Extracellular matrix

The extracellular matrix is composed of collagen and, in some cases, elastic fibres, embedded in a highly hydrated but stiff ground substance (Fig. 5.1). The components are unique to cartilage, and endow it with unusual mechanical properties. The ground substance is a firm gel, rich in carbohydrates and predominantly acidic. The chemistry of the ground substance is complex. It consists mainly of water and dissolved salts, held in a meshwork of long interwoven proteoglycan molecules together with various other minor constituents, mainly proteins or glycoproteins.

Fig. 5.1 Fine structural organization of hyaline cartilage matrix. Depicted are large proteoglycan complexes and type II collagen fibres (cross-banded and of different diameters). Proteoglycan complexes bind to the surface of these fibres via their monomeric sidechains and link them together. The arrangement of glycosaminoglycans and core protein of the proteoglycan monomer is illustrated in the expansion.

Hyaline cartilage

Hyaline cartilage has a homogeneous glassy, bluish opalescent appearance. It has a firm consistency and some elasticity. Costal, nasal, some laryngeal, tracheobronchial, all temporary (developmental) and most articular, cartilages are hyaline. The arytenoid cartilage changes from hyaline at its base, to elastic cartilage at its apex. Size, shape and arrangement of cells, fibres and proteoglycan composition vary at different sites and with age. The chondrocytes are flat near the perichondrium and rounded or angular, deeper in the tissue. They are often grouped in pairs, sometimes more, forming cell nests (isogenous cell groups) which are daughter cells of a common parent chondroblast: apposing cells have a straight outline. The matrix is typically basophilic (Fig. 5.2) and metachromatic, particularly in the lacunar capsule, where recently formed, territorial matrix borders the lacuna of a chondrocyte. The paler-staining interterritorial matrix between cell nests is older synthetically. Fine collagen fibres are arranged in a basket-like network (Fig. 5.3), but are often absent from a narrow zone immediately surrounding the lacuna. An isogenous cell group, together with the enclosing pericellular matrix, is sometimes referred to as a chondron.

Fig. 5.2 Sections through hyaline cartilage. A, Low-power view of human rib, showing perichondrium (P, left), young chondroblasts (Cb) embedded in pale-staining interterritorial matrix and mature chondrocytes (Cc) embedded in the basophilic interterritorial matrix (centre and right). B, Higher magnification of hyaline cartilage in human bronchial wall, showing isogenous groups of chondrocytes (C). Note the more deeply-stained basophilic zones (B) (rich in acidic proteoglycans) around the cell clusters, with older, paler-staining matrix (M) between clusters.

Fig. 5.3 Electron micrograph of chondroblasts in rabbit femoral condylar cartilage. The central cell has an active euchromatic nucleus with a prominent nucleolus, and its cytoplasm contains concentric cisternae of rough endoplasmic reticulum, scattered mitochondria, lysosomes and glycogen aggregates. The plasma membrane bears numerous short filopodia which project into the surrounding matrix. The latter shows a delicate feltwork of collagen fibrils within finely granular interfibrillary material. No pericellular lacuna is present; the matrix separates the central chondroblast from the cytoplasm of two adjacent chondroblasts (left, and crescentic profile).

(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.

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).

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.

Fig. 5.15 Main features of the microstructure of mature lamellar bone. Areas of compact and trabecular (cancellous) bone are included. Note the general construction of the osteons; distribution of the osteocyte lacunae; Haversian canals and their contents; resorption spaces. Different views of the structural basis of bone lamellation.

Fig. 5.16 A, Osteons in a dry ground transverse section of bone. Concentric lamellae surround the central Haversian canal of each complete osteon; they contain the dark lacunae of osteocytes and the canaliculi which are occupied in life by their dendrites. These canaliculi interconnect with canaliculi of osteocytes in adjacent lamellae. Incomplete (interstitial) lamellae (e.g. centre field) are the remnants of osteons remodelled by osteoclast erosion. B, High-power view of osteocytes within lamellae; a Haversian canal is seen on the right.

(B, Photograph by Sarah-Jane Smith.)

Fig. 5.17 Osteons in a dry ground longitudinal section of bone. The central Haversian canals (H: tubular structures, mainly dark) show transverse nutrient (Volkmann’s) canals (V) which form bridges between adjacent osteons and their blood vessels.

