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Advanced Drug Delivery Reviews 58 (2006) 128 – 149 www.elsevier.com/locate/addr

Osteoarthritis: Pathobiology—targets and ways for therapeutic interventionB T. Aigner a,*, A. Sachse b, P.M. Gebhard a, H.I. Roach c a Institute of Pathology, University of Leipzig, Liebigstraße 26, 04103 Leipzig, Germany Department of Orthopedics, University of Jena, Klosterlausnitzerstr. 81, 07607 Eisenberg, Germany Bone and Joint Research Group, Division of Developmental Origins of Health and Disease, University of Southampton, CF86, MP 817, General Hospital, Southhampton, SO16 6YD, UK b

Received 30 September 2005; accepted 30 January 2006 Available online 6 March 2006

Abstract Osteoarthritis is first and foremost the ongoing destruction of the articular cartilages of joints. Therefore, the extracellular matrix and the cells of the articular cartilages are the primary targets of osteoarthritis therapy. This tries to inhibit enzymatic destruction of the extracellular cartilage matrix as well as the modification of the cellular phenotype of the chondrocytes: cell degeneration and cell death are alongside anabolic activation and stabilization of the cellular phenotype of major interest. However, apart from the cartilage and its cells, other tissues of the joints are also important for the symptoms of the disease, which basically all originate outside the articular cartilage. In addition, changes in the subchondral bone as well as the synovial capsule and membrane are important at least for the progression of the disease process. All the named tissues offer different directions and ways for therapeutic intervention. D 2006 Elsevier B.V. All rights reserved. Keywords: Cartilage; Drugs; Joints; Chondrocytes; Senescence; Cartilage regeneration

Contents 1. 2.

Introduction—the scenario of osteoarthritis . . . . . . . . . . . . . . . . Joint physiology—functioning of an organ system . . . . . . . . . . . . 2.1. The joint capsule–the synovial membrane–the synovial fluid . . . 2.2. The articular cartilage . . . . . . . . . . . . . . . . . . . . . . . The maintenance of the joint–the maintenance of the extracellular matrix

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This review is part of the Advanced Drug Delivery Reviews theme issue on bDrug Delivery in Degenerative Joint DiseaseQ, Vol. 58/2, 2006. * Corresponding author. Tel.: +49 341 97 15036; fax: +49 341 97 15019. E-mail address: thomas.aigner@medizin.uni-leipzig.de (T. Aigner).

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Osteoarthritis–disease of an organ system–tissues to be targeted . . 4.1. The synovial membrane, the joint capsule and inflammation 4.2. The subchondral bone . . . . . . . . . . . . . . . . . . . . 4.3. The cartilage matrix . . . . . . . . . . . . . . . . . . . . . 4.4. The cartilage cells (chondrocytes) . . . . . . . . . . . . . . 4.5. Anabolic and catabolic mediators . . . . . . . . . . . . . . 4.6. Chondrocyte proliferation and death. . . . . . . . . . . . . 5. Cartilage repair: option for therapeutical intervention . . . . . . . 5.1. Evaluation of repair tissue . . . . . . . . . . . . . . . . . . 6. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction—the scenario of osteoarthritis Osteoarthritis, the degeneration of the joints, is the most common disabling condition in the Western world. Clinically, degeneration affects mostly the large weight bearing joints of the legs (i.e. hips and knees), but can in principle affect any joint of the body including, notably, the finger joints. Osteoarthritis is not a single disease entity, but represents a disease group with rather different underlying pathophysiological mechanisms. In this respect, primary osteoarthritis has to be distinguished from secondary forms of the disease, which are due to traumatic events, endocrine or metabolic disorders, etc. Clinically, pain as well as loss of joint functioning is the major issue leading to a significantly reduced quality of life for patients suffering from the disease. The enormous frequency of this disease makes osteoarthritic joint disease (to delete) one of the most expensive conditions in the Western world, both in terms of direct as well as indirect costs. So far, the treatment options are rather limited and mostly restricted to symptomatic approaches (i.e. exercise, physiotherapy, etc.) until–in the end–the joint needs to be replaced by endoprosthetic joint surgery. Thus, there is a high need for development of disease modifying agents in order to improve quality of life as well as to reduce the enormous socio-economic burdens of the disease. However, despite intensive efforts over several decades, the success of diseasemodifying approaches have so far been rather limited. This review focuses on the major basic biological principles of the joint tissues in physiology as well as degenerative pathology. It will indicate potential tissues and targets for therapeutic intervention. Of

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note, the various tissues involved in the degeneration process also implicate different strategies to deliver potential therapeutic agents.

2. Joint physiology—functioning of an organ system Joints are highly specialized organs that allow repetitive pain-free and largely frictionless movements. These properties are provided by the articular cartilage and its extracellular matrix, which under physiological conditions is capable of sustaining high cyclic loading. Articular cartilage covers the joint surfaces and is mainly responsible for the unique biomechanical properties of the joints. Joints are, however, complex composites of different types of connective tissue including (subchondral) bone, cartilage surfaces, ligaments and the joint capsule (Fig. 1). Thus, the capsule, together with the ligaments, is extremely important for the mechanical stability of the joint as a whole. If mal-aligned, the cartilage is loaded abnormally and degenerates rather dramatically, as seen in misalignment syndromes of the joints. All the different joint tissues together provide their own functional capacities in order to allow the correct functioning of the joint. In contrast to parenchymal organs, such as liver, heart, brain, kidney, etc., the connective tissues of the joints do not function directly via their cells, but their extracellular matrix. This means that e.g. the liver provides the body with its benefits via the capacity of the liver cells to metabolize nutrients or to synthesize serum proteins, etc. The intercellular matrix of liver mostly relates to keeping the cells together and, thus, to provide the

