CHAPTER 5 Functional anatomy of the musculoskeletal system

The skeletal system consists of the specialized supporting connective tissues of the bony skeleton and the associated tissues of joints, including cartilage. Cartilage is the fetal precursor tissue in the development of many bones; it also supports non-skeletal structures, as in the ear, larynx and tracheobronchial tree. Bone provides a rigid framework which protects and supports most of the soft tissues of the body and acts as a system of struts and levers which, through the action of attached skeletal muscles, permits movement of the body. Bones of the skeleton are connected with each other at joints which, according to their structure, allow varying degrees of movement. Some joints are stabilized by fibrous tissue connections between the articulating surfaces, while others are stabilized by tough but flexible ligaments. Skeletal muscles are attached to bone by strong flexible, but inextensible, tendons which insert into bone tissue. The entire assembly forms the musculoskeletal system; all its cells are related members of the connective tissue family and are derived from mesenchymal stem cells.

CARTILAGE

During early fetal life the human skeleton is mostly cartilaginous, but is subsequently largely replaced by bone. In adults, cartilage persists at the surfaces of synovial joints, in the walls of the larynx and epiglottis, trachea, bronchi, nose and external ears. Developmental replacement by bone is a complex process: cells in cartilaginous growth plates – which lie between ossifying epiphyses and the diaphyses of long bones (and elsewhere) – continue to proliferate, increasing the length of the bones concerned until they eventually ossify, when growth ceases.

MICROSTRUCTURE OF CARTILAGE

Cartilage is a type of load-bearing connective tissue. It has a low metabolic rate and its vascular supply is confined to its surface or to large cartilage canals. It has a capacity for continued and often rapid interstitial and appositional growth, and a high resistance to tension, compression and shearing, with some resilience and elasticity. Cartilage is covered by a fibrous perichondrium except at its junctions with bone and at synovial surfaces, which are lubricated by a secreted nutrient synovial fluid.

The cells of cartilage are chondroblasts and chondrocytes. Like connective tissues generally, the extracellular matrix is a dominant component and gives the tissue its distinguishing characteristics. The extracellular matrix of cartilage varies in appearance, composition and in the nature of its fibres in the different types of cartilage, namely, hyaline cartilage, white fibrocartilage and yellow elastic cartilage. A densely cellular cartilage, with thin septa of matrix between its cells, is typical of early embryonic cartilage. Hyaline cartilage is the prototypical form but it varies more in composition and properties according to age and location, than either elastic or fibrocartilage. Hyaline cartilage may become calcified as part of the normal process of bone development, or as an age-related, degenerative change.

Cartilage cells occupy small lacunae in the matrix which they secrete. Young cells (chondroblasts) are smaller, often flat and irregular in contour, and bear many surface processes, which fit into complementary recesses in the matrix. Newly generated chondroblasts often retain intercellular contacts, including gap junctions. These are lost when daughter cells are separated by the synthesis of new matrix. As cartilage cells mature, they lose the ability to divide and become metabolically less active. Some authors reserve the name chondrocytes for such cells, but this term is commonly employed, as it is here, to denote all cartilage cells embedded in matrix. Mature chondrocytes enlarge with age and become more rounded. The ultrastructure of chondrocytes is typical of cells which are active in making and secreting proteins. The nucleus is round or oval, euchromatic and possesses one or more nucleoli. The cytoplasm is filled with rough endoplasmic reticulum, transport vesicles and Golgi complexes, and contains many mitochondria and frequent lysosomes, together with numerous glycogen granules, intermediate filaments (vimentin) and pigment granules. When these cells mature to the relatively inactive chondrocyte stage, the nucleus becomes heterochromatic, the nucleolus smaller, and the protein synthetic machinery much reduced: the cells may also accumulate large lipid droplets.

Cartilage is often described as totally avascular. Most cartilage cells are usually distant from exchange vessels, which are mostly perichondrial, and so nutrient substances and metabolites diffuse along concentration gradients across the matrix between the perichondrial capillary network and chondrocytes. This limitation is reflected in the fact that most living cartilage tissue is restricted to a few millimetres in thickness. Cartilage cells situated further than this from a nutrient vessel do not survive, and their surrounding matrix typically becomes calcified. In the larger cartilages and during the rapid growth of some fetal cartilages, vascular cartilage canals penetrate the tissue at intervals, providing an additional source of nutrients. In some cases these canals are temporary structures, but others persist throughout life.

(Preparation by courtesy of Susan Smith, Department of Anatomy, GKT School of Medicine, London.)

After adolescence, hyaline cartilages are prone to calcification, especially in costal and laryngeal sites. In costal cartilage, the matrix tends to fibrous striation, especially in old age when cellularity diminishes. The xiphoid process and the cartilages of the nose, larynx and trachea (except the elastic cartilaginous epiglottis and corniculate cartilages) resemble costal cartilage in microstructure. The regenerative capacity of hyaline cartilage is poor.

