Osteoarthritis: how is articular cartilage organized?

Normal articular cartilage performs two main functions: absorbing pressure by deformation during mechanical loading and providing smoothness of the articular surfaces, which allows to minimize friction during joint movements. This is ensured by the unique structure of articular cartilage, which consists of chondroitins immersed in the extracellular matrix (ECM).

Normal adult articular cartilage can be divided into several layers or zones: the superficial or tangential zone, the transitional zone, the deep or radial zone, and the calcified zone. The layer between the superficial and transitional zones and especially between the transitional and deep zones has no clear boundaries. The junction between the noncalcified and calcified articular cartilage is called the "wavy border" - a line visible when staining decalcified tissue. The calcified zone of cartilage constitutes a relatively constant proportion (6-8%) of the total cartilage cross-sectional height. The total thickness of articular cartilage, including the calcified cartilage zone, varies depending on the load on a particular area of the articular surface and on the type of joint. Intermittent hydrostatic pressure in the subchondral bone plays an important role in maintaining the normal structure of cartilage by slowing ossification.

Chondrocytes make up approximately 2-3% of the total tissue mass; in the superficial (tangential) zone they are located along, and in the deep (radial) zone - perpendicular to the cartilage surface; in the transition zone, chondrocytes form groups of 2-4 cells scattered throughout the matrix. Depending on the zone of articular cartilage, the density of chondrocytes varies - the highest cell density is in the superficial zone, the lowest - in the calcified zone. In addition, the density of cell distribution varies from joint to joint, it is inversely proportional to the thickness of the cartilage and the load experienced by the corresponding area.

The most superficially located chondrocytes are discoid and form several layers of cells in the tangential zone located below a narrow strip of matrix; the deeper located cells of this zone tend to have more uneven contours. In the transitional zone, chondrocytes are spherical, sometimes they combine into small groups scattered in the matrix. Chondrocytes of the deep zone are predominantly ellipsoid in shape, grouped into radially located chains of 2-6 cells. In the calcified zone, they are distributed even more sparsely; some of them are necrotic, although most are viable. The cells are surrounded by noncalcified matrix, the intercellular space is calcified.

Thus, human articular cartilage consists of hydrated ECM and cells immersed in it, which make up 2-3% of the total tissue volume. Since cartilage tissue does not have blood or lymphatic vessels, interaction between cells, delivery of nutrients to them, and removal of metabolic products are carried out by diffusion through the ECM. Despite the fact that chondrocytes are very active metabolically, they do not normally divide in adults. Chondrocytes exist in an oxygen-free environment, and their metabolism is believed to be predominantly anaerobic.

Each chondrocyte is considered as a separate metabolic unit of cartilage, isolated from neighboring cells, but responsible for the production of ECM elements in the immediate vicinity of the donated cell and the maintenance of its composition.

The ECM is divided into three sections, each with a unique morphological structure and a specific biochemical composition. The ECM immediately adjacent to the chondrocyte basement membrane is called the pericellular, or lacunar, matrix. It is characterized by a high content of proteoglycan aggregates associated with the cell by the interaction of hyaluronic acid with CD44-like receptors, and a relative absence of organized collagen fibrils. Directly adjacent to the pericellular matrix is the territorial, or capsular, matrix, which consists of a network of intersecting fibrillar collagens that encapsulates individual cells or (sometimes) groups of cells, forming a chondron, and probably provides specialized mechanical support for the cells. Contact of chondrocytes with the capsular matrix is achieved through numerous cytoplasmic processes rich in microfilaments, as well as through specific matrix molecules such as ancorin and CD44-like receptors. The largest and most distant section of the ECM from the chondrocyte basement membrane is the interterritorial matrix, which contains the greatest number of collagen fibrils and proteoglycans.

The division of the ECM into compartments is more clearly defined in adult articular cartilage than in immature articular cartilage. The relative size of each compartment varies not only between joints but even within the same cartilage. Each chondrocyte produces a matrix that surrounds it. According to research, chondrocytes of mature cartilage tissue exert active metabolic control over their pericellular and territorial matrices, and they exert less active control over the interterritorial matrix, which may be metabolically “inert.”

