INTRODUCTION — The skeleton is a highly dynamic organ that constantly undergoes changes and regeneration. It consists of specialized bone cells, mineralized and unmineralized connective tissue matrix, and spaces that include the bone marrow cavity, vascular canals, canaliculi, and lacunae containing osteocytes. Bone also contains water, which represents at least 25 percent of its wet weight and provides much of its unique strength and resilience.
The skeleton has both structural and metabolic functions:
●Its structural function is critical for locomotion, respiration, and protection of internal organs. The structural connection between the skeleton and the hematopoietic system is particularly intimate; these two systems share both cells and local regulatory factors.
●Its metabolic function is largely as a storehouse for calcium, phosphorus, and carbonate, and it can contribute to buffering changes in hydrogen ion concentration.
This topic will review how the skeleton develops and how the processes of bone formation and resorption are regulated. The pathogenesis of osteoporosis is reviewed separately. (See "Pathogenesis of osteoporosis".)
SKELETAL COMPONENTS — The vertebrate skeleton is subdivided into an axial and an appendicular component. The axial component comprises the skull, spine, sternum, and the ribs. The appendicular component comprises long bones and their appendices. There are two major types of bone in the adult skeleton (figure 1):
●Cortical bone is dense and compact. It constitutes the outer part of all skeletal structures. The lamellae may be extensive (circumferential) or tightly packed in concentric circles in osteons. Cortical bone comprises 80 percent of the skeletal weight. Its major function is to provide mechanical strength and protection, but it can participate in metabolic responses, particularly when there is severe or prolonged mineral deficit.
●Trabecular (cancellous) bone is found inside the long bones (particularly at the ends), throughout the bodies of the vertebrae, and in the inner portions of the pelvis and other large flat bones. Trabecular bone is an important contributor to mechanical support, particularly in the vertebrae. It is also more metabolically active than cortical bone and provides the initial supplies of mineral in acute deficiency states.
SKELETAL DEVELOPMENT — Flat bones, including the vault of the skull, most of the upper facial skeleton, parts of the mandible, clavicle, and the pelvis, as well as the collar of long bones, arise from the direct conversion of undifferentiated mesenchymal cells into bone in a process called intramembranous bone formation. The remaining axial and appendicular bone is formed by a multistep process that requires the sequential formation and degradation of cartilaginous structures that serve as a template for the developing bones. Bone formation arising from a cartilaginous template is referred to as endochondral bone formation (figure 2).
Cartilage is unique among skeletal tissues in its capacity to grow interstitially, ie, by division of chondrocytes. Longitudinal bone growth occurs at the growth plates and involves elongation of the cartilaginous template as a result of the division of chondrocytes as well as increases in their size. The chondrocytes enlarge, form columns, and undergo apoptosis. Then marrow cells enter the space formerly occupied by these cells and the cartilage in between the columns becomes mineralized. Osteoblasts settle on the cartilaginous template and lay down extracellular matrix (osteoid) that calcifies into woven bone. With time, woven bone is resorbed by osteoclasts and replaced with mature trabecular bone made by osteoblasts. This process is responsible for the formation of the spongy, honeycomb-like cancellous bone, and it allows for the formation and expansion of the bone marrow cavity simultaneously with the increase in bone size.
Long bones, especially the femur and tibia, are subjected to most of the load during daily activities and they are crucial for skeletal mobility. They grow primarily by elongation of the diaphysis, with an epiphysis at the ends of the growing bone. The ends of epiphyses are covered with a hyaline cartilage ("articular cartilage"). The longitudinal growth of long bones is a result of endochondral ossification at the epiphyseal plate. Bone growth in length is stimulated by the production of a wide variety of hormones including growth hormone (GH) and insulin-like growth factor-1 (IGF-1), sex steroids, thyroxine (T4), and parathyroid hormone (PTH). (See 'Systemic and local regulators of bone cells' below.)
Skeletal development also requires the formation of blood vessels in the bone, and vascular endothelial growth factor (VEGF) signaling is critical for the conversion of avascular cartilage to highly vascular bone [1,2].
Cartilage proliferation and differentiation — The initial formation of the skeleton, first as cartilage and then as bone, requires the sequential activity of a large number of genes. Genes of the homeobox family (Hox, MSX, DLX) [3-6] are responsible for the initial differentiation as well as the maintained growth of skeletal tissues. A complex interaction involving parathyroid hormone-related peptide (PTHRP) and the Indian hedgehog (IHH) genes is critical for the development and regulation of the cartilage growth plate [7,8]. Notch signaling is also involved in chondrocyte proliferation and differentiation into bone [9,10]. Bone morphogenetic proteins (BMPs) stimulate bone formation, but orderly growth requires that there also be periods when growth is inhibited or unwanted skeletal tissue is removed.
Phosphate has long been known to play an important role in mineralization, but it also can affect cartilage proliferation and differentiation [11]. The most important factor for phosphate regulation is probably fibroblast growth factor 23 (FGF23) that possibly, along with other phosphatonins, plays a role in skeletal development as well as interacting with calcium regulatory hormones in phosphorus homeostasis in bone, kidney, and intestine [12,13].