Articular hyaline cartilage

Articular hyaline cartilage covers articular surfaces in synovial joints (Fig. 5.4). It provides an extremely smooth, resistant surface bathed by synovial fluid, which allows almost frictionless movement. Its elasticity, together with that of other articular structures, dissipates stresses, and gives the whole articulation some flexibility, particularly near extremes of movement. Articular cartilage is particularly effective as a shock-absorber, and resists the large compressive forces generated by weight transmission, especially during movement.

Fig. 5.4 Articular cartilage from the anterior region of the lateral femoral condyle of a young adult human female. Shown are the articular surface (top), articular cartilage and subchondral bone (below). Note the changes in size and spatial distribution of articular chondrocytes through the thickness of the cartilage. 3-D digital volumetric fluorescence imaging of serially sectioned, eosin-Y and acridine orange-stained tissue.

(Provided by courtesy of Professor Robert L Sah, Drs Won C Bae, Kyle D Jadin, Benjamin L Wong, Kelvin W. Li and Mrs Barbara L. Schumacher, Department of Bioengineering and Whitaker Institute of Biomedical Engineering, University of California, San Diego.)

Articular cartilage does not ossify. It varies from 1 to 7 mm in thickness and is moulded to the shape of the underlying bone, indeed it often accentuates and modifies the surface geometry of the bone. It is thickest centrally on convex osseous surfaces, and the reverse is true of concave surfaces. Its thickness decreases from maturity to old age. The surface of articular cartilage lacks a perichondrium; synovial membrane overlaps and then merges into its structure circumferentially (see Fig. 5.32).

Fig. 5.32 A section of a synovial joint and its associated highly vascular (red) synovial membrane in a human fetal hand. The two articular cartilage surfaces (A, arrowed) are separated on the right by a layer of synovial fluid (S) secreted by the synovial membrane (SM) which extends a short distance into the joint space from the capsule (C).

Adult articular cartilage shows a structural zonation with increasing depth from the surface. The arrangement of collagen fibres has been variously described as plexiform, helical, or in the form of serial arcades which radiate from the deepest zone to the surface, where they follow a short tangential course before returning radially. If the surface of an articular cartilage is pierced by a needle, a longitudinal split-line remains after withdrawal. For any given joint, the patterns of split-lines are constant and distinctive and follow the predominant directions of collagen bundles in tangential zones of cartilage. These patterns may reveal tension trajectories set up in surrounding cartilage during joint movement.

Zone 1 is the superficial or tangential layer. The free articular surface is a thin, cell-free layer, 3 μm thick, which contains fine collagen type II fibrils covered superficially by a protein coating. The cells are small, oval or elongated and parallel to the surface, relatively inactive, and surrounded by fine tangential fibres. The collagen fibres deeper within this zone are regularly tangential, their diameters and density increase with depth. Zone 2 is the transitional or intermediate layer. The cells are larger, rounder, and are either single or in isogenous groups. Most are typical active chondrocytes, surrounded by oblique collagen fibres. Deeper still, in the radiate layer (zone 3), cells are large, round and often disposed in vertical columns, with intervening radial collagen fibres. As elsewhere, the cells, either singly or in groups, are encapsulated in pericellular matrix which has fine fibrils and contains fibronectin and types II, IX and XI collagen. The deepest layer or calcified layer (zone 4) lies adjacent to the subchondral bone (hypochondral osseous lamina) of the epiphysis. The adjacent surfaces show reciprocal fine ridges, grooves and interdigitations, which, with the confluence of their fibrous arrays, resist shearing stresses produced by postural changes and muscle action. The junction between zones 3 and 4 is called the tidemark. With age, articular cartilage thins and degenerates by advancement of the tidemark zone, and the replacement of calcified cartilage by bone.

Concentrations of GAGs vary according to site and, in particular, with age. The proportion of keratan sulphate increases linearly with depth, mainly in the older matrix between cell nests, whereas chondroitin sulphates are concentrated around lacunae. The turnover rates of GAGs in cartilage are faster than those of collagen, and the smaller, more soluble GAGs turn over fastest. Turnover decreases with age and distance from the cells. The proteoglycan turnover time is estimated at nearly 5 years for adult human articular cartilage.

The sequence of structural features outlined above is also typical of cartilaginous growth plates (see p. 95). During radial epiphysial growth, the extension of endochondral ossification into overlying calcified cartilage starts with the development of isogenous groups followed by the appearance of hypertrophic cells arranged in vertical columns. This ceases in maturity, but the zones persist throughout life. The same terminal mechanism also occurs in bones which lack epiphyses.

Cells of articular cartilage are capable of cell division, but mitoses are rare except in young bones and damage is not repaired in the adult. Superficial cells are lost progressively from normal young joint surfaces, and they are replaced by cells from deeper layers. Degenerating cells may occur in any of the four zones. This probably accounts for the progressive reduction in cellularity of cartilage with advancing age, particularly in superficial layers.

