- Email: [email protected]
Metabolic Kinetics of Proteoglycans by Embryonic Chick Sternal Cartilage in Culture
Archives of Biochemistry and Biophysics Vol. 367, No. 2, July 15, pp. 225–232, 1999 Article ID abbi.1999.1246, available online at http://www.idealibrary.com on
Metabolic Kinetics of Proteoglycans by Embryonic Chick Sternal Cartilage in Culture Hongxiang Liu, 1 James A. Bee, 2 and Peter Lees Department of Veterinary Basic Sciences, The Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, United Kingdom
Received December 29, 1998, and in revised form April 9, 1999
Explant cultures of embryonic chick sternum have been widely studied, but the kinetics of biosynthesis of proteoglycans by this tissue in culture has not been characterized. Caudal cartilaginous portions of 16day-old embryonic chick sterna were cultured for 8 days. Histological examination showed that the fresh cartilage contained morphologically homogenous chondrocytes, which were embedded in a uniform extracellular matrix. After culture for 8 days, the histological appearance of the explant remained unchanged but the tissue increased in size with time as indicated by a progressive increase in DNA content and in the content of glycosaminoglycan and collagen. Rates of degradation and release from the tissue of proteoglycans labeled in ovo with 35S were first order during culture, as were the unlabeled proteoglycans. Proteoglycan synthesis was high during the first 2 days of culture, and this then gradually decreased from this high level during the following 2 days. Synthesis was then maintained at a constant level for the remainder of the culture period. After culture for 2 and 7 days, the proteoglycans synthesized by the explants were identical to the preexisting proteoglycans in hydrodynamic size, glycosaminoglycan chain size, and ability to form aggregates. These findings suggest that the embryonic chick sterna maintained a stable cartilage phenotype during the extended culture periods. The initial rapid rate of matrix turnover was probably attributable to an adaptation of the tissue to ex ovo culture conditions and the subsequent maintenance of cellular activities at a lower level indicated the establishment of a steady-state rate of metabolism. © 1999 Academic Press
1
To whom correspondence should be addressed at Department of Histopathology, University College London Medical School, Rockfeller Building, University Street, London WC1E 6JJ, UK. Fax: (0) 44 (0) 171 387 3674. E-mail: [email protected]. 2 Deceased. 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Key Words: proteoglycan; embryonic chick sternal cartilage; explant culture.
Much current knowledge regarding cartilage metabolism has been obtained using in vitro explant cultures derived from either embryonic and immature or mature tissues, and explant cultures of developing chick sternum have been widely used. The embryonic chick sternum contains two types of cartilage and chondrocytes; the cephalic large-celled hypertrophic cartilage region and the caudal small-celled region which retains its cartilaginous structure and contains an homogenous chondrocyte population and matrix composition (1– 4). This tissue is readily available, easily dissected, and can be aged with absolute accuracy. Because of its anatomic features, sampling variation in preparing explant cultures is minimized. The intact tissue can be cultured without the need for fragmentation, since whole sternum incorporates sulfate into glycosaminoglycans (GAGs) 3 to essentially the same extent as its fragments (5). Therefore, caudal embryonic chick sternum provides a convenient and reproducible tissue source for cartilage explant culture, and this culture system has been used to study the development of cartilage and the regulation of chondrocyte metabolism and differentiation (2– 4, 6 –16). However, very little is known about the catabolic and anabolic behavior of embryonic chick sternal cartilage in explant culture. Studies have shown that the response of chondrocytes to applied stimuli may be dependent on the metabolic status of the tissue (14, 17– 22). This study, therefore, aimed to characterize the metabolic kinetics of embryonic chick sternal cartilage 3 Abbreviations used: GAGs, glycosaminoglycans; PG, proteoglycan; CS, chondroitin sulfate; ANOVA, analysis of variance.
225
226
LIU, BEE, AND LEES
FIG. 1. Histological organisation of freshly isolated cephalic (a) and caudal (b) regions of day 16 embryonic chick sternum and sterna explanted to culture for 2 (c) and 8 (d) days. Tissues were processed in paraffin wax, sectioned at right angles to the long axis of the sterna, and stained with safranin O and Mayer’s hematoxylin (3200).
in culture and to define culture conditions that enable the maintenance of phenotype and metabolic state of the chondrocytes. We examined the morphology and for the first time measured quantitative and qualita-
tive changes in the synthesis and turnover of proteoglycan (PG), a major cartilage matrix component, in 16-day-old embryonic chick sternal cartilage cultured for 8 days in the presence of 10% fetal calf serum.
PROTEOGLYCAN TURNOVER BY CHICK STERNAL CARTILAGE IN EXPLANT CULTURE
227
MATERIALS AND METHODS Materials. Ham’s F12 medium was obtained from Sigma Chemical Co. (Poole, Dorset, UK). Fetal calf serum was from ICN Flow (Irvine, Scotland, UK). Carrier-free [ 35S]sulfuric acid was from Amersham International plc (Amersham, UK). Papain was from Sigma Chemical Co. Chondroitinase ABC was from ICN Biochemicals (Cleveland, OH). Dimethylmethylene Blue was from Aldrich Chemical Co. (Gillingham, Dorset, UK). Safranin O was supplied by Geoge T. Gurr Ltd. (London, UK). Sepharose CL-2B and Sepharose CL-6B were supplied by Pharmacia (Uppsala, Sweden). Cellogel cellulose acetate strips were from Whatman (Chemetron, Italy). All other reagents were of analytical reagent grade and supplied by BDH or Sigma Chemical Co. Explant culture. Embryonic chick sternal cartilage was cultured as reported previously (13, 15–16). Briefly, 16-day-old White Leghorn embryonic chick sternum was dissected free of surrounding and adherent tissues and the caudal region corresponding to hyaline cartilage was recovered as explant (3). The explants were randomly placed in 35-mm tissue culture dishes, each containing three or four pieces of tissue, and cultured in Ham’s F12 medium, supplemented with 10% fetal calf serum, 0.1 mg/ml ascorbate, 50 IU/ml penicillin, and 50 mg/ml streptomycin in a humidified atmosphere at 37°C under 5% CO 2 in air for periods up to 8 days. The medium was changed daily. At the end of each culture period, explants and media were collected and the daily collected media were pooled for each culture period and stored, with collected explants, at 220°C. Determination of DNA, GAG, and collagen content. Freshly dissected sternal cartilages and cartilage explants and media harvested after culture for 1 to 8 days were digested with papain (16). Aliquots of papain digests were measured for DNA content by a fluorometric method (23), for collagen as hydroxyproline content following hydrolysis of samples (24), and for GAG content by reaction with dimethylmethylene blue (25). The remainder of the papain digests were further treated with chondroitinase ABC to determine the proportion of the enzyme susceptible chondroitin sulfate (CS) in the mixture (25). Determination of PG synthesis and degradation. Sternal cartilages were cultured for periods from 1 to 8 days as described above. To determine PG synthesis, explants were radiolabeled by incubation with [ 35S]sulfate for the final 24 h of each culture period. Labeling at each time point was performed by replacing the culture medium from a single batch of medium containing 5 mCi/ml [ 35S]sulfuric acid to ensure that all cultures at each time period were labeled with the same specific radioactivity. Explants and media were harvested at the end of the each culture period and digested with papain, and the amounts of [ 35S]sulfated PGs retained by the explants or released into the media were measured by cetylpyridinium chloride precipitation (26). PG degradation was determined by measurement of the in vitro breakdown of preexisting PGs (13). The preexisting PGs were labeled in ovo by injection of 10 mCi [ 35S]sulfuric acid onto the chorioallantoic membrane through windowed egg shell on the sixth day of embryonic development. Following administration of the isotope, the windows were sealed and eggs were returned to the incubator to permit continued development. Ten days later, biosynthetically radiolabeled sternal cartilages were isolated from the embryos. The cartilages were either analyzed immediately for radioactivity retained in the tissues or cultured from 1 to 8 days as described above. At the end of each culture period, the explants and media were harvested and the daily collected media pooled. The radioactivity of [ 35S]sulfated PGs released into the medium and retained by the explants during the course of the culture was measured as described above and the relative percentage of prelabeled PGs remaining in the tissue after each day of the culture was calculated. Analysis of hydrodynamic size distribution of [ 35S]sulfated PGs. Cartilages were labeled for preexisting PGs and new PGs synthesized after culture for 2 and 7 days as described above, except that
FIG. 2. DNA, collagen, and GAG content of sternal cartilage in explant culture. Day 16 embryonic chick sternal cartilage was explanted to culture for periods up to 8 days and DNA (a), collagen (b), and GAG (c) content of the explant and the relative percentage of total GAGs released into medium (d) before and during the culture were determined. each culture comprised 20 pieces of cartilage maintained in a 60-mm culture dish containing 10 ml culture medium. The recovered cartilages were diced and extracted with 4 M guanidine chloride in 0.05
228
LIU, BEE, AND LEES TABLE I
Relative Proportion of CS in GAGs from Freshly Dissected Sternal Cartilage and Cartilage Explants Cultured for 8 Days Days in culture
% of total GAGs as CS
Mean SD
0
1
2
3
4
5
6
7
8
92.6 1.8
88.5 2.0
92.3 1.1
87.6 2.9
88.8 1.9
87.9 2.8
88.1 2.4
89.0 2.3
88.4 2.1
Note. Day 16 embryonic chick sternal cartilage was explanted to culture for periods up to 8 days. The explants were recovered after each culture period, digested with papain, and measured for the content of GAGs. Aliquot papain digest was further treated with chondroitinase ABC and CS composition of total GAGs was determined.