Osteoblasts
Differentiation — Osteoblasts form the connective tissue matrix, which mineralizes and becomes bone. The precursors of osteoblasts are multipotent mesenchymal stem cells, which also give rise to bone marrow stromal cells, chondrocytes, muscle cells, and adipocytes (figure 3) [14-16]. Importantly, the osteoblasts responsible for bone formation in different physiologic, pathologic, or pharmacologic conditions are obtained from numerous different sources, including chondrocytes of the growth plate, stromal cells of the bone marrow, quiescent bone lining cells that reside on the bone surface, specialized fibroblasts in sutures and periodontal ligaments, and pericytes adherent to the endothelial layer of vessels [17,18]. Mesenchymal stem cell progenitors of osteoblasts residing on the outer surface of blood vessels are critical for the formation of blood vessels as well [19-21]. The process of differentiation of progenitors to mature osteoblasts (ie, the cells capable of synthesizing the bone matrix) is accomplished by a sequential progression from the least differentiated and most proliferative cell to terminally differentiated cell that can no longer undergo mitosis. At the beginning of the process, mesenchymal stem cells with unlimited self-renewal capacity undergo asymmetric divisions resulting in not only other stem cells, but also daughter cells with limited self-renewal and extensive proliferation capacity. Progressively, the proliferative capacity decreases as differentiation increases.
Runx2 — Runx2, a member of the runt homology domain transcription factor family, is critical for the initiation of the osteoblast differentiation process and the commitment of multipotential precursors to osteoprogenitors [22-24]. Runx2 is the earliest differentiation marker of the osteoblast lineage. Together with Runx3, Runx2 is also required for the maturation of hypertrophic chondrocytes. Deletion of Runx2 results in the development of a complete cartilaginous skeleton, but no transformation to bone. Hence, the animals die at birth because their soft ribcage cannot support respiration. Heterozygous deletion of one Runx2 gene results in a human genetic disease called cleidocranial dysplasia [25,26]. Osterix, another transcription factor and a zinc-finger protein, acts downstream of Runx2 and is responsible for the transition of osteoprogenitors to preosteoblasts [27].
The nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma is a crucial cellular switch in the process of the commitment of the multipotential mesenchymal osteoprogenitors. Activation of PPAR-gamma by its natural ligands (eg, prostaglandin J2 or free fatty acids) or synthetic ligands, like the diabetes drug rosiglitazone, suppresses differentiation of the uncommitted progenitors toward the osteoblast lineage while simultaneously promoting differentiation toward the adipocyte lineage [28].
Cultured preosteoblasts and osteoblasts have a reproducible temporal pattern of collagen and alkaline phosphatase synthesis followed by synthesis of osteocalcin, a protein containing gamma-carboxylated glutamic acid that is produced by cells of osteoblast lineage [29]. In vivo, fully differentiated osteoblasts express the genes for all three of these proteins and for other specialized proteins such as osteopontin and bone sialoprotein.
Osteoblasts are functionally and morphologically heterogeneous [30]. During rapid phases of bone formation, they may be columnar in shape, with abundant endoplasmic reticulum, and they synthesize collagen rapidly. When bone formation rates are slower, they are flatter and less metabolically active. Fully differentiated osteoblasts are oriented so that the collagen and noncollagen proteins are laid down on the bone surface in forms suitable for mineralization. Mineralization is delayed for several days, which allows time for collagen cross-linking and the development of large, strong fibers.
Wnt signaling pathway — The Wnt signaling pathway is another critical regulator of skeletal development and mass, working in part through the stimulation of Runx2 gene expression [31]. Activation of the canonical Wnt signaling involves the formation of a complex between Wnt proteins, frizzled and low-density lipoprotein receptor-related protein 5 (LRP5) or LRP6 receptors [32,33]. This complex in turn leads to phosphorylation and inactivation of glycogen synthase kinase (GSK)-3beta, inhibition of beta-catenin degradation, and subsequent accumulation of beta-catenin in the nucleus [34]. Nuclear beta-catenin binds the Tcf/Lef family of transcription factors and induces target gene expression [35].
Genetic studies in mice in which the activity of beta-catenin was manipulated have established that beta-catenin is indeed essential for the specification of osteoblasts from mesenchymal precursor cells through effects on both the proliferation and maturation of the precursor pool [36-38]. Moreover, it is now clear that beta-catenin suppresses the alternative chondrocyte fate of the precursor cells [39]. Besides its role in promoting osteoblastogenesis, deletion or activation of beta-catenin at a later stage of osteoblast differentiation leads to an increase or suppression of osteoclastogenesis, respectively, through regulation of osteoprotegerin (OPG) [40,41]. Modulation of beta-catenin levels at this later stage of osteoblast differentiation had no apparent effect on osteoblast function.
Osteocytes are the predominant cellular source of the Wnt antagonist sclerostin, a limiting factor for osteoblast generation and bone mass accrual that mediates the homeostatic adaptation of bone to mechanical loading [42]. Sclerostin is the product of the SOST gene and a negative regulator of Wnt signaling [43]. It binds to both LRP5 and LRP6 and prevents activation of the Wnt receptor complex [44,45]. This results in inhibition of bone formation [46]. In addition to sclerostin, the DKK family members, particularly DKK-1 (Dickkopf-1), inhibit the Wnt pathway by binding to the LPR5/6 receptor [47]. Wnt signaling can also be blocked by other proteins, such as soluble frizzled-related protein, that bind to Wnt ligands [48].
