Articular cartilage matrix is heterogeneous and contains a core matrisome of key extra-cellular matrix (ECM) proteins, of which the large aggregating proteoglycan aggrecan and collagen types II, IX, and XI are the major structural and functional constituents.
The collagen network of cartilage confers tensile strength, and large aggregating proteoglycans, such as aggrecan, provide resistance to compression.
Adult articular chondrocytes are nonmitotic cells that survive at low oxygen tension in an acidic and nutrient-challenged microenvironment in the absence of a vascular supply.
In response to trauma or inflammation, the metabolic activity of the chondrocyte is increased in response to catabolic and anabolic factors that regulate remodeling of the ECM.
Under physiologic conditions, the chondrocyte maintains low-turnover repair of proteoglycans, but the repair capacity, responses to anabolic factors, cell survival, and quality of the matrix decline with age.
Chondroprogenitor cells can be derived from multiple tissue sources, including bone marrow, synovium, and adipose tissue. There is emerging evidence for chondroprogenitors in adult articular cartilage, but their capacity for replacing chondrocytes or for repairing damaged matrix is not well established.
Hyaline cartilage, including the articular cartilage of diarthrodial joints, consists of a single cellular component, the chondrocyte, which is embedded in a unique and complex matrix. Adult articular chondrocytes are considered to be fully differentiated cells that maintain matrix constituents in a low-turnover state of equilibrium. Chondrocytes serve diverse functions during development and postnatal life. In the embryo, chondrocytes arise from mesenchymal progenitors from diverse sources, including the cranial neural crest of the neural ectoderm, cephalic mesoderm, sclerotome of the paraxial mesoderm, and somatopleure of the lateral plate mesoderm, depending upon the ultimate location of the cartilage. The chondrocyte synthesizes the templates, or cartilage anlagen, through a process termed chondrogenesis.
After mesenchymal condensation and chondroprogenitor cell (CPC) differentiation, chondrocytes undergo proliferation, terminal differentiation to chondrocyte hypertrophy, and apoptosis through a process termed endochondral ossification, whereby the hypertrophic cartilage is replaced by bone. A similar sequence of events occurs in the postnatal growth plate and leads to rapid growth of the skeleton. Processes that control the different stages of skeletal development are described in Chapter 1 .
The permanent cartilage of articular surfaces, airways, ears, and nose persists throughout life. In adults, the anatomic distribution of cartilage is restricted primarily to the joints, trachea, and nasal septum, where the major function is structural support. In joints, cartilage has the additional function of providing low-friction articulation. Adult articular cartilage comprises a specialized matrix of collagens, proteoglycans, and other cartilage-specific and nonspecific proteins. Adult articular chondrocytes, remnants of the resting or reserve chondrocytes that laid down the original cartilage matrix during chondrogenesis, are inactive metabolically, owing partially to the absence of a vascular supply and innervation in the tissue. However, there is accumulating evidence to suggest that chondrocytes undergo behavioral alterations and metabolic reprogramming in degenerative and inflammatory joint diseases. The clinical importance of the adult chondrocyte resides in its capacity to respond to mechanical stimuli, growth factors, and cytokines that may influence normal homeostasis in a positive or negative manner.
Chondrocytes play important roles in the cellular taxonomy of arthritic diseases. In rheumatoid arthritis (RA), cartilage destruction occurs primarily in areas contiguous with the proliferating synovial pannus, although evidence indicates that the chondrocyte can respond to the inflammatory milieu and participate in degrading its own extra-cellular matrix (ECM). In osteoarthritis (OA), the chondrocyte plays a key role by reacting to structural changes in the surrounding cartilage matrix through the production of catabolic cytokines and anabolic factors, which act in an autocrine-paracrine manner. The chondrocyte has limited capacity, which declines with age, to regenerate the normal cartilage architecture with zonal variations in the matrix network that was formed originally. This chapter focuses on the structure and function of normal articular cartilage and the role of the chondrocyte in maintaining cartilage homeostasis and responding to adverse environmental insults that may modify cartilage integrity.
Normal articular cartilage is a specialized tissue characterized macroscopically by its milky, shelled-almond (hyaline) appearance. It is an avascular tissue nourished by diffusion from the vasculature of the subchondral bone and from the synovial fluid. Articular cartilage is more than 70% water, and it is hypocellular compared with other tissues; chondrocytes constitute only 1% to 2% of its total volume. Most of the dry weight of cartilage consists of two components: type II collagen and the large aggregating proteoglycan, aggrecan. Several “minor” collagens and small proteoglycans also contribute to the unique structural organization of the cartilage matrix.
Among the organic constituents, collagen, primarily fibrillar type II, accounts for approximately 15% to 25% of the wet weight and about half of the dry weight except in the superficial zone, where it represents most of the dry weight. Proteoglycans, primarily aggrecan, account for 10% of the wet weight and about 25% of the dry weight. The highly cross-linked type II collagen–containing fibrils form a systematically oriented network that traps the highly negatively charged proteoglycan aggregates. Histochemical analysis of cartilage shows that proteoglycans can be stained reliably with Safranin O, Toluidine blue, or Alcian blue, although at low-substrate concentrations these methods are not stoichiometric. Collagen also can be stained efficiently, but differentiation of collagen types requires immunostaining with specific antibodies. Less attention has been paid to less abundant and minor collagens, including types IV, VI, IX, X, XI, XII, XIII, and XIV.
Despite its thinness (≤7 mm) and apparent homogeneity, mature articular cartilage is a heterogeneous tissue with four distinct regions: (1) the superficial tangential (or gliding) zone, (2) the middle (or transitional) zone, (3) the deep (or radial) zone, and (4) the calcified cartilage zone, which is located immediately below the tidemark and above the subchondral bone ( Fig. 3.1 ). In the superficial zone, there are thin collagen fibrils in tangential array, and it contains a high concentration of the small proteoglycan decorin and a low concentration of aggrecan. The middle zone, comprising 40% to 60% of cartilage weight, consists of radial bundles of collagen fibrils that are thicker than in other zones. In the deep zone, the collagen fibrils become more perpendicular to the surface.