It has been estimated that there are 21 million osteons in the adult skeleton. In transverse section they are round or ellipsoidal, varying from 100 to 400 μm in diameter. A medium-sized osteon contains about 30 lamellae, each approximately 3 μm thick. Each osteon is permeated with the canaliculi of its resident osteocytes, and these form pathways for diffusion of nutrients, gases, etc. between the vascular system and the osteocytes. The maximum diameter of an osteon ensures that no osteocyte is more than 200 μm from a blood vessel, a distance that may be a limiting factor in cellular survival. The spaces between osteons contain interstitial lamellae which are the fragmentary remains of osteons and the partially eroded circumferential lamellae of older bone (see below).

The central Haversian canals of osteons vary in size, with a mean diameter of 50 μm; those near the marrow cavity are somewhat larger. Each canal contains one or two capillaries lined by fenestrated endothelium and surrounded by a basal lamina which also encloses typical pericytes. They usually contain a few unmyelinated and occasional myelinated axons. The bony surfaces of osteonic canals are perforated by the openings of osteocyte canaliculi and are lined by collagen fibres.

Haversian canals communicate with each other and directly or indirectly with the marrow cavity via vascular (nutrient) channels called Volkmann’s canals, which run obliquely or at right angles to the long axes of the osteons (Fig. 5.17). The majority of these channels appear to branch and anastomose, but some join large vascular connections with vessels in the periosteum and the medullary cavity.

Osteons are distinguished from their neighbours by a cement line which contains little or no collagen, and is strongly basophilic because it has a high content of glycoproteins and proteoglycans. Cement lines mark the limit of bone erosion prior to the formation of a new osteon, and are therefore also known as reversal lines. Canaliculi occasionally pass through cement lines, and so provide a route for exchange between interstitial bone lamellae and vascular channels within osteons. Basophilic lines can occur in the absence of erosion: they indicate where bony growth has been interrupted and then resumed and are called resting lines.

Each lamella consists of a sheet of mineralized matrix that contains collagen fibres of similar orientation locally, running in branching bundles 2–3 μm thick, and often extending the full width of a lamella. This interconnecting, three-dimensional construction increases the strength of the bone. The orientation of the collagen fibres and associated mineral crystals differs in adjacent lamellae: the difference varies between 0° and 90°, and is clearly shown by polarized light microscopy. A less perfect packing of collagen fibrils into bundles occurs at the borders of lamellae, where intermediate and random orientations predominate. The main direction of the collagen fibres within osteons of long bone shafts varies: the fibres are more longitudinal at sites which are subjected predominantly to tension, and more oblique at sites subjected mostly to compression. The peripheral lamellae of osteons contain more transverse fibres at any site in a diaphysis.

Trabecular bone

The organization of trabecular (cancellous, spongy) bone is basically lamellar, as shown most clearly under polarized light (Fig. 5.18). It takes the form of branching and anastomosing curved plates, tubes and bars of various widths and lengths which surround marrow cavities and are lined by endosteal tissue (Fig. 5.8, Fig. 5.9). Their thickness ranges from 50 to 400 μm. In general, bone lamellae are oriented parallel with the adjacent bone surface, and the arrangement of cells and matrix is similar to that found in circumferential and osteonic bone. Thick trabeculae and regions close to compact bone may contain small osteons, but blood vessels do not otherwise lie within the bony tissue, and osteocytes therefore rely on canalicular diffusion from adjacent medullary vessels. In young bone, calcified cartilage may occur in the cores of trabeculae, but this is generally replaced by bone during subsequent remodelling.

Fig. 5.18 Trabecular bone in a bone marrow sample taken from the human posterior iliac crest. A, Irregular trabeculae of bone, surrounded by bone marrow haemopoietic and adipose tissue (haematoxylin and eosin stain); B, the same field viewed under polarized light, demonstrating lamellar, non-osteonic bone with lamellae oriented in different directions in different regions. Osteocytes are just visible, embedded in the solid matrix.

