Comparative Skeletal Structure

Clinton Rubin, State University of New York, Stony Brook, New York, USA Mani Alikhani, State University of New York, Stony Brook, New York, USA Janet Rubin, Emory University, Atlanta, Georgia, USA
The sophisticated organization of the skeleton achieves a structure that can withstand the extremes of functional load-bearing. The growth, development and repair of the skeletal structure is realized through the tightly regulated remodelling of bone tissue, orchestrated by cells that specifically form or resorb the matrix.

Introductory article
Article Contents
. Bone . Macroscopic Structure of Bone . Microscopic Organization . Bone Cells . Composition of Bone Matrix . Development of the Skeleton . Modelling and Remodelling . Mechanical Properties of Bone . Cartilage . Ligament . Tendon

Bone
Bone is a highly specialized form of connective tissue that provides an internal support system and facilitates locomotion. The rst vertebrates appeared on Earth in the Ordovician period, some 500 million years ago, and since that time bone has evolved to become a complex living tissue in which the extracellular matrix is mineralized, conferring marked rigidity and strength to the skeleton while still maintaining some degree of elasticity. In addition to understanding the palaeontological record, the study of bone is important because diseases of the skeleton, such as osteoporosis, are major health concerns that aect tens of millions of people. Clearly, an improved understanding of the basic science of bone will provide a tremendous opportunity to develop novel approaches to treat and reverse these crippling diseases. Bone regulates its mass and architecture to meet two critical and competing responsibilities: one structural, the other metabolic. In the rst case, the skeleton protects vital organs of the cranial and abdominal cavities; it encloses the blood-forming elements of the bone marrow, and facilitates locomotion. Second, the skeleton serves as a mineral reservoir that contains 99% of the bodys total calcium, 85% of its phosphorus, and 66% of its magnesium key elements for the function of the organism. Bone is a composite material comprised of a mineralized matrix interspersed with an organic scaold consisting primarily of collagen. There are three dierent cell types found in the bone tissue: osteoblasts, which form bone, osteocytes, which maintain bone, and osteoclasts, which are multinucleated giant cells responsible for the resorption of bone. As a structure, bone possesses several remarkable attributes, with high tensile and compressive strength similar in magnitude to that of cast iron, while, at the same time, being a relatively lightweight material, and thus ecient during movement. Bone, as an idealized structural material, can readily be appreciated by considering the forces and impact involved

. Summary

during the gallop of a thoroughbred racehorse. The canon bone (third metacarpal, towards the foot of the front limb), with a cross-sectional area of approximately 2.5 cm of mineralized tissue, successfully holds up an animal weighing 500 kg and travelling at about 50 km per hour. Yet, with all its strength, hardness and resilience, bone is a dynamic living material, constantly being renewed and reconstructed throughout the lifetime of the individual. Indeed, bone is the only tissue in the body that has the capacity to heal without forming a scar. Bone even has the ability to adapt its structure relative to its functional demands, exemplifying the paradigm of form follows function. In the positive direction, this plasticity is evidenced by the 40% more bone in the professional tennis players serving arm compared with the arm that simply throws the ball into the air. On the negative side, there is the drastic osteoporosis that results from long-term space ight, creating a signicant hurdle to extended exploration of space. The structural success of skeletal morphology can be examined eectively at various levels: by its gross anatomy and functional responsibility, by its ultrastructural morphology (cortical or cancellous), by its microscopic organization (lamellar or woven) and on the basis of its development and repair, processes achieved through endochondral ossication or intramembranous ossication.

Macroscopic Structure of Bone

On the basis of their gross appearance, bones are typically classied as either long or at. Long bones include the bones of the axial skeleton (e.g. tibia, femur, radius, ulna and humerus); at bones include the skull bones plus the sternum, scapula and pelvis. Each long bone can be
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divided into three distinct anatomical areas. The region at the end of a long bone is called the epiphysis. The metaphysis is the area just beneath the epiphysis, where the bone becomes more slender. The long shaft of the bone is the diaphysis. Characteristic of all bones are a dense outer sheet of cortical bone (alternatively referred to as cortical bone) and a central medullary cavity, which is lled with either red or yellow bone marrow. The marrow cavity is interrupted throughout its length, particularly in the epiphyseal and metaphyseal regions, by a reticular network of trabecular bone (alternatively referred to as cancellous or spongy bone). These internal trabeculae act as well-banded reinforcement rods, or internal scaolding, to buttress the outer compact bone as it becomes more slender at the articulating surfaces. Despite the dierences in their appearance, under the microscope cortical and trabecular bone can be seen to have the same basic histological structure (Figure 1). Surrounding every compact bone is an osteogenic connective tissue membrane, the periosteum, which consists of two distinct layers. The inner layer, adjacent to the actual bone surface, consists of osteoblasts and their precursors, as well as a rich microvasculature. The outer layer, which is more brous, gives rise to the Sharpey bres that penetrate the bone matrix, binding the periosteum to bone and aiding in anchoring tendons, ligaments and muscles to the bone. A single layer of bone cells, a syncitium referred to as the endosteum, covers the internal surface of bone. The endosteum, considerably thinner than the periosteum, physically separates the bone surface from the bone marrow within. The principal functions of periosteum and endosteum are nutrition of osseous tissue and provision of a continuous supply of new osteoblasts for repair or growth of bone.

