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The ultrastructural distribution of proteoglycans in normal and rachitic growth cartilage from the mandibular condyle of the rat
A&S oral Biol. Vol. 33, No. 5, pp. 379-381, 1988 Printed in Great Britain. AlI rights reserved
Copyright 0
0003-9969/88 $3.00 + 0.00 1988 Pergamon Press plc
SHORT COMMUNICATION THE ULTRASTRUCTURAL DISTRIBUTION OF PROTEOGLYCANS IN NORMAL AND RACHITIC GROWTH CARTILAGE FROM THE MANDIBULAR CONDYLE OF THE RAT J. APPLETON Electron Microscope Unit, Department of Dental Surgery, School of Dental Surgery, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England, U.K. Summary-The mandibular condylar cartilages from normal and rachitic weanling rats were stained with ruthenium red to demonstrate the distribution of proteoglycan. In the rachitic animal there was an increased concentration of ruthenium-red positive granules in the pericellular region of chondrocytes from the extended hypertrophic zone when compared with the controls. These aggregates may represent unmodified proteoglycan which excludes calcium and thereby prevents matrix calcification.
Low phosphate vitamin D-dependent rickets in the rat is a long-established model for investigation of disturbed patterns of calcification (McCollum et al., 1922). In the rachitic rat there is an increase in the depth of the hypertrophic zone of the epiphyseal growth plate, a fall in the level of serum calcium and reduced cartilage-matrix calcification (Durkin, Healey and Irving, 1971; Morris, 1981). Biochemical investigations show t.hat the matrix of normal growth cartilage contains la.rge proteoglycan aggregates. A marked difference between florid and healing rickets, however, is a large reduction in the ability of proteoglycan to aggregate which suggests that proteoglycan may be degraded with the onset of calcification (Reinholt et al., 1985; Campo and Romano, 1986). I have now examined the distribution of proteoglycan in normal and rachitic growth cartilage as demonstrated by ruthenium-red staining. Ruthenium red and #other cationic dyes are known to prevent extraction of proteoglycan during aqueous fixation and staining (Hunziker, Hermann and Schenk, 1983; Famum Schenk and Wilsman, 1983; Hunziker and Schenk, 1984; Shepard and Mitchell, 1985). Twelve black-and-white male weanling rats were used. The animals were kept in stainless-steel cages in a room deprived of uv. light. They were divided into 2 groups of 6 consistmg of 3 males and 3 females. The animals were between 21 and 25 days old and had been born to dams receiving a laboratory cube diet. One group was maintained for 4 weeks on a phosphate- and vitamin D-deficient diet and the other on a control diet containing calcium, phosphorus and vitamin D. The animals were given distilled de-ionized water ad libitum and fed twice daily in order to avoid starvation-induced calcification within the cartilage (Howell et al., 1968). After 4 weeks the animals were killed and their mandibular condy:lar growth cartilage rapidly removed by bisectin the condylar process. The carti-
lages were fixed for 1 h in 2.5 per cent cacodylatebuffered glutaraldehyde (pH 7.4) containing 0.05 per cent ruthenium red, washed in buffer containing 0.05 per cent ruthenium red and post-fixed in 1 per cent osmium tetroxide containing 0.05 per cent ruthenium red. The cartilages were then routinely processed and embedded in low-viscosity Spurr resin. Thin sections were mounted on copper grids and examined in a Jeol 1OOCX Temscan electron microscope operating at 60 kV. In the control animals there were large proteoglycan granules in the immediate pericellular region and the chondrocyte membrane was closely apposed to the surrounding matrix. The interterritorial matrix contained smaller and evenlydispersed proteoglycan granules (Fig. 1). At the junction of the pericellular matrix with the interterritorial matrix there were parallel, chain-like configurations of proteoglycan granules at right angles to the chondrocyte membrane. There were. small groups of crystallites in the inter-territorial matrix which may initially have been associated with the matrix vesicles sometimes visible in the background (Fig. 1). Calcification proceeded into the inter-territorial matrix and not into the pericellular region containing the larger proteoglycan granules (Fig. 2). In the extended hypertrophic zone of the rachitic animal there was considerable aggregation and increased accumulation of proteoglycan granules in $he pericellular of granules chondrocyte
region (Fig. 3). Sometimes large clumps were associated with depressions of.the membrane (Fig. 4). In the in&-
