SECTION I Basic Concepts 1

Structure and Function

Alan M. Rosenberg, Ross E. Petty

Rheumatic diseases involve abnormalities of multiple organs and response to fluctuating mechanical loads, and as a normal physiolog- systems, but it is predominantly the musculoskeletal system that is ical process throughout life. It is continuously generating new blood affected. The musculoskeletal system consists of bones, , carti- cells and contributing to the maintenance of the body’s biochemical lage, tendons, , muscles, and associated connective tissues. and energy balance. This chapter is an overview of normal musculoskeletal biology. In other sections, pathology of the musculoskeletal system and other sys- Bone tems pertinent to rheumatic diseases, including immune, vascular, and Bone is a composite of organic and inorganic materials. The hardness neurological systems, are discussed in relevant contexts. of bone is a consequence of the interaction of organic constituents such as collagen, proteoglycans, and matrix proteins, which confer ten- THE SKELETON sile strength and permit flexibility to withstand stress, and inorganic materials such as hydroxylapatite and other calcium and phosphate The musculoskeletal system is derived from embryonic mesoderm, a salts, which account for bone’s compressive strength. layer that first appears at approximately the fourth week of gestation. As the embryonic neural crest closes to create the notochord, cells from Classification of Bones the dorsal margins migrate to form the teeth and some facial bones, Bones are categorized based on either their respective shapes and cartilage, and connective tissue of the anterior skull.1 Mesodermal cells attendant functions or the process by which they become ossified. arising from the prechordal region (located rostral to the notochord) Bone classification based on shape and function.All bones form cartilage and bone for the posterior skull, paraxial mesodermal share similar ultrastructural characteristics, but gross anatomical shapes cells (somites) generate the axial skeleton, and lateral plate mesoder- differ, reflecting adaptations to their respective functions. Long bones, mal cells form the appendicular skeleton.1 with a greater length than width, have a thick wall of cortical bone to As with all large organisms, the human endoskeleton provides a withstand and adapt to fluctuating stresses of weight-bearing and to rigid framework, allowing movement to occur with power and pre- allow for rotation and leverage. Short, cuboid-shaped bones, such as cision. The axial skeleton comprises the skull, mandible, auditory carpal bones, support and facilitate movement. Broad flat bones, such as ossicles, hyoid bone, vertebral column, sternum, and ribs, and the cranial bones, sternum, and ribs, are thin and curvilinear; they serve as appendicular skeleton includes the limbs and limb girdles. Ossification attachment sites for musculature and they shield vital organs. Irregular- of the fibrous membranes and hyaline cartilage that constitute the shaped bones protect the face and spinal cord. Sesamoid bones develop primordial skeletal scaffold begins at approximately the seventh gesta- as smooth, round ossifications to counteract compressive forces within tional week, and by birth it is almost, but not entirely, complete. tendons; patellae are the only sesamoid bones consistently present The skeleton is the framework that maintains body shape; sup- in humans, although the hallux sesamoid bone of the foot and the ports muscles and connective tissues, neurovascular networks, and pisiform of the wrist are usually present as well. skin; protects the body’s vital organs; and makes movement possible Bone classification based on ossification process.Bone is through articulations and muscle and tendon attachments. The skel- formed by either intramembranous or endochondral ossification. eton is the repository for hematopoietic and mesenchymal progeni- Intramembranous ossification occurs in facial, cranial, and clavicular tor cells, a source of energy derived from yellow marrow adipocytes, bones as mesenchymal cells within the unossified skeleton coalesce and a mediator of the immune response, and functions as an endocrine then differentiate into either capillaries or osteoblasts. The osteoblasts organ contributing to calcium, phosphate, and glucose homeosta- secrete unmineralized osteoid matrix into which mineral salts (notably sis.2-7 Further, the musculoskeletal system aids other bodily functions, calcium and phosphorus) are then deposited. When ossified, osteoid including digestion by mastication, respiration by moistening inspired produced around capillaries becomes honeycomb-like trabecular bone air passing through bone sinuses, and communication by way of audi- (also referred to as cancellous or spongy bone), which encroaches on the tory ossicles for hearing and the larynx for vocalization. vasculature, compacting it into red marrow. The skeleton is a dynamic organ both anatomically and physio- Endochondral ossification occurs in most appendicular and axial logically. It is modified structurally during growth and maturation, in skeleton bones. Endochondral ossification involves the progressive