Microscopic Organization
At the microscopic level, bone can be generalized into two specic morphologies: the disorganized, hypercellular woven bone, and the highly organized, relatively hypocellular, lamellar bone. Woven bone is the rst bone tissue to emerge in embryonic development as well as the rst mineralizing tissues to appear in repair process such as fracture healing. Woven bone has an irregular, disorganized pattern of collagen orientation and osteocyte distribution, indicative of the rapid manner in which it is laid down. While woven bone is characteristic of embryonic development and early stages of repair; it is also found in the healthy adult skeleton at ligament and tendon insertions. Woven bone also is evident in specic disease states which aect local bone sites such as osteogenic sarcoma or metastatic cancerous lesions. It serves a critical wound-healing role by rapidly lling osseous defects, a callus that provides the initial continuity for fractures and osteotomy segments. Within a few weeks of being deposited, woven bone is most often reabsorbed and replaced during remodelling by lamellar bone. It is this highly orchestrated process of repair and remodelling that allows bone fractures to repair without leaving a scar. Lamellar, or mature, bone characteristically shows collagen bres arranged in lamellae (37 mm thick) that are parallel to one another or concentrically organized around a vascular canal. The whole complex of concentric lamellae, or plates, of bone that surround a canal containing blood vessels, nerves and loose connective tissue, is called an osteon (Figure 2). Osteons are typically 200 300 mm in diameter, consisting of up to 20 lamellar plates. Within these mineralized plates are lacunae, small lakes that house osteocytes, the cells residing within the bone tissue. To provide added tensile strength, each lamella consists of collagen bres organized such that they are parallel to one another. Importantly, the orientation of the collagen in adjacent lamellar plates is distinct from that of its neighbour, adding tremendous strength to the composite material, a strategy similar to that used in plywood to add rigidity. The arrangement of the collagen matrix (Figure 3) ultimately determines the orientation of the bone mineral crystals. Mineralization proceeds and extends over the collagen matrix, with the long axis of the hydroxyapatite crystal parallel to the collagen bre, a process that also adds strength to the tissue. In addition to concentric lamellae, two other type of lamellae are recognized. Circumferential lamellae enclose the entire bone, forming its outer perimeter. Interstitial lamellae are interspersed between adjacent concentric lamellae and ll the spaces between them. They are actually fragments of preexisting concentric lamellae, or remnants of the early stages of woven bone. An osteon that has formed de novo is recognized as a primary osteon. If an osteon is established within the cortex via bone resorption, a process that succeeds in

Figure 1 Bone can be categorized into two morphological components: cortical and cancellous bone. The dense cortical bone envelopes the entire structure, while cancellous or trabecular bone is typically found towards the ends of the bone. The internal spaces of bone are filled with marrow. Reprinted, with permission, from Lynch SE, Genco RJ and Marx RE (1999) Tissue Engineering: Applications in Maxillofacial Surgery and Periodontics. Quintessence.

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osteoblasts, dependent on local factors such as cytokines. These osteoprogenitor cells persist throughout postnatal life and are found on or near all of the free surfaces of bones such as the inner layer of the periosteum or the endosteum. Osteoprogenitor cells are most active during the growth and development of bones but are reactivated in adult life in the repair of bone fractures, and when adaptive or remodelling processes stimulate the need for bone formation. Osteoblasts and osteocytes are thought to be incapable of division. Thus, as the population of osteoblasts depletes, they are replaced by proliferation and dierentiation of the osteoprogenitor cells.

Osteoblasts
Osteoblasts (Figure 3) are mononucleated cells, derived from the mesenchyme, that synthesize both collagenous and noncollagenous bone proteins, which all together makes up the organic matrix of bone called the osteoid. Embryonic undierentiated mesenchymal cells can give rise to cartilage, bone, muscle cells or adipocytes, dependent on the inuence of hormones and growth factors. Osteoblasts are connected to one another via extended cell processes that are interconnected via gap junctions, establishing a single continuous blanket of cells on the bone surface, all of which are in direct communication with neighbouring cells. The active osteoblast is a plump cuboidal cell with an eccentrically placed or polarized nucleus, which resides away from that part of the cell nearest to the mineralized surface. The cytoplastic elements of the osteoblast include abundant endoplasmic reticulum, a well developed Golgi body, and numerous free ribosomes that are responsible for the basophilia seen in sections stained with haematoxylin and eosin. In bone remodelling, osteoblasts are transiently active at sites of reformation of bone. These features help to distinguish osteoblasts from the mesenchymal precursors (preosteoblasts), which are also found on bone surfaces (Figure 4). These bone-lining cells are attened spindleshaped cells with oval nuclei and few organelles. Bonelining cells cover bone surfaces, particularly in the adult skeleton, that are quiescent in terms of bone formation and resorption. In certain circumstances, such as when exposed to growth factors or other anabolic stimuli, they proliferate and dierentiate into osteoblasts. The osteoblastic lineage comprises several cells at dierent stages of dierentiation, including osteoprogenitor cells, preosteoblasts and osteoblasts according to the progressive expression of markers of the osteoblast phenotype. Preosteoblasts express markers such as alkaline phosphatase, osteopontin and collagen type I, whereas mature postmitotic osteoblasts express osteocalcin. A temporal sequence of expression of genes encoding osteoblast phenotype markers has dened three distinct
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Figure 2 Diagram depicting a section of the cortical shaft of a long bone, showing the arrangement of the lamellae in the osteons, the interstitial lamellae, and the outer and inner circumferential lamellae. The outer surface is protected by the periosteum, while the inner surface is covered by the endosteum. Within the cortical shell, the branching out of the buttressing trabeculae can be seen. Reprinted, with permission, from Bloom W and Fawcett DW (1986) A Textbook of Histology, 11th edn. Saunders.

replacing preexisting bone tissue, it is referred to as a secondary osteon, or haversian system. The bulk of bone tissue within the adult human is haversian, as it has been remodelled and replaced over the lifetime of the skeleton. In the adult skeleton, when bone tissue needs to be repaired or replaced, it undergoes a process of remodelling. Bone remodelling is an intricate, interdependent, highly orchestrated process that begins by the removal of tissue by osteoclasts and the tightly coupled replacement of tissue by osteoblasts. In the adult skeleton, approximately 5% of the skeleton is constantly involved in bone remodelling, or the turnover of bone tissue.

Bone Cells
Bone is composed, in essence, of four dierent cell types: osteoblasts, osteoclasts, osteocytes and the bone-lining cells. Osteoblasts, osteocytes and bone-lining cells originate from local osteoprogenitor cells, whereas osteoclasts arise from the fusion of mononuclear precursors, which originate in the various haemopoietic tissues. Osteoprogenitor cells develop from embryonic mesenchymal stem cells and are able to proliferate and dierentiate into chondroblasts (cartilage-forming cells) or

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Comparative Skeletal Structure

Figure 3 Bone may be categorized into three microstructural components: (1) bone cells, which include osteoblasts, osteocytes and osteoclasts (stained with a modified Goldner trichrome stain); (2) an organic matrix consisting of collagenous and noncollagenous factors, such as the bone morphogenetic proteins (the mineralized matrix has been removed and cells have been coloured green to distinguish them from the organic framework); (3) an inorganic component consisting primarily of calcium and phosphate; this component has been stylized as an array of hexagonal crystals. Reprinted, with permission, from Lynch SE, Genco RJ and Marx RE (1999) Tissue Engineering: Applications in Maxillofacial Surgery and Periodontics. Quintessence.