Cell density progressively decreases from the surface to the deep zone, where it is one-half to one-third the density of that in the superficial zone; chondrocytes in the deep and middle zones have cell volumes that are twice those of superficial chondrocytes. Cell morphology also changes across the different cartilage zones; cells in the superficial zone are relatively small, elongated in shape, aligned parallel to the surface, and lack an extensive pericellular matrix (PCM). Chondrocytes in the middle zone are spherical and do not exhibit an organized orientation relative to the surface. Cells in the deep zone exhibit extensive PCM deposition with chondrons in groups of three or more cells arranged in columns perpendicular to the articulating surface.
Water is 75% to 80% of the wet weight in the superficial zone and progressively decreases to 65% to 70% with increasing depth. Compared with the middle and deep zones, greater amounts of collagen relative to proteoglycans are present in the superficial zone, and type I collagen may be present in addition to type II collagen. With increasing depth, the proportion of proteoglycan increases to 50% of the dry weight in the deep zone. The calcified zone is formed as a result of endochondral ossification and persists after growth plate closure with the histologically defined tidemark defining the boundary with the articular cartilage. The calcified zone serves as an important mechanical buffer between uncalcified articular cartilage and subchondral bone ( Fig. 3.2 ).
The physical properties of articular cartilage are determined by the unique fibrillar collagen network, which provides tensile strength, interspersed with proteoglycan aggregates that bestow compressive resilience. Proteoglycans are associated with large quantities of water bound to the hydrophilic glycosaminoglycans (GAGs). This proteoglycan-rich ECM, with its tightly bound water, provides a high degree of resistance to deformation by compressive forces. The capacity to resist compressive forces is associated with the ability to extrude water as the cartilage compresses. When compression is released, the proteoglycans (now depleted of balancing counter ions that were removed with the water) contain sufficient fixed charge to reabsorb osmotically the water and small solutes into the matrix, which then rebounds to its original dimensions.
Structure-Function Relationships of Cartilage Matrix Components
ECM components synthesized by chondrocytes include highly cross-linked fibrils of triple-helical type II collagen molecules that interact with other collagens, aggrecan, small proteoglycans, and other cartilage-specific and nonspecific matrix proteins ( Table 3.1 ). The importance of these structural proteins may be observed in heritable disorders, such as chondrodysplasias, or in transgenic animals in which mutations or deficiencies in cartilage genes result in cartilage abnormalities. Deficiencies or disruptions in genes that encode the cartilage-specific collagens result, in some cases, in premature OA. Knowledge of the composition of the cartilage matrix has permitted the development of methods for identifying molecular markers in serum and synovial fluid that can be used to monitor changes in cartilage metabolism and to assess cartilage damage in OA or RA. Changes in the structural composition of cartilage can markedly affect its biomechanical properties.
|Molecule||Structure||Function and Location|
|Type II||[α1(II)] 3 ; fibril-forming||Tensile strength; major component of collagen fibrils|
|Type IX||[α1(IX)α2(IX)α3(IX)]; single CS or DS chain; α1(II) gene encodes α3(IX); FACIT||Tensile properties, interfibrillar connections; cross-links to surface of collagen fibril, NC4 domain projects into matrix|
|Type XI||[α1(XI)α2(XI)α3(XI)]; fibril-forming||Nucleation/control of fibril formation; within collagen fibril|
|Type VI||[α1(VI)α2(VI)α3(VI)]; microfibrils||Forms microfibrillar network, binds hyaluronan, biglycan, decorin; pericellular|
|Type X||[α1(X)] 3 ; hexagonal network||Support for endochondral ossification; hypertrophic zone and calcified cartilage|
|Type XII||[α1(XII)] 3 ; FACIT large cruciform NC3 domain||Associated with type I collagen fibrils in perichondrium and articular surface|
|Type XIV||[α1(XIV)] 3 ; FACIT||Associated with type I collagen; superficial zone|
|Type XVI||[α1(XVI)] 3 ; FACIT||Integrates with collagen II/XI fibrils|
|Type XXVII||Col27a1 gene: 156 kb, 61 exons||Fibril-forming; developing cartilage|
|Aggrecan||255 kDa core protein; CS/KS side chains; C-terminal EGF and lectin-like domains||Compressive stiffness through hydration of fixed charge density; binding through G1 domain to HA stabilized by link protein|
|Versican||265-370 kDa core protein; CS/DS side chains; C-terminal EGF, C-type lectin, and CRP-like domains||Low levels in articular cartilage throughout life; calcium-binding and selectin-like properties|
|Perlecan||400-467 kDa core protein; HS/CS side chains; no HA binding||Cell-matrix adhesion; pericellular|
|Biglycan||38 kDa; LRR core protein with two DS chains (76 kDa)||Binds collagen VI and TGF-β; pericellular|
|Decorin||36.5 kDa; LRR core protein with one CS or DS side chain (100 kDa)||Controls size/shape of collagen fibrils, binds collagen II and TGF-β; interterritorial|
|Asporin||40 kDa; LRR core protein; N-terminal extension of 15 aspartate residues||Binds collagen, modulates TGF-β function|
|Fibromodulin||42 kDa; containing KS chains in central LRR region and N-terminal tyrosine sulfate domains||Same as decorin|
|Lumican||38 kDa; structure similar to fibromodulin||Same as decorin|
|PRELP||44 kDa; LRR core protein; proline-rich and arginine-rich N-terminal binding domain for heparin and HS||Mediates cell binding through HS in syndecan|
|Chondroadherin||45 kDa; LRR core protein without N-terminal extension||Binding to cells via α2β1 integrin|
|Hyaluronic acid (HA; hyaluronan)||1000-3000 kDa||Retention of aggrecan within matrix|
|Link protein||38.6 kDa||Stabilizes attachment of aggrecan G1 domain to HA|
|Cartilage oligomeric matrix protein (COMP)||550 kDa; five 110-kDa subunits; thrombospondin-like||Interterritorial in articular cartilage; stabilizes collagen network or promotes collagen fibril assembly; calcium binding|
|Cartilage matrix protein (CMP, or matrilin-1); matrilin-3||Three 50 kDa subunits with vWF and EGF domains||Tightly bound to aggrecan in immature cartilage|
|Cartilage intermediate-layer protein (CILP)||92 kDa; homology with nucleotide pyrophosphohydrolase without active site||Restricted to middle/deep zones of cartilage; increase in early and late osteoarthritis|