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normal

Osteoarthritis (joint deformation)

articular cartilage

destructed cartilage

joint capsule

capsular fibrosis osteophyte formation

synovial membrane

synovial hyperplasia

(subchondral) bone

(subchondral) bone remodelling and sclerosis

Fig. 1. Schematic representation of the main structures of a healthy (left side) and degenerated (right side) joint in osteoarthritis: in particular the articular cartilage is lost or severely thinned, the (subchondral) bone is sclerotic and the joint capsule thickened and the synovial membrane activated.

structural coherence (and importantly also orientation) of the organ. Obviously, no significant mechanical load is applied to the liver nor to other parenchymal organs. This is very much in contrast to connective tissues which mostly function via their mechanical properties. Accordingly, in parenchymal organs more than 95% of the tissue volume consists of the cells, whereas in connective tissues and in particular in articular cartilage more than 95% of the volume consists of their functional element, the extracellular matrix (Fig. 2). 2.1. The joint capsuleâ&#x20AC;&#x201C;the synovial membraneâ&#x20AC;&#x201C;the synovial fluid The synovial capsule and in particular the synovial membrane (i.e. the synovial lining cell layer) represent important portions of the joint as an organ. As already mentioned, it is the capsule together with the ligaments which provide the mechanical stability of the joint and determine the flexibility of the possible movement. Overflexibility (e.g. after traumatic ligament rupture) clearly

increases the risk for joint degeneration with time [1]. The synovial membrane with its metabolically highly active surface cells (synoviocytes) plays a crucial role in nourishing the chondrocytes as well as removing metabolites and (matrix) degradation products from the synovial space: this is partly performed by the intrinsic metabolic activity of these cells, partly by diffusion of serum components into the joint space. It is the synoviocytes that maintain the basic metabolic homeostasis of the joints. Furthermore, the synoviocytes produce large amounts of hyaluronic acid, which provides the joint surfaces with its gliding capacity. Otherwise, the synovial fluid constitutes the major medium for substances diffusing between the articular cartilage and the synoviocytes (nutrients and metabolites from the chondrocytes as well as oxygen molecules and many other components). 2.2. The articular cartilage The articular cartilage is a highly specialized and uniquely designed biomaterial that forms the smooth,

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chondrocytes

extracellular matrix functional element

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articular cartilage

reactive element

Fig. 2. Articular cartilage mainly consists mainly of extracellular matrix (more than 95% of tissue volume), its functional element. Interspersed in between the abundant matrix are the cells, the chondrocytes, which are, however, the living (i.e. reacting) element of the articular cartilage tissue.

gliding surface of the diarthrodial joints (Fig. 3b,f). It is largely an avascular, aneural and alymphatic matrix, which is synthesized by sparsely distributed resident cellsâ&#x20AC;&#x201D;the chondrocytes (Fig. 2). The major constituents of the articular cartilage matrix are collagens, proteoglycans as well as the very heterogeneous group of non-collagenous non-proteoglycaneous proteins [2]. In fact, most of the physiological (wet) weight of articular cartilage comes from water bound to the proteoglycans (namely aggrecan). The cartilage matrix can be subdivided according to different cartilage zones based on the arrangement of the cells and the matrix fibrils (i.e. superficial, radial, deep and calcified). Also, the cartilage matrix can be split up into different compartments depending on its relationship to the cells: whereas the pericellular matrix is immediate to the cells, the interterritorial matrix compartment represents the major portion of the cartilage matrix away from the cells. The territorial matrix is defined as the cell-associated matrix located between the pericellular and the interterritorial matrix components, but no real biochemical characterization is so far available. The interterritorial cartilage matrix consists of two basic components: a fibrillar and an extrafibrillar matrix. The fibrillar matrix is a network consisting

mainly of collagen type II together with other collagens, predominantly types IX, XI, and XVI [3,4]. Collagen type XI is located in the core of the collagen type II fibrils and is thought to be involved in fibril initiation and limiting fibril diameter [5]. Collagen type IX as fibril-associated collagens with interrupted triple helices (FACIT)-collagen (for overview, see Gelse et al. [6]) is located periodically along the surface of collagen type II fibrils in antiparallel direction [7] and might be responsible via its Nterminal globular domain for crosslinking the collagen network with itself, but also to the non-collagenous matrix [8,9]. Of note, the so-called btype collagen II fibrilsQ also contain many non-collagenous protein components in addition to the collagens, such as small proteoglycans, cartilage matrix protein, and many other only partially defined [3]. The nonfibrillar component of hyaline articular cartilage consists predominantly of highly sulfated aggrecan monomers, which are attached to hyaluronic acid via link protein and form very large aggregates [10]. In terms of the physical properties of the cartilage matrix, tensile strength comes from the collagen network, which hinders expansion of the viscoelastic aggrecan component and, thus, provides compressive stiffness of the tissue [11]. On the other hand, the

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aggrecan–hyaluronan aggregates bind high amounts of intercellular water due to their extensive fixed charges and are responsible for the elasticity of the tissue. Thus, under compression, the cartilage matrix is compliant, but rapidly regains its elasticity upon unloading as water molecules are drawn back into the matrix by the strongly hydrophilic aggrecan aggregates [12]. Apart from the collagen fibrils and the aggrecan aggregates, cartilage matrix also contains a large number of other components that are important for matrix cohesion and for regulation of chondrocyte function. In particular, these molecules are important in cross-linking the major collagen network of the cartilage matrix as such with the proteoglycans inbetween. Thus, a large number of small non-aggregating proteoglycans such as decorin, biglycan, and fibromodulin have been detected in cartilage matrix, some of them directly associated with the collagen fibrils [13,14]. Most investigations have so far analyzed the (patho)biochemistry of the interterritorial cartilage matrix. Much less attention has been paid to the changes of the pericellular cartilage matrix. At present, only the changes in type VI collagen have been rather well investigated. Type VI collagen is concentrated in the pericellular matrix in normal and diseased cartilage [15–19]. Own ultrastructural studies have shown a physical overlap of the type VI collagen network with the type II collagen matrix, supporting the notion that type VI collagen is one central molecular component forming a mechanical interface between the rigid type II matrix and the cells.

