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Connective tissue function and malfunction: A biomechanical perspective
Pathology (1988), 20, pp. 93-104
The Third George Swanson Christie Memorial Lecture CONNECTIVE TISSUE FUNCTION AND MALFUNCTION: A BIOMECHANICAL PERSPECTIVE NEILD. BROOM
BiomechanicsLaboratory, School of Engineering, University of Auckland, Auckland, New Zealand
Key words: Connective tissue, collagen, biomechanics.
Accepted October 23, 1987
INTRODUCTION The soft connective tissues of the body are nearly all involved in the task of load-bearing. They are, in essence, living engineering structures with detailed architecture and composition accurately reflecting particular mechanical roles. In the world of man-made engineering structures the same principles apply. The material properties and shape of the component similarly reflect the functional loads predicted from design considerations. There is, however, a major difference between the soft connective tissues of the body and those materials commonly used in man-made engineering structures; this is the matter of stiffness or compliance. Most engineering structures function by virtue of their intrinsic stiffness or rigidity. Under normal loading conditions there is virtually no detectable deformation unless of course catastrophic failure occurs. Materials such as steel, concrete, timber, ceramics and even most structural plastics are first rigid, fairly strong and, finally, cheap! This property of stiffness, also characteristic of some body tissues such as bone and dentine, is a requirement if components are to be assembled with proper clearances in both the unloaded and loaded states.* We cannot build motor car bodies with accurately fitting doors, with or without their full quota of passengers, using floppy or compliant materials. Steel is used because it is both stiff and cheap. Unfortunately it is also prone to ‘disease’ - corrosion! By contrast, the soft connective tissues actually function normally over enormous ranges of deformation or strain. Body movement is dependent on these structural tissues being both tough and compliant. This means their constituent components will undergo considerable spatial or morphological re-arrangement as loads are applied and removed. If we are to understand how the component parts are exploited to produce a particular connective tissue with a specific mechanical role, these large functional variations in morphology must be considered, even though they will not be evident when viewing a single histological section of the processed tissue. In fact we ~~
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*For a lucid and entertaining treatment of this whole area of materials suitability, see Gordon.’
must examine the tissue directly, in a state as close as possible to its in vivo physiological condition, and while simultaneously applying and removing loads. How can such observation be made? With a combination of high resolution differential interference contrast light microscopy (DIC) and mechanical manipulation it is possible to examine a variety of soft connective tissues in ways that yield fundamental insights into the relationship between structural arrangement and mechanical function. The DIC imaging system permits direct imaging of optically resolvable structures without requiring the tissue to be stained or modified in any way from its normal physiological state. This technique, when combined with an appropriate means of loading, can provide a direct visualization of the response of the structural elements in the tissue to physiological loading. For the remainder of this lecture two things will be attempted. First I would like to illustrate how models relating detailed structure to mechanical function can be derived for complex connective tissues using data obtained from both theoretical engineering analysis and biomechanical techniques. I shall illustrate this first aspect of the lecture using the example of leaflet function within the aortic valve. Second, the model development for a very different connective tissue, articular cartilage, will be described. Here the intention is to show how the healthy cartilage matrix derives its splendid compressive load-bearing properties from complex structural interactions, and that these interactions can only be observed by using indirect biomechanical procedures. I shall then attempt to demonstrate how a dramatic loss of load-bearing ability appears to be related to subtle changes in these hidden structural interactions. This latter aspect will be the pathological connection in the lecture. ELUCIDATING THE BIOMECHANICAL PRINCIPLES GOVERNING THE FUNCTION OF THE AORTIC VALVE Leaflet structure The leaflets of the aortic valve are highly differentiated connective tissue structures comprising largely collagen, elastin and glycosaminoglycans. Mechanically they are required to close completely under minimal reverse flow to ensure that the valve is fully competent. The pressure drop across the closed valve generates large stresses in
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the leaflets. The fibrous structures within them must therefore be capable of distributing these stresses to the sinus boundaries of the valve. Fig. 1 shows an excised aortic leaflet and a series of through-thickness sections to illustrate the complex structural arrangement at the macroscopic level. Three major structural layers have been identified in the On the ventricular side there is an elastin-rich planar layer termed the ventricularis, in which the elastin fibres form a diffuse interconnecting meshwork approximately aligned in the radial direction perpendicular to the leaflet free margin (Fig. 2a). There is also a minor amount of collagen in the ventricularis lying approximately parallel to the free margin, i.e., the cirumferential direction. The aortic side contains the collagen-rich corrugated fibrosa. Here the fibres are arranged approximately circumferentially, and in the relaxed state adopt an acute waveform or crimp (Fig 3a). Between the ventricularis and-fibrosa is a central layer of loose connective tissue, rich in mucopolysaccharide, termed the spongiosa.
Fig. 1 Excised porcine aortic leaflet and associated section profiles. ( x 3).
Fig. 2a Diffuse network of elastin fibres in ventricularis of aortic valve approximately aligned in radial direction (see arrow). (DIC, x800).
Theoretical studies of leaflet mechanics Because there is such a non-uniform arrangement of the fibrous structures, as described above, the leaflet exhibits considerable anisotropy in its mechanical properties. In some theoretical studies of the aortic valve Christie and Medland6 showed that the deformations and stresses within the leaflet could be computed with considerable accuracy using a method of finite element stress analysis. They were able to model the behaviour of the leaflet geometry taking into account its anisotropic properties.
Fig. 2b Elastin network as in Fig. 2a but stretched radially (DIC, x800).
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radial sample A fibrosa severed
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20 40 60 p c t strain
Fig. 3a Circumferential array of acutely crimped collagen fibres in fibrosa of relaxed aortic leaflet. (DIC, x 528).
Their studies demonstrated that with an isotropic leaflet property, where there is no difference between radial and circumferential compliance, the adjacent leaflets show virtually no tendency to move radially inwards and form a coapting competent seal when a back pressure is applied. However as the circumferential stiffness of the leaflet is gradually increased relative to the radial stiffness, the downward movement of the leaflet is restricted and instead it moves radially inwards and seals against its neighbouring leaflets. Further, this mutual coaptive support provided by each neighbouring leaflet has the effect of reducing the stresses in the leaflet corners at the points of suspension from the aortic wall - the commissures. These mechanical predictions of Christie and Medland provided an important conceptual framework within which the actual structural and mechanical properties of the multilayered leaflet structure could be analysed.
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Fig. 4 Load-strain curves obtained from radial strips of fresh porcine aortic leaflet before and following selective severing of fibrosa (sample A) and ventricularis (sample B). From Broom and Christie.’ Reproduced with permission of Yorke Medical Books.
An experimental study of the load-bearing layers in the aortic valve leaflet The respective mechanical roles of the layers comprising the leaflet thickness can be determined by monitoring the tensile load-strain response of circumferential and radial samples both before and following severing of the fibrosa or ventricularis with a sharp scalpel blade. This procedure allows differentiation between load carried by each layer in both directions, which can then be related to the arrangement of fibres in each layer.’ Fig. 3b Collagen fibres as in Fig. 3a but with aortic valve inflated to 80 mmHg pressure. (DIC, x 528).
