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© 2003 The Journal of Bone and Joint Surgery, Inc.
The Biology of the Growth Plate
• Chondrocytes in the growth plate undergo a complex series of molecular, biochemical, and morphological changes during the process of differentiation. • Longitudinal growth depends on both proliferation and hypertrophy of chondrocytes in the growth plate. • Integrated growth factor signaling pathways control the key transition between proliferation and hypertrophy that is required for completion of the maturation process. • Terminal chondrocyte maturation is associated with programmed cell death and the secretion of matrix and other factors that promote matrix calcification and vascular invasion. • Systemic hormones interact with local growth factors and signaling pathways to influence the rate of growth and lead to closure of the growth plate. In order for paired human limbs to reach the same adult length and proportions, the longitudinal growth of the skeleton must be tightly regulated. The regulation of longitudinal growth at the growth plate occurs generally through the intimate interaction of circulating systemic hormones and locally produced peptide growth factors, the net result of which is to trigger changes in gene expression by growth plate chondrocytes. These molecular events lead to precisely orchestrated alterations in chondrocyte size, extracellular matrix components, secreted enzymes and growth factors, and receptor expression. The culmination of these events is calcification of the matrix, chondrocyte apoptosis, and completion of endochondral bone formation. This review will highlight some of the advances in genetics and cell biology in the past decade that have begun to illuminate some of the important regulatory mechanisms governing the biology of the growth plate.
The growth plate can be divided into a series of anatomic zones that distinguish unique morphological and biochemical stages during the process of chondrocyte differentiation. In the resting zone, the ratio of extracellular matrix to cell volume is high and the cells are in a relatively quiescent state. In the proliferating zone, chondrocytes assume a flattened appearance, begin to divide, and become organized into columns. In the zone of maturation, the synthesis of extracellular matrix allows the recently divided cells to separate from each other. This extracellular matrix consists predominantly of collagens and proteoglycans as well as other noncollagenous proteins. Type-II collagen is the primary collagen species in the growth plate, although type-IX and type-XI collagen also are highly expressed and have important functions. Type-IX collagen molecules surround the surface of the type-II collagen fibrils, to which they are covalently cross-linked. It is postulated that type-IX collagen mediates the interaction of type-II collagen with other extracellular matrix components in cartilage. Mutations in type-II collagen are associated with a number of skeletal dysplasias in humans, including spondyloepiphyseal dysplasia, Kniest dysplasia, and Stickler syndrome 1 . Type-IX collagen mutations have been identified in a subset of patients with multiple epiphyseal dysplasia 2 , while mutations in type-XI collagen have been linked to some forms of Stickler syndrome 3 . Aggrecan, the large aggregating proteoglycan of cartilage, is the principal proteoglycan molecule in the cartilage matrix and provides the osmotic properties necessary for cartilage to resist compressive loads. Decorin and biglycan, two smaller proteoglycan molecules, also may serve important functions. Decorin, for example, coats the outside of the collagen fibrils and may play a role in regulating collagen fibrillogenesis. Cartilage oligomeric protein (COMP) is a critical noncollagenous protein found in the extracellular matrix. COMP is an extracellular calcium-binding glycoprotein belonging to the thrombospondin family. The COMP molecule is composed of five flexible arms with a large globular domain at the end of each arm, resembling a bouquet of flowers. Mutations in COMP have been linked to pseudoachondroplasia as well as some forms of multiple epiphyseal dysplasia 4-6 . In the hypertrophic zone, cell division ceases and the chondrocytes begin to terminally differentiate. Terminal differentiation is associated with a large increase in cell volume, a marked increase in alkaline phosphatase enzyme activity, and synthesis and secretion of type-X collagen, a unique short-chain collagen found only in the hypertrophic zone of the growth plate ( Figs. 1-A and 1-B ). Although the exact function of type-X collagen in the growth plate remains unclear, mutations in the type-X collagen gene have been found to cause Schmid metaphyseal chondrodysplasia 7 . Surprisingly, transgenic mice lacking type-X collagen show only subtle alterations in hematopoiesis and growth plate architecture, but no obvious skeletal phenotype 8 .