periods: a growth period (proliferation), a period of matrix development and a mineralization period. A period of active proliferation is reected by mitotic activity with expression of cell cycle and cell growth-regulated genes (e.g. histone, c-myc, c-fos and AP-1 (alkaline phosphatase) activity). During this proliferation period, and fundamental to development of the bone cell phenotype, several genes associated with formation of the extracellular matrix (type I collagen, bronectin and transforming growth factor (TGF) b) are actively expressed, and these proteins are ultimately key to maintaining the intricate balance between bone formation and resorption. In bone, type I collagen, a heterotrimer of one a2 (I) and two a1 (I) chains, makes up about 90% of the total organic matrix. It is the collagen, with its organization, that provides a great deal of the tensile strength of bone. Therefore, even subtle variations in the composition or content of the collagen can have devastating eects. For example, mutations in the gene coding for the a1 (I) chain, as well as mutations in the gene coding for the a2 (I) chain, can cause osteogenesis imperfecta, an aiction that makes bone very susceptible to fracture. Immediately after the downregulation of osteoblast proliferation reected by the decline in deoxyribonucleic
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acid (DNA) synthesis and histone gene expression, the expression of alkaline phosphatase, a protein associated with the bone cell phenotype, increases greater than 10fold. During this period the extracellular matrix undergoes a series of modications in composition and organization that facilitate the mineralization of the matrix. The onset of mineralization is also signalled by the induction of several other genes, such as osteopontin and osteocalcin. It is likely that dierentiation along the osteoblast lineage involves osteoblast-specic transcription factors (OSFs) that have yet to be identied. OSF2 is a cis-acting element in the promoter of the mouse osteocalcin gene that binds a factor present only in nuclear extracts of osteoblasts and confers osteoblast-specic activity. Several studies have indicated that Osf2/Cbfa1 is encoded by the gene for Cbfa1. Mutation in OSF2/CBFA1 can result in the abnormal skeletogenesis seen in cleidocranial dysplasia and transgenic mice who have Cbfal knocked out are unable to ossify their cartilaginous skeletons. In addition to secreting several matrix components, including type I collagen, proteoglycans, osteocalcin, osteonectin and osteopontin, osteoblasts also produce growth factors that have important autocrine and paracrine eects on bone growth and remodelling (Figure 4).

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Hematopoetic stem cell (CFU-GM)

Monocytic precursor CFU-M Macrophage

+ Resorption RANKL MCSF Interleukins

+ Formation BMPs Growth factors Interleukins

Osteoblast precursors Osteoclast precursors Osteoclast Osteoblasts Bone and bone matrix Resorption pit

Figure 4 Osteoclasts (red) and osteoblasts (dark green) interact through cytokines released into the bone micromilieu. Macrophages secrete macrophage colony-stimulating factor (MCSF), various interleukins and tumour necrosis factor, all of which promote osteoclast differentiation from haematopoietic stem cells, from the colony forming unit for granulocyte-macrophages (CFU-GM) and the CFU-M (CFU for macrophage) to terminal osteoclast phenotype. Osteoblasts interact by expressing factors which affect osteoclasts, mainly RANKL and MCSF, as well as factors affecting bone mineralization and progression of their own phenotype, such as insulin-like growth factors and basic fibroblast growth factors. Importantly, disuse will also upregulate osteoclast activity, while increases in mechanical factors will elevate bone formation.

They also have surface receptors for a variety of hormones, vitamins and cytokines, which inuence their activity. As the osteoblasts produce osteoid, they often become engulfed in their own matrix and dierentiate into osteocytes.

Osteocytes
The osteocyte (Figure 3) is a mature osteoblast entrapped within the bone matrix, and is now believed to be largely responsible for maintaining the viability of the tissue. Not only can osteocytes sense alterations in physical or chemical conditions (and thus orchestrate the recruitment of osteoblasts and osteoclasts), it has been recently demonstrated that these cells can both synthesize and resorb matrix, thus modifying their own immediate physical and chemical environment. Osteocytes reside in lacunae within the matrix and are 11by far the most abundant cells of mature bone. As osteoblasts become entombed in their own matrix and dierentiate into osteocytes, the cells diminish in size and lose many parts of their cytoplasmic organelles. Within each osteon, osteocytes are connected to each other via long, slender, branched cytoplasmic processes and gap junctions. These cytoplasmic extensions pass through a network of catacombs, called canaliculi, which connect adjacent lacunae. These interconnecting canaliculi are ideal pathways for chemical, electrical and stress generated uid communication through the dense bone matrix.

Osteocytes express cellcell channels called connexins, through which small molecules such as second messengers can pass, suggesting that networks of osteocytes are in intricate communication with one another. Considering the skeletons ability to adapt to changes in its functional environment, it is important to consider the means by which the bone tissue can perceive and respond to mechanical signals. Given their preponderance in bone, and their intricate three-dimensional network, osteocytes are most probably the key mechanosensor element in bone. Loading of bone results in strain, or deformation, in the matrix. This deformation may evoke cellular responses, either directly or indirectly via uid shear stress produced by increased uid ow in the lacunocanalicular system or electrical strain potential. Osteocytes may orchestrate the overall remodelling response by secreting key factors, such as prostaglandins, nitric oxide and insulin-like growth factors (IGFs). These, and certainly other yet unknown factors, activate the bone remodelling system of osteoblasts and osteoclasts, and thus demonstrate the balance between bone forming and resorbing cells. Recent evidence suggests that osteocytes can metabolically manipulate their environment more or less independent of surface resorption and accretion. This ability is important to the cellular regulation of calcium exchange. Most of the bone crystals, buried away from the endosteal and periosteal bone surfaces, appear to be unavailable to eect the necessary mineral exchange with extracellular uid, making it dicult to explain the immediate exchange
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of bone mineral with the extracellular uid. There is, however, a vast surface area on the Haversian canal and lacunar walls, and an even larger area on the canalicular walls, where bone mineral exchange with extracellular uid can take place. Nevertheless, when major reorganization of bone tissue is called for, the bone resorption is performed by the osteoclast.