|Glycoprotein (gp)-39, YKL-40, or chitinase 3-like protein 1 (CH3L1)||39 kDa; chitinase homology||Marker of cartilage turnover; chondrocyte proliferation; superficial zone of cartilage|
|Fibronectin||Dimer of 220 kDa subunits||Cell attachment and binding to collagen and proteoglycans; increased in osteoarthritis cartilage|
|Tenascin-C||Six 200-kDa subunits forming hexabrachion structure||Binds syndecan-3 during chondrogenesis; angiogenesis|
|Superficial zone protein (SZP), lubricin, or proteoglycan (PRG) 4||225 kDa, 200 nm length||Joint lubrication; superficial zone only|
|CD44||Integral membrane protein with extra-cellular HS/CS side chains||Cell-matrix interactions; binds HA|
|Syndecan-1, -3, -4||N-terminal HS attachment site; cytoplasmic tyrosine residues||Syndecan-3 is receptor for tenascin-C during cartilage development; cell-matrix interactions|
|Annexin V (anchorin CII)||34 kDa; homology to calcium-binding proteins calpactin and lipocortin||Cell surface attachment to type II collagen; calcium binding|
|Integrins (α1, α2, α3, α5, α6, α10; β1, β3, β5)||Two noncovalently linked transmembrane glycoproteins (α and β subunits)||Cell-matrix binding: α1β1/collagen I or VI, α2β1 or α3β1/collagen II, α5β1/fibronectin; intra-cellular signaling|
|Discoidin domain receptor 2||Receptor tyrosine kinase||Binds native type II collagen fibrils; Ras/ERK signaling|
|Transient receptor potential vanilloid 4 (TRPV4)||Ca 2+ channel||Mechanosensor|
|Connexin 43||ATP release channel||Mechanosensor; primary cilia|
The major component of the collagen network in adult articular cartilage is the triple-helical type II collagen molecule, which is composed of three identical α chains (α1[II]) 3 . These molecules are assembled in fibrils in a quarter-stagger array that can be observed by electron microscopy. These fibrils are thinner than type I collagen–containing fibrils in skin because of the higher numbers of hydroxylysine residues that can form cross-links and the presence of other collagen and noncollagen components in the fibril. Type IIB collagen in articular cartilage is a product of alternative splicing and lacks a 69 amino acid, cysteine-rich domain of the amino-terminal propeptide, which is encoded by exon 2 in the human type II collagen gene (COL2A1). This domain is found in type IIA procollagen, which is expressed by CPCs during development, and in the amino propeptides of other interstitial collagen types, and may play a feedback-inhibitory role in collagen biosynthesis. The reappearance of type IIA collagen in the midzone PCM and type X collagen, the hypertrophic chondrocyte marker, in the deep zone of OA cartilage suggests reversion to a developmental phenotype in an attempt to repair the damaged matrix.
Although collagens VI, IX, XI, XII, and XIV are quantitatively minor components, they may have important structural and functional properties and could represent a unique opportunity for the development of future biomarker tools for studying ECM repair and remodeling, especially in a regenerative context. Collagens IX and XI are relatively specific to cartilage, whereas collagens VI, XII, and XIV are widely distributed in other connective tissues. Collagen VI is present as microfibrils in the PCM and may play a role in cell attachment, and it interacts with other matrix proteins, such as hyaluronan, perlecan, biglycan, monomers or small aggregates of aggrecan, and type IX collagen, which are located there exclusively or at higher amounts than in the interterritorial matrix. There are small amounts of collagen III in cartilage, and collagens VI and III may increase in OA cartilage.
Type IX collagen is a proteoglycan and a collagen because it contains a chondroitin sulfate chain attachment site in one of the noncollagen domains. The helical domains of the type IX collagen molecule form covalent cross-links with type II collagen telopeptides and are attached to the fibrillar surface, as observed by electron microscopy. Type IX collagen may function as a structural intermediate between type II collagen fibrils and the proteoglycan aggregates, serving to enhance the mechanical stability of the fibril network and resist the swelling pressure of the trapped proteoglycans. Destruction of type IX collagen accelerates cartilage degradation and loss of function.
The α3 chain of type XI collagen has the same primary sequence as the α1(II) chain, and the heterotrimeric type XI collagen molecule is buried in the same fibril as type II collagen. Type XI collagen may have a role in regulating fibril diameter. The more recently discovered nonfibrillar fibril-associated collagens with interrupted triple helices (FACIT), XII and XIV, which are structurally related to type IX collagen, do not form fibrils by themselves but co-aggregate with fibril-forming collagens and modulate the packing of collagen fibers through domains projecting from their surfaces.
The major proteoglycan in articular cartilage is the large aggregating proteoglycan aggrecan, which consists of a core protein of 225 to 250 kDa with covalently attached side chains of GAGs, including approximately 100 chondroitin sulfate chains, 30 keratan sulfate chains, and shorter N -linked and O -linked oligosaccharides. Link protein, a small glycoprotein, stabilizes the noncovalent linkage between aggrecan and hyaluronic acid (also called hyaluronan ) to form the proteoglycan aggregate that may contain 100 aggrecan monomers. The G1 and G2 N-terminal globular domains of aggrecan and its C-terminal G3 domain have distinct structural properties that function as integral parts of the aggrecan core protein and contribute cleavage products that accumulate with age or in OA. The G2 domain is separated from G1 by a linear interglobular domain and has two proteoglycan tandem repeats. The G3 domain contains sequence homologies to epidermal growth factor, lectin, and complement regulatory protein, and participates in growth regulation, cell recognition, intra-cellular trafficking, ECM assembly, and stabilization. About half of the aggrecan molecules in adult cartilage lack the G3 domain, probably as a result of proteolytic cleavage during matrix turnover. Small quantities of other large proteoglycans are found in cartilage, including versican, which forms aggregates with hyaluronic acid, and perlecan, which is nonaggregating; however, these proteoglycans function primarily during skeletal development, where versican is expressed in prechondrogenic condensations, and perlecan is expressed in the cartilage anlagen after expression of type II collagen and aggrecan.