Matrix vesicles, formed by budding of the chondrocyte plasma membrane, are deposited into the surrounding extracellular matrix in the hypertrophic zone and serve as a nidus for mineralization. Mineralization of the cartilage extracellular matrix occurs in a somewhat directional pattern, with the longitudinal septa of cartilage matrix between the columns of hypertrophic chondrocytes being the favored sites of mineral deposition ( Figs. 1-A and 1-B ). The mineral deposited consists primarily of poorly crystalline hydroxyapatite. In the zone of vascular invasion, invading capillary loops from the metaphysis break through the last transverse septum of mineralized cartilage to enter the hypertrophic chondrocyte lacuna. Approximately two-thirds of the longitudinal septa are actively resorbed by chondroclasts, while the remaining one-third serve as a template for deposition of bone matrix by osteoblasts. These "mixed spicules," containing both mineralized cartilage and bone matrix, are known as primary trabeculae and are subsequently remodeled in the metaphysis to trabeculae of lamellar bone, or secondary trabeculae 9,10 . A peripheral ring of fibrocartilage encircles these growth plate zones to provide structural support and increasing width of the physis. The ossification groove of Ranvier is a wedge-shaped area of chondrocyte progenitor cells that contributes reserve zone cells to allow the physis to expand its width as the bone grows longer. The perichondrial fibrous ring of LaCroix is a band of fibrous tissue that merges with the periosteum of the bone to provide mechanical support in response to compression, tension, or shear loads on the physis.
Chondrocytes synthesize and secrete a characteristic matrix into the extracellular space. Therefore the factors that regulate the synthesis of cartilage matrix are also many of the same factors responsible for regulating the process of chondrogenesis—namely, the conversion of mesenchymal stem cells into cells that elaborate cartilage matrix. As is the case for myogenesis, adipogenesis, and osteogenesis, the differentiation of mesenchymal cells into chondrocytes during chondrogenesis is regulated by the activity of a DNA transcription factor that controls the expression of the principal genes encoding the extracellular matrix proteins of cartilage. In the case of cartilage, this transcription factor is Sox 9, which is required for expression of several chondrocyte-specific matrix proteins, including type-II collagen, type-IX collagen, type-XI collagen, and aggrecan 11 . Sox 9 binds to specific enhancer regions in the promoters of these target genes in conjunction with two other related proteins, L-Sox 5 and Sox 6, in order to activate gene transcription 12 . In chimeric mice, cells with no functional Sox-9 genes fail to differentiate into chondrocytes because of a block at the stage of mesenchymal condensation. This indicates that, in addition to regulating the synthesis of extracellular matrix proteins, Sox 9 may also control the expression of cell surface proteins involved in the condensation process 13 . Mutations in Sox 9 have been linked to the human skeletal malformation syndrome, campomelic dysplasia 11 .
Proliferation occurs in a narrow band of cells located in the proliferating region of the growth plate. Histomorphometric studies of rats have shown that one layer of hypertrophic cells is eliminated from the growth plate every three hours; therefore, proliferation must produce eight new cells in each chondrocyte column per day 9,10 . Recent experiments have suggested that the proliferation of chondrocytes in the growth plate is under the control of a local feedback loop primarily involving three signaling molecules synthesized by growth plate chondrocytes: parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), and transforming growth factor-beta (TGF-ß). This feedback loop acts to regulate the rate at which the growth plate cells leave the proliferative zone of the physis and irreversibly commit to being terminally differentiated hypertrophic cells. Cells in the periarticular region of the long bones produce PTHrP; however, the PTHrP receptor is found primarily in the prehypertrophic cells and lower proliferating zone cells. PTHrP delays hypertrophic differentiation in these lower proliferating zone cells by maintaining cells in a prehypertrophic phenotype. Growth plate cells that are beginning to undergo hypertrophic differentiation secrete Ihh, which relays a signal back through the perichondrium to the periarticular cells to increase production of PTHrP 14 . This perichondrial relay involves the receptors for Ihh, patched and gli, which are located primarily in the cells of the perichondrium, as well as TGF-ß produced by perichondrial cells in response to Ihh. TGF-ß then acts on the perichondrial and periarticular cells to increase PTHrP synthesis 15,16 and also can act directly on chondrocytes to inhibit hypertrophy 17 . This increase in PTHrP synthesis in the periarticular cells is transmitted to the late proliferating cells expressing the PTHrP receptor, which slow the production of Ihh-producing cells, thereby controlling the pace of hypertrophic differentiation 18 ( Fig. 2 ).
