The Role of the Growth Plate in Longitudinal Bone Growth 1 M. PINES and S. HURWITZ Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250 Israel (Received for publication August 5, 1990)
1991 Poultry Science 70:1806-1814 INTRODUCTION
Longitudinal bone growth, which determines the size and shape of the body frame, proceeds at a rate distinct from that of muscle and other tissues and is controlled by specific mechanisms. Unlike growth of most tissues, that of bone involves, in addition to tissue accretion, significant tissue destruction. The initial step in longitudinal bone growth involves deposition of cartilage ratiier than of bone. Next, the formed cartilage degrades due to calcification and invasion of bone-forming cells, which in turn form typical osseous tissue within the template previously created by cartilage. These generative-degenerative changes occur at the ends of the long bones, within the epiphyseal growth plate. Irregular metabolism at the growth plate area is associated in poultry with various problems such as dwarfism and dyschondroplasia (for review see Sauveur, 1984; Leach and Gay, 1987). The present communication
Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. Number 3025-E, 1990 series.
deals only with the regulation of growth and metabolism in this region. GROWTH PLATE CELLULARTTY
The cartilaginous epiphyseal plate lies between the epiphysis and the shaft of the long bones. Its cyto-architectural structure remains unchanged from early fetal life to the time of skeletal maturity. The cellular population of the growth plate is composed mosdy of chondrocytes, which are arranged in columns parallel to the long axis of the bone. Passing from the epiphyseal to the diaphysial side of the plate several zones are found: 1) the resting or the reserve zone, containing stem cells; 2) the proliferative zone, with stacks of flattened cells (cell division occurs mostly in a longitudinal direction and leads to cell column formation; 3) the hypertrophic zone, with hypertrophied chondrocytes; and 4) the degenerative zone, with a calcifying matrix and invading capillaries. Although the anatomic regions of the growth plate are defined by a terminology mat implies discrete structural and functional zones, in reality a gradual transition among these cellular components is observed. The major interspecies differences in the
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ABSTRACT The epiphyseal growth plate is the main site of longitudinal growth of the long bones. At this site, cartilage is formed by the proliferation and hypertrophy of cells and synthesis of the typical extracellular matrix. The formed cartilage is then calcified, degraded, and replaced by osseous tissue. Proliferation and differentiation of cartilage cells (i.e., chondrocytes) as studied mostly in culture, is regulated by various endocrine, paracrine, and autocrine agents such as growth hormone, insulin-like growth factor-I (IGF-I), trarisfonning growth factor (TGE-f$), and vitamin D metabolites (1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol). Avian chondrocyte proliferation is enhanced by agents which use adenosine 3':5'-cyclic monophosphate as a second messenger, such as parathyroid hormone or prostaglandin-^, and is depressed by guanosine 3':5'-cyclic monophosphate agonists, such as atrial natriuretic peptide. Several of the regulating agents also affect synthesis of the main extracellular components (i.e., collagen and proteoglycans) and their transfer to the extracellular space. Cartilage calcification involves matrix vesicles secreted by the chondrocytes at a specific stage. Calcification probably involves some initial nucleation agent and participation of phosphatases. During sexual maturation, the growth plate closes by an unknown mechanism and longitudinal bone growth ceases. Disorders in the metabolism of the controlling agents or the cellular responses in growth plate may lead to several deformities classified as dysplasias. In poultry, this class of disorders is represented by chondrodystrophy and dyschondroplasia. (Key words: bone growth, growth factors, epiphyseal growth plate, cartilage, chondrocytes)
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FIGURE 1. Avian and mammalian growth plate. The avian growth plate (B), when compared with the mammalian (A), contains longer columns of cells that become randomly oriented. In the hypertrophic and calcified zones, cell columns are no longer apparent. More cells are found in each zone and the metaphysial blood vessels penetrate deeper into the growth plate. The magnification for Parts A and B was 28 and 112 x, respectively (Courtesy of M. Silbermann).
growth plate may be found in the relative number of cells in each zone, the overall height of the growth plate (these two factors reflecting of growth rate), or replacement of the hypertrophic zone with a zone of fibrocartilage. Each chondrocyte, once formed, remains in a spatially fixed location throughout its cellular life cycle and accomplishes all of its physiological functions at the same discrete site. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during any particular phase of its life. Chondrocytes within the different regions show varied cell morphology as well as biochemical activities, including secretion of various extracellular matrix components (Reinholt et al., 1985) and activities of various enzymes. Alkaline phosphatase is of particular interest because its appearance marks the onset of hypertrophy and calcification (Vaananen, 1980). Longitudinal bone growth occurs as a consequence of chondrocyte proliferation and hypertrophy. The process begins with the division of the stem cells at the top of each column to produce the cells of the proliferative zone. These proliferative chondrocytes divide; the highest rate of division occurs in the
middle of the proliferative zone. The rates of cellular turnover were estimated for the rat at eight cells per column per day (Hunziker et al. 1987). In response to some unknown signal, proliferation ceases and cell hypertrophy begins. During hypertrophy, cellular volume and height increase by factors of 4 to 10, thus contributing significantly to the longitudinal growth of the bone (Hunziker et al., 1987). The rate of division of the stem cells, proliferation rate of the chondrocytes, the size of the proliferation zone, and the degree of cellular hypertrophy are controlled precisely, because they determine the size and shape of the individual long bones. Avian Growth Plate The avian growth plate, when compared with that of mammals, contains longer columns of cells that become randomly oriented (Figure 1). In the hypertrophic and calcified zones, cell columns are no longer apparent. More cells are found in each zone and the metaphysial blood vessels penetrate deeper into the growtJh plate (Leach and Gay, 1987). The growth plate of 4- to 7-wk-old chickens contains approximately 200 cells per column (Howlett, 1979) as compared with 25 cells in the rat (Kember,
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PINES AND HURWTTZ
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FIGURE 2. Avian chondrocytes in culture. A = 8 days; B = 11 days; C and D = 17 days after plating. Magnification for Parts A to C was 100 X and for Part D, 200 x. After 2 to 3 days in culture, some of the adhering cells became polygonal and a few had a fibroblast-like appearance. The latter disappeared with time, so that after 11 days most of the cells were polygonal in shape (from Pines and Hurwitz, 1988).