1 2 SECTION I Basic Concepts

Circumferential lamellae

Concentric lamellae of osteon Osteon

Osteocytes Central (haversian) canal

Neurovascular bundle

Perforating (Volkmann’s) canal

Periosteum Compact bone Spongy bone Fig. 1.1 A depiction of the ultrastructural characteristics of bone. For clarity, 2 or 3 concentric lamellae are shown to represent the osteon; normally, the osteon is composed of 5 to 20 concentric lamellae. (Drawing by Alexandru Margarit and labeling by Alan Rosenberg. Printed with permission. All rights reserved.) replacement of the primordial cartilaginous scaffold with bone through as the bones of the leg, tend to be cylindrical with a thick collar of cor- a sequence in which chondrocytes proliferate, mature, and enlarge. By tical bone and a marrow cavity. 8 weeks gestational age some mesenchymal cells have differentiated Type I collagen constitutes 90% of bone matrix. The triple heli- into cartilage-producing chondrocytes that create the cartilaginous cal strands of collagen, when densely packed into parallel alignments skeletal framework. Building the skeletal scaffold begins as mesen- (lamellae), confer optimal tensile strength to accommodate mechani- chymal stem cells form condensations at the site of future bone. Cells cal loading. Other organic constituents of bone contribute to regulat- within the condensations do not differentiate directly into bone-pro- ing mineralization, growth, and energy metabolism. ducing osteoblasts; instead, they become proliferating chondrocytes, The circular osteon is the fundamental physiological unit of most which synthesize the extracellular matrix comprising predominantly mammalian compact bone (Fig. 1.1). The microstructure of the osteon type II collagen, some type IX and XI collagens, and proteoglycans. is characterized by 5 to 20 concentric layers of compact (cortical) bone Then, as mature, hypertrophied chondrocytes, they secrete a matrix that encircle a central haversian canal (first identified by 17th-century rich in type X collagen, which provides the supporting framework for English physician Clopton Havers), through which neurovascular bun- endochondral ossification. dles traverse to supply the adjacent osteocytes (Fig. 1.1). Osteons, mea- As the cartilage matrix ossifies, blood flow to the chondrocytes is suring a few millimeters in length and less than 0.25 mm in diameter, impeded, the cells die, and adjacent cartilage breaks down, leaving a tend to orient to the long axis of the bone. Osteons are characteristically medullary cavity into which new vessels invade to deliver bone-pro- present in mature bone that has undergone new bone growth, remodel- ducing osteoblasts. While ossification continues from the primary ing, or repair. Osteons and their associated intra-haversian blood vessels ossification center in the diaphysis, cartilage continues to be produced communicate by interconnecting transverse Volkmann canals (named at the ends of bones as the epiphyses. At birth, cartilage remains at the for 19th-century German physiologist Alfred Volkmann). articular surface and the growth plate (the physis) situated between the Deeper layers of bone are made of spongy bone, the normal physio- epiphysis and the diaphysis. A mature long bone includes the epiphysis logical consequence of bone breakdown induced by osteoclasts. at the end of the bone, the diaphysis (the shaft of the bone), and the interposing metaphysis, which represents the region of the previous Bone Cells cartilaginous growth plate. Apophyses, such as the tibial tuberosity, Bone contains four cell types: mesenchymal stem cells, osteoblasts, posterior calcaneus, greater and lesser trochanters, and iliac crests, like osteocytes, and osteoclasts. Mesenchymal stem cells, located in the epiphyses, are the sites of new bone formation but do not contribute to intertrabecular loose connective tissue adjacent to vascular channels bone length; instead, they generate new bone in response to tendinous and in the periosteum, have the potential to differentiate into cells of or ligamentous traction. bone (osteoblasts, osteocytes, and osteoclasts), cartilage (chondro- cytes), muscle (myocytes), or fat (adipocytes). The Morphology and Physiology of Bone The differentiation of mesenchymal stem cells into osteoclasts is Bone includes an outer layer of thick, compact, cortical bone and an regulated by a wide array of cytokines, growth factors, and hormones inner layer of interlacing ossified struts (trabeculae). Alignment of the that, respectively, promote or inhibit osteoclastogenesis (Fig. 1.2).8-11 trabecular network with the long axis of the bone confers maximal Groups of osteoblasts, which assemble into clusters to form the osteon, tensile strength and compressive tolerance so that less bone mass is produce the organic constituents of bone, mainly collagen and to a required. Bones subjected to predominantly tensile and compressive lesser extent osteocalcin and osteopontin. Osteocalcin is a hormone forces, such as vertebrae, have a thin cortex of compact bone; they that acts on pancreatic beta cells to promote insulin production in exert their support by their shapes and trabecular architecture. Bones response to leptin and can activate bone-based adipocytes to regulate that are required to tolerate torsion, bending, and shearing forces, such insulin responsiveness.12 Thus, there is an interaction between bone CHAPTER 1 Structure and Function 3

Fig. 1.2 A simplified illustration of activation of molecular signaling cascades by soluble factors in the bone marrow that regulate mesenchymal stem cell differentiation into bone-producing cells. Cytokines, growth factors, and hormones within the bone marrow microenvironment (BMME) bind to their respective cognate receptors on the mesenchymal stem cell plasma membrane, initiating activation of signaling pathways that promote gene expression for differentiation of mesenchymal stem cells into cells that regulate osteoclas- togenesis. More detailed discussion of soluble factors in the bone marrow microenvironment that regu- late osteoclastogenesis is provided by Amarasekara et al.8 and Sobacchi et al.9 cAMP, Cyclic adenosine monophosphate; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GH, growth hor- mone; IGF-1, insulin-like growth factor 1; IL, interleukin; JAK2, Janus kinase 2; MAPK, mitogen-activated protein kinase; MSC, mesenchymal stem cell; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor κB; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKB, protein kinase B; PKCα, protein kinase C alpha; PLCγ, phospholipase C gamma; PTH, parathyroid hormone; p38, protein; RANKL, receptor activator of NF-κB ligand; SMAD, small mothers against decapentaplegic; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor-beta; TNF, tumor necrosis factor; VEGF, vascular endothe- lial growth factor.

development, fat cell function, and insulin production. Osteopontin surfaces of bone, such as the epiphyses. Osteoclasts also participate participates in bone remodeling by securing osteoclasts to the inor- in the conversion of cancellous bone into lamellar (compact) bone ganic (mineral) matrix of bone.13 by creating spaces in which osteons can develop. Osteoclasts degrade After becoming entrapped in fully formed bone, osteoblasts become bone by secreting lysosomal enzymes, resulting in calcium release to osteocytes within intramatrix cavities (lacunae). The mechanosensory maintain mineral homeostasis. osteocytes begin mineralizing hydroxylapatite, the bone’s organic matrix. Osteocytes are interconnected by cytoplasmic tendrils that traverse a net- Bone Growth work of canaliculi through which nutrients and metabolites are delivered After birth, longitudinal bone growth occurs from the epiphyseal to ensure osteocyte viability. Osteocytes, the most abundant bone cells, plate, which has four zones: reserve (resting), young proliferating car- maintain the physiological balance between bone formation by osteo- tilage, maturing cartilage with hypertrophic chondrocytes, and calci- blasts and bone resorption by osteoclasts, thus preserving bone integrity fying matrix (Fig. 1.3). The epiphyseal side of the plate is retained as and mediating bone remodeling in response to mechanical stresses. articular cartilage, whereas on the diaphyseal side, the epiphysis ossifies Osteoclasts mediate resorption at the inner surfaces of bone, such to increase bone length. The dense cortex of long bones is enveloped by as the marrow cavity and cancellous bone trabeculae, and the outer an outer layer of periosteum and an inner layer of endosteum. 4 SECTION I Basic Concepts