Recent genetic experiments in mice have confirmed this primary role of PTHrP in controlling the transition between chondrocyte proliferation and differentiation. Transgenic mice lacking either PTHrP or the PTH/PTHrP receptor show evidence of dwarfism resulting from accelerated differentiation and premature hypertrophy 19 . Conversely, mice in which PTHrP is overexpressed in the growth plate also exhibit dwarfism, but this dwarfism is due to marked slowing of the rate of differentiation 20 . In humans, mutations in the PTH/PTHrP receptor that result in a constitutively active PTHrP signal have been identified as the cause of Jansen metaphyseal chondrodysplasia, a dwarfing condition associated with delays in growth plate mineralization and hypercalcemia 21 . It is not likely that the periarticular cartilage is a relevant source of PTHrP in the adolescent animal because of the large distance between the growth plate and the articular surface 22 . Furthermore, recent studies have indicated that Ihh is not produced by the growth plates of postnatal animals, which suggests a role for other signals as regulators of chondrocyte differentiation in adolescent animals 23 . Evidence is accumulating to implicate TGF-ß as the key inhibitor of chondrocyte differentiation in adolescence. In vitro, TGF-ß is a potent inhibitor of maturation, including cell hypertrophy, Type-X collagen expression, and alkaline phosphatase activity 17,22,24,25 . TGF-ß actions on the cell are mediated in part by a specific transcription factor, Smad 3 22,24 . Mice deficient in Smad 3 have a completely normal skeleton at birth, but by three weeks of age they begin to exhibit cartilage abnormalities, including premature hypertrophy of both growth plate and articular chondrocytes and disorganization of the growth plate columns, resulting in decreased longitudinal growth 26 . The effects on articular cartilage mimic the changes observed during the development of osteoarthritis. Therefore, although TGF-ß signaling through Smad 3 is not critical for development of a normal skeleton at birth, it is essential for normal postnatal growth and development. TGF-ß is secreted by chondrocytes in an inactive form bound to a latency molecule, underlining the importance of defining the factors leading to TGF-ß activation in the growth plate 26 . Mechanisms defined so far include matrix metalloproteinases, other proteases, and acidic conditions, such as those present during osteoclastic bone resorption 27-29 . Although this PTHrP-Ihh-TGF-ß feedback loop currently appears to be the primary regulator of cell proliferation in the growth plate, it is also likely that this regulatory network is modulated by other systemic and local signaling molecules that have been previously shown to have effects on cell proliferation in the growth plate. For example, genetic disruption of the murine fibroblast growth factor receptor-3 (FGFR-3), which binds to at least nine members of the fibroblast growth factor family, results in prolonged endochondral bone growth with expansion of the proliferating and hypertrophic zones of the growth plate 30,31 . An activating mutation in the FGFR-3 receptor has also been identified as the cause of achondroplasia, a dwarfing condition in which proliferation of growth plate cells is markedly reduced 32 . It is therefore likely that FGF signaling is able to modulate the PTHrP-Ihh regulatory feedback loop. In support of this notion is the fact that transgenic mice overexpressing the FGFR-3 gene in growth plate cartilage show markedly reduced proliferation of growth plate cells associated with downregulation of Ihh expression 33 . Another important growth factor is insulin-like growth factor-I (IGF-I), which is an autocrine factor that stimulates increased rates of cell division 34 . In addition to its effects on circulating levels of IGF-I, growth hormone increases the local synthesis of IGF-I in growth plate cells, which then leads to increased rates of cell division 34 . Transgenic mice lacking a functional IGF-I gene demonstrate severe growth retardation as well as profound defects in the development of major organ systems, including bone, muscle, and reproductive, and many die within twenty-four hours after birth 35 . Administration of growth hormone to these IGF-I-null mice has no effect on skeletal growth. Likewise, mice in which the growth hormone gene has been deleted show evidence of reduced bone growth that can be restored by administration of IGF-I 36 . In humans, mutations in the growth hormone receptor result in Laron syndrome, a hereditary dwarfism associated with truncal obesity and low serum IGF-I levels 37 . Recently, a new line of transgenic mice lacking only IGF-I derived from the liver was created 38 . These mice have serum levels of IGF-I that are 75% lower than those of normal mice; yet they demonstrate normal longitudinal bone growth. Therefore IGF-I produced locally by growth plate chondrocytes in response to growth hormone is the critical source for maintenance of normal postnatal skeletal growth. In part, IGF-I may mediate some of its effects through other growth factors since it has been shown to enhance the effects of TGF-ß on proliferation 39 . One possible mechanism for integrating the effects of signaling molecules like PTHrP, TGF-ß, IGF-I, and FGF