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Cartilage Turnover in Embryonic Chick Tibial Explant Cultures1
Cartilage Turnover in Embryonic Chick Tibial Explant Cultures1 M. W. Orth,2 T. L. Peters, and K. A. Chlebek-Brown Department of Animal Science, Michigan State University, East Lansing, Michigan 48824-1225 ansims involved in the regulation of growth plate cartilage turnover. In our cultures, chondrocytes mature and then die, completely degrading the cartilage in approximately 16 d. The matrix undergoes a predictable pattern of degradation in which proteoglycans followed by collagen are removed. Increases in matrix metalloproteinase activity and nitric oxide production are detected in cartilage concurrently with release of proteoglycans into media. Inhibitors of nitric oxide inhibit nitric oxide production and proteoglycan degradation, suggesting that nitric oxide, at least in part, regulates growth plate cartilage turnover in the explant culture. Information gained from using this explant culture will aid in understanding the regulation of growth plate cartilage turnover in vivo and potentially help determine the cause of bone growth diseases such as tibial dyschondroplasia.
ABSTRACT Growth plate cartilage regulates the rate of growth and ultimate length of several bones in the skeleton. Chondrocytes within the growth plate proliferate, differentiate, enlarge, and die. The extracellular matrix undergoes synthesis, reorganization, and eventually degradation. The majority of research in growth plate physiology has focused on the proliferation and differentiation of chondrocytes as well as proteins they produce for the extracellular matrix. However, little is known about the transition from hypertrophic to apoptotic chondrocytes or the regulation of terminal degradation of cartilage prior to bone formation. An explant culture has been developed to study cartilage differentiation using 12-d-old embryonic chick tibiae. We have modified the explant culture and are using it to further elucidate mech-
(Key words: cartilage, apoptosis, nitric oxide, tibia, metalloproteinases) 2000 Poultry Science 79:990–993
degradation of the extracellular matrix by matrix metalloproteinases (MMP) is required for apoptosis (Lund et al., 1996; Werb et al., 1996). Metaphyseal blood vessels penetrate lacunae very rapidly (approximately 15 min) after apoptotic chondrocytes are removed (Farnum and Wilsman, 1989). Targeted inactivation of the gelatinase B/MMP-9 gene in mice revealed a requirement of this enzyme for vascularization of the growth plate (Vu et al., 1998). The incomplete vascularization in MMP-9-deficient mice was attributed to reduced apoptosis (turnover) of hypertrophic chondrocytes. Dysregulation of chondrocyte apoptosis and vascularization resulted in progressive lengthening of the growth plate. The development of growth plate diseases, such as tibial dyschondroplasia (TD), could also involve impairment of apoptosis (Ohyama et al., 1997). Apoptosis is accompanied by a complex cascade of events that includes increased mitochondrial membrane permeability, intracellular calcium, and activity of several Ca++/Mg++-dependent enzymes such as calpain, transglutaminase, and the endonuclease(s) responsible for internucleosomal DNA cleavage. Numerous factors, such as glucocorticoids, tumor necrosis factor-α, ionizing radia-
INTRODUCTION Chondrocytes within the growth plate will undergo proliferation, differentiation, and apoptosis in a relatively short period of time during peak growth periods in birds reared for meat production. The coordination of these events with vascularization of hypertrophic cartilage by metaphyseal blood vessels is critical to ensure proper long bone growth development. Research efforts have provided important information on understanding the role of growth factors, cellular signals, and extracellular matrix proteins in growth plate cartilage synthesis and maturation (Orth, 1999). However, relatively little is known about regulation of chondrocyte apoptosis and degradation of the extracellular matrix prior to bone matrix synthesis. Apoptosis occurs in hypertrophic chondrocytes adjacent to subchondral bone (Hatori et al., 1995). Chondrocyte apoptosis may be coordinated with matrix degradation (Gibson, 1998). During mammary gland involution,
Received for publication December 15, 1999. Accepted for publication February 29, 2000. 1 Salary and research support provided by the Michigan Agricultural Experiment Station and USDA Animal Health Formula Funds. 2 To whom correspondence should be addressed: orthm@pilot. msu.edu.