Relationship between radial stiffness and leaflet structure With tensile loading of the radially cut sections
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'fS"j, IibrOSa severe(
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Fig. 5 Load-strain curves obtained from circumferential strips of fresh porcine aortic leaflet before and following selective severing of fibrosa (sample A) and ventricularis (sample B). From Broom and Christie.' Reproduced with permission of Yorke Medical Books.
the corrugated fibrosa straightens out, while the planar ventricularis simply extends. The load-strain responses are shown in Fig. 4.In the intact condition strains of more than 70 per cent (pct) are reached before significant strainlocking occurs. Severing of the fibrosa has negligible influence on the mechanical response as indicated from the reload curve (sample A), whereas severing of the ventricularis greatly increases the amount of available radial extension (sample B). This demonstrates that it is the ventricularis, and not the fibrosa, that determines the stiffness of the leaflet in the radial direction. Structurally this increased radial compliance is provided by the diffuse radial array of elastin fibres in the ventricularis. They are intrinsically more elastic than the collagen fibres and also become more radially aligned with stress (see Figs 2a, b).
Fig. 7a Amorphous appearance of general matrix of normal articular cartilage as revealed by DIC light microscopy. Chondrocyte columns are aligned in radial direction. ( x 485).
the intact tissue there is a region of high compliance up to about 20 pct extension, followed by rapid stiffening or strain-locking. The reload curves obtained after severing either the fibrosa (sample A) or the ventricularis (sample B) are virtually identical to the intact tissue curves, but displaced a small amount along the strain axis. This mechanical response clearly indicates that both the fibrosa and the ventricularis cooperate in circumferential load-bearing. Severing of either layer results in an immediate off-loading onto the other with little reduction
Relationship between circurnferential stiffness and leaflet structure The mechanical responses of two different circumferential sections are shown in Fig. 5 . For
Fibrosa spongiosa
ventnculans
Fig. 6 Model showing the complex structural arrangement of the three layers comprising the aortic leaflet.
Fig. 7b Ultrastructural view of cartilage general matrix similar to that shown in Fig. 7a. The collagen fibrils lie in oblique orientations about a radial mean direction. (TEM, x 11,300).
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Fig. 8a Prominent radial, fibrous texture in general matrix of human osteoarthritic cartilage. (DIC, x 198).
Fig. 8b Ultrastructural appearance of fibrous texture shown in Fig. 8a. ( ~ 9 2 0 0 ) .
in stiffness. The rapid increase in stiffness beyond about 20 pct strain is a direct consequence of the straightening out of the initially crimped collagen fibres that are circumferentially aligned in both the fibrosa and ventricularis (Figs 3a, b).
The primary load-bearing layer is the collagen-rich corrugated fibrosa in which the fibres are aligned circumferentially and incorporate an acute waveform or crimp. The straightening out of this crimp provides the rapid strain-limiting or stiffening of the leaflet in its circumferential direction. The lesser amount of similarly aligned and crimped collagen in the ventricularis explains the offloading effect when the fibrosa is severed in the circumferential slices. The ventricularis comprises in effect a ‘raft’ of elastin fibrils permitting extensive stretching in the radial
A biomechanical model of the aortic leaflet From the mechanical and structural data it is possible to develop a model that integrates leaflet structure and function. A ‘unit cell’ of the multilayered leaflet structure is shown in Fig. 6 .
Fig. 9a Prominent crimped radial fibrous texture in softened cartilage from central region of bovine tibia1 plateau. (DIC, ~ 4 1 5 ) .
Fig. 9b Ultrastructure of crimped fibrous texture as in Fig. 9a showing fibril aggregation. ( x 17,300).
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Fig. 9d
Fig. 9c Prominent radial fibrous texture from softened bovine tibial plateau. (DIC, x445).
Ultrastructure of aligned fibrous structure as in Fig. 9b.
( x 10,400).
direction. The fibrosa accommodates this large radial extension by means of its large radial corrugations. As the ventricularis extends radially the corrugations flatten without disrupting the integrity of the circumferentially aligned parallel arrays of collagen fibres in the fibrosa. The middle spongiosa layer, lacking a directional fibrous structure, is sufficiently loose and compliant to accommodate there large shape changes in the leaflet during normal function and probably ties the two outer load-bearing layers into a coherent whole.' The leaflet model therefore illustrates how a complex configuration of structural elements possessing uniquely
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Fig. 10 Schematic illustrating principle of simultaneous microcompression and DIC optical microscopy. T = cartilage specimen; U =upper glass surface; L = lower glass surface; I = indenter; A = anvil/spacer; Y =optical axis. From Broom and P o ~ l e Reproduced .~ with permission.
different mechanical properties can be used to produce a composite biological structure whose anisotropic mechanical properties are harnessed for a particular functional application. BIOMECHANICAL PRINCIPLES GOVERNING THE FUNCTION OF ARTICULAR CARTILAGE The second connective tissue I wish to consider in this lecture is articular cartilage. Its functional role within the body is principally a compressive one and as such is in striking contrast to the membrane-type biaxial loadbearing structure of the aortic leaflet.
Articular cartilage structure This ability of articular cartilage to transmit compressive loads is achieved through the interaction of two principal constituents, collagen and proteoglycan, and these possess fundamentally different mechanical properties. The tension-bearing collagen fibrils cannot individually sustain compressive stresses without extensive collapse; the hydrated proteoglycan complexes are weak in shear. Yet, when appropriately integrated, these two components yield a structural tissue capable of transmitting repeatedly high levels of both compressive and shear loads. In articular cartilage exhibiting normal mechanical stiffness the collagen component cannot be resolved by light microscopy (Fig. 7a). This is a consequence of the small diameter of the collagen fibrils and their spatial arrangement as revealed by transmission electron microscopy (Fig. 7b). However, in cartilage that is noticeably soft, e.g., osteoarthritic cartilage or cartilage from the central tibial plateau region of large mature mammals, there is an increased tendency for fibrils to form radial arrays frequently aggregating into larger groups termed fibres. These fibres are visible at the light microscopy
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COMPRESSIVE STRAIN pct
Fig. 1 1 Compressive s t r e d s t r a i n curves for normal and softened bovine articular cartilage. From Broom." Reproduced with permission.
level, and ultrastructural examination clearly reveals their aggregated fibrillar make-up. (Figs 8, 9).
Micromechanical methods of determining structure/function relationships in cartilage It is possible to examine directly the response of the fully hydrated cartilage matrix to compressive loading using a combination of micro-compression and simultaneous DIC light microscopy (Fig. 10). This experimental procedure mimics reasonably closely the in vivo loading on the joint, particularly if the cartilage slice is still attached to its subchondral constraint. The chondrocytes and the fibrils where they have aggregated into fibres can be observed directly as they respond to the applied load.'-'' The s t r e d s t ra in curves in Fig. 11 illustrate the dramatic difference in compressive stiffness between normal and softened cartilage matrices. The mechanically 'soft' response is directly associated with the large scale in-phase collapse of the parallel arrays of fibres under compression12 (Fig. 12). It can therefore be inferred that the collagenous architecture within the matrix profoundly influences its response to mechanical loading. A thorough elucidation of the biomechanical principles governing the function of the healthy tissue is required if we are to understand more fully the processes involved in its degeneration. The microcompression experiment described above provides a quantitative measure of mechanical performance and also some limited correlation with structural response. The compressive response of cartilage is, however, a generalized one, reflecting the integrated contribution of all the individual structural elements. Consequently application of the microcompression technique cannot yield the fundamental structural
Fig. 12 Response of softened radial fibrous array in middle zone of osteoarthritic human cartilage to radial compression. a , relaxed; b, compressed in direction indicated by arrows. (DIC, x 230). From Broom." Reproduced with permission.