1960). In Leghorns, a more orderly transition between zones was observed than in broilers (Reiland et al., 1978). In turkeys, the individual columns are less distinguished than in the mammalian growth plate and are not widely separated. Fifty percent more columns are found per unit of lateral measurement in the turkey than in the rabbit growth plate and 60% more cells per unit of vertical measurement (Wise and Jennings, 1973). The proliferative zone of the turkey growth plate is approximately twice as deep as the mammalian one. A higher proliferative zone was found in a heavy turkey strain than in a light strain (LeBlanc et al., 1986). Growth Plate Cells in Culture In vivo experimentation is hardly suitable for studying short-term metabolic control in the growth plate as test agents cannot be applied directly to the cells and rapid temporal sampling is at best difficult and in most cases impossible. Growth plate explants of mouse condyle (Silbermann et al., 1987) have been successfully used to investigate hormonal control of growth and metabolism. This system preserves in vitro the in vivo architecture of the area and is advanta-
geous in studying responses of a cellular population while situated next to its natural neighbors. However, difficulties in preparation limit the practical scope of experimentation with this preparation. Furthermore, it is not possible in this system to evaluate the response of a single cell population without the confounding interactions with others. These difficulties are eliminated when using cell culture techniques. Cells are harvested from the growth plate and can be used in the form of fresh isolates or as cultured. The latter technique has been used in the present study for chicken growth plate chondrocytes (Figure 2). CHONDROCYTE PROLIFERATION
Effect of Growth Hormone and Insulin-like Growth Factor-I There is no question that growth hormone (GH) stimulates longitudinal bone growth, but whether the hormone acts directly on the growth plate or indirectly by regulating the circulating levels of insulin-like growth factorI (IGF-I) remains in question. The IGF-I hypothesis implies that GH regulates IGF-I synthesis by nonskeletal tissues such as the
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SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
Involvement of Cyclic Nucleotides Cyclic nucleotides serve as cellular second messengers to a variety of hormones. Adenosine 3':5' cyclic monophosphate (cAMP) generated by adenylate cyclase mediates signals for catecholamines such as epinephrine (Gilman, 1984), to parathyroid hormone (PTH) (Pines et al., 1983), and to dopamine (Attie et al., 1980), whereas guanosine 3':5' cyclic monophosphate acid (cGMP) mediates the message of a more recently discovered peptide hormone, atrial natriuretic peptide (ANP), which is secreted by the heart atria and is involved in the regulation of Na + homeostasis (Laragh and Atlas, 1988). Growth-plate chondrocytes exhibit adenylate cyclase activity (Pines et al., 1989) which can be activated by receptor agonists such as PTH or
prostaglandin E2, and nonreceptor agonists such as cholera toxin or forskolin (Pines and Hurwitz, 1988). An increase in cAMP accumulation caused by any of these agonists stimulates chondrocyte proliferation (Lewinson and Silbermann, 1986; Pines and Hurwitz, 1988). Moreover, chondrocytes that lose their proliferative activity with aging can be restimulated by PTH (Livne et al., 1989). In contrast to cAMP, elevation of cGMP by ANP, which activates plasma membrane guanylate cyclase (Pines and Hurwitz, 1989), inhibits basal and PTH-stimulated chondrocyte proliferation (Figure 3, Pines and Hurwitz, 1988). Effect of Vitamin D Metabolites, Other Hormones, and Growth Factors Cartilage cells possess receptors for both 1,25-[dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] (Conoletal, 1980) andcanhydroxylate 25-hydroxycholecalciferol in either the 1 or 24 position (Garabedian et al, 1978). The 24,25-(OH)2D3 was found to be essential for normal tibial epiphyseal growth (Goodwin et al, 1978) and induced DNA synthesis and activity of ornithine decarboxylase (an enzyme associated with cell proliferation) in chicken cartilage in vitro (Binderman and Somjen, 1984). However, 1,25-(OH)2D3 inhibited DNA synthesis in rat condylar cartilage (Silbermann et al, 1987) and stimulated avian and mammalian cartilage growth in vitro (Burch et al, 1988). Although other hormones such as thyroid hormones (Burch and Van Wyk, 1987; Lewinson et al, 1989) and sex hormones (Corvol et al, 1987) augment chondrocyte proliferation, interleukin1 attenuated proliferation (Iwamoto et al, 1989a). GROWTH PLATE EXTRACELLULAR MATRIX
The principal components of the extracellular matrix are collagen and proteoglycans. Five genetically distinct collagen types have been shown to be associated with the epiphyseal cartilage, namely, Types II, VI, DC, X, and XI. Among these, Type X collagen is synthesized exclusively by hypertrophic chondrocytes in the part of the growth plate destined for matrix mineralization (Kwan et al, 1986, 1989). The distribution of Type H collagen mRNA suggests that this molecule is synthesized primarily by cells of the proliferating zone and by newly formed hypertrophic
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liver (Froesch et al, 1985). With the recent recognition that GH regulates the production of IGF-I in various tissues, autocrine and paracrine functions of IGF-I have been suggested as important components of GH action (D'Ercole et al, 1984). Hypophysectomy resulted in a time-dependent decrease in the rate of longitudinal bone growth. Local administration of GH into the proximal tibial epiphyseal plate stimulated longitudinal bone growth on the injected side (Isaksson et al., 1982). Local administration of IGF-I also affected epiphyseal cartilage without any synergism with GH (Isgaard et al., 1986). Cultured chondrocytes possess IGF-I and GH receptors (Eden et al., 1983; Trippel et al., 1983) and respond to both peptides by increased DNA synthesis (Madsen et al., 1983). The GH and IGF-I appear to stimulate chondrocytes at different stages of maturation: GH affects the chondroprogenitor cells and IGF-I affects the proliferative chondrocytes. Direct stimulation by GH sensitizes the prechondrocytes and young differentiating cells to IGF-I. Concomitantly, the IGF-I gene is expressed, resulting in an increase in IGF-I synthesis in the differentiating cells. The locally produced IGF-I interacts with receptors on the proliferative chondrocytes by either an autocrine or a paracrine mechanism (for review, see Isaksson et al., 1987). Thus GH stimulates longitudinal growth by 1) directly stimulating the differentiated epiphyseal growth plate precursor cells and 2) stimulating the local production of and increasing the cell responsiveness of IGF-I.
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hPTH
hPTH+hANP
FIGURE 3. Effect of human parathyroid hormone (h PTH) and human atrial natriuretic peptide (h ANP) on avian chondrocyte proliferation. Cells were incubated for a total 48 h with the hormones in 1 mL Dulbecco's Modified Eagle's Medium containing 5% fetal calf serum. After 24 h, the medium was replaced with fresh medium containing the hormones. At the end of the incubation period, the hormone-containing medium was discarded and fresh medium containing 1 uCi/mL [H]thymidine was added for 4 h and the DNA-bound [ H]thymidine was evaluated, dpm = disintigration per minute, (from Pines and Hurwitz, 1988).