Articular cartilages derive nutrients by diffusion from vessels of the synovial membrane, synovial fluid and hypochondral vessels of an adjacent medullary cavity, some capillaries from which penetrate and occasionally traverse the calcified cartilage. The contributions from these sources are uncertain and may change with age. Small molecules freely traverse articular cartilage, with diffusion coefficients about half those in aqueous solution. Larger molecules have diffusion coefficients inversely related to their molecular size. The permeability of cartilage to large molecules is greatly affected by variations in its GAG, and hence water, content, e.g. a three-fold increase multiplies the diffusion coefficient a hundred-fold.

Fibrocartilage

Fibrocartilage is dense, fasciculated, opaque white fibrous tissue. It contains fibroblasts and small interfascicular groups of chondrocytes. The cells are ovoid and surrounded by concentrically striated matrix (Fig. 5.5). When present in quantity, as in intervertebral discs, fibrocartilage has great tensile strength and appreciable elasticity. In lesser amounts, as in articular discs, the glenoid and acetabular labra, and the cartilaginous lining of bony grooves for tendons and some articular cartilages, it provides strength, elasticity and resistance to repeated pressure and friction. It is resistant to degenerative change.

Fig. 5.5 White fibrocartilage in a late fetal intervertebral disc (human). Chondroblasts lie between coarse collagen type I fibres (blue) derived from the anulus fibrosus. Mallory’s triple stain.

Fibrocartilage is unlike other types of cartilage in that it contains a considerable amount of type I (general connective tissue) collagen which is synthesized by the fibroblasts in its matrix. It is perhaps best regarded as a mingling of the two types of tissue, e.g. where a ligament or tendinous tissue inserts into hyaline cartilage, rather than as a separate type of cartilage. However, fibrocartilage in joints often lacks type II collagen altogether, and so possibly represents a distinct class of connective tissue.

The articular surfaces of bones which ossify in mesenchymal membranes (e.g. squamous temporal, mandible and clavicle) are covered by white fibrocartilage. The deep layers, adjacent to hypochondral bone, resemble calcified regions of the radial zone of hyaline articular cartilage. The superficial zone contains dense parallel bundles of thick collagen fibres, interspersed with typical dense connective tissue fibroblasts and little ground substance. Fibre bundles in adjacent layers alternate in direction, as they do in the cornea. A transitional zone of irregular bundles of coarse collagen and active fibroblasts separates the superficial and deep layers. The fibroblasts are probably involved in elaboration of proteoglycans and collagen, and may also constitute a germinal zone for deeper cartilage. Fibre diameters and types may differ at different sites according to the functional load.

Elastic cartilage

Elastic cartilage occurs in the external ear, corniculate cartilages, epiglottis and apices of the arytenoids. It contains typical chondrocytes, but its matrix is pervaded by yellow elastic fibres, except around lacunae (where it resembles typical hyaline matrix with fine type II collagen fibrils) (Fig. 5.6). Its elastic fibres are irregularly contoured and show no periodic banding. Most sites in which elastic cartilage occurs have vibrational functions, such as laryngeal sound wave production, or the collection and transmission of sound waves in the ear. Elastic cartilage is resistant to degeneration; it can regenerate to a limited degree following traumatic injury, e.g. the distorted repair of a ‘cauliflower ear’.

Fig. 5.6 Elastic cartilage, stained to demonstrate elastin fibres (blue-black). Chondroblasts and larger chondrocytes are embedded in the matrix, which also contains collagen type II fibres.

DEVELOPMENT AND GROWTH OF CARTILAGE

Cartilage is usually formed in embryonic mesenchyme. Mesenchymal cells proliferate and become tightly packed: the shape of their condensation foreshadows that of the future cartilage. They also become rounded, with prominent round or oval nuclei and a low cytoplasm: nucleus ratio. Adjacent cells are linked by gap junctions. Each cell next secretes a basophilic halo of matrix, composed of a delicate network of fine type II collagen filaments, type IX collagen and cartilage proteoglycan core protein, i.e. it differentiates into a chondroblast (Fig. 5.7). In some sites, continued secretion of matrix separates the cells, producing typical hyaline cartilage. Elsewhere, many cells become fibroblasts: collagen synthesis predominates and chondroblastic activity appears only in isolated groups or rows of cells which become surrounded by dense bundles of collagen fibres to form white fibrocartilage. In yet other sites, the matrix of early cellular cartilage is permeated first by anastomosing oxytalan fibres, and later by elastin fibres. In all cases, developing cartilage is surrounded by condensed mesenchyme which differentiates into a bilaminar perichondrium. The cells of the outer layer become fibroblasts and secrete a dense collagenous matrix lined externally by vascular mesenchyme. The cells of the inner layer contain differentiated, but mainly resting, chondroblasts or prechondroblasts.

Fig. 5.7 Highly cellular fetal cartilage, human phalanx. The cells are small and almost uniform in distribution. Mallory’s triple stain.