The nonaggregating small proteoglycans are not specific to cartilage, but in cartilage they serve specific roles in matrix structure and function, primarily by modulating collagen-fibril formation. Of the more than 10 leucine-rich repeat (LRR) proteoglycans discovered so far, only osteoadherin is not present in cartilage. The 24-amino acid central LRR domain is conserved, but the N-terminal and C-terminal domains have patterns of cysteine residues involved in intrachain disulfide bonds that distinguish the four subfamilies: (1) biglycan, decorin, fibromodulin, and lumican; (2) keratocan and proline and arginine-rich end leucine-rich repeat protein (PRELP); (3) chondroadherin; and (4) epiphycan/PG-Lb and mimecan/osteoglycin. Biglycan may have two GAG chains—chondroitin sulfate or dermatan sulfate, or both—attached near the N-terminus through two closely spaced serine-glycine dipeptides. Decorin contains only one chondroitin sulfate or dermatan sulfate chain. Fibromodulin and lumican contain keratan sulfate chains linked to the central domain of the core protein and several sulfated tyrosine residues in the N-terminus. Negatively charged GAG side chains contribute to the fixed charge density of the matrix and, together with the highly anionic tyrosine-sulfation sites, permit multiple-site linkage between adjacent collagen fibrils, stabilizing the network. Decorin, the most extensively studied LRR proteoglycan, binds to collagens II, VI, XII, and XIV, and to fibronectin and thrombospondin. Biglycan, decorin, and fibromodulin bind transforming growth factor (TGF)-β and the epidermal growth factor receptor and may modulate growth, remodeling, and repair. PRELP and chondroadherin may regulate cell-matrix interactions through binding to syndecan and α2β1 integrin.
PRG4 (proteoglycan 4), also known as lubricin and superficial zone proteoglycan (SZP), is a large surface-active mucinous proteoglycan synthesized and secreted by chondrocytes located at the surface of articular cartilage and by some synovial lining cells. PRG4 plays an important role in cartilage integrity in the synovial joint by providing boundary lubrication at the cartilage surface and contributing to the elastic absorption and energy dissipation of synovial fluid. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice with a genetic deficiency of PRG4. In healthy synovial joints, PRG4 molecules coat the cartilage surface, providing boundary lubrication and preventing cell and protein adhesion, inhibiting caspase-3 activation, thus preventing chondrocyte apoptosis. The chondroprotective properties of PRG4 may be exploited to provide new therapeutic options for reducing friction in degenerative and inflammatory joint disorders.
Other Extra-cellular Matrix and Cell Surface Proteins
Several other noncollagenous matrix proteins may play important roles in determining cartilage matrix integrity. Cartilage oligomeric protein (COMP), a member of the thrombospondin family, is a disulfide-bonded, pentameric, 550 kDa, calcium-binding protein that constitutes approximately 10% of the noncollagenous, nonproteoglycan protein in normal adult cartilage. COMP is located in the interterritorial matrix of adult articular cartilage, where it interacts with the COL3 and NC4 domains of type IX collagen that protrude from the fibril, stabilizing the collagen network. COMP is pericellular in the proliferating region of the growth plate, where it may have a role in cell-matrix interactions. The cartilage matrix protein (or matrilin-1) and matrilin-3 are expressed in cartilage at certain stages of development. Matrilin-1 is present in the PCM of adult articular cartilage, and matrilin-1, -2, and -3 may be upregulated in articular cartilage during OA.
Tenascin-C, a glycoprotein that is regulated in development, is characteristic of nonossifying cartilage. Similar to fibronectin, alternative splicing of tenascin-C mRNA gives rise to different protein products at different stages of chondrocyte differentiation. Both proteins are increased in OA cartilage and may serve specific functions in remodeling and repair. The cartilage intermediate-layer protein (CILP) is expressed by chondrocytes in the middle to deep zones of articular cartilage as a precursor protein. When cleaved during secretion, CILP has structural similarities with nucleotide pyrophosphohydrolase, although it lacks the catalytic site, and it may play a role in pyrophosphate metabolism and calcification. Asporin is related to decorin and biglycan and, similar to those other LRR proteins, may interact with and sequester growth factors such as TGF-β. YKL-40/HC-gp39, also known as chitinase 3-like protein 1, is found only in the superficial zone of normal cartilage and stimulates proliferation of chondrocytes and synovial cells. Chitinase 3-like protein 1 is induced by inflammatory cytokines and may function as a feedback regulator because it inhibits cytokine-induced cellular responses. Synthesis or release of these proteins or fragments is often increased in cartilage that is undergoing repair or remodeling, and they have been investigated as markers of cartilage damage in arthritis.
Morphology, Classification, and Normal Function of Chondrocytes
The characteristic feature of the chondrocyte embedded in cartilage matrix is its rounded or polygonal morphology. The exception occurs at tissue boundaries, such as the articular surfaces of joints, where chondrocytes may be flattened or discoid. Intra-cellular features, including a rough endoplasmic reticulum (ER), a juxtanuclear Golgi apparatus, and deposition of glycogen, are characteristic of a synthetically active cell. The cell density of full-thickness, human, adult, femoral condyle cartilage is maintained at 14.5 (±3.0) × 10 3 cells/mm 2 from age 20 to 30 years. Although senescence of chondrocytes occurs with aging, mitotic figures are not observed in normal adult articular cartilage.
Chondrocytes possess mitochondria with varying degrees of structural and functional heterogeneity. Mitochondria are important for cartilage development and are primarily associated with cellular energetics and metabolism, but they are also important mediators of calcium accumulation needed for ECM calcification, especially in epiphyseal chondrocytes. Chondrocyte mitochondrial impairment and dysfunction have been implicated in degenerative processes and OA. Mitochondrial degeneration can occur in response to oxidative damage, contributing to the age-related loss of chondrocyte function.
Chondrocytes exhibit different behaviors depending on their position within the different cartilage layers, and these zonal differences in biosynthetic properties may persist in primary chondrocyte cultures. The primary cilia are important for spatial orientation of cells in developing growth plate and are sensory organelles in chondrocytes. Primary cilia are centers for Wnt and hedgehog signaling and contain mechanosensitive receptors, including the transient receptor potential vanilloid 4 (TRPV4), a Ca 2+ permeable, nonselective cation channel, and connexin 43, an adenosine triphosphate (ATP) permeable gap junction channel involved in ATP release.