cells. Relatively little or no Type II collagen is made by mature hypertrophic chondrocytes, particularly those present in the calcifying cartilage (Sandberg and Vuorio, 1987). In contrast to Types U and X, which have specific localization, Type IX collagen is distributed throughout the growth plate matrix (MullerGlauser et al, 1986). Chondrocyte collagen synthesis is regulated by growth factors (Redini et al, 1988) and hormones such as the PTH (Figure 4, Pines et al, 1990). Proteoglycans are macromolecules present in varying amounts in all connective tissues (for review, see Ruoslahti, 1988). These macromolecules are constructed of a protein core onto which long side chain sugars such as chondroitin sulfate, keratan sulfate, or dermatan sulfate are attached. Chondroitin sulfate contains repeating units of n-acetyl-galactoseamine and glucoronic acid, whereas keratin sulfate consists of sulfated galactose and glucoseamine. The protein and sugar moieties are linked by link proteins with hyaluronic acid to form aggregates. The maturation of the chondrocytes that occurs along the growth plate is accompanied by changes in the level of proteoglycan synthesis, in the proportion of proteoglycan aggregates and in the size of proteoglycan monomers (Campo and Romano, 1986). For example, hypertrophic chondro-
cytes produce less proteoglycan than cells from resting or proliferative zones (Pawlowski et al, 1987, Makower et al, 1988). The amount and the structure of the proteoglycans synthesized by chondrocytes are affected by hormones and growth factors. Different growth factors cause synthesis of proteoglycans of different sizes (Makower et al, 1988). Moreover, IGF-I stimulates proteoglycan synthesis in chondrocytes isolated from the resting and proliferative zones but not from the hypertrophic zone, whereas epidermal growth factor stimulates proteoglycan synthesis from all three zones. The IGF-I (Kemp et al, 1988) and transforming growth factor P (Redini et al., 1988) augmented glycosaminoglycan synthesis of chondrocytes. CARTILAGE CALCIFICATION
The process of cartilage calcification involves different interacting matrix molecules and is carefully regulated at the cellular level. Whatever the precise mechanism, it appears mat some nucleating agent is required to initiate calcification. Crystal growth can then proceed by spontaneous accretion of calcium salts at any such nucleus. Although collagen has been suggested as active in nucleation in bone (Glimcher, 1989), it does not appear to
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SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
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initiate calcification in cartilage because the crystals of hydroxyapatite bear no consistent spatial orientation relative to the collagen fibers. The two matrix components, matrix vesicles (Anderson, 1989) and the proteoglycan monomers (Poole et al, 1989), have been implicated as initiators of calcification. Matrix vesicles, first described by Bonucci (1967) and Anderson (1969), are cell-derived, membranous 100- to 200-nm particles, in which calcium phosphate mineral is deposited (Anderson, 1969). The first mineral deposits are often in close apposition to the inner leaflet of the vesicle membrane (Anderson, 1989). These vesicles contain a high concentration of Ca-binding acidic phospholipids (Peress et al, 1974; Wuthier, 1975; Glaser and Conrad 1981) and phosphatases, which yield orthophosphate for nascent mineral formation (Hsu, 1983; Siegel et al., 1983; Chin et al., 1986). After initiation of calcification, crystals of hydroxyapatite begin to accumulate within the confines of the matrix vesicle membrane (Anderson, 1989). The contribution of matrix vesicles to initiation of calcification is, however, controversial, since some studies demonstrated mineral deposition in areas poor in or devoid of matrix vesicles (Landis and Glimcher, 1982; Poole et al, 1984). Using electron spec-
troscopic imaging techniques, Arsenault et al. (1988) found that the extracellular matrix contains high levels of Ca associated with proteoglycans and related matrix proteins. Phosphate, however, appeared restricted to sites of matrix vesicles associated with mineral deposition. Thus, matrix vesicles could act to deliver phosphate ions to sites of calcification. In recent reports it has been proposed that the C-terminal peptide of Type II collagen synthesized primarily by the cells of the proliferating zone and newly formed hypertrophic cells (Sandberg and Vuorio, 1987), acts as a nucleation center for the Ca-P crystals (Poole et al, 1982, 1989). This 35-kDa protein was found mainly in the calcification sites of the lower hypertrophic zone (Poole et al, 1984) and binds avidly to hydroxyapatite (Choi et al., 1983). Histological studies showed that following the formation of the calcified cartilage the chondrocytes vanish and are replaced by bone cells. Recently, it has been suggested that hypertrophic chondrocytes are not the terminal stage of development but can become osteoblasts (Shapiro and Boyde, 1987). The demonstration that hypertrophic chondrocytes could switch from synthesis of Type II collagen to Type I collagen supports this hypothesis.
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PINES AND HURWITZ
foundly incomplete (Boden et al, 1987). In osteochondromatosis, for example, loss of columnar and matrical integrity at the growth plate periphery can cause short stature. In endochondromatosis, some of the longitudinal columns fail to undergo chondroosseous replacement and are left within the metaphysis, but the others continue with their longitudinal elongation (Ogden and Rosenberg, 1988). Some skeletal dysplasias are characterized by biochemical abnormalities. For example, in Kniest dysplasia the impaired production of collagen Type II results in imperfect collagen fibrils formation (Poole et al, 1988), spondyloepiphyseal dysplasia (Murray et al, 1989) or proteoglycans defects (Pedrini-Mille et al, 1984).
inhibited by various matrix proteins such as proteoglycans (Dziewaitkowski and Majznerski, 1985), osteocalcin (Romberg et al, 1986), phosphoproteins, and osteonectin (Menanteau et al, 1982).
Anderson, H. C , 1969. Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41:59-72. Anderson, H. C , 1989. Biology of disease: Mechanism of mineral formation in bone. Lab. Invest 60:320-330. Arsenault, A. L., F. P. Ottensmeyer, and I. B. Heath, 1988. An electron microscopic and spectroscopic study of murine epiphyseal cartilage: Analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution. J. Ultrastruct. Mol. Struct. Res. 98:32-^7. Attie, M. F., E. M. Brown, D. G. Gardner, A. M. Spiegel and G. D. Aurbach, 1980. Characterization of the dopamine-responsive adenylate cyclase of bovine parathyroid cells and its relationship to parathyroid hormone secretion. Endocrinology 107:1776-1781. Binderman, I., and D. Somjen, 1984. 24,25-dihydroxycholecalciferol induces the growth of chick cartilage in vitro. Endocrinology 115:430-432. Boden, S. D., F. S. Kaplan, M. D. Fallon, R. Ruddy, J. Belik, E. Zachai, and J. Ellis, 1987. Metatrophic dwarfism. Uncoupling of endochondral and perichondral growth. J. Bone Jt Surg. 69A:174-184. Bonucci, E., 1967. Fine structure of early cartilage calcification. J. Ultrastruct. Res. 20:33-50. Burch, W. M., M. Lopez-Carlos, M. R. Uskokovic, and M. K. Drezner, 1988. 1,25-dihydroxyvitamin D3 stimulates avian and mammalian cartilage growth in vitro. J. Bone Miner. Res. 3:87-91. Burch, W. M., and J. J. Van Wyk, 1987. Triiodothyronine stimulates cartilage growth and maturation by different mechanisms. Am. J. Physiol. 252:E177-E182. Campo, R. D., and J. E. Romano, 1986. Changes in cartilage proteoglycans associated with calcification. Calcif. Tissue Int. 39:175-184. Chin, J. E., E. M. Elaine, M S. Kemick, and R. E. Wuthier, 1986. Effect of synthetic human parathyroid hormone on the levels of alkaline phosphatase activity and formation of alkaline-rich matrix vesicles by primary cultures of chicken epiphyseal growth plate chondrocytes. Bone Miner. 1:421^36. Choi, H. U., L. H. Tang, T. L. Johnson, S. Pal, L. Rosenberg, A. Riener, and A. R. Poole, 1983.