As mentioned earlier, articular cartilage mainly consists of extensive ECM synthesized and regulated by chondrocytes. Tissue macromolecules and their concentrations change throughout life in accordance with changing functional needs. However, it remains unclear whether cells synthesize the entire matrix simultaneously or in certain phases in accordance with physiological needs. The concentration of macromolecules, the metabolic balance between them, their relationships and interactions determine the biochemical properties and, therefore, the function of articular cartilage within a single joint. The main component of the ECM of adult articular cartilage is water (65-70% of the total mass), which is firmly bound within it due to the special physical properties of cartilage tissue macromolecules that are part of collagens, proteoglycans and non-collagen glycoproteins.

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Biochemical composition of cartilage

Collagen fibers consist of molecules of the fibrillar protein collagen. In mammals, collagen accounts for a quarter of all proteins in the body. Collagen forms fibrillar elements (collagen fibrils) consisting of structural subunits called tropocollagen. The tropocollagen molecule has three chains that form a triple helix. This structure of the tropocollagen molecule, as well as the structure of the collagen fiber, when these molecules are located parallel in the longitudinal direction with a constant shift of about 1/4 of the length and provide high elasticity and strength to the tissues in which they are located. Currently, 10 genetically different types of collagen are known, differing in the chemical structure of the α-chains and / or their set in the molecule. The best studied first four types of collagen are capable of forming up to 10 molecular isoforms.

Collagen fibrils are part of the extracellular space of most connective tissues, including cartilage. Entangled within the insoluble three-dimensional network of intersecting collagen fibrils are other more soluble components such as proteoglycans, glycoproteins, and tissue-specific proteins; these are sometimes covalently linked to the collagen elements.

Collagen molecules organized into fibrils constitute about 50% of the organic dry residue of cartilage (10-20% of native cartilage). In mature cartilage, about 90% of collagens are type II collagens, which are found only in some tissues (e.g., vitreous body, embryonic dorsal cord). Type II collagen belongs to class I (fibril-forming) collagen molecules. In addition to it, mature human articular cartilage also contains collagens of types IX, XI, and a small amount of type VI. The relative amount of type IX collagen fibers in collagen fibrils decreases from 15% in fetal cartilage to about 1% in mature bovine cartilage.

Type I collagen molecules consist of three identical polypeptide a, (II)-chains synthesized and secreted as precursor procollagen. Once the finished collagen molecules are released into the extracellular space, they form fibrils. In mature articular cartilage, type II collagen forms fibrillar arcades in which the "thicker" molecules are located in the deep layers of the tissue, and the "thinner" ones are horizontally located in the superficial layers.

An exon encoding a cysteine-rich N-terminal propeptide has been found in the procollagen type II gene. This exon is expressed not in mature cartilage, but in the early stages of development (prechondrogenesis). Due to the presence of this exon, the procollagen type II molecule (type II A) is longer than collagen type II. Probably, the expression of this type of procollagen inhibits the accumulation of elements in the ECM of articular cartilage. It may play a certain role in the development of cartilage pathology (e.g., inadequate reparative response, osteophyte formation, etc.).

The network of type II collagen fibrils provides the function of resistance to stretching and is necessary for maintaining the volume and shape of the tissue. This function is enhanced by covalent and cross-links between collagen molecules. In the ECM, the enzyme lysyl oxidase forms an aldehyde from hydroxylysine, which is then converted to the multivalent amino acid hydroxylysyl-pyridinoline, which forms cross-links between the chains. On the one hand, the concentration of this amino acid increases with age, but in mature cartilage it remains virtually unchanged. On the other hand, in articular cartilage, an increase in the concentration of cross-links of various types formed without the participation of enzymes is found with age.

About 10% of the total amount of collagens in cartilage tissue are the so-called minor collagens, which largely determine the unique function of this tissue. Collagen type IX belongs to class III short-helix molecules and to a unique group of FACIT collagens (Fibril-Associated Collagen with Interrupted Triple-helices). It consists of three genetically different chains. One of them, the _a2 chain, is glycosylated simultaneously with chondroitin sulfate, which makes this molecule a proteoglycan. Both mature and immature hydroxypyridine cross-links are found between the helical segments of collagen type IX and collagen type II. Collagen IX can also function as an intermolecular-interfibrillar "connector" (or bridge) between adjacent collagen fibrils. Collagen IX molecules form cross-links with each other, which increases the mechanical stability of the fibrillar three-dimensional network and protects it from the effects of enzymes. They also provide resistance to deformation, limiting the swelling of proteoglycans located inside the network. In addition to the anionic CS chain, the collagen IX molecule contains a cationic domain, which imparts a large charge to the fibril and a tendency to interact with other matrix macromolecules.