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Fig. 13 Schematic representation of radial and transverse notch geometries and their different loading configurations. From Broom.'4 Reproduced with permission.
principles and mechanisms upon which this integrated response depends. Yet, because disease processes may well target specific components within the matrix, these structural principles require detailed elucidation. The matrix of articular cartilage is inhomogeneous, exhibiting major structural differences in directions parallel and perpendicular to the surface (these are referred to as the transverse and radial directions respectively). Any biomechanical interpretation must therefore
Fig. 14 Radial notch propagation through normal matrix of bovine articular cartilage: (a) unstressed radial notch; (b) extensive radial notch propagation results in fibres being drawn out transversely across rupture front with chondrocytes also being released. (Both DIC, x 198).
take into account this anisotropic property. Also, in the initial stages of this research it was judged that the important structural principles underlying the functional properties of cartilage would be revealed only if we could observe the structural response of the matrix to extensional loading in each of the two primary directions as separate experiments. Stressing cartilage in the transverse direction is simple and has been performed by other ~ 0 r k e r s . But, l ~ how to stress in tension the matrix in the radical direction initially posed a seemingly insoluble experimental problem. There seemed no obvious way of effectively gripping a sample of matrix whose length along the axis of tension would be no more than the thickness of the layer of cartilage, i.e., 1.0-1.5 mm. And further, how could this be achieved while simultaneously observing the tissue's structural response microscopically? A solution to this problem was in fact obtained by devising a novel micro-notch technique. l4 Full depth slices 100 to 200 vm in thickness were taken from scallops of fresh cartilage removed from the subchondral bone. The notching geometrics for both the radial and transverse directions and their respective modes of loading in tension are illustrated in Fig. 13. The notched fully hydrated slices, sandwiched between two optically flat glass surfaces in a microtensile d e ~ i c ecan , ~ be observed under load using high resolution DIC optical microscopy.
New structural insights into articular cartilage function What new insights into the biomechanics of healthy cartilage can be obtained with the micro-notch technique? First, it enables the structurally related mechanical anisotropy of the matrix to be measured directly. By notching adjacent slices of tissue such that both the radial and transverse notch roots are located at identical zonal depths and then loading to the first onset of rupture the matrix extensivity in the transverse direction was shown to be up t o ten times greater than that in the radial direction. l 5 This strongly suggested that the collagen arrangement in the general matrix of cartilage has a profound strain-limiting effect in the radial direction only. Second, the mechanisms of notch propagation involving matrix rupture are completely different in the two direction^.'^ With increasing load the radial notch always advances steadily in the radial direction by a process in which aggregates of fibrils or fibres are repeatedly drawn out 'from the adjacent matrix, transversely stressed across the notch root, and finally fractured (Fig. 14). By contrast, the transverse notch in the healthy matrix propagates in an unstable manner. In fact further advance of the initial notch in the transverse direction by matrix rupture will not take place. Instead the onset of matrix rupture always involves an immediate sideways deflection of the notch tip into a true radial mode either up towards the articular surface or downwards in the subchondral direction, or simultaneously in both directions (Fig. 15). This rupture behaviour of the normal matrix clearly demonstrates that any attempt to extend further the transverse notch in the transverse direction will require rupture across the fibril 'grain' or texture, whereas radial notch
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Fig. 15 Transverse notch propagation through normal matrix as in Fig. 14: (a) unstressed transverse notch; (b) rupture at the stressed transverse notch occurs via a radially deflected mechanism. Note virtual absence of radial extension in the matrix beyond the highly stressed band closest to the notch root. (Both DIC, x 198).
propagation is along this ‘grain’, and hence its stable propagation at a lower stress than that required to rupture across the fibrillar ‘grain’. Ultrastructual studies of the change in orientation of the collagen fibrils in the highly stressed matrix immediately below the notch root have revealed contrasting morphological responses between the radial and transverse ge~matries.’~Because the two different notch geometries actually permit loading of the fibrillar structure either along or across its primary orientation, ultrastructural studies of changes in the fibrillar structure with this directional stressing have proven useful in further elucidating the basic ‘knit’ of the collagen fibrils in the general matrix. In the highly stressed matrix immediately below the transverse notch root the fibrils become closely packed
and aligned as parallel radial arrays (Fig. 16a). However, in the stressed radial notch region the fibrils are initially transverse in the matrix at the immediate notch root, but quickly change to an acutely kinked radial arrangement in the matrix below (Fig. 16b). These biomechanical and ultrastructural studies provide convincing evidence that the fibrillar architecture in the genetal matrix of cartilage is a coherently ordered arrangement of fibrils developed from well defined structural relationships. It cannot be viewed simply as a mass of fibrillar elements with varying orientations about a radial mean direction depending on the zonal depth. The experimental evidence suggests that the fibrillar architecture or ‘knit’ in the general matrix incorporates the following structural principles: (1) The overall arrangement of fibrils is radial and they
Fig. 16 (a) Pronounced radial alignment of collagen fibrils in the highly stressed band of matrix at the transverse notch root as in Fig. 15b. Arrows indicate radial stressing direction. (TEM, x 18,690). (b) Acutely kinked and collapsed fibrillar structure in matrix below stressed radial notch root as in Fig. 14b. Arrows indicate transverse stressing direction. These structures should be compared with the underformed matrix ultrastructure as shown in Fig. 7b. (TEM, x 12,700).
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Fig. 17 A 2-dimensional wire mesh analogue of the interconnecting goemetry of radial elements proposed for the arrangement of fibrils in the general matrix of articular cartilage. Note the rapid strain-locking in the radial direction (cf A and B) versus the considerable extension permitted in the transverse direction (cf A and C).
appear to be continuous or nearly so for considerable distances through the cartilage depth. This provides the extremely high strength in the radial direction. The fibrils are required to be axially fractured if transverse rupture is to occur. Hence the phenomenon of a radially deflected rupture path with the transverse notch (Fig. 15). (2) The fibrils, as they pass radially up through the cartilage depth, are repeatedly deflected sideways to
Pathology (1988). 20, April
form an arrangement of obliquely oriented segments in a crude zig-zag configuration. This secondary morphology accounts for the strain-locking effect we observe when the matrix is stressed radially as in the transverse notch experiment (see Figs 15b and 16a). (3) Some form of interfibril link or connection is assumed to exist to give the overall radial array of fibrils structural cohesion in the transverse direction. It is the transverse extension of these interconnected radial fibrils that accounts for the much greater degree of extension in the transverse direction relative to the radial, hence the development of an acutely kinked arrangement with transverse stressing of the matrix in the radial notch experiment (Fig. 16b). These structural concepts have been modelled recently with a 3-dimensional physical mode1I6 and are illistrated in 2-dimensions with the wire mesh analogue in Fig. 17. The model shows how, beginning with an interconnected geometry of radial elements, there is rapid strain locking radially, but very considerable transverse extensivity. It is proposed that within such an interconnected collagenous structure the proteoglycan component is contained. The entire fibrillar structure is kept taut by the swelling capacity of the hydrated proteglycans, the oblique interconnecting collagen segments effectively creating a braced structure that is resistant to both compression and shear.” The model also appears to explain the presence of the long transverse fibres drawn out across the propagating radial notch root (Fig. 14b). As rupture proceeds matrix material directly under the notch will move laterally to either side, displacing entire arrays of fibrils out of their original overall radial orientation. Many fibrils will be deflected to the side opposite to which they belong as radial elements in the unruptured matrix. They will therefore be stressed transversely across the notch and finally rupture.
Fig. 18 Impact-induced structural transformation in normal bovine articular cartilage: (a) undeformed matrix; (b) impacted matrix exhibiting well-developed crimped fibrous structure. Chondrocyte alignment is radial. (Both DIC, x 566).
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Fig. 19 Enzymatically induced structural transformation in normal bovine articular cartilage: (a) matrix appearance after trypsin pretreatment; (b) well-developed crimped, radial fibrous structure following partial digestion of (a) with bacterial collagenase. (Both DIC, x 275).