Classification: Cell Origin and Differentiation
Chondrocytes arise in the embryo from mesenchymal origin during chondrogenesis, which is the earliest phase of skeletal development involving mesenchymal cell recruitment, migration, and condensation and differentiation of mesenchymal CPCs. As described in detail in Chapter 1 , chondrogenesis results in the formation of cartilage anlagen, or templates, at sites where skeletal elements form. This process is controlled by cell-cell and cell-matrix interactions and by growth and differentiation factors that initiate or suppress cellular signaling pathways and transcription of specific genes in a temporospatial manner ( Fig. 3.3 ).
Vertebrate limb development is controlled by interacting patterning systems involving fibroblast growth factor (FGF), hedgehog, bone morphogenetic protein (BMP), TGF-β, Wnt, and Notch pathways. Wnt signaling, via the canonical β-catenin pathway and activation of TCF/Lef transcription factors, functions in a cell-autonomous manner to induce osteoblast differentiation and suppress chondrocyte differentiation in early chondroprogenitors. During chondrogenesis, Wnt/β-catenin acts at two stages: at low levels to promote chondroprogenitor differentiation and later at high levels to promote chondrocyte hypertrophic differentiation and subsequent endochondral ossification. The transcription factor, Sry-type high-mobility group box 9 (Sox9), is an early marker of the differentiating chondrocyte that is required for the onset of expression of type II collagen, aggrecan, and other cartilage-specific matrix proteins, such as type IX collagen. Two other members of the SOX family, L-Sox5 and Sox6, are not present in early mesenchymal condensations but are required during overt chondrocyte differentiation, forming heterodimers that induce transcription more efficiently than Sox9 by itself. At different times during development, SOX proteins interact with SMADs (signaling through mammalian homologs of Drosophila mothers against decapentaplegic), which are functionally redundant and active in differentiating chondrocytes. Other transcription factors such as Gata4/5/6 and Nkx3.2 may interact directly or indirectly with Sox9 to upregulate the expression of COL2A1 aggrecan (ACAN), and other cartilage-specific genes at early stages of chondrogenesis.
In the embryonic or postnatal epiphyseal growth plates, molecules that promote matrix remodeling and angiogenesis facilitate endochondral ossification, whereby bone replaces the calcified cartilaginous matrix in the hypertrophic zone (see Chapter 1 ). Differentiated chondrocytes that remain in the reserve, or resting, zone become the cartilage elements in articular joints, or they can proliferate and undergo the complex process of terminal differentiation to hypertrophy marked by type X collagen. Indian hedgehog and parathyroid hormone–related protein (PTHrP) transiently induce proliferation and repress differentiation, determining the number of cells that enter the hypertrophic maturation pathway. The Runt domain transcription factor, Runx2 (also known as core binding factor or Cbfa1 ), serves as a positive regulatory factor in chondrocyte maturation to the hypertrophic phenotype and subsequent osteogenesis. Runx2 is expressed in the adjacent perichondrium and in prehypertrophic chondrocytes, but less in late hypertrophic chondrocytes, and is required for expression of type X collagen and other markers of terminal differentiation.
Numerous other transcription factors positively or negatively regulate chondrocyte terminal differentiation by controlling, in part, the expression or activity of Runx2. BMP-induced Smad1 and interactions between Smad1 and Runx2 are required for the induction of chondrocyte hypertrophy. Histone deacetylase 4 (HDAC4), which is expressed later in prehypertrophic chondrocytes, prevents premature chondrocyte hypertrophy by interacting with Runx2 and inhibiting its activity. The hypoxia-inducible factor (HIF)-1α contributes to chondrocyte survival during hypertrophic differentiation, owing partially to its regulation of vascular endothelial cell growth factor (VEGF) expression, and it prevents premature hypertrophy by directly suppressing Runx2 activity. The transcription factors involved in regulating gene expression during late-stage hypertrophy include Runx3, myocyte enhancer factor (MEF) 2C and 2D, Foxa2 and Foxa3, and Zfp521, in addition to Runx2.
One major function of the chondrocyte is growth of the skeleton through increasing cell proliferation, production of ECM, and cell volume through hypertrophy. Lineage tracing studies indicate that Sox9-expressing cells are precursors for both articular and growth plate chondrocytes. Furthermore, cells expressing Tgfbr2, the gene encoding the TGF-β receptor (R) II in the interzone, can be traced to synovial lining, superficial meniscus, and ligaments and may persist as reserve progenitor cells that could later participate in regeneration. Finally, Gdf5, matrilin-1, and PTHrP appear to specify the eventual border between the articular cartilage surface and the joint space. After cessation of growth, the resting chondrocyte remains as part of the supporting structures in articular, tracheal, and nasal cartilages, indicating that the fate of a chondrocyte depends on origin and location.
Adult human articular cartilage contains CPCs that retain their expansion capacity with the potential of reproducing the structural and biomechanical properties of healthy articular cartilage. CPCs are stem/progenitor cells capable of chondrogenic differentiation and can be derived from multiple tissue sources including articular cartilage, synovium, and adipose tissue. CPCs reside not only in the superficial zone of articular cartilage but also in other zones of articular cartilage and in the neighboring tissues. They have been classified as mesenchymal stem cells (MSCs) and have been postulated to play a role in responses to cartilage injury. They can be identified by their colony forming ability, proliferative potential, telomere dynamics, multipotency, and expression of stem cell markers. Therefore, they have potential applications in cartilage tissue engineering and may have clinical applications in cartilage repair.
Normal Function of the Adult Articular Chondrocyte
The mature articular chondrocyte embedded in its ECM is a resting cell with no detectable mitotic activity and a low rate of synthetic activity, exemplified by long half-lives of the major ECM components. For example, the half-life of the aggrecan core protein is close to 25 years whereas the half-life of type II collagen has been calculated to be 100 years. Because articular cartilage is avascular, the chondrocyte must rely on diffusion from the articular surface or subchondral bone for exchange of nutrients and metabolites. Chondrocytes maintain active membrane transport systems for exchange of cations, including Na + , K + , Ca 2+ , and H + , whose intra-cellular concentrations fluctuate with load and changes in the composition of the cartilage matrix. The chondrocyte cytoskeleton is composed of actin, tubulin, and vimentin filaments, and the composition of these filament systems varies in the different cartilage zones.