3. The maintenance of the joint–the maintenance of the extracellular matrix In normal human articular cartilage, aggrecan core and link proteins exist as a heterogeneous population

[20–22] differing in size and composition as a result of differential post-translational glycosylation and proteolysis [23]. Normal proteolytic aggrecan turnover is highly regulated and is most probably implemented by the action of MMPs, particularly MMP-3 [24]. However, a second cleavage site, the daggrecanaseT site, has also been described in the interglobular domain and contributes to the proteolytic degradation of aggrecan [25–27]. In normal adult articular cartilage, the turnover of aggrecan is not excessive and the half life of aggrecan monomers and aggregates ranges from days to months, depending on the particular matrix compartment [28]. Subdomains of the aggrecan core protein have even been shown to persist even for years [29]. The collagen type II network is extremely stable. The tightly wound triple helices that constitute the collagen fibres are further stabilized by a high-degree of crosslinking, which steadily increases with age [30]. Fibre destabilization can only be brought about by cleavage of the triple helix due to the action of collagenases, with MMP-1 and MMP-13 being the most important for type II collagen [31,32]. Whatever the turnover rates of the various components of the cartilage matrix are (and many turnover rates are completely unknown), a balanced turnover of the matrix is needed for its proper functioning and preservation.

4. Osteoarthritis–disease of an organ system–tissues to be targeted Primary osteoarthritis of the large weight-bearing joints is generally the result of an imbalance between applied mechanical stress and the physico-chemical ability of the articular cartilage to resist this stress. In the end, osteoarthritis results from the destruction and failure of the extracellular matrix, the functional element of articular cartilage. However, osteoarthritis is a disease of the joint as an organ system, not only of

Fig. 3. (a) Radiographic appearance of hip osteoarthritis displaying distorted joint architecture, loss of joint space as well as osteophyte formation in the joint margins. (b–d) Macroscopic appearance of femoral condyles of the knee: normal (a), early degenerated (b), and severely damaged (c). (e) Arthroscopic picture of a cartilage defect of the femoral condyle within the knee joint (courtesy from Dr. Eger, Rummelsberg). (f–i) Histomorphology of normal (f: smooth surface, normal cell distribution), moderately damaged (g: fissuring, some matrix loss, some cellular cloning; d: loss of proteoglycans in the upper zones demonstrated by the toluidine blue stain), and severely osteoarthritic cartilage (i: most matrix lost, subchondral bone partly exposed).

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the articular cartilage. This includes all connective tissues within and around the joints as well as the respective musculature, the nervous system and even portions of the body more remote from the joint site, such as the central nervous system. The latter is in particular important for the symptomatic aspects of the disease and in fact the innervation and the processing are important to pain, the major symptom of the disease process. In terms of joint tissues, clearly the synovial capsule including the synovial membrane plays a very important role in the scenario of joint functioning and tissue maintenance. Overall, the recognition that the joint does not only consist of articular cartilage, but also a number of adjacent tissues is not only important for the understanding of joint physiology, but also of joint pathology. All these tissues are more or less affected by degenerative changes or their consequences and–in the context of this volume even more importantly–are potential targets for therapeutic intervention. Thus, it is not only the articular chondrocytes and their surrounding matrix with their particular biology and their remote positioning with all the problems of accessibility, etc., but also structures like the synovial membrane and the bone which are rather easy to reach in terms of oral drug treatment. 4.1. The synovial membrane, the joint capsule and inflammation Osteoarthritis research is traditionally concentrating on the understanding of events within the degenerated articular cartilage as the tissue in which the initiating events presumably take place. Changes occurring in the synovial membrane are largely neglected in this respect, at least as far as diseasemodifying therapy is concerned. The synovial changes are generally interpreted as largely secondary to the degeneration of the articular cartilage and not pathogenetically involved to a relevant extent in the disease process. However, the synovial capsule and, in particular, the synovial lining cells represent an important portion of the joint as an organ. As already mentioned, it is the capsule together with the ligaments which provide the mechanical stability of the joint and determine the allowed flexibility of movements. Thus, thickening of the collagen plate within the joint capsule, a typical feature in many

osteoarthritic patients at least in the late stages, reduces significantly the movement properties of their joints (Fig. 4d,e). Thus, capsular fibrosis is centrally responsible for joint stiffening, which is, after pain, the second biggest symptomatic issue of osteoarthritic joint degeneration. We and others have shown in studies on synovial specimens derived from patients suffering from early and advanced osteoarthritis that all cases of clinically significant osteoarthritic joint disease are associated with some significant synovial pathology [33,34]. This supports the notion that there is a direct relation between clinical symptoms and the synovial reaction in osteoarthritis. However, the possibility that these changes are involved at least in the progression of the disease should, therefore, be considered [35]. Most likely, the initial event in osteoarthritic synoviopathy is molecular cartilage detritus, which exceeds the bphysiologicalQ molecular detritus derived from articular cartilage during normal matrix turnover. Synoviocyte activation, proliferation, and synovial hyperplasia presumably all represent reactive changes to increased demands for clearance of molecular debris in the synovial fluid of the joint [36–38]. This might explain [38,39] the increase in the proportion of CD68-positive type A synoviocytes, which have phagocytic capacity, in the synovial lining layer [40–43]. According to the literature, two subforms of synovial reaction pattern are distinguished in osteoarthritic joint degeneration: synovial hyperplasia (Fig. 4d) and the detritus-rich synovitis found in end-stage disease (Fig. 4h,i) [44,45]. The latter is [36,44] due to abundant macromolecular cartilage and bone detritus (i.e. avital bone and cartilage fragments incorporated into the synovial membrane). The most interesting subform among osteoarthritic synoviopathies in terms of pathogenesis might be the inflammatory osteoarthritic synoviopathy, which can show moderately extensive lymphocytic infiltrates (Fig. 4j,g) [34,46, 47], very much resembling less severe rheumatoid synovitis. It is intriguing to speculate whether this subform reflects also some kind of autoimmune aspect of a subset of osteoarthritic patients. This clearly opens up the option of anti-inflammatory therapy of osteoarthritis at least for some patients. In most instances, however, hyperplastic osteoarthritic synoviopathy is the form of synovial reaction