Remodelling

Remodelling of the interior of a bone depends upon the balance of resorption and deposition of bone, i.e. on the balanced activities of osteoclasts and osteoblasts. Osteoclasts first excavate a cylindrical tunnel by concerted action. A ‘cutting cone’ is formed by groups of osteoclasts moving at 50 μm/day, followed by osteoblasts which fill in the space so created. The osteoblasts deposit new osteoid matrix concentrically around a centrally ingrowing blood vessel, starting at the peripheral surface of the tunnel. This forms a ‘closing cone’ with 4000 osteoblasts/mm². Deposition of successive, concentric lamellae follows, as cohorts of osteoblasts become embedded in the matrix they secrete, and are succeeded by new osteoblasts which line the free surface thus created, and secrete the next layer. In this way the walls of resorption cavities are lined with new lamellar matrix, and the vascular channels are progressively narrowed (Fig. 5.19). The pattern and extent of remodelling is dictated by the mechanical loads applied to the bone.

Fig. 5.19 Bone remodelling. Longitudinal and cross-sections of a time line illustrating the formation of an osteon. Osteoclasts cut a cylindrical channel through bone. Osteoblasts follow, laying down bone on the surface of the channel until matrix surrounds the central blood vessel of the newly formed osteon (closing cone of a new osteon).

A hypermineralized basophilic cement line marks a site of reversal from resorption to deposition. Formation of osteons does not end with growth but continues variably throughout life. Remnants of circumferential lamellae of old osteons form interstitial lamellae between newer osteons (Fig. 5.15, Fig. 5.16A).

It has been estimated that approximately 10% of the adult bony skeleton turns over each year by the process of remodelling. The degree of remodelling varies with age and the number of osteons and osteon fragments have therefore been used in attempts to estimate the age of skeletal material at death.

Periosteum, endosteum and bone marrow

The outer surface of bone is covered by a condensed, fibrocollagenous layer, the periosteum. The inner surface is lined by a thinner, more cellular, endosteum. Osteoprogenitor cells, osteoblasts, osteoclasts and other cells important in the turnover and homeostasis of bone tissue lie in these layers.

The periosteal layer is tethered to the underlying bone by extrinsic collagen fibres, Sharpey’s fibres, which penetrate deep into the outer cortical bone tissue. It is absent from articular surfaces, and from the points of insertion of tendons and ligaments (entheses) (see Fig. 5.46). The periosteum is highly active during fetal development, when it generates osteoblasts for the appositional growth of bone. These cells form a layer, two to three cells deep, between the fibrous periosteum and new woven bone matrix. Osteoprogenitor cells within the mature periosteum are indistinguishable morphologically from fibroblasts. Periosteum is important in the repair of fractures: where it is absent, e.g. within the joint capsule of the femoral neck, fractures are slow to heal.

Fig. 5.46 The microstructure of bone at entheses. A, B, The cortical shell of bone (short arrows) is very thin at fibrocartilaginous attachment sites. In these examples showing the attachment of the tendons of triceps brachii (TB) and of fibularis longus (FL), it is approximately the same thickness as the underlying trabeculae (T). Note that in A, the superficial trabeculae (long arrows) are aligned along the direction of pull of the tendon of triceps. C, In marked contrast, the layer of cortical bone (CB) at the fibrous attachment site of pronator teres (PT) to the mid-shaft of the radius, is much thicker. D, Higher power view of the cortical calcified shell of tissue at a fibrocartilaginous attachment site (the Achilles tendon), which consists of bone (B) and calcified fibrocartilage (CF). In this specimen, there are two tidemarks, TM1 and TM2, associated with the cortical shell of calcified tissue. TM1 is adjacent to the zone of uncalcified fibrocartilage (UF), and marks the mechanical boundary between hard and soft tissues. TM2 lies nearer the bone and indicates an earlier phase of calcification. Note the relative straightness of the tidemarks but the highly irregular interface between calcified fibrocartilage and bone (arrows), which is important in anchoring the tendon to the bone. Sections of human cadaveric bone stained with Masson’s trichrome.

(Photographs provided by courtesy of Professor Michael Benjamin from sections cut and stained by S. Redman.)

In resting adult bone, quiescent osteoblasts and osteoprogenitor cells are present chiefly on the endosteal surfaces, which act as the principal reservoir of new bone-forming cells for remodelling or repair. The endosteum provides a surface of approximately 7.5 m², thought to be important in calcium homeostasis. It is formed by flattened osteoblast precursor cells and reticular (type III collagen) fibres, and lines all the internal cavities of bone, including the Haversian canals. It overlies the endosteal circumferential lamellae, and encloses the medullary cavity.