Disruption in the Wnt signaling pathway results in profound changes in skeletal homeostasis [49]. As examples:
●Mutations in WNT1 impair bone formation [50]. A heterozygous missense mutation in WNT1 (c. 652T to G) causes severe, early-onset, dominantly inherited osteoporosis, and a homozygous nonsense mutation (c. 884C to A) causes a severe form of osteogenesis imperfecta.
●Loss- or gain-of-function mutations in LRP5 causes osteoporosis-pseudoglioma syndrome [51] or the hereditary high-bone-mass trait, respectively [52-54].
●Genetic disorders in which sclerostin production is decreased are associated with increased bone mass [55], and antibodies to sclerostin can increase bone formation [56].
●The anabolic effects of PTH and mechanical loading may involve enhanced Wnt signaling via downregulation of sclerostin activation of LRP5 [57-60].
●Inactivation of DKK family members can increase bone mass [61].
●The Wnt pathway is also a major regulator of joint remodeling and tumor necrosis factor (TNF)-alpha induction of DKK-1 results in the joint erosion characteristically seen in rheumatoid arthritis [47].
Apoptosis — Sixty to 80 percent of osteoblasts die by apoptosis. The remaining become either lining cells that spread out and cover quiescent bone surfaces or are entombed individually in lacunae of the mineralized matrix as osteocytes. The lining cells remain connected to the osteocytes, which may be necessary for transmitting the activation signal during remodeling.
An increase in reactive oxygen species (ROS) has been implicated in the decreased bone formation associated with advancing age [62]. In line with this evidence, oxidative stress caused by increased ROS production in osteoblasts stimulates apoptosis and decreases bone formation [63-65]. To protect against oxidative stress, organisms ranging from prokaryotes to mammals scavenge ROS by a network of overlapping mechanisms including various forms of superoxide dismutases (SODs) and catalase as well as thiol-containing oligopeptides with redox-active sulfhydryl moieties, the most abundant of which are glutathione and thioredoxin [66]. Reduction of these scavenging mechanisms, along with increased mitochondrial respiratory chain leakage and increased activity of membrane oxidases, are the three main mechanisms for oxidative stress.
FoxO transcription factors are another important defense mechanism against oxidative stress, serving primarily to maintain the integrity of long-lived cells including tissue stem cells. Consistent with this, global deletion of FoxOs recapitulates the effects of old age on the murine skeleton in young mice within five weeks, suggesting that FoxO-dependent oxidative defense provides a mechanism to handle the oxygen free radicals constantly generated by the aerobic metabolism of osteoblasts and is thereby indispensable for bone mass homeostasis throughout life. While defending against oxidative stress, FoxOs attenuate Wnt signaling and its potent influence on bone mass [67,68]. Indeed, in the setting of oxidative stress, the limited pool of beta-catenin in osteoblast progenitors is diverted from Wnt/Tcf- to FoxO-mediated transcription [67]. This diversion leads to suppressed osteoblastogenesis and is associated with the old age-dependent decrease in osteoblast number and bone formation in the murine skeleton [67,68], and it may contribute to several other age-related pathologies [69-73].
Osteocytes — In the process of their entombment within the mineralized matrix, osteocytes undergo a dramatic morphologic transformation that includes the development of an average of 50 slender cytoplasmic processes that radiate from the cell body and give osteocytes the resemblance of neuronal cells [74]. The processes run like buried cable along narrow canaliculi and are linked by gap junctions with the processes of neighboring osteocytes, as well as cells present on the bone surface, including the lining cells and cellular elements of the bone marrow and the endothelial cell of the bone marrow vasculature (figure 4). Unlike osteoclasts and osteoblasts, which are relatively short lived and transiently present only on a small fraction of the bone surface, osteocytes are deployed throughout the skeleton, are long lived, and are far more abundant than either osteoclasts (1000 times) or osteoblasts (10 times) [75].
Osteocytes and their dendritic processes are surrounded by a gel-like matrix that is in continuity with the peripheral circulation [76,77]. Transport of solutes through the lacunar-canalicular system is accommodated in part by hydraulic vascular pressure and in part by diffusion and convection induced by mechanical forces. Transmission of fluid shear stresses to the cytoskeleton by the matrix is essential for the ability of osteocytes to sense mechanical loading and choreograph the adaptation of bone to the prevailing mechanical forces. Cilia tether the osteocytes to the collagen, allowing them to sense fluid shifts. Osteocytes secrete growth factors that regulate bone formation. Mature osteocytes tonically secrete sclerostin to maintain an inhibition of bone formation, but when mechanical forces are applied to the bone, the osteocytes stop secreting sclerostin and bone formation is initiated on the bone surface.
Osteoclasts
Development — Osteoclasts are terminally differentiated, multinucleated cells that are uniquely capable of digesting calcified bone matrix. They are formed by fusion of mononuclear precursors of the monocyte/macrophage lineage (figure 3) [78,79]. The receptor activator of nuclear factor kappa-B ligand (RANKL) and the macrophage colony-stimulating factor (M-CSF) are two cytokines that are essential for the development, function, and survival of osteoclasts [80,81], and nuclear factor of activated T cells 1 (NFATc1) is the master transcription factor responsible for osteoclast differentiation and function [81,82]. Osteocytes are the main cellular source of the RANKL that is required for osteoclast generation during bone remodeling [83]. However, it is currently unclear whether during remodeling RANKL is produced by apoptotic osteocytes or live osteocytes responding to the death of their neighbors [84].