What then is the relationship between cartilage structure and its malfunction? As noted earlier, a consistent feature of abnormally soft cartilage is the appearance in the matrix of radially aligned aggregates of fibrils or fibres, easily resolved at the optical microscope level. These readily collapse in phase with compressive loads (Fig. 12). This response is consistent with an absence of structural bracing assumed to be associated with the interconnecting fibrillar structure of the normal matrix. The oblique interconnecting arrangement of fibril segments will of course represent a high energy configuration dependent on the maintenance of the interfibril linkage. Is structural softening then a direct consequence of this interfibril link being degraded? Unfortunately we have no way of examining structurally a particular region of cartilage prior to the development of abnormal softening. However, there are two additional pieces of experimental evidence that strongly support the theory that the interfibril linkage is directly implicated. First, repeated impact loading of fresh cartilage-on-bone in vitro can transform the interconnecting arrangement of fibrils comprising the general matrix of normal cartilage into a strongly aligned radial configuration of fibres incorporating the characteristic waveform or crimp" (Fig. 18). This stress-induced transformation produces a collagenous arrangement virtually identical to that observed in the naturally softened cartilage (Fig. 9a). It is also consistent with the type of structural response we would expect from dynamic overloading. Rapidly applied compressive stresses would prevent the time-dependent flow of water from under the site of compression to regions of lower stress. The matrix will instead respond as a 'hard' solid generating high internal stresses sufficient to disrupt the interfibril linkage. This would, in turn, allow a relaxation of the high energy interconnecting
network with consequent alignment of fibrils in their primary radial configuration. The second piece of experimental evidence supporting the concept that an interfibril link is directly involved in the maintenance of the high energy interconnecting configuration of fibrils comes from some very recent enzymatic studies.'* By first removing a large fraction of the proteoglycan component using either testicular hyaluronidase or trypsin digestion, followed by a limited attack on the collagen using bacterial collagenase, it is possible to transform the high energy interconnecting fibrillar arrangement comprising the normal matrix, into a strongly aligned radial configuration (Fig. 19). This degraded fibrillar structure is mechanically soft, and again virtually identical to that observed in the osteoarthritic matrix (Fig. 8a) and in the softened cartilage on the tibia1 plateau (Figs 9a and 9c). A key requirement for the above enzymatic transformation to occur is some limited collagenolytic action. This COLLAGEN NETWORK TRANSFORMATION I N ARTICULAR C A R T I L A G E
DEGENERATION
TRAUMA
.,
b
E N Z V Y A T I C DIGESTION T"I.II*?COLL.TI*I.S
Fig. 20 Schematic illustration of 3-fold transformation pathway from the pseudo-random fibrillar arrangement to a radially aligned crimped configuration. From Broom.'8 Reproduced with permission.
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would suggest that the interfibril link is intimately associated with the collagen fibrils, but what it actually is has still be to determined. It is therefore argued that the interconnected, oblique arrangement of fibril segments in association with the hydrated ground-substance provides a braced structural system with excellent load-bearing characteristics. A variety of factors can however induce a major structural transformation in the collagenous architecture of articular cartilage. Naturally occurring disease or degenerative processes, dynamic overloading or selective enzymatic attack can all transform this normal architectural arrangement of fibrils into a radially aligned and aggregated configuration, poorly braced and therefore lacking in load-bearing ability (Fig. 20). This multipathed transformation is, I believe, convincing evidence that the fibrillar architecture of the articular cartilage matrix is a highly specialized configuration constrained by forces still not well understood. Elucidation of these factors should represent a significant advance in our understanding of the degenerative process. CONCLUDING COMMENTS In this presentation I have endeavoured to show how complex connective tissue systems can be examined in a manner that reveals fundamental insights into their biomechanical properties. A key requirement in this approach is that the tissues be observed microscopically in their fully hydrated condition, while being simultaneously manipulated using specific loading configurations that selectively stress key structural components within the tissue. Having clarified, at least in part, the biomechanical principles relating to two quite different structural connective tissues, the problem of abnormal mechanical function in articular cartilage and its structural origin has been considered.
ACKNOWLEDGEMENTS I would like to thank the Australasian Society for Experimental Pathology for their invitation to present this Third George Swanson Christie Memorial Lecture. In particular my appreciation to Dr Kathryn Ham whose courageous idea it was to let an engineer loose before an audience of pathologists! The research activities reflected in this lecture have been supported by funding from the Medical Research Council of New Zealand.
References 1. Gordon JE. The New Science of Strong Materials. London: Penguin
Books, 1976, Chp 2. 2. Sauren AAHJ, Kuijpers W, Van Streenhoven AA, Veldpaus FE. Aortic valve histology and its relation to mechanics - preliminary report. J Biomech 1980; 13: 97-104. 3. Ferrans VJ, Spray TL, Billingham ME, Roberts WC. Structural changes in porcine xenografts used as substitute cardiac valves. Am J Cardiol 1978; 41: 1159-84. 4. Clark RE, Finke EH. Scanning electron microscopy of human aortic leaflets in stressed and relaxed states. J Thorac Cardiovasc Surg 1974; 67: 792-804. 5 . Mohri H, Reichenbeck DD, Merendino KA. Biology of homologous and heterologous aortic valves. In: Ross DN, Wooler GH, eds. Biological tissue in heart valve replacement. London: Butterworths, 1972; 137-42. 6. Christie GW, Medland IC. A non-linear finite element stress analysis of bioprosthetic heart valves. In: Gallagher RW, Simon BR, Johnson PC, Gross JF, eds. Finite elements in biomechanics. New York: John Wiley, 1982; 153-79. 7. Broom ND, Christie GW. The structure/function relationship of fresh and glutaraldehyde-fixed aortic valve leaflets. In: Cohn L, Gallucci V, eds. Cardiac bioprostheses - Proceedings of the 2nd international symposium. New York: Yorke Medical, 1982; 477-91. 8. Broom ND, Myers DB. A study of the structural response of wet hyaline cartilage to various loading situations. Connect Tiss Res 1980; 7: 227-37. 9. Broom ND, Poole CA. A functional-morphological study of the tidemark region of articular cartilage maintained in a non-viable physiological condition. J Anat 1982; 135: 65-82. 10. Broom ND, Poole CA. Articular cartilage collagen and
proteoglycans: their functional interdependency. Arthritis Rheum 1983; 26: 111-9.
1 1 . Broom ND. Abnormal softening in articular cartilage: its relationship to the collagen framework. Arthritis Rheum 1982; 25: 1209-16. 12. Broom ND. The altered biomechanical state of human femoral head osteoarthritic articular cartilage. Arthritis Rheum 1984; 27: 1028-39. 13. Roth V, Mow VC. The intrinsic tensile behaviour of the matrix
14. 15.
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of bovine articular cartilage and its variation with age. J Bone Joint Surg 1980; 62A: 1102-17. Broom ND. Further insights into the structural principles governing the function of articular cartilage. J Anat 1984; 139: 275-94. Broom ND. The collagenous architecture of articular cartilage a synthesis of ultrastructure and mechanical function. J Rheum 1986; 13: 142-52. Broom ND, Marra DL. New structural concepts of articular cartilage demonstrated with a physical model. Connect Tiss Res 1985; 14: 1-8. Broom ND. Structural consequences of traumatising articular cartilage. Ann Rheum Dis 1986; 45: 225-34. Broom ND. An enzymatically induced structural transformation in articular cartilage: its significance with respect to matrix breakdown. Arthritis Rheum 1987, In press.