Cartilage grows by interstitial and appositional mechanisms. Interstitial growth is the result of continued mitosis of early chondroblasts throughout the tissue mass and is obvious only in young cartilage, where plasticity of the matrix permits continued expansion. When a chondroblast divides, its descendants temporarily occupy the same lacuna. They are soon separated by a thin septum of secreted matrix, which thickens and further separates the daughter cells. Continuing division produces isogenous groups. Appositional growth is the result of continued proliferation of the cells that form the internal, chondrogenic layer of the perichondrium. Newly formed chondroblasts secrete matrix around themselves, creating superficial lacunae beneath the perichondrium. This continuing process adds additional surface, while the entrapped cells participate in interstitial growth. Apposition is thought to be most prevalent in mature cartilages, but interstitial growth must persist for long periods in epiphysial cartilages. Relatively little is known about the factors which determine the overall shape of a cartilage.

BONE

Bone, and the struts and levers which it forms, is exquisitely adapted to resist stress with suitable resilience, support the body and provide leverage for movement. It is a highly vascular mineralized connective tissue: the great majority of its cells are embedded in an extracellular matrix composed of organic materials (about 40% dry weight in mature bone) and inorganic salts rich in calcium and phosphate.

MACROSCOPIC ANATOMY OF BONE

Macroscopically, living bone is white. Its texture is either dense like ivory (compact bone), or honeycombed by large cavities (trabecular, cancellous or spongy bone), where the bony element is reduced to a latticework of bars and plates (trabeculae) (Fig. 5.8, Fig. 5.9). Compact bone is usually limited to the cortices of mature bones (cortical bone) and is of great importance in providing their strength. Its thickness and architecture vary for different bones, according to their overall shape, position and functional roles. The cortex plus the hollow medullary canal of long bones allows combination of strength with low weight. Cancellous bone is usually internal, giving additional strength to cortices and supporting the bone marrow. Bone forms a reservoir of metabolic calcium (99% of body calcium is in the bony skeleton) and phosphate which is under hormonal and cytokine control.

Fig. 5.8 Vertical section 2 cm below the anterosuperior border of the iliac crest (female, 42 years). The cancellous bone consists of intersecting curved plates and struts. Osteonal (Haversian) canals can just be seen in the two cortices (C) at this magnification.

Fig. 5.9 Trabecular bone at different sites in the proximal part of the same human femur. All fields are at the same scale. A, Subcapital part of the neck; B, Greater trochanter; C, Rim of the articular surface of the head. Note the wide variation in thickness, orientation and spacing of the trabeculae.

(Original photographs from Whitehouse WJ, Dyson ED 1974 Scanning electron microscope studies of trabecular bone in the proximal end of the human femur. J Anat 118: 417–414, by permission from Blackwell Publishing.)

The proportions of compact to cancellous bone vary greatly. In long bones, the diaphysis consists of a thick cylinder of compact bone with a few trabeculae and spicules on its inner surface. It encloses a large central medullary or marrow cavity that communicates freely with the intratrabecular spaces of the expanded bone ends. In other bones, especially flat bones such as the ribs, the interior is uniformly cancellous, and compact bone forms the surface. The cavities are usually filled with marrow, either red haemopoietic or yellow adipose, according to age and site. However, in some bones of the skull, notably the mastoid process of the temporal bone, and the paranasal sinuses of the maxilla, sphenoid and ethmoid, many of the internal cavities are filled with air, i.e. they are pneumatized.

Bones vary not only in their primary shape but also in lesser surface details, or secondary markings, which appear mainly in postnatal life. Most bones display features such as elevations and depressions (fossae), smooth areas and rough ridges. Numerous names are used to describe these secondary features. Some articular surfaces are called fossae (e.g. the glenoid fossa); lengthy depressions are grooves or sulci (e.g. the humeral bicipital sulcus); a notch is an incisura, and an actual gap is a hiatus. A large projection is termed a process or, if elongated and slender or pointed, a spine. A curved process is a hamulus or cornu (e.g. the pterygoid hamuli of the sphenoid bone and the cornua of the hyoid). A rounded projection is a tuberosity or tubercle, and occasionally a trochanter. Long elevations are crests, or lines, if they are less developed; crests are wider and present boundary edges or lips. An epicondyle is a projection close to a condyle and is usually a site where the common tendon of a superficial muscle group or the collateral ligament of the adjacent joint are attached. The terms protuberance, prominence, eminence and torus are less often applied to certain bony projections. The expanded proximal ends of many long bones are often termed the ‘head’ or caput (e.g. humerus, femur, radius). A hole in bone is a foramen, and becomes a canal when lengthy. Large holes may be called apertures or, if covered largely by connective tissue, fenestrae. Clefts in or between bones are fissures. A lamina is a thin plate; larger laminae may be called squamae (e.g. the temporal squama). Large areas on many bones are featureless and often smoother than articular surfaces, from which they differ because they are pierced by many visible vascular foramina. This texture occurs where muscle is directly attached to bone, and small blood vessels pass through the foramina from bone to muscle, and perhaps vice versa. Areas covered only by periosteum are similar, but vessels are less numerous.