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Fig. 4. (a, b) Histological appearance of normal synovial membrane with flat, non-activated synovial lining cells at the surface (b: detail). (c) Typical picture of hyperplastic synoviopathy with numerous synovial villi. (d, e) Typical picture of fibrotic synoviopathy with a very much thickened and fibrotic capsule (note at the surface also some hyperplastic synovial villi; e: collagen stain (van Giesonâ&#x20AC;&#x2122;s stain)). (f, g) Inflammatory synovitis in osteoarthritic patients with a minor to moderate lymphocytic infiltrate, partly organized in lymph follicles (g). (h, i) Detritus-rich synovitis, which is a typical feature of rapid-progressive end-stage disease with numerous bone and cartilage particles intermixed with fibrin and partly incorporated into the synovial stroma. Large fragments get successively degraded by osteoclast-type multinuclear giant cells (i: here labeled with CD68, a marker of phagocyting cells).

found in early osteoarthritic patients (Fig. 4c). This shows only moderate synovial hyperplasia without significant capsular fibrosis and lacks significant inflammatory infiltrates or macromolecular detritus [34,48]. The synovial lining layer shows mostly a

slight to moderate proliferation and activation of the cells. Although this situation, an inflammatory component, is missing, the proliferation and activation of the synovial lining cells might generate significant problems for the articular cartilage as these cells are

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able to secrete matrix-degrading proteases (MMPs) and catabolic cytokines (IL-1, TNF-a) [49,50]. It is intriguing to speculate that the cartilage matrix catabolism might be at least in part induced or promoted by catabolic mediators such as IL-1h and TNF-a secreted by the activated synoviocytes. Basically, inflammatory cytokines such as IL-1h and TNFa are top candidates for therapeutic intervention, because both are not only able to down-regulate matrix anabolism in articular chondrocytes [51], but also to induce expression and secretion of matrix degrading proteases [52â&#x20AC;&#x201C;54]. Alternatively, antagonistic molecules such as the IL-1-receptor-antagonist might be down-regulated [55]. In fact, Smith et al. were able to demonstrate that the expression and synthesis of IL-1 and TNF-a correlates with ongoing matrix destruction [48]. Thus, again, targeting inflammatory and degradative activity of synoviocytes represents an interesting therapeutic target for early stage disease. In most cases of osteoarthritis [34,56], capsular fibrosis (Fig. 4d,e) is found mostly in the late stage specimens and has very detrimental effects due to shortening of the capsule, which is responsible for part of the symptoms, in particular joint stiffness. Additionally, it presumably further increases the biomechanical stress on the already damaged articular cartilage by increasing tension stress all over the joint. The fact that joint capsules of osteoarthritic joints have an enormous tendency to thicken and to become fibrotic, which (after pain) represents the second most important clinical problem, opens up the field of antifibrotic therapy for the osteoarthritic disease condition. 4.2. The subchondral bone Another important tissue often neglected in osteoarthritis research is the subchondral bone [57], although it is unclear yet whether pathological changes within the subchondral bone tissue (e.g. sclerosis) can precede changes in the articular cartilage (e.g. bone mass as risk factor of osteoarthritis [58]) or subchondral bone changes are secondary adaptation processes following changes in the biomechanical properties of the cartilage [59]. Significant changes in terms of increased thickness of the subchondral bone plate as well as underlying trabe-

cules are already apparent in the early stages of the disease. Thus, active new bone formation is found at multiple foci in early- to mid-stage patients [60]. In later stages, severe bone remodeling processes take place in particular in areas of advanced destruction of the overlying articular cartilage. Apart from extensive bone sclerosis, significant aseptic bone necrosis is a common feature of advanced osteoarthritic joint degeneration. In areas of total cartilage destruction (i.e. eburnated bone plate), synovial fluid gets access to the bone marrow and induces fibrocytic and even chondrometaplastic changes of mesenchymal precursor cells. This leads to the characteristic bcartilagenodulesQ or btuftsQ, which are frequently found in these areas in late stage disease. At least in moderate to advanced lesions, the changes in the subchondral bone represent one tissue responsible for the osteoarthritic joint pain [61,62]. Thus, the osteoarthritic bone represents one interesting target tissue for symptomatic treatment at least in late stage patients. Also, in earlier disease stages, modification of bone remodeling might be a way to prevent subchondral stiffening, which might as such reduce the progression of cartilage destruction and maybe also inhibit osteophyte formation [63]. 4.3. The cartilage matrix Macroscopically, osteoarthritic cartilage is often yellowish or brownish and is typically soft. The surface shows roughening in the early stages and overt fibrillation and matrix loss in the later stages until the eburnated subchondral bone plate is visible. Apart from the degradation of molecular components, destabilization of supramolecular structures also takes place. For example destabilization of the collagen network results in microscopically and finally macroscopically visible matrix destruction. Both mechanical wear and enzymatic degradation appear to play a pivotal role during the disease process. Together, these result in the destruction of cartilage matrix on the molecular (e.g. proteoglycan depletion) and the macromolecular (e.g. network loosening), the microscopic (e.g. fissuring) and the macroscopic level (e.g. cartilage tear). The destruction of articular cartilage and the loss of its biomechanical function are largely due to the destruction and loss of the interterritorial cartilage