The Third George Swanson Christie Memorial Lecture CONNECTIVE TISSUE FUNCTION AND MALFUNCTION: A BIOMECHANICAL PERSPECTIVE NEILD. BROOM
BiomechanicsLaboratory, School of Engineering, University of Auckland, Auckland, New Zealand
Key words: Connective tissue, collagen, biomechanics.
Accepted October 23, 1987
INTRODUCTION The soft connective tissues of the body are nearly all involved in the task of load-bearing. They are, in essence, living engineering structures with detailed architecture and composition accurately reflecting particular mechanical roles. In the world of man-made engineering structures the same principles apply. The material properties and shape of the component similarly reflect the functional loads predicted from design considerations. There is, however, a major difference between the soft connective tissues of the body and those materials commonly used in man-made engineering structures; this is the matter of stiffness or compliance. Most engineering structures function by virtue of their intrinsic stiffness or rigidity. Under normal loading conditions there is virtually no detectable deformation unless of course catastrophic failure occurs. Materials such as steel, concrete, timber, ceramics and even most structural plastics are first rigid, fairly strong and, finally, cheap! This property of stiffness, also characteristic of some body tissues such as bone and dentine, is a requirement if components are to be assembled with proper clearances in both the unloaded and loaded states.* We cannot build motor car bodies with accurately fitting doors, with or without their full quota of passengers, using floppy or compliant materials. Steel is used because it is both stiff and cheap. Unfortunately it is also prone to ‘disease’ - corrosion! By contrast, the soft connective tissues actually function normally over enormous ranges of deformation or strain. Body movement is dependent on these structural tissues being both tough and compliant. This means their constituent components will undergo considerable spatial or morphological re-arrangement as loads are applied and removed. If we are to understand how the component parts are exploited to produce a particular connective tissue with a specific mechanical role, these large functional variations in morphology must be considered, even though they will not be evident when viewing a single histological section of the processed tissue. In fact we ~~
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~
*For a lucid and entertaining treatment of this whole area of materials suitability, see Gordon.’
must examine the tissue directly, in a state as close as possible to its in vivo physiological condition, and while simultaneously applying and removing loads. How can such observation be made? With a combination of high resolution differential interference contrast light microscopy (DIC) and mechanical manipulation it is possible to examine a variety of soft connective tissues in ways that yield fundamental insights into the relationship between structural arrangement and mechanical function. The DIC imaging system permits direct imaging of optically resolvable structures without requiring the tissue to be stained or modified in any way from its normal physiological state. This technique, when combined with an appropriate means of loading, can provide a direct visualization of the response of the structural elements in the tissue to physiological loading. For the remainder of this lecture two things will be attempted. First I would like to illustrate how models relating detailed structure to mechanical function can be derived for complex connective tissues using data obtained from both theoretical engineering analysis and biomechanical techniques. I shall illustrate this first aspect of the lecture using the example of leaflet function within the aortic valve. Second, the model development for a very different connective tissue, articular cartilage, will be described. Here the intention is to show how the healthy cartilage matrix derives its splendid compressive load-bearing properties from complex structural interactions, and that these interactions can only be observed by using indirect biomechanical procedures. I shall then attempt to demonstrate how a dramatic loss of load-bearing ability appears to be related to subtle changes in these hidden structural interactions. This latter aspect will be the pathological connection in the lecture. ELUCIDATING THE BIOMECHANICAL PRINCIPLES GOVERNING THE FUNCTION OF THE AORTIC VALVE Leaflet structure The leaflets of the aortic valve are highly differentiated connective tissue structures comprising largely collagen, elastin and glycosaminoglycans. Mechanically they are required to close completely under minimal reverse flow to ensure that the valve is fully competent. The pressure drop across the closed valve generates large stresses in
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the leaflets. The fibrous structures within them must therefore be capable of distributing these stresses to the sinus boundaries of the valve. Fig. 1 shows an excised aortic leaflet and a series of through-thickness sections to illustrate the complex structural arrangement at the macroscopic level. Three major structural layers have been identified in the On the ventricular side there is an elastin-rich planar layer termed the ventricularis, in which the elastin fibres form a diffuse interconnecting meshwork approximately aligned in the radial direction perpendicular to the leaflet free margin (Fig. 2a). There is also a minor amount of collagen in the ventricularis lying approximately parallel to the free margin, i.e., the cirumferential direction. The aortic side contains the collagen-rich corrugated fibrosa. Here the fibres are arranged approximately circumferentially, and in the relaxed state adopt an acute waveform or crimp (Fig 3a). Between the ventricularis and-fibrosa is a central layer of loose connective tissue, rich in mucopolysaccharide, termed the spongiosa.
Fig. 1 Excised porcine aortic leaflet and associated section profiles. ( x 3).
Fig. 2a Diffuse network of elastin fibres in ventricularis of aortic valve approximately aligned in radial direction (see arrow). (DIC, x800).
Theoretical studies of leaflet mechanics Because there is such a non-uniform arrangement of the fibrous structures, as described above, the leaflet exhibits considerable anisotropy in its mechanical properties. In some theoretical studies of the aortic valve Christie and Medland6 showed that the deformations and stresses within the leaflet could be computed with considerable accuracy using a method of finite element stress analysis. They were able to model the behaviour of the leaflet geometry taking into account its anisotropic properties.
Fig. 2b Elastin network as in Fig. 2a but stretched radially (DIC, x800).
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radial sample A fibrosa severed
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20 40 60 p c t strain
Fig. 3a Circumferential array of acutely crimped collagen fibres in fibrosa of relaxed aortic leaflet. (DIC, x 528).
Their studies demonstrated that with an isotropic leaflet property, where there is no difference between radial and circumferential compliance, the adjacent leaflets show virtually no tendency to move radially inwards and form a coapting competent seal when a back pressure is applied. However as the circumferential stiffness of the leaflet is gradually increased relative to the radial stiffness, the downward movement of the leaflet is restricted and instead it moves radially inwards and seals against its neighbouring leaflets. Further, this mutual coaptive support provided by each neighbouring leaflet has the effect of reducing the stresses in the leaflet corners at the points of suspension from the aortic wall - the commissures. These mechanical predictions of Christie and Medland provided an important conceptual framework within which the actual structural and mechanical properties of the multilayered leaflet structure could be analysed.
80
11
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radial sample B 15 ventric-
intact tissue
L
0
severed
v
Q
0
I 20
40 60 p c t strain
80
100
Fig. 4 Load-strain curves obtained from radial strips of fresh porcine aortic leaflet before and following selective severing of fibrosa (sample A) and ventricularis (sample B). From Broom and Christie.’ Reproduced with permission of Yorke Medical Books.
An experimental study of the load-bearing layers in the aortic valve leaflet The respective mechanical roles of the layers comprising the leaflet thickness can be determined by monitoring the tensile load-strain response of circumferential and radial samples both before and following severing of the fibrosa or ventricularis with a sharp scalpel blade. This procedure allows differentiation between load carried by each layer in both directions, which can then be related to the arrangement of fibres in each layer.’ Fig. 3b Collagen fibres as in Fig. 3a but with aortic valve inflated to 80 mmHg pressure. (DIC, x 528).