RANKL interacts with a receptor on osteoclast precursors that is identical to the receptor involved in interaction of T-cells and dendritic cells called RANK (figure 5). NFATc1 is induced by RANKL and coactivated by immunoglobulin-like receptors and their associated adapter proteins. RANKL can also bind to a protein called OPG or osteoclastogenesis inhibitory factor [85-89]. Administration of a single dose of osteoprotegerin or an antibody to RANKL to postmenopausal women results in rapid reduction of biochemical markers of bone turnover and increase in BMD [90,91].
ROS, in contrast to their negative effects on osteoblasts, are a critical requirement for RANKL-induced osteoclast generation, activation, and survival [92-94]. RANKL increases ROS through a cascade involving TRAF6, Rac1, and NADPH oxidase (figure 5) [92,95-99]. ROS also play an important role in M-CSF-induced monocyte survival [100]. Conversely, decreased ROS levels caused by the administration of the antioxidant N-acetyl cysteine (NAC), inhibition of NADPH oxidase (Nox1), a dominant negative rac1, or overexpression of glutathione peroxidase (Gpx) prevents RANKL-induced JNK, p38, and ERKs activation and osteoclastogenesis [92,99]. Several different antioxidants prevent the increased bone resorption that follows loss of sex steroids [65,101]. Moreover, mitochondrial biogenesis and ROS production coupled with iron uptake through transferrin receptor 1 (TfR1) and iron supply to mitochondrial respiratory proteins are essential for the bone-resorbing function of osteoclasts and the bone loss caused by estrogen deficiency [102].
Function — As they mature, osteoclasts undergo dramatic reorganization of their cytoskeleton. Filamentous actin (F-actin) is first organized into podosomes, highly dynamic structures that mediate cell adhesion and migration of osteoclasts. When osteoclasts adhere on bone, F-actin forms a ring-like structure (actin-ring) at the sealing zone, a tight adhesion structure where the osteoclast plasma membrane is juxtaposed to bone [20]. The sealing zone surrounds a specialized plasma membrane domain, the ruffled border, thus forming an isolated resorptive microenvironment between the osteoclast and the underlying bone matrix. The ruffled border is generated by the fusion of secretory vesicles with the bone apposing plasma membrane. During this process, protons and lysosomal enzymes (predominantly cathepsin K) are vectorially secreted into the resorption lacuna to dissolve bone mineral and digest organic matrix, respectively [103].
The functional differentiation of osteoclasts involves the expression of several genes. Deletion of the c-src, c-fos, and PU-1 genes results in an osteopetrotic phenotype associated with inadequate formation or activity of osteoclasts [104]. Critical functional proteins include: integrin receptors for sealing zone attachment, a hydrogen-potassium ATPase for hydrogen ion secretion, chloride channels, and cathepsin K. Mononuclear osteoclast precursors express the genes for these proteins and for calcitonin receptors during differentiation.
The final activation step probably involves binding of integrins to proteins on the mineralized bone surface that are either already present or are secreted by the osteoclasts [105]. The life span of osteoclasts is probably limited because of programmed death of the nuclei (apoptosis). Estrogen and transforming growth factor (TGF)-beta may decrease bone resorption by stimulating apoptosis [106-108].
Osteoclasts remove mineral and matrix to a limited depth on the trabecular surface or within cortical bone. It is unclear what stops this process, but high local concentrations of calcium or substances released from the matrix may be involved [109,110].
●Among the stimulators of osteoclast generation (some of which act indirectly) are calcitriol, PTH, TNF-alpha, prostaglandin E2, interleukin (IL)-1, IL-6, IL-11, and IL-17.
●Among the inhibitors of osteoclast generation are several interleukins (IL-4,-12,-13,-18). Interferon-gamma (IFNg) appears to have a direct inhibitory effect and an indirect stimulatory effect [111,112].
BIOCHEMISTRY OF BONE MINERAL MATRIX — Bone is composed of an orderly collagen matrix on which calcium and phosphate are deposited in the form of hydroxyapatite. Collagen is deposited in a lamellar fashion and strengthened by multiple crosslinks, both within and between the triple-helical collagen molecules. These crosslinks are pyridinolines that are resistant to degradation and are released during bone resorption, either as free or peptide forms that can be measured in serum and urine. (See "Bone physiology and biochemical markers of bone turnover".)
The matrix also contains noncollagen proteins that are critical for regulating mineralization and strengthening the collagen backbone [113]. The calcium-binding proteins include osteocalcin (bone Gla protein) and matrix Gla protein, which contain gamma carboxyglutamic acid and are vitamin K-dependent, similar to many clotting factors (see "Vitamin K-dependent clotting factors: Gamma carboxylation and functions of Gla"). Osteocalcin increases bone quality and strength by adjusting the alignment of biological apatite crystallites parallel to collagen fibrils; however, it plays no role in bone formation, resorption, or bone mass acquisition [114]. Earlier suggestions that osteocalcin may be a hormone have been contested by preclinical animal studies as well as clinical evidence in humans [115]. Bone sialoprotein and osteopontin bind both calcium and collagen and may play a role in the adherence of osteoclasts to the bone surface.
The bone mineral is composed of complex, often incomplete, crystals of hydroxyapatite. The crystals may contain carbonate, fluoride, and a variety of trace minerals, depending on the environment in which the skeleton grows. They are relatively small, which is appropriate for a structure that can undergo strain with minimal microdamage. Mineralization is probably limited not only by the packing of the collagen fibers, but also by substances present on the bone crystal surface such as pyrophosphate.