Chondrocyte metabolism operates at low oxygen tension within the cartilage matrix, ranging from 10% at the surface to less than 1% in the deep zone. The consumption of oxygen by cartilage on a per-cell basis is only 2% to 5% of that in liver or kidney, although the amounts of lactate produced are comparable. Classical studies of oxygen consumption and glucose/lactate metabolism in cartilage suggest a shift in the major energy generating pathways as the oxygen environment is altered.
Compared with highly metabolic cells such as neurons and cardiomyocytes, chondrocytes do not normally contain abundant mitochondria. Thus, energy metabolism depends strongly on glucose supply, and energy requirements may be modulated by mechanical stress. Glucose serves as the major energy source for chondrocytes and as an essential precursor for GAG synthesis. Facilitated glucose transport in chondrocytes is mediated by several distinct glucose transporter proteins (GLUTs) that may be constitutively expressed or induced by cytokines. Chondrocytes express GLUT1, GLUT3, and several other glucose transporters. GLUT1 is an abundant constitutively expressed GLUT that can be induced by hypoxia and pro-inflammatory cytokines. GLUT3 is responsive to hypoxia, growth factors, and cytokines. ATP-sensitive potassium [K(ATP)] channels can sense intra-cellular ATP/ADP (adenosine diphosphate) levels, being essential components of a glucose-sensing apparatus that couples glucose metabolism to ATP availability in chondrocytes. K(ATP) channels sense intra-cellular ATP and regulate the abundance of GLUT1 and GLUT3 according to functional demand. Proteomic studies of chondrocytes have identified these and other intra-cellular proteins known to be involved in cell organization, energy protein fate, metabolism, and cell stress. The relative expression of these proteins may determine the capacity of chondrocytes to survive in cartilage matrix and to modulate metabolic activity in response to environmental changes.
When cultured in a range of oxygen tensions between severe hypoxia (0.1% oxygen) and normoxia (21% oxygen), chondrocytes adapt to low oxygen tension by upregulating HIF-1α. Hypoxia via HIF-1α can stimulate chondrocytes to express GLUTs, angiogenic factors such as VEGF, and numerous genes associated with cartilage anabolism and chondrocyte differentiation. In the growth plate, hypoxia and HIF-1α are associated with type II collagen production. HIF-1α is expressed in normal and OA articular cartilage, where it maintains tonic activity during physiologic hypoxia in the deeper layers associated with increased proteoglycan synthesis. In contrast to other tissues, however, HIF-1α is not completely degraded in cartilage when normoxic conditions are applied. Long-term systemic hypoxia (13%) may downregulate collagen and aggrecan gene expression in articular cartilage, whereas hyperoxia (55% oxygen) may increase the breakdown of cartilage collagens in articular cartilage in the presence of vascularized rheumatoid synovium. By modulating the intra-cellular expression of survival factors such as HIF-1α, chondrocytes have a high capacity to survive in the avascular cartilage matrix and to respond to environmental changes.
The chondrocyte maintains a steady-state metabolism secondary to equilibrium between anabolic and catabolic processes, resulting in the normal turnover of matrix molecules. As mentioned earlier, normal adult articular cartilage exhibits low turnover of type II collagen and aggrecan core protein, whereas the GAGs on aggrecan are more readily replaced. Other cartilage ECM components, including biglycan, decorin, COMP, tenascins, and matrilins, incorporated previously into the matrix during development also may be synthesized by chondrocytes under low-turnover conditions. Regional differences in the remodeling activities of chondrocytes have been noted, however, and matrix turnover may be more rapid in the immediate pericellular zones. The metabolic potential of these cells is indicated by their capacity to proliferate in culture and to synthesize matrix proteins after enzymatic release from the cartilage of even elderly individuals. The complex composition of the articular cartilage matrix is more difficult for the chondrocyte to replicate if severe damage to the collagen network occurs.
Interactions of Chondrocytes With the Extra-cellular Matrix
Chondrocytes in vivo respond to structural changes in the cartilage ECM. The ECM not only provides a framework for chondrocytes suspended within it, but its constituents interact with cell surface receptors to provide signals that regulate many chondrocyte functions ( Fig. 3.4 ).
The most prominent of the ECM receptors are the integrins, which are heterodimeric transmembrane receptors consisting of α and β subunits that link or “integrate” the ECM with the cytoskeleton. Integrins bind specifically to different cartilage matrix components and induce the formation of intra-cellular signaling complexes that regulate cell proliferation, differentiation, survival, and matrix remodeling. Integrins also may serve as mechanoreceptors that mediate responses to normal and abnormal loading of cartilage. Chondrocytes express many different integrins that interact with cartilage ECM ligands, although most are not specific to this cell type. They include integrins that are receptors for collagen (α1β1, α2β1, α3β1, α10β1), fibronectin (α5β1, αvβ3, αvβ5), and laminin (α6β1). The integrin α1β1 has broader ligand specificity than the other collagen-binding integrins and mediates chondrocyte adhesion to pericellular type VI collagen and to the cartilage matrix protein, matrilin-1. The α2β1 integrin also binds to chondroadherin. The αv-containing integrins bind to vitronectin and osteopontin, in addition to serving as alternative fibronectin receptors. The α5β1 and αvβ3 integrins serve as receptors for different conformations of COMP.
Because α1β1, α2β1, and α10β1 are receptors for cartilage-specific type II collagen, there is great interest in determining whether they mediate differential responses of chondrocytes to changes in the ECM resulting from normal loading or pathologic changes. The α5β1 integrin is the prominent integrin in human adult articular cartilage. Depending on the method of analysis, adult chondrocytes also express α1β1 and αvβ5 integrins accompanied by weaker expression of α3β1 and αvβ3. Normal adult articular chondrocytes express little or no α2β1, whereas expression of α2β1 and α3β1 integrins is associated with a proliferative phenotype, as in fetal chondrocytes and in chondrosarcoma and chondrocyte cell lines. In growth plate chondrocytes, α5β1, αvβ5, and α10β1 are important for joint formation, chondrocyte proliferation, hypertrophy, and survival. Knockout of the β1 integrin subunit results in severe growth plate abnormalities and chondrodysplasias, whereas α1 integrin knockout mice develop spontaneous OA without growth plate abnormalities. In contrast, knockout of the α5 integrin subunit during joint development did not alter the synovial joints, but protected mice from surgically induced OA. Importantly, α10β1 is the critical collagen receptor in skeletal development.