Fig. 1.3 Histology of the epiphysis. Cells at the top, the epiphyseal side, undergo rapid mitosis to increase the thickness of the epiphyseal plate resulting in increased bone length. In the hypertrophic zone, matrix is deposited and, as matrix accumulates, cells in the vicinity die. (The histological image, to which radiograph and labeling have been added, is attributed to the copyright holder, Springer Publishing, and appeared in Horvai A. Anatomy and histology of cartilage In TM Links, Editor, Cartilage Imaging, Springer, 2011, Figure 1-8. Reprinted with permission.)

The stages of skeletal maturation are reflected by the radiographic differentiate into chondroprogenitor cells.20 In this way, a continuous appearance of secondary ossification centers in long bones and physeal supply of chondrocytes is available during growth. closure. The Tanner-Whitehouse14,15 and Greulich-Pyle16 protocols Bone growth is accompanied by coincident angiogenesis, a process remain the conventional methods for assessing skeletal maturation regulated by bone cells.21,22 Osteocytes, which have dendritic processes (bone age) using left hand and wrist radiographs, although newer connected to blood vessels, induce angiogenesis by the vascular endo- assessment methods have been considered.17 Bone growth at the phy- thelial growth factor and mitogen-activated phosphorylated kinase sis is governed by thyroid, growth, and sex hormones. Peak bone mass signaling pathways.21 development during adolescence is determined by a variety of factors, Growth factors are delivered to the physis through three routes of including diet, physical activity, and synergistic effects of growth hor- arterial supply: nutrient arteries, a metaphyseal-epiphyseal network, mone and insulin-like growth factor. During the most rapid phases and a periosteal system (Fig. 1.4). In the resting state, bones receive of adolescent growth, testosterone promotes cell division resulting in approximately 10% to 15% of the cardiac output.23 Pathological states, widening of the physis, and estrogen promotes calcification and clo- such as inflammation, are associated with increased blood flow sure of the physis.18,19 Completion of ossification of the iliac apophyses and acceleration of bone growth. is temporally related to cessation of the appendicular skeleton growth, The nutrient blood vessels (together with nerves) penetrate the cor- although vertebral height may continue to increase and contribute to tex through nutrient foramina, enter the medullary cavity, and then increasing height until the third decade of life. branch into ascending and descending arterioles that travel through The endochondral ossification that leads to longitudinal bone the haversian system to supply blood at high-pressure flow to the inner growth during childhood and adolescence requires the continuous two-thirds of mature bone (Fig. 1.4). The metaphyseal-epiphyseal vas- availability of chondrocytes at the growth plate. During fetal and early cular supply is derived from arteries arising from the periarticular vas- neonatal periods, chondroprogenitor cells are progressively depleted. cular plexus that include metaphyseal, perichondral, and epiphyseal In contrast, as secondary ossification centers develop later in the bone arteries. The periosteal vascular flow is a low-pressure system supply- growth process, chondrocytes in the physis begin to acquire charac- ing the outer one-third of bone through the haversian and Volkmann teristics of stem cells and, under the influence of two signaling path- canals. Evidence indicates that an elaborate and dense transcortical ways (hedgehog and mammalian target of rapamycin complex 1), capillary network is the terminal pathway for blood delivery to bone.24 CHAPTER 1 Structure and Function 5

Fig. 1.4 Distribution of blood supply in growing bone. (Image design concept inspired by GJ Tortora and B Derrickson, Principles of Anatomy & Physiology, 14th Edition, John Wiley and Sons, page 175, Figure 6-4. Image drawn by Alexandru Margarit and labeled by Alan Rosenberg and reprinted with permission. All rights reserved.)

Circumferential growth, an increase in bone width, results from differentiation and activity of osteoclasts. Wnt/β-catenin signaling appositional subperiosteal membranous ossification on the bone prods mesenchymal stem cells to differentiate into osteoblasts, chon- surface. drocytes, or osteocytes. The Wnt/β-catenin pathway enhances bone mass by promoting stem cell renewal and osteoblastogenesis, suppress- Bone Remodeling ing osteoblast and osteocyte apoptosis, and sensing mechanical load- Maintaining the integrity of mature bone, repairing damaged bone, ing. Bone remodeling also occurs in response to endocrine influences and releasing calcium from bone are mediated by a shared process such as thyroid, growth, and sex hormones. called bone remodeling. Bone remodeling, which occurs continuously throughout life, is more rapid during childhood and adolescence. The Bone and the Immune and Inflammatory Systems regulation of bone modeling during development and subsequent New knowledge about the important connections between the skeletal bone remodeling that occurs after birth requires direct interactions and immune systems has fostered osteoimmunology as a research and between osteoblasts and osteoclasts, endocrine and neurological con- clinical field.3,27-30 Bone and immune and inflammatory systems are trols, and innate immune and inflammatory regulators. integrally related in both physiological and pathological circumstances. The metabolic events that initiate bone remodeling are unclear, but Immune and inflammatory cells and mediators arise from pro- the death of osteocytes is likely to be an early event in the repair/remod- genitor cells in bone marrow. As an example, the receptor activator of eling cascade. After osteocytes die, osteoclasts degrade bone matrix, the nuclear factor (NF)-κB ligand (RANKL), a member of the tumor forming lacunae into which mesenchymal cells migrate. Intralacunae necrosis factor (TNF) superfamily derived from osteocytes, primor- mesenchymal cells differentiate into osteoblasts resulting in new bone dial osteoblasts, and T cells, is a cytokine that links bone with immune matrix formation and subsequent mineralization. Depending on bone response.31 RANKL mediates osteoclast differentiation by transmitting size and patient age, this remodeling process might take up to 6 months. its signal through its cognate receptor, RANK. RANK and RANKL gene Bone remodeling occurs continually even in adults; it is estimated that the knockout mice, having no osteoclasts, have a phenotype characterized entire healthy adult skeleton is remodeled several-fold during a lifetime. by severe osteopetrosis and immune deficiency associated with absent The controlled balance between bone-forming osteoblasts and lymph nodes.32-35 RANKL is expressed by osteocytes and hypertrophic bone-resorbing osteoclasts is fundamental to sustaining bone health. chondrocytes in response to factors that promote bone production, Intercellular signaling by the wingless-related integrated site (Wnt) including vitamin D, parathyroid hormone, and prostaglandins. The family of glycoproteins regulates the transcription cofactor β-cat- binding of a RANK receptor on osteocytes to its ligand RANKL on enin, which is required for signaling the expression of genes involved osteoblasts is a key inducer of bone resorption. Osteoprotegerin, pro- in development.25,26 The Wnt/β-catenin signaling pathway (referred duced by osteoblasts, inhibits osteoclastogenesis by serving as a decoy to as the canonical Wnt signaling pathway) is crucial for growth and receptor for RANKL and thus inhibits bone resorption. In addition development of a wide array of tissues, including bone. Wnt signaling to its role in bone metabolism, RANKL/RANK also participate in is integral for skeletal formation and embryonic limb development, immune activation, immune tolerance, and development of thymus chondrogenesis, production of bone matrix by osteoblasts, and the and lymph node tissues.33,36 6 SECTION I Basic Concepts