Abbreviation Key: LPS = lipopolysaccharides; MMP = matrix metalloproteinase; TD = tibial dyschondroplasia.
990
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBLEMS IN POULTRY
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tion, and growth factor withdrawal, can trigger apoptosis, depending upon the cell types. The mechanisms that initiate apoptosis of hypertrophic chondrocytes are not well understood. Transglutaminase, an enzyme that crosslinks intracellular proteins during apoptosis, is expressed in hypertrophic chondrocytes. Parathyroid hormone-related protein decreases expression of the antiapoptotic protein bcl-2 in hypertrophic chondrocytes (Amling et al., 1997). In studies of MMP-9-deficient mice, Vu et al. (1998) suggested that MMP-9 activity liberates an apoptotic/angiogenic signal from the extracellular matrix of growth plate cartilage.
TIBIAL EXPLANT CULTURE Our laboratory, along with others, has developed an explant culture system to study apoptosis and extracellular matrix degradation by using tibiae collected from Day 12 chick embryos (Cole et al., 1992, 1993; Orth et al., 1997). The tibiae collected at this stage of development comprise developing cartilage, a bony sheath, and marrow. The cartilage at both ends of the tibia contain a proliferative and hypertrophic zone similar to that found in posthatch growth plate cartilage (Schmid and Linsenmeyer, 1985). The explant culture maintains the interactions of bone and marrow that are present in vivo and are required for complete cartilage degradation (Cole et al., 1992). When stimulated by lipopolysaccharides (LPS) or other catabolic agents, cultured tibiae produce proteolytic enzymes that degrade the two major structural components of the extracellular matrix, proteoglycan followed by collagen. Although the specific enzymes that mediate LPS-induced growth plate extracellular matrix degradation are not known, MMP activity in conditioned media peaks simultaneously with collagen release (Cole et al., 1993). In addition, we have recently found that MMP activity increases in the cartilage matrix just prior to proteoglycan release into the media (Orth et al., 1999). Studies of growth plate cartilage turnover in vivo in normal animals are complicated by several factors. In vivo, a chondrocyte’s life cycle is short (24 to 48 h in young broiler chickens) and few apoptotic chondrocytes exist because they are quickly resorbed so that vascularization can occur. In the explant culture, we can follow the development of chondrocytes from the proliferative to apoptotic stage over an extended period of time (10 to 14 d). Apoptotic cells remain in the cartilage until the surrounding matrix has been degraded. Our histological analysis of cultured tibiae revealed that chondrocytes within the proliferative zone first undergo hypertrophy and then later apoptosis (Chlebek-Brown, 1996). In a preliminary experiment we have found that apopain (caspase-3) activity, an intracellular enzyme involved in apoptosis increased on Day 6 of culture in the cartilage, suggesting that the cells have terminally differentiated (Figure 1). Furthermore, breakdown of the extracellular matrix in the growth plate can be difficult to quantitate in vivo and is limited to a specific timepoint, usually when the animal is killed. However, in culture, matrix-
FIGURE 1. Apopain (Caspase-3) activity (an indicator of terminal differentiation) in cartilage during 12 d of culture.
derived by-products are released into media that can then be analyzed. Thus, we believe our explant culture will help elucidate the regulation of growth plate cartilage turnover in vivo.
THE ROLE OF NITRIC OXIDE IN CARTILAGE TURNOVER We propose that nitric oxide could be an important regulator of growth plate cartilage turnover. Nitric oxide, a lipophilic molecule with a short half-life (<15 s), is an important intracellular or intercellular signaling molecule during numerous physiological events, including neurotransmission, blood clotting, macrophage cytotoxicity, and angiogenesis (Bredt and Snyder, 1994). Nitric oxide synthase catalyzes nitric oxide production from the terminal guanidino nitrogen of L-arginine. The enzyme has a constitutive and inducible form. Macrophage and chondrocytes contain the inducible form. Interleukin-1 and LPS induce synthesis of the enzyme (Stadler et al., 1991). In a macrophage cell line, interferon-γ stimulates nitric oxide synthesis (Melillo et al., 1995). Nitric oxide activates MMP, suppresses proteoglycan synthesis, and induces apoptosis in human articular chondrocytes (Ha¨ uselmann et al., 1994; Blanco et al., 1995; Murrell et al., 1995). In cultured rat chondrocytes, it inhibits cell proliferation (Kondo et al., 1993). The intracellular signaling properties of nitric oxide in chondrocytes are mediated, at least in part, via activation of guanylate cyclase, resulting in increased formation of the second messenger cGMP (Khatib et al., 1997). In vivo and clinical studies support the catabolic nature of nitric oxide in adult articular cartilage. Patients with osteoarthritis or rheumatoid arthritis have higher concentrations of nitric oxide catabolites in their serum and urine than age-matched individuals without clinical signs of arthritis (Melillo et al., 1995; Grabowski et al., 1996; Pel-
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ORTH ET AL.