Cellular binding to immobilized ECM proteins or integrin receptor aggregation with activating antibodies can promote numerous intra-cellular signaling events. As in other cell types, integrin signaling is mediated by interaction with intra-cellular protein tyrosine kinases, such as pp125 focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), which interact with the integrin cytoplasmic tail and induce a conformational change in the receptor subunits. Changes in organization of the cytoskeleton are associated with the formation of integrin-signaling complexes, which contain scaffolding proteins such as talin, paxillin, and α-actinin, in addition to FAK, Pyk2, and the integrin-linked kinase (ILK). Mice lacking ILK in cartilage display chondrodysplasia, a phenotype similar to that of cartilage-specific β1 integrin knockout mice. Developing chondrocytes express and secrete the protein integrin-β-like 1 (ITGBL1), which regulates integrin signaling to promote cartilage formation. Decreased ITBGL1 is observed in arthritic cartilage, and its ectopic expression reduces the severity of surgically induced OA in mice.
Cooperative signaling among integrins and growth factors is a fundamental mechanism in the regulation of cellular functions. Integrin aggregation and receptor occupancy enhance phosphorylation of growth factor receptors and activation of mitogen-activated protein kinases (MAPKs), notably extra-cellular signal-regulated kinase (ERK)-1 or ERK-2. Integrins mediate the effects of mechanical forces through activation of cell signaling, a process termed mechanotransduction .
Normal chondrocytes use α5β1 as a mechanoreceptor. As the primary fibronectin receptor, α5β1 plays a role in cartilage degradation by binding to fibronectin fragments that upregulate matrix metalloproteinases (MMPs) such as MMP-3 and MMP-13. Chondrocyte binding to fibronectin fragments also increases the production of cytokines, chemokines, and other catabolic or inflammatory mediators through a mechanism requiring reactive oxygen species (ROS).
Other Cell Surface Receptors on Chondrocytes
Other integral membrane proteins found in chondrocytes include cell determinant 44 (CD44), annexins, syndecans, and discoidin domain receptor 2 (DDR2). CD44 is a receptor for hyaluronan that in turn binds multiple aggrecan proteoglycan monomers to form a gel-like PCM. Through specific interactions with hyaluronan, CD44 has a role in assembly, organization, and maintenance of the chondrocyte PCM. CD44 expression is upregulated in chondrocytes in articular cartilage from RA patients and in experimental OA. Chondroprotective agents and anabolic factors may ameliorate cartilage matrix destruction by reducing CD44 fragmentation that disrupts hyaluronan-CD44 interactions and has adverse effects on the PCM.
Annexin V, also known as annexin CII, is a 34 kDa integral membrane protein that binds type II collagen and shares extensive homology with the calcium-binding proteins calpactin and lipocortin. Annexins II, V, and VI have been detected in chondrocytes, where they likely play roles in physiologic mineralization of skeletal tissues and in pathologic mineralization of articular cartilage. Annexin V was first detected in chick cartilage and described as a type II collagen–binding protein that anchors chondrocytes to the ECM. In growth plate chondrocytes, annexins are required for calcium ion uptake and subsequent mineralization. Annexin A6 is highly expressed in OA cartilage and plays a role in catabolic signaling.
Syndecans have important roles during cartilage development and homeostasis. Syndecans link to the cell surface via glycosyl phosphatidylinositol and bind growth factors, proteinases and their inhibitors, and matrix molecules through heparan sulfate side chains on the extra-cellular domain. Syndecan-1, syndecan-3, and syndecan-4 are upregulated in OA cartilage. Syndecan-4 is a positive effector of aggrecanase activity through controlling the synthesis of the stromelysin, MMP-3.
In contrast to integrins, which bind collagen fragments, DDR2 binds specifically to type II and X collagen fibrils, leading to activation of its integral receptor tyrosine kinase. DDR2 is upregulated in OA cartilage and induces specifically the expression of MMP-13 associated with cleavage of type II collagen. The serine proteinase, high temperature requirement A1 (HTRA1), which is induced by TGF-β and increased in OA articular cartilage, is responsible for disrupting the PCM, thereby exposing DDR2 to activation by fibrillar type II collagen. Contributing to this process is connective tissue growth factor (CTGF), a latent TGF-β binding protein that controls the matrix sequestration and activation of TGF-β in mechanically injured cartilage.
Angiogenic and Antiangiogenic Factors
Adult articular cartilage is among the few avascular tissues in mammalian organisms; its matrix composition and the presence of angiogenesis inhibitors make it resistant to vascular angiogenesis and invasion by inflammatory and neoplastic cells. Troponin I, MMP inhibitors, chondromodulin-I, and endostatin, a 20 kDa proteolytic fragment of type XVIII collagen, all function as endogenous angiogenic inhibitors in cartilage. In conditions in which extensive remodeling of ECM occurs, as in arthritis, the cartilage becomes susceptible to invasion by vascular endothelial and mesenchymal cells from the synovium and subchondral bone. VEGF, which is an essential mediator of angiogenesis during endochondral ossification (see Chapter 1 ), is induced by inflammatory cytokines, hypoxia, and mechanical overload. In OA, in which abnormal biomechanics and joint effusions cause severe hypoxia, chondrocytes produce VEGF, inducing angiogenesis at the chondro-osseous junction and contributing to expansion of the calcified cartilage, tidemark duplication, and cartilage thinning. TGF-β-mediated angiogenesis in the subchondral bone may be among the earliest events driving OA, where microcracks produced by mechanical stress and exacerbation of naturally occurring pores provide conduits for vascular invasion into the calcified cartilage and diffusion of small molecules. In RA, ingrowth of blood vessels and synovial pannus into cartilage contributes to degradation of the cartilage matrix.
Gene expression profiling analyses comparing inflamed and noninflamed areas of synovium from the same OA patients identified STC1, encoding stanniocalcin-1, a molecule that plays roles in angiogenic sprouting via the VEGF/VEGF receptor 2 pathway, as the most highly upregulated gene in inflamed synovial membrane. Articular cartilage is aneural, and the sensory nerve fibers observed in the vascular channels associated with osteochondral angiogenesis may constitute a potential source of symptomatic pain. The clinical importance of these observations is supported by the report that VEGF blockade with bevacizumab inhibits post-traumatic OA in a rabbit model with pain relief associated with prevention of both synovitis and angiogenesis.