Fig. 1.5 Categories and examples of diarthrodial joints. (Skeletal image drawings by Alexandru Margarit and reprinted with permission. The composite figure is reprinted with permission of Alan Rosenberg and Alexan- dru Margarit. All rights reserved.)

The processes of bone turnover and inflammation are regulated flat or slightly curvilinear. Bony projections and ligaments limit move- by shared mediators, including cytokines and transcription factors.3,37 ment in this type of joint. Carpal bones (except the capitate articulating Bone marrow–derived T lymphocytes are integral to regulating bone with the lunate and scaphoid), tarsal joints (except the talus and the remodeling and the response of bone cells to parathyroid hormone.37 navicular), acromioclavicular joints, and spinal facet joints are exam- Osteoclastic-mediated bone resorption is generated by T cell–derived ples of plane diarthrodial joints. cytokines.8 T cells also promote bone formation by direct interaction Spheroidal (ball-and-socket) joints: A spheroidal articulation is one with bone cells in part by activating Wnt signaling in the osteoblast in which a proximal cup-like indentation of one bone accepts the cell lineage. T helper 17 (Th17) cells stimulate bone resorption by round, convex surface of the other. These joints have a wide range of promoting osteoclast differentiation.38 Th17 cells promote osteoclas- movements, including flexion/extension, adduction/abduction, rota- togenesis in rheumatic diseases by elaborating TNF, interleukin (IL)-1, tion, and circumduction. The and shoulder are spheroidal joints. IL-6, IL-17, and RANKL. IL-17 stimulates the release of RANKL by Cotylic (ellipsoid; cotyloid) joints: The configuration of cotylic joints osteoblasts and osteocytes and upregulates RANK expression on osteo- resembles spheroidal joints, but the articulating surfaces are more cytes. By producing antiinflammatory cytokines IL-4, IL-10, and trans- elliptical and, as a consequence, have a more limited range of motion forming growth factor-beta (TGF-β), T regulatory cells (Tregs) inhibit as they lack rotation in the axial plane. The convex surface of the coty- osteoclastogenesis and thus promote bone formation. lic joint has an ovoid shape that articulates with an elliptical concavity. Cotylic joints may be simple, where there is only one articulating sur- Joints face, or compound, where there is more than one articulating surface. Joint Classifications The metacarpal-phalangeal joint is a simple cotylic joint and the radio- Joints and articulations, which represent the anatomical structures by carpal joint, a compound cotylic joint. which bones interface, can be assigned to one of three broad catego- Hinge joints: Hinge joints have movement restricted to a single, ries: (1) synarthroses, (2) amphiarthroses, or (3) diarthrodial joints. flexion-extension plane and are reinforced by collateral ligaments that Synarthroses (as in the skull) are joints in which there is no movement; prevent lateral/medial movements. The humeroulnar and interphalan- the related bones are in direct contact and are held together by inter- geal joints are hinge joints. locking fibrous seams. Amphiarthroses are partially moveable joints in Condylar joints: Condylar joints are characterized by two bones which bones are separated by fibrocartilage discs, as in the interverte- articulating with each other by two separate articulating surfaces. bral articulations, or by interosseus fibrous tissue, as in the distal tibio- Although condylar joints predominantly move in flexion and exten- fibular articulation.Diarthrodial joints are freely movable, facilitated sion, their configuration allows for some degree of rotational move- by smooth cartilage at the ends, lined with , have ment. There are two condylar joints in the body: the knee, which is an interosseus fluid-filled cavity, and are encased in a fibrous capsule. enclosed in a single joint space, and the temporomandibular joint in Diarthrodial joints can be classified further into seven subcategories: which the right and left articulating surfaces are separated widely. In plane, spheroidal (ball-and-socket), cotylic (ellipsoid, cotyloid), hinge, the clinical context the two sides of the temporomandibular joint are condylar, trochoid (pivot), and sellar (saddle) (Fig. 1.5).39 considered as separate joints; however, the fact that one side cannot Plane joints: Plane joints involve gliding one bone over another function independently of the other indicates that the joint is, strictly without any angular or rotary movement. The articulating surfaces are speaking, a single condylar joint. CHAPTER 1 Structure and Function 7