letier et al., 1996). In dogs, daily oral supplementation of N-iminoethyl-L-lysine, a selective inhibitor of inducible nitric oxide synthase, reduced the progression of experimentally induced arthritis (Pelletier et al., 1998). Dogs that were treated with N-iminoethyl-L-lysine had decreased MMP activity in their affected articular cartilage and decreased synovial fluid concentrations of the proinflammatory and catabolic agents, interleukin-1 and prostaglandin E2, compared to those that received no treatment. In articular cartilage, nitric oxide apparently induces biological responses that can lead to irreversible degradation of cartilage. In growth plate cartilage, it might have the same impact but with the purpose of facilitating vascularization and bone formation. Just recently, expression of nitric oxide synthase mRNA has been detected in neonatal hypertrophic and degenerating chondrocytes of the epiphyseal growth plate in rat femurs (Hukkanen et al., 1999). The authors suggest that nitric oxide might help regulate the turnover of chondrocytes. Results of recent experiments in our laboratory with the tibial explant culture suggest that nitric oxide stimulates turnover of developing cartilage. In our cultures, proteoglycan release from the cartilage in the tibiae coincides with a peak in nitric oxide production (Orth, 1999). Two different inhibitors of nitric oxide synthesis, S-methylisothiourea and pyrrolidine dithiocarbamate, completely inhibited LPS-induced nitric oxide production and cartilage degradation. Furthermore, upregulated nitric oxide production coincides with the transition of chondrocytes from hypertrophy to apoptosis during the culture period (Chlebek-Brown and Orth, unpublished observations). Our research supports the literature suggesting that nitric oxide upregulates activity or expression of MMP (Murrell et al., 1995; Sasaki et al., 1998). As previously mentioned, we have found that nitric oxide production increases in tibial explant cultures concurrently with proteoglycan release into media. However, those results were based on sampling of media every 2 d. Therefore, we conducted an experiment with more frequent sampling of media to precisely determine the temporal relationship between nitric oxide production and proteoglycan release. Approximately 50 tibiae were cultured in medium without LPS and chicken serum for 6 d. On the sixth day, LPS and chicken serum were added. Aliquots of conditioned media were collected every 8 h over a 48-h period, and concentrations of nitric oxide and proteoglycan were determined. The results (repeated twice with the same outcome) show that LPS stimulated nitric oxide production before proteoglycan release (Figure 2). One interpretation is that LPS stimulates chondrocyte nitric oxide production, which, in turn, increases MMP activity, leading to the enzymatic release of proteoglycans from the extracellular matrix. In a preliminary experiment, nitric oxide donors were added to serum-free cultures (each treatment was done in duplicate) on Day 6 to determine if exogenous nitric oxide will increase proteoglycan release into the media. LPS was added as a positive control, and medium alone was added as a negative control. Cultures were then incubated an additional 48 h. LPS addition
FIGURE 2. The temporal relationship between nitric oxide (NO) production and proteoglycan (PG) concentrations in conditioned medium after addition of lipopolysaccharide (LPS) and chicken serum to tibiae that were cultured for 6 d in serum-free medium.