M sodium acetate buffer, pH 5.8, and the extracts were dialyzed against 0.01 M sodium acetate buffer, pH 6.8 (16, 27). The samples were then eluted from Sepharose CL-2B columns under dissociative conditions to determine size distribution of 35S-labeled PG monomers and under associative conditions to determine ability of 35S-labeled PGs to form aggregates with excess exogenous hyaluronic acid (16, 27). To determine size distribution of 35S-labeled GAGs, peak fractions from Sepharose CL-2B chromatography under associative conditions were pooled, digested with papain, and eluted on Sepharose CL-6B columns. In addition, an aliquot of the above papain digests was subjected to cellulose acetate electrophoresis on Cellogel strips to determine the degree of sulfation and charge density of residual and newly synthesized sulfated GAGs (16, 28, 29). Histology. The morphology of freshly dissected whole sterna and cultured sternal explants from 16-day-old chick embryos was studied histologically. The tissues were fixed immediately after dissection, or after culture for 2 or 8 days, for 24 h in buffered formaldehyde sublimate. Following standard histological procedures, tissues were embedded in paraffin wax, serially sectioned at 7 mm on a rotary microtome at right angles to the long axis of the sternum, and stained with safranin O for GAGs and Mayer’s hematoxylin for general morphology (30). Data analysis. All experiments were performed in at least triplicate. All quantitative data were normalized, where applicable, by reference to DNA content. Data are expressed as means 6 SD of at least three measurements. Statistical analysis was performed by t test, ANOVA, or correlation using Microsoft Excel 5.0.
RESULTS
Histological morphology. Transverse sections of day 16 fresh sternum or day 16 sternal explants cultured for 2 and 8 days are shown in Fig. 1. In the cephalic region of day 16 fresh sternum (Fig. 1a), most chondrocytes were small and surrounded by an amorphous matrix but the central area exhibited a slightly more advanced stage of development, indicating an early sign of chondrocyte hypertrophy. In the caudal region of the same sternum (Fig. 1b); however, all chondrocytes were small and round except on the surface of the sternum where they were flattened. The chondrocytes were uniformly distributed in the matrix, which was intensely and uniformly stained with safranin O throughout the full thickness of the keel of the sternum. After 2 days of culture (Fig. 1c), the matrix staining was slightly weaker and deep chondrocytes were less evenly distributed. However, chondrocytes
remained small, round and uniform in size. After culture for 8 days (Fig. 1d), the histological morphology of the explant resembled that of the explant cultured for 2 days, except that the tissue had grown markedly. These findings suggested that the present sternal explants maintained their morphological characteristics during the experimental period. DNA, GAG, and collagen content. DNA, GAG, and collagen content of sternal cartilage at day 0 and the end of each culture period from 1 to 8 days was measured. The DNA content per initial wet weight increased progressively with time over the first 6 days of culture (r 2 5 0.97; r, coefficient of determination), attaining 138% of the initial level on day 6 (P , 0.01) (Fig. 2a). It then remained at a similar level for the remainder of the culture period. The collagen content per microgram of DNA increased linearly with time over the culture period (r 2 5 0.85, Fig. 2b). By day 8 the collagen content was increased by 30% (P , 0.05). The GAG content per microgram of DNA also increased linearly with time during culture (r 2 5 0.99) (Fig. 2c), but the rate and extent of increase were greater than those of the collagen content. By day 8 the GAG content was 2.5 times its initial value. These data are consistent with the morphological finding that the sternal cartilage continued to grow in culture. The increase in matrix production resulted not only from an increased cell population but also from increased biosynthetic activity of the cells. The release of collagen and GAG from explants into the media were also investigated. While the release of collagen was not measurable, the cumulative release of GAGs increased linearly with the duration of the culture period (r 2 5 0.996) (data not shown). When the percentage release of total GAG content was plotted against time, release rate was no longer linear, the rate of release decreasing with time (Fig. 2d). Digestion with chondroitinase ABC showed that 92.6% of GAGs in day 0 explants was composed of CS and this composition varied insignificantly between 87.6 and 92.3% (P . 0.05) during 8 days of culture (Table I). This range was similar to that recorded by
PROTEOGLYCAN TURNOVER BY CHICK STERNAL CARTILAGE IN EXPLANT CULTURE
FIG. 3. Synthesis and degradation of PGs by sternal cartilage in explant culture. To determine PG synthesis, day 16 embryonic chick sternal cartilage was explanted to culture for periods up to 8 days and [ 35S]sulfate was added to the cultures for the final 24h of each culture period. The rate of [ 35S]sulfate incorporation was measured as recoveries of radioactivity from the explant (a) and the medium (b). To determine PG degradation, sternal cartilage was labeled in ovo with [ 35S]sulfate at day 6 of the embryonic development, isolated at day 16, and explanted to culture for periods up to 8 days. The recoveries of preexisting PGs from explant and collected medium
229
other workers using embryonic chick cartilage at the equivalent developmental stage (31–33). The chondroitinase ABC-resistant content was not further analyzed in the present study. According to studies on embryonic or newly hatched chick cartilage, these minor GAG components were mainly keratan sulfate (32–34). PG synthesis and degradation. Sternal explants showed a high rate of PG synthesis during the first 2 days of culture (Fig. 3a). The rate decreased to 70.5% of the day 1 value on the 3rd day (P , 0.01), decreasing further to 43% of the day 1 value on the fourth day, and then remaining at this reduced level on subsequent days. Throughout the culture period, a small proportion (3–9%) of newly synthesized 35S-labeled PGs was released from explants into the medium (Fig. 3b). However, the overall release of 35S-labeled PGs was relatively constant and not correlated with the synthetic rate. Six-day-old chick embryos were administered with [ 35S]sulfate and the amount of 35S-labeled PGs recovered from sternal cartilage after 4, 7, and 10 days of in ovo labeling was 14,775 6 944, 19,397 6 1,818 and 21,186 6 1,640, respectively (mean cpm per sternum 6 SD of three sterna), increasing with embryonic development. However, the amount of 35S-labeled PGs expressed as cpm per mg wet weight did not differ significantly from each other (3,281 6 704, 3,003 6 377, and 2,843 6 277). Therefore, 35S-labeled PGs present in sternal explants at day 0 of culture were considered to represent the native pool of PGs in the tissue and their rate of release from explant during culture was measured as in vitro degradation. The release rate of preexisting 35S-labeled PGs decreased gradually over the 8-day culture period (Fig. 3c). When the amount of preexisting 35S-labeled PGs remaining in the tissue was plotted as a semilogarithmic function of time, the degradation rate was found to be first order (Fig. 3d). The in vitro half-life (t 1/2) of the 35S-labeled PGs, defined as the time required for 50% of the radioactivity present in the tissue on day 0 to be released into the medium, was approximately 15 days. Structures of preexisting and newly synthesized PGs. Preexisting 35S-labeled PGs and new 35S-labeled PGs synthesized after culture for 2 or 7 days were extracted from tissues and analyzed by chromatography on Sepharose CL-2B (Fig. 4a). 35S-labeled PGs were eluted as large monomers under dissociative conditions and as aggregates under associative conditions. Both the size distribution and the ability to form aggregates of
were summed and the percentage of preexisting [ 35S]sulfated PGs remaining in the explant after each culture period was plotted against time arithmetically (c) or as a semi-log degradation plot verses arithmetic time scale (d).