Trochoid (pivot) joints: Trochoid joints have movement limited Vasculogenesis within the synovium begins during embryonic to rotation around one axis. The mobile bone rotates within a ring development. Normal synovial blood flow is governed locally by formed in the adjacent bone by a concavity and associated ligaments. autocrine and paracrine influences and extrinsically by neuronal and The atlantoaxial and proximal and distal radioulnar joints are exam- humoral factors. With inflammation, there is accelerated angiogenesis ples of this type of articulation. and increased blood flow to the synovium. The inflamed synovial tis- Sellar (saddle) joints: Sellar joints are distinguished by each bone sue is proportionally more proliferative than the vasculature, resulting of the articulation having convex and concave surfaces that recipro- in a relatively smaller vascular network and consequent synovial mem- cally interlock. Like cotylic joints, they have a wide array of movements brane hypoxia. Intraarticular hypoxia results in a switch in synovial except for axial rotation. The first carpometacarpal joint (the articula- cells from a resting metabolic state to a markedly increased metabolic tion between the first metacarpal of the thumb and the trapezium) and state to ensure cell survival. As a result, metabolic intermediates accu- the sternoclavicular joints are sellar joints. mulate and promote hypoxia-induced, proinflammatory signaling pathways.43 Diarthrodial Joint Structures The synovial membrane microvasculature is linked to the vascu- Development of diarthrodial joints. Embryonic development of lature of the periarticular bone and periosteum. The microvascular synovial joints is regulated by genes and signaling networks that promote network includes arterioles and capillaries in the intimal region and cartilage condensation (growth differentiation factor-5), cavitation venules and lymphatics in the subintima. The dense neural network (WNT9A), and tissue differentiation.40 The diarthrodial joint anlage follows blood vessels into the synovial lining. These nerve fibers tend to comprises a three-layered interzone. The dense chondrogenic middle be slow conducting fibers resulting in sensations of aching and burning zone is the site of cavitation, the future joint space, which occurs at 8 in inflammatory states. weeks gestational age.41 The various joint components, including joint .Synovial fluid, ordinarily clear, transparent, capsule, synovial membrane, menisci, and intraarticular ligaments and pale yellow, and viscous, is a plasma transudate derived from tendons, are derived from interzone mesenchymal cells. the subintimal capillary network and serves to nourish mature Anatomy of diarthrodial joints. Diarthrodial joints are enveloped avascular cartilage and lubricate, protect, and regulate temperature by a capsule of dense fibrous tissue that can be reinforced by adjacent within joints. Hyaluronic acid in the fluid accounts for normal ligaments and tendons. The capsule, which attaches to bones of synovial fluid’s high viscosity. Hyaluronic acid appears to work diarthrodial joints by fibrocartilaginous insertions, provides stability synergistically with lubricin, a synovial fluid glycoprotein, to to the joint, limits joint mobility, and contains the intraarticular fluid. lubricate the articulating surfaces.44,45 The synovial fluid also serves The synovium attaches to the cartilage-bone junction. In healthy as a shock-absorbing cushion interposed between articulating joints the synovium does not impinge on the articular surface; when bones. Some synovial fluid coats the cartilage surface and some is inflamed, the synovial membrane may proliferate and encroach on the absorbed into cartilage; when the cartilage is compressed, fluid is articular surface as inflammatory pannus. expressed to reduce friction. The small amount of synovial fluid in Synovium. The synovial membrane (synovium), a tissue of a healthy joint reduces compressive shock by dispersing pressure ectodermal origin, lines the inner surface of the diarthrodial joint evenly across the articular surface. capsule and intraarticular ligaments, tendons, and fat pads but not The attributes of synovial fluid quantity, color, clarity, and viscosity articular cartilage or menisci. The synovial membrane is configured can be easily assessed at the bedside to help distinguish normal from with pouches and folds which, together with its inherent deformability inflammatory, septic, and hemorrhagic synovial fluid. The volume of and property of nonadherence to adjacent tissues, allows unhindered synovial fluid in a healthy joint is small (<3.5 mL in an adult knee). movement. It is the source of synovial fluid that nourishes and The fluid is clear, colorless, or pale yellow; it has a high viscosity, a lubricates the joint cartilage. neutral pH (7.4), a protein concentration of 1.7 to 2.1 g/dL, and a glu- There are two synovial membrane layers: the inner intima and cose level that is within approximately 10 mg/dL of the serum level.46-50 the outer subintima. The intima, composed of two to three layers of There are no red blood cells and fewer than 200/mm3 white blood cells. a mixture of macrophage and fibroblast cells, is interposed between Ingress of larger molecules, such as complement, is limited. Synovial the joint cavity and the subintima to which it is loosely attached. fluid is devoid of elements of the coagulation pathway, and, conse- There is no basement membrane delineating the thin intima from quently, normal synovial fluid does not clot.51 the subintima; rather, the intima rests loosely on intermingling col- Articular cartilage. Normal cartilage on the articular surface of lagen fibrils, vascular and lymphatic vessels, and nerves of the sub- diarthrodial joints has a glassy, translucent, pearl-white appearance, intima with a film of hyaluronic acid interposed between the two giving rise to the term hyaline cartilage (from the Latin hyalinus layers. This loose network accounts for the synovial lining’s physi- and Greek hyalinos meaning of glass or crystal). In addition to ological permeability. Normal synovial lining is also replete with a providing structural form, hyaline cartilage minimizes friction within wide variety of adhesion molecules, vasculature, and cells. Adhesion articulations and, because of its inherent compressibility, protects molecules help ensure that crucial components are not lost during from mechanical loads. The cartilage margins blend with the synovial compression and shearing movements. Adhesion molecules also are membrane and with metaphyseal periosteum. Hyaline cartilage involved in the recruitment of inflammatory cells under patholog- includes chondrocytes, which become progressively less abundant with ical states. advancing age, within an extracellular matrix that is predominantly Synovial lining cells within the synovial intimal layer can be type A type II collagen. The cell volume of adult cartilage is less than 2%.52 macrophage-like synoviocytes or type B fibroblast-like synoviocytes. During cartilage development and fetal and postnatal growth, cartilage Synovial membrane macrophages scavenge intraarticular debris has abundant metaphyseal and epiphyseal vascularity. In children, the through phagocytosis and participate in antigen-mediated immune epiphyseal cartilage is vascular until approximately 18 months of age responses. Synovial membrane fibroblasts produce the main joint after which transphyseal vessels regress, leaving remnant epiphyseal lubricants, lubricin and hyaluronic acid,42 as well as cytokines, pros- vascular canals. These canals can increase in size with advancing age and taglandins, collagen, proteoglycans, and metalloproteinases and their can become more conspicuous in the presence of inflammation.53,54 cognate inhibitors. Articular cartilage is aneural. 8 SECTION I Basic Concepts