to the medium caused an 88% increase in proteoglycan release from the cartilage relative to medium alone. Addition of glyco-SNAP-2 (25 µM) and Spermine NONOate (50 µM) caused increases of 55 and 43%, respectively, in proteoglycan release over medium alone. Addition of these concentrations of nitric oxide donors resulted in nitric oxide levels similar to those found during LPS stimulated cartilage degradation. Our preliminary evidence supports a role for nitric oxide in the regulation of MMP activity. We hypothesize that induction of nitric oxide synthesis in hypertrophic chondrocytes increases apoptosis in an autocrine or paracrine fashion and facilitates matrix degradation by upregulating activity of MMP in the surrounding cartilage (Figure 3). Two potential physiological cell signals for the upregulation of nitric oxide synthesis that we have tested in preliminary experiments include proteolytic fragments of fibronectin and interleukin-6. However, their importance in growth plate turnover in vivo is unclear.
CONCLUSIONS We feel the chick tibial explant system provides a good model for understanding growth plate cartilage turnover.
FIGURE 3. A proposed model for the role of nitric oxide in growth plate cartilage turnover.
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBLEMS IN POULTRY
We have focused primarily on biochemical analyses but are in the process of using molecular biology tools, such as in situ hybridization, to further elucidate the role of nitric oxide in the growth plate. Another potential benefit is that this system could serve as an in vitro model for long bone growth abnormalities such as TD and osteochondrosis. Dithiocarbamates will induce TD in birds and inhibit cartilage degradation in our explants. Preliminary research suggests calcium-deficient media (mimicking rickets in animals) will lead to impaired cartilage differentiation. Thus, the explant system might help elucidate the underlying causes of growth plate diseases like TD.
REFERENCES Amling, M., L. Neff, S. Tanaka, D. Inoue, K. Kuida, E. Weir, W. M. Philbrick, A. E. Broadus, and R. Baron, 1997. Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development. J. Cell Biol. 136:205–213. Blanco, F. J., R. L. Ochs, H. Schwarz, and M. Lotz, 1995. Chondrocyte apoptosis induced by nitric oxide. Am. J. Pathol. 146:75–85. Bredt, D. S., and S. H. Snyder, 1994. Nitric oxide: A physiologic messenger molecule. Annu. Rev. Biochem. 63:175–195. Chlebek-Brown, K. A., 1996. Cell death and proteoglycan loss during hypertrophic cartilage degradation of avian tibiae. Ph.D. Dissertation. Rush University, Chicago, IL. Cole, A. A., T. Boyd, L. L. Luchene, K. E. Kuettner, and T. M. Schmid, 1993. Type X collagen degradation in long-term serum-free culture of the embryonic chick tibia following production of active collagenase and gelatinase. Dev. Biol. 159:528–534. Cole, A. A., L. J. Luchene, T. F. Linsenmeyer, and T. M. Schmid, 1992. The influence of bone and marrow on cartilage hypertrophy and degradation during 30-day serum-free culture of the embryonic chick tibia. Dev. Dyn. 193:277–285. Farnum, C. E., and N. J. Wilsman. 1989. Cellular turnover at the chondro-osseous junction of growth plate cartilage: Analysis by serial sections at the light microscopical level. J. Orthop. Res. 7:654–666. Gibson, G., 1998. Active role of chondrocyte apoptosis in endochondral ossification. Microsc. Res. Tech. 43:191–204. Grabowski, P. S., A. J. England, R. Dykhuizen, M. Copland, N. Benjamin, D. M. Reid, and S. H. Ralston, 1996. Elevated nitric oxide production in rheumatoid arthritis. Arthritis Rheum. 39:643–647. Hatori, M., K. J. Klatte, C. C. Teixeira, and I. M. Shapiro. 