230
LIU, BEE, AND LEES 35
S-labeled PGs synthesized after culture for either 2 or 7 days were identical to those of preexisting 35S-labeled PGs. Peak fractions from associative chromatography were pooled, digested with papain to release GAG chains, and subjected to chromatography on Sepharose CL-6B (Fig. 4b). This showed that the preexisting and newly synthesized 35S-labeled GAG chains were similar in their size distribution. Further analysis of 35Slabeled GAGs by cellulose acetate electrophoresis showed that the migration distance of radiolabeled GAG chains was identical to that of alcian blue-stained resident GAGs in all cultures and no difference was detected in the mobilities of GAGs between preexisting and newly synthesized samples (Fig. 4c). The migration rate of individual GAGs depends solely on their degree of sulfation (35). Hence, the newly synthesized GAGs were sulfated and charged to the same degree as preexisting GAGs. DISCUSSION
The fresh caudal portion of 16-day-old embryonic chick sternum was shown to contain homogenous chondrocytes closely embedded in a uniform extracellular matrix which stained strongly with safranin O. This morphology is consistent with previous findings on chick sternal cartilage at a similar developmental stage (1– 4). When cultured in the presence of 10% fetal calf serum for periods up to 8 days, sternal cartilage maintained not only the tissue integrity and chondrocyte homogeneity but also the differentiated phenotype of the chondrocytes. The preexisting PGs labeled with 35 S in ovo exhibited a cartilage-specific structure, being similar in monomer size, ability to aggregate with hyaluronic acid, GAG chain length, and degree of sulfation to those described in previous studies of in ovo PGs from the same species (33) and to those described in mammalian hyaline cartilage of various sources (36 – 38). When tissues were explanted to culture, these characteristics of the PGs synthesized after culture for 2 and 7 days were not changed. In contrast, the PGs synthesized by rabbit articular cartilage explants were larger than normal in size of both PG monomers and GAG chains (39). In addition, collagen synthesized by chick sternal explants after culture for 2 or 7 days was predominantly cartilage-specific type II collagen (14).
FIG. 4. Structures of preexisting and newly synthesized PGs. Sternal cartilage was labeled in ovo or in vitro with [ 35S]sulfate and preexisting 35S-labeled PGs (day 0), and new 35S-labeled PGs synthe-
sized after culture for 2 and 7 days were extracted with 4 M guanidine and eluted from Sepharose CL-2B columns (a) under associative (—) and dissociative ( z z z ) conditions. The peak fractions (indicated by bars) were pooled from associative chromatography, digested with papain, and eluted from Sepharose CL-6B columns (b). An aliquot of papain digests was further analyzed on Cellogel strips by cellulose acetate electrophoresis (c). Strips were either sectioned for detecting radioactivity (line profiles) or stained with alcian blue (denoted by the bars) following digestion with papain.
PROTEOGLYCAN TURNOVER BY CHICK STERNAL CARTILAGE IN EXPLANT CULTURE
There was no evidence for the synthesis of either type I or type X collagen (14) which appears during endochondral bone formation of developing chick limbs (2, 3, 40). This is in contrast to the dedifferentiation of isolated chondrocytes in monolayer culture (41– 43). The kinetics of sternal cartilage metabolism in culture was characterized quantitatively in this study for the first time. In contrast to most cartilage explants in culture, which do not grow as a consequence of density limitation and the physical restrictions imposed by their geometry (44), embryonic sternal explants grew readily with time, as indicated by the progressive increase in their DNA content. Similar findings have been reported in organ cultures of embryonic chick limb rudiments (31, 45). In addition to the increase in tissue cellularity, the metabolic activity of chondrocytes was high, as indicated by the linear increase in production of GAG and collagen per DNA with time, with GAG production increasing more rapidly than collagen. Chondroitinase ABC digestion showed that the GAGs in the cultured explants were indistinguishable from those in the fresh tissue in their CS composition. An interesting finding was that the release over time of GAGs from cultured sternal cartilage explants was low compared to that of rabbit (46), pig (47), bovine (48), and human (49) articular cartilages. This might be related to the relatively intact architecture of the explant and the rapid deposition of GAGs in the tissue. Using [ 35S]sulfate labeling, the rate of new 35S-labeled PG synthesis and the rate of preexisting 35Slabeled PG degradation were both shown to be high during the initial culture period. After 3– 4 days the rates of synthesis and degradation were reduced and then maintained at a lower but stable level for the remaining culture period. The kinetics of degradation of preexisting 35S-labeled PGs was first order, similar to that of unlabeled GAGs and also similar to previous findings on bovine articular cartilage in culture (48, 50, 51). However, the release of newly synthesized (less than 1 day old) PGs was constant as previously found in other cartilage cultures (47). The synthesis and degradation of PGs are dependent on continued cellular activity. However, the decreased rates of PG synthesis and degradation observed after the initial period of culture in this study were not due to decreased chondrocyte viability or activity, since DNA content and the content of GAGs and collagen per microgram of DNA continued to increase linearly. The consistent release of both unlabeled and labeled PGs suggests that the higher synthetic rate of PGs during the initial culture period was stimulated by the rapid release of this component from the matrix at a time when the tissue was adapting to culture conditions and the subsequent decrease in GAG synthesis was thus balanced by reduced matrix release. This finding is consistent with general knowledge that the homeo-
231
static maintenance of PGs in cartilage involves coordinated synthesis and degradation (52, 53). The activation of PG synthesis by loss of GAG from the tissue has been previously reported in organ cultures of embryonic chick cartilage (31, 54, 55) as well as in young and mature mammalian articular cartilage (46, 56, 57). It has also been described in human and experimental animal models of osteoarthritis (58 – 60) and is not serum dependent (39). In contrast, addition of PG to cartilage cultures inhibits further PG synthesis (39, 61). The explant cultures of embryonic chick sternal cartilage were shown to be dissimilar to the explant cultures of articular cartilage derived from the rabbit (46), bovine (50, 57, 62), or pig (47). In these species, under similar culture conditions, the steady-state metabolism of PGs requires either sustained or increased rates of PG synthesis to maintain extracellular PG concentration at a level lower than or similar to that of fresh tissue without significant change in collagen and DNA content. Since the response of chondrocytes to applied stimuli may be dependent on the metabolic status of the tissue (14, 17–22), this study suggests that a short period of equilibration might be essential when embryonic chick sternal cartilage is used to study cartilage metabolism. ACKNOWLEDGMENTS This research was supported by Electro-Biology, Inc. (Upper Pond Road, Parsippany, NJ 07054-1079). The authors would like to thank Professor Michael T. Bayliss and Dr. Michael F. Dean of the Royal Veterinary College, University of London, UK, for their advice in the preparation of this manuscript.