second stage is the modification of procollagen in the Golgi apparatus and its packaging into secretory vesicles. The third stage is the forma- tion of the collagen molecule in the extracellular space by cleavage of the procollagen. The final step is the cross-linking between the collagen molecules to stabilize the supramolecular collagen structure. Collagen is distinguished from other proteins by its high content of proline (Pro), hydroxyproline (Hyp), and glycine (Gly), which occu- pies every third amino acid position. Most of the proline in collagen is present in the sequence Gly-Pro-Xaa, where Xaa (an unspecified amino acid) is frequently alanine or hydroxyproline. The stereochem- ical constraints imposed by proline and hydroxyproline result in the repeating tripeptide sequences to form the triple-helical configuration. Glycine is essential for ensuring the stability of the triple-helical con- formation. Tropocollagen, the basic unit of collagen, is a triple-heli- cal structure. The three polypeptide chains (except for the amino and carboxy termini) form a left-handed helix, while the tropocollagen superhelix coils to the right. Tropocollagen assembles into collagen fibrils, which are then bundled (as in tendon and bone), stacked (as in cornea), or loosely connected (as in interstitial tissue) to form the Fig. 1.6 Histological section of normal articular cartilage stained with hematoxylin and eosin. Healthy articular cartilage includes three zones: structural fibrous tissue within respective organs. 56 superficial, intermediate, and deep zones. The tidemark demarcates Although there are an estimated 28 different collagen types, type the deep zone from the calcified zone and the underlying subchondral I collagen accounts for more than 90% of all collagen. Type II collagen bone. (The histological image, to which labeling has been added, is has the most restricted tissue distribution, being confined to hyaline reproduced with permission of Dr. Andrew Horvai.) cartilage, intervertebral discs, and the vitreous of the eye. Collagen and cartilage proteases: Proteinases, endopeptidase enzymes Healthy articular cartilage is mostly water (70%). Type II collagen that cleave by hydrolyzing peptide bonds in the middle of the molecule, and aggrecan, an aggregate of proteoglycans, account for most of the are integral to ensuring bone and cartilage homeostasis.57 Endopeptidases dry weight of cartilage; other collagen types and unaggregated proteo- are classified based on the chemical group that is targeted for hydrolysis. glycans contribute minimally to cartilage volume. Endopeptidases that act extracellularly in a neutral pH are serine and Macroscopically, hyaline cartilage appears as a homogenous tissue. metalloproteinases, and those acting intracellularly in an acid pH are Microscopically, cartilage has four discrete layers (Fig. 1.6). The most aspartate, cysteine, and threonine proteinases. Extracellular homeostasis superficial region (the gliding or tangential zone) is characterized by is regulated partly by an array of proteolytic enzymes that account for flattened chondrocytes, fine collagen fibrils arranged in a tangential continual physiological bone remodeling or degradation in pathological array, a high concentration of small molecular weight proteoglycans states such as inflammation. Matrix metalloproteinases (and their respec- (decorin),55 and a low concentration of aggrecan. The middle (tran- tive inhibitors, tissue inhibitors of matrix metalloproteinases) are clas- sitional) zone, accounting for half the cartilage mass, includes round sified into five broad categories (collagenases, gelatinases, stromelysins, chondrocytes surrounded by thick strands of collagen fibers arranged matrilysins, and membrane type) that relate to the substrate they target, radially. In the deep zone, chondrocytes are aligned in columns. The their structure, and their location within the cell.58 number of chondrocytes gradually decreases from the superficial to Proteoglycans: Proteoglycans are glycosylated proteins to which a the deeper layers, but the volume of each cell progressively increases multitude of anionic glycosaminoglycans chains (e.g., chondroitin through to the deeper zones. Water content progressively decreases sulfate, keratan sulfate, and heparan sulfate) are bound. Proteoglycans from the superficial to deeper zones. Collagen is the most abundant may link to each other to form aggrecan, or to hyaluronic acid or other and proteoglycan content is the least abundant in the superficial zone; matrix proteins such as collagen. Hydrodynamics is the predominant proteoglycans become proportionally more abundant in the deeper physiological function of proteoglycans as cationic water molecules zones accounting for 50% of dry weight. As bone growth finishes, a bind to the negatively charged glycosaminoglycan sulfates. Aggregates calcified zone, formed by endochondral ossification, persists to pro- of proteoglycans, which provide resistance to compression, are distrib- vide a protective buffer between the soft uncalcified articular cartilage uted among the networks of collagen fibers and confer tensile strength and the underlying subchondral bone. to the articular cartilage. The water molecules bound to the hydrophilic At some sites, such as the pubic , the annulus fibrosus of proteoglycans within cartilage provide resistance to deformation on the intervertebral discs, the triangular fibrocartilage of the wrist, and compression. As cartilage is compressed, water is extruded but quickly the temporomandibular joint, fibrocartilage rather than hyaline carti- reabsorbed, allowing the cartilage to regain is precompressive form. lage is interposed between bones of the respective joints. An essential biological role of heparan sulfate proteoglycan in the Extracellular matrix. extracellular matrix is to bind an array of cytokines to regulate their Collagen. Collagen (from the Greek kόlla for glue and gen for availability and mobility. The relative abundance of various proteo- producing), the dominant constituent of connective tissue, is the most glycans varies with age; with advancing age, proteoglycan aggregates abundant and ubiquitously distributed extracellular structural protein increase, keratin sulfate increases, and chondroitin sulfate decreases. in the body, accounting for approximately one-third of the human body’s total protein content. TENDONS After transcription and translation of the procollagen α chains, four distinct stages occur for the assembly of collagen fibrils. The first stage Tendons provide the anatomical connection between bone and muscle. is the transportation of the α chains into the rough endoplasmic retic- The tendon allows a large mass of muscle to focus its force on a small ulum where they are modified to form triple-helical procollagen. The area of bone, or if the tendon is split, to distribute the force of the CHAPTER 1 Structure and Function 9

