Condensation of mesenchymal cells, which differentiate into chondrocytes, results in formation of the cartilage anlagen, which provides the template for the developing skeleton.
During development of the synovial joint, growth differentiation factor-5 regulates interzone formation, and interference with movement of the embryo during development impairs joint cavitation.
Members of the bone morphogenetic protein/transforming growth factor-β, fibroblast growth factor, and Wnt families and the parathyroid hormone–related peptide/Indian hedgehog axis are essential for joint development and growth plate formation.
The synovial lining of diarthrodial joints is a thin layer of cells lacking a basement membrane and consisting of two principal cell types—macrophages and fibroblasts.
The articular cartilage receives its nutritional requirements via diffusion from the synovial fluid, and interaction of the cartilage with components of the synovial fluid contributes to the unique low-friction surface properties of the articular cartilage.
Classification of Joints
Human joints, which provide the structures by which bones join with one another, may be classified according to the histologic features of the union and the range of joint motion. Three classes of joint design exist: (1) synovial or diarthrodial joints ( Fig. 1.1 ), which articulate with free movement, have a synovial membrane lining the joint cavity, and contain synovial fluid; (2) amphiarthroses, in which adjacent bones are separated by articular cartilage or a fibrocartilage disk and are bound by firm ligaments, permitting limited motion (e.g., the pubic symphysis, intervertebral disks of vertebral bodies, distal tibiofibular articulation, and sacroiliac joint articulation with pelvic bones); and (3) synarthroses, which are found only in the skull (suture lines) where thin, fibrous tissue separates adjoining cranial plates that interlock to prevent detectable motion before the end of normal growth, yet permit growth in childhood and adolescence.
Joints also can be classified according to the connective tissues that join opposing bones. Symphyses have a fibrocartilaginous disk separating bone ends that are joined by firm ligaments (e.g., the symphysis pubis and intervertebral joints). In synchondroses, the bone ends are covered with articular cartilage, but no synovium or significant joint cavity is present (e.g., the sternomanubrial joint). In syndesmoses, the bones are joined directly by fibrous ligaments without a cartilaginous interface (the distal tibiofibular articulation is the only joint of this type outside the cranial vault).
Synovial joints are classified further according to their shapes, which include ball-and-socket (hip), hinge (interphalangeal), saddle (first carpometacarpal), and plane (patellofemoral) joints. These configurations reflect function, with the shapes and sizes of the opposing surfaces determining the direction and extent of motion. The various designs permit flexion, extension, abduction, adduction, or rotation. Certain joints can act in one (humeroulnar), two (wrist), or three (shoulder) axes of motion.
This chapter concentrates on the developmental biology and relationship between structure and function of a “prototypic,” “normal” human diarthrodial joint—the joint in which arthritis is most likely to develop. Most of the research performed concerns the knee because of its accessibility, but other joints are described when appropriate.
Developmental Biology of the Diarthrodial Joint
Skeletal development is initiated by the differentiation of mesenchymal cells that arise from three embryonic sources: (1) neural crest cells of the neural ectoderm that give rise to craniofacial bones; (2) the sclerotome of the paraxial mesoderm, or somite compartment, which forms the axial skeleton; and (3) the somatopleure of the lateral plate mesoderm, which yields the skeleton of the limbs. The appendicular skeleton develops in the human embryo from limb buds, which are first visible at approximately 4 weeks of gestation. Structures resembling adult joints are generated at approximately 4 to 7 weeks of gestation. Many other crucial phases of musculoskeletal development follow, including vascularization of epiphyseal cartilage (8 to 12 weeks), appearance of villous folds in synovium (10 to 12 weeks), evolution of bursae (3 to 4 months), and the appearance of periarticular fat pads (4 to 5 months).
The upper limbs develop approximately 24 hours earlier than the analogous portions of the lower limbs. Proximal structures, such as the glenohumeral joint, develop before more distal ones, such as the wrist and hand. Consequently, insults to embryonic development during limb formation affect a more distal portion of the upper limb than of the lower limb. Long bones form as a result of replacement of the cartilage template by endochondral ossification. The stages of limb development are shown in Fig. 1.2 The developmental sequence of the events occurring during synovial joint formation and some of the regulatory factors and extra-cellular matrix components involved are summarized in Fig. 1.3 . The three main stages in joint development are interzone formation, cavitation, and morphogenesis, as described in detail in several reviews.
Interzone Formation and Joint Cavitation
The structure of the developing synovial joint and the process of joint cavitation have been described in many classic studies performed on the limbs of mammalian and avian embryos. In the human embryo, cartilage condensations can be detected at stage 17, when the embryo is small—approximately 11.7 mm long. In the region of the future joint, after formation of the homogeneous chondrogenic interzone at 6 weeks (stages 18 and 19), a three-layered interzone is formed at approximately 7 weeks (stage 21), which consists of two chondrogenic, perichondrium-like layers that cover the opposing surfaces of the cartilage anlagen (embryonic pre-chondrogenic cell clusters) and are separated by a narrow band of densely packed cellular blastema that remains and forms the interzone. Cavitation begins in the central interzone at about 8 weeks (stage 23).
Although the cellular events associated with joint formation have been recognized for many years, only recently have the genes regulating these processes been elucidated. These genes include growth differentiation factor (GDF)-5 (also known as cartilage-derived morphogenetic protein-1) and Wnt14 (also known as Wnt9a), which are involved in early joint development. Two major roles have been proposed for Wnt14. First, it acts at the onset of joint formation as a negative regulator of chondrogenesis. Second, it facilitates interzone formation and cavitation by inducing the expression of GDF-5; autotaxin; lysophosphatidic acid; the bone morphogenetic protein (BMP) antagonist, chordin; and the hyaluronan receptor, CD44. Paradoxically, application of GDF-5 to developing joints in mouse embryo limbs in organ culture causes joint fusion, suggesting that temporospatial interactions among distinct cell populations are important for the correct response. The current view is that GDF-5 is required at the early stages of condensations, where it stimulates recruitment and differentiation of chondrogenic cells, and later, when its expression is restricted to the interzone. Recent evidence from one study sheds light on the temporospatial sequence of events, and it suggests that there is a continuous recruitment of new GDF-5 expressing cells into the interzone during joint development that leads to lineage divergence at different stages. Early recruited GDF-5+ cells preferentially populate the developing epiphysis, and later recruited cells undergo chondrogenesis and contribute more to the developing articular surface.