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matrix, which result from an imbalance between degradation and de novo synthesis of matrix components on the molecular and supramolecular level in spite of the compensatory attempts of the chondrocytes. So far, our knowledge focuses on changes of the two major components of the interterritorial cartilage matrix, the collagen network and the interwoven proteoglycan aggregates. Loss of aggrecan and its fixed (negative) charges is characteristic of the early stages of cartilage degeneration, whereas the overall collagen content remains rather constant until the very end of the disease process [64,65]. Although overall no collagen is lost, loosening of the collagen network is a major feature in cartilage degeneration. It is still unknown, what comes first: the loss of proteoglycans or the loosening of the collagen network, as each affects the other. As shown by Maroudas [66], a loosening of the collagen network leads to loss of proteoglycans and loss of proteoglycans leads to a mechanical overload and, thus, damage and loosening of the collagen network. In particular, the latter appears to be responsible for the hyperhydration of articular cartilage in the early phases of the disease process, macroscopically visible as softening and swelling of the osteoarthritic articular cartilage [66]. Degradation processes appear to be specifically prominent in the surface zone and around the chondrocytes in osteoarthritic cartilage [67,68]. Enhanced levels of many metalloproteinases including MMP-2 [69,70], MMP-7 [71], MMP-8 [72,73], MMP-9 [74,75] MMP-13 [32,52,70,76], MMP-14 (membrane-type I MMP [77]), ADAMTS-4 and -5 [78,79], ADAM-10 [80] and ADAM-15 [81], have been reported to accompany the increased matrix degradation in osteoarthritic cartilage. So far it is rather enigmatic which proteases are really crucial for the degradation of the various cartilage matrix components although e.g. MMP-13 is certainly a top candidate for primary collagen fibril degradation [32,76]. Recently, ADAMTS-5 was shown to be responsible for aggrecanase-mediated proteoglycan degradation, at least in mice [82,83]. This supports previous analysis of mRNA expression of proteases which suggested ADAMTS-5 to be the only significantly expressed aggrecanase up-regulated in osteoarthritic chondrocytes in situ [52]. Apart from alterations of cartilage components, expression of molecules that are not present in normal

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articular cartilage also appear to be important phenomena. Thus, the composition of the proteoglycans has been shown to change in osteoarthritic cartilage. We and others reported the appearance of molecules in osteoarthritic cartilage such as tenascin [84], collagen types IIA [85] and III [86], which are not normally present in articular cartilage. At the moment, it is a matter of speculation whether the newly synthesized type IIA and III procollagens have any special functional role in osteoarthritic cartilage or represent functionless matrix deposits. The latter would sooner or later become degraded and diffuse out into the cartilage matrix and finally the synovial fluid. Type X collagen becomes a prominent component in the very deep and calcified cartilage zones [87], where it might be directly involved in the ongoing calcification process in these zones, which is characteristic for osteoarthritic cartilage degeneration. Poole et al. [18,68,88,89] have shown that the type VI positive pericellular matrix is severely altered in osteoarthritic cartilage. Both increased synthesis as well as enhanced degradation of collagen type VI is well documented in osteoarthritic cartilage [90,91]. Ultrastructural analysis showed abundant cross-striated fibrous type VI collagen aggregates in particular around osteoarthritic chondrocytes [17,19,90] which most likely reflects partial degradation of type VI collagen. Such a transformational conformation of resident type VI collagen rather than some sort of net loss of collagen type VI molecules might be the primary reason of a significant functional derangement of the pericellular cartilage matrix in osteoarthritic cartilage. The consequences of the derangement of the pericellular matrix in osteoarthritic cartilage are unknown. It might impede proper cellâ&#x20AC;&#x201C;matrix interaction e.g. via integrins [92â&#x20AC;&#x201C;94] and the cell might lose its protective basket against compression forces. Alterations in the type VI collagen microenvironment can also influence the synthetic activity of chondrocytes and, thus, modulate e.g. proteoglycan synthesis. 4.4. The cartilage cells (chondrocytes) Despite the importance of the extracellular matrix for the functioning of articular cartilage, the cells are not bfunctionlessQ in connective tissues, as they are the only viable players within the tissue. Thus, they are centrally responsible for the balanced turnover of

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the extracellular matrix, which is necessary for maintenance of the integrity of the extracellular cartilage. This, in turn, requires appropriate anabolic activity of the chondrocytes. During the osteoarthritic disease process, the cellular reaction patterns are altered. At first sight these are rather pleomorphic, but can be basically summarized in three categories. First, the chondrocytes can undergo cell death (see Section 4.6) or they can proliferate to compensate for cell loss or to increase their synthetic activity. Secondly, chondrocytes activate or deactivate their synthetic–anabolic activity by increasing or decreasing anabolic gene expression. Lastly, chondrocytes undergo phenotypic modulation implicating an overall severely altered gene expression profile of the cells in the diseased tissue. In addition, osteoarthritic chondrocytes are heterogeneous and nearly all observed cellular changes are region- and zone-specific, and also dependant on the degradation stage. A number of biochemical studies have demonstrated an enhanced synthesis of extracellular matrix components [95–97], in particular in the middle zones of low-grade OA. Chondrocytes attempt to repair the damaged matrix by increasing their anabolic activity. The fact that type II collagen expression appears to be much more up-regulated than aggrecan, for example [96,98,99] mimics fetal [100] and osteophytic cartilage [101]. In both of these tissues, chondrocytes have to synthesize and assemble a new extracellular matrix consisting largely of collagen type II. Both are in contrast to normal articular cartilage in which chondrocytes only need to control tissue homeostasis by maintaining a stable matrix composition. This mainly involves the control of proteoglycan turnover, whereas collagen type II turnover is presumably nearly zero [102]. Despite the increased biosynthetic activity of chondrocytes, a net loss of proteoglycan content is one of the hallmarks of all stages of osteoarthritic cartilage degeneration [64]. This leads to the assumption that overall enzymatic degradation of matrix components might be the reason for the metabolic imbalance in osteoarthritic cartilage. However, most studies were based on an overall measurement of matrix composition within the whole osteoarthritic cartilage, which did not allow to detect differences between cells of different cartilage zones. In situ analyses showed that the loss of fixed charges occurs