Collagen type XI accounts for only 2-3% of the total collagen mass. It belongs to class I (fibril-forming) collagens and consists of three different α-chains. Together with collagen types II and IX, collagen type XI forms heterotypic fibrils of articular cartilage. Molecules of collagen type XI have been detected within collagen fibrils of type II using immunoelectromicroscopy. They probably organize collagen type II molecules, controlling the lateral growth of fibrils and determining the diameter of the heterotypic collagen fibril. In addition, collagen XI is involved in the formation of cross-links, but even in mature cartilage, the cross-links remain in the form of immature divalent ketoamines.

Small amounts of collagen type VI, another member of class III short-helix molecules, are found in articular cartilage. Collagen type VI forms various microfibrils and is probably concentrated in the capsular matrix of the chondron.

Proteoglycans are proteins to which at least one glycosaminoglycan chain is covalently attached. Proteoglycans are among the most complex biological macromolecules. Proteoglycans are most abundant in the ECM of cartilage. "Entangled" within a network of collagen fibrils, hydrophilic proteoglycans perform their main function - they impart to cartilage the ability to deform reversibly. It is assumed that proteoglycans also perform a number of other functions, the essence of which is not completely clear.

Aggrecan is the major proteoglycan of articular cartilage, comprising approximately 90% of the total proteoglycan mass in the tissue. Its 230 kD core protein is glycosylated by multiple covalently linked glycosaminoglycan chains and N-terminal and C-terminal oligosaccharides.

The glycosaminoglycan chains of articular cartilage, which constitute about 90% of the total mass of macromolecules, are keratan sulfate (a sequence of the sulfated disaccharide N-acetyl glucosamino lactose with multiple sulfated sites and other monosaccharide residues such as sialic acid) and chondroitin sulfate (a sequence of the disaccharide N-acetyl galactosamine glucuronic acid with a sulfate ester attached to every fourth or sixth carbon atom of N-acetyl galactosamine).

The core protein of aggrecan contains three globular (G1, G2, G3) and two interglobular (E1 and E2) domains. The N-terminal region contains the G1 and G2 domains separated by the E1 segment, which is 21 nm long. The C3 domain, located at the C-terminal region, is separated from G2 _by a longer (about 260 nm) E2 segment, which carries more than 100 chains of chondroitin sulfates, about 15-25 chains of keratin sulfates, and O-linked oligosaccharides. N-linked oligosaccharides are found mainly within the G1 and C2 domains and the E1 segment, as well as near the G3 _region. Glycosaminoglycans are grouped in two regions: the longest (the so-called chondroitin sulfate-rich region) contains chondroitin sulfate chains and about 50% keratan sulfate chains. The keratan sulfate-rich region is located on the E2 _segment near the G1 domain and precedes the chondroitin sulfate-rich region. Aggrecan molecules also contain phosphate esters, located primarily on the xylose residues that attach the chondroitin sulfate chains to the core protein; they are also found on serine residues of the core protein.

The C-terminal segment of the C3 _domain is highly homologous to lectin, allowing proteoglycan molecules to be fixed in the ECM by binding to certain carbohydrate structures.

Recent studies have identified an exon encoding an EGF-like subdomain within G3 _. Using anti-EGF polyclonal antibodies, the EGF-like epitope was localized within a 68-kD peptide in human articular cartilage aggrecan. However, its function remains to be elucidated. This subdomain is also found in adhesion molecules that control lymphocyte migration. Only about a third of aggrecan molecules isolated from mature human articular cartilage contain an intact C3 _domain; this is likely because aggrecan molecules can be enzymatically reduced in size in the ECM. The fate and function of the cleaved fragments are unknown.

The main functional segment of the aggrecan molecule is the glycosaminoglycan-bearing E2 _segment. The region, rich in keratan sulfates, contains the amino acids proline, serine, and threonine. Most of the serine and threonine residues are O-glycosylated with N-acetylgalactosamine residues; they initiate the synthesis of certain oligosaccharides that are incorporated into the keratan sulfate chains, thereby lengthening them. The remainder of the E2 _segment contains more than 100 serine-glycine sequences in which serine provides attachment to xylosyl residues at the beginning of the chondroitin sulfate chains. Typically, both chondroitin-6-sulfate and chondroitin-4-sulfate exist simultaneously within the same proteoglycan molecule, their ratio varying depending on the localization of the cartilage tissue and the age of the person.