The distribution of collagen types and proteoglycans in developing avian and rodent joints is characterized histologically and by immunohistochemistry and in situ hybridization. The matrix produced by mesenchymal cells in the interzone is rich in types I and III collagen, and during condensation, production switches to types II, IX, and XI collagens that typify the cartilaginous matrix. The messenger RNAs (mRNAs) encoding the small proteoglycans biglycan and decorin may be expressed at this time, but the proteins do not appear until after cavitation in the regions destined to become articular cartilage. The interzone regions are marked by the expression of genes encoding type IIA collagen by chondrocyte progenitors in the perichondrial layers, type IIB and XI collagens by differentiated chondrocytes in the cartilage anlagen, and type I collagen in the interzone and in the developing capsule and perichondrium ( Fig. 1.4 ).
The interzone region contains cells in two outer layers, where they are destined to differentiate into chondrocytes and become incorporated into the epiphyses, and in a thin intermediate zone where they are programmed to undergo joint cavitation and may remain as articular chondrocytes. These early chondrocytes all arise from the same population of progenitors, but unlike the other chondrocytes of the anlagen, they do not activate matrilin-1 expression and are destined to form the articular surface. As cavitation begins in this zone, fluid and macromolecules accumulate in this space and create a nascent synovial cavity. Blood vessels appear in the surrounding capsulosynovial blastemal mesenchyme before separation of the adjacent articulating surfaces. Although it was first assumed that these interzone cells undergo necrosis or programmed cell death (apoptosis), many investigators have found no evidence of DNA fragmentation preceding cavitation. In addition, no evidence exists that metalloproteinases are involved in loss of tissue strength in the region undergoing cavitation. Instead, the actual joint cavity seems to be formed by mechanospatial changes induced by the synthesis and secretion of hyaluronan via uridine diphosphoglucose dehydrogenase (UDPGD) and hyaluronan synthase. Interaction of hyaluronan and CD44 on the cell surface modulates cell migration, but the accumulation of hyaluronan and the associated mechanical influences force the cells apart and induce rupture of the intervening extra-cellular matrix by tensile forces. This mechanism accounts, partially, for observations that joint cavitation is incomplete in the absence of movement. Equivalent data from human embryonic joints are difficult to obtain, but in all large joints in humans, complete joint cavities are apparent at the beginning of the fetal period.
Cartilage Formation and Endochondral Ossification
The skeleton develops from the primitive, avascular, densely packed cellular mesenchyme, termed the skeletal blastema. Common precursor mesenchymal cells divide into chondrogenic, myogenic, and osteogenic lineages that determine the differentiation of cartilage centrally, muscle peripherally, and bone. The surrounding tissues, particularly epithelium, influence the differentiation of mesenchymal progenitor cells to chondrocytes in the cartilage anlagen. The cartilaginous nodules appear in the middle of the blastema, and simultaneously cells at the periphery become flattened and elongated to form the perichondrium. In the vertebral column, cartilage disks arise from portions of the somites surrounding the notochord, and nasal and auricular cartilage and the embryonic epiphysis form from the perichondrium. In the limb, the cartilage remains as a resting zone that later becomes the articular cartilage, or it undergoes terminal hypertrophic differentiation to become calcified (growth plate formation) and is replaced by bone (endochondral ossification). The latter process requires extra-cellular matrix remodeling and vascularization (angiogenesis). These events are controlled exquisitely by cellular interactions with the surrounding matrix, growth and differentiation factors, and other environmental factors that initiate or suppress cellular signaling pathways and transcription of specific genes in a temporospatial manner.
Condensation and Limb Bud Formation
Formation of the cartilage anlage occurs in four stages: (1) cell migration, (2) aggregation regulated by mesenchymal-epithelial cell interactions, (3) condensation, and (4) chondrocyte differentiation. Interactions with the epithelium determine mesenchymal cell recruitment and migration, proliferation, and condensation. The aggregation of chondroprogenitor mesenchymal cells into precartilage condensations was first described by Fell and depends on signals initiated by cell-cell and cell-matrix interactions, the formation of gap junctions, and changes in the cytoskeletal architecture. Before condensation, the prechondrocytic mesenchymal cells produce extra-cellular matrix that is rich in hyaluronan and type I collagen and type IIA collagen, which contains the exon-2–encoded aminopropeptide found in noncartilage collagens. The initiation of condensation is associated with increased hyaluronidase activity and the transient upregulation of versican, tenascin, syndecan, the cell adhesion molecules, neural cadherin (N-cadherin), and neural cell adhesion molecule (NCAM), which facilitate cell-cell interactions.
Before chondrocyte differentiation, the cell-matrix interactions are facilitated by the binding of fibronectin to syndecan, thus downregulating NCAM and setting the condensation boundaries. Increased cell proliferation and extra-cellular matrix remodeling, with the disappearance of type I collagen, fibronectin, and N-cadherin, and the appearance of tenascins, matrilins, and thrombospondins, including cartilage oligomeric matrix protein (COMP), initiate the transition from chondroprogenitor cells to a fully committed chondrocyte. N-cadherin and NCAM disappear in differentiating chondrocytes and are detectable later only in perichondrial cells. As discussed previously, recent evidence suggests that during joint development there is a continuous recruitment of new GDF-5–expressing cells into the interzone. These cells preferentially populate the developing epiphysis, and later, recruited cells undergo chondrogenesis and contribute to the developing articular surface.
Much of the current understanding of limb bud development is based on early studies in chickens and recently in mice. The regulatory events are controlled by interacting patterning systems involving homeobox (Hox) transcription factors and fibroblast growth factor (FGF), hedgehog, transforming growth factor-β (TGF-β)/BMP, and Wnt pathways, each of which functions sequentially over time (see Fig. 1.3 ). The HoxA and HoxD gene clusters are crucial for the early events of limb patterning in the undifferentiated mesenchyme, as they are required for the expression of FGF-8 and Sonic hedgehog (Shh), which modulate the proliferation of cells within the condensations. BMP-2, BMP-4, and BMP-7 coordinately regulate the patterning of limb elements within the condensations depending on the temporal and spatial expression of BMP receptors and BMP antagonists, such as noggin and chordin, as well as the availability of BMP- and TGF-β–induced SMADs (signaling mammalian homologues of Drosophila mothers against decapentaplegic). BMP signaling is required for the formation of precartilaginous condensations and for the differentiation of precursors into chondrocytes, acting, in part, by opposing FGF actions. Growth of the condensation ceases when noggin inhibits BMP signaling and permits differentiation to chondrocytes. The cartilage formed serves as a template for formation of cartilaginous elements in the vertebra, sternum, and rib, and for limb elongation or endochondral bone formation.