REFERENCES 1. Fell, H. B. (1956) in The Biochemistry of Physiology of Bone (Bourne, G. H., Ed.), pp. 401– 441, Academic Press, New York. 2. Gibson, G. J., and Flint, M. H. (1985) J. Cell Biol. 101, 277–284. 3. Reginato, A. M., Lash, J. W., and Jimenez, S. A. (1986) J. Biol. Chem. 261, 2897–2904. 4. Tschan, T., Bohme, K., Zenke, G., Winterhalter, K. H., and Bruckner, P. (1993) J. Biol. Chem. 268, 5156 –5161. 5. Audhya, T. K., and Gibson, K. D. (1974) Endocrinology 95, 1614 – 1620. 6. Jimenez, S. A., Yankowski, R., and Reginato, A. M. (1986) Biochem. J. 233, 357–367. 7. McClure, J., Bates, G. P., Rowston, H., and Grant, M. E. (1988) Bone Miner. 3, 235–247. 8. Thom, J. R., and Morris, N. P. (1991) J. Biol. Chem. 266, 7262– 7269. 9. Pacifici, M., Golden, E. B., Adams, S. L., and Shapiro, I. M. (1991) Exp. Cell Res. 192, 266 –270. 10. Mettal, D., Brune, K., and Mollenhauer, J. (1992) Biochim. Biophys. Acta 1138, 85–92. 11. Chen, P., Vukicevic, S., Sampath, T. K., and Luyten, F. P. (1993) Biochim. Biophys. Res. Commun. 197, 1253–1259. 12. Cornelissen, M., Thierens, H., and de Ridder, L. (1993) Tissue Cell 25, 343–350.
232
LIU, BEE, AND LEES
13. Bee, J. A., Liu, H., Clarke, N., and Abbott, J. (1994) in Biomechanics and Cells (Lyall, F., and El Haj, A. J., Eds.), pp. 244 –269, Society for Experimental Biology Seminar Series 54, Cambridge University Press, Cambridge. 14. Liu, H. (1996) Ph.D. Thesis, University of London. 15. Liu, H., Abbot, J., and Bee, J. A. (1996) Osteoarthritis Cartilage 4, 63–76. 16. Liu, H., Lees, P., Abbott, J., and Bee, J. A. (1997) Biochim. Biophys. Acta 1336, 303–314. 17. Handley, C. J., Ng, C. K., and Curtis, A. J. (1990) in Methods in Cartilage Research (Maroudas, A., and Kuettner, K., Eds.), pp.105–108, Academic Press, London. 18. Norton, L. A. (1985) Reconstr. Surg. Traumatol. 19, 70 – 86. 19. Murray, J. C., and Farndale, R. W. (1985) Biochim. Biophys. Acta 838, 98 –105. 20. Farndale, R. W., and Murray, J. C. (1985) Calcif. Tissue Int. 37, 178 –182. 21. Guzelsu, N., Salkind, A. J., Shen, X., Patel, U., Thaler, S., and Berg, R. A. (1994) Bioelectromagnetics 15, 115–131. 22. Ciombor, D. Mck., and Aaron, R. K. (1994) Trans. Orthop. Res. Soc. 19, 461. 23. Royce, P. M., and Lowther, D. A. (1979) Connect. Tissue Res. 6, 215–221. 24. Woessner, J. F., Jr. (1977) in Methodology of Connective Tissue Research (Hall, D. A., Ed.), pp. 247–251, Joynson-Bruvvers, Oxford. 25. Farndale, R. W. (1980) Biochim. Biophys. Acta 883, 173–177. 26. Saklatvala, J. (1986) Nature 322, 547–549. 27. Bayliss, T. M., and Ali, S. Y. (1978) Biochem. J. 169, 123–132. 28. Wessler, E. (1971) Anal. Biochem. 41, 67– 69. 29. Tyler, J. A. (1989) Biochem. J. 260, 543–548. 30. Rosenberg, L. (1971) J. Bone Joint Surg. 53A, 69 – 82. 31. Hardingham, T. E., Fitton-Jackson, S., and Muir, H. (1972) Biochem. J. 129, 101–112. 32. Sobue, M., and Takeuchi, J. (1979) Calcif. Tissue Int. 27, 269 –273. 33. Carrino, D. A., and Caplan, A. I. (1985) J. Biol. Chem. 260, 122–127. 34. Vasan, N. S. (1981) J. Embryol. Exp. Morphol. 63, 181–191. 35. Wasteson, A., and Lindahl, U. (1972) Biochem. J. 125, 903–908. 36. Hardingham, T. E. (1977) in The Methodology of Connective Tissue Research (Hall, D. A., Ed.), pp. 153–166, Joynson-Bruvvers, Oxford. 37. Bayliss, M. T. (1984) in Connective Tissue Matrix (Hukins, D. W. L., Ed.), pp. 55– 88, MacMillan, Hong Kong. 38. Carney, S. L., and Muir, H. (1988) Physiol. Rev. 68, 858 –910.
39. Sandy, J. D., Brown, H. L. G., and Lowther, D. A. (1980) Biochem. J. 188, 119 –130. 40. van der Mark, K., and van der Mark, H. (1977) J. Bone Joint Surg. 59B, 458 – 464. 41. Mayne, R., Vail, M. S., and Miller, E. J. (1975) Proc. Natl. Acad. Sci. USA 72, 4511– 4515. 42. Benya, P. D., Padilla, S. R., and Nimni, M. E. (1978) Cell 15, 1313–1321. 43. Liu, H., Lee, Y.-W., and Dean, M. F. (1998) Biochim. Biophys. Acta 1425, 505–515. 44. Freshney, R. I. (1994) Culture of Animal Cells, A Manual of Basic Technique, 3rd ed., p. 5, Wiley–Liss, New York. 45. Gwatkin, R. B. L., and Biggers, J. D. (1963) J. Exp. Zool. 152, 149. 46. Sandy, J. D., and Plaas, A. H. K. (1986) J. Orthop. Res. 4, 263–272. 47. Tyler, J. A. (1985) Biochem. J. 227, 869 – 878. 48. Morales, T. I., Wahl, L. M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 6720 – 6729. 49. Jubb, R. W., and Saklatvala, J. (1985) Br. J. Rheumatol. 24(Suppl. 1), 156 –157. 50. Hascall, V. C., Handley, C. J., McQuillan, D. J., Robinson, H. C., and Lowther, D. A. (1983) Arch. Biochem. Biophys. 224, 206 –223. 51. Luyten, F. P., Hascall, V. C., Nissley, S. P., Morales, T. I., and Reddi, A. H. (1988) Arch. Biochem. Biophys. 267, 416 – 425. 52. Hardingham, T. E. (1988) in Control of Tissue Damage (Glauert, A., Ed.), pp. 215–219, Elsevier, Amsterdam. 53. Hardingham, T. E., and Bayliss, M. T. (1990) Semin. Arthritis Rheum. 20, 12–23. 54. Fitton-Jackson, S. (1970) Proc. R. Soc. Ser. B 175, 405– 453. 55. Muir, H. (1980) in The Joint and Synovial Fluid (Sokoloff, L., Ed.), pp. 27–94, Academic Press, New York. 56. Sandy, J. D., Brown, H. L. G., and Lowther, D. A. (1978) Biochim. Biophys. Acta 543, 536 –544. 57. Hascall, V. C., Luyten, F. P., Plaas, A. H. K., and Sandy, J. D. (1990) in Methods in Cartilage Research (Maroudas, A., and Kuettner, K., Eds.), pp. 108 –112, Academic Press, London. 58. Mankin, H. J., and Lippiello, L. (1970) J. Bone Joint Surg. 52A, 424 – 434. 59. Eronen, I., Videman, T., Friman, C., and Michelsson, J. E. (1978) Acta Orthop. Scand. 49, 329 –334. 60. Rizkalla, G., Reiner, A., Bogoch, E., and Poole, A. R. (1992) J. Clin. Invest. 90, 2268 –2277. 61. Wiebkin, O., and Muir, H. (1973) FEBS Lett. 37, 42– 46. 62. Morales, T. I., and Roberts, A. B. (1988) J. Biol. Chem. 263, 12828 –12831.