BONE MODELING AND REMODELING — During development and growth, the skeleton is sculpted to achieve its shape and size by the removal of bone from one site and deposition at a different one; this process is called modeling. After the skeleton has reached maturity, regeneration continues in the form of a periodic replacement of old bone with new at the same location [116]. This process is called remodeling and is responsible for the complete regeneration of the adult skeleton every 10 years.
Bone remodeling begins early in skeletal development. Endochondral remodeling in the primary spongiosa converts the relatively weak spicules of calcified cartilage into strong trabecular bone. Haversian remodeling in the cortex occurs in larger bones and is probably necessary to maintain the viability of cells far from the bone surface. Haversian remodeling is probably also important in repairing fatigue damage to the skeleton.
Removal of bone (resorption) is the task of osteoclasts. Formation of new bone is the task of osteoblasts. Both of these processes are controlled by the osteocytes [75].
Modeling — Growth of the skeleton and changes in bone shape are produced by modeling. Linear growth during childhood and adolescence occurs by growth of cartilage at the end plates, followed by endochondral bone formation (figure 2). The width of the bones increases by periosteal apposition. During childhood, this is accompanied by endosteal resorption. The endosteal (or inner) surface is in contact with the marrow; thus, endosteal resorption results in a concomitant enlargement of the marrow cavity.
During puberty and early adult life, endosteal apposition and trabecular thickening provide maximum skeletal mass and strength (peak bone mass) [117]. These processes are influenced by locally and systemically produced factors and mechanical forces.
Remodeling — The purpose of remodeling in the adult skeleton is unclear but most likely it serves to remove dead osteocytes, maintain oxygen and nutrient supply and the appropriate level of matrix hydration, and repair fatigue damage, thus preventing excessive aging and its consequences. Remodeling, with positive balance, does occur in the growing skeleton as well. Its purpose, quite different from that proposed for the adult skeleton, is to expand the marrow cavity while increasing trabecular thickness [118].
The stages of the remodeling cycle (resorption, reversal, formation) have different lengths. Resorption probably continues for approximately two weeks. The reversal phase may last up to four or five weeks, while formation can continue for four months until the new bone structural unit is fully formed. Because bone resorption precedes and is faster than bone formation, when there is a sudden increase in the remodeling rate, there is always a temporary imbalance, termed the remodeling space.
Bone multicellular unit — Most of the adult skeleton consists of bone that is remodeled periodically by temporal anatomic units comprising osteoclasts and osteoblasts, termed bone multicellular units (BMU) (picture 1) [118]. All osteoclasts and osteoblasts belong to a BMU [118]. Although during modeling, one cannot distinguish anatomical units analogous to BMU per se, sculpting of the growing skeleton requires spatial and temporal orchestration of the destination of osteoblasts and osteoclasts, albeit with different rules and coordinates to those operating in the BMU of the remodeling skeleton.
The BMU, approximately 1 to 2 mm long and 0.2 to 0.4 mm wide, comprises a team of osteoclasts in the front, a team of osteoblasts in the rear, a central vascular capillary, a nerve supply, and associated connective tissue. In healthy human adults, three to four million BMUs are initiated per year and approximately one million are operating at any moment. Each BMU begins at a particular place and time (origination) and advances toward a target, which is a region of bone in need of replacement, and for a variable distance beyond its target (progression) and eventually comes to rest (termination) [119].
In cortical bone, the BMU (also termed osteon) travels through the bone, excavating and replacing a tunnel (Haversian canal). In cancellous bone, the BMU moves across the trabecular surface, excavating and replacing a trench (Howship's lacunae). In both situations, the cellular components of the BMUs maintain a well-orchestrated spatial and temporal relationship with each other. Osteoclasts adhere to bone and subsequently remove it by acidification and proteolytic digestion. As the BMU advances, osteoclasts leave the resorption site and osteoblasts move in to cover the excavated area and begin the process of new bone formation by secreting osteoid, which is eventually mineralized into new bone. In normal adults, bone resorption and bone formation are tightly balanced so that the amount of bone formed in new BMUs equals the amount of bone resorbed.
One or more capillaries are always in close proximity to the teams of the osteoclasts and osteoblasts that remodel bone and are most likely the conduits by which osteoclast precursors reach the site that is targeted for remodeling [20,120]. In cancellous bone, blood vessels are juxtaposed to a canopy of flat cells that separates the BMU from the bone marrow [121]. Low oxygen tension, developed as the bone matrix expands, stimulates the expression of endothelial growth factors by osteoblasts and initiates a neovasculogenic program [1,122,123].
Signaling pathways — Hypertrophic chondrocytes and osteocytes are essential sources of the receptor activator of nuclear factor kappa B ligand (RANKL) that controls mineralized cartilage resorption and bone remodeling, respectively [83,124]. Newer studies of cultured cells show that osteocytes express a much higher amount of RANKL and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts, and mice with deletion of the RANKL gene in osteocytes have increased bone mass. Contrary to the older paradigm, RANKL produced by mature osteoblasts or their progenitors does not seem to contribute to adult bone remodeling. The rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself. This evidence provides a mechanistic explanation for the view that at least some remodeling is a targeted (as opposed to stochastic) process in which cells that can sense mechanical strains [125] or matrix damage [126] act as beacons for excavation and repair of a specific region of bone. Similarly, hypertrophic chondrocytes may sense signals that initiate transformation of the cartilage scaffold into bony trabeculae. In both cases, the cells that can best detect the need for matrix removal directly control the process.