GROWTH PLATE CLOSURE
During sexual maturation, when bone reaches its ultimate length, growth plate cells diminish in number and the growth plate loses its functionality. The mechanisms responsible for growth plate closure remain unknown. It may be hypothesized that each of the stem cells is programmed to a certain number of divisions. Once this limit has been reached, proliferation ceases and the growth plate closes. In the rat, each stem cell gives rise to 30 cells, and 40 to 50 cell divisions of the chondroprogenitor cells were sufficient to account for normal growth of rat tibia (Kember, 1978). Gonadal hormones probably play a key role in the demise of the growth plate. Administration of sex hormones to immature animals produces an initial acceleration of growth followed by premature cessation of growth and growth plate closure (Kember, 1978). ABNORMAL GROWTH PLATE
The growth plate may be involved in a variety of morphological and physiological deformities referred to as skeletal dysplasias. In metatrophic dwarfism the maturation of proliferating chondrocytes into hypertrophic cells was either completely absent or pro-
REFERENCES
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The events involving matrix synthesis and calcification are under hormonal control. Both l,25-(OH)2D3 and 24,25-(OH)2D3 are required for maximal synthesis of C-propeptide of collagen Type H (Hinek and Poole, 1988). The 24,25-(OH)2D3 is needed for maximal expression of alkaline phosphatase activity (Hale et al, 1986; Schwartz et al, 1988). The 24,25-(OH)2D3 inhibits chondrocyte phospholipase A 2 , which acts on membrane phospholipid (Schwartz et al, 1988). Receptors for l,25-(OH)2D3 were found in rabbit hypertrophic chondrocytes (Iwamoto et al, 1989b). Parathyroid hormone inhibits matrix vesicle alkaline phosphatase activity (Chin et al, 1986). Once crystal nucleation has occurred, calcification can proceed, resulting in crystal growth. The rate of this process is regulated by Ca 2+ and HPO4 - concentrations and can be
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
hormone on longitudinal bone growth. Endocr. Rev. 8:426-438. Isgaard, J., A. Nilsson, A. Lindahl, J. O. Jansson, and O.G.P. Isaksson, 1986. Effect of local administration of GH and IGF-I on longitudinal bone growth in rats. Am. J. Physiol. 250:E367-E372. Iwamoto, M., T. Koike, K. Nakashima, K. Sato, and Y. Kato, 1989a. Interleukin 1: a regulator of chondrocyte proliferation. Immunol. Lett. 21:153-156. Iwamoto, M. K., K. Sato, A. Nakashima, A. Shimazu, and Y. Kato, 1989b. Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor. Dev. Biol. 136: 500-508. Kember, N. F., 1960. Cell division in endochondral ossification. J. Bone Jt Surg. 42B:824-839. Kember, N. F., 1978. Cell kinetics and the control of growth in long bones. Cell Tissue Kinet. 11: 477-485. Kemp, S. F., G. L. Kearns, W. G. Smith, and M. J. Elders, 1988. Effects of IGF-I on the synthesis and processing of glucosaminoglycans in cultured chick chondrocytes. Acta Endocrinol. 119:245-250. Kwan, A.P.L., I. R. Dickson, A. J. Freemont, and M. E. Grant 1989. Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage. J. CeU. Biol. 109:1849-1856. Kwan, AX.P., A. J. Freemont, and M. E. Grant 1986. Immunoperoxidase localization of type X collagen in chick tibia. Biosci. Rep. 6:155-162. Landis, W. J., and M. J. Glimcher, 1982. Electron optical and analytical observation of rat growth plate cartilage prepared by ultracrypmicrotomy: The failure to detect a mineral phase in matrix vesicles and the identification of heterodispersed particles as the initial solid phase of calcium phosphate deposited in the extracellular matrix. J. Ultrastruct. Res. 78: 227-268. Laragh, J. H., and S. A. Atlas, 1988. Atrial natriuretic hormone: A regulator of blood pressure and volume homeostasis. Kidney Int. 34:S64-S71. Leach, R. M, and C. V. Gay, 1987. Role of epiphyseal cartilage in endochondral bone formation. J. Nutr. 117:784-790. LeBlanc, B., M Wyers, F. Cohn-Bendit J. M. Legall, E. Thibault and J. M. Florent 1986. Histology and histomorphometry of the tibia growth in two turkey strains. Poultry Sci. 65:1787-1795. Lewinson, D., Z. Harel, P. Shenzer, M. Silbennann, and Z. Hochberg, 1989. Effect of thyroid hormone and growth hormone on recovery from hypothyroidism of epiphyseal growth plate cartilage and its adjacent bone. Endocrinology 124:937-945. Lewinson, D., and M. Silbennann, 1986. Parathyroid hormone stimulates proliferation of chondroprogenitor cells in vitro. Calcif. Tissue Int. 38:155-162. Livne, E., A. Weiss, and M. Silbennann, 1989. Articular chondrocytes lose their proliferative activity with aging yet can restimulate by PTH (1-84), PGEi, and dexamethasone. J. Bone Miner. Res. 4:539-548. Madsen, K., U. Friburg, P. Roos, S. Eden, and O. Isaksson, 1983. Growth hormone stimulates the proliferation of culture chondrocytes from rabbit ear and rat rib growth cartilage. Nature 304:545-547. Makower, A. M., J. Wroblewski, and A. Pawlowski, 1988. Effect of IGF-I, EGF, and FGF on proteoglycans synthesis by fractionated chondrocytes of rat rib
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Isolation and characterization of a 35,000 molecular weight subunit fetal cartilage matrix protein. J. Biol. Chem. 258:655-661. Corvol, M., A. Carracosa, L. Tsagris, O. Blanchard, and R. Rappaport, 1987. Evidence for a direct in vitro action of sex steroids on rabbit cartilage cells during skeletal growth: Influence of age and sex. Endocrinology 120:1422-1429. Corvol, M., A. Ulmann, and M. Garabedian, 1980. Specific nuclear uptake of 24,25(OH)2D3 is a vitamin D3 metabolite biologically active in cartilage. Fed. Eur. Biochem. Soc. Lett 116:273-276. D'Ercole, A. J., A. D. Stiles, and L. E. Underwood, 1984. Tissue concentrations of somatomedin C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc. Natl. Acad. Sci. USA 81:935-939. Dziewaitkowski, D. D., and L. L. Majznerski, 1985. Role of proteoglycans in endochondral ossification: Inhibition of calcification. Calcif. Tissue Int. 37: 560-564. Eden, S. O., G. P. Isaksson, K. Madsen, and U. Friberg, 1983. Specific binding of growth hormone to isolated chondrocytes from rabbit ear and epiphyseal plate. Endocrinology 112:1127-1129. Froesch, E. R., C. Schmid, J. Schawander, and J. Zapf, 1985. Action of insulin-growth factors. Annu. Rev. Physiol. 47:443-467. Garabedian, M., M. B. DuBois, M. T. Corvol, E. Pezant, and S. Balsan, 1978. Vitamin D and cartilage. I. In vitro metabolism of 25-hydroxycholecalciferol by cartilage. Endocrinology 102:1262-1268. Gilman, A. G., 1984. Guanine nucleotide-binding regulatory proteins and dual control of adenylate cyclase. J. Clin. Invest. 73:1-4. Glaser, J. H., and H. E. Conrad, 1981. Formation of matrix vesicles by cultured chick embryo chondrocytes. J. Biol. Chem. 256:12607-12611. Glimcher, M. J., 1989. Mechanism of calcification: Role of collagen fibrils and collagen-phosphoprotein complexes in vitro and in vivo. Anat Rec. 224:139—153. Goodwin, D., D. Noff, and S. Edelstein, 1978. 