territorial matrix the proteoglycan granules were not evenly distributed and there was some clumping. Where matrix vesicles were present they contained only small crystallites and the vesicular membrane . . was intact (Fig. 4). Cationic dyes, such as ruthenium red, prevent the aqueous
379
extraction
of proteoglycans
by the
for-
J. APPLETON
380
mation of cross-linkages within and between proREFERENCES teoglycan molecules. Therefore it has been suggested Campo R. D. and Roman0 J. E. 119%) Changes in cartilage that in chondrocytes linkages are produced between proteogly~ans associated with calcification. Cafe. Tiss. anionic compounds in the membrane and proteoInt. 39, 1755184. giycans of the matrix so that in hypertrophic chonDurkin J. F. Healey and Irving J. T. (1971) Comparison and drocytes the membrane remains intact and closely changes in the articular mandibular condylar and growth adherent to the matrix (Hunziker et al., 1983; plate cartilages during the onset and healing of rickets in rats. Archs oral Bid. 16, 689-706. Hunziker and Schenk, 1984). This differs from Fart-mm C. E. and W&man N. J. (1983) Pericellular matrix normal aqueous fixation where the membrane may of growth plate chondrocytes: a study using post~xation be discontinuous and detached from the matrix to with osmium-ferrocyanide. J. Hisfochem. Cyrochem. 31, produce a distinct lacuna (Hunziker and Schenk, 765-775. 1984). This linkage effect was observed in my study Howell D. S., Pita J. C., Marzuez J. F. and Madruga J. E. but additionally, in the rachitic animal, there was a (1968) Partition of calcium phosphate and protein in the significant increase in the concentration and aggrefluid phase aspirated at calcifying sites in epiphyseal gation of ruthenium red-positive, proteoglycan grancartilage. J. din. Invest. 47, 1121-l 132. ules, particularly in the pericellular region of the Hunziker E. B. and Schenk R. K. (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution chondrocytes, when compared with the controls. This and low temperature embedding. II Intercellular matrix was accompanied by an increase in the depth of the ultrastructure-preservation of proteoglycans in their hypertrophic zone and a decrease in matrix native state. J. Cell Biol. 98, 271-282. calcification. It is possible, therefore, that these granHunziker E. B. and Graber W. (1986) Differential extraction ules may represent an accumulation of unmodified of proteoglycans from cartilage tissue matrix compartproteoglycan aggregated so that calcium ions are ments in isotonic buffer salt solutions and commercial excluded and so preventing matrix calcification. This tissue culture media. J. ~~s~oehern. Cyrochem. 34, is well illustrated in Fig. 2 where calcification has 1149-l 153. progressed into the inter-territorial region but not Hunziker E. B., Hermann W. and Schenk R. K. (1983) Ruthenium hexamine trichoride (RHT)-mediated interinto the pericellular region where proteoglycan granaction between plasmalemmal components and periules are relatively large and more closely packed. cellular matrix proteoglycans is responsible for the preserHunziker and Graber (1986) have shown that there vation of chondrocytic plasma membranes in situ during are differences in the chemical behaviour of procartilage fixation. j. Hikochem. Cyrochem. 31, 717-727: teoglycans from pericellular, territorial and interMcCollum E. B.. Simmonds N.. Shiolev P. G. and Park E. territorial compartments, as demonstrated by their A. (1922) Studies on experimentairi~kets. A delicate test differential extraction with various buffers and tissuefor calcium depositing substances. J. biol. Chem. 51, culture media. Proteoglycans may, therefore, be re41-49. garded as regulators of matrix calcification but the Morris D. C. (1981) Ultrastructural localisation of calcium in the condylar cartilage of the rat mandible. PhD thesis, mechanisms by which the turnover of proteoglycans University of Liverpool. is regulated are unknown (Reinholt et al., 1985). Pita J. C., Muller F. J. and Howell D. S. (1979) Structural Several theoretical mechanisms have been proposed changes of sulphated proteoglycans of growth cartilage of including a suggestion that lyzozyme, the activity of rats during endochondral ossification. In: Glyconjugute which is almost doubled in the hypertrophic zone Research (Edited by Jeanlow R. and Gregory J.) Vol. II, 72 h after the induction of healing, produces a breakpp. 743-746. Academic Press, New York. down of large aggregates (Pita, Muller and Howell, Reinholt F. P., Engfeldt B., HeinegHrd D. and Hjerpe A. 1979). (1985). Proteoglycans and glycosaminoglycans of epi-
Acknowledgemenr-The technical assistance Chesters is gratefully acknowledged.