The structure of aggrecan molecules in the human articular cartilage matrix undergoes a number of changes during maturation and aging. Aging-related changes include a decrease in hydrodynamic size due to a change in the average length of chondroitin sulfate chains, and an increase in the number and length of keratan sulfate chains. A number of changes in the aggrecan molecule are also caused by the action of proteolytic enzymes (e.g., aggrecanase and stromelesin) on the core protein. This results in a progressive decrease in the average length of the core protein of the aggrecan molecule.

Aggrecan molecules are synthesized by chondrocytes and secreted into the ECM, where they form aggregates stabilized by linker protein molecules. This aggregation involves highly specific noncovalent and cooperative interactions between a glucuronic acid strand and nearly 200 aggrecan and linker protein molecules. Glucuronic acid is an extracellular, nonsulfated, high-molecular-weight linear glycosaminoglycan composed of multiple sequentially linked N-acetylglucosamine and glucuronic acid molecules. The paired loops of the G1 domain of aggrecan reversibly interact with five sequentially located hyaluronic acid disaccharides. The linker protein, which contains similar (highly homologous) paired loops, interacts with the C1 domain and the hyaluronic acid molecule and stabilizes the aggregate structure. The C1 domain - hyaluronic acid - binding protein complex forms a highly stable interaction that protects the G1 domain and the binding protein from the action of proteolytic enzymes. Two molecules of the binding protein with a molecular weight of 40-50 kDa have been identified; they differ from each other in the degree of glycosylation. Only one molecule of the binding protein is present at the site of the hyaluronic acid - aggrecan bond. The third, smaller, molecule of the binding protein is formed from larger ones by proteolytic cleavage.

About 200 aggrecan molecules can bind to one molecule of hyaluronic acid to form an aggregate 8 μm long. In the cell-associated matrix, consisting of pericellular and territorial compartments, the aggregates maintain their association with the cells by binding (via a hyaluronic acid thread) to CD44-like receptors on the cell membrane.

Formation of aggregates in the ECM is a complex process. Newly synthesized aggrecan molecules do not immediately exhibit the ability to bind to hyaluronic acid. This may serve as a regulatory mechanism allowing newly synthesized molecules to reach the interterritorial zone of the matrix before being immobilized into large aggregates. The number of newly synthesized aggrecan molecules and binding proteins capable of forming aggregates by interacting with hyaluronic acid decreases significantly with age. In addition, the size of aggregates isolated from human articular cartilage significantly decreases with age. This is partly due to a decrease in the average length of hyaluronic acid molecules and aggrecan molecules.

Two types of aggregates have been established in articular cartilage. The average size of the first type of aggregates is 60 S, while that of the second type (rapidly precipitating "superaggregates") is 120 S. The latter is distinguished by an abundance of molecules of the binding protein. The presence of these superaggregates may play a major role in the functioning of the tissue; during tissue restoration after limb immobilization, higher concentrations of them are found in the middle layers of articular cartilage, while in a joint affected by osteoarthrosis, their sizes are significantly reduced in the early stages of the disease.

In addition to aggrecan, articular cartilage contains a number of smaller proteoglycans. Biglycan and decorin, molecules that carry dermatan sulfates, have molecular weights of about 100 and 70 kDa, respectively; the mass of their core protein is about 30 kDa.

In human articular cartilage, the biglycan molecule contains two chains of dermatan sulfate, whereas the more common decorin contains only one. These molecules constitute only a small fraction of the proteoglycans in articular cartilage, although they may be as numerous as large aggregated proteoglycans. Small proteoglycans interact with other macromolecules in the ECM, including collagen fibrils, fibronectin, growth factors, etc. Decorin is primarily localized to the surface of collagen fibrils and inhibits collagen fibrillogenesis. The core protein is tightly retained with the cell-binding domain of fibronectin, thereby likely preventing the latter from binding to cell surface receptors (integrins). Because both decorin and biglycan bind to fibronectin and inhibit cell adhesion and migration, as well as thrombus formation, they are capable of inhibiting tissue repair processes.

Fibromodulin of articular cartilage is a proteoglycan with a molecular weight of 50-65 kD associated with collagen fibrils. Its core protein, homologous to the core proteins of decorin and biglycan, contains a large number of tyrosine sulfate residues. This glycosylated form of fibromodulin (previously called the 59 kD matrix protein) may participate in the regulation of the formation and maintenance of the structure of collagen fibrils. Fibromodulin and decorin are located on the surface of collagen fibrils. Thus, as indicated earlier, an increase in fibril diameter should be preceded by selective removal of these proteoglycans (as well as type IX collagen molecules).