Osteoclasts
Osteoclasts (see Figure 3) are the major resorptive cells of bone and are characterized by their large size and multiple nuclei. They are specialized monocyte/macrophage family members that dierentiate from haematopoietic precursors. Whereas monocytes are mononuclear cells, osteoclasts form from the fusion of monocytes. Terminal dierentiation in this lineage is characterized by the acquisition of mature phenotypic markers, such as the calcitonin receptor, tartrate-resistant acid phosphatase (TRAP), morphological conversion into large multinucleated cells, and the capacity to form resorption lacunae on bone. An osteoclast is a strongly polarized cell with a paucity of rough endoplasmic reticulum, a moderate number of ribosomes, numerous smooth vesicles, and well developed mitochondria. These cells occupy shallow concavities, called Howship lacunae, which are created by the erosive action of the osteoclast on the underlying bone. Osteoclasts typically attach only to fully mineralized bone matrix, and are not able to resorb bone that is not fully mineralized. The osteoclast has three dierent domains: rued border, sealing zone and smooth basolateral border. The rued border is the central, highly infolded area of the plasma membrane where secretion for bone resorption takes place. Osteoclasts appearing some distance from the surface of bone do not have rued borders and are called inactive or resting osteoclasts. The sealing zone is a microlamentrich, organelle-free area of the plasma membrane that surrounds the rued border and serves as the point of anchoring of the osteoclast to the underlying bone matrix achieved by integrin attachment. This sealing zone creates a closed subosteoclastic compartment, or pocket, between the cell and bone that permits a conned space subject to resorption. Osteoclasts not only attach to the bone: they can move along the surface, thus increasing their range of action. During the movement of the osteoclast, the seal must remain intact in order for resorption to continue. Once activated, osteoclasts resorb bone by isolating an area of bone under the region of cell attachment. The osteoclasts then actively reduce the pH of the local environment by production of hydrogen ions through the carbonic anhydrase system. The lowered pH increases the solubility of the apatite crystals, in essence dissolving the mineral. After the mineral is removed, the organic components of the matrix are hydrolysed through acidic
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proteolytic digestion. The reconstruction of bone requires seven to ten generations of osteoblasts to follow and ll the resorption space. Absence of osteoclasts, or a population of dysfunctional osteoclasts, leads to osteopetrosis, or marble bone disease, which can be fatal in childhood. Devoid of osteoclasts, the development of bone through endochondral ossication fails to create a marrow cavity, thereby eliminating the space necessary for the production of all haematopoietic elements. Many factors are involved in regulating osteoclast recruitment from marrow precursors. Two factors predominate: MCSF and RANKL. MCSF is necessary to support proliferation, early dierentiation and survival of the monocytic progenitors of osteoclasts. Loss of MCSF action engenders deciencies in both osteoclast and macrophage recruitment, as well as dierentiation. MCSF alone is unable to generate osteoclasts in in vitro culturesystems; further progression into the osteoclast lineage requires the presence of RANLK which binds to RANK receptors on the monocytic precursors, inducing late dierentiation and fusion. Expression of RANKL on the surface of bone stromal and osteoblast cells is controlled by hormones that regulate calcium balance, such as PTH and vitamin D. Increased PTH causes increased expression of RANKL, leading to increased osteoclastogenesis. RANKL can also be expressed on lymphocytes and may be responsible for hypercalcaemia associated with haematopathologic diseases. Bone cells also secrete a protein called osteoprotegerin (OPG), which serves as a decoy receptor for RANKL, preventing its action on monocytic progenitors. Overexpression of OPG is thus associated with decreased bone turnover. Many other factors are capable of regulation of osteoclast formation, including cfos, a ubiquitous nuclear transcription factor whose absence prevents late osteoclast dierentiation, and TNFa, which can support osteoclastogenesis even in the absence of RANKL. Systemic factors such as vitamin D, parathyroid hormone and tumour necrosis factor can enhance the development of osteoclasts from haematopoietic progenitor cells in the presence of stromal elements from bone, largely by up-regulating bone expression of RANKL, but also aecting cytokines such as the interleukins. Together, the osteoblasts, osteoclasts and osteocytes work together to lay down, maintain and remodel the bone matrix.

Composition of Bone Matrix

The composite structure of bone allows it to withstand compressive and tensile stresses, as well as bending and torsional moments. The inorganic phase of bone, with hydroxyapatite crystals arrayed in a protein matrix, provides the ability to resist compression. Individual calcium phosphate crystals of multiple sizes are imbedded

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in and around the brils of the collagen type I lattice. Hydroxyapatite crystal, while eectively resisting compressive loads, has a poor ability to withstand tensile loads. As in concrete, a material that excels at resisting compression but is poor in resisting tension, tensile elements (e.g. steel reinforcing rods) are added to create a composite material that can cope with complex loading environments. As discussed, in the case of bone, this tensile strength arises from collagen brils organized into lamellae. The collagen orientation between adjacent lamellae can rotate by as much as 908, permitting the tissue to resist forces and moments acting from several dierent directions, much like the added strength in plywood realized by the distinct orientation of the bres in each specic ply. While the ultrastructural organization is, to a certain extent, dened by the genome, the functional environment also contributes to the distribution of lamellae, as well as the osteons that house them. This directed deposition of collagen adds to the anisotropy of the bone. Given that more than 80% of functional strains are due to bending (and thus a high percentage of strain is tensile), the structural quality of the bone may ultimately be determined by the quality of the collagen and the organization of the microarchitecture. Recent studies have shown that collagen itself deteriorates with age, and undoubtedly contributes to the declining material properties of the skeleton. Before its calcication, newly synthesized bone matrix is essentially completely organic and is called osteoid. Collagen type I is the predominant organic component in bone, accounting for approximately 94% of the unmineralized matrix. Collagen bres are embedded in a ground substance which is rich in proteoglycans, chondroitin sulfate, keratan sulfate and hyaluronic acid. Other noncollagenous proteins most often found in bone are found in osteoid, and account for approximately 4% of its weight. These include glycoproteins and phosphoproteins such as osteonectin; sialoproteins, which are predominantly osteopontin; bone Gla protein, also called BGP (bone gamma-carboxyglutamic acid-containing protein) or osteocalcin; and bone morphogenetic protein. Extracts of bone also include enzymes, hormones, growth factors and other metabolites essential for bone metabolism. It is even thought that many of these molecules serve as coupling factors, which, when released from the bone matrix during osteoclastic resorption, serve to stimulate recruitment and dierentiation of osteoblasts. The inorganic component of bone (see Figure 3) is composed principally of a calcium phosphate mineral analogous to crystalline calcium hydroxyapatite, Ca10(PO4)6(OH)2. This crystallite structure is available for the exchange of ions. Thus magnesium and sodium can substitute in the calcium position, uoride and chloride in the hydroxyl position, and carbonate in both the hydroxyl and phosphate positions. These small amounts

of impurities in hydroxyapatite may alter certain physical properties of the crystal, such as solubility. For example, uoride substitution decreases the solubility of the crystallites, whereas carbonate increases it. Many investigators believe that the impurities of the crystalline matrix are critical to enabling the osteoclast to resorb the tissue.