During the last few years, it has become evident that osteocytes are the choreographers of the remodeling process on the bone surface by virtue of their ability to: sense worn-out bone, direct the homing of osteoclasts (and perhaps osteoblasts) to the site that is in need of remodeling, produce the RANKL and sclerostin that regulate osteoclast and osteoblast generation, respectively (figure 6), and control and modify the mineralization of the matrix produced by osteoblasts [127]. Mechanical forces sustain osteocyte survival. Conversely, diminished mechanical forces eliminate the necessary signals that sustain osteocyte survival, thereby leading to their apoptosis. Moreover, osteocytes are critical mediators of the homeostatic adaptation of bone to mechanical forces. On the other hand, disruption of the integrity of the osteocyte network leads to inappropriate remodeling and in and of itself compromises bone strength independently of changes in bone mass [128]. Furthermore, osteocytes control and modify the mineralization of the matrix produced by osteoblasts by secreting factors such as matrix extracellular phosphoglycoprotein (MEPE) and the phosphaturic hormone fibroblast growth factor 23 (FGF23) [127,129].
Resorption — The bone remodeling cycle begins with osteoclast generation and recruitment to a particular site. Under physiologic conditions, such site may be in need for repair, while under pathologic conditions, it may be randomly and inappropriately targeted.
Reversal — After osteoclastic resorption is completed, there is a reversal phase in which mononuclear cells, possibly of monocyte/macrophage lineage, appear on the bone surface. These cells could prepare the surface for new osteoblasts to begin bone formation. A layer of glycoprotein-rich material is laid down on the resorbed surface, the so-called "cement line," to which the new osteoblasts can adhere. Osteopontin may be a key protein in this process [130]. The cells at the reversal site may also provide signals for osteoblast differentiation and migration.
Formation — The formation phase follows, with successive waves of osteoblasts laying down bone until the resorbed bone is completely replaced and a new bone structural unit is fully formed. When this phase is complete, the surface is covered with flattened lining cells, and there is a prolonged resting period with little cellular activity on the bone surface until a new remodeling cycle begins.
Mineralization — The newly formed osteoid begins to mineralize after approximately two weeks. This process involves accumulation of matrix molecules. Mineralization occurs rapidly at first, then more slowly. It takes several years for a bone structural unit to become fully mineralized.
ABNORMAL BONE REMODELING — In normal adults, bone resorption and bone formation are tightly balanced so that the amount of bone formed in new bone multicellular units (BMUs) equals the amount of bone resorbed. The mechanism(s) of normal balancing is unknown but probably involves several factors released from the bone matrix during osteoclastic bone resorption, such as transforming growth factor (TGF)-beta [131-133], factors produced by osteocytes, such as sclerostin, as well as mechanical needs or hormonal changes. High or low rates of remodeling with an imbalance between resorption and formation can be associated with decreased or increased bone mass.
The rate of bone remodeling is very high during bone growth with total body bone formation exceeding resorption, but slows considerably thereafter and the volume of bone formed by each BMU declines with advancing age [62]. Normal parathyroid hormone (PTH) production is an important determinant of the basal rate of remodeling, and patients with congenital, autoimmune, or surgical hypoparathyroidism have low remodeling rates [134]. Other hormonal changes such as menopause or loss of androgens in castrated males, hyperthyroidism, or primary hyperparathyroidism accelerate the rate of remodeling. In the case of the loss of sex steroid, the increased rate of remodeling is transient and returns to baseline within 5 to 10 years (unless there is vitamin D deficiency and secondary hyperparathyroidism), perhaps because of compensatory mechanisms. An oversupply of osteoclasts relative to the need for remodeling or an undersupply of osteoblasts relative to the need for cavity repair are the seminal pathophysiological cellular changes in the most common bone diseases, including osteoporosis [135,136].
Low bone mass — An oversupply of osteoclasts can result in excessive resorption depth, which can lead to complete loss of trabecular structures. The resulting perforations and discontinuities convert the normal plate-like trabecular architecture to a much weaker system of rods, some of which have free, disconnected ends (image 1 and figure 7). There is no template for bone formation at the perforations, and no formation occurs at the free ends of the trabeculae, perhaps because they are not subject to mechanical stress. Complete removal of template also occurs physiologically. As long bones grow, the metaphyseal trabecular bone area decreases as the secondary spongiosa is completely resorbed and the marrow space expands.
Bone thinning can also occur without perforation when the extent of resorption or the number of resorbing sites is increased. Osteoblasts normally completely fill the resorption cavity [137]; if this does not occur, the BMU is incomplete, which results in decreased "wall thickness" in trabecular bone. This is the histologic hallmark of osteoporosis in older patients or patients treated with glucocorticoids [138], and it is the result of a decrease in the amount of bone formed by the team of osteoblasts within each BMU.