Molecular Signals in Cartilage Morphogenesis and Growth Plate Development
The cartilage anlagen grow by cell division, deposition of extra-cellular matrix, and apposition of proliferating cells from the inner chondrogenic layer of the perichondrium. The nuclear transcription factor Sox9 is one of the earliest markers expressed in cells undergoing condensation and is required for the subsequent stage of chondrogenesis characterized by the deposition of matrix-containing collagens II, IX, and XI and aggrecan. The expression of SOX proteins depends on BMP signaling via BMPR1A and BMPR1B, which are functionally redundant and active in chondrocyte condensations, but not in the perichondrium. Sox5 and Sox6 are required for the expression of Col9a1, aggrecan, link protein, and Col2a1 during chondrocyte differentiation. The runt-domain transcription factor, Runx2 (also known as core binding factor, Cbfa1), is expressed in all condensations including those that are destined to form bone.
Throughout chondrogenesis, the balance of signaling by BMPs and FGFs determines the rate of proliferation and the pace of the differentiation. In the long bones, long after condensation, BMP-2, BMP-3, BMP-4, BMP-5, and BMP-7 are expressed primarily in the perichondrium, but only BMP-7 is expressed in the proliferating chondrocytes. BMP-6 is found later, exclusively in hypertrophic chondrocytes along with BMP-2. More than 23 FGFs have been identified thus far. The specific ligands that activate each FGF receptor (FGFR) during chondrogenesis in vivo have been difficult to identify because the signaling depends on the temporal and spatial location of not only the ligands but also the receptors. FGFR2 is upregulated early in condensing mesenchyme and is present later in the periphery of the condensation along with FGFR1, which is expressed in surrounding loose mesenchyme. FGFR3 is associated with proliferation of chondrocytes in the central core of the mesenchymal condensation and overlaps with FGFR2. Proliferation of chondrocytes in the embryonic and postnatal growth plate is regulated by multiple mitogenic stimuli, including FGFs, which converge on cyclin D1.
Early studies indicated that FGFR3 could serve as a master inhibitor of chondrocyte proliferation via Stat1 and the cell cycle inhibitor p21. FGFR3 activation downregulates AKT activity to decrease proliferation, and MEK activation leads to decreased chondrocyte differentiation. The physiologic FGFR3 ligands are not known, but FGF-9 and FGF-18 are good candidates because they bind FGFR3 in vitro and are expressed in the adjacent perichondrium and periosteum, forming a functional gradient. FGF-18–deficient mice have an expanded zone of proliferating chondrocytes similar to that in FGFR3-deficient mice, and FGF-18 can inhibit Indian hedgehog (Ihh) expression. As the growth plate develops, FGFR3 disappears and FGFR1 is upregulated in the prehypertrophic and hypertrophic zones, where FGF-18 and FGF-9 regulate vascular invasion by inducing vascular endothelial growth factor (VEGF) and VEGFR1 and terminal differentiation.
The proliferation of chondrocytes in the lower proliferative and prehypertrophic zones is under the control of a local negative feedback loop involving signaling by parathyroid hormone–related protein (PTHrP) and Ihh. Ihh expression is restricted to the prehypertrophic zone, and the PTHrP receptor is expressed in the distal zone of periarticular chondrocytes. The adjacent, surrounding perichondrial cells express the Hedgehog receptor patched (Ptch), which, upon Ihh binding, similar to Shh in the mesenchymal condensations, activates Smo and induces Gli transcription factors, which can feedback regulate Ihh target genes in a positive ( Gli1 and Gli2 ) or negative (Gli3) manner. Ihh induces expression of PTHrP in the perichondrium, and PTHrP signaling stimulates cell proliferation via its receptor expressed in the periarticular chondrocytes. Recent evidence indicates that Ihh also acts independently of PTHrP on periarticular chondrocytes to stimulate differentiation of columnar chondrocytes in the proliferative zone, whereas PTHrP acts by preventing premature differentiation into prehypertrophic and hypertrophic chondrocytes, suppressing premature expression of Ihh. Ihh and PTHrP, by transiently inducing proliferation markers and repressing differentiation markers, function in a temporospatial manner to determine the number of cells that remain in the chondrogenic lineage versus the number that enter the endochondral ossification pathway. Components of the extra-cellular matrix also contribute to regulation of the different stages of growth plate development, including chondrogenesis and terminal differentiation, by interacting with signaling molecules and chondrocyte cell surface receptors.
Endochondral Ossification
The development of long bones from the cartilage anlagen occurs by a process termed endochondral ossification, which involves terminal differentiation of chondrocytes to the hypertrophic phenotype, cartilage matrix calcification, vascular invasion, and ossification (see Fig. 1.3 ). This process is initiated when the cells in the central region of the anlage begin to hypertrophy, increasing cellular fluid volume by almost 20 times. Ihh plays a pivotal role in regulating endochondral bone formation by synchronizing perichondrial maturation with chondrocyte hypertrophy, which, in turn, is essential for initiating the process of vascular invasion. Ihh is expressed in prehypertrophic chondrocytes as they exit the proliferative phase and enter the hypertrophic phase, at which time they begin to express hypertrophic chondrocyte markers type X collagen and alkaline phosphatase. These cells are responsible for laying down the cartilage matrix that subsequently undergoes mineralization. Wnt/β-catenin signaling promotes chondrocyte maturation by a BMP-2–mediated mechanism and induces chondrocyte hypertrophy partly by enhancing matrix metalloproteinase (MMP) expression and potentially by enhancing Ihh signaling and vascularization.