Metabolic Kinetics of Proteoglycans by Embryonic Chick Sternal Cartilage in Culture Hongxiang Liu, 1 James A. Bee, 2 and Peter Lees Department of Veterinary Basic Sciences, The Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, United Kingdom
Received December 29, 1998, and in revised form April 9, 1999
Explant cultures of embryonic chick sternum have been widely studied, but the kinetics of biosynthesis of proteoglycans by this tissue in culture has not been characterized. Caudal cartilaginous portions of 16day-old embryonic chick sterna were cultured for 8 days. Histological examination showed that the fresh cartilage contained morphologically homogenous chondrocytes, which were embedded in a uniform extracellular matrix. After culture for 8 days, the histological appearance of the explant remained unchanged but the tissue increased in size with time as indicated by a progressive increase in DNA content and in the content of glycosaminoglycan and collagen. Rates of degradation and release from the tissue of proteoglycans labeled in ovo with 35S were first order during culture, as were the unlabeled proteoglycans. Proteoglycan synthesis was high during the first 2 days of culture, and this then gradually decreased from this high level during the following 2 days. Synthesis was then maintained at a constant level for the remainder of the culture period. After culture for 2 and 7 days, the proteoglycans synthesized by the explants were identical to the preexisting proteoglycans in hydrodynamic size, glycosaminoglycan chain size, and ability to form aggregates. These findings suggest that the embryonic chick sterna maintained a stable cartilage phenotype during the extended culture periods. The initial rapid rate of matrix turnover was probably attributable to an adaptation of the tissue to ex ovo culture conditions and the subsequent maintenance of cellular activities at a lower level indicated the establishment of a steady-state rate of metabolism. © 1999 Academic Press
1
To whom correspondence should be addressed at Department of Histopathology, University College London Medical School, Rockfeller Building, University Street, London WC1E 6JJ, UK. Fax: (0) 44 (0) 171 387 3674. E-mail: [email protected]. 2 Deceased. 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Key Words: proteoglycan; embryonic chick sternal cartilage; explant culture.
Much current knowledge regarding cartilage metabolism has been obtained using in vitro explant cultures derived from either embryonic and immature or mature tissues, and explant cultures of developing chick sternum have been widely used. The embryonic chick sternum contains two types of cartilage and chondrocytes; the cephalic large-celled hypertrophic cartilage region and the caudal small-celled region which retains its cartilaginous structure and contains an homogenous chondrocyte population and matrix composition (1– 4). This tissue is readily available, easily dissected, and can be aged with absolute accuracy. Because of its anatomic features, sampling variation in preparing explant cultures is minimized. The intact tissue can be cultured without the need for fragmentation, since whole sternum incorporates sulfate into glycosaminoglycans (GAGs) 3 to essentially the same extent as its fragments (5). Therefore, caudal embryonic chick sternum provides a convenient and reproducible tissue source for cartilage explant culture, and this culture system has been used to study the development of cartilage and the regulation of chondrocyte metabolism and differentiation (2– 4, 6 –16). However, very little is known about the catabolic and anabolic behavior of embryonic chick sternal cartilage in explant culture. Studies have shown that the response of chondrocytes to applied stimuli may be dependent on the metabolic status of the tissue (14, 17– 22). This study, therefore, aimed to characterize the metabolic kinetics of embryonic chick sternal cartilage 3 Abbreviations used: GAGs, glycosaminoglycans; PG, proteoglycan; CS, chondroitin sulfate; ANOVA, analysis of variance.
225
226
LIU, BEE, AND LEES
FIG. 1. Histological organisation of freshly isolated cephalic (a) and caudal (b) regions of day 16 embryonic chick sternum and sterna explanted to culture for 2 (c) and 8 (d) days. Tissues were processed in paraffin wax, sectioned at right angles to the long axis of the sterna, and stained with safranin O and Mayer’s hematoxylin (3200).
in culture and to define culture conditions that enable the maintenance of phenotype and metabolic state of the chondrocytes. We examined the morphology and for the first time measured quantitative and qualita-
tive changes in the synthesis and turnover of proteoglycan (PG), a major cartilage matrix component, in 16-day-old embryonic chick sternal cartilage cultured for 8 days in the presence of 10% fetal calf serum.
PROTEOGLYCAN TURNOVER BY CHICK STERNAL CARTILAGE IN EXPLANT CULTURE
227
MATERIALS AND METHODS Materials. Ham’s F12 medium was obtained from Sigma Chemical Co. (Poole, Dorset, UK). Fetal calf serum was from ICN Flow (Irvine, Scotland, UK). Carrier-free [ 35S]sulfuric acid was from Amersham International plc (Amersham, UK). Papain was from Sigma Chemical Co. Chondroitinase ABC was from ICN Biochemicals (Cleveland, OH). Dimethylmethylene Blue was from Aldrich Chemical Co. (Gillingham, Dorset, UK). Safranin O was supplied by Geoge T. Gurr Ltd. (London, UK). Sepharose CL-2B and Sepharose CL-6B were supplied by Pharmacia (Uppsala, Sweden). Cellogel cellulose acetate strips were from Whatman (Chemetron, Italy). All other reagents were of analytical reagent grade and supplied by BDH or Sigma Chemical Co. Explant culture. Embryonic chick sternal cartilage was cultured as reported previously (13, 15–16). Briefly, 16-day-old White Leghorn embryonic chick sternum was dissected free of surrounding and adherent tissues and the caudal region corresponding to hyaline cartilage was recovered as explant (3). The explants were randomly placed in 35-mm tissue culture dishes, each containing three or four pieces of tissue, and cultured in Ham’s F12 medium, supplemented with 10% fetal calf serum, 0.1 mg/ml ascorbate, 50 IU/ml penicillin, and 50 mg/ml streptomycin in a humidified atmosphere at 37°C under 5% CO 2 in air for periods up to 8 days. The medium was changed daily. At the end of each culture period, explants and media were collected and the daily collected media were pooled for each culture period and stored, with collected explants, at 220°C. Determination of DNA, GAG, and collagen content. Freshly dissected sternal cartilages and cartilage explants and media harvested after culture for 1 to 8 days were digested with papain (16). Aliquots of papain digests were measured for DNA content by a fluorometric method (23), for collagen as hydroxyproline content following hydrolysis of samples (24), and for GAG content by reaction with dimethylmethylene blue (25). The remainder of the papain digests were further treated with chondroitinase ABC to determine the proportion of the enzyme susceptible chondroitin sulfate (CS) in the mixture (25). Determination of PG synthesis and degradation. Sternal cartilages were cultured for periods from 1 to 8 days as described above. To determine PG synthesis, explants were radiolabeled by incubation with [ 35S]sulfate for the final 24 h of each culture period. Labeling at each time point was performed by replacing the culture medium from a single batch of medium containing 5 mCi/ml [ 35S]sulfuric acid to ensure that all cultures at each time period were labeled with the same specific radioactivity. Explants and media were harvested at the end of the each culture period and digested with papain, and the amounts of [ 35S]sulfated PGs retained by the explants or released into the media were measured by cetylpyridinium chloride precipitation (26). PG degradation was determined by measurement of the in vitro breakdown of preexisting PGs (13). The preexisting PGs were labeled in ovo by injection of 10 mCi [ 35S]sulfuric acid onto the chorioallantoic membrane through windowed egg shell on the sixth day of embryonic development. Following administration of the isotope, the windows were sealed and eggs were returned to the incubator to permit continued development. Ten days later, biosynthetically radiolabeled sternal cartilages were isolated from the embryos. The cartilages were either analyzed immediately for radioactivity retained in the tissues or cultured from 1 to 8 days as described above. At the end of each culture period, the explants and media were harvested and the daily collected media pooled. The radioactivity of [ 35S]sulfated PGs released into the medium and retained by the explants during the course of the culture was measured as described above and the relative percentage of prelabeled PGs remaining in the tissue after each day of the culture was calculated. Analysis of hydrodynamic size distribution of [ 35S]sulfated PGs. Cartilages were labeled for preexisting PGs and new PGs synthesized after culture for 2 and 7 days as described above, except that
FIG. 2. DNA, collagen, and GAG content of sternal cartilage in explant culture. Day 16 embryonic chick sternal cartilage was explanted to culture for periods up to 8 days and DNA (a), collagen (b), and GAG (c) content of the explant and the relative percentage of total GAGs released into medium (d) before and during the culture were determined. each culture comprised 20 pieces of cartilage maintained in a 60-mm culture dish containing 10 ml culture medium. The recovered cartilages were diced and extracted with 4 M guanidine chloride in 0.05
228
LIU, BEE, AND LEES TABLE I
Relative Proportion of CS in GAGs from Freshly Dissected Sternal Cartilage and Cartilage Explants Cultured for 8 Days Days in culture
% of total GAGs as CS
Mean SD
0
1
2
3
4
5
6
7
8
92.6 1.8
88.5 2.0
92.3 1.1
87.6 2.9
88.8 1.9
87.9 2.8
88.1 2.4
89.0 2.3
88.4 2.1
Note. Day 16 embryonic chick sternal cartilage was explanted to culture for periods up to 8 days. The explants were recovered after each culture period, digested with papain, and measured for the content of GAGs. Aliquot papain digest was further treated with chondroitinase ABC and CS composition of total GAGs was determined.