exactly in the upper zones of osteoarthritic cartilage, in which the cells down-regulate their expression of matrix components including aggrecan, whereas the cells of the deeper zones are still activated [30,99,103]. This explains, at least in part, the loss in proteoglycan content in this zone, if one assumes that the diffusion capacity of aggrecan monomers is limited and enhanced synthesis in one zone cannot compensate for the synthetic failure in adjacent zones. Notably, even in specimens with a very high Mankin’s grade (N 8), implicating an advanced disease state, some chondrocytes still express aggrecan, link protein, and collagen type II mRNA were observed [99]. Thus, even in advanced stages of cartilage degeneration, some chondrocytes keep their capacity to be anabolically active. Phenotypic changes are a central feature of chondrocytes. This is known from many studies of chondrocyte differentiation in vivo in growth plate cartilage, but even more so from analyses of chondrocyte behavior in vitro (for review, see Cancedda et al. [104]). Thus, the chondrocyte phenotype is not stable in vitro, in particular in monolayer culture. Several factors such as retinoic acid, bromodeoxyuridine, or interleukin-1 induce socalled bdedifferentiationQ or modulation of chondrocytes to fibroblast-like cells. They stop expressing aggrecan and collagen type II, even though they are still very active cells, and express collagen types I, III, and V, for example [105–107]. This example clearly demonstrates the implications of phenotypic alterations of chondrocytes: despite potentially high synthetic activity, bdedifferentiatedQ chondrocytes do not express cartilage-specific genes such as aggrecan or type II collagen. Most data suggest that the major phenotypic alterations are initially observed in the superficial zone of early-stage osteoarthritic cartilage, where chondrocytes express de novo abnormal, non-chondrocytic genes; in particular, they express the enzymes required for degrading the matrix that surrounds the cells as well as many of the cytokines and growth factors relevant for turning on the catabolic processes within cartilage. The degradative phenotype may involve epigenetic mechanisms: for example loss of DNA methylation of the promoters of MMP-3, -9, -13 and ADAMTS-4 was found to be associated with enzyme expression [108,109] (for review of the

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importance of methylation events in chondrocytes, see Aigner [110]). In addition, as the superficial zone becomes degraded with advancing osteoarthritis, these cells, originally only at the surface, are now presumably also found in the intermediate and deep zones, which suggests that some propagation mechanism exists so that increasingly more chondrocytes become degradative. Both heritability and propagation of the degradative phenotype thus may constitute reasons, among others, for the continuing catabolism of the cartilage matrix. Therapeutic interventions directed at preventing the propagation of the degradative phenotype would therefore seem promising for future treatment. 4.5. Anabolic and catabolic mediators Anabolic and catabolic activation of cells as well as phenotypic alterations are largely the result of the exposure of the cells to various cytokines and growth factors: mainly IGF-1, BMPs and TGFhs on the anabolic, and TNF-a and IL-1h on the catabolic side (Fig. 5). The roles of other mediators such as hepatocyte growth factor (HGF) remain rather controversial [111,112]. TGF-h is one of the most potent mediators of cartilage matrix synthesis [113]. It up-regulates the expression of several types of collagens and proteoglycans (PG). Other members of the TGF-h superfamily, the bone morphogenetic proteins (BMPs), are also known to stimulate cartilage matrix synthesis. In particular, BMP-2 and BMP-7 have been shown to stimulate anabolic activity of chondrocytes as well as being present in articular cartilage [114â&#x20AC;&#x201C;117]. IGF-I was the first identified growth factor with potent anabolic effects on chondrocytes [118]. Chondrocytes express IGF-I and the concentration of IGF-I increases in osteoarthritic synovial fluid and in osteoarthritic cartilage. Opposing the anabolic effects of growth factors are pro-inflammatory cytokines, primarily IL-1h and TNF-a. Their role in the progression of osteoarthritis has attracted considerable attention (for review, see Goldring [119] and Fernandes et al. [120]). These cytokines stimulate the production of degradative enzymes and suppress protein synthesis [121,122]. Many other factors are also involved in the maintenance of the homeostasis of the cells and the

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osteoarthritis: imbalance of cartilage matrix turnover by chondrocytes

IGF BMP

anabolism aggrecan collagen type VI collagen type IX link protein

Il-1 TNFa

catabolism MMP-2,9 MMP-3 MMP-13 MMP-14

ADAMTS-1 ADAMTS-4 ADMATS-5

Fig. 5. Osteoarthritis exhibits an imbalance in cartilage matrix turnover. Whereas the extracellular matrix (electron micrograph left hand side) of articular cartilage represents the bfunctional elementQ (i.e. the essential component providing its biomechanic properties), the cells of the tissue (i.e. the chondrocytes: electron micrograph right-hand side) represent the active players within the process: they synthesize most of the matrix degrading proteases and fully provide anabolic activity. Even most of the anabolic and catabolic stimulatory factors, many of them not yet fully identified, are synthesized by the chondrocytes themselves in an auto- and paracrine manner. Altogether, this motivates functional genomics of articular chondrocytes in terms of understanding the cellular gene expression patterns as one valuable approach for understanding and manipulating a disease of a biochemically altered extracellular matrix (i.e. osteoarthritis). (BMP: bone morphogenetic protein; IGF: insulin-like growth factor; IL-1h: interleukin 1h; TNF-a: tumor necrosis factor-a.)

matrix turnover in articular cartilage such as chemokines, nitric oxide and oxygen radicals. Clearly, in particular the latter appear to be highly interesting targets for osteoarthritis therapy, but the reader is referred for more detailed insights into these in recent reviews on these topics [123â&#x20AC;&#x201C;125]. 4.6. Chondrocyte proliferation and death Several studies [25,64,126,127] have clearly shown that there is (a very low) proliferative activity in osteoarthritic chondrocytes in contrast to normal articular chondrocytes, which do not show any proliferative activity (Fig. 6). Another explanation for osteoarthritic cartilage degeneration would be a mere loss of viable cells at the beginning and during the disease process (for