Runx2 serves as an essential positive regulatory factor in chondrocyte maturation to hypertrophy. In Runx2-deficient mice, chondrocyte hypertrophy and terminal differentiation is blocked, and as a result endochondral ossification does not proceed. It is expressed in the adjacent perichondrium and in prehypertrophic chondrocytes and less in late hypertrophic chondrocytes, overlapping with Ihh, COL10A1, and BMP-6. IHH induces Gli transcription factors, which interact with Runx2 and BMP-induced SMADs to regulate transcription and expression of COL10A1. A member of the myocyte enhancer factor (MEF) 2 family, MEF2C, stimulates hypertrophy partly by increasing Runx2 expression. The class II histone deacetylase, HDAC4, prevents premature hypertrophy by directly suppressing the activities of Runx2 and MEF2C. HDAC4 is in turn regulated by PTHrP and salt-inducible kinase 3 (SIK3). Sox9, FOXA2 and FoxA3, Runx3, Zfp521, and peroxisome proliferator-activated receptor γ (PPARγ ) are also important transcriptional regulators of chondrocyte hypertrophy. MMP-13, a downstream target of Runx2, is expressed by terminal hypertrophic chondrocytes, and MMP-13 deficiency results in significant interstitial collagen accumulation, leading to the delay of endochondral ossification in the growth plate with increased length of the hypertrophic zone.
Runx2 also is required for transcription activation of COL10A1, the gene encoding type X collagen, which is the major matrix component of the hypertrophic zone in the embryo and in the postnatal growth plate. Mutations in the COL10A1 gene are associated with the dwarfism observed in human chondrodysplasias. These mutations affect regions of the growth plate that are under great mechanical stress, and the defect in skeletal growth may be due partly to alteration of the mechanical integrity of the pericellular matrix in the hypertrophic zone, although a role for defective vascularization also is proposed. The extra-cellular matrix remodeling that accompanies chondrocyte terminal differentiation is thought to induce an alteration in the environmental stress experienced by hypertrophic chondrocytes, which eventually undergo apoptosis. Whether chondrocyte hypertrophy with cell death is the ultimate fate of hypertrophic chondrocytes or whether hypertrophy is a transient process that precedes osteogenesis is a subject of debate. However, recent genetic lineage tracing studies suggest that hypertrophic chondrocytes can survive at the chondro-osseous junction and become osteoblasts and osteocytes.
Cartilage is an avascular tissue, and because the developing growth plate is relatively hypoxic, hypoxia inducible factor (HIF)-1α is important for survival as chondrocytes transition to hypertrophy. Under normoxia, the cell content of HIF-1α, -2α, and -3α is low because of oxygen-dependent hydroxylation by prolyl-hydroxylases, resulting in ubiquitination and degradation by the proteasome. In contrast, under hypoxia, prolyl-hydroxylase activity is reduced and the α subunits heterodimerize with the constitutive β-subunit members known as aryl hydrocarbon receptor nuclear translocators (ARNTs). HIFs are transcription factors that bind to hypoxia-responsive elements (HREs) in responsive genes. HIF-2α regulates endochondral ossification processes by directly targeting HREs within the promoters of the COL10A1, MMP13, and VEGFA genes.
Vascular invasion of the hypertrophic zone is required for the replacement of calcified cartilage by bone. VEGF acts as an angiogenic factor to promote vascular invasion by specifically activating local receptors, including Flk1, which is expressed in endothelial cells in the perichondrium or surrounding soft tissues; neuropilin 1 (Npn1), which is expressed in late hypertrophic chondrocytes; or Npn2, which is expressed exclusively in the perichondrium. VEGF is expressed as three different isoforms: VEGF188, a matrix-bound form, is essential for metaphyseal vascularization, whereas the soluble form, VEGF120 (VEGFA), regulates chondrocyte survival and epiphyseal cartilage angiogenesis, and VEGF164 can be either soluble or matrix bound and may act directly on chondrocytes via Npn2. VEGF is released from the extra-cellular matrix by MMPs, including MMP-9, membrane-type (MT)1-MMP (MMP-14), and MMP-13. MMP-9 is expressed by endothelial cells that migrate into the central region of the hypertrophic cartilage. MMP-14, which has a broader range of expression than MMP-9, is essential for chondrocyte proliferation and secondary ossification, whereas MMP-13 is found exclusively in late hypertrophic chondrocytes. Perlecan (Hspg2), a heparan sulfate proteoglycan in cartilage matrix, is required for vascularization in the growth plate through its binding to the VEGFR of endothelial cells, permitting osteoblast migration into the growth plate.
A number of ADAM (a disintegrin and metalloproteinase) proteinases are also emerging as important regulators in growth plate development. For example, ADAM10 is a principle regulator of Notch signaling, which modulates endochondral ossification via RBPjk in chondrocytes and promotes osteoclastogenesis at the chondro-osseous junction by regulating endothelial cell organization in the developing bone vasculature. ADAM17 is the critical proteinase mediating cellular shedding of TNF but also the epidermal growth factor receptor (EGFR) ligands, including TGF-α. The EGFR signaling pathway induced by EGF and TGF-α plays a crucial role in the remodeling of the growth plate, where inactivation of EGFR results in the inability of hypertrophic chondrocytes to degrade the surrounding collagen matrix and to attract osteoclasts to invade and remodel the advancing growth plate under control of the osteoclast differentiation factor receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Mice lacking ADAM17 in chondrocytes (Adam17ΔCh) show an expanded hypertrophic zone in the growth plate, essentially phenocopying mice with defects in EGFR signaling in chondrocytes. Tight regulation of EGFR signaling is important for cartilage and joint homeostasis, as shown in mice with cartilage-specific deletion of the mitogen-inducible gene 6 (MIG-6), a scaffold protein that binds EGFR and targets it for internalization and degradation. These events of cartilage matrix remodeling and vascular invasion are required for the migration and differentiation of osteoclasts and osteoblasts, which remove the mineralized cartilage matrix and replace it with bone.
Development of Articular Cartilage
In the vertebrate skeleton, cartilage is the product of cells from three distinct embryonic lineages. Craniofacial cartilage is formed from cranial neural crest cells; the cartilage of the axial skeleton (intervertebral disks, ribs, and sternum) forms from paraxial mesoderm (somites); and the articular cartilage of the limbs is derived from the lateral plate mesoderm. In the developing limb bud, mesenchymal cells form condensations in digital zones, followed by chondrocyte differentiation and maturation, whereas undifferentiated mesenchymal cells in the interdigital web zones undergo cell death. Embryonic cartilage is destined for one of several fates: it can remain as permanent cartilage (as on the articular surfaces of bones), or it can provide a template for the formation of bones by endochondral ossification. During development, cells in the cartilage anlage resembling the shape of the future bone undergo chondrocyte maturation expanding from the central site of the original condensation toward the ends of the forming bones. During joint cavitation, the peripheral interzone is absorbed into each adjacent cartilaginous zone, evolving into the articular surface. The articular surface is destined to become a specialized cartilaginous structure that does not normally undergo vascularization and ossification.