on the proliferation of growth plate chondrocytes is through modulation of the cell division cycle. The cell cycle is exquisitely regulated and is characterized by several sequential phases. Cells pass through an initial growth phase (G1) of the cell cycle before entering into S phase, in which DNA is replicated in preparation for another obligatory round of cell division. Alternatively, cells may withdraw from the cell cycle and undergo terminal differentiation 40,41 . The passage of cells across this critical G1/S restriction point is controlled by phosphorylation of the retinoblastoma protein (Rb) or the closely related proteins p107 and p130 42-47 . Simultaneous deletion of both p107 and p130 genes in mice results in deregulated chondrocyte growth, defective endochondral bone development, shortened limbs, and neonatal death 48 . The phosphorylation, or activation state, of the Rb proteins is controlled by the action of a family of cyclin-dependent kinases (CDKs) that require binding of cyclin proteins for their kinase activity 42-47 . Several inhibitory proteins have been found that also compete with the cyclin subunits for binding to their respective CDKs, thereby preventing Rb phosphorylation and passage into S phase. For growth plate cells to continue proliferation, the balance between cyclins and CDK inhibitors must be weighted in favor of maintaining Rb (or the related proteins p107 and p130) in a hyperphosphorylated state 49 . Elevated levels of cyclin D1, for example, result in stimulation of CDK activity, Rb hyperphosphorylation, and chondrocyte proliferation. Recently it has been demonstrated that both PTHrP and TGF-ß are able to stimulate transcription of the cyclin D1 gene through specific regulatory sites in the cyclin D1 promoter region 50 . Conversely, expression of the CDK inhibitors p21 waf-1 and p27 kip-1 have been found to be elevated in growth plate cells undergoing hypertrophy in vivo 51 and in growth plate cells induced to stop proliferating and to terminally differentiate in response to thyroid hormone 52 . In contrast, studies 53,54 have shown that TGF-ß regulates cell cycle progression in other cell types by reducing the expression of the cyclin-dependent kinase inhibitors p15 ink4B and p27 kip-1 . This important reciprocal relationship between the cell cycle regulators and the events of proliferation and differentiation dictates that when the balance of cyclins and cyclin-dependent kinase inhibitors favor Rb family hyperphosphorylation, proliferation occurs. In contrast, when the balance is tipped toward inhibitors of the cyclin-dependent kinases, Rb family members remain hypophosphorylated and chondrocytes undergo terminal differentiation. These basic cell cycle events are likely to be coordinately regulated by important signaling events mediated by hormonal and growth factor-receptor interactions.
Chondrocyte maturation is marked by related physical and biochemical changes that occur in a spatial and temporal pattern 40,55 . The progression of chondrocytes through the resting, proliferating, and hypertrophy stages of differentiation that culminates in matrix calcification and programmed cell death occurs within twenty-four hours in rapidly growing animals 42 . During differentiation, chondrocytes have a five to tenfold increase in intracellular volume 42 . This is not a passive swelling of the cell but instead reflects an active process marked by an increase in the number of intracellular organelles, including mitochondria and the endoplasmic reticulum 42 . The factors that stimulate cellular enlargement are not clear, but they probably involve alterations in ion channels that lead to an ingress of water. Chondrocyte hypertrophy has an important role in the longitudinal growth of the skeleton 43 . It has been shown that the increase in chondrocyte height is responsible for 44% to 59% of long-bone growth, with the remainder due to matrix synthesis and chondrocyte proliferation 44,45 . Furthermore, the differential growth of various bones appears to be related to differences in the size of hypertrophic chondrocytes. Chondrocytes in bones with rapid growth, such as the femur, increase more in size than do chondrocytes in growth plates in bones such as the radius, which grow less rapidly 44,45 . The factors that control these local differences in hypertrophic cell size have not been defined, but they probably involve an interaction of both local and systemic factors 45 . Hypertrophic chondrocytes also have an important metabolic function in that they prepare the extracellular matrix for calcification. Terminally differentiated hypertrophic chondrocytes uniquely express type-X collagen as well as high levels of the enzyme alkaline phosphatase 46-49 ( Figs. 1-A and 1-B ). Alkaline phosphatase is essential for calcification of the matrix 50 and is present in a high concentration in matrix vesicles, which are small membrane vesicles secreted by hypertrophic chondrocytes into the surrounding matrix 50,51 . These matrix vesicles are believed to be the first sites of calcium hydroxyapatite nucleation. Alkaline phosphatase increases the concentration of phosphate ions, which are necessary for this calcification process. Indeed, the absence of alkaline phosphatase, as observed in hypophosphatasia, is associated with