Vascular supply and lymphatic drainage

The osseous circulation supplies bone tissue, marrow, perichondrium, epiphysial cartilages in young bones, and, in part, articular cartilages. The vascular supply of a long bone depends on several points of inflow which feed complex and regionally variable sinusoidal networks within the bone. The sinusoids drain to venous channels which leave through all surfaces that are not covered by articular cartilage. The flow of blood through cortical bone in the shafts of long bones is mainly centrifugal (Fig. 5.20).

Fig. 5.20 The main features of the blood supply of a long bone. Note the contrasting supplies of the diaphysis, metaphysis and epiphysis, and their connections with periosteal, endosteal, muscular and periarticular vessels. The expansion shows part of the diaphysis in more detail. The marrow cavity contains a large central venous sinus, a dense network of medullary sinusoids, and longitudinal medullary arteries and their circumferential rami. Longitudinally oblique transcortical capillaries emerge through minute ‘cornet-shaped’ foramina to become confluent with the periosteal capillaries and venules. The obliquity of the cortical capillaries is emphasized for clarity. Not to scale.

One or two main diaphysial nutrient arteries enter the shaft obliquely through nutrient foramina which lead into nutrient canals. Their sites of entry and angulation are almost constant and characteristically directed away from the dominant growing epiphysis. Nutrient arteries do not branch in their canals, but divide into ascending and descending branches in the medullary cavity which approach the epiphyses, dividing repeatedly into smaller helical branches close to the endosteal surface. The endosteal vessels are vulnerable during operations which involve passing metal implants into the medullary canal, e.g. intramedullary nailing for fractures. Near the epiphyses they are joined by terminal branches of numerous metaphysial and epiphysial arteries. The former are direct branches of neighbouring systemic vessels, the latter come from periarticular vascular arcades formed on non-articular bone surfaces. Numerous vascular foramina penetrate bones near their ends, often at fairly specific sites; some are occupied by arteries, but most contain thin-walled veins. Within bone, the arteries are unusual in consisting of endothelium with only a thin layer of supportive connective tissue. The epiphysial and metaphysial arterial supply is richer than the diaphysial supply.

Medullary arteries in the shaft give off centripetal branches which feed a hexagonal mesh of medullary sinusoids that drain into a wide, thin-walled central venous sinus. They also possess cortical branches which pass through endosteal canals to feed fenestrated capillaries in osteons (Haversian systems). The central sinus drains into veins which retrace the paths of nutrient arteries, sometimes piercing the shaft elsewhere as independent emissary veins. Cortical capillaries follow the pattern of Haversian canals, and are mainly longitudinal with oblique connections via Volkmann’s canals (Fig. 5.17). At bone surfaces, cortical capillaries make capillary and venous connections with periosteal plexuses (Fig. 5.20). The latter are formed by arteries from neighbouring muscles which contribute vascular arcades with longitudinal links to the fibrous periosteum. From this external plexus a capillary network permeates the deeper, osteogenic periosteum. At muscular attachments, periosteal and muscular plexuses are confluent and the cortical capillaries then drain into interfascicular venules.

In addition to the centrifugal supply of cortical bone, there is an appreciable centripetal arterial flow to outer cortical zones from periosteal vessels. The large nutrient arteries of epiphyses form many intraosseous anastomoses, their branches passing towards the articular surfaces within the trabecular spaces of the bone. Near the articular cartilages these form serial anastomotic arcades (e.g. three or four in the femoral head), which give off end-arterial loops. These often pierce the thin hypochondral compact bone to enter, and sometimes traverse, the calcified zone of articular cartilage, before returning to the epiphysial venous sinusoids.

In immature long bones the supply is similar, but the epiphysis is a discrete vascular zone. Epiphysial and metaphysial arteries enter on both sides of the growth cartilage; there are few, if any, anastomoses between them. Growth cartilages probably receive a supply from both sources, and from an anastomotic collar in the adjoining periosteum. Occasionally, cartilage canals are incorporated into a growth plate. Metaphysial bone is nourished by terminal branches of metaphysial arteries and by primary nutrient arteries of the shaft which form terminal blind-ended sprouts or sinusoidal loops in the zone of advancing ossification. Young periosteum is more vascular, its vessels communicate more freely with those of the shaft than their adult counterparts, and they give off more metaphysial branches.