24,25-dihydroxyvitamin D is a metabolite of vitamin D essential for bone formation. Nature 276:517-519. Hale, L. V., M.L.S. Kemick, and R. E. Wuthier, 1986. Effect of vitamin D metabolites on the expression of alkaline phosphatase activity by epiphyseal hypertrophic chondrocytes in primary culture. J. Bone Miner. Res. 1:489-495. Hinek, A., and A. R. Poole, 1988. The influence of vitamin D metabolites on the calcification of cartilage matrix and the C-propeptide of type II collagen (chondrocalcin). J. Bone Miner. Res. 3: 421-429. Howlett, C. R., 1979. The fine structure of the proximal growth plate of avian tibia. J. Anat. 128:377-399. Hsu, H.H.T., 1983. Purification and partial characterization of ATP-pyrophosphohydrolase from fetal bovine epiphyseal cartilage. J. Biol. Chem. 258:3463-3468. Hunziker, E. B., R. K. Schenk, and L. M. Cruz-Orive, 1987. Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J. Bone Jt. Surg. 69A: 162-173. Isaksson, O., J. O. Jansson, and I.A.M. Gause, 1982. Growth hormone stimulates longitudinal bone growth directly. Science 216:1237-1239. Isaksson, O.G.P., A. Lindahl, A. Nilsson, and J. Isgaard, 1987. Mechanism of the stimulatory effect of growth
1813
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PINES AND HURWITZ Hollister, L. Muray, and D. Rimoin, 1988. Kniest dysplasia is characterized by an apparent abnormal processing of the C-propeptide of type II cartilage collagen resulting in imperfect fibril assembly. J. Clin. Invest. 81:579-589. Redini, F., P. Galera, A. Mauviel, G. Loyau, and J. P. Pujol, 1988. Transforming growth factor p stimulates collagen and glycosaminoglycan biosynthesis in cultured rabbit articular chondrocytes. Fed. Eur. Biochem. Soc. Lett. 234:172-176. Reiland, S., S. E. Olsson, P. W. Poulos, and K. Elwinger, 1978. Normal and pathological skeletal development in broiler and Leghorn chickens. A comparative investigation. Acta Radio. Suppl. 358:277-298. Reinholt, F. P., B. Engfelt, D. Heinegard, and A. Hjerpe, 1985. Proteoglycans and glycosaminoglycans of epiphyseal chondrocytes in florid and healing low phosphate, vitamin D deficient rickets. Collagen Relat. Res. 5:55-64. Romberg, R. W., P. G. Wemess, B. L. Riggs, and M. G. Mann, 1986. Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium binding proteins. Biochemistry 25:1176-1180. Ruoslahti, E., 1988. Structure and biology of proteoglycans. Annu. Rev. Cell. Biol. 4:229-255. Sandberg, M., and E. Vuorio, 1987. Localization of type I, n and in collagen mRNAs in developing human skeletal tissue it situ hybridization. J. Cell Biol. 104: 1077-1084. Sauveur, B., 1984. Dietary factors as causes of leg abnormalities in poultry—a review. World Poult Sci. J. 40:195-206. Schwartz, Z., D. L. Schlader, L. D. Swain, and B. D. Boyan, 1988. Direct effect of 1,25-dihyrdroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on growth zone and resting zone chondrocytes membrane alkaline phosphatase and phospholipase-A2 specific activity. Endocrinology 123:2878-2884. Shapiro, 1. M., and A. Boyde, 1987. Mineralization of normal and rachitic chick growth cartilage: Vascular canals, cartilage calcification and osteogenesis. Scanning Electron Microsc. 1:599-606. Siegel, S. A., C. F. Hummel, and R. P. Carty, 1983. The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification. J. Biol. Chem. 258: 8601-8607. Silbermann, M, K. Von Der Mark, N. Mirsky, M. Van Menxel, and D. Lewinson, 1987. Effect of increased doses of 1,25-dihydroxyvitamin D3 on matrix and DNA synthesis in condylar cartilage of suckling mice. Calcif. Tissue Int. 41:95-104. Trippel, S. B., J. J. Van Wyk, M. B. Foster, M E. Svoboda, 1983. Characterization of specific somatomedin-C receptor on isolated bovine growth plate chondrocytes. Endocrinology 112:2128-2135. Vaananen, H. K., 1980. Immunohistochemistry localization of alkaline phosphatase in the chicken epiphyseal growth plate. Histochemistry 65:143-148. Wise, D. R„ and A. R. Jennings, 1973. The development and morphology of the growth plates of two long bones of the turkey. Res. Vet. Sci. 14:161-166. Wuthier, R. E., 1975. Lipid composition of isolated cartilage cells, membranes and matrix vesicles. Biochim. Biophys. Acta 409:128-143.
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growth plate. Exp. Cell Res. 179:498-506. Menanteau, J., W. F. Newman, and M. W. Newman, 1982. A study of bone proteins which can prevent hydroxyhapatite formation. Metab. Bone Dis. Relat. Res. 4:157-161. Muller-Glauser, W. B., B. Humbel, M. Glatt, P. Stauli, K. H. Winterhalter, and P. Bruckner, 1986. On the role of type IX collagen in the extracellular matrix cartilage: Type IX collagen is localized to intersection of collagen fibrils. J. Biol. Chem. 102: 1931-1939. Murray, L. W., J. Bautista, P. L. James, and D. L. Rimoin, 1989. Type U collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias. Am. J. Hum. Genet. 45:5-15. Ogden, J. A., and L. C. Rosenberg, 1988. Defining the growth plate. Pages 1-15 in: Behavior of the Growth Plate. K. H. Uhthoff and J. J. Wiley, ed. Raven Press, New York, NY. Pawlowski, A., A. M. Makower, K. Madsen, J. Wroblewski, and U. Friberg, 1987. Cell fractions from rat rib growth cartilage: Biochemical characterization of matrix molecules. Exp. Cell Res. 164: 211-222. Pedrini-Mille, A., J. A. Maynard, and V. A. Pedrini, 1984. Pseudoachondroplasia: Biochemical and histochemical studies of cartilage. J. Bone Jt. Surg. 66: 1401-1414. Peress, N. S., H. C. Anderson, and S. W. Sajdera, 1974. The lipids of matrix vesicles from bovine fetal epiphyseal cartilage. Calcif. Tissue Res. 14:275-281. Pines, M., I. Granot, and S. Hurwitz, 1990. Cyclic AMPdependent inhibition of collagen synthesis in avian epiphyseal cartilage cells: effect of chicken and human parathyroid hormone and parathyroid hormone-related peptide. Bone Miner. 9:23-33. Pines, M., and S. Hurwitz, 1988. The effect of parathyroid hormone and atrial natriuretic peptide on cyclic nucleotides production and proliferation of avian epiphyseal growth plate chondropiogenitor cells. Endocrinology 123:360-365. Pines, M., and S. Hurwitz, 1989. Atrial natriuretic peptide and sodium nitroprusside stimulate cyclic GMP accumulation by avian skin fibroblasts and epiphyseal growth-plate chondroprogenitor cells. J. Endocrinol. 120:319-324. Pines, M., D. Polin, and S. Hurwitz, 1983. Urinary cyclic AMP excretion in birds: Dependence on parathyroid hormone activity. Gen. Comp. Endocrinol. 49:90-96. Pines, M., B. Yosif, and S. Hurwitz, 1989. Modulation of the responsiveness of the adenylate cyclase system in avian chondroprogenitor cells by pertussis toxin, PTH and PGE2. J. Bone Miner. Res. 4:743-750. Poole, A. R., Y. Matsui, A. Hinek, and E. R. Lee, 1989. Cartilage macromolecules and the calcification of cartilage matrix. Anat. Rec. 224:167-179. Poole, A. R., I. Pidoux, and A. Reiner, 1982. An immunoelectron microscopic study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage. J. Cell Biol. 93:921-937. Poole, A. R., I. Pidoux, A. Reiner, H. Choi, and L. C. Rosenberg, 1984. Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation. J. Cell Biol. 98:532-539. Poole, A. R., I. Pidoux, A. Riener, L. Rosenberg, D.