decreased mineralization of the matrix and widening of the growth plate as well as defective mineralization of bone 50 . Chondrocyte differentiation and hypertrophy are essential steps in longitudinal growth; therefore, identification of the regulators of these processes is an area of intense investigation. Several paradigms have emerged from research to date: (1) chondrocytes in the endochondral ossification pathway are primed to complete maturation spontaneously and undergo hypertrophy; (2) negative regulators of differentiation and hypertrophy are critically important; and (3) it is likely that the mechanisms that control this process during embryologic development are different from, or complementary to, those controlling the process during adolescent development 23,52,53 . Both in vitro and in vivo models of chondrocyte differentiation have shown that, in the absence of inhibitory factors, chondrocytes undergo hypertrophy spontaneously. There is substantial evidence that the bone morphogenetic proteins (BMPs) and their receptors are responsible for the spontaneous completion of maturation 52-56 . Isolated chondrocytes in culture are induced to undergo hypertrophy and increase the expression of hypertrophic markers including type-X collagen and alkaline phosphatase activity in the presence of BMPs 52,54,56,57 . In contrast, inhibition of BMP signaling prevents chondrocyte maturation 56,58 . In vivo studies have demonstrated that expression of either of the BMP antagonists noggin or chordin in the developing chick limb bud prevents chondrocyte hypertrophy and expression of genes associated with maturation 59,60 . Other factors that induce chondrocyte maturation also appear to act through BMP signaling. Thyroxine induces type-X collagen synthesis and other maturational characteristics in growth plate chondrocytes in culture through induction of BMP-2, an effect that can be blocked by addition of the BMP antagonist noggin 61 . Similarly, the induction of chondrocyte differentiation by retinoic acid appears to be related to effects on BMP signaling. Retinoic acid has recently been shown to induce the expression of the BMP signaling molecules Smad 1 and Smad 5 in chondrocytes, making retinoic acid-treated chondrocytes more sensitive to BMP-mediated signaling events 62 . Similar to the role of Sox 9 in chondrogenesis, the transcription factor core binding factor-1 (CBFA-1) appears to have a critical role during the process of chondrocyte hypertrophy and terminal differentiation 63 . Although CBFA-1 is able to induce terminal differentiation in chondrocytes, mice without the CBFA-1 gene have an absence of hypertrophic chondrocytes in some growth plates while hypertrophy proceeds normally in other growth plates; this suggests that other transcription factors are involved in this process 64,65 . The transcription factors in the BMP signaling pathway, Smad 1, Smad 5, and Smad 8, also appear to mediate hypertrophy 62,66 and have recently been shown to interact with CBFA-1 at the type-X collagen promoter to induce the expression of this gene 66 . It is likely that the growth disturbances that arise from hormonal abnormalities and toxic agents are partly related to disturbances in the autocrine signaling loops in the growth plate. Recent work has shown that the inhibition of skeletal growth in children exposed to lead, along with associated morphological abnormalities of the growth plates, may be due to an alteration in chondrocyte responses to PTHrP and TGF-ß 67-69 . Lead interferes with the inhibitory effect of PTHrP and TGF-ß on chondrocyte differentiation, and thus it alters the normal regulatory events that control the rate of chondrocyte hypertrophy 69 . Better understanding of the molecular and cellular events involved with hypertrophy will improve therapies for congenital and acquired diseases involving the growth plate.
Chondrocytes are metabolically active cells that secrete and maintain a highly specialized matrix. The function of this tissue is to promote calcification of cartilage that serves as a template for bone formation by osteoblasts. This mineralization of cartilage occurs primarily in the matrix located between distinct hypertrophic chondrocyte columns and not in the interzone between hypertrophic chondrocytes in the same column 70 ( Figs. 1-A and 1-B ). Matrix vesicles are the initial sites of mineralization in the hypertrophic region of the growth plate and are critical components of the calcification process 51,71 . Matrix vesicles are 100-nm-diameter extracellular membrane-invested particles, released by budding from the surfaces of chondrocytes, osteoblasts, and odontoblasts 51 . Matrix vesicle accumulation of calcium appears to depend on a family of calcium channel molecules referred to as annexins 72,73 . Annexin II, V, and VI are present with the lipid bilayer of matrix vesicles and are required for accumulation of calcium in these structures. Ca ++ channel blockers specific for annexins block their uptake of calcium 73 . Type-II and type-X collagen bind to matrix vesicles as a result of interactions with annexin V. In the absence of annexin V, these collagens do not interact with matrix vesicles 73 . Furthermore, type-II and type-X collagen both stimulate the activity of the annexin-V calcium channel 73 . Thus, one of the