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Fig. 6. Schematic representation of basic chondrocyte reaction pattern and main factors influencing it.

review, see Aigner and Kim [128] and Aigner et al. [129]). Cell death can be broadly divided into apoptosis (physiological or pathological) and necrosis (pathological, due to injury, causing inflammation). Apoptosis has evolved as a mechanism to eliminate, without inflammation, surplus, abnormal, or dysfunctional cells whose survival and proliferation would be detrimental. Apoptosis is thus normally a beneficial process, although aberrant apoptosis can occur in pathological states. This is clearly disadvantageous when it leads to the elimination of healthy cells. Many studies have considered whether cell death plays a role in the pathology of osteoarthritis (for review, see Aigner et al. [129]), because articular chondrocytes cannot self-renew and cell loss would therefore be permanent. However, opinions on the prevalence and importance of chondrocyte death for osteoarthritis pathology differ widely. Early studies [130,131] estimated the incidence of cell death as 25â&#x20AC;&#x201C;50%, but the data were obtained with isolated chondrocytes and therefore may have measured a predisposition to death rather than actual incidence. Estimates of cell death based on in vivo staining also vary widely, from 21% [132] to less than 1%, as shown in own studies [25]. Opinions also vary as to the type of cell death. Most authors classify chondrocyte death as due to apoptosis,

even though injury or mechanical damage by themselves would lead to a high level of necrosis. More recently, it has been shown that chondrocytes undergo a variant of classical apoptosis, termed dchondroptosisT [129,133], a mode of cell death that leads to selfelimination by the chondrocytes and thus obviates the need for phagocytosis of the apoptotic bodies. Consistent with this self-elimination process is the presence of empty lacunae in higher-grade OA samples [25,129] as well as the presence of hypocellular regions. It is the authorsâ&#x20AC;&#x2122; view that chondroptosis or chondronecrosis have only a limited impact on the pathology of early osteoarthritis or aging of human articular cartilage [64]. The only zone, in which a large number of empty lacunae were found, was the calcified cartilage layer. The continuous progression of cartilage calcification in osteoarthritic cartilage might explain, at least partly, the increasing number of empty lacunae reported, particularly in high-grade lesions [134,135].

5. Cartilage repair: option for therapeutical intervention At the margins of joints, in particular in osteoarthritic joint disease, frequently (osteo)cartilaginous

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exogenous (therapeutic) repair strategies in mind. The question, whether secondary cartilage formed in oldaged people can be compared with the high-quality original hyaline articular cartilage is relevant for all efforts using secondary cartilaginous repair tissue to treat cartilage defects. Central for the basic understanding of osteophytic tissue is the analysis of the developmental steps during osteophyte formation. Thus, although it is clear that osteophyte development is a continuous process and many osteophytes show different stages in various portions at the same time, one can define basic steps based on the cellular phenotype and the matrix composition of the predominating tissue (Fig. 7) [139]: Initially, mesenchymal precursor cells derived either from periosteum or synovium initiate chondrogenic differentiation. This results in fibrocartilage composed of both fibrous and cartilaginous matrix components. In early osteophytes, endochondral ossification is initiated. The deepest cell layer becomes hypertrophic and resembles very much the

outgrowths appear ((chondro-)osteophytes). They are best considered as a process of secondary chondroneogenesis in the adult [101]. Osteophytes derive from mesenchymal precursor cells within periosteal or synovial tissue and often merge with or overgrow the original articular cartilage [136,137]. Thus, in this process, mesenchymal precursor cells differentiate into chondrocytes. A similar, but less structured process is observed in the areas of the eburnated bone, in which the articular cartilage is completely torn off. Here, mesenchymal multipotential stem cells of the bone marrow undergo also chondrogenic differentiation: metaplastic cartilage in forms of nodules or btuftsQ is found either within the bone marrow or at the naked bone surface [138]. Osteophytes could be considered as endogenous repair attempts in degenerating joints and might be a physiological response to mechanical overloading by increasing the articulating joint surface. Even if their supportive effect within the joints is doubtful, their chondrogenic potential is of interest, especially having

a stage of osteophyte development normal

III

GAGs Col2 Col 10

Fig. 7. Osteophyte development can be subdivided into five stages with different structural organization although many osteophytes show different stages simultaneously in different areas. Stage I (early chondrophytes) shows first chondrocytic differentiation of previously undifferentiated mesenchymal precursor cells. Stage II (chondrophytes) shows extensive areas of newly formed cartilage, but no (endochondral) bone formation is observed. Stage III (early osteophytes) shows an arrangement as the fetal growth plate cartilage whereas stage IV (mature osteophytes) shows a structure most resembling hyaline articular cartilage physiologically covering the joint surfaces.

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lowest cells found in the growth plate [140]. Mature osteophytes are characterized by the predominance of a hyaline-cartilage like extracellular matrix. At a first glance, mature osteophytes can, macroscopically and histologically, be easily mistaken for original articular cartilage. Indeed, this misconception reflects to some degree the fact that chondrocytic cells in osteophytes are able to construct an extracellular matrix containing all the typical components of hyaline articular cartilage such as collagen types II, IX, and XI as well as aggrecan [101,139]. The zonal distribution also resembles that found in adult articular cartilage with collagen type VI concentrated in the pericellular matrix [18]. Although hyaline zones in osteophytes resemble articular cartilage in terms of structural composition, there are, nevertheless, certain differences such as a more random cellular arrangement, the lack of a distinct tide mark, and a missing linear subchondral bone plate. Furthermore, the proper alignment of the described matrix components, which is obligatory for the cartilage to be able to resist high mechanical forces, has not yet been investigated yet at the ultrastructural level. To date, the molecular mechanisms in the development of osteophytes are largely unknown. Mechanical or biochemical stimuli could play a central role, but it is more likely that growth factors play a dominant role in the induction and promotion of osteophyte formation. For example, the exogenous application of TGF-h and BMP-2 into knee joints of adult mice leads to significant osteophyte formation [141,142]. Thus, members of the TGF-h superfamily, like BMP-2 or -7, strongly initiate chondrogenesis [143,144] while FGFs and IGF-1 predominantly promote chondrocyte proliferation [145]. All of them might be involved in initiation and progression of osteophyte development [146â&#x20AC;&#x201C;149]. The growth of osteophytes confirms that adult mesenchymal cells still possess the potential to differentiate into chondrocytes expressing all major cartilage matrix components similar to physiological articular chondrocytes. This shows that (high quality) hyaline cartilage can derive from mesenchymal progenitor cells which show full cartilage forming and proliferating potency even in the adult.