M sodium acetate buffer, pH 5.8, and the extracts were dialyzed against 0.01 M sodium acetate buffer, pH 6.8 (16, 27). The samples were then eluted from Sepharose CL-2B columns under dissociative conditions to determine size distribution of 35S-labeled PG monomers and under associative conditions to determine ability of 35S-labeled PGs to form aggregates with excess exogenous hyaluronic acid (16, 27). To determine size distribution of 35S-labeled GAGs, peak fractions from Sepharose CL-2B chromatography under associative conditions were pooled, digested with papain, and eluted on Sepharose CL-6B columns. In addition, an aliquot of the above papain digests was subjected to cellulose acetate electrophoresis on Cellogel strips to determine the degree of sulfation and charge density of residual and newly synthesized sulfated GAGs (16, 28, 29). Histology. The morphology of freshly dissected whole sterna and cultured sternal explants from 16-day-old chick embryos was studied histologically. The tissues were fixed immediately after dissection, or after culture for 2 or 8 days, for 24 h in buffered formaldehyde sublimate. Following standard histological procedures, tissues were embedded in paraffin wax, serially sectioned at 7 mm on a rotary microtome at right angles to the long axis of the sternum, and stained with safranin O for GAGs and Mayer’s hematoxylin for general morphology (30). Data analysis. All experiments were performed in at least triplicate. All quantitative data were normalized, where applicable, by reference to DNA content. Data are expressed as means 6 SD of at least three measurements. Statistical analysis was performed by t test, ANOVA, or correlation using Microsoft Excel 5.0.
RESULTS
Histological morphology. Transverse sections of day 16 fresh sternum or day 16 sternal explants cultured for 2 and 8 days are shown in Fig. 1. In the cephalic region of day 16 fresh sternum (Fig. 1a), most chondrocytes were small and surrounded by an amorphous matrix but the central area exhibited a slightly more advanced stage of development, indicating an early sign of chondrocyte hypertrophy. In the caudal region of the same sternum (Fig. 1b); however, all chondrocytes were small and round except on the surface of the sternum where they were flattened. The chondrocytes were uniformly distributed in the matrix, which was intensely and uniformly stained with safranin O throughout the full thickness of the keel of the sternum. After 2 days of culture (Fig. 1c), the matrix staining was slightly weaker and deep chondrocytes were less evenly distributed. However, chondrocytes
remained small, round and uniform in size. After culture for 8 days (Fig. 1d), the histological morphology of the explant resembled that of the explant cultured for 2 days, except that the tissue had grown markedly. These findings suggested that the present sternal explants maintained their morphological characteristics during the experimental period. DNA, GAG, and collagen content. DNA, GAG, and collagen content of sternal cartilage at day 0 and the end of each culture period from 1 to 8 days was measured. The DNA content per initial wet weight increased progressively with time over the first 6 days of culture (r 2 5 0.97; r, coefficient of determination), attaining 138% of the initial level on day 6 (P , 0.01) (Fig. 2a). It then remained at a similar level for the remainder of the culture period. The collagen content per microgram of DNA increased linearly with time over the culture period (r 2 5 0.85, Fig. 2b). By day 8 the collagen content was increased by 30% (P , 0.05). The GAG content per microgram of DNA also increased linearly with time during culture (r 2 5 0.99) (Fig. 2c), but the rate and extent of increase were greater than those of the collagen content. By day 8 the GAG content was 2.5 times its initial value. These data are consistent with the morphological finding that the sternal cartilage continued to grow in culture. The increase in matrix production resulted not only from an increased cell population but also from increased biosynthetic activity of the cells. The release of collagen and GAG from explants into the media were also investigated. While the release of collagen was not measurable, the cumulative release of GAGs increased linearly with the duration of the culture period (r 2 5 0.996) (data not shown). When the percentage release of total GAG content was plotted against time, release rate was no longer linear, the rate of release decreasing with time (Fig. 2d). Digestion with chondroitinase ABC showed that 92.6% of GAGs in day 0 explants was composed of CS and this composition varied insignificantly between 87.6 and 92.3% (P . 0.05) during 8 days of culture (Table I). This range was similar to that recorded by
PROTEOGLYCAN TURNOVER BY CHICK STERNAL CARTILAGE IN EXPLANT CULTURE
FIG. 3. Synthesis and degradation of PGs by sternal cartilage in explant culture. To determine PG synthesis, day 16 embryonic chick sternal cartilage was explanted to culture for periods up to 8 days and [ 35S]sulfate was added to the cultures for the final 24h of each culture period. The rate of [ 35S]sulfate incorporation was measured as recoveries of radioactivity from the explant (a) and the medium (b). To determine PG degradation, sternal cartilage was labeled in ovo with [ 35S]sulfate at day 6 of the embryonic development, isolated at day 16, and explanted to culture for periods up to 8 days. The recoveries of preexisting PGs from explant and collected medium
229
other workers using embryonic chick cartilage at the equivalent developmental stage (31–33). The chondroitinase ABC-resistant content was not further analyzed in the present study. According to studies on embryonic or newly hatched chick cartilage, these minor GAG components were mainly keratan sulfate (32–34). PG synthesis and degradation. Sternal explants showed a high rate of PG synthesis during the first 2 days of culture (Fig. 3a). The rate decreased to 70.5% of the day 1 value on the 3rd day (P , 0.01), decreasing further to 43% of the day 1 value on the fourth day, and then remaining at this reduced level on subsequent days. Throughout the culture period, a small proportion (3–9%) of newly synthesized 35S-labeled PGs was released from explants into the medium (Fig. 3b). However, the overall release of 35S-labeled PGs was relatively constant and not correlated with the synthetic rate. Six-day-old chick embryos were administered with [ 35S]sulfate and the amount of 35S-labeled PGs recovered from sternal cartilage after 4, 7, and 10 days of in ovo labeling was 14,775 6 944, 19,397 6 1,818 and 21,186 6 1,640, respectively (mean cpm per sternum 6 SD of three sterna), increasing with embryonic development. However, the amount of 35S-labeled PGs expressed as cpm per mg wet weight did not differ significantly from each other (3,281 6 704, 3,003 6 377, and 2,843 6 277). Therefore, 35S-labeled PGs present in sternal explants at day 0 of culture were considered to represent the native pool of PGs in the tissue and their rate of release from explant during culture was measured as in vitro degradation. The release rate of preexisting 35S-labeled PGs decreased gradually over the 8-day culture period (Fig. 3c). When the amount of preexisting 35S-labeled PGs remaining in the tissue was plotted as a semilogarithmic function of time, the degradation rate was found to be first order (Fig. 3d). The in vitro half-life (t 1/2) of the 35S-labeled PGs, defined as the time required for 50% of the radioactivity present in the tissue on day 0 to be released into the medium, was approximately 15 days. Structures of preexisting and newly synthesized PGs. Preexisting 35S-labeled PGs and new 35S-labeled PGs synthesized after culture for 2 or 7 days were extracted from tissues and analyzed by chromatography on Sepharose CL-2B (Fig. 4a). 35S-labeled PGs were eluted as large monomers under dissociative conditions and as aggregates under associative conditions. Both the size distribution and the ability to form aggregates of
were summed and the percentage of preexisting [ 35S]sulfated PGs remaining in the explant after each culture period was plotted against time arithmetically (c) or as a semi-log degradation plot verses arithmetic time scale (d).