Tendons are usually attached at roughened bone surfaces. Wherever any aggregation of collagen in a muscle reaches bone, surface irregularities correspond in form and extent to the pattern of such ‘tendinous fibres’. Such markings are almost always elevated above the general surface, as if ossification advanced into the collagen bundles from periosteal bone. How such secondary markings are induced is uncertain but they may result from the continued incorporation of new collagen fibres into the bone, perhaps necessary for minor functional adjustment. Evidence suggests that their prominence may be related to the power of the muscles involved and they increase with advancing years, as if the pull of muscles and ligaments exercised a cumulative effect over a limited area. Surface markings delineate the shape of attached connective tissue structures, whether these are an obvious tendon, intramuscular tendon or septum, aponeurosis, or tendinous fibres mediating what is otherwise a direct muscular attachment. These markings may be facets, ridges, nodules, rough areas or complex mixtures: they afford accurate indications of the junctions of bone with muscles, tendons, ligaments or articular capsules.

When a muscle is apparently attached directly to bone, its fibres do not themselves adhere directly to periosteum or bone. The route of transmission of tension from contracting muscle to bone is through the connective tissue that encapsulates (epimysium) and pervades (perimysium and endomysium) all muscles. These two forms of attachment of muscles, which are at the extremes of a range of admixtures, differ in the density of collagen fibres between muscle and bone. Where collagen is visibly concentrated, markings appear on the bone surface. In contrast, the multitude of microscopic connective tissue ties of direct attachment that occur over a larger area do not visibly mark the bone, and so it appears smooth to unaided vision and touch.

Bones display articular surfaces at synovial joints with their neighbours: if small, these are termed facets or foveae, larger, knuckle-shaped surfaces are condyles, and a trochlea is grooved like a pulley. Articular surfaces are smooth and adapted in shape to the movement of particular joints. In life they are covered by articular cartilage; they are smooth partly because they lack the vascular foramina typical of most other bone surfaces. Large tendons, e.g. those of adductor magnus and subscapularis, are attached to facets which lack the regular contours of articular surfaces, but which resemble them in texture, because they are poorly vascularized. These facets are sometimes depressed, but they may surmount large elevations, e.g. the humeral tubercles.

MICROSTRUCTURE OF BONE

Bone contains a mineralized extracellular matrix; specialized cells including osteoblasts, osteocytes and osteoclasts; and components of the periosteum, endosteum and marrow. These components will be described in detail below, first individually, and then in terms of their overall organization.

Bone matrix

Bone matrix is the mineralized extracellular material of bone; like general connective tissues, it consists of a ground substance in which numerous collagen fibres are embedded, usually ordered in parallel branching arrays (Fig. 5.10). In mature bone, the matrix is moderately hydrated, and 10–20% of its mass is water. Of its dry weight, 60–70% is made up of inorganic, mineral salts (mainly microcrystalline calcium and phosphate hydroxides, hydroxyapatite (see below), approximately 30% is collagen and the remainder is non-collagenous protein and carbohydrate, mainly conjugated as glycoproteins. The proportions of these components vary with age, location and metabolic status.

Fig. 5.10 Scanning electron micrograph of collagen fibres on the surface of human trabecular bone. Note branching fibres (female, 2 months, sixth rib).

Collagen

The collagen that is found in bone closely resembles that of many other connective tissues, and is mainly type I: there are trace amounts of type V which is thought to regulate fibrillogenesis. However, its molecular structure is unlike that of collagen in general connective tissue: it displays internal covalent cross-linkages, and the transverse spacings within its fibrils are somewhat larger. The cross-links make it stronger and chemically more inert, and the internal gaps provide the space for deposition of minerals. Up to two-thirds of the mineral content of bone is thought to be located within collagen fibrils. Crystal formation is probably initiated in the hole zones, which are gaps between the ends of tropocollagen subunits.

Collagen contributes greatly to the mechanical strength of bone, although its precise role in bone mechanics has yet to be clarified. As well as contributing to the tensile, compressive and shearing strengths of bone, the small degree of elasticity shown by collagen imparts a measure of resilience to the tissue, and helps to resist fracture when bone is mechanically loaded.

Collagen fibres are synthesized by osteoblasts, polymerize from tropocollagen extracellularly, and become progressively more cross-linked as they mature. In primary bone, they form a complex interwoven meshwork of non-lamellar woven or bundle bone, which in most sites is almost entirely replaced by regular laminar arrays of nearly parallel collagen fibres (lamellar bone). Partially mineralized collagen networks can be seen within osteoid on the outer and internal surfaces of bone, and in the endosteal linings of vascular canals. Collagen fibres from the periosteum are incorporated in cortical bone (extrinsic fibres), and anchor this fibrocellular layer at its surface. Terminal collagen fibres of tendons and ligaments are incorporated deep into the matrix of cortical bone. They may be interrupted by new osteons during cortical drift (modelling) and turnover (remodelling), and remain as islands of interstitial lamellae or even trabeculae.