1995. End labeling studies of fragmented DNA in the avian growth plate: Evidence of apoptosis in terminally differentiated chondrocytes. J. Bone Miner. Res. 10:1960–1968. Ha¨ uselmann, H. J., L. Oppliger, B. A. Michel, M. StefanovicRacic, and C. H. Evans, 1994. Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture. FEBS Lett. 352:361–364. Hukkanen, M.V.J., L.A.M. Platts, I. F. De Marticorena, M. O’Shaughnessy, I. Macintyre, and J. M. Polak, 1999. Developmental regulation of nitric oxide synthase expression in rat skeletal bone. J. Bone Miner. Res. 14:868–877. Khatib, A. M., G. Siegfried, M. Quintero, and D. R. Mitrovic, 1997. The mechanism of inhibition of DNA synthesis in artic-
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ular chondrocytes from young and old rats by nitric oxide. Nitric Oxide 1:218–225. Kondo, S., N. Ishiguro, H. Iwata, I. Nakashima, and K. Isobe, 1993. The effects of nitric oxide on chondrocytes and lymphocytes. Biochem. Biophys. Res. Comm. 197:1431–1437. Lund, L. R., J. Romer, N. Thomasset, H. Solberg, C. Pyke, M. J. Bissell, K. Dano, and Z. Werb, 1996. Two distinct phases of apoptosis in mammary gland involution: Proteinase-independent and -dependent pathways. Development 122:181– 193. Melillo, G., T. Musso, A. Sica, L. S. Taylor, G. W. Cox, and L. Varesio, 1995. A hyposia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J. Exp. Med. 182:1683–1693. Murrell, G.A.C., D. Jang, and R. J. Williams, 1995. Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem. Biophys. Res. Comm. 206:15–21. Ohyama, K., C. Farquharson, C. C. Whitehead, and I. M. Shapiro, 1997. Further observations on programmed cell death in the epiphyseal growth plate: Comparison of normal and dyschondroplastic epiphyses. J. Bone Miner. Res. 12:1647– 1656. Orth, M. W., 1999. The regulation of growth plate cartilage turnover. J. Anim. Sci. 77(Suppl. 2):183–189. Orth, M. W., K. A. Chlebek, A. A. Cole, and T. M. Schmid, 1997.Tetracycline derivatives inhibit cartilage degradation in cultured embryonic chick tibiae. Res. Vet. Sci. 67:11–14. Orth, M. W., J. I. Fenton, and K. A. Chlebek-Brown, 1999. Biochemical characterization of cartilage degradation in embryonic chick tibial explant cultures. Poultry Sci. 78:1596–1600. Pelletier, J. P., D. Jovanovic, J. C. Fernandes, P. Manning, J. R. Connor, M. G. Currie, J. A. Di Battista, and J. Martel Pelletier, 1998. Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum. 41:1275–1286. Pelletier, J. P., F. Mineau, P. Ranger, G. Tardif, and J. Martel Pelletier, 1996. The increased synthesis of inducible nitric oxide inhibits IL-1ra synthesis by human articular chondrocytes: Possible role in osteoarthritic cartilage degradation. Osteoarthritis Cart. 4:77–84. Sasaki, K., T. Hattori, T. Fujisawa, K. Takahashi, H. Inoue, and M. Takigawa, 1998. Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes. J. Biochem. 123:431–439. Schmid, T. M., and T. F. Linsenmeyer, 1985. Developmental acquisition of type X collagen in the embryonic chick tibiotarsus. Dev. Biol. 107:373–381. Stadler, J., M. Stefanovic-Racic, T. R. Billiar, R. D. Curran, L. A. McIntyre, H. I. Georgescu, R. L. Simmons, and C. H. Evans, 1991. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharides. J. Immunol. 147:3915–3920. Vu, T. H., J. M. Shipley, G. Bergers, J. E. Berger, J. A. Helms, D. Hanahan, S. D. Shapiro, R. M. Senior, and Z. Werb, 1998. MMP-9/gelatinase B is a key regulatory of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411–422. Werb, Z., J. Ashkenas, A. MacAuley, and J. F. Wiesen, 1996. Extracellular matrix remodeling as a regular of stromal-epithelial interactions during mammary gland development, involution and carcinogenesis. Braz. J. Med. Biol. Res. 29:1087–1097.