230
LIU, BEE, AND LEES 35
S-labeled PGs synthesized after culture for either 2 or 7 days were identical to those of preexisting 35S-labeled PGs. Peak fractions from associative chromatography were pooled, digested with papain to release GAG chains, and subjected to chromatography on Sepharose CL-6B (Fig. 4b). This showed that the preexisting and newly synthesized 35S-labeled GAG chains were similar in their size distribution. Further analysis of 35Slabeled GAGs by cellulose acetate electrophoresis showed that the migration distance of radiolabeled GAG chains was identical to that of alcian blue-stained resident GAGs in all cultures and no difference was detected in the mobilities of GAGs between preexisting and newly synthesized samples (Fig. 4c). The migration rate of individual GAGs depends solely on their degree of sulfation (35). Hence, the newly synthesized GAGs were sulfated and charged to the same degree as preexisting GAGs. DISCUSSION
The fresh caudal portion of 16-day-old embryonic chick sternum was shown to contain homogenous chondrocytes closely embedded in a uniform extracellular matrix which stained strongly with safranin O. This morphology is consistent with previous findings on chick sternal cartilage at a similar developmental stage (1– 4). When cultured in the presence of 10% fetal calf serum for periods up to 8 days, sternal cartilage maintained not only the tissue integrity and chondrocyte homogeneity but also the differentiated phenotype of the chondrocytes. The preexisting PGs labeled with 35 S in ovo exhibited a cartilage-specific structure, being similar in monomer size, ability to aggregate with hyaluronic acid, GAG chain length, and degree of sulfation to those described in previous studies of in ovo PGs from the same species (33) and to those described in mammalian hyaline cartilage of various sources (36 – 38). When tissues were explanted to culture, these characteristics of the PGs synthesized after culture for 2 and 7 days were not changed. In contrast, the PGs synthesized by rabbit articular cartilage explants were larger than normal in size of both PG monomers and GAG chains (39). In addition, collagen synthesized by chick sternal explants after culture for 2 or 7 days was predominantly cartilage-specific type II collagen (14).
FIG. 4. Structures of preexisting and newly synthesized PGs. Sternal cartilage was labeled in ovo or in vitro with [ 35S]sulfate and preexisting 35S-labeled PGs (day 0), and new 35S-labeled PGs synthe-
sized after culture for 2 and 7 days were extracted with 4 M guanidine and eluted from Sepharose CL-2B columns (a) under associative (—) and dissociative ( z z z ) conditions. The peak fractions (indicated by bars) were pooled from associative chromatography, digested with papain, and eluted from Sepharose CL-6B columns (b). An aliquot of papain digests was further analyzed on Cellogel strips by cellulose acetate electrophoresis (c). Strips were either sectioned for detecting radioactivity (line profiles) or stained with alcian blue (denoted by the bars) following digestion with papain.
PROTEOGLYCAN TURNOVER BY CHICK STERNAL CARTILAGE IN EXPLANT CULTURE
There was no evidence for the synthesis of either type I or type X collagen (14) which appears during endochondral bone formation of developing chick limbs (2, 3, 40). This is in contrast to the dedifferentiation of isolated chondrocytes in monolayer culture (41– 43). The kinetics of sternal cartilage metabolism in culture was characterized quantitatively in this study for the first time. In contrast to most cartilage explants in culture, which do not grow as a consequence of density limitation and the physical restrictions imposed by their geometry (44), embryonic sternal explants grew readily with time, as indicated by the progressive increase in their DNA content. Similar findings have been reported in organ cultures of embryonic chick limb rudiments (31, 45). In addition to the increase in tissue cellularity, the metabolic activity of chondrocytes was high, as indicated by the linear increase in production of GAG and collagen per DNA with time, with GAG production increasing more rapidly than collagen. Chondroitinase ABC digestion showed that the GAGs in the cultured explants were indistinguishable from those in the fresh tissue in their CS composition. An interesting finding was that the release over time of GAGs from cultured sternal cartilage explants was low compared to that of rabbit (46), pig (47), bovine (48), and human (49) articular cartilages. This might be related to the relatively intact architecture of the explant and the rapid deposition of GAGs in the tissue. Using [ 35S]sulfate labeling, the rate of new 35S-labeled PG synthesis and the rate of preexisting 35Slabeled PG degradation were both shown to be high during the initial culture period. After 3– 4 days the rates of synthesis and degradation were reduced and then maintained at a lower but stable level for the remaining culture period. The kinetics of degradation of preexisting 35S-labeled PGs was first order, similar to that of unlabeled GAGs and also similar to previous findings on bovine articular cartilage in culture (48, 50, 51). However, the release of newly synthesized (less than 1 day old) PGs was constant as previously found in other cartilage cultures (47). The synthesis and degradation of PGs are dependent on continued cellular activity. However, the decreased rates of PG synthesis and degradation observed after the initial period of culture in this study were not due to decreased chondrocyte viability or activity, since DNA content and the content of GAGs and collagen per microgram of DNA continued to increase linearly. The consistent release of both unlabeled and labeled PGs suggests that the higher synthetic rate of PGs during the initial culture period was stimulated by the rapid release of this component from the matrix at a time when the tissue was adapting to culture conditions and the subsequent decrease in GAG synthesis was thus balanced by reduced matrix release. This finding is consistent with general knowledge that the homeo-
231
static maintenance of PGs in cartilage involves coordinated synthesis and degradation (52, 53). The activation of PG synthesis by loss of GAG from the tissue has been previously reported in organ cultures of embryonic chick cartilage (31, 54, 55) as well as in young and mature mammalian articular cartilage (46, 56, 57). It has also been described in human and experimental animal models of osteoarthritis (58 – 60) and is not serum dependent (39). In contrast, addition of PG to cartilage cultures inhibits further PG synthesis (39, 61). The explant cultures of embryonic chick sternal cartilage were shown to be dissimilar to the explant cultures of articular cartilage derived from the rabbit (46), bovine (50, 57, 62), or pig (47). In these species, under similar culture conditions, the steady-state metabolism of PGs requires either sustained or increased rates of PG synthesis to maintain extracellular PG concentration at a level lower than or similar to that of fresh tissue without significant change in collagen and DNA content. Since the response of chondrocytes to applied stimuli may be dependent on the metabolic status of the tissue (14, 17–22), this study suggests that a short period of equilibration might be essential when embryonic chick sternal cartilage is used to study cartilage metabolism. ACKNOWLEDGMENTS This research was supported by Electro-Biology, Inc. (Upper Pond Road, Parsippany, NJ 07054-1079). The authors would like to thank Professor Michael T. Bayliss and Dr. Michael F. Dean of the Royal Veterinary College, University of London, UK, for their advice in the preparation of this manuscript.