roles of type-X collagen, which is present only in hypertrophic cartilage, may be to facilitate the deposition of calcium in the matrix. Recent studies of animals have demonstrated that uremia, which retards growth in children, is associated with an increase in the width of hypertrophic cartilage as well as a decrease in the deposition of both type-II and type-X collagen, alterations in collagen fibril architecture, and defective mineralization 74 . Matrix vesicles also contain enzymes, including alkaline phosphatase and matrix metalloproteinases. The role of alkaline phosphatase in matrix mineralization is not certain, but it probably involves the important step of metabolism of pyrophosphate to yield two molecules of orthophosphate 51 . Whereas pyrophosphate is a known inhibitor of hydroxyapatite crystal formation, pyrophosphate hydrolysis (resulting in two molecules of orthophosphate) stimulates mineralization of matrix vesicles 51 . Hypophosphatasia is a heritable disease characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase, and it results in rickets because of decreased calcification of the matrix 50 . Electron microscopic studies have recently shown that matrix vesicles in patients with rickets maintain their ability to concentrate calcium and phosphate internally and to initiate mineral formation 50 . However, in the absence of alkaline phosphatase, there is retarded extravesicular calcium-hydroxyapatite crystal propagation 50 . It is well known that vitamin-D deficiency also results in defective mineralization and widening of the growth plate and that these effects are mediated through classic vitamin D-receptor-dependent mechanisms as well as through a membrane-mediated signaling mechanism 75 . Vitamin D increases alkaline phosphatase and matrix metalloproteinase (MMP) activity in chondrocytes 75 . However, some of the effects of vitamin D on the growth plate are secondary to hormonal and metabolic effects. In mice lacking the classic vitamin-D receptor, rickets develop, with thickening of the growth plate and decreased mineralization 76 . However, in the vitamin D-receptor-ablated mice with preservation of normal mineral ion homeostasis, growth plate morphology and width are normal 76 . Therefore, a principal action of vitamin D on the growth plate is its role in intestinal calcium absorption. The skeletal consequences of the absence of the vitamin-D receptor appear to be related to impaired intestinal calcium absorption and/or the resultant secondary hyperparathyroidism and hypophosphatemia 76 . Matrix metalloproteinases are responsible for catabolism and turnover of the matrix and are induced during the process of chondrocyte hypertrophy 27,77 . MMP-13 is produced by hypertrophic chondrocytes and is secreted in matrix vesicles. Mineralization of the matrix is associated with a marked increase in the cleavage of type-II collagen by MMPs 27,78,79 . Matrix vesicles also contain TGF-ß, which is present in a latent form but is activated by MMP-13. The increased levels of active TGF-ß present in the growth plate at the onset of mineralization are believed to be due, in part, to the presence of MMP-13 in matrix vesicles 27 . Additionally, MMPs are critical for angiogenesis in the growth plate and thus are necessary for normal calcification and bone formation 80 . MMP-9 is expressed in chondroclasts and in stromal cells at the junction of hypertrophic cartilage and bone 81 . Mice without MMP-9 have defective angiogenesis, reduced chondrocyte apoptosis, widening of the hypertrophic zone, and decreased mineralization of the matrix 81 . The effects of the MMPs on angiogenesis may be related to a decrease in catabolism of the matrix, release of important growth factors, or other effects, but these relationships have not yet been clarified. Proteoglycans are major components of the extracellular matrix in cartilage. Like articular cartilage, the growth plate contains large aggregating proteoglycans and is thus characterized as hyaline cartilage. Although it was initially thought that the content of aggregating proteoglycans is reduced in the hypertrophic region, it has now been established that increased concentrations of aggregating proteoglycans are present at the onset of calcification 82,83 . However, there are changes in the relative content of the proteoglycan monomers, their degree of sulfation, and their size during maturation that are probably important 83 . In contrast, there is an increase in the concentration of some of the smaller, nonaggregating proteoglycans during chondrocyte hypertrophy. The mRNA and protein for the proteoglycan biglycan are observed in hypertrophic chondrocytes and the surrounding matrix but not in other areas of the epiphysis 84 , suggesting an association of this proteoglycan with mineralization. Cartilage also contains components that inhibit calcification of the extracellular matrix. The best characterized is matrix Gla protein (MGP), a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. MGP is expressed by proliferative and late hypertrophic chondrocytes but not by the intervening chondrocytes 85 . MGP inhibits calcification both in vitro and in vivo 85,86 . MGP-deficient mice have inappropriate calcification of the growth plate that leads to short stature, osteopenia, and fractures 86 .