Roles of Growth and Differentiation (Anabolic) Factors in Normal Cartilage Metabolism
Growth and differentiation factors generally are considered positive regulators of homeostasis in mature articular cartilage because of their capacity to stimulate chondrocyte anabolic activity and, in some cases, inhibit catabolic activity. The best-characterized anabolic factors in the context of their production and action in articular cartilage include insulin-like growth factor I (IGF-I) and members of the FGF and TGF-β/BMP families. The PTHrP, Ihh, and the Wnt/β-catenin pathways have been implicated in maintenance of cartilage homeostasis or OA disease processes. Many of these factors also regulate chondrogenesis and endochondral ossification during skeletal development (see Chapter 1 ). In adult cartilage, the expression and/or activity of growth factors declines with age, which is a risk factor for OA.
Insulin-like Growth Factor
IGF-I was first described as somatomedin C, a serum factor controlling sulfate incorporation by articular cartilage in vitro, and it was later found to have the specific capacity to stimulate or maintain chondrocyte phenotype in vitro by promoting the synthesis of type II collagen and aggrecan. IGF-I is a competence factor for cell proliferation that is categorized more appropriately as a differentiation factor because its limited mitogenic activity seems to depend on the presence of other growth factors, such as FGF-2, a progression factor. IGF-I is considered an essential mediator of cartilage homeostasis through its capacity to stimulate proteoglycan synthesis, promote chondrocyte survival, and oppose the activities of catabolic cytokines in cooperation with other anabolic factors such as BMP-7. IGF-I and insulin can activate cell signaling via the IGF-I tyrosine kinase receptor or the type I insulin receptor at concentrations proportional to their binding affinities. A study in rats showed that delivery of IGF-I to chondrocytes using unique nanocarriers reduces the severity of surgically induced OA.
Specific IGF-binding proteins (IGFBPs) that do not recognize insulin also regulate IGF-I activity. Chondrocytes at different stages of differentiation express IGF-I and IGF receptors and different arrays of IGFBPs, providing a unique system by which IGF-I can exert different regulatory effects on these cells. IGFBP-2 and IGFBP-5 are positive regulators that increase proteoglycan synthesis in chondrocytes, whereas binding of IGFBP-3 to IGF-I negatively regulates the anabolic functions of IGF-I. IGFBP-3 may also directly inhibit chondrocyte proliferation in an IGF-independent manner.
In OA cartilage, the normal anabolic function of IGF-I may be disrupted because chondrocytes from animals with experimental arthritis and from patients with OA are hyporesponsive to IGF-I, despite normal or increased IGF-I receptor levels. This hyporesponsiveness has been attributed to excessive levels of reactive oxygen species seen in aged and OA cartilage, which alter the cell signaling response patterns to IGF-1.
Fibroblast Growth Factor
Members of the FGF family, including FGF-2, FGF-4, FGF-8, FGF-9, FGF-10, and FGF-18, together with the FGF receptors, FGFR1, FGFR2, FGFR3, and FGFR4, coordinate patterning and cell proliferation during chondrogenesis and endochondral ossification in embryonic and postnatal growth plates. The most extensively studied is FGF-2, or basic FGF, which is a potent mitogen for adult articular chondrocytes, but findings on its effects on the synthesis of cartilage matrix are contradictory, showing stimulation, inhibition, or no effect on proteoglycan synthesis.
The FGFs are generally considered homeostatic factors in joint tissues. FGF-2 stored in the adult cartilage matrix is released with mechanical injury or with loading, suggesting a mechanism for modulating chondrocyte proliferation and anabolic activity. Different FGF receptors in cartilage may mediate opposing effects: FGF-2 promotes cartilage protection via FGFR3 and cartilage destruction via FGFR1. Of the four receptors, FGFR1 and 3 are the most abundant, and the ratio of FGFR3 to FGFR1 is reduced in OA cartilage. Furthermore, cartilage-specific deletion of the FGFR1 gene attenuates articular cartilage degeneration in mice.
In cartilage development, FGF-18 negatively regulates chondrocyte proliferation and terminal differentiation in the growth plate via FGFR3. In articular cartilage, FGF-18 has a role in the maintenance of homeostasis via FGFR3 and protects against loading-induced damage in cartilage explants ex vivo. In vivo, intra-articular administration of FGF-18 protects against cartilage damage in a rat model of injury-induced arthritis. Thus, there is considerable interest in the potential of anabolic factors such as FGF-18 for enhancing cartilage regeneration with tissue engineering approaches. A proof-of-concept trial with human recombinant FGF-18, sprifermin, showed statistically significant, dose-dependent improvement in prespecified secondary structural end points by magnetic resonance imaging (MRI), radiographic joint space narrowing, and Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain scores. Recent findings in pre-clinical models using sprifermin suggest clinical efficacy.
Transforming Growth Factor-β/Bone Morphogenetic Protein Superfamily
Activities of the TGF-β/BMP superfamily in the skeleton were first discovered by Marshall Urist as constituents of demineralized bone that induced new bone formation when implanted into extraskeletal sites in rodents. These bioactive morphogens subsequently were extracted, purified, and cloned and were found to regulate the early commitment of mesenchymal cells to chondrogenic and osteogenic lineages during cartilage development and endochondral bone formation ( Table 3.2 ). The TGF-β/BMP superfamily includes activins, inhibins, müllerian duct inhibitory substance, nodal, glial-derived neurotrophic factor, OP-1 (or BMP-7), and growth differentiation factors (GDFs), also called cartilage-derived morphogenetic proteins (CDMPs). In addition to regulating cartilage condensation and chondrocyte differentiation, members of this superfamily play key roles in site specification and cavitation of synovial joints (see Chapter 1 ) and in the development of other organ systems. Many of these factors, including BMP-2, BMP-6, BMP-7, BMP-9, TGF-β, and CDMP-1, can induce chondrogenic differentiation of mesenchymal progenitor cells in vitro. They also may have direct effects on mature articular chondrocytes in vivo and in vitro and are important for cartilage maintenance.