High bone mass — Remodeling imbalance can also occur because of failure of the resorptive process. This can result in dense bones (osteopetrosis or osteosclerosis) and impaired hematopoiesis. As described above, macrophage colony-stimulating factor (M-CSF, CSF-1) is required to initiate osteoclast differentiation. There are osteopetrotic mice that do not make M-CSF, and the osteopetrotic gene locus maps to the same region of chromosome 3 as the M-CSF gene [139]. The applicability of this mechanism to osteopetrosis in humans is unknown. However, other genetic abnormalities have been identified that lead to osteosclerosis, including defects in the genes for ion transport in osteoclasts [140-143].
In humans, the most common mutation linked to osteopetrosis is a defect in the osteoclast-specific proton-pump subunit (TCIRG1); 60 percent of patients with severe autosomal recessive osteopetrosis have this mutation [144]. Other clinically significant mutations have been identified in CLCN7, a gene that encodes an osteoclast-specific chloride channel; OSTM1, a gene that may be involved in Wnt signaling; and in CAII, the carbonic anhydrase II gene. These mutations are associated with normal or elevated numbers of abnormally functioning osteoclasts [144-146]. In contrast, mutations in receptor activator of nuclear factor kappa-B ligand (RANKL) result in an osteoclast poor form of osteopetrosis [147].
In addition to increases in bone density due to impaired resorption, bone density can be increased by greater stimulation of bone formation or by a combination of the two. This probably is the mechanism for increased bone mass in so-called autosomal dominant type I osteopetrosis in which an activating mutation in the low-density lipoprotein receptor-related protein 5 (LRP5) gene leads to activation of the Wnt signaling pathway and promotion of osteoblastogenesis [53,148]. Sclerostin is a major antagonist of the Wnt signaling pathway (see 'Wnt signaling pathway' above). Loss-of-function mutations in the gene for sclerostin (SOST) have been described and are also associated with high bone mass [149,150]. The anabolic effect of intermittent PTH administration may also be due in part to suppression of sclerostin, decreased osteoblast and osteocyte apoptosis, and decreased oxidative stress [151].
SYSTEMIC AND LOCAL REGULATORS OF BONE CELLS — Many systemic hormones, cytokines, growth factors, and local signals influence the birth, death, and function of bone cells. The major systemic regulators are the calcium-regulating hormones, parathyroid hormone (PTH), calcitriol, growth hormone (GH)/insulin-like growth factor-1 (IGF-1), glucocorticoids, thyroid hormones, and sex hormones. Other factors, such as IGFs, have both systemic and local effects, and some have mainly or solely local effects, particularly prostaglandins, transforming growth factor (TGF)-beta, bone morphogenetic proteins (BMPs), and cytokines.
Parathyroid hormone — Parathyroid hormone (PTH) is the most important regulator of calcium homeostasis. It maintains serum calcium concentrations by stimulating bone resorption, increasing renal tubular calcium reabsorption, and increasing renal calcitriol production. PTH stimulates bone formation when given intermittently, but inhibits collagen synthesis at high concentrations [152,153]. It stimulates bone resorption when given (or secreted) continuously, a process mediated by osteoclasts. It also stimulates gene expression in these cells and increases the production of several local factors, including interleukin (IL)-6, IGF-1, an IGF-binding protein (IGF-BP-5), and prostaglandins. The effects of PTH on bone development and remodeling, as well as renal vitamin D activation and organismal calcium homeostasis, result from PTH receptor 1 signaling and inhibition of salt-inducible kinases Sik2 and Sik3 via cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylation [154,155]. (See "Parathyroid hormone secretion and action".)
Calcitriol — Calcitriol increases intestinal calcium and phosphorus absorption, thereby promoting bone mineralization. At high concentrations, under conditions of calcium and phosphate deficiency, it also stimulates bone resorption, thereby helping to maintain the supply of these ions to other tissues. Calcitriol stimulates osteoclastogenesis in cell cultures, but animals lacking vitamin D have relatively normal bone growth and remodeling during development, as long as calcium and phosphate are maintained [156].
Sex steroids — Both estrogens and androgens have profound effects on bone homeostasis. Estrogen acts directly on cells of both the osteoblastic and osteoclastic lineage [107,108,157] and influences skeletal development in all individuals. In late puberty, estrogens decrease bone turnover by inhibiting bone resorption and they are necessary for epiphyseal closure in both females and males. Thus, males with genetic loss of estrogen receptors or of the aromatase enzyme that converts androgen to estrogen have delayed bone age and decreased bone accrual and delayed epiphyseal closure [158]. (See "Pathogenesis of osteoporosis".)
Calcitonin — Calcitonin inhibits osteoclasts and therefore bone resorption in pharmacologic doses. However, its physiologic role is minimal in the adult human skeleton. Its pharmacologic effects are transient, probably because of receptor downregulation. As a result, it is only transiently effective in treating hypercalcemia due to excessive bone resorption. (See "Treatment of hypercalcemia".)
Growth hormone and IGFs — The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) system and IGF-2 are important for skeletal growth, especially growth at the cartilaginous end plates and endochondral bone formation. The actions of the IGFs are in part determined by the availability of the various IGF-BPs; IGF-BP-3 is the major determinant of serum IGF concentrations, while IGF-BP-5 may facilitate and IGF-BP-4 may inhibit the local actions of IGFs.
TGF-beta — Transforming growth factor (TGF)-beta and the BMP family of proteins consist of at least 10 proteins that are produced by many different cells and that have multiple actions on growth and development [159-161]. TGF-beta can inhibit bone resorption and stimulate bone formation. BMP-2 and other members of this family increase osteoblast differentiation and bone formation when injected subcutaneously or intramuscularly.