Large irregular bones, e.g. the scapula and innominate, receive a periosteal supply. In addition, they often have large nutrient arteries which penetrate directly into their cancellous bone: the two systems anastomose freely. Short bones receive numerous fine vessels which supply their compact and cancellous bone and medullary cavities from the periosteum. Arteries enter vertebrae close to the bases of their transverse processes. Each vertebral medullary cavity drains to two large basivertebral veins which converge to a foramen on the posterior surface of the vertebral body. Flatter cranial bones are supplied by numerous periosteal or mucoperiosteal vessels. Large thin-walled veins run tortuously in cancellous bone. Lymphatic vessels accompany periosteal plexuses but have not been convincingly demonstrated in bone.

Innervation

Nerves are most numerous in the articular extremities of long bones, vertebrae and larger flat bones, and in periosteum. Fine myelinated and unmyelinated axons accompany nutrient vessels into bone and marrow and lie in the perivascular spaces of Haversian canals. Bone has a complex autonomic and sensory innervation; osteoblasts possess receptors for several neuropeptides that are found in the nerves which supply bone, e.g. neuropeptide Y, calcitonin gene-related peptide, vasoactive intestinal peptide and substance P.

Intramembranous ossification

Intramembranous ossification is the direct formation of bone (membrane bone) within highly vascular sheets or ‘membranes’ of condensed primitive mesenchyme (Fig. 5.21). At centres of ossification, mesenchymal stem cells differentiate into osteoprogenitor cells which proliferate around the branches of a capillary network, forming incomplete layers of osteoblasts in contact with the primitive bone matrix. The cells are polarized because they secrete a fine mesh of collagen fibres and ground substance, osteoid, from the surface which faces away from the blood vessels. The earliest crystals appear in association with extracellular matrix vesicles produced by the osteoblasts; crystal formation subsequently extends into collagen fibrils in the surrounding matrix, producing an early labyrinth of woven bone, the primary spongiosa. As layers of calcifying matrix are added to these early trabeculae, the osteoblasts enclosed by matrix come to lie within primitive lacunae. New osteocytes retain intercellular contact by means of their fine cytoplasmic processes (dendrites) and, as these elongate, matrix condenses around them to form canaliculi.

Fig. 5.21 Intramembranous ossification forming the nasal bones of a 7-month human fetus. Islands of bone (solid pink matrix [M], enclosing osteocytes) enlarge through the deposition of new matrix by osteoblasts (arrows). They subsequently fuse and are remodelled by osteoclasts to form mature lamellar bone.

As matrix secretion, calcification and enclosure of osteoblasts proceed, the trabeculae thicken and the intervening vascular spaces become narrower. Where bone remains trabecular, the process slows and the spaces between trabeculae become occupied by haemopoietic tissue. Where compact bone is forming, trabeculae continue to thicken and vascular spaces continue to narrow. Meanwhile the collagen fibres of the matrix, secreted on the walls of the narrowing spaces between trabeculae, become organized as parallel, longitudinal or spiral bundles, and the cells they enclose occupy concentric sequential rows. These irregular, interconnected masses of compact bone each have a central canal, and are called primary osteons (primary Haversian systems). They are later eroded, together with the intervening woven bone, and replaced by generations of mature (secondary) osteons.

While these changes are occurring, mesenchyme condenses on the outer surface to form a fibrovascular periosteum. Bone is laid down increasingly by new osteoblasts which differentiate from osteoprogenitor cells in the deeper layers of the periosteum. Modelling of the growing bone is achieved by varying rates of resorption and deposition at different sites.

Endochondral ossification

The hyaline cartilage model which forms during embryogenesis is a miniature template of the bone (cartilage bone) that will subsequently develop. It becomes surrounded by a condensed, vascular mesenchyme or perichondrium, which resembles the mesenchymal ‘membrane’ in which intramembranous ossification occurs. Its deeper layers contain osteoprogenitor cells.

The first appearance of a centre of primary ossification (Fig. 5.22) occurs when chondroblasts deep in the centre of the primitive shaft enlarge greatly, and their cytoplasm becomes vacuolated and accumulates glycogen. Their intervening matrix is compressed into thin, often perforated, septa. The cells degenerate and may die, leaving enlarged and sometimes confluent lacunae (primary areolae) whose thin walls become calcified during the final stages (Fig. 5.23

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