Recent evidence indicates that postnatal maturation of the articular cartilage involves an appositional growth mechanism originating from progenitor cells at the articular surface rather than an interstitial mechanism. During formation of the mature articular cartilage, the differentiated articular chondrocytes synthesize the cartilage-specific matrix molecules, such as type II collagen and aggrecan (see Chapter 3 ). Through the processes described previously, the articular joint spaces are developed and lined on all surfaces either by cartilage or by synovial lining cells. These two different tissues merge at the enthesis, the region at the periphery of the joint where the cartilage melds into bone, and where ligaments and the capsule are attached. In the postnatal growth plate, the differentiation of the perichondrium also is linked to the differentiation of the chondrocytes in the epiphysis to form the different zones of the growth plate, contributing to longitudinal bone growth. Once the growth plate closes in the human joint, the adult articular cartilage must be maintained by the resident chondrocytes with low-turnover production of matrix proteins.
Development of the Joint Capsule, Synovial Lining, Menisci, and Intracapsular Ligaments
The interzone and the contiguous perichondrial envelope, of which the interzone is a part, contain the mesenchymal cell precursors that give rise to other joint components, including the joint capsule, synovial lining, menisci, intracapsular ligaments, and tendons. The external mesenchymal tissue condenses as a fibrous capsule. The peripheral mesenchyme becomes vascularized and is incorporated as the synovial mesenchyme, which differentiates into a pseudomembrane at about the same time as cavitation begins in the central interzone (stage 23, approximately 8 weeks). The menisci arise from the eccentric portions of the articular interzone. In common usage, the term synovium refers to the true synovial lining and the subjacent vascular and areolar tissue, up to—but excluding—the capsule. Synovial lining cells can be distinguished as soon as the multiple cavities within the interzone begin to coalesce. At first, these cells are exclusively fibroblast-like (type B) cells.
The synovial lining cells express the hyaluronan receptor CD44 and UDPGD, the levels of which remain elevated after cavitation. This increased activity likely contributes to the high concentration of hyaluronan in joint fluids. As the joint cavity increases in size, synovial-lining cell layers expand by proliferation of fibroblast-like cells and recruitment of macrophage-like (type A) cells from the circulation. In developing human temporomandibular joints, these type A cells can be detected by 12 weeks of gestation. Further synovial expansion results in the appearance of synovial villi at the end of the second month, early in the fetal period, which greatly increases the surface area available for exchange between the joint cavity and the vascular space. Cadherin 11 is an additional molecule expressed by synovial lining cells. It is essential for establishment of synovial lining architecture during development, where its expression correlates with cell migration and tissue outgrowth of the synovial lining.
The development and cellular composition of the synovium are discussed later.
The role of innervation in the developing joint is not well understood. A dense capillary network develops in the subsynovial tissue, with numerous capillary loops that penetrate into the true synovial lining layer. The human synovial microvasculature is already innervated by 8 weeks of gestation (stage 23), around the time of joint cavitation. Evidence of neurotransmitter function is not found until much later, however, with the appearance of the sensory neuropeptide substance P at 11 weeks. The putative sympathetic neurotransmitter, neuropeptide Y, appears at 13 weeks of gestation, along with the catecholamine-synthesizing enzyme tyrosine hydroxylase. The finding that the Slit2 gene, which functions for the guidance of neuronal axons and neurons, is expressed in the mesenchyme and in peripheral mesenchyme of the limb bud (stages 23 to 28) suggests that innervation is an integral part of synovial joint development.
Development of Nonarticular Joints
In contrast to articular joints, the temporomandibular joint develops slowly, with cavitation at a crown-rump length of 57 to 75 mm (i.e., well into the fetal stage). This slow development may occur because this joint develops in the absence of a continuous blastema and involves the insertion between bone ends of a fibrocartilaginous disk that arises from muscular and mesenchymal derivatives of the first pharyngeal arch. However, many of the same genes as those involved in articular joint development are involved in morphogenesis and growth of the temporomandibular joint.
The development of other types of joints, such as synarthroses, is similar to that of diarthrodial joints except that cavitation does not occur, and synovial mesenchyme is not formed. In these respects, synarthroses and amphiarthroses resemble the “fused” peripheral joints induced by paralyzing chicken embryos, and they may develop as they do because relatively little motion is present during their formation.
The intervertebral disk consists of a semiliquid nucleus pulposus (NP) in the center, surrounded by a multilayered fibrocartilaginous annulus fibrosus (AF), which is sandwiched between the cartilaginous end plates (EPs). Between the EPs lies the vertebral body consisting of the growth plate, which later disappears, and the primary and secondary centers of ossification that fuse together. The cells in the NP arise from the embryonic notochord, and the notochord orchestrates somatogenesis, from which arises the ventral mesenchymal sclerotome that forms the AF of the intervertebral disk, as well as the vertebral bodies and ribs. The NP acts as the center for controlling cell differentiation in the AF and EP through Shh signaling, which is regulated by Wnt signaling and, in turn, promotes growth and differentiation through downstream transcription factors, Brachyury and Sox9, and gene expression of extra-cellular matrix components. The proteoglycans and collagens expressed during development of the intervertebral disk have been mapped and reflect the complex structure-function relationships that allow flexibility and resistance to compression in the spine.
Organization and Physiology of the Mature Joint
The unique structural properties and biochemical components of diarthrodial joints make them extraordinarily durable load-bearing devices. The mature diarthrodial joint is a complex structure, influenced by its environment and mechanical demands (see Chapter 6 ). Joints have structural differences that are determined by their different functions. The shoulder joint, which demands an enormous range of motion, is stabilized primarily by muscles, whereas the hip, which requires motion and antigravity stability, has an intrinsically stable ball-and-socket configuration. The components of the “typical” synovial joint are the synovium, muscles, tendons, ligaments, bursae, menisci, articular cartilage, and subchondral bone. The anatomy and physiology of muscles are described in detail in Chapter 5 .