ABSTRACT Growth plate cartilage regulates the rate of growth and ultimate length of several bones in the skeleton. Chondrocytes within the growth plate proliferate, differentiate, enlarge, and die. The extracellular matrix undergoes synthesis, reorganization, and eventually degradation. The majority of research in growth plate physiology has focused on the proliferation and differentiation of chondrocytes as well as proteins they produce for the extracellular matrix. However, little is known about the transition from hypertrophic to apoptotic chondrocytes or the regulation of terminal degradation of cartilage prior to bone formation. An explant culture has been developed to study cartilage differentiation using 12-d-old embryonic chick tibiae. We have modified the explant culture and are using it to further elucidate mech-
(Key words: cartilage, apoptosis, nitric oxide, tibia, metalloproteinases) 2000 Poultry Science 79:990–993
degradation of the extracellular matrix by matrix metalloproteinases (MMP) is required for apoptosis (Lund et al., 1996; Werb et al., 1996). Metaphyseal blood vessels penetrate lacunae very rapidly (approximately 15 min) after apoptotic chondrocytes are removed (Farnum and Wilsman, 1989). Targeted inactivation of the gelatinase B/MMP-9 gene in mice revealed a requirement of this enzyme for vascularization of the growth plate (Vu et al., 1998). The incomplete vascularization in MMP-9-deficient mice was attributed to reduced apoptosis (turnover) of hypertrophic chondrocytes. Dysregulation of chondrocyte apoptosis and vascularization resulted in progressive lengthening of the growth plate. The development of growth plate diseases, such as tibial dyschondroplasia (TD), could also involve impairment of apoptosis (Ohyama et al., 1997). Apoptosis is accompanied by a complex cascade of events that includes increased mitochondrial membrane permeability, intracellular calcium, and activity of several Ca++/Mg++-dependent enzymes such as calpain, transglutaminase, and the endonuclease(s) responsible for internucleosomal DNA cleavage. Numerous factors, such as glucocorticoids, tumor necrosis factor-α, ionizing radia-
INTRODUCTION Chondrocytes within the growth plate will undergo proliferation, differentiation, and apoptosis in a relatively short period of time during peak growth periods in birds reared for meat production. The coordination of these events with vascularization of hypertrophic cartilage by metaphyseal blood vessels is critical to ensure proper long bone growth development. Research efforts have provided important information on understanding the role of growth factors, cellular signals, and extracellular matrix proteins in growth plate cartilage synthesis and maturation (Orth, 1999). However, relatively little is known about regulation of chondrocyte apoptosis and degradation of the extracellular matrix prior to bone matrix synthesis. Apoptosis occurs in hypertrophic chondrocytes adjacent to subchondral bone (Hatori et al., 1995). Chondrocyte apoptosis may be coordinated with matrix degradation (Gibson, 1998). During mammary gland involution,
Received for publication December 15, 1999. Accepted for publication February 29, 2000. 1 Salary and research support provided by the Michigan Agricultural Experiment Station and USDA Animal Health Formula Funds. 2 To whom correspondence should be addressed: orthm@pilot. msu.edu.
Abbreviation Key: LPS = lipopolysaccharides; MMP = matrix metalloproteinase; TD = tibial dyschondroplasia.
990
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBLEMS IN POULTRY
991
tion, and growth factor withdrawal, can trigger apoptosis, depending upon the cell types. The mechanisms that initiate apoptosis of hypertrophic chondrocytes are not well understood. Transglutaminase, an enzyme that crosslinks intracellular proteins during apoptosis, is expressed in hypertrophic chondrocytes. Parathyroid hormone-related protein decreases expression of the antiapoptotic protein bcl-2 in hypertrophic chondrocytes (Amling et al., 1997). In studies of MMP-9-deficient mice, Vu et al. (1998) suggested that MMP-9 activity liberates an apoptotic/angiogenic signal from the extracellular matrix of growth plate cartilage.