Development of the Skeleton

Skeletal development requires the exquisite coordination of many distinctive cell types and the orchestration of their cellular growth, dierentiation, apoptosis, production of extracellular matrix and remodelling. Formation of the skeleton (ossication) occurs by either a direct (intramembranous) or an indirect (endochondral) process. Intramembranous ossication occurs during embryonic development by the direct transformation of mesenchymal cells into osteoblasts. The rst event of this process is the migration of undierentiated mesenchymal cells into an area destined to become bone. Mesenchymal cells condense, the surrounding tissue becomes vascularized, and cells dierentiate directly into osteoblasts. Intramembranous ossication begins in the centre of the mesenchymal cell concentration. The osteoblasts then synthesize the osteoid in which hydroxyapatite crystals are formed by calcium and phosphate ion enrichment. Collagen molecules secreted by the osteoblasts during embryonic bone deposition polymerize extracellularly to form numerous randomly oriented brils throughout the trabeculae. The early intramembranous bone, in which the collagen bres run in all directions, is described as woven bone. Intramembranous ossication occurs primarily in the neurocranium (which forms a protective case around the brain) and viscerocranium (which forms the skeleton of the face) and part of the clavicle. In endochondral ossication, the condensed embryonic mesenchyme transforms rst into a cartilage anlage (a population of cells that constitutes the beginning of a tissue), which reects both the position and form of the eventual bone at that site. During mouse embryonic development the undierentiated cells initially express mesenchymal matrix (e.g. collagen types I and III) but, gradually, cells in the central region dramatically change their phenotype and become large and round. As these prechondrocytes dierentiate further, their cytoplasmic volume, endoplasmic reticulum and Golgi complex increase and they switch from the production of mesenchymal matrix to the production of cartilaginous matrix (e.g. collagen types II, IX, XI and matrix Gla protein) and several other matrix proteins. These chondrocytes then undergo a programme of dierentiation, which includes hypertrophy, expression of type X collagen and decreased expression of type II collagen. The cartilage anlage grows rapidly through appositional and interstitial growth, and
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resembles the future bone in shape, but, as it grows in size, the cartilage anlage becomes too big to support the chondrocytes purely through diusion, and the hypertrophic cells begin to die o and ossify. Through endochondral ossication, cells from the perichondrium dierentiate into osteoblasts and begin to deposit a thin layer of bone, appearing as a collar, around the cartilage (Figure 5). Concurrently, mononuclear cells (monocytes) dierentiate into chondroclasts, which begin to break down the ossifying cartilage. From the perichon-

drium, the invasion of vascular channels begins, a key process called angiogenesis. As the hypertrophic chondrocytes continue to die through apoptosis, the osteoblasts brought in by the blood vessels start the process of depositing bone tissue just as if bone were forming on the surface of adult bone. The development of bone through endochondral ossication, therefore, allows the rapid expansion of a bone template, through both interstitial and appositional growth. This swelling could not occur if the tissue was originally bone, as the mineralized matrix restricts growth only to surfaces. The replacement of the dying cartilage with mineralizing bone, however, allows skeleton to full its responsibilities as a calcium reservoir and a weight-bearing structure.

Mineralization of bone tissue

The mineralization of bone begins approximately 1015 days after the organic osteoid matrix has been laid down. At this point, mineral increases almost immediately to 70% of the ultimate content, whereas deposition of the nal 30% takes several months. While the actual process of mineralization is not completely understood, it is believed that two mechanisms are involved. The rst involves a structure called the matrix vesicle, and the second is heterogeneous nucleation. Matrix vesicles are small, round, extracellular lipidbilaminar bound organelles, which bud from hypertrophic chondrocytes or osteoblasts undergoing the process of apoptosis as well as from cell processes originating from the plasma membrane. There is a denite polarity to the vesicles, with mineralization occurring in a predictable and organized way adjacent to the requisite phosphatases on the inner leaet of the membrane. The matrix vesicles contain high levels of alkaline phosphatase, adenosine triphosphatase (ATPase), inorganic pyrophosphatase, 5nucleotidase and ATP-pyrophosphohydrolase, in addition to phospholipids (especially phosphatidylserine), which have a strong anity for calcium ions. It is believed that these ions accumulate in the matrix vesicle because of their anity for the phospholipids and a membrane-bound calcium pump. At a point of supersaturation, nucleation of the mineral begins. Alkaline phosphatase, a biosynthetic product of osteoblasts, is present in very high concentrations during development and osteoid production. The regulatory role of this disulde-linked dimer is not known, but its presence may increase the local concentration of P (phosphate) and thereby facilitate hydroxyapatite deposition. Increasing the concentration of P in the micromilieu exceeds the local solubility product and catalyses deposition along the inner leaet of the vesicle. Following this accretion, the destruction of the vesicles membrane has been attributed to an increasing concentration of lysophospholipids within

Figure 5 Development of a long bone as shown in longitudinal sections (A J), and in cross-sections A, B, C and D. Pale blue is cartilage; purple, calcified cartilage; black, bone; red, arteries. A, The original cartilage model of the bone; B, a periosteal collar of bone appears before any calcification of cartilage occurs; C, cartilage begins to calcify; D, vascular mesenchyme enters the calcified cartilage and divides it into two zones of ossification (E and F); G, blood vessels and mesenchyme penetrate the epiphyseal cartilage and the epiphyseal ossification centre develops within it; H, a similar ossification centre develops in the lower epiphyseal cartilage; as the bone ceases to grow in length, the lower epiphyseal plate disappears first (I) and then the upper epiphyseal plate (J). The marrow cavity then becomes continuous throughout the length of the bone, and the blood vessels of the diaphysis, metaphyses and epiphyses intercommunicate. Reprinted, with permission, from Bloom W and Fawcett DW (1986) A Textbook of Histology, 11th edn. Saunders.

