is achieved solely through short-range sideways deflection of short lengths of the collagen fibers (condition 1). This model accounts for the gradual change in morphology from the normal to the softened sites as well as the wide range in morphologies reported in the literature (conditions 2 and 3). The model is consistent with the observed, very large differences in mechanical compliance of the different regions in articular cartilage (condition 4). Lastly, it explains the increased thickness of the softened tissue compared with that of normal tissue (condition 5). As the fibers lose their short-range randomness, their increased length will permit the articular cartilage to thicken radially under the swelling pressure of the remaining proteoglycans. The coarse wave-form or crimp, frequently observed in the collagenous arrays in the softened tissue, probably results from more long-range interactions between adjacent fibers (a property of many aligned collagenous structures, e.g., fibrocartilage, tendon, ligaments, pericardium). This relatively large crimp can be accounted for geometrically within the length of the radial arrays and still yield an overall increase in thickness of the softened articular cartilage. The reduced level of metachromatic staining in the softened tissue was in accordance with the above model, reflecting either a removal or breakdown of the proteoglycan aggregates. And, although the model has not been discussed with particular reference to the superficial layers of articular cartilage, the significant disruption of the softened tissue compared with that of the normal surface was strongly indicative of a major

8 1216 BROOM breakdown in the bonding of the softened tissue matrix. The complete removal of these weakened superficial layers could expose the subsurface radial arrays to direct erosion (Figure 8). Lastly, the development of osteoarthritis seems to be related to the presence of radially aligned fibers in the middle zone of articular cartilage (8,10,16). An increase both in the degree of hydration (swelling) and the level of extractable proteoglycans is also observed (6,17,18). This would suggest that, in the degenerate tissue, the proteoglycans are less effectively immobilized in the collagen network, reflecting a reduction in the collagen-proteoglycan interactions (6). This interpretation is consistent with the model proposed in this paper. ACKNOWLEDGMENT Mr. D. L. Marra of the School of Engineering, University of Auckland, provided technical assistance during the course of this research REFERENCES Weightman B, Kempson GE: Load carriage, Adult Articular Cartilage. Edited by MAR Freeman. London, Pitman Medical, 1979, pp Mow VC, Lai WM: Mechanics of animal joints. Ann Rev Fluid Mech 11: , 1979 Broom ND, Myers DB: Fibrous waveforms or crimp in surface and subsurface layers of hyaline cartilage maintained in its wet functional condition. Connect Tissue Res 7: , 1980 Broom ND, Myers DB: A study of the structural response of wet hyaline cartilage to various loading situations. Connect Tissue Res 7: , Broom ND, Poole CAP A functional-morphological study of the tidemark region of articular cartilage maintained in a nonviable physiological condition. J Anat (in press) Muir H: Molecular approach to the understanding of osteoarthrosis. Ann Rheum Dis 36: , 1977 McDevitt C, Gilbertson E, Muir H: An experimental model of osteoarthritis: early morphological and biochemical changes. J Bone Joint Surg 59B:24-35, 1977 Meachim G: Ways of cartilage breakdown in human and experimental osteoarthrosis, The Aetiopathogenesis of Osteoarthrosis. Edited by G Nuki. London, Pitman Medical, 1980, pp Bloom W, Fawcett DW A Textbook of Histology. Ninth edition. Philadelphia, Saunders, 1968, p 214 Clarke IC: Articular cartilage: a review and scanning electron microscope study. J Bone Joint Surg 53B: , 1971 Aspden RM, Hukins DWL: Collagen organization in articular cartilage, determined by X-ray diffraction, and its relationship to tissue function. Proc R Soc Lond B , 1981 Muir IHM: Biochemistry, Adult Articular Cartilage. 12. Edited by MAR Freeman. London, Pitman Medical, 1979, pp Lane JM, Weiss C: Review of articular cartilage collagen research. Arthritis Rheum , Minns RJ, Steven FS: The collagen fibril organization in human articular cartilage. J Anat 123: , Meachim G, Stockwell RA: The matrix, Adult Articular Cartilage. Edited by MAR Freeman. London, Pitman Medical, 1979, pp Weiss C: Ultrastructural characteristics of osteoarthritis. Fed Proc 32: , Brandt KD, Palmoski M: Organization of ground substance proteogl ycans in normal and osteoarthritic knee cartilage. Arthritis Rheum , Maroudas A, Venn M: Chemical composition and swelling of normal and osteoarthritic femoral head cartilage. Ann Rheum Dis 36: , 1977