Non-collagenous organic components

Small amounts of various complex macromolecules are attached to collagen fibres and surrounding bone crystals. These are secreted by osteoblasts and young osteocytes and include osteonectin, osteocalcin, the bone proteoglycans biglycan and decorin, the bone sialoproteins osteopontin and thrombospondin, many growth factors including transforming growth factor β (TGF-β), proteases and protease inhibitors, often in a latent form. The functions of some of these molecules are described with osteoblasts (see below).

Bone minerals

Bone minerals are the inorganic constituents of the bone matrix. They confer the hardness and much of the rigidity of bone, and are the main reason that bone is easily seen on radiographs (bone has to be 50% mineralized to be visible on radiographs produced with a standard X-ray unit). The mineral substances of bone are mostly acid-soluble. If they are removed, using calcium chelators such as citrates or ethylene diamine tetra-acetic acid (EDTA), the bone retains its shape but becomes highly flexible.

The mineral portion of mature bones is composed largely of crystals made of a substance generally referred to as hydroxyapatite (but with an important carbonate content, and a lower Ca/P ratio than pure hydroxyapatite (Ca₁₀ (PO₄)₆ (OH)₂), together with a small amount of calcium phosphate. Bone crystals are small but have a large surface area. They take the form of thin plates or leaf-like structures and range in size up to 150 nm long × 80 nm wide × 5 nm thick, although most are half that size. They are often packed quite closely together, with their long axes nearly parallel to the axes of the collagen fibrils. The narrow gaps between the crystals contain associated water and organic macromolecules.

The major ions which make up the mineral part of bone include calcium, phosphate, hydroxyl and carbonate. Less numerous ions are citrate, magnesium, sodium, potassium, fluoride, chloride, iron, zinc, copper, aluminium, lead, strontium, silicon and boron, many of which are present only in trace quantities. Fluoride ions can substitute for hydroxyl ions, and carbonate can substitute for either hydroxyl or phosphate groups. Group IIA cations, e.g. radium, strontium and lead, all readily substitute for calcium and are therefore known as bone-seeking cations. Since they can be either radioactive or chemically toxic, their presence in bone, where they may be close to haemopoietic bone marrow, may cause illness and characteristic appearances on X-rays.

The concentration of mineral in young osteons is low but increases with age: it is highest in the older, more peripheral, lamellae. Mineral distribution is uniform in established, highly mineralized, osteons. Mineralization normally reaches 70–80% in 3 weeks. Immature woven bone mineralizes faster and can be identified from adjacent lamellar bone by its higher degree of mineralization. Osteons may show one or more highly mineralized arrest lines within their walls.

Osteoblasts

Osteoblasts are derived from osteoprogenitor (stem) cells of mesenchymal origin, which are present in the bone marrow and other connective tissues. They proliferate and differentiate, stimulated by bone morphogenetic proteins (BMPs), into osteoblasts prior to bone formation. Osteoblasts are basophilic, roughly cuboidal mononuclear cells 15–30 μm across. Ultrastructurally, they have features typical of protein-secreting cells. They are found on the forming surfaces of growing or remodelling bone, where they constitute a covering layer (Fig. 5.11). In relatively quiescent adult bones they appear to be present mostly on endosteal rather than periosteal surfaces, but they also occur deep within compact bone where osteons are being remodelled. They are responsible for the synthesis, deposition and mineralization of the bone matrix, which they secrete. Once embedded in the matrix, they become osteocytes.

Fig. 5.11 Osteoblasts (Ob) covering the free surfaces of developing bone in a human fetal hand. Deep to the layer of osteoblasts in the lower field is a layer of osteoid matrix (Os, pale blue) which has yet to be mineralized. Osteocytes (Oc) are shown within lacunae in mineralized matrix (red).

Osteoblasts contain prominent bundles of actin, myosin and other cytoskeletal proteins which are associated with the maintenance of cell shape, attachment and motility. Their plasma membranes display many extensions, some of which contact neighbouring osteoblasts and embedded osteocytes at intercellular gap junctions. This arrangement facilitates coordination of the activities of groups of cells, e.g. in the formation of large domains of parallel collagen fibres.

Osteoblasts synthesize and secrete organic matrix, i.e. type I collagen, small amounts of type V collagen, and numerous other macromolecules involved in bone formation and resorption. Collagen synthesis occurs in the rough endoplasmic reticulum and Golgi apparatus, and type I collagen is secreted as monomers which assemble into the triple helical procollagen extracellularly. Other glycoprotein products include osteocalcin, which is required for bone mineralization, binds hydroxyapatite and calcium, and is used as a marker of new bone formation; osteonectin, a phosphorylated glycoprotein which binds strongly to hydroxyapatite and collagen – it may play a role in initiating hydroxyapatite crystallization, and may also be a cell adhesion factor; RANKL, the cell surface ligand for RANK (receptor for activation of nuclear factor kappa B), which is an osteoclast progenitor receptor (see below); osteoprotegerin (a soluble, high affinity decoy ligand for RANKL) which restricts osteoclast differentiation; the bone proteoglycans biglycan and decorin which bind TGF-β; bone sialoproteins, osteopontin and thrombospondin, which mediate osteoclast adhesion to bone surfaces via binding to osteoclast integrins; latent proteases and growth factors, including BMPs. TGF-β is secreted by osteoblasts as well as osteoclasts: it is activated in the acid conditions of the ruffled border zone of the osteoclast, and may be a coupling factor for stimulating new bone formation at resorption sites.