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the matrix vesicles, which suggests that vesicles are programmed to self-destruct. Following dissolution of the matrix vesicle membrane, the hydroxyapatite crystals are exposed to the extravesicular environment, where additional mineral accretes to the newly formed crystal. The crystal is then believed to move chemotactically toward and to bind preferentially at the gap between collagen brils, precipitated by the nesting osteonectin and bronectin. Mineralization proceeds and extends over the collagen matrix, with the long axis of the hydroxyapatite crystal parallel to the collagen bre. In the second mechanism, apatite crystallites are deposited in the gap zones at the ends of collagen molecules, where the mineral rst appears. Initially, these gaps are lled with proteoglycans, which bind to calcium. The proteoglycans are removed enzymatically, leaving behind the calcium. At next step, phosphoproteins bind to the collagen. Dephosphorylation of the phosphoproteins (as a result of alkaline phosphatase activity) provides the additional phosphate ions for nucleation and crystal growth.

Modelling and Remodelling

Both trabecular and cortical bone grow, change and turnover through two fundamentally distinct mechanisms: modelling and remodelling. Bone modelling typically refers solely to the deposition of new bone without bone resorption. Bone remodelling, in contrast, is a specic, coupled sequence of resorption and formation events that replaces previously existing bone. As this constant turnover and replenishment of damaged tissue is critical to retaining bone strength, it is clear that disruption of these processes through age and disease will ultimately compromise the structural viability of the skeleton.

The mechanism for internal remodelling of dense compact bone is via axially oriented cuttinglling cones. The cuttinglling cone has a front of osteoclasts that bore through the bone, followed by a tail of osteoblasts that coordinately lay down osteoid and begin the formation of the new secondary osteon (Figure 6). A group of osteoblasts, osteocytes and osteoclasts that are linked and participate in remodelling (activation, resorption and formation) of a discrete area of bone is called a bone modelling unit (BMU). Traumatic or surgical wounds usually results in an intense, but localized, modelling and remodelling response. This process, referred to as a regional acceleratory phenomenon (RAP), is an important aspect of the postoperative healing process. In contrast, the slow but persistent remodelling of adult bone involves approximately 5% of skeleton at any given time, progressing through activation of a resting site, resorption of the bone, reversal of the process nishing with formation to ll in the pocket of excavated bone (ARRF). Both modelling and remodelling are controlled by an interaction of metabolic and mechanical signals. Bone modelling is patterned by temporal expression of structural genes followed by the integrated biomechanical control of functional applied loads. However, hormones and other metabolic agents have a strong secondary inuence, particularly during growth and advanced ageing. Paracrine and autocrine mechanisms, such as local growth factors and prostaglandins, are capable of temporarily overriding the mechanical control mechanism during wound healing. Remodelling responds to metabolic mediators such as parathyroid hormone and oestrogen, primarily by varying the rate of bone turnover. Certainly, a combination of mechanical signals, such as duration, magnitude and distribution of loads, also inuence remodelling and the form and function of bone.

Resorption cavity

Osteoblasts form new bone

Advanced filling cone

Completed secondary osteon

Figure 6 The cutting filling cone has a head of osteoclasts that cut through the bone, and a tail of osteoblasts that form a new secondary osteon. The velocity through bone is determined by measuring between two tetracycline labels (1 and 2) administered 1 week apart. Reprinted, with permission, from Graber T and Vanarsdall RL (2000) Orthodontics: Current Principles and Techniques, 3rd edn. Mosby.

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Comparative Skeletal Structure

Mechanical Properties of Bone

Bone, as an organ, needs to be both sti (to resist deformation) and tough (to prevent crack propagation). However, there is a compromise between these two objectives, as they are attained through a balance between the resiliency to crack propagation provided by collagen and the resiliency to deformation provided by mineral. For normal tensile or compressive loading, the stiness of the material, or elastic modulus, for human haversian bone is approximately 17.0 GPa (gigapascals, or 109 pascals) in the longitudinal direction, 11.5 GPa in the transverse direction and 3.3 GPa in shear. The degree of mineralization (young bone) or porosity (old bone) compromises the stiness of the bone and thereby lowers the elastic modulus. However, the eective modulus of the bone can compensate for decreased stiness through changes in morphology, such as the periosteal expansion seen with ageing. Bone tissue has the capacity to adapt to its functional environment. The term adaptive bone remodelling refers to changes in bone mass and geometry in response to an alteration in the bones mechanical environment. The most familiar examples are increased bone mass with certain kinds of exercise (such as jumping or tournament-level tennis playing) and bone loss following disuse (such as bed rest or space ight-induced disuse). Although vertebrate design and function are diverse, at the level of small volumes of tissue all loads and bending moments resolve into strain. Strain, a dimensionless unit of change in length divided by its original length, is used in bone physiology as 10 2 6 strain, or microstrain. The yield strain of bone, or the degree of deformation at which the bone does not recover elastically, is approximately 7000 microstrain; that is, a 0.7% change in length causes irreversible damage to the tissue. Ultimate strain in bone, or the degree of deformation at which the material actually fractures, is 15 000 microstrain. Strain generated in long bones by vigorous activity rarely exceed peaks of 3000 microstrain, indicating a safety factor to failure of approximately 23-fold. The similarity in peak strain magnitude of 20003500 microstrain has been called dynamic strain similarity, and suggests that skeletal morphology and locomotion character combine to elicit a very specic and perhaps benecial level of strain. It is also important to emphasize that the great majority of strain elicited in bone is due to bending ( 4 85%). Therefore, assumption that skeletal elements are subject solely to compression must be approached with caution. Although the nature of this structurefunction relationship is only poorly understood, it has been proposed that bone remodelling is continually inuenced by the level and distribution of the functional strains within the bone. Alteration in bone mass, turnover and internal replacement are sensitive to changes in the magnitude, distribution and rate of strain generated within the bone tissue. For example, static load applied continuously on bone
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produces an eect on remodelling activity that is no dierent from that produced by disuse. For a loading regimen to be anabolic, it must be dynamic in nature; static loads do not inuence bone morphology. In parallel with studies trying to identify pharmacological agents to inhibit the bone loss that follows the menopause or ageing process, several investigators are attempting to dene exercise studies or mechanical interventions that will serve as strong anabolic agents to resist osteoporosis.