REFERENCES 1. Fell, H. B. (1956) in The Biochemistry of Physiology of Bone (Bourne, G. H., Ed.), pp. 401– 441, Academic Press, New York. 2. Gibson, G. J., and Flint, M. H. (1985) J. Cell Biol. 101, 277–284. 3. Reginato, A. M., Lash, J. W., and Jimenez, S. A. (1986) J. Biol. Chem. 261, 2897–2904. 4. Tschan, T., Bohme, K., Zenke, G., Winterhalter, K. H., and Bruckner, P. (1993) J. Biol. Chem. 268, 5156 –5161. 5. Audhya, T. K., and Gibson, K. D. (1974) Endocrinology 95, 1614 – 1620. 6. Jimenez, S. A., Yankowski, R., and Reginato, A. M. (1986) Biochem. J. 233, 357–367. 7. McClure, J., Bates, G. P., Rowston, H., and Grant, M. E. (1988) Bone Miner. 3, 235–247. 8. Thom, J. R., and Morris, N. P. (1991) J. Biol. Chem. 266, 7262– 7269. 9. Pacifici, M., Golden, E. B., Adams, S. L., and Shapiro, I. M. (1991) Exp. Cell Res. 192, 266 –270. 10. Mettal, D., Brune, K., and Mollenhauer, J. (1992) Biochim. Biophys. Acta 1138, 85–92. 11. Chen, P., Vukicevic, S., Sampath, T. K., and Luyten, F. P. (1993) Biochim. Biophys. Res. Commun. 197, 1253–1259. 12. Cornelissen, M., Thierens, H., and de Ridder, L. (1993) Tissue Cell 25, 343–350.
232
LIU, BEE, AND LEES
13. Bee, J. A., Liu, H., Clarke, N., and Abbott, J. (1994) in Biomechanics and Cells (Lyall, F., and El Haj, A. J., Eds.), pp. 244 –269, Society for Experimental Biology Seminar Series 54, Cambridge University Press, Cambridge. 14. Liu, H. (1996) Ph.D. Thesis, University of London. 15. Liu, H., Abbot, J., and Bee, J. A. (1996) Osteoarthritis Cartilage 4, 63–76. 16. Liu, H., Lees, P., Abbott, J., and Bee, J. A. (1997) Biochim. Biophys. Acta 1336, 303–314. 17. Handley, C. J., Ng, C. K., and Curtis, A. J. (1990) in Methods in Cartilage Research (Maroudas, A., and Kuettner, K., Eds.), pp.105–108, Academic Press, London. 18. Norton, L. A. (1985) Reconstr. Surg. Traumatol. 19, 70 – 86. 19. Murray, J. C., and Farndale, R. W. (1985) Biochim. Biophys. Acta 838, 98 –105. 20. Farndale, R. W., and Murray, J. C. (1985) Calcif. Tissue Int. 37, 178 –182. 21. Guzelsu, N., Salkind, A. J., Shen, X., Patel, U., Thaler, S., and Berg, R. A. (1994) Bioelectromagnetics 15, 115–131. 22. Ciombor, D. Mck., and Aaron, R. K. (1994) Trans. Orthop. Res. Soc. 19, 461. 23. Royce, P. M., and Lowther, D. A. (1979) Connect. Tissue Res. 6, 215–221. 24. Woessner, J. F., Jr. (1977) in Methodology of Connective Tissue Research (Hall, D. A., Ed.), pp. 247–251, Joynson-Bruvvers, Oxford. 25. Farndale, R. W. (1980) Biochim. Biophys. Acta 883, 173–177. 26. Saklatvala, J. (1986) Nature 322, 547–549. 27. Bayliss, T. M., and Ali, S. Y. (1978) Biochem. J. 169, 123–132. 28. Wessler, E. (1971) Anal. Biochem. 41, 67– 69. 29. Tyler, J. A. (1989) Biochem. J. 260, 543–548. 30. Rosenberg, L. (1971) J. Bone Joint Surg. 53A, 69 – 82. 31. Hardingham, T. E., Fitton-Jackson, S., and Muir, H. (1972) Biochem. J. 129, 101–112. 32. Sobue, M., and Takeuchi, J. (1979) Calcif. Tissue Int. 27, 269 –273. 33. Carrino, D. A., and Caplan, A. I. (1985) J. Biol. Chem. 260, 122–127. 34. Vasan, N. S. (1981) J. Embryol. Exp. Morphol. 63, 181–191. 35. Wasteson, A., and Lindahl, U. (1972) Biochem. J. 125, 903–908. 36. Hardingham, T. E. (1977) in The Methodology of Connective Tissue Research (Hall, D. A., Ed.), pp. 153–166, Joynson-Bruvvers, Oxford. 37. Bayliss, M. T. (1984) in Connective Tissue Matrix (Hukins, D. W. L., Ed.), pp. 55– 88, MacMillan, Hong Kong. 38. Carney, S. L., and Muir, H. (1988) Physiol. Rev. 68, 858 –910.
39. Sandy, J. D., Brown, H. L. G., and Lowther, D. A. (1980) Biochem. J. 188, 119 –130. 40. van der Mark, K., and van der Mark, H. (1977) J. Bone Joint Surg. 59B, 458 – 464. 41. Mayne, R., Vail, M. S., and Miller, E. J. (1975) Proc. Natl. Acad. Sci. USA 72, 4511– 4515. 42. Benya, P. D., Padilla, S. R., and Nimni, M. E. (1978) Cell 15, 1313–1321. 43. Liu, H., Lee, Y.-W., and Dean, M. F. (1998) Biochim. Biophys. Acta 1425, 505–515. 44. Freshney, R. I. (1994) Culture of Animal Cells, A Manual of Basic Technique, 3rd ed., p. 5, Wiley–Liss, New York. 45. Gwatkin, R. B. L., and Biggers, J. D. (1963) J. Exp. Zool. 152, 149. 46. Sandy, J. D., and Plaas, A. H. K. (1986) J. Orthop. Res. 4, 263–272. 47. Tyler, J. A. (1985) Biochem. J. 227, 869 – 878. 48. Morales, T. I., Wahl, L. M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 6720 – 6729. 49. Jubb, R. W., and Saklatvala, J. (1985) Br. J. Rheumatol. 24(Suppl. 1), 156 –157. 50. Hascall, V. C., Handley, C. J., McQuillan, D. J., Robinson, H. C., and Lowther, D. A. (1983) Arch. Biochem. Biophys. 224, 206 –223. 51. Luyten, F. P., Hascall, V. C., Nissley, S. P., Morales, T. I., and Reddi, A. H. (1988) Arch. Biochem. Biophys. 267, 416 – 425. 52. Hardingham, T. E. (1988) in Control of Tissue Damage (Glauert, A., Ed.), pp. 215–219, Elsevier, Amsterdam. 53. Hardingham, T. E., and Bayliss, M. T. (1990) Semin. Arthritis Rheum. 20, 12–23. 54. Fitton-Jackson, S. (1970) Proc. R. Soc. Ser. B 175, 405– 453. 55. Muir, H. (1980) in The Joint and Synovial Fluid (Sokoloff, L., Ed.), pp. 27–94, Academic Press, New York. 56. Sandy, J. D., Brown, H. L. G., and Lowther, D. A. (1978) Biochim. Biophys. Acta 543, 536 –544. 57. Hascall, V. C., Luyten, F. P., Plaas, A. H. K., and Sandy, J. D. (1990) in Methods in Cartilage Research (Maroudas, A., and Kuettner, K., Eds.), pp. 108 –112, Academic Press, London. 58. Mankin, H. J., and Lippiello, L. (1970) J. Bone Joint Surg. 52A, 424 – 434. 59. Eronen, I., Videman, T., Friman, C., and Michelsson, J. E. (1978) Acta Orthop. Scand. 49, 329 –334. 60. Rizkalla, G., Reiner, A., Bogoch, E., and Poole, A. R. (1992) J. Clin. Invest. 90, 2268 –2277. 61. Wiebkin, O., and Muir, H. (1973) FEBS Lett. 37, 42– 46. 62. Morales, T. I., and Roberts, A. B. (1988) J. Biol. Chem. 263, 12828 –12831.