TIBIAL EXPLANT CULTURE Our laboratory, along with others, has developed an explant culture system to study apoptosis and extracellular matrix degradation by using tibiae collected from Day 12 chick embryos (Cole et al., 1992, 1993; Orth et al., 1997). The tibiae collected at this stage of development comprise developing cartilage, a bony sheath, and marrow. The cartilage at both ends of the tibia contain a proliferative and hypertrophic zone similar to that found in posthatch growth plate cartilage (Schmid and Linsenmeyer, 1985). The explant culture maintains the interactions of bone and marrow that are present in vivo and are required for complete cartilage degradation (Cole et al., 1992). When stimulated by lipopolysaccharides (LPS) or other catabolic agents, cultured tibiae produce proteolytic enzymes that degrade the two major structural components of the extracellular matrix, proteoglycan followed by collagen. Although the specific enzymes that mediate LPS-induced growth plate extracellular matrix degradation are not known, MMP activity in conditioned media peaks simultaneously with collagen release (Cole et al., 1993). In addition, we have recently found that MMP activity increases in the cartilage matrix just prior to proteoglycan release into the media (Orth et al., 1999). Studies of growth plate cartilage turnover in vivo in normal animals are complicated by several factors. In vivo, a chondrocyte’s life cycle is short (24 to 48 h in young broiler chickens) and few apoptotic chondrocytes exist because they are quickly resorbed so that vascularization can occur. In the explant culture, we can follow the development of chondrocytes from the proliferative to apoptotic stage over an extended period of time (10 to 14 d). Apoptotic cells remain in the cartilage until the surrounding matrix has been degraded. Our histological analysis of cultured tibiae revealed that chondrocytes within the proliferative zone first undergo hypertrophy and then later apoptosis (Chlebek-Brown, 1996). In a preliminary experiment we have found that apopain (caspase-3) activity, an intracellular enzyme involved in apoptosis increased on Day 6 of culture in the cartilage, suggesting that the cells have terminally differentiated (Figure 1). Furthermore, breakdown of the extracellular matrix in the growth plate can be difficult to quantitate in vivo and is limited to a specific timepoint, usually when the animal is killed. However, in culture, matrix-
FIGURE 1. Apopain (Caspase-3) activity (an indicator of terminal differentiation) in cartilage during 12 d of culture.
derived by-products are released into media that can then be analyzed. Thus, we believe our explant culture will help elucidate the regulation of growth plate cartilage turnover in vivo.
THE ROLE OF NITRIC OXIDE IN CARTILAGE TURNOVER We propose that nitric oxide could be an important regulator of growth plate cartilage turnover. Nitric oxide, a lipophilic molecule with a short half-life (<15 s), is an important intracellular or intercellular signaling molecule during numerous physiological events, including neurotransmission, blood clotting, macrophage cytotoxicity, and angiogenesis (Bredt and Snyder, 1994). Nitric oxide synthase catalyzes nitric oxide production from the terminal guanidino nitrogen of L-arginine. The enzyme has a constitutive and inducible form. Macrophage and chondrocytes contain the inducible form. Interleukin-1 and LPS induce synthesis of the enzyme (Stadler et al., 1991). In a macrophage cell line, interferon-γ stimulates nitric oxide synthesis (Melillo et al., 1995). Nitric oxide activates MMP, suppresses proteoglycan synthesis, and induces apoptosis in human articular chondrocytes (Ha¨ uselmann et al., 1994; Blanco et al., 1995; Murrell et al., 1995). In cultured rat chondrocytes, it inhibits cell proliferation (Kondo et al., 1993). The intracellular signaling properties of nitric oxide in chondrocytes are mediated, at least in part, via activation of guanylate cyclase, resulting in increased formation of the second messenger cGMP (Khatib et al., 1997). In vivo and clinical studies support the catabolic nature of nitric oxide in adult articular cartilage. Patients with osteoarthritis or rheumatoid arthritis have higher concentrations of nitric oxide catabolites in their serum and urine than age-matched individuals without clinical signs of arthritis (Melillo et al., 1995; Grabowski et al., 1996; Pel-