Articular cartilage contains a number of proteins in the ECM that are neither proteoglycans nor collagens. They interact with other macromolecules to form a network that includes most of the ECM molecules.

Ancorin, a 34 kD protein, is localized on the surface of chondrocytes and in the cell membrane, mediating interactions between the cell and the matrix. Due to its high affinity for type II collagen, it can act as a mechanoreceptor, transmitting a signal about changed pressure on the fibril to the chondrocyte.

Fibronectin is a component of most cartilaginous tissues and differs slightly from plasma fibronectin. Fibronectin is believed to promote matrix integration by interacting with cell membranes and other matrix components, such as type II collagen and thrombospondin. Fibronectin fragments have a negative effect on chondrocyte metabolism: they inhibit aggrecan synthesis and stimulate catabolic processes. High concentrations of fibronectin fragments have been found in the joint fluid of patients with osteoarthritis, so they may participate in the pathogenesis of the disease at late stages. Fragments of other matrix molecules that bind to chondrocyte receptors are likely to have similar effects.

Oligomeric matrix protein of cartilage (OMPC), a member of the thrombospondin superfamily, is a pentamer with five identical subunits with a molecular weight of about 83 kDa. They are found in large quantities in articular cartilage, especially in the layer of proliferating cells in growing tissue. Therefore, it is possible that OMPC is involved in the regulation of cell growth. They are found in much lower concentrations in the ECM of mature articular cartilage. Matrix proteins also include:

• basic matrix protein (36 kDa), which has high affinity for chondrocytes, may mediate cell-cell interactions in the ECM, such as during tissue remodeling;
• GP-39 (39 kDa) is expressed in the superficial layer of articular cartilage and in the synovial membrane (its functions are unknown);
• 21 kD protein is synthesized by hypertrophied chondrocytes, interacts with type X collagen, and can function in the “wavy line” zone.

In addition, it is evident that chondrocytes express non-glycosylated forms of small non-aggregated proteoglycans at certain stages of cartilage development and under pathological conditions, but their specific function is currently under study.

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Functional properties of articular cartilage

Aggrecan molecules provide articular cartilage with the ability to undergo reversible deformation. They demonstrate specific interactions within the extracellular space and undoubtedly play an important role in the organization, structure and function of the ECM. In cartilage tissue, aggrecan molecules reach a concentration of 100 mg/ml. In cartilage, aggrecan molecules are compressed to 20% of the volume they occupy in solution. A three-dimensional network formed by collagen fibrils gives the tissue its characteristic shape and prevents an increase in the volume of proteoglycans. Within the collagen network, immobile proteoglycans carry a large negative electrical charge (they contain a large number of anionic groups), which allows them to interact with mobile cationic groups of the interstitial fluid. Interacting with water, proteoglycans provide the so-called swelling pressure, which is counteracted by the collagen network.

The presence of water in the ECM is very important. Water determines the volume of the tissue; bound to proteoglycans, it provides resistance to compression. In addition, water provides transport of molecules and diffusion in the ECM. The high density of negative charge on large proteoglycans fixed in the tissue creates the "excluded volume effect". The pore size of the intraconcentrated solution of proteoglycans is so small that the diffusion of large globular proteins into the tissue is sharply limited. The ECM repels small negatively charged proteins (e.g., chloride ions) and large proteins (such as albumin and immunoglobulins). The size of the cells within the dense network of collagen fibrils and proteoglycans is comparable only to the size of some inorganic molecules (e.g., sodium and potassium, but not calcium).

In the ECM, some water is present in collagen fibrils. The extrafibrillar space determines the physicochemical and biomechanical properties of cartilage. The water content in the intrafibrillar space depends on the concentration of proteoglycans in the extrafibrillar space and increases with a decrease in the concentration of the latter.

The fixed negative charge on proteoglycans determines the ionic composition of the extracellular medium, which contains free cations in high concentration and free anions in low concentration. As the concentration of aggrecan molecules increases from the superficial to the deep zone of cartilage, the ionic environment of the tissue changes. The concentration of inorganic ions in the ECM creates high osmotic pressure.