Relationship between radial stiffness and leaflet structure With tensile loading of the radially cut sections
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'fS"j, IibrOSa severe(
intact
0
5
20 40 6C p c t strain
p c t strain
Fig. 5 Load-strain curves obtained from circumferential strips of fresh porcine aortic leaflet before and following selective severing of fibrosa (sample A) and ventricularis (sample B). From Broom and Christie.' Reproduced with permission of Yorke Medical Books.
the corrugated fibrosa straightens out, while the planar ventricularis simply extends. The load-strain responses are shown in Fig. 4.In the intact condition strains of more than 70 per cent (pct) are reached before significant strainlocking occurs. Severing of the fibrosa has negligible influence on the mechanical response as indicated from the reload curve (sample A), whereas severing of the ventricularis greatly increases the amount of available radial extension (sample B). This demonstrates that it is the ventricularis, and not the fibrosa, that determines the stiffness of the leaflet in the radial direction. Structurally this increased radial compliance is provided by the diffuse radial array of elastin fibres in the ventricularis. They are intrinsically more elastic than the collagen fibres and also become more radially aligned with stress (see Figs 2a, b).
Fig. 7a Amorphous appearance of general matrix of normal articular cartilage as revealed by DIC light microscopy. Chondrocyte columns are aligned in radial direction. ( x 485).
the intact tissue there is a region of high compliance up to about 20 pct extension, followed by rapid stiffening or strain-locking. The reload curves obtained after severing either the fibrosa (sample A) or the ventricularis (sample B) are virtually identical to the intact tissue curves, but displaced a small amount along the strain axis. This mechanical response clearly indicates that both the fibrosa and the ventricularis cooperate in circumferential load-bearing. Severing of either layer results in an immediate off-loading onto the other with little reduction
Relationship between circurnferential stiffness and leaflet structure The mechanical responses of two different circumferential sections are shown in Fig. 5 . For
Fibrosa spongiosa
ventnculans
Fig. 6 Model showing the complex structural arrangement of the three layers comprising the aortic leaflet.
Fig. 7b Ultrastructural view of cartilage general matrix similar to that shown in Fig. 7a. The collagen fibrils lie in oblique orientations about a radial mean direction. (TEM, x 11,300).
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Fig. 8a Prominent radial, fibrous texture in general matrix of human osteoarthritic cartilage. (DIC, x 198).
Fig. 8b Ultrastructural appearance of fibrous texture shown in Fig. 8a. ( ~ 9 2 0 0 ) .
in stiffness. The rapid increase in stiffness beyond about 20 pct strain is a direct consequence of the straightening out of the initially crimped collagen fibres that are circumferentially aligned in both the fibrosa and ventricularis (Figs 3a, b).
The primary load-bearing layer is the collagen-rich corrugated fibrosa in which the fibres are aligned circumferentially and incorporate an acute waveform or crimp. The straightening out of this crimp provides the rapid strain-limiting or stiffening of the leaflet in its circumferential direction. The lesser amount of similarly aligned and crimped collagen in the ventricularis explains the offloading effect when the fibrosa is severed in the circumferential slices. The ventricularis comprises in effect a ‘raft’ of elastin fibrils permitting extensive stretching in the radial
A biomechanical model of the aortic leaflet From the mechanical and structural data it is possible to develop a model that integrates leaflet structure and function. A ‘unit cell’ of the multilayered leaflet structure is shown in Fig. 6 .
Fig. 9a Prominent crimped radial fibrous texture in softened cartilage from central region of bovine tibia1 plateau. (DIC, ~ 4 1 5 ) .
Fig. 9b Ultrastructure of crimped fibrous texture as in Fig. 9a showing fibril aggregation. ( x 17,300).
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Fig. 9d
Fig. 9c Prominent radial fibrous texture from softened bovine tibial plateau. (DIC, x445).
Ultrastructure of aligned fibrous structure as in Fig. 9b.
( x 10,400).
direction. The fibrosa accommodates this large radial extension by means of its large radial corrugations. As the ventricularis extends radially the corrugations flatten without disrupting the integrity of the circumferentially aligned parallel arrays of collagen fibres in the fibrosa. The middle spongiosa layer, lacking a directional fibrous structure, is sufficiently loose and compliant to accommodate there large shape changes in the leaflet during normal function and probably ties the two outer load-bearing layers into a coherent whole.' The leaflet model therefore illustrates how a complex configuration of structural elements possessing uniquely
U
L
Fig. 10 Schematic illustrating principle of simultaneous microcompression and DIC optical microscopy. T = cartilage specimen; U =upper glass surface; L = lower glass surface; I = indenter; A = anvil/spacer; Y =optical axis. From Broom and P o ~ l e Reproduced .~ with permission.
different mechanical properties can be used to produce a composite biological structure whose anisotropic mechanical properties are harnessed for a particular functional application. BIOMECHANICAL PRINCIPLES GOVERNING THE FUNCTION OF ARTICULAR CARTILAGE The second connective tissue I wish to consider in this lecture is articular cartilage. Its functional role within the body is principally a compressive one and as such is in striking contrast to the membrane-type biaxial loadbearing structure of the aortic leaflet.
Articular cartilage structure This ability of articular cartilage to transmit compressive loads is achieved through the interaction of two principal constituents, collagen and proteoglycan, and these possess fundamentally different mechanical properties. The tension-bearing collagen fibrils cannot individually sustain compressive stresses without extensive collapse; the hydrated proteoglycan complexes are weak in shear. Yet, when appropriately integrated, these two components yield a structural tissue capable of transmitting repeatedly high levels of both compressive and shear loads. In articular cartilage exhibiting normal mechanical stiffness the collagen component cannot be resolved by light microscopy (Fig. 7a). This is a consequence of the small diameter of the collagen fibrils and their spatial arrangement as revealed by transmission electron microscopy (Fig. 7b). However, in cartilage that is noticeably soft, e.g., osteoarthritic cartilage or cartilage from the central tibial plateau region of large mature mammals, there is an increased tendency for fibrils to form radial arrays frequently aggregating into larger groups termed fibres. These fibres are visible at the light microscopy
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COMPRESSIVE STRAIN pct
Fig. 1 1 Compressive s t r e d s t r a i n curves for normal and softened bovine articular cartilage. From Broom." Reproduced with permission.
level, and ultrastructural examination clearly reveals their aggregated fibrillar make-up. (Figs 8, 9).
Micromechanical methods of determining structure/function relationships in cartilage It is possible to examine directly the response of the fully hydrated cartilage matrix to compressive loading using a combination of micro-compression and simultaneous DIC light microscopy (Fig. 10). This experimental procedure mimics reasonably closely the in vivo loading on the joint, particularly if the cartilage slice is still attached to its subchondral constraint. The chondrocytes and the fibrils where they have aggregated into fibres can be observed directly as they respond to the applied load.'-'' The s t r e d s t ra in curves in Fig. 11 illustrate the dramatic difference in compressive stiffness between normal and softened cartilage matrices. The mechanically 'soft' response is directly associated with the large scale in-phase collapse of the parallel arrays of fibres under compression12 (Fig. 12). It can therefore be inferred that the collagenous architecture within the matrix profoundly influences its response to mechanical loading. A thorough elucidation of the biomechanical principles governing the function of the healthy tissue is required if we are to understand more fully the processes involved in its degeneration. The microcompression experiment described above provides a quantitative measure of mechanical performance and also some limited correlation with structural response. The compressive response of cartilage is, however, a generalized one, reflecting the integrated contribution of all the individual structural elements. Consequently application of the microcompression technique cannot yield the fundamental structural
Fig. 12 Response of softened radial fibrous array in middle zone of osteoarthritic human cartilage to radial compression. a , relaxed; b, compressed in direction indicated by arrows. (DIC, x 230). From Broom." Reproduced with permission.