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letier et al., 1996). In dogs, daily oral supplementation of N-iminoethyl-L-lysine, a selective inhibitor of inducible nitric oxide synthase, reduced the progression of experimentally induced arthritis (Pelletier et al., 1998). Dogs that were treated with N-iminoethyl-L-lysine had decreased MMP activity in their affected articular cartilage and decreased synovial fluid concentrations of the proinflammatory and catabolic agents, interleukin-1 and prostaglandin E2, compared to those that received no treatment. In articular cartilage, nitric oxide apparently induces biological responses that can lead to irreversible degradation of cartilage. In growth plate cartilage, it might have the same impact but with the purpose of facilitating vascularization and bone formation. Just recently, expression of nitric oxide synthase mRNA has been detected in neonatal hypertrophic and degenerating chondrocytes of the epiphyseal growth plate in rat femurs (Hukkanen et al., 1999). The authors suggest that nitric oxide might help regulate the turnover of chondrocytes. Results of recent experiments in our laboratory with the tibial explant culture suggest that nitric oxide stimulates turnover of developing cartilage. In our cultures, proteoglycan release from the cartilage in the tibiae coincides with a peak in nitric oxide production (Orth, 1999). Two different inhibitors of nitric oxide synthesis, S-methylisothiourea and pyrrolidine dithiocarbamate, completely inhibited LPS-induced nitric oxide production and cartilage degradation. Furthermore, upregulated nitric oxide production coincides with the transition of chondrocytes from hypertrophy to apoptosis during the culture period (Chlebek-Brown and Orth, unpublished observations). Our research supports the literature suggesting that nitric oxide upregulates activity or expression of MMP (Murrell et al., 1995; Sasaki et al., 1998). As previously mentioned, we have found that nitric oxide production increases in tibial explant cultures concurrently with proteoglycan release into media. However, those results were based on sampling of media every 2 d. Therefore, we conducted an experiment with more frequent sampling of media to precisely determine the temporal relationship between nitric oxide production and proteoglycan release. Approximately 50 tibiae were cultured in medium without LPS and chicken serum for 6 d. On the sixth day, LPS and chicken serum were added. Aliquots of conditioned media were collected every 8 h over a 48-h period, and concentrations of nitric oxide and proteoglycan were determined. The results (repeated twice with the same outcome) show that LPS stimulated nitric oxide production before proteoglycan release (Figure 2). One interpretation is that LPS stimulates chondrocyte nitric oxide production, which, in turn, increases MMP activity, leading to the enzymatic release of proteoglycans from the extracellular matrix. In a preliminary experiment, nitric oxide donors were added to serum-free cultures (each treatment was done in duplicate) on Day 6 to determine if exogenous nitric oxide will increase proteoglycan release into the media. LPS was added as a positive control, and medium alone was added as a negative control. Cultures were then incubated an additional 48 h. LPS addition
FIGURE 2. The temporal relationship between nitric oxide (NO) production and proteoglycan (PG) concentrations in conditioned medium after addition of lipopolysaccharide (LPS) and chicken serum to tibiae that were cultured for 6 d in serum-free medium.
to the medium caused an 88% increase in proteoglycan release from the cartilage relative to medium alone. Addition of glyco-SNAP-2 (25 µM) and Spermine NONOate (50 µM) caused increases of 55 and 43%, respectively, in proteoglycan release over medium alone. Addition of these concentrations of nitric oxide donors resulted in nitric oxide levels similar to those found during LPS stimulated cartilage degradation. Our preliminary evidence supports a role for nitric oxide in the regulation of MMP activity. We hypothesize that induction of nitric oxide synthesis in hypertrophic chondrocytes increases apoptosis in an autocrine or paracrine fashion and facilitates matrix degradation by upregulating activity of MMP in the surrounding cartilage (Figure 3). Two potential physiological cell signals for the upregulation of nitric oxide synthesis that we have tested in preliminary experiments include proteolytic fragments of fibronectin and interleukin-6. However, their importance in growth plate turnover in vivo is unclear.
CONCLUSIONS We feel the chick tibial explant system provides a good model for understanding growth plate cartilage turnover.
FIGURE 3. A proposed model for the role of nitric oxide in growth plate cartilage turnover.
SYMPOSIUM: SKELETAL BIOLOGY AND RELATED PROBLEMS IN POULTRY
We have focused primarily on biochemical analyses but are in the process of using molecular biology tools, such as in situ hybridization, to further elucidate the role of nitric oxide in the growth plate. Another potential benefit is that this system could serve as an in vitro model for long bone growth abnormalities such as TD and osteochondrosis. Dithiocarbamates will induce TD in birds and inhibit cartilage degradation in our explants. Preliminary research suggests calcium-deficient media (mimicking rickets in animals) will lead to impaired cartilage differentiation. Thus, the explant system might help elucidate the underlying causes of growth plate diseases like TD.
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