Cartilage
Cartilage is a special type of connective tissue which has a sti and rm, but not hard, extracellular matrix. In contrast to bone, cartilage is neither vascular nor calcied. Cartilage provides three basic functions. It gives exible support in appropriate anatomical places (the nasal tip, ear lobe, thoracic cage, tracheal rings); it is a pressure-tolerant, extremely low friction tissue located in specic skeletal areas where direct pressure and articulation occur (e.g. surfaces of bones); and it functions as a site of rapid growth in conjunction with many bones undergoing elongation. Cartilage consists of cells called chondrocytes, and an extensive extracellular matrix composed of bres and ground substance. Chondrocytes synthesize and secrete the extracellular matrix, and the cells themselves are located in matrix cavities, called lacunae, just as in bone. It is the extracellular matrix with its embedded bres that not only gives cartilage its resiliency, but also allows it to bear weight and to achieve considerable tensile strength. Collagen, hyaluronic acid, proteoglycans and small amounts of several glycoproteins are the principal macromolecules present in all types of cartilage matrix. Cartilage has no vasculature, lymphatic network or innervation of its own. Chondrocytes are nourished by diusion of oxygen and nutrition through the matrix from blood vessels located in surrounding connective tissues. Because diusion is the only source for oxygen and nutrition, the maximum width of the cartilage is limited. However, vessels may pass through cartilage without supplying it directly. Cartilage, like bone, derives from the mesenchyme. The rst modication observed is the rounding up of the mesenchymal cells, which retract their extensions, multiply rapidly, and form mesenchymal condensations. The cells formed by this direct dierentiation of mesenchymal cells are called chondroblasts. Synthesis and deposition of the matrix then begin to separate the chondroblasts from one another. The dierentiation of cartilage takes place from the centre outward; therefore, the more central cells have characteristics of chondrocytes, while the peripheral cells are typical chondroblasts. The growth of cartilage is attributable to two processes: interstitial growth, resulting from the mitotic division of preexisting chondrocytes, and appositional growth, resulting from the dierentiation of perichondrial cells.

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Comparative Skeletal Structure

Like bone, cartilage is sensitive to mechanical stimuli. The synthesis of the extracellular matrix and maintenance of the tissue benets from strain, promoting diusion and eliciting electrical potential. However, too much strain can be damaging to the cartilage, causing degeneration and leading to osteoarthritis. Based on the amount of bre and ground substance, and the type of bres, three varieties of cartilage are distinguished: hyaline cartilage, elastic cartilage and brocartilage.

Except in the articular cartilage of joints, all hyaline cartilage is covered by a layer of dense connective tissue, the perichondrium, which is essential for the growth and maintenance of cartilage. It is rich in collagen type I bres and contains numerous chondroblasts that easily dierentiate into chondrocytes.

Elastic cartilage and fibrocartilage

Elastic cartilage is typically found in regions requiring a exible form of support: the auricle of the ear, the walls of the external auditory canals and eustachian tubes, the epiglottis, and the cuneiform cartilage in the larynx. Basically, elastic cartilage is identical to hyaline cartilage except that, in addition to collagen brils, it contains an abundant network of ne elastin bres. Fibrocartilage has characteristics intermediate between those of dense connective tissue and hyaline cartilage. It is found in intervertebral discs, in attachments of certain ligaments to the cartilaginous surface of bones, and in the pubic symphysis. Chondrocytes occur singly or in rows between large bundles of collagenous bres. Fibrocartilage matrix contains a great number of coarse type I collagen bres, contributing to the elasticity, toughness, resiliency and strength of the structure.

Hyaline cartilage
Hyaline cartilage is the most common form of cartilage. As discussed, it forms the majority of the temporary skeleton, as the cartilage anlage, until it is gradually replaced by bone. Further, the epiphyseal plate, located between the metaphysis and the epiphysis of growing long bones, is composed of hyaline cartilage, which is responsible for the longitudinal growth of bone. In adults, hyaline cartilage is found in the nasal septum, larynx, trachea and bronchi of the respiratory system, on the articulating surfaces of bones (articular cartilage) and on the ventral ends of the ribs where they attach to the sternum. A dening characteristic of adult skeleton is that the growth plate has been mineralized, preventing further growth of long bones. The extracellular matrix of hyaline cartilage contains primarily type II collagen, but several other types are also present. Type XI collagen forms a core for the type II collagen bril, and contributes to the control of bril growth. Type IX collagen, which is really a form of a proteoglycans molecule, is found periodically along the type II collagen bril and is covalently cross-linked to it. Collagen types VI, X, XII and XIV have also been discovered in cartilage. Cartilage proteoglycans contain chondroitin 4-sulfate, chondroitin 6-sulfate and keratan sulfate, covalently linked to core proteins. These core proteins are in turn the attachment points for glycosaminoglycans (GAGs). Most of these proteoglycans are noncovalently associated with long molecules of hyaluronic acid, forming proteoglycan aggregates (aggrecan) that interact with collagen. The high content of water bound to the negative charges of GAGs serve, in essence, as a shock absorber that is of great functional importance, especially in articular cartilages. In fact, it is believed that age-related degeneration of GAGs contributes to the progressive destruction of cartilage in the sense that it loses its resiliency to impact-loading. In contrast to bone, once damaged, cartilage is not able to repair itself unfortunately this is why so many athletes require surgery in midlife. The amount of strain that causes cartilage to fail is not known, but it is believed that this tissue is rarely strained more than 25%. Another important component of cartilage matrix is the glycoprotein chondronectin, a macromolecule that promotes the adherence of chondrocytes to matrix collagen.