Growth and development in all organisms requires the proliferation, differentiation, and then removal of cells. Apoptosis is the mechanism through which cells undergo programmed death, a process necessary for the homeostasis of most organs, including the growth plate 87-89 . The cells in the growth plate that undergo apoptosis are terminally differentiated chondrocytes 90-94 . The role of terminally differentiated chondrocytes is to prepare the matrix for calcification, which then acts as a template for osteoblastic bone formation. Once the cartilage matrix calcifies, death and removal of terminally differentiated hypertrophic chondrocytes provides space for the ingress of vascular channels and bone-marrow stromal cells 94-97 . While it was initially thought that hypertrophic chondrocytes died through a passive process involving depletion of nutrients and oxygen tension in the hypertrophic region of the growth plate, it is now recognized that the process is an active and regulated event. Thus, chondrocytes in the hypertrophic region of the growth plate have morphological features similar to those of other cells undergoing apoptosis 94 . The morphological events result from activation of a set of enzymes that target and metabolize important intracellular structures 98,99 . Morphological findings in cells undergoing programmed death include condensation of the nuclear chromatin, cell shrinkage, and plasma membrane blebbing 87,100 . In contrast to necrotic cells, which lyse and release degradative enzymes into the local environment, apoptotic cells are rapidly recognized and taken up by neighboring or phagocytic cells but do not induce inflammation as necrotic cells do 100 . The enzymes that initiate apoptosis are called caspases 88,98,99 . Caspases are a family of cysteine proteases that cleave target proteins with high specificity. All cells contain caspases in their cytoplasm in an inactive, or zymogenic, form and are therefore primed to undergo apoptosis. In addition to the caspases, a series of inhibitor molecules also exist that can block caspase activation. Recently, several protein families with this activity, including the bcl-2 family of proteins, have been identified 98,101 . Bcl-2 stabilizes the mitochondria and prevents the release of cytochrome c, while another member, BAX, stimulates the release of cytochrome c and leads to apoptosis 98,101 . The relative concentration of the inhibitory protein, bcl-2, and the stimulator, BAX, appears to be a critical determinant of whether a cell undergoes apoptosis or continues to survive. The relative levels of these proteins have been shown to be important not only in the pathogenesis and progression of cancer, but also in the homeostasis of normal tissue 101 . The mechanisms regulating physiologic cell death in the growth plate have not been well defined but probably involve cell interactions with extracellular matrix, growth factors, and cytokines. Mineralization of the matrix is associated with the release of phosphate ions. In vitro experiments have shown chondrocytes that have increased apoptosis in the presence of elevated phosphate concentrations and that the effect depends on the maturational state of the cells; differentiated chondrocytes are more sensitive to increased phosphate levels than are less differentiated cells 93 . The increase in phosphate concentration is associated with abnormalities in mitochondrial function. Chondrocytes have loss of mitochondrial membrane potential and greater reliance on glycolysis with progression through hypertrophy. It has been hypothesized that phosphate triggers apoptosis in these energy-compromised cells by promoting a mitochondrial membrane transition, leading to the release of cytochrome c and other pro-apoptotic factors and thereby inducing the death process 102 . It is also clear that local growth factors regulate programmed cell death and that abnormalities in these factors or their signaling molecules are associated with some of the developmental diseases involving the growth plate. Normally, growth factor receptors require binding of a specific growth factor to be activated. A gene mutation that causes the receptor to be activated at all times, even in the absence of the specific growth factor, is called an activating mutation. Achondroplasia, the most common cause of human dwarfism, results from an activating mutation of FGFR-3. Normally, the growth factor FGF-2 binds to FGFR-3 and leads to a reduction in proliferation and an increase in apoptosis of growth plate chondrocytes 103 . In transgenic mice, both the FGFR-3 activating mutation and overexpression of FGF-2 in chondrocytes result in growth abnormalities that mimic the human condition of achondroplasia 103 . Recently, it has been shown that the transcription factor STAT1 is responsible for the FGF effects. In mice without STAT1, the growth plate abnormalities and early apoptosis associated with FGF-2 overexpression are corrected 103 , implicating this specific signal, which is downstream of FGFR-3, as a regulator of apoptosis. PTHrP is a potent inhibitor of apoptosis and probably mediates this action by upregulation of the apoptosis inhibitor bcl-2 104,105 . In contrast, the adverse effects of glucocorticoids and radiation on skeletal growth are mediated in part by an increase in apoptosis 106,107 . Animals treated with a ten-day course of glucocorticoids have increased rates of apoptosis in hypertrophic chondrocytes in vivo and have reduced width of the growth plate 106 . Similarly, radiation stimulates apoptosis and inhibits the expression of bcl-2 while stimulating a fivefold increase in caspase-3 levels 107 . Thus, radiation favors the expression of factors that positively regulate apoptosis. Understanding the normal and pathological events involved in apoptosis of growth plate chondrocytes will lead to novel therapeutic strategies to protect the growing skeleton from the detrimental effects of radiation therapy and other toxic agents 107 .