zone appears as a tidemark demarcating the uncalcified from calci- fied fibrocartilage; the tidemark zone serves as a mechanical interface between soft and hard tissue. The third zone, calcified fibrocartilage, comprises mostly type II collagen with some aggrecan, types I and X collagen, and chondrocytes. The fourth zone is bone. A unique T cell lineage within entheseal sites contributes to the patho- genesis of enthesitis. IL-23, produced in response to Th17 cells, is an essential cytokine in the development of inflammation at entheses, acting on the resident T cells to promote IL-17–mediated and IL-22–mediated inflammation and bone remodeling at the enthesopathic site.61,62

BURSAE Thin-walled bursal sacs, which either appear during embryonic devel- Fig. 1.7 Histological section, stained with Masson trichrome, showing opment contemporaneously with synovial membranes or develop a normal . The red vertical lines on the left are collagen tendon postnatally, have ultrastructure resembling synovial tissue but are fibers. The blue color in the middle of the image is fibrocartilage. The less vascular. By cushioning and facilitating gliding between points of red band on the right is the transition zone (tidemark) from fibrocarti- pressure, bursae prevent damage that would otherwise be incurred by lage at the attachment site to bone. The white on the far right is fat the rubbing of one tissue against another. Most bursae in the human within the bone cavity. (Image reproduced from http://www.enthesis. body are in the vicinity of large joints such as the shoulder, hip, and info/anatomy/enthesis_insertion.html with permission from Dr. Dennis knee. The location of bursae can be subfascial, subligamentous, sub- G. McGonagle.) tendinous, or submuscular. Bursae may increase in size, or new bursal sacs (adventitious bursa) may develop in response to inflammation or trauma. In the presence of intraarticular inflammation, communica- muscle to multiple bones. The tendon comprises predominantly type tion between deep bursae and the joint space may develop. As exam- I collagen (85% of dry weight) and small amounts of type III collagen. ples, the lateral subtendinous bursa of the gastrocnemius muscle can Tendon fibers are orientated longitudinally along the lines of stress communicate with the knee joint, the with the gle- within a proteoglycan, elastin, neurovascular, and fibroblast matrix. nohumeral joint, and the iliopsoas bursa with the hip joint. Superficial Decorin, the most abundant proteoglycan in the tendon, establishes bursae, such as the olecranon bursa and the , are less cross-linking between collagen fibers to facilitate the transfer of loads likely to form a conduit to the respective joint spaces. among fibers. The hierarchical tendon structure includes microfibrils, subfibrils, fibrils, fascicles, and the main tendon unit. The movement of SKELETAL MUSCLE many tendons, particularly large ones, is facilitated by gliding through tubes comprising an inner synovial sheath and an outer fibrous sheath; As strong and functional as the skeleton is, its operations would be mesenchymal cells from the synovial sheath produce hyaluronic acid impossible without the support and activities of muscles. The more that promotes lubrication to facilitate movement. Fibroblasts within than 600 human muscles account for approximately 40% of the body’s the tendons produce collagens, proteoglycans, and matrix metallopro- mass. During the third gestational week, the paraxial mesoderm under- teinases and their inhibitors that mediate tendon damage and repair. goes somitogenesis, yielding spherical somites adjacent to each side of the neuronal groove. Skeletal muscle forms from these somites and LIGAMENTS AND FASCIA mononucleated myoblasts differentiate into multinucleated myotubes. Entire muscles are surrounded and secured by the epimysium, a Ligaments and fascia provide connections between bones, conferring sheath of connective tissue (Fig. 1.8). Within each muscle are bun- stability and constraining movement. Although resembling tendons dles of muscle fibers (fascicles) that function in concert. Fascicles are structurally, ligaments have more elastin and type I collagen fibers. cloaked with a connective sheath, the perimysium. Within each fasci- Ligaments are longitudinally orientated, as in tendons, but tend to be cle, individual muscle fibers are each surrounded by a loose connec- crimped, allowing for the ability to stretch and recoil. tive tissue, the endomysium, through which terminal nerve fibers and capillaries pass. The perimysium and endomysium blend together as ENTHESES the muscle fibers merge with tendons, thus helping to reinforce the attachment of muscle to tendon. Muscle fibers each contain a nucleus, The attachment of tendons and ligaments to bone occurs at the enthe- a plasma membrane (the sarcolemma), and cytoplasm (sarcoplasm). sis (from the Greek insertion), a distinct, specialized interface where Inside each muscle cell are myofibrils, protein bundles comprising tendon and fibers gradually blend into bone tissue.59,60 The myofilamentous fibers (sarcomeres). entheseal connection can take the form of either a dense fibrous con- nective tissue or, more commonly, fibrocartilage. Fibrous entheses Classification of Skeletal Muscles Based on Shape are characterized by mineralized collagen fibers inserting directly into The variety of shapes and configurations of skeletal muscles reflects bone or periosteal tissue, entering into a broad section of the diaphysis their respective mechanical actions. Fusiform muscles, such as the (e.g., the deltoid insertion into the humerus). Fibrocartilage entheses biceps brachii, are centrally thick with tapered ends and have muscle are characterized by a four-zone gradient, transitioning from uncalci- fibers aligned in parallel along the long axis. Parallel muscles, such as fied tendon to bone (Fig. 1.7). The tendon/ligament, comprising types the sartorius and sternocleidomastoid, also have muscle fibers aligned I and III collagen fibers, elastin, proteoglycans, and fibroblasts, transi- in parallel. They are longer and less strong than fusiform muscles tions to zone two, an uncalcified fibrocartilaginous zone comprising but can endure relatively longer loading. Pennate (feather-like) mus- types I, II, and III collagens, aggrecan, and chondrocytes. This second cles have fascicles that are obliquely aligned to the orientation of the 10 SECTION I Basic Concepts