Synovium
The synovium, which lines the joint cavity, is the site of production of synovial fluid that provides the nutrition for the articular cartilage and lubricates the cartilage surfaces. The synovium is a thin membrane between the fibrous joint capsule and the fluid-filled synovial cavity that attaches to skeletal tissues at the bone-cartilage interface and does not encroach on the surface of the articular cartilage. It is divided into functional compartments: the lining region (synovial intima), the subintimal stroma, and the neurovasculature ( Fig. 1.5 ). The synovial intima, also termed synovial lining, is the superficial layer of the normal synovium that is in contact with the intra-articular cavity. The synovial lining is loosely attached to the subintima, which contains blood vessels, lymphatics, and nerves. Capillaries and arterioles generally are located directly underneath the synovial intima, whereas venules are located closer to the joint capsule.
A transition from loose to dense connective tissue occurs from the joint cavity to the capsule. Most cells in the normal subintimal stroma are fibroblasts and macrophages, although adipocytes and occasional mast cells are present. These compartments are not circumscribed by basement membranes but nonetheless have distinct functions; they are separated from each other by chemical barriers, such as membrane peptidases, which limit the diffusion of regulatory factors between compartments. Synovial compartments are unevenly distributed within a single joint. Vascularity is high at the enthesis where synovium, ligament, and cartilage coalesce. Far from being a homogeneous tissue in continuity with the synovial cavity, synovium is highly heterogeneous, and synovial fluid may be poorly representative of the tissue-fluid composition of any synovial tissue compartment. In rheumatoid arthritis, the synovial lining of diarthrodial joints is the site of the initial inflammatory process. This lesion is characterized by proliferation of the synovial lining cells, increased vascularization, and infiltration of the tissue by inflammatory cells, including B and T lymphocytes, plasma cells, and activated macrophages (see Chapter 75 ).
Synovial Lining
The synovial lining, a specialized condensation of mesenchymal cells and extra-cellular matrix, is located between the synovial cavity and stroma. In normal synovium, the lining layer is two to three cells deep, although intra-articular fat pads usually are covered by only a single layer of synovial cells, and ligaments and tendons are covered by synovial cells that are widely separated. At some sites, lining cells are absent, and the extra-cellular connective tissue constitutes the lining layer. Such “bare areas” become increasingly frequent with advancing age. Although the synovial lining is often referred to as the synovial membrane, the term membrane is more correctly reserved for endothelial and epithelial tissues that have basement membranes, tight intercellular junctions, and desmosomes. Instead, synovial lining cells lie loosely in a bed of hyaluronate interspersed with collagen fibrils; this is the macromolecular sieve that imparts the semipermeable nature of the synovium. The absence of any true basement membrane is a major determinant of joint physiology.
Early electron microscopic studies characterized lining cells as macrophage-derived type A and fibroblast-derived type B cells. High UDPGD activity and CD55 are used to distinguish type B synovial cells, whereas nonspecific esterase and CD68 typify type A cells. Normal synovium is lined predominantly by fibroblast-like synoviocytes, whereas macrophage-like synovial cells compose only 10% to 20% of lining cells (see Fig. 1.5 ).
Type A, macrophage-like synovial cells contain vacuoles, a prominent Golgi apparatus, and filopodia, but they have little rough endoplasmic reticulum. These cells express numerous cell surface markers of the monocyte-macrophage lineage, including CD16, CD45, CD11b/CD18, CD68, CD14, CD163, and the immunoglobulin (Ig)G Fc receptor, FcγRIIIa. Synovial intimal macrophages are phagocytic and may provide a mechanism by which particulate matter can be cleared from the normal joint cavity. Similar to other tissue macrophages, these cells have little capacity to proliferate and are likely localized to the joint during development. The op/op osteopetrotic mouse that is deficient in macrophages because of an absence of macrophage colony-stimulating factor also lacks synovial macrophages, suggesting that type A synovial cells are of a common lineage with other tissue macrophages. Although they represent only a small percentage of the cells in the normal synovium, the macrophages are recruited from the circulation during synovial inflammation, potentially from subchondral bone marrow through vascular channels near the enthesis.
The type B, fibroblast-like synoviocytes contain fewer vacuoles and filopodia than type A cells and have abundant protein-synthetic organelles. Similar to other fibroblasts, lining cells express genes encoding extra-cellular matrix components, including collagens, sulfated proteoglycans, fibronectin, fibrillin-1, and tenascin, and they express intra-cellular and cell surface molecules, such as vimentin and CD90 (Thy-1). They have the potential to proliferate, although proliferation markers are rarely seen in normal synovium. In contrast to stromal fibroblasts, synovial intimal fibroblasts express UDPGD and synthesize hyaluronan, an important constituent of synovial fluid. They also synthesize lubricin, which, together with hyaluronan, is necessary for the low-friction interaction of cartilage surfaces in the diarthrodial joint. Synovial lining cells bear abundant membrane peptidases on their surface that are capable of degrading a wide range of regulatory peptides, such as substance P and angiotensin II.
Normal synovial lining cells also express a rich array of adhesion molecules, including CD44, the principal receptor for hyaluronan; vascular cell adhesion molecule (VCAM)-1; intercellular adhesion molecule (ICAM)-1; and CD55 (decay-accelerating factor). They are essential for cellular attachment to specific matrix components in the synovial lining region, preventing loss into the synovial cavity of cells subjected to deformation and shear stresses during joint movement. Adhesion molecules such as VCAM-1 and ICAM-1 potentially are involved in the recruitment of inflammatory cells during the evolution of arthritis. Cadherins mediate cell-cell adhesion between adjacent cells of the same type. The identification of cadherin-11 as a key adhesion molecule that regulates the formation of the synovial lining during development and the synoviocyte function postnatally has provided the opportunity to examine its role in inflammatory joint disease. Cadherin-11 is highly expressed in fibroblast-like cells at the pannus-cartilage interface in rheumatoid synovium, where it plays a role in the invasive properties of the synovial fibroblasts, and treatment with a cadherin-11 antibody or a cadherin-11 fusion protein reduces synovial inflammation and cartilage erosion in an animal model of arthritis.
Of interest, recent studies have highlighted the development and expansion of distinct synovial fibroblast populations in inflammatory arthritis, and data support a key role for these cells in the pathogenesis and maintenance of joint inflammation in rheumatoid arthritis. The roles of synovitis and synovial angiogenesis are also of current interest in relation to the severity and progression of pain and joint damage in osteoarthritis (OA).