Ligament
Ligaments are tough bands of brous connective tissue that tether bones together and support organs. Ligaments originate and insert on bone. From a skeletal point of view, their principal function is to maintain correct bone and joint geometry. The teres ligament helps to contain the femoral head in the pelvis, while ligaments such as the cruciates and collaterals bind the bones around the knee together. Ligaments, together with their associated joint capsules (composed of similar material), are often referred to as passive joint stabilizers, and together with the articular contours determine a joints range of motion. A second function of ligamentous tissue is proprioception. Proprioceptive receptors in ligament permit the sensing of joint position, the monitoring of ligament tension and integrity, and the initiation of protective reexes. Ligament cells are called broblasts, which also derive from mesenchymal stem cells. They are generally oriented longitudinally along the length of the ligament body. Fibroblasts are responsible for synthesizing and degrading the ligament matrix in response to various stimuli. The matrix comprises virtually the entire body of the ligament. It consists of water, collagen, proteoglycans, bronectin, elastin, actin and a few other glycoproteins. Water makes up approximately two-thirds of the wet weight of a ligament. Collagen (primarily type I with some type III) comprises approximately 7080% of the dry weight of
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Comparative Skeletal Structure

ligament, and elastin a further 1015%. In contrast to bone being subject to strains of 0.3% (3000 microstrain), ligaments can stretch up to 5% (50 000 microstrain) before they are damaged.

Tendon
Tendons are anatomically and functionally discrete, very complex, dense collagenous structures that connect muscles to bones. Collagen molecules combine to form ordered units of microbrils, subbrils and brils. These units are arranged in closely packed, highly ordered, parallel bundles that are oriented in a distinct longitudinal pattern, with proteoglycans and glycoproteins in association with water incorporated in a matrix, binding the brils together. Tendons contain a relatively poor blood supply and few cells, making the tissue relatively hypometabolic. Tendons contain a small number of nociceptive and proprioceptive nerves, particularly near their attachment sites. One of these specialized sensory endings in tendon is called the Golgi tendon organ, which senses tension and provides feedback control that alters muscle activity. Golgi tendon organs normally have a somewhat steady rate of impulse transmission to the spinal cord, one that is proportional to the degree of muscle tension. However, if the tension is suddenly increased, an intense response of the tendon organ results in a reex inhibitory eect on the muscle that presumably prevents the development of potentially destructive excessive tension. Energy storage is another important function of tendon. The distinctly kinked morphology of the collagen within the tendon is believed to facilitate the elastic storage and recovery of mechanical strain energy, much like a spring. In a runner, the tendons of the lower limb muscles are stretched when the ball of the foot strikes the ground, and the kinetic energy is stored in the elastic components of the tissue. At lift-o, the recoil of the tendon to its original length helps to push the foot o the ground at the beginning of the next stride. Unlike muscle contraction, the elastic energy of tendon recoil requires no energy input. The composition of tendon is similar to that of ligament. Collagen comprises as much as 85% of tendon dry weight; of this 95% is type I and 5% type III and/or type V. Unlike ligament, elastin constitutes less than 3% of tendon dry weight.

mineralization, growth and adaptation, all contribute to this elaborate balance of modelling and remodelling. Systemic conditions such as ageing or the menopause may interfere with any number of these processes, ultimately contributing to the aetiology of diseases such as osteoporosis. Thus, caution must be exercised when considering interventions that inuence a single aspect of this elaborate balance (e.g. calcitonin disruption of osteoclast activity) which may, in turn, interrupt the ability of remodelling to function appropriately. Hopefully, it is clear that the osteoblast, osteocyte and osteoclast together, and not alone, are responsible for the skeletons success as a dynamic mineral reservoir and structural material extraordinaire.

Further Reading
Alsina M, Guise T and Roodman GD (1996) Cytokine regulation of bone cell dierentiation. Vitamins and Hormones 52: 6398. Baron R, Ravesloot JH, Neef L et al. (1993) Cellular and molecular biology of the osteoclast. In: Noda M (ed.) Cellular and Molecular Biology of Bone, pp. 445495. San Diego: Academic Press. Bilezikian JP, Raisz LG and Rodan GA (eds) (1996) Principles of Bone Biology. San Diego: Academic Press. Buckwalter JA, Einhorn TA and Sheldon RS (eds) (2000) Orthopaedic Basic Science. Rosemont, Illinois: American Academy of Orthopaedic Surgeons. Cowin SC (1989) Bone Mechanics. Boca Raton, FL: CRC Press. Ducy P and Karsenty G (1998) Genetic control of cell dierentiation in the skeleton. Current Opinion in Cell Biology 10: 614619. Ferguson CM, Miclau T, Hu D, Alpern E and Helms JA (1998) Common molecular pathways in skeletal morphogenesis and repair. Annals of the New York Academy of Sciences 857: 3342. Hall BK and Miyake T (1992) The membranous skeleton: the role of cell condensations in vertebrate skeletogenesis. Anatomy and Embryology 186: 107124. Lian B and Stein GS (1992) Concepts of osteoblast growth and dierentiation: basis for modulation of bone cell development and tissue formation. Critical Reviews in Oral Biology and Medicine 3(3): 269305. Martin RB, Burr DB and Sharkey NA (1998) Skeletal Tissue Mechanics. New York: Springer. Niiweide PJ, Ajubi NE and Aarden EM (1998) Biology of osteocytes. Advances in Organ Biology 5B: 529542. Owen TA, Aronow M, Shalhoub V, Lian JB and Stein GS (1990) Progressive development of the rat osteoblast phenotype in vitro reciprocal relationships in expression of genes associated with osteoblast proliferation and dierentiation during formation of the bone extracellular matrix. Journal of Cellular Physiology 143: 420 430. Reddi AH (1994) Bone and cartilage dierentiation. Current Opinion in Genetics and Development 4(5): 737744. Rubin CT (1984) Skeletal strain and the functional strain signicance of bone architecture. Calcied Tissue International 36: S11S18. Rubin CT and Lanyon LE (1984) Dynamic strain similarity in vertebrates: an alternative to limb bone scaling. Journal of Theoretical Biology 107: 321327. Rubin CT and Rubin J (2000) Biology, physiology and morphology of bone. In: Harris E, Ruddy S and Sledge C (eds) Kellys Textbook of Rheumatology, 6th edn, pp. 16111634. Philadelphia, PA: WB Saunders.

Summary
A brief overview of the biology, physiology and morphology of bone and connective tissues, as well as the local and systemic factors that inuence their growth, development, maintenance and repair, has been provided. The process of cell proliferation and dierentiation, matrix synthesis and
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ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net