of Mm J.
physeal cartilage in florid and healing low phosphate, vitamin D deficiency rickets. Coil. Relut. Res. 5, 55-64, Shepard N. and Mitchell N. (1985) Ultrastru~tural modifications of proteoglycans coincident with mineralisation in local regions of rat growth plate. J. Bone Surg 6723, 455-w.
Plate 1 Fig. I. The pericellular region of a hypertrophic chondrocyte from a control animal showing large proteoglycan granules associated with the pericelluiar region fp) and smaller granules scattered throughout the territorial (T) and inter-territorial (IT) matrix. Crystallites are associated with vesicular structures. Ruthenium red, x 18,000 Fig. 2. Deeper in the hypertrophic zone of the control cartilage, calcification has extended within the territorial matrix but not into the pericellular region (P). Ruthenium red, x 27,000 Fig. 3. Hypertrophic cartilage from a rachitic animal showing electron-dense pericellular zone (1) and localized membrane accumulations of proteoglycan granules (It). Ruthenium red, x 3,500 Fig. 4. Pericellufar region from hy~rtrophic chondrocyte of a rachitic animal showing a~~ulations of granules in the pericellular region (7). There is no vesicle or matrix calcitication. Ruthenium red, x 18,000
Distribution
of proteoglycans
Plate
1
381
Copyright 0
0003-9969/88 $3.00 + 0.00 1988 Pergamon Press plc
SHORT COMMUNICATION THE ULTRASTRUCTURAL DISTRIBUTION OF PROTEOGLYCANS IN NORMAL AND RACHITIC GROWTH CARTILAGE FROM THE MANDIBULAR CONDYLE OF THE RAT J. APPLETON Electron Microscope Unit, Department of Dental Surgery, School of Dental Surgery, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England, U.K. Summary-The mandibular condylar cartilages from normal and rachitic weanling rats were stained with ruthenium red to demonstrate the distribution of proteoglycan. In the rachitic animal there was an increased concentration of ruthenium-red positive granules in the pericellular region of chondrocytes from the extended hypertrophic zone when compared with the controls. These aggregates may represent unmodified proteoglycan which excludes calcium and thereby prevents matrix calcification.