Synovial Vasculature
The subintimal synovium contains blood vessels, providing the blood flow that is required for solute and gas exchange in the synovium itself and for the generation of synovial fluid. The avascular articular cartilage also depends on nutrition in the synovial fluid, derived from the synovial vasculature. The vascularized synovium behaves similar to an endocrine organ, generating factors that regulate synoviocyte function and serving as a selective gateway that recruits cells from the circulation during stress and inflammation. Finally, synovial blood flow plays an important role in regulating intra-articular temperature.
The synovial vasculature can be divided on morphologic and functional grounds into arterioles, capillaries, and venules. In addition, lymphatics accompany arterioles and larger venules. Arterial and venous networks of the joint are complex and are characterized by arteriovenous anastomoses that communicate freely with blood vessels in periosteum and periarticular bone. As large synovial arteries enter the deep layers of the synovium near the capsule, they give off branches, which bifurcate again to form “microvascular units” in the subsynovial layers. The synovial lining region, the surfaces of intra-articular ligaments, and the entheses (the angle of ligamentous insertions into bone) are particularly well vascularized.
The distribution of synovial vessels, which were formed largely as a result of vasculogenesis during development of the joint, displays considerable plasticity. In inflammatory arthritis, the density of blood vessels decreases relative to the growing synovial mass, creating a hypoxic and acidotic environment. Angiogenic factors such as VEGF, acting via VEGF receptors 1 and 2 (Flt1 and Flk2), and basic FGF promote proliferation and migration of endothelial cells, a process that is facilitated by matrix-degrading enzymes and adhesion molecules such as integrin αvβ3 and E-selectin, expressed by activated endothelial cells. Vessel maturation is facilitated by angiopoietin-1 acting via the Tie-2 receptor. The angiogenic molecules are restricted to the capillary epithelium in normal synovium, but their levels are elevated in inflamed synovium in perivascular sites and areas remote from vessels.
Regulation of Synovial Blood Flow
Synovial blood flow is regulated by intrinsic (autocrine and paracrine) and extrinsic (neural and humoral) systems. Locally generated factors, such as the peptide vasoconstrictors angiotensin II and endothelin-1, act on adjacent arteriolar smooth muscle to regulate regional vascular tone. Normal synovial arterioles are richly innervated by sympathetic nerves containing vasoconstrictors, such as norepinephrine and neuropeptide Y, and by “sensory” nerves that also play an efferent vasodilatory role by releasing neuropeptides, such as substance P and calcitonin gene–related peptide (CGRP). Arterioles regulate regional blood flow. Capillaries and postcapillary venules are sites of fluid and cellular exchange. Correspondingly, regulatory systems are differentially distributed along the vascular axis. Angiotensin-converting enzyme, which generates angiotensin II, is localized predominantly in arteriolar and capillary endothelia and decreases during inflammation. Specific receptors for angiotensin II and for substance P are abundant on synovial capillaries, with lower densities on adjacent arterioles. Dipeptidyl peptidase IV, a peptide-degrading enzyme, is specifically localized to the cell membranes of venular endothelium. The synovial vasculature is not only functionally compartmentalized from the surrounding stroma but also highly specialized along its arteriovenous axis. Other unique characteristics of the normal synovial vasculature include the presence of inducible nitric oxidase synthase–independent 3-nitrotyrosine, a reaction product of peroxynitrite, and the localization of the synoviocyte-derived CXCL12 chemokine on heparan sulfate receptors on endothelial cells, suggesting physiologic roles for these molecules in normal vascular function.
Joint Innervation
Dissection studies have shown that each joint has a dual nerve supply consisting of specific articular nerves that penetrate the capsule as independent branches of adjacent peripheral nerves and articular branches that arise from related muscle nerves. The definition of joint position and the detection of joint motion are monitored separately and by a combination of multiple inputs from different receptors in varied systems. Nerve endings in muscle and skin and in the joint capsule mediate sensation of joint position and movement. Normal joints have afferent (sensory) and efferent (motor) innervations consisting of both unmyelinated and sensory thick myelinated A fibers in ligaments, fibrous capsule, menisci, and adjacent periosteum, where they are thought to function primarily as sensors for pressure and movements. Sensory A and C fibers terminate as free nerve endings in the fibrous capsule, adipose tissue, ligaments, menisci, and the adjacent periosteum, where they are thought to act as nociceptors and contribute to the regulation of synovial microvascular function.
In normal synovium, a dense network of fine unmyelinated nerve fibers follows the courses of blood vessels and extends into the synovial lining layers. These nerve fibers are largely unmyelinated and are slow-conducting fibers; they may transmit diffuse, burning, or aching pain sensation. Sympathetic nerve fibers surround blood vessels, particularly in the deeper regions of synovium, and contain and release classic neurotransmitters, such as norepinephrine, and neuropeptides that are markers of sensory nerves including substance P, CGRP, neuropeptide Y, and vasoactive intestinal peptide. Substance P and CGRP, in particular, have been implicated in modulating inflammation and the pain pathway in OA. In addition, substance P is released from peripheral nerve terminals into the joint, and specific, G protein–coupled receptors for substance P are localized to microvascular endothelium in normal synovium. Abnormalities in neuropeptide release in arthritis may contribute to changes in vascular permeability and the failure of synovial inflammation to resolve. The expression of substance P and CGRP are upregulated by nerve growth factor (NGF), which belongs to a family of neurotrophins that regulate neuronal growth during embryonic development. In addition to promoting nerve growth and mediating pain perception, NGF can act together with VEGF to promote blood vessel formation. Angiogenesis and nerve growth thus are linked by common pathways involving NGF, VEGF, and neuropeptides such as CGRP, neuropeptide Y, and semaphorins.
Afferent nerve fibers from the joint play an important role in the reflex inhibition of muscle contraction. Trophic factors generated by motor neurons, such as the neuropeptide CGRP, are important in maintaining muscle bulk and a functional neuromuscular junction. Decreases in motor neuron trophic support during articular inflammation probably contribute to muscle wasting in arthritic conditions. Inflammation and excessive local neuropeptide release may result in the loss of nerve fibers, and synovial tissue proliferation without concomitant growth of new nerve fibers may lead to an apparent partial denervation of synovium in arthritis. However, there is also evidence from humans and pre-clinical arthritis models of sprouting of sensory nerves in synovium, and ingrowth of neurovascular channels at the osteochondral junction. Overall, aberrant innervation of synovium and other joint structures during arthritis development may contribute to changes in synovial joint homeostasis, motor neuron trophic support, and pain.