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Transverse n o t c h
Fig. 13 Schematic representation of radial and transverse notch geometries and their different loading configurations. From Broom.'4 Reproduced with permission.
principles and mechanisms upon which this integrated response depends. Yet, because disease processes may well target specific components within the matrix, these structural principles require detailed elucidation. The matrix of articular cartilage is inhomogeneous, exhibiting major structural differences in directions parallel and perpendicular to the surface (these are referred to as the transverse and radial directions respectively). Any biomechanical interpretation must therefore
Fig. 14 Radial notch propagation through normal matrix of bovine articular cartilage: (a) unstressed radial notch; (b) extensive radial notch propagation results in fibres being drawn out transversely across rupture front with chondrocytes also being released. (Both DIC, x 198).
take into account this anisotropic property. Also, in the initial stages of this research it was judged that the important structural principles underlying the functional properties of cartilage would be revealed only if we could observe the structural response of the matrix to extensional loading in each of the two primary directions as separate experiments. Stressing cartilage in the transverse direction is simple and has been performed by other ~ 0 r k e r s . But, l ~ how to stress in tension the matrix in the radical direction initially posed a seemingly insoluble experimental problem. There seemed no obvious way of effectively gripping a sample of matrix whose length along the axis of tension would be no more than the thickness of the layer of cartilage, i.e., 1.0-1.5 mm. And further, how could this be achieved while simultaneously observing the tissue's structural response microscopically? A solution to this problem was in fact obtained by devising a novel micro-notch technique. l4 Full depth slices 100 to 200 vm in thickness were taken from scallops of fresh cartilage removed from the subchondral bone. The notching geometrics for both the radial and transverse directions and their respective modes of loading in tension are illustrated in Fig. 13. The notched fully hydrated slices, sandwiched between two optically flat glass surfaces in a microtensile d e ~ i c ecan , ~ be observed under load using high resolution DIC optical microscopy.
New structural insights into articular cartilage function What new insights into the biomechanics of healthy cartilage can be obtained with the micro-notch technique? First, it enables the structurally related mechanical anisotropy of the matrix to be measured directly. By notching adjacent slices of tissue such that both the radial and transverse notch roots are located at identical zonal depths and then loading to the first onset of rupture the matrix extensivity in the transverse direction was shown to be up t o ten times greater than that in the radial direction. l 5 This strongly suggested that the collagen arrangement in the general matrix of cartilage has a profound strain-limiting effect in the radial direction only. Second, the mechanisms of notch propagation involving matrix rupture are completely different in the two direction^.'^ With increasing load the radial notch always advances steadily in the radial direction by a process in which aggregates of fibrils or fibres are repeatedly drawn out 'from the adjacent matrix, transversely stressed across the notch root, and finally fractured (Fig. 14). By contrast, the transverse notch in the healthy matrix propagates in an unstable manner. In fact further advance of the initial notch in the transverse direction by matrix rupture will not take place. Instead the onset of matrix rupture always involves an immediate sideways deflection of the notch tip into a true radial mode either up towards the articular surface or downwards in the subchondral direction, or simultaneously in both directions (Fig. 15). This rupture behaviour of the normal matrix clearly demonstrates that any attempt to extend further the transverse notch in the transverse direction will require rupture across the fibril 'grain' or texture, whereas radial notch
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Fig. 15 Transverse notch propagation through normal matrix as in Fig. 14: (a) unstressed transverse notch; (b) rupture at the stressed transverse notch occurs via a radially deflected mechanism. Note virtual absence of radial extension in the matrix beyond the highly stressed band closest to the notch root. (Both DIC, x 198).
propagation is along this ‘grain’, and hence its stable propagation at a lower stress than that required to rupture across the fibrillar ‘grain’. Ultrastructual studies of the change in orientation of the collagen fibrils in the highly stressed matrix immediately below the notch root have revealed contrasting morphological responses between the radial and transverse ge~matries.’~Because the two different notch geometries actually permit loading of the fibrillar structure either along or across its primary orientation, ultrastructural studies of changes in the fibrillar structure with this directional stressing have proven useful in further elucidating the basic ‘knit’ of the collagen fibrils in the general matrix. In the highly stressed matrix immediately below the transverse notch root the fibrils become closely packed
and aligned as parallel radial arrays (Fig. 16a). However, in the stressed radial notch region the fibrils are initially transverse in the matrix at the immediate notch root, but quickly change to an acutely kinked radial arrangement in the matrix below (Fig. 16b). These biomechanical and ultrastructural studies provide convincing evidence that the fibrillar architecture in the genetal matrix of cartilage is a coherently ordered arrangement of fibrils developed from well defined structural relationships. It cannot be viewed simply as a mass of fibrillar elements with varying orientations about a radial mean direction depending on the zonal depth. The experimental evidence suggests that the fibrillar architecture or ‘knit’ in the general matrix incorporates the following structural principles: (1) The overall arrangement of fibrils is radial and they
Fig. 16 (a) Pronounced radial alignment of collagen fibrils in the highly stressed band of matrix at the transverse notch root as in Fig. 15b. Arrows indicate radial stressing direction. (TEM, x 18,690). (b) Acutely kinked and collapsed fibrillar structure in matrix below stressed radial notch root as in Fig. 14b. Arrows indicate transverse stressing direction. These structures should be compared with the underformed matrix ultrastructure as shown in Fig. 7b. (TEM, x 12,700).
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Fig. 17 A 2-dimensional wire mesh analogue of the interconnecting goemetry of radial elements proposed for the arrangement of fibrils in the general matrix of articular cartilage. Note the rapid strain-locking in the radial direction (cf A and B) versus the considerable extension permitted in the transverse direction (cf A and C).
appear to be continuous or nearly so for considerable distances through the cartilage depth. This provides the extremely high strength in the radial direction. The fibrils are required to be axially fractured if transverse rupture is to occur. Hence the phenomenon of a radially deflected rupture path with the transverse notch (Fig. 15). (2) The fibrils, as they pass radially up through the cartilage depth, are repeatedly deflected sideways to
Pathology (1988). 20, April
form an arrangement of obliquely oriented segments in a crude zig-zag configuration. This secondary morphology accounts for the strain-locking effect we observe when the matrix is stressed radially as in the transverse notch experiment (see Figs 15b and 16a). (3) Some form of interfibril link or connection is assumed to exist to give the overall radial array of fibrils structural cohesion in the transverse direction. It is the transverse extension of these interconnected radial fibrils that accounts for the much greater degree of extension in the transverse direction relative to the radial, hence the development of an acutely kinked arrangement with transverse stressing of the matrix in the radial notch experiment (Fig. 16b). These structural concepts have been modelled recently with a 3-dimensional physical mode1I6 and are illistrated in 2-dimensions with the wire mesh analogue in Fig. 17. The model shows how, beginning with an interconnected geometry of radial elements, there is rapid strain locking radially, but very considerable transverse extensivity. It is proposed that within such an interconnected collagenous structure the proteoglycan component is contained. The entire fibrillar structure is kept taut by the swelling capacity of the hydrated proteglycans, the oblique interconnecting collagen segments effectively creating a braced structure that is resistant to both compression and shear.” The model also appears to explain the presence of the long transverse fibres drawn out across the propagating radial notch root (Fig. 14b). As rupture proceeds matrix material directly under the notch will move laterally to either side, displacing entire arrays of fibrils out of their original overall radial orientation. Many fibrils will be deflected to the side opposite to which they belong as radial elements in the unruptured matrix. They will therefore be stressed transversely across the notch and finally rupture.
Fig. 18 Impact-induced structural transformation in normal bovine articular cartilage: (a) undeformed matrix; (b) impacted matrix exhibiting well-developed crimped fibrous structure. Chondrocyte alignment is radial. (Both DIC, x 566).
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Fig. 19 Enzymatically induced structural transformation in normal bovine articular cartilage: (a) matrix appearance after trypsin pretreatment; (b) well-developed crimped, radial fibrous structure following partial digestion of (a) with bacterial collagenase. (Both DIC, x 275).