Extracellular fluid is supersaturated with respect to the basic calcium phosphates, yet mineralization is not a widespread phenomenon. Osteoblasts play a significant role in the mineralization of osteoid, the unmineralized organic matrix. They secrete osteocalcin which binds calcium weakly, but at levels sufficient to concentrate the ion locally. They also contain membrane-bound vesicles, 0.1–0.2 μm in diameter, which contain alkaline phosphatase (which can cleave phosphate ions from various molecules to elevate concentrations locally), and pyrophosphatase (which degrades inhibitory pyrophosphate in the extracellular fluid). The vesicles bud off from the cell surfaces of the osteoblasts into newly formed osteoid and are the sites of initiation of hydroxyapatite crystal formation in newly forming bone (see below). Crystals are then released into the osteoid matrix by an unknown mechanism. Some alkaline phosphatase reaches the blood circulation where it can be detected in conditions of rapid bone formation or turnover.

Osteoblasts play a key role in the hormonal regulation of bone resorption, since they express receptors for parathyroid hormone (PTH), 1,25-dihydroxy vitamin D3 and other promoters of bone resorption. During bone resorption, osteoblasts promote osteoclast differentiation via PTH-activated expression of cell surface RANKL, which binds to RANK on immature osteoclasts, establishes cell–cell contact and triggers contact-dependent osteoclast differentiation. In the presence of PTH, osteoblasts also downregulate secretion of osteoprotegerin, a soluble decoy ligand with higher affinity for RANKL. In conditions favouring bone deposition, secreted osteoprotegerin blocks RANKL binding to RANK and restricts numbers of mature osteoclasts. (For a recent review, see Blair et al 2007.)

Bone-lining cells are flattened epithelioid cells found on the surfaces of adult bone that is not undergoing active deposition or resorption, and are generally considered to be quiescent osteoblasts or osteoprogenitor cells. They form the outer boundary of the marrow tissue on the endosteal surface of marrow cavities, are present on the periosteal surface, and line the system of vascular canals within osteons.

Osteocytes

Osteocytes constitute the major cell type of mature bone, and are scattered within its matrix, interconnected by numerous dendritic processes to form a complex cellular network (Fig. 5.12). They are derived from osteoblasts and are enclosed within their matrix but, unlike chondrocytes, they do not divide. Bone growth is appositional: new layers are added only to pre-existing surfaces and so, again unlike chondrocytes, osteocytes enclosed in lacunae do not secrete new matrix. The rigidity of mineralized bone matrix prevents internal expansion, which means that interstitial growth, which is characteristic of most tissues, does not occur in bone. Osteocytes retain contacts with each other and with cells at the surfaces of bone (osteoblasts and bone-lining cells) throughout their lifespan.

Fig. 5.12 Osteocyte lacunae shown at high magnification in a dry ground section of lamellar bone. The territories of three osteocytes are shown. Their branching dendrites contact those of neighbouring cells via the canaliculi seen here within the bone matrix. Several other osteocyte lacunae are present, out of the focal plane in this section, and tangential to the osteon axis.

Mature, relatively inactive, osteocytes possess an ellipsoid cell body with the longest axis (25 μm) parallel to the surrounding bony lamella. The rather narrow rim of cytoplasm is faintly basophilic, contains relatively few organelles and surrounds an oval nucleus. Osteocytes in woven bone are larger and more irregular in shape (Fig. 5.13).

Fig. 5.13 Human parietal bone (male neonate) showing primary osteonal bone (grey) and woven bone (white) containing many connecting osteocyte lacunae (black). Internal resorption of the bone has produced large irregular dark spaces (trabecularization).

Numerous fine dendritic processes emerge from the cell body of each osteocyte and branch a number of times. They contain bundles of microfilaments and some smooth endoplasmic reticulum. At their distal tips they contact the processes of adjacent cells, i.e. other osteocytes and, at surfaces, osteoblasts and bone-lining cells. They form communicating gap junctions with these cells which means that they are in electrical and metabolic continuity.

Bone matrix surrounds the cell bodies and processes. There appears to be a variable space filled with extracellular fluid between each osteocyte and its enclosing wall. Each cell body lies in a lacuna from which many narrow, branched channels extend. These channels or canaliculi are 0.5–0.25 μm wide, and contain the dendritic processes of the osteocytes: they provide a route for the diffusion of nutrients, gases and waste products between the osteocytes and the blood vessels. Canaliculi do not usually extend through and beyond the reversal line surrounding an osteon and so do not communicate with neighbouring systems. The walls of lacunae may be lined with a variable (0.2–2 μm) layer of unmineralized organic matrix.