Mechanisms of joint pain have been reviewed in detail. In a noninflamed joint, most sensory nerve fibers do not respond to movement within the normal range; these fibers are referred to as silent nociceptors. In an inflamed joint, however, these nerve fibers become sensitized by mediators such as bradykinin, neurokinin 1, NGF and prostaglandins, and the resulting peripheral sensitization leads to pain during normal joint movement, a characteristic symptom of arthritis. Pain sensation is upregulated or downregulated further in the central nervous system, at the level of the spinal cord and in the brain, by central sensitization and “gating” of nociceptive input. A poor correlation often exists between the severity of apparent joint disease and perceived pain in people with chronic arthritis, and this may be a sign of sensitization either at the peripheral or central levels.
Synovial Fluid and Nutrition of Joint Structures
The volume and composition of synovial fluid are determined by the properties of the synovium and its vasculature. A normal joint contains a small quantity of fluid (2.5 mL in the knee), sufficient to coat the synovial and cartilage surfaces. Tendon sheath fluid and synovial fluid are biochemically similar. Both are essential for the nutrition and lubrication of adjacent avascular structures, including tendons and articular cartilage, and for limiting adhesion formation and maintaining movement. Characterization and measurement of synovial fluid constituents have proven useful for the identification of locally generated regulatory factors, markers of cartilage turnover, and the metabolic status of the joint, as well as for the assessment of the effects of therapy on cartilage homeostasis. However, interpretation of such data requires an understanding of the generation and clearance of synovial fluid and its various components.
Generation and Clearance of Synovial Fluid
Synovial fluid concentrations of a protein represent the net contributions of synovial blood flow, plasma concentration, microvascular permeability, and lymphatic removal and its production and consumption within the joint space. Synovial fluid is a mixture of a protein-rich ultrafiltrate of plasma and hyaluronan synthesized by synoviocytes. Generation of this ultrafiltrate depends on the differences between intracapillary and intra-articular hydrostatic pressures and between colloid osmotic pressures of capillary plasma and synovial tissue fluid. Fenestrations (i.e., small pores covered by a thin membrane) in the synovial capillaries and the macromolecular sieve of hyaluronic acid facilitate rapid exchange of small molecules, such as glucose and lactate, assisted—in the case of glucose—by an active transport system. Proteins are present in synovial fluid at concentrations inversely proportional to molecular size, with synovial fluid albumin concentrations being about 45% of those in plasma. Concentrations of electrolytes and small molecules are equivalent to those in plasma.
Synovial fluid is cleared through lymphatics in the synovium, assisted by joint movement. In contrast to ultrafiltration, lymphatic clearance of solutes is independent of molecular size. In addition, constituents of synovial fluid, such as regulatory peptides, may be degraded locally by enzymes, and low-molecular-weight metabolites may diffuse along concentration gradients into plasma. The kinetics of delivery and removal of a protein must be determined (e.g., using albumin as a reference solute) to assess the significance of its concentration in the joint.
Hyaluronic acid is synthesized by fibroblast-like synovial lining cells, and it appears in high concentrations in synovial fluid at around 3 g/L, compared with a plasma concentration of 30 μg/L. Lubricin, a glycoprotein that assists articular lubrication, is another constituent of synovial fluid that is generated by the lining cells. It is now believed that hyaluronan functions in fluid-film lubrication, whereas lubricin is the true boundary lubricant in synovial fluid (see later discussion). Because the volume of synovial fluid is determined by the amount of hyaluronan, water retention seems to be the major function of this large molecule.
Despite the absence of a basement membrane, synovial fluid does not mix freely with extra-cellular synovial tissue fluid.Hyaluronan may trap molecules within the synovial cavity by acting as a filtration screen on the surface of the synovial lining, resisting the movement of synovial fluid out from the joint space. Synovial fluid proteins have a rapid turnover time (around 1 hour in normal knees), and equilibrium is not usually reached among all parts of the joint. However, the turnover time for hyaluronan in the normal joint (13 hours) is an order of magnitude slower than that of small solutes and proteins, so association with hyaluronan may result in trapping of solutes within synovial fluid. Tissue fluid around fenestrated endothelium reflects plasma ultrafiltrate most closely, with a low content of hyaluronate compared with synovial fluid. Locally generated or released peptides, such as endothelin and substance P, may attain much higher perivascular concentrations than those measured in synovial fluid.
In normal joints, intra-articular pressures are slightly subatmospheric at rest (0 to −5 mm Hg). During exercise, hydrostatic pressure in the normal joint may decrease further. Repeated abnormal mechanical stresses can interrupt synovial perfusion during joint movement, particularly in the presence of a synovial effusion. Resting intra-articular pressures in rheumatoid joints are around 20 mm Hg, whereas during isometric exercise, they may increase to greater than 100 mm Hg, well above capillary perfusion pressure and, at times, above arterial pressure.
Synovial Fluid as an Indicator of Joint Function
In the absence of a basement membrane separating synovium or cartilage from synovial fluid, measurements of synovial fluid may reflect the activity of these tissues. A wide range of regulatory factors and products of synoviocyte metabolism and cartilage breakdown may be generated locally within the joint, resulting in marked differences between the composition of synovial fluid and plasma ultrafiltrate. Because little capacity exists for the selective concentration of solutes in synovial fluid, solutes that are present at higher concentrations than in plasma are probably synthesized locally. It is necessary, however, to know the local clearance rate to determine whether the solutes present in synovial fluid at lower concentrations than in plasma are generated locally. Because clearance rates from synovial fluid may be slower than those from plasma, synovial fluid levels of drugs or urate may remain elevated after plasma levels have declined.
Plasma proteins are less effectively filtered in inflamed synovium, perhaps because of increased size of endothelial cell fenestrations or because interstitial hyaluronate-protein complexes are fragmented by enzymes associated with the inflammatory process. Concentrations of proteins, such as α2 macroglobulin (the principal proteinase inhibitor of plasma), fibrinogen, and IgM, are elevated in synovial fluid from patients with arthritis (see Fig. 1.6 ), as are associated protein-bound cations. Membrane peptidases may limit the diffusion of regulatory peptides from their sites of release into synovial fluid. In inflammatory arthritis, fibrin deposits may additionally retard flow between the tissue and the liquid phase.