What then is the relationship between cartilage structure and its malfunction? As noted earlier, a consistent feature of abnormally soft cartilage is the appearance in the matrix of radially aligned aggregates of fibrils or fibres, easily resolved at the optical microscope level. These readily collapse in phase with compressive loads (Fig. 12). This response is consistent with an absence of structural bracing assumed to be associated with the interconnecting fibrillar structure of the normal matrix. The oblique interconnecting arrangement of fibril segments will of course represent a high energy configuration dependent on the maintenance of the interfibril linkage. Is structural softening then a direct consequence of this interfibril link being degraded? Unfortunately we have no way of examining structurally a particular region of cartilage prior to the development of abnormal softening. However, there are two additional pieces of experimental evidence that strongly support the theory that the interfibril linkage is directly implicated. First, repeated impact loading of fresh cartilage-on-bone in vitro can transform the interconnecting arrangement of fibrils comprising the general matrix of normal cartilage into a strongly aligned radial configuration of fibres incorporating the characteristic waveform or crimp" (Fig. 18). This stress-induced transformation produces a collagenous arrangement virtually identical to that observed in the naturally softened cartilage (Fig. 9a). It is also consistent with the type of structural response we would expect from dynamic overloading. Rapidly applied compressive stresses would prevent the time-dependent flow of water from under the site of compression to regions of lower stress. The matrix will instead respond as a 'hard' solid generating high internal stresses sufficient to disrupt the interfibril linkage. This would, in turn, allow a relaxation of the high energy interconnecting
network with consequent alignment of fibrils in their primary radial configuration. The second piece of experimental evidence supporting the concept that an interfibril link is directly involved in the maintenance of the high energy interconnecting configuration of fibrils comes from some very recent enzymatic studies.'* By first removing a large fraction of the proteoglycan component using either testicular hyaluronidase or trypsin digestion, followed by a limited attack on the collagen using bacterial collagenase, it is possible to transform the high energy interconnecting fibrillar arrangement comprising the normal matrix, into a strongly aligned radial configuration (Fig. 19). This degraded fibrillar structure is mechanically soft, and again virtually identical to that observed in the osteoarthritic matrix (Fig. 8a) and in the softened cartilage on the tibia1 plateau (Figs 9a and 9c). A key requirement for the above enzymatic transformation to occur is some limited collagenolytic action. This COLLAGEN NETWORK TRANSFORMATION I N ARTICULAR C A R T I L A G E
DEGENERATION
TRAUMA
.,
b
E N Z V Y A T I C DIGESTION T"I.II*?COLL.TI*I.S
Fig. 20 Schematic illustration of 3-fold transformation pathway from the pseudo-random fibrillar arrangement to a radially aligned crimped configuration. From Broom.'8 Reproduced with permission.
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would suggest that the interfibril link is intimately associated with the collagen fibrils, but what it actually is has still be to determined. It is therefore argued that the interconnected, oblique arrangement of fibril segments in association with the hydrated ground-substance provides a braced structural system with excellent load-bearing characteristics. A variety of factors can however induce a major structural transformation in the collagenous architecture of articular cartilage. Naturally occurring disease or degenerative processes, dynamic overloading or selective enzymatic attack can all transform this normal architectural arrangement of fibrils into a radially aligned and aggregated configuration, poorly braced and therefore lacking in load-bearing ability (Fig. 20). This multipathed transformation is, I believe, convincing evidence that the fibrillar architecture of the articular cartilage matrix is a highly specialized configuration constrained by forces still not well understood. Elucidation of these factors should represent a significant advance in our understanding of the degenerative process. CONCLUDING COMMENTS In this presentation I have endeavoured to show how complex connective tissue systems can be examined in a manner that reveals fundamental insights into their biomechanical properties. A key requirement in this approach is that the tissues be observed microscopically in their fully hydrated condition, while being simultaneously manipulated using specific loading configurations that selectively stress key structural components within the tissue. Having clarified, at least in part, the biomechanical principles relating to two quite different structural connective tissues, the problem of abnormal mechanical function in articular cartilage and its structural origin has been considered.
ACKNOWLEDGEMENTS I would like to thank the Australasian Society for Experimental Pathology for their invitation to present this Third George Swanson Christie Memorial Lecture. In particular my appreciation to Dr Kathryn Ham whose courageous idea it was to let an engineer loose before an audience of pathologists! The research activities reflected in this lecture have been supported by funding from the Medical Research Council of New Zealand.
References 1. Gordon JE. The New Science of Strong Materials. London: Penguin
Books, 1976, Chp 2. 2. Sauren AAHJ, Kuijpers W, Van Streenhoven AA, Veldpaus FE. Aortic valve histology and its relation to mechanics - preliminary report. J Biomech 1980; 13: 97-104. 3. Ferrans VJ, Spray TL, Billingham ME, Roberts WC. Structural changes in porcine xenografts used as substitute cardiac valves. Am J Cardiol 1978; 41: 1159-84. 4. Clark RE, Finke EH. Scanning electron microscopy of human aortic leaflets in stressed and relaxed states. J Thorac Cardiovasc Surg 1974; 67: 792-804. 5 . Mohri H, Reichenbeck DD, Merendino KA. Biology of homologous and heterologous aortic valves. In: Ross DN, Wooler GH, eds. Biological tissue in heart valve replacement. London: Butterworths, 1972; 137-42. 6. Christie GW, Medland IC. A non-linear finite element stress analysis of bioprosthetic heart valves. In: Gallagher RW, Simon BR, Johnson PC, Gross JF, eds. Finite elements in biomechanics. New York: John Wiley, 1982; 153-79. 7. Broom ND, Christie GW. The structure/function relationship of fresh and glutaraldehyde-fixed aortic valve leaflets. In: Cohn L, Gallucci V, eds. Cardiac bioprostheses - Proceedings of the 2nd international symposium. New York: Yorke Medical, 1982; 477-91. 8. Broom ND, Myers DB. A study of the structural response of wet hyaline cartilage to various loading situations. Connect Tiss Res 1980; 7: 227-37. 9. Broom ND, Poole CA. A functional-morphological study of the tidemark region of articular cartilage maintained in a non-viable physiological condition. J Anat 1982; 135: 65-82. 10. Broom ND, Poole CA. Articular cartilage collagen and
proteoglycans: their functional interdependency. Arthritis Rheum 1983; 26: 111-9.
1 1 . Broom ND. Abnormal softening in articular cartilage: its relationship to the collagen framework. Arthritis Rheum 1982; 25: 1209-16. 12. Broom ND. The altered biomechanical state of human femoral head osteoarthritic articular cartilage. Arthritis Rheum 1984; 27: 1028-39. 13. Roth V, Mow VC. The intrinsic tensile behaviour of the matrix
14. 15.
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of bovine articular cartilage and its variation with age. J Bone Joint Surg 1980; 62A: 1102-17. Broom ND. Further insights into the structural principles governing the function of articular cartilage. J Anat 1984; 139: 275-94. Broom ND. The collagenous architecture of articular cartilage a synthesis of ultrastructure and mechanical function. J Rheum 1986; 13: 142-52. Broom ND, Marra DL. New structural concepts of articular cartilage demonstrated with a physical model. Connect Tiss Res 1985; 14: 1-8. Broom ND. Structural consequences of traumatising articular cartilage. Ann Rheum Dis 1986; 45: 225-34. Broom ND. An enzymatically induced structural transformation in articular cartilage: its significance with respect to matrix breakdown. Arthritis Rheum 1987, In press.