Fig. 1.9 Photomicrograph of normal muscle histology. Type I fibers are pale and type II fibers are dark.

absorbed by the myosin head (which is connected to the neck and tail portions of the myosin molecule) transmits energy at the site of bind- ing to actin. Actin activates the enzyme ATP triphosphatase (ATPase) in the myosin head, resulting in pivoting and bending of the myosin to pull the actin toward the sarcomere and in sarcomere shorting and muscle contraction. The heterogeneity of muscle function and biochemical character- istics is reflected in the classification of muscle types. Muscles can be categorized as type I (slow) fibers or type II (fast fibers) (Fig. 1.9). Type I fibers are narrower, have poorly defined myofibrils, are irregular in size, Fig. 1.8 Depiction of muscle ultrastructure. (Artwork by Alexandru Mar- have thick Z bands, and are rich in mitochondria and oxidative enzymes garit; labeling by Alan Rosenberg. Printed with permission. All rights reserved.) but poor in phosphorylases. Type II fibers have fewer mitochondria and are poor in oxidative enzymes but rich in phosphorylases and glycogen. Individual muscles differ in the proportion of each fiber type. Skeletal muscles also can be classified into three groups (I, IIA, IIX) tendon, thus allowing greater force but with a limited range of motion. based on the myosin heavy-chain isoform they contain. The rapidity Pennate muscles tend to have a high density of muscle fibers and are with which ATP hydrolyzes and hence the speed of muscle contraction therefore strong. Pennate muscles can insert into the tendon at one site depends on the myosin head isoform; a myosin head isoform that is (unipennate) such as the lumbricals or at two sites (bipennate) such associated with a slower ATP hydrolysis rate (e.g., isoform I) is asso- as the rectus femoris, or they can be multipennate such as the deltoid. ciated with slower muscle fiber contractions, whereas isoforms with Convergent muscles have an origin (usually proximal) that is wider faster hydrolysis (e.g., isoform IIX) allow for faster contractions. Most than the insertion such as pectoralis major. Circular muscles (sphinc- muscles contain mixtures of different myosin head isotypes in varying ter muscles) surround openings such as the orbicularis oris. proportions accounting for the heterogeneity of contraction character- The Function of Skeletal Muscle istics among respective muscles. Most often, skeletal muscle groups are situated in pairs around joints to exert antagonistic movements such as flexion and extension. KEY REFERENCES Muscles contract when stimulated by somatic motor neurons in the 1. Berendsen AD, Olsen BR. Bone development. Bone. 2015;80:14–18. brainstem and spinal cord. In response to nerve impulses, chemical 2. DiGirolamo DJ, Clemens TL, Kousteni S. The skeleton as an endocrine energy in the form of adenosine triphosphate (ATP) in the muscle, organ. Nat Rev Rheumatol. 2012;8(11):674–683. derived from aerobic oxidation of carbohydrates and fats and anaer- 3. Okamoto K, Takayanagi H. Osteoimmunology. Cold Spring Harb Perspect obic reactions, is converted to mechanical energy. Sarcomeres, the Med. 2019;9:a031245. pii. muscle’s functional units responsible for contraction, include thick, 4. Guntur AR, Rosen CJ. Bone as an endocrine organ. Endocr Pract. thin, and elastic myofilaments, and it is movement between thick and 2012;18(5):758–762. thin filaments that effects muscle contraction/relaxation Fig.( 1.8). 5. Li Q, Wu Y, Kang N. Marrow adipose tissue: its origin, function, Myosin molecules are the predominant component of thick fila- and regulation in bone remodeling and regeneration. Stem Cells Int. ments. Thin filaments have two molecules of actin; each actin mole- 2018;2018:7098456. cule is distributed as a string of beads with the beads representing the 6. Mori G, D’Amelio P, Faccio R, Brunetti G. The interplay between the bone and the immune system. Clin Dev Immunol. 2013;2013:720504. globular portion of the actin molecule. Each actin bead can bind to 8. Amarasekara DS, Yun H, Kim S, Lee N, Kim H, Rho J. Regulation of osteo- one myosin molecule. Elastin myofilaments course down the thick clast differentiation by cytokine networks. Immune Netw. 2018;18(1):e8. myofilament and extend out the ends to anchor to the noncontractile 9. Sobacchi C, Palagano E, Villa A, Menale C. Soluble factors on stage to Z discs, which serve as the site of attachment of elastic muscle fibers. direct mesenchymal stem cells fate. Front Bioeng Biotechnol. 2017;5:32. The region between each Z disc is the sarcomere, which is responsi- 11. Takada I, Kouzmenko AP, Kato S. Wnt and PPARgamma ­signaling in osteo- ble for the dark and light striations in skeletal muscle. ATP energy blastogenesis and adipogenesis. Nat Rev Rheumatol. 2009;5(8):442–447. CHAPTER 1 Structure and Function 11

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