1991 Poultry Science 70:1806-1814 INTRODUCTION
Longitudinal bone growth, which determines the size and shape of the body frame, proceeds at a rate distinct from that of muscle and other tissues and is controlled by specific mechanisms. Unlike growth of most tissues, that of bone involves, in addition to tissue accretion, significant tissue destruction. The initial step in longitudinal bone growth involves deposition of cartilage ratiier than of bone. Next, the formed cartilage degrades due to calcification and invasion of bone-forming cells, which in turn form typical osseous tissue within the template previously created by cartilage. These generative-degenerative changes occur at the ends of the long bones, within the epiphyseal growth plate. Irregular metabolism at the growth plate area is associated in poultry with various problems such as dwarfism and dyschondroplasia (for review see Sauveur, 1984; Leach and Gay, 1987). The present communication
Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. Number 3025-E, 1990 series.
deals only with the regulation of growth and metabolism in this region. GROWTH PLATE CELLULARTTY
The cartilaginous epiphyseal plate lies between the epiphysis and the shaft of the long bones. Its cyto-architectural structure remains unchanged from early fetal life to the time of skeletal maturity. The cellular population of the growth plate is composed mosdy of chondrocytes, which are arranged in columns parallel to the long axis of the bone. Passing from the epiphyseal to the diaphysial side of the plate several zones are found: 1) the resting or the reserve zone, containing stem cells; 2) the proliferative zone, with stacks of flattened cells (cell division occurs mostly in a longitudinal direction and leads to cell column formation; 3) the hypertrophic zone, with hypertrophied chondrocytes; and 4) the degenerative zone, with a calcifying matrix and invading capillaries. Although the anatomic regions of the growth plate are defined by a terminology mat implies discrete structural and functional zones, in reality a gradual transition among these cellular components is observed. The major interspecies differences in the
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ABSTRACT The epiphyseal growth plate is the main site of longitudinal growth of the long bones. At this site, cartilage is formed by the proliferation and hypertrophy of cells and synthesis of the typical extracellular matrix. The formed cartilage is then calcified, degraded, and replaced by osseous tissue. Proliferation and differentiation of cartilage cells (i.e., chondrocytes) as studied mostly in culture, is regulated by various endocrine, paracrine, and autocrine agents such as growth hormone, insulin-like growth factor-I (IGF-I), trarisfonning growth factor (TGE-f$), and vitamin D metabolites (1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol). Avian chondrocyte proliferation is enhanced by agents which use adenosine 3':5'-cyclic monophosphate as a second messenger, such as parathyroid hormone or prostaglandin-^, and is depressed by guanosine 3':5'-cyclic monophosphate agonists, such as atrial natriuretic peptide. Several of the regulating agents also affect synthesis of the main extracellular components (i.e., collagen and proteoglycans) and their transfer to the extracellular space. Cartilage calcification involves matrix vesicles secreted by the chondrocytes at a specific stage. Calcification probably involves some initial nucleation agent and participation of phosphatases. During sexual maturation, the growth plate closes by an unknown mechanism and longitudinal bone growth ceases. Disorders in the metabolism of the controlling agents or the cellular responses in growth plate may lead to several deformities classified as dysplasias. In poultry, this class of disorders is represented by chondrodystrophy and dyschondroplasia. (Key words: bone growth, growth factors, epiphyseal growth plate, cartilage, chondrocytes)
1807
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
B
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FIGURE 1. Avian and mammalian growth plate. The avian growth plate (B), when compared with the mammalian (A), contains longer columns of cells that become randomly oriented. In the hypertrophic and calcified zones, cell columns are no longer apparent. More cells are found in each zone and the metaphysial blood vessels penetrate deeper into the growth plate. The magnification for Parts A and B was 28 and 112 x, respectively (Courtesy of M. Silbermann).
growth plate may be found in the relative number of cells in each zone, the overall height of the growth plate (these two factors reflecting of growth rate), or replacement of the hypertrophic zone with a zone of fibrocartilage. Each chondrocyte, once formed, remains in a spatially fixed location throughout its cellular life cycle and accomplishes all of its physiological functions at the same discrete site. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during any particular phase of its life. Chondrocytes within the different regions show varied cell morphology as well as biochemical activities, including secretion of various extracellular matrix components (Reinholt et al., 1985) and activities of various enzymes. Alkaline phosphatase is of particular interest because its appearance marks the onset of hypertrophy and calcification (Vaananen, 1980). Longitudinal bone growth occurs as a consequence of chondrocyte proliferation and hypertrophy. The process begins with the division of the stem cells at the top of each column to produce the cells of the proliferative zone. These proliferative chondrocytes divide; the highest rate of division occurs in the
middle of the proliferative zone. The rates of cellular turnover were estimated for the rat at eight cells per column per day (Hunziker et al. 1987). In response to some unknown signal, proliferation ceases and cell hypertrophy begins. During hypertrophy, cellular volume and height increase by factors of 4 to 10, thus contributing significantly to the longitudinal growth of the bone (Hunziker et al., 1987). The rate of division of the stem cells, proliferation rate of the chondrocytes, the size of the proliferation zone, and the degree of cellular hypertrophy are controlled precisely, because they determine the size and shape of the individual long bones. Avian Growth Plate The avian growth plate, when compared with that of mammals, contains longer columns of cells that become randomly oriented (Figure 1). In the hypertrophic and calcified zones, cell columns are no longer apparent. More cells are found in each zone and the metaphysial blood vessels penetrate deeper into the growtJh plate (Leach and Gay, 1987). The growth plate of 4- to 7-wk-old chickens contains approximately 200 cells per column (Howlett, 1979) as compared with 25 cells in the rat (Kember,
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PINES AND HURWTTZ
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FIGURE 2. Avian chondrocytes in culture. A = 8 days; B = 11 days; C and D = 17 days after plating. Magnification for Parts A to C was 100 X and for Part D, 200 x. After 2 to 3 days in culture, some of the adhering cells became polygonal and a few had a fibroblast-like appearance. The latter disappeared with time, so that after 11 days most of the cells were polygonal in shape (from Pines and Hurwitz, 1988).