Low phosphate vitamin D-dependent rickets in the rat is a long-established model for investigation of disturbed patterns of calcification (McCollum et al., 1922). In the rachitic rat there is an increase in the depth of the hypertrophic zone of the epiphyseal growth plate, a fall in the level of serum calcium and reduced cartilage-matrix calcification (Durkin, Healey and Irving, 1971; Morris, 1981). Biochemical investigations show t.hat the matrix of normal growth cartilage contains la.rge proteoglycan aggregates. A marked difference between florid and healing rickets, however, is a large reduction in the ability of proteoglycan to aggregate which suggests that proteoglycan may be degraded with the onset of calcification (Reinholt et al., 1985; Campo and Romano, 1986). I have now examined the distribution of proteoglycan in normal and rachitic growth cartilage as demonstrated by ruthenium-red staining. Ruthenium red and #other cationic dyes are known to prevent extraction of proteoglycan during aqueous fixation and staining (Hunziker, Hermann and Schenk, 1983; Famum Schenk and Wilsman, 1983; Hunziker and Schenk, 1984; Shepard and Mitchell, 1985). Twelve black-and-white male weanling rats were used. The animals were kept in stainless-steel cages in a room deprived of uv. light. They were divided into 2 groups of 6 consistmg of 3 males and 3 females. The animals were between 21 and 25 days old and had been born to dams receiving a laboratory cube diet. One group was maintained for 4 weeks on a phosphate- and vitamin D-deficient diet and the other on a control diet containing calcium, phosphorus and vitamin D. The animals were given distilled de-ionized water ad libitum and fed twice daily in order to avoid starvation-induced calcification within the cartilage (Howell et al., 1968). After 4 weeks the animals were killed and their mandibular condy:lar growth cartilage rapidly removed by bisectin the condylar process. The carti-
lages were fixed for 1 h in 2.5 per cent cacodylatebuffered glutaraldehyde (pH 7.4) containing 0.05 per cent ruthenium red, washed in buffer containing 0.05 per cent ruthenium red and post-fixed in 1 per cent osmium tetroxide containing 0.05 per cent ruthenium red. The cartilages were then routinely processed and embedded in low-viscosity Spurr resin. Thin sections were mounted on copper grids and examined in a Jeol 1OOCX Temscan electron microscope operating at 60 kV. In the control animals there were large proteoglycan granules in the immediate pericellular region and the chondrocyte membrane was closely apposed to the surrounding matrix. The interterritorial matrix contained smaller and evenlydispersed proteoglycan granules (Fig. 1). At the junction of the pericellular matrix with the interterritorial matrix there were parallel, chain-like configurations of proteoglycan granules at right angles to the chondrocyte membrane. There were. small groups of crystallites in the inter-territorial matrix which may initially have been associated with the matrix vesicles sometimes visible in the background (Fig. 1). Calcification proceeded into the inter-territorial matrix and not into the pericellular region containing the larger proteoglycan granules (Fig. 2). In the extended hypertrophic zone of the rachitic animal there was considerable aggregation and increased accumulation of proteoglycan granules in $he pericellular of granules chondrocyte
region (Fig. 3). Sometimes large clumps were associated with depressions of.the membrane (Fig. 4). In the in&-
territorial matrix the proteoglycan granules were not evenly distributed and there was some clumping. Where matrix vesicles were present they contained only small crystallites and the vesicular membrane . . was intact (Fig. 4). Cationic dyes, such as ruthenium red, prevent the aqueous
379
extraction
of proteoglycans
by the
for-
J. APPLETON
380
mation of cross-linkages within and between proREFERENCES teoglycan molecules. Therefore it has been suggested Campo R. D. and Roman0 J. E. 119%) Changes in cartilage that in chondrocytes linkages are produced between proteogly~ans associated with calcification. Cafe. Tiss. anionic compounds in the membrane and proteoInt. 39, 1755184. giycans of the matrix so that in hypertrophic chonDurkin J. F. Healey and Irving J. T. (1971) Comparison and drocytes the membrane remains intact and closely changes in the articular mandibular condylar and growth adherent to the matrix (Hunziker et al., 1983; plate cartilages during the onset and healing of rickets in rats. Archs oral Bid. 16, 689-706. Hunziker and Schenk, 1984). This differs from Fart-mm C. E. and W&man N. J. (1983) Pericellular matrix normal aqueous fixation where the membrane may of growth plate chondrocytes: a study using post~xation be discontinuous and detached from the matrix to with osmium-ferrocyanide. J. Hisfochem. Cyrochem. 