Glucocorticoids — Inhibition of bone formation is the major cause of glucocorticoid-induced osteoporosis and may be due to accelerated apoptosis of osteoblasts and osteocytes [162]. (See "Clinical features and evaluation of glucocorticoid-induced osteoporosis".)
Thyroid hormones — Thyroid hormones stimulate both bone resorption and formation. Thus, bone turnover is increased in hyperthyroidism, and bone loss can occur. (See "Bone disease with hyperthyroidism and thyroid hormone therapy".)
Cytokines — Cytokines, produced by bone cells and adjacent hematopoietic and vascular cells, have multiple regulatory actions in the skeleton [163-168]. Many of these factors have been implicated in the bone loss associated with ovariectomy in rodents. Regulation may occur as a result of both varying production of agonists and of changes in the receptors or binding proteins (receptor antagonists) for these factors.
Fibroblast growth factors — Fibroblast growth factors (FGFs) are another family of proteins involved in skeletal development. Mutations in the receptors for these factors result in abnormal skeletal phenotypes, such as achondroplasia [160]. Other growth factors such as vascular endothelial growth factor (VEGF) are produced in bone and may play a role in bone remodeling.
Other — A number of other factors play an important role in bone metabolism [159,161,169-172]. As examples, prostaglandins, leukotrienes, and nitric oxide may be critical in the rapid responses of bone cells to inflammation and mechanical forces. Prostaglandins have biphasic effects on bone resorption and formation, but the dominant effects in vivo are stimulatory [170]. Prostaglandin production can be increased by impact loading and by inflammatory cytokines. Nitric oxide can inhibit osteoclast function [173], while leukotrienes stimulate bone resorption [174].
SUMMARY
●Skeletal structure and function – The skeleton is a highly dynamic organ that constantly undergoes changes and regeneration. It consists of specialized bone cells, mineralized and unmineralized connective tissue matrix, and spaces that include the bone marrow cavity, vascular canals, canaliculi, and lacunae containing osteocytes. The skeleton has both structural and metabolic functions. (See 'Introduction' above.)
●Skeletal development – Flat bones, including the vault of the skull, most of the upper facial skeleton, parts of the mandible, clavicle, and the pelvis, as well as the collar of long bones, arise from the direct conversion of undifferentiated mesenchymal cells into bone in a process called intramembranous bone formation. Axial and appendicular bone is formed from a cartilaginous template and is termed endochondral bone formation (figure 2). (See 'Skeletal development' above.)
●Osteoblasts – The precursors of osteoblasts are multipotent mesenchymal stem cells (figure 3). The process of differentiation of these progenitors to mature osteoblasts (ie, the cells capable of synthesizing the bone matrix) is accomplished by a sequential progression from the least differentiated and most proliferative cell to terminally differentiated cell that can no longer undergo mitosis. Runx2 and the Wnt signaling pathway are critical for the initiation of the osteoblast differentiation process and the commitment of multipotential precursors to osteoprogenitors. (See 'Osteoblasts' above.)
●Osteoclasts – Osteoclasts remove mineral and matrix to a limited depth on the trabecular surface or within cortical bone. They are formed by fusion of mononuclear precursors of the monocyte/macrophage lineage. The receptor activator of nuclear factor kappa B ligand (RANKL) and the macrophage colony-stimulating factor (M-CSF) are two cytokines that are essential for the development, function, and survival of osteoclasts. (See 'Osteoclasts' above.)
●Bone modeling and remodeling
•Modeling – During development and growth, the skeleton is sculpted to achieve its shape and size by the removal of bone from one site and deposition at a different one; this process is called modeling. (See 'Modeling' above.)
•Remodeling – After the skeleton has reached maturity, regeneration continues in the form of a periodic replacement of old bone with new at the same location, a process called remodeling. (See 'Remodeling' above.)
All osteoclasts and osteoblasts belong to a unique temporary structure known as a bone multicellular unit (BMU) (picture 1). Osteoclasts adhere to bone and subsequently remove it by acidification and proteolytic digestion. As the BMU advances, osteoclasts leave the resorption site and osteoblasts move in to cover the excavated area and begin the process of new bone formation by secreting osteoid, which is eventually mineralized into new bone. In normal adults, bone resorption and bone formation are tightly balanced so that the amount of bone formed in new BMUs equals the amount of bone resorbed. (See 'Bone multicellular unit' above.)
●Abnormal bone remodeling – An oversupply of osteoclasts relative to the need for remodeling or an undersupply of osteoblasts relative to the need for cavity repair are the seminal pathophysiological cellular changes in the most common bone diseases, including osteoporosis (image 1 and figure 7). (See 'Abnormal bone remodeling' above.)
●Systemic and local regulators of bone cells – Several systemic hormones, cytokines, growth factors, and local signals influence the birth, death, and function of bone cells. The major systemic regulators are the calcium-regulating hormones, parathyroid hormone (PTH), calcitriol, growth hormone (GH)/insulin-like growth factor-1 (IGF-1), glucocorticoids, thyroid hormones, and sex hormones. Other factors, such as IGFs, have both systemic and local effects, and some have mainly or solely local effects, particularly prostaglandins, transforming growth factor (TGF)-beta, bone morphogenetic proteins (BMPs), and cytokines. (See 'Systemic and local regulators of bone cells' above.)
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