In well-vascularized bone, osteocytes are long-lived cells which actively maintain the bone matrix. The average lifespan of an osteocyte varies with the metabolic activity of the bone and the likelihood that it will be remodelled, but is measured in years. Old osteocytes may retract their processes from the canaliculi; when they die, their lacunae and canaliculi may become plugged with cell debris and minerals, which hinders diffusion through the bone. Dead osteocytes occur commonly in interstitial bone and the inner regions of trabecular bone which escape surface remodelling, and are particularly noticeable by the second and third decades. Bones which experience little turnover, e.g. the auditory ossicles, are most likely to contain aged osteocytes and low osteocyte viability.

Osteocytes play an essential role in the maintenance of bone: their death leads to the resorption of the matrix by osteoclast activity. They remain responsive to parathyroid hormone and 1,25(OH)₂ vitamin D₃, and it is possible that they are involved in mineral exchange at adjacent bone surfaces. Osteocytes themselves are often mineralized.

Osteoclasts

Osteoclasts are large (40 μm or more) polymorphic cells containing up to 20 oval, closely packed nuclei. They lie in close contact with the bone surface in resorption bays (Howship’s lacunae). Their cytoplasm contains numerous mitochondria and vacuoles, many of which are acid phosphatase-containing lysosomes. The rough endoplasmic reticulum is relatively sparse given the size of cell, but the Golgi complex is extensive. The cytoplasm also contains numerous coated transport vesicles and microtubule arrays involved in the transport of the vesicles between the Golgi stacks and the ruffled membrane, which is the highly infolded cell surface of active osteoclasts at sites of local bone resorption. A well-defined zone of actin filaments and associated proteins occurs beneath the ruffled membrane around the circumference of the resorption bay, in a region termed the sealing zone.

Functionally, osteoclasts are responsible for the local removal of bone during bone growth and subsequent remodelling of osteons and surface bone (see Fig. 5.25). They cause demineralization by proton release, which creates an acidic local environment, and organic matrix destruction by releasing lysosomal (cathepsin K) and non-lysosomal (e.g. collagenase) enzymes. Factors stimulating osteoclasts to resorb bone include osteoblast-derived signals; cytokines from other cells, e.g. macrophages and lymphocytes; blood-borne factors, e.g. parathyroid hormone and 1,25(OH)₂ vitamin D₃ (calcitriol). Calcitonin, produced by C cells of the thyroid follicle, reduces osteoclast activity.

Fig. 5.25 Endochondral ossification in human fetal bone. Spicules of cartilage remnant (pale blue) serve as surfaces for the deposition of osteoid (dark blue), shown in the upper half of the field. Mineralized, woven bone is stained red. Three large multinucleate osteoclasts are seen centre right, further eroding cartilage and remodelling the developing bone. Blood sinusoids and haemopoietic tissue (below) fill the spaces between areas of ossification. Heidenhain’s azan trichrome preparation.

Osteoclasts arise by fusion of monocytes derived from the bone marrow or other haemopoietic tissue. They probably share a common ancestor with macrophages within the granulocyte–macrophage lineage (see Fig. 4.12) but it is thought that they subsequently follow a distinct differentiation pathway.

Osteons

The mechanical properties of bone, particularly its strength and resilience, are dependent on the general composition of its matrix. Woven and lamellar bone display two quite distinct types of organization.

In woven, or bundle, bone, the collagen fibres and bone crystals are irregularly arranged. The diameters of the fibres vary, so that fine and coarse fibres intermingle, producing the appearance of the warp and weft of a woven fabric. Woven bone is typical of young fetal bones, but is also seen in adults during excessively rapid bone remodelling and repair of fractures (Fig. 5.14). It is formed by highly active osteoblasts during development, and is stimulated in the adult by fracture, growth factors, or prostaglandin E₂.

Fig. 5.14 Electron micrograph of woven bone from a failed fracture of human distal tibia. Two osteoblasts (O) lie on the free surface (top). Newly synthesized collagenous osteoid matrix (M) is seen in the centre field, with a mineralization front (electron-dense area) below (arrows).

Lamellar bone makes up almost all of an adult osseous skeleton (Fig. 5.15, Fig. 5.16). The precise arrangement of lamellae varies from site to site, particularly between compact cortical bone and the trabecular bone within. In many bones a few lamellae form continuous circumferential layers at the outer (periosteal) and inner (endosteal) surfaces. However, by far the greatest proportion of lamellae are arranged in concentric cylinders around neurovascular channels called Haversian canals, to form the basic units of bone tissue which are the Haversian systems or osteons. Osteons usually lie parallel with each other (Fig. 5.17) and, in elongated bones such as those of the appendicular skeleton, with the long axis of the bone. They may also spiral, branch or intercommunicate, and some end blindly.