5.1. Evaluation of repair tissue Many approaches (for review, see Mainil-Varlet [150]) have been followed in order to promote external cartilage repair either by implanting autologous chondrocytes or chondrocyte precursor cells. Whatever therapeutical method is used, the important issue, in terms of outcome measurement, is not only clinical symptom evaluation but also the question what tissue is formed in terms of composition and function. Both biochemical composition and biomechanical function are closely related as the newly formed tissue has to fulfill the biomechanical needs of a specific connective tissue, i.e. articular cartilage, which it has to substitute. Thus, although the final functional outcome remains the main criterion for the success of a procedure, the tissue type formed appears to be the major prerequisite for the final success. Basically, three types or levels of repair tissues (besides the complete absence of repair tissue at all) can be distinguished [151]: fibrous tissue, fibrocartilage, and hyaline-like repair cartilage of varying resemblance to original articular cartilage [152]. All these types of tissue resemble the different stages of osteophyte development [139], making this in vivo phenomenon so interesting for cartilage repair biology. Fibrous repair tissue can be regarded as a failed repair attempt as it is unlikely to represent a successful long-term restoration of the joint function. Cells produce a matrix with a poor content of glycosaminoglycans and with abundance of type I collagen [153,154]. Type II collagen is not present or represents only a very minor fraction. Since this repair tissue lacks the unique properties of articular cartilage, which are required for a successful participation in the articulating processes of joints, fibrous repair tissue fails when exposed to mechanical load [152]. Fibrocartilage is the most common form of repair tissue achieved. In terms of structure and composition, fibrocartilage has an intermediate position in between fibrous and hyaline-like cartilaginous tissue. The content of glycosaminoglycans does not reach the abundance found in hyaline articular cartilage, but is increased compared to fibrous tissue. The matrix of fibrocartilage is composed of both type I collagen, typical for fibrous tissue, and of type II collagen, typical for hyaline cartilage [152,155,156].

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Thus, although at first sight the joint surface and the macroscopical integrity of the cartilage appears to be largely restored, also fibrocartilage does not possess the biomechanical properties of articular cartilage, which are needed for tolerating long-term constant mechanical loading and movement. Therefore, therapeutic attempts leading to fibrocartilagenous repair tissue are also prone to suffer from long-term deterioration with fibrillation, swelling, loss of cells, and finally the loss of the repair tissue itself [153–155]. Under certain conditions, a rather complete chondrogenic differentiation and remodeling process was reported that generated repair cartilage which shared great similarity with normal articular cartilage [156– 158]. Macroscopically, glossy surfaces of the restored defects suggested highly effective repair yielding the restoration of tissue resembling healthy articular cartilage. Histochemical analysis confirmed an abundance of water-binding glycosaminoglycans within the extracellular matrix of this repair tissue. The cells displayed a spherical shape typical for chondrocytes and were embedded in lacunar spaces. The extracellular matrix of these repair tissues was shown to contain types II, IX, and XI collagens. The absence of type I collagen indicated a rather complete transformation or differentiation of the implanted cells into a functional chondrocytic phenotype forming hyaline cartilage-like repair tissue [153,157,159,160]. Despite the similarities of this repair tissue to normal articular cartilage, which are apparent at first glance, there might still be subtle differences: In normal articular cartilage, the structural organization of the collagen network has a typical zonal pattern. In repair cartilage, the fibres appear to be more randomly distributed and the cellular density and cellular arrangement often differs significantly from that of normal articular cartilage [152,161–163]. Additionally, repair cartilage often lacks a clear tide mark which separates the upper portions of articular cartilage from the underlying calcified cartilage and bone [158,160]. Of note, long-term studies of repair cartilage revealed that a structural reorientation is possible. The continuous adaptation to mechanical forces seems to be able to generate a more zonal specific pattern also in repair cartilage. But even hyaline repair cartilage often fails in the long run. Similar to chondrocytes in vitro and osteoarthritic chondrocytes

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in vivo, chondrocyte-like cells in repair cartilage tend to change their cellular phenotype. They change their gene expression pattern and start to produce a fibrous matrix containing type I collagen leading to a tissue which is more susceptible to detrimental mechanical influences [152,158,164,165].

6. Perspectives Clearly, genomics, defined either as specific gene defects or as a rather undefined genetic background, plays an important role for the initiation and the progression of the osteoarthritic disease process [166]. Responsible genes have, however, been difficult to identify and might be different for different joints (for review, see Loughlin [167]). Still this will be one important focus of future research and will certainly provide new targets for therapeutic intervention. Also, better understanding of the biochemistry of the articular cartilage matrix as well as further elucidation of the cell biology of the articular chondrocytes will provide more in depth insights into the disease process and render new, maybe more clearly defined targets for drug development in the future. In this respect, functional genomics of osteoarthritis [168,169], not discussed in this chapter, offers a great opportunity to draw molecular portraits of the involved cells and the disease process as such and represents, thus, a very promising tool. The plethora of directions how to manipulate the disease process based on the pathobiology of osteoarthritis as known at the moment will be supplemented by emerging new knowledge taking advantage of the more modern methodologies available nowadays.

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