1960). In Leghorns, a more orderly transition between zones was observed than in broilers (Reiland et al., 1978). In turkeys, the individual columns are less distinguished than in the mammalian growth plate and are not widely separated. Fifty percent more columns are found per unit of lateral measurement in the turkey than in the rabbit growth plate and 60% more cells per unit of vertical measurement (Wise and Jennings, 1973). The proliferative zone of the turkey growth plate is approximately twice as deep as the mammalian one. A higher proliferative zone was found in a heavy turkey strain than in a light strain (LeBlanc et al., 1986). Growth Plate Cells in Culture In vivo experimentation is hardly suitable for studying short-term metabolic control in the growth plate as test agents cannot be applied directly to the cells and rapid temporal sampling is at best difficult and in most cases impossible. Growth plate explants of mouse condyle (Silbermann et al., 1987) have been successfully used to investigate hormonal control of growth and metabolism. This system preserves in vitro the in vivo architecture of the area and is advanta-
geous in studying responses of a cellular population while situated next to its natural neighbors. However, difficulties in preparation limit the practical scope of experimentation with this preparation. Furthermore, it is not possible in this system to evaluate the response of a single cell population without the confounding interactions with others. These difficulties are eliminated when using cell culture techniques. Cells are harvested from the growth plate and can be used in the form of fresh isolates or as cultured. The latter technique has been used in the present study for chicken growth plate chondrocytes (Figure 2). CHONDROCYTE PROLIFERATION
Effect of Growth Hormone and Insulin-like Growth Factor-I There is no question that growth hormone (GH) stimulates longitudinal bone growth, but whether the hormone acts directly on the growth plate or indirectly by regulating the circulating levels of insulin-like growth factorI (IGF-I) remains in question. The IGF-I hypothesis implies that GH regulates IGF-I synthesis by nonskeletal tissues such as the
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SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
Involvement of Cyclic Nucleotides Cyclic nucleotides serve as cellular second messengers to a variety of hormones. Adenosine 3':5' cyclic monophosphate (cAMP) generated by adenylate cyclase mediates signals for catecholamines such as epinephrine (Gilman, 1984), to parathyroid hormone (PTH) (Pines et al., 1983), and to dopamine (Attie et al., 1980), whereas guanosine 3':5' cyclic monophosphate acid (cGMP) mediates the message of a more recently discovered peptide hormone, atrial natriuretic peptide (ANP), which is secreted by the heart atria and is involved in the regulation of Na + homeostasis (Laragh and Atlas, 1988). Growth-plate chondrocytes exhibit adenylate cyclase activity (Pines et al., 1989) which can be activated by receptor agonists such as PTH or
prostaglandin E2, and nonreceptor agonists such as cholera toxin or forskolin (Pines and Hurwitz, 1988). An increase in cAMP accumulation caused by any of these agonists stimulates chondrocyte proliferation (Lewinson and Silbermann, 1986; Pines and Hurwitz, 1988). Moreover, chondrocytes that lose their proliferative activity with aging can be restimulated by PTH (Livne et al., 1989). In contrast to cAMP, elevation of cGMP by ANP, which activates plasma membrane guanylate cyclase (Pines and Hurwitz, 1989), inhibits basal and PTH-stimulated chondrocyte proliferation (Figure 3, Pines and Hurwitz, 1988). Effect of Vitamin D Metabolites, Other Hormones, and Growth Factors Cartilage cells possess receptors for both 1,25-[dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] (Conoletal, 1980) andcanhydroxylate 25-hydroxycholecalciferol in either the 1 or 24 position (Garabedian et al, 1978). The 24,25-(OH)2D3 was found to be essential for normal tibial epiphyseal growth (Goodwin et al, 1978) and induced DNA synthesis and activity of ornithine decarboxylase (an enzyme associated with cell proliferation) in chicken cartilage in vitro (Binderman and Somjen, 1984). However, 1,25-(OH)2D3 inhibited DNA synthesis in rat condylar cartilage (Silbermann et al, 1987) and stimulated avian and mammalian cartilage growth in vitro (Burch et al, 1988). Although other hormones such as thyroid hormones (Burch and Van Wyk, 1987; Lewinson et al, 1989) and sex hormones (Corvol et al, 1987) augment chondrocyte proliferation, interleukin1 attenuated proliferation (Iwamoto et al, 1989a). GROWTH PLATE EXTRACELLULAR MATRIX
The principal components of the extracellular matrix are collagen and proteoglycans. Five genetically distinct collagen types have been shown to be associated with the epiphyseal cartilage, namely, Types II, VI, DC, X, and XI. Among these, Type X collagen is synthesized exclusively by hypertrophic chondrocytes in the part of the growth plate destined for matrix mineralization (Kwan et al, 1986, 1989). The distribution of Type H collagen mRNA suggests that this molecule is synthesized primarily by cells of the proliferating zone and by newly formed hypertrophic
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liver (Froesch et al, 1985). With the recent recognition that GH regulates the production of IGF-I in various tissues, autocrine and paracrine functions of IGF-I have been suggested as important components of GH action (D'Ercole et al, 1984). Hypophysectomy resulted in a time-dependent decrease in the rate of longitudinal bone growth. Local administration of GH into the proximal tibial epiphyseal plate stimulated longitudinal bone growth on the injected side (Isaksson et al., 1982). Local administration of IGF-I also affected epiphyseal cartilage without any synergism with GH (Isgaard et al., 1986). Cultured chondrocytes possess IGF-I and GH receptors (Eden et al., 1983; Trippel et al., 1983) and respond to both peptides by increased DNA synthesis (Madsen et al., 1983). The GH and IGF-I appear to stimulate chondrocytes at different stages of maturation: GH affects the chondroprogenitor cells and IGF-I affects the proliferative chondrocytes. Direct stimulation by GH sensitizes the prechondrocytes and young differentiating cells to IGF-I. Concomitantly, the IGF-I gene is expressed, resulting in an increase in IGF-I synthesis in the differentiating cells. The locally produced IGF-I interacts with receptors on the proliferative chondrocytes by either an autocrine or a paracrine mechanism (for review, see Isaksson et al., 1987). Thus GH stimulates longitudinal growth by 1) directly stimulating the differentiated epiphyseal growth plate precursor cells and 2) stimulating the local production of and increasing the cell responsiveness of IGF-I.
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PINES AND HURWITZ
hANP
hPTH
hPTH+hANP
FIGURE 3. Effect of human parathyroid hormone (h PTH) and human atrial natriuretic peptide (h ANP) on avian chondrocyte proliferation. Cells were incubated for a total 48 h with the hormones in 1 mL Dulbecco's Modified Eagle's Medium containing 5% fetal calf serum. After 24 h, the medium was replaced with fresh medium containing the hormones. At the end of the incubation period, the hormone-containing medium was discarded and fresh medium containing 1 uCi/mL [H]thymidine was added for 4 h and the DNA-bound [ H]thymidine was evaluated, dpm = disintigration per minute, (from Pines and Hurwitz, 1988).
cells. Relatively little or no Type II collagen is made by mature hypertrophic chondrocytes, particularly those present in the calcifying cartilage (Sandberg and Vuorio, 1987). In contrast to Types U and X, which have specific localization, Type IX collagen is distributed throughout the growth plate matrix (MullerGlauser et al, 1986). Chondrocyte collagen synthesis is regulated by growth factors (Redini et al, 1988) and hormones such as the PTH (Figure 4, Pines et al, 1990). Proteoglycans are macromolecules present in varying amounts in all connective tissues (for review, see Ruoslahti, 1988). These macromolecules are constructed of a protein core onto which long side chain sugars such as chondroitin sulfate, keratan sulfate, or dermatan sulfate are attached. Chondroitin sulfate contains repeating units of n-acetyl-galactoseamine and glucoronic acid, whereas keratin sulfate consists of sulfated galactose and glucoseamine. The protein and sugar moieties are linked by link proteins with hyaluronic acid to form aggregates. The maturation of the chondrocytes that occurs along the growth plate is accompanied by changes in the level of proteoglycan synthesis, in the proportion of proteoglycan aggregates and in the size of proteoglycan monomers (Campo and Romano, 1986). For example, hypertrophic chondro-
cytes produce less proteoglycan than cells from resting or proliferative zones (Pawlowski et al, 1987, Makower et al, 1988). The amount and the structure of the proteoglycans synthesized by chondrocytes are affected by hormones and growth factors. Different growth factors cause synthesis of proteoglycans of different sizes (Makower et al, 1988). Moreover, IGF-I stimulates proteoglycan synthesis in chondrocytes isolated from the resting and proliferative zones but not from the hypertrophic zone, whereas epidermal growth factor stimulates proteoglycan synthesis from all three zones. The IGF-I (Kemp et al, 1988) and transforming growth factor P (Redini et al., 1988) augmented glycosaminoglycan synthesis of chondrocytes. CARTILAGE CALCIFICATION
The process of cartilage calcification involves different interacting matrix molecules and is carefully regulated at the cellular level. Whatever the precise mechanism, it appears mat some nucleating agent is required to initiate calcification. Crystal growth can then proceed by spontaneous accretion of calcium salts at any such nucleus. Although collagen has been suggested as active in nucleation in bone (Glimcher, 1989), it does not appear to
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SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
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-10
PTH, Log M FIGURE 4. Effect of parathyroid hormone (PTH) on [3H]proline incorporation into protein exported from avian growth plate chondrocytes in culture. Cells were incubated for 24 h with [3H]proline in the presence or absence of PTH. • = collagenase digestible proteins; O = noncollagenase digestible proteins, dpm = disintegration per minute, (from Pines et al., 1990).