The material properties of cartilage depend on the interaction of collagen fibrils, proteoglycans and the liquid phase of the tissue. Structural and compositional changes associated with the discrepancy between the processes of synthesis and catabolism, degradation of macromolecules and physical trauma significantly affect the material properties of cartilage and change its function. Since the concentration, distribution and macromolecular organization of collagens and proteoglycans change depending on the depth of the cartilage zone, the biomechanical properties of each zone vary. For example, the superficial zone with its high concentration of collagen, tangentially located fibrils, and relatively low concentration of proteoglycans has the most pronounced properties to resist stretching, distributing the load uniformly over the entire surface of the tissue. In the transition and deep zones, the high concentration of proteoglycans imparts to the tissue the property of withstanding compressive load. At the level of the "wavy line" the material properties of the cartilage change sharply from the pliable non-calcified zone to the more rigid mineralized cartilage. In the "wavy line" region the tissue strength is provided by the collagen network. The underlying cartilage sections are not crossed by collagen fibrils; in the area of the osteochondral junction the tissue strength is provided by the special contours of the boundary between the non-calcified and calcified cartilage zones in the form of irregular finger-like outgrowths, which "closes" the two layers and prevents their separation. Calcified cartilage is less dense than the subchondral bone, thus it functions as an intermediate layer that softens the compressive load on the cartilage and transfers it to the subchondral bone.

During loading, a complex distribution of three forces occurs - extension, shear and compression. The articular matrix is deformed due to the expulsion of water (as well as cell metabolism products) from the load zone, the concentration of ions in the interstitial fluid increases. The movement of water directly depends on the duration and force of the applied load and is delayed by the negative charge of proteoglycans. During tissue deformation, proteoglycans are pressed more tightly against each other, thereby effectively increasing the density of the negative charge, and the intermolecular forces that repel the negative charge in turn increase the resistance of the tissue to further deformation. Ultimately, the deformation reaches an equilibrium in which the external loading forces are balanced by internal resistance forces - swelling pressure (interaction of proteoglycans with ions) and mechanical stress (interaction of proteoglycans and collagens). When the load is removed, the cartilage tissue acquires its original shape by absorbing water along with nutrients. The initial (pre-loading) shape of the tissue is achieved when the swelling pressure of the proteoglycans is balanced by the resistance of the collagen network to their spread.

The biomechanical properties of articular cartilage are based on the structural integrity of the tissue - a collagen-proteoglycan composition as a solid phase and water and dissolved ions as a liquid phase. Unloaded, the hydrostatic pressure of articular cartilage is about 1-2 atm. This hydrostatic pressure can increase in vivo to 100-200 atm per millisecond during standing and to 40-50 atm during walking. In vitro studies have shown that hydrostatic pressure of 50-150 atm (physiological) leads to a moderate increase in cartilage anabolism over a short period of time, and over 2 h leads to a loss of cartilage fluid, but does not cause any other changes. The question of how quickly chondrocytes respond in vivo to this type of load remains unresolved.

The induced decrease in hydration with the subsequent increase in proteoglycan concentration leads to the attraction of positively charged ions such as H ⁺ and Na ⁺. This leads to a change in the overall ionic composition and pH of the ECM and chondrocytes. Long-term exercise induces a decrease in pH and, at the same time, a decrease in proteoglycan synthesis by chondrocytes. It is possible that the influence of the extracellular ionic environment on synthetic processes is also partly related to its influence on the ECM composition. Newly synthesized aggrecan molecules mature into aggregated forms later in a weakly acidic environment than under normal conditions. It is likely that a decrease in pH around chondrocytes (e.g., during exercise) allows more newly synthesized aggrecan molecules to reach the interterritorial matrix.

When the load is removed, water returns from the synovial cavity, carrying nutrients for the cells. In cartilage affected by osteoarthritis, the concentration of proteoglycans is reduced, therefore, during the load, water moves not only vertically into the synovial cavity, but also in other directions, thereby reducing the nutrition of chondrocytes.

Immobilization or mild loading results in a marked decrease in cartilage synthesis and proteoglycan content, whereas increased dynamic loading results in a moderate increase in proteoglycan synthesis and content. Strenuous exercise (20 km/day for 15 weeks) in dogs induced changes in proteoglycan content, particularly a sharp decrease in their concentration in the superficial zone. Some reversible cartilage softening and subchondral bone remodeling occurred. However, severe static loading caused cartilage damage and subsequent degeneration. In addition, loss of ECM aggrecan initiates the abnormal changes characteristic of osteoarthritis. Loss of aggrecan results in water attraction and swelling of the small amount of proteoglycan remaining. This dissolution of aggrecan contributes to a decrease in the local fixed charge density and ultimately leads to a change in osmolarity.