The growth plate is essentially an avascular structure that relies on diffusion of both oxygen and nutrients from vascular arcades located on the metaphyseal side of the growth plate for cell metabolism 96 . The metaphyseal vascular channels are found in compartments bounded by calcified cartilage beneath the last row of hypertrophic chondrocytes. These vascular channels are aligned along the longitudinal axis of the bone and contain an ascending and descending capillary system 95 . It has been recognized in recent years that vascular invasion is a pivotal event in the regulation of endochondral ossification and is necessary for normal bone formation 108 . Vascular endothelial growth factor (VEGF) appears to be the factor responsible and necessary for vascular ingrowth into the growth plate 109 . VEGF is a 44-kD protein that targets vascular endothelial cells and stimulates their proliferation and migration and ultimately the formation of blood vessels 109-111 . VEGF is expressed by hypertrophic chondrocytes in the growth plate, but it is absent in resting and proliferating chondrocytes 111-114 . In animals, inhibition of VEGF function by an oral inhibiting agent or an injected genetically engineered protein that blocks activation of the receptor for VEGF leads to loss of vascular invasion 109,114 . This inhibition of vascular invasion in the absence of VEGF leads to profound disturbances in the architecture of the growth plate and affects longitudinal growth. Calcified cartilage persists as a result of a decrease in the recruitment and differentiation of osteoclasts and/or chondroclasts 114 , with widening of the hypertrophic region and diminution of trabecular bone formation. Although chondrocyte proliferation, differentiation, and maturation apparently remain normal, elimination of terminally differentiated hypertrophic chondrocytes is diminished. In contrast, on cessation of anti-VEGF treatment, there is a return of normal growth plate structure and function with resumption of capillary invasion, restoration of bone growth, resorption of hypertrophic cartilage, and normalization of growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Hypertrophic chondrocytes also express the receptor for VEGF, suggesting that this factor may have an autocrine role in these cells, although the nature of the direct effect on chondrocytes is not known 111 . Other factors have also been found to have a role in angiogenesis, although their effects may be related to modulation of VEGF expression 112 . Studies have shown that growth factors that inhibit maturation, such as PTHrP, prevent angiogenesis, while factors that accelerate chondrocyte hypertrophy, such as the transcription factor core binding factor-1(CBFA-1), induce angiogenesis 20,115 . Basic fibroblast growth factor (bFGF) is produced by chondrocytes and has known angiogenic properties. Infusion of bFGF into the proximal tibial growth plate of a rabbit accelerates vascular invasion and ossification of growth plate cartilage 116 . There is evidence to suggest that endothelial cells may also influence the terminal differentiation of chondrocytes. In cell culture, chondrocytes can be induced to undergo terminal differentiation when co-cultured with vascular endothelial cells. This activity is due to a factor secreted by these cells, and it is unique to endothelial cells 108 . Similarly, in a model of cartilage explants cultured on a chick chorioallantoic membrane, chondrocyte hypertrophy was found to occur in regions adjacent to several blood vessels. These findings suggest that cumulative release of diffusible factors from more than one vessel triggers chondrocyte hypertrophy 97 . The nature of the diffusible factors from endothelial tissues that contain this activity has not been determined. Similarly, although it has been recognized for years that enhanced growth occurs following diaphyseal fracture and that this is probably due to increased blood flow, the molecular events underlying this phenomenon are not understood.
As the skeleton approaches maturity, the rate of longitudinal bone growth diminishes and proliferation of growth plate chondrocytes decreases. This decreased growth rate is associated with structural changes in the physis, including a gradual decline in growth plate width due to the reduced height of the proliferative and hypertrophic zones as well as reduced hypertrophic cell size and column density 117 . In humans and some other mammals, the growth plate is completely resorbed following puberty, resulting in fusion of the epiphysis to the metaphysis.
Recently, it has become evident that this process of physeal closure is primarily under the control of estrogen in both sexes. In patients with genetic mutations in either the gene encoding the aromatase enzyme that converts androgen to estrogen or the gene encoding the estrogen receptor- The molecular mechanisms involved in estrogen-mediated physeal closure remain incompletely characterized. Experiments on rabbits, in which the growth plates resorb following sexual maturation as they do in humans, have suggested that estrogen may exert its effect by promoting a process of programmed replicative senescence in growth plate chondrocytes, rather than by accelerating vascular invasion or ossification 117 . Once the proliferative potential of growth plate cells is exhausted, epiphyseal fusion may occur spontaneously.
The growth of long bones is a result of a precisely orchestrated and tightly regulated series of biological steps that culminate in the formation of a calcified cartilage matrix that is subsequently resorbed and replaced by lamellar bone. The regulation of these critical cellular events occurs through integration of signals from both systemic hormones and locally produced growth factors that are subsequently relayed to the nucleus and converted into alterations in gene expression. Elucidation of the cellular and molecular pathways that regulate the process of endochondral ossification at the growth plate is an obligatory step in devising rational treatment strategies that will address the myriad of genetic, developmental, and traumatic abnormalities of growth plate function that occur in children.
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Introduction
Morphology of the Growth...
, the physes fail to close at the time of sexual maturation, and these patients show evidence of increased height due to longitudinal bone growth well into adulthood