initiate calcification in cartilage because the crystals of hydroxyapatite bear no consistent spatial orientation relative to the collagen fibers. The two matrix components, matrix vesicles (Anderson, 1989) and the proteoglycan monomers (Poole et al, 1989), have been implicated as initiators of calcification. Matrix vesicles, first described by Bonucci (1967) and Anderson (1969), are cell-derived, membranous 100- to 200-nm particles, in which calcium phosphate mineral is deposited (Anderson, 1969). The first mineral deposits are often in close apposition to the inner leaflet of the vesicle membrane (Anderson, 1989). These vesicles contain a high concentration of Ca-binding acidic phospholipids (Peress et al, 1974; Wuthier, 1975; Glaser and Conrad 1981) and phosphatases, which yield orthophosphate for nascent mineral formation (Hsu, 1983; Siegel et al., 1983; Chin et al., 1986). After initiation of calcification, crystals of hydroxyapatite begin to accumulate within the confines of the matrix vesicle membrane (Anderson, 1989). The contribution of matrix vesicles to initiation of calcification is, however, controversial, since some studies demonstrated mineral deposition in areas poor in or devoid of matrix vesicles (Landis and Glimcher, 1982; Poole et al, 1984). Using electron spec-
troscopic imaging techniques, Arsenault et al. (1988) found that the extracellular matrix contains high levels of Ca associated with proteoglycans and related matrix proteins. Phosphate, however, appeared restricted to sites of matrix vesicles associated with mineral deposition. Thus, matrix vesicles could act to deliver phosphate ions to sites of calcification. In recent reports it has been proposed that the C-terminal peptide of Type II collagen synthesized primarily by the cells of the proliferating zone and newly formed hypertrophic cells (Sandberg and Vuorio, 1987), acts as a nucleation center for the Ca-P crystals (Poole et al, 1982, 1989). This 35-kDa protein was found mainly in the calcification sites of the lower hypertrophic zone (Poole et al, 1984) and binds avidly to hydroxyapatite (Choi et al., 1983). Histological studies showed that following the formation of the calcified cartilage the chondrocytes vanish and are replaced by bone cells. Recently, it has been suggested that hypertrophic chondrocytes are not the terminal stage of development but can become osteoblasts (Shapiro and Boyde, 1987). The demonstration that hypertrophic chondrocytes could switch from synthesis of Type II collagen to Type I collagen supports this hypothesis.
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O
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foundly incomplete (Boden et al, 1987). In osteochondromatosis, for example, loss of columnar and matrical integrity at the growth plate periphery can cause short stature. In endochondromatosis, some of the longitudinal columns fail to undergo chondroosseous replacement and are left within the metaphysis, but the others continue with their longitudinal elongation (Ogden and Rosenberg, 1988). Some skeletal dysplasias are characterized by biochemical abnormalities. For example, in Kniest dysplasia the impaired production of collagen Type II results in imperfect collagen fibrils formation (Poole et al, 1988), spondyloepiphyseal dysplasia (Murray et al, 1989) or proteoglycans defects (Pedrini-Mille et al, 1984).
inhibited by various matrix proteins such as proteoglycans (Dziewaitkowski and Majznerski, 1985), osteocalcin (Romberg et al, 1986), phosphoproteins, and osteonectin (Menanteau et al, 1982).
Anderson, H. C , 1969. Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41:59-72. Anderson, H. C , 1989. Biology of disease: Mechanism of mineral formation in bone. Lab. Invest 60:320-330. Arsenault, A. L., F. P. Ottensmeyer, and I. B. Heath, 1988. An electron microscopic and spectroscopic study of murine epiphyseal cartilage: Analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution. J. Ultrastruct. Mol. Struct. Res. 98:32-^7. Attie, M. F., E. M. Brown, D. G. Gardner, A. M. Spiegel and G. D. Aurbach, 1980. Characterization of the dopamine-responsive adenylate cyclase of bovine parathyroid cells and its relationship to parathyroid hormone secretion. Endocrinology 107:1776-1781. Binderman, I., and D. Somjen, 1984. 24,25-dihydroxycholecalciferol induces the growth of chick cartilage in vitro. Endocrinology 115:430-432. Boden, S. D., F. S. Kaplan, M. D. Fallon, R. Ruddy, J. Belik, E. Zachai, and J. Ellis, 1987. Metatrophic dwarfism. Uncoupling of endochondral and perichondral growth. J. Bone Jt Surg. 69A:174-184. Bonucci, E., 1967. Fine structure of early cartilage calcification. J. Ultrastruct. Res. 20:33-50. Burch, W. M., M. Lopez-Carlos, M. R. Uskokovic, and M. K. Drezner, 1988. 1,25-dihydroxyvitamin D3 stimulates avian and mammalian cartilage growth in vitro. J. Bone Miner. Res. 3:87-91. Burch, W. M., and J. J. Van Wyk, 1987. Triiodothyronine stimulates cartilage growth and maturation by different mechanisms. Am. J. Physiol. 252:E177-E182. Campo, R. D., and J. E. Romano, 1986. Changes in cartilage proteoglycans associated with calcification. Calcif. Tissue Int. 39:175-184. Chin, J. E., E. M. Elaine, M S. Kemick, and R. E. Wuthier, 1986. Effect of synthetic human parathyroid hormone on the levels of alkaline phosphatase activity and formation of alkaline-rich matrix vesicles by primary cultures of chicken epiphyseal growth plate chondrocytes. Bone Miner. 1:421^36. Choi, H. U., L. H. Tang, T. L. Johnson, S. Pal, L. Rosenberg, A. Riener, and A. R. Poole, 1983.
GROWTH PLATE CLOSURE
During sexual maturation, when bone reaches its ultimate length, growth plate cells diminish in number and the growth plate loses its functionality. The mechanisms responsible for growth plate closure remain unknown. It may be hypothesized that each of the stem cells is programmed to a certain number of divisions. Once this limit has been reached, proliferation ceases and the growth plate closes. In the rat, each stem cell gives rise to 30 cells, and 40 to 50 cell divisions of the chondroprogenitor cells were sufficient to account for normal growth of rat tibia (Kember, 1978). Gonadal hormones probably play a key role in the demise of the growth plate. Administration of sex hormones to immature animals produces an initial acceleration of growth followed by premature cessation of growth and growth plate closure (Kember, 1978). ABNORMAL GROWTH PLATE
The growth plate may be involved in a variety of morphological and physiological deformities referred to as skeletal dysplasias. In metatrophic dwarfism the maturation of proliferating chondrocytes into hypertrophic cells was either completely absent or pro-
REFERENCES
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The events involving matrix synthesis and calcification are under hormonal control. Both l,25-(OH)2D3 and 24,25-(OH)2D3 are required for maximal synthesis of C-propeptide of collagen Type H (Hinek and Poole, 1988). The 24,25-(OH)2D3 is needed for maximal expression of alkaline phosphatase activity (Hale et al, 1986; Schwartz et al, 1988). The 24,25-(OH)2D3 inhibits chondrocyte phospholipase A 2 , which acts on membrane phospholipid (Schwartz et al, 1988). Receptors for l,25-(OH)2D3 were found in rabbit hypertrophic chondrocytes (Iwamoto et al, 1989b). Parathyroid hormone inhibits matrix vesicle alkaline phosphatase activity (Chin et al, 1986). Once crystal nucleation has occurred, calcification can proceed, resulting in crystal growth. The rate of this process is regulated by Ca 2+ and HPO4 - concentrations and can be
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
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