31, produce a distinct lacuna (Hunziker and Schenk, 765-775. 1984). This linkage effect was observed in my study Howell D. S., Pita J. C., Marzuez J. F. and Madruga J. E. but additionally, in the rachitic animal, there was a (1968) Partition of calcium phosphate and protein in the significant increase in the concentration and aggrefluid phase aspirated at calcifying sites in epiphyseal gation of ruthenium red-positive, proteoglycan grancartilage. J. din. Invest. 47, 1121-l 132. ules, particularly in the pericellular region of the Hunziker E. B. and Schenk R. K. (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution chondrocytes, when compared with the controls. This and low temperature embedding. II Intercellular matrix was accompanied by an increase in the depth of the ultrastructure-preservation of proteoglycans in their hypertrophic zone and a decrease in matrix native state. J. Cell Biol. 98, 271-282. calcification. It is possible, therefore, that these granHunziker E. B. and Graber W. (1986) Differential extraction ules may represent an accumulation of unmodified of proteoglycans from cartilage tissue matrix compartproteoglycan aggregated so that calcium ions are ments in isotonic buffer salt solutions and commercial excluded and so preventing matrix calcification. This tissue culture media. J. ~~s~oehern. Cyrochem. 34, is well illustrated in Fig. 2 where calcification has 1149-l 153. progressed into the inter-territorial region but not Hunziker E. B., Hermann W. and Schenk R. K. (1983) Ruthenium hexamine trichoride (RHT)-mediated interinto the pericellular region where proteoglycan granaction between plasmalemmal components and periules are relatively large and more closely packed. cellular matrix proteoglycans is responsible for the preserHunziker and Graber (1986) have shown that there vation of chondrocytic plasma membranes in situ during are differences in the chemical behaviour of procartilage fixation. j. Hikochem. Cyrochem. 31, 717-727: teoglycans from pericellular, territorial and interMcCollum E. B.. Simmonds N.. Shiolev P. G. and Park E. territorial compartments, as demonstrated by their A. (1922) Studies on experimentairi~kets. A delicate test differential extraction with various buffers and tissuefor calcium depositing substances. J. biol. Chem. 51, culture media. Proteoglycans may, therefore, be re41-49. garded as regulators of matrix calcification but the Morris D. C. (1981) Ultrastructural localisation of calcium in the condylar cartilage of the rat mandible. PhD thesis, mechanisms by which the turnover of proteoglycans University of Liverpool. is regulated are unknown (Reinholt et al., 1985). Pita J. C., Muller F. J. and Howell D. S. (1979) Structural Several theoretical mechanisms have been proposed changes of sulphated proteoglycans of growth cartilage of including a suggestion that lyzozyme, the activity of rats during endochondral ossification. In: Glyconjugute which is almost doubled in the hypertrophic zone Research (Edited by Jeanlow R. and Gregory J.) Vol. II, 72 h after the induction of healing, produces a breakpp. 743-746. Academic Press, New York. down of large aggregates (Pita, Muller and Howell, Reinholt F. P., Engfeldt B., HeinegHrd D. and Hjerpe A. 1979). (1985). Proteoglycans and glycosaminoglycans of epi-
Acknowledgemenr-The technical assistance Chesters is gratefully acknowledged.
of Mm J.
physeal cartilage in florid and healing low phosphate, vitamin D deficiency rickets. Coil. Relut. Res. 5, 55-64, Shepard N. and Mitchell N. (1985) Ultrastru~tural modifications of proteoglycans coincident with mineralisation in local regions of rat growth plate. J. Bone Surg 6723, 455-w.
Plate 1 Fig. I. The pericellular region of a hypertrophic chondrocyte from a control animal showing large proteoglycan granules associated with the pericelluiar region fp) and smaller granules scattered throughout the territorial (T) and inter-territorial (IT) matrix. Crystallites are associated with vesicular structures. Ruthenium red, x 18,000 Fig. 2. Deeper in the hypertrophic zone of the control cartilage, calcification has extended within the territorial matrix but not into the pericellular region (P). Ruthenium red, x 27,000 Fig. 3. Hypertrophic cartilage from a rachitic animal showing electron-dense pericellular zone (1) and localized membrane accumulations of proteoglycan granules (It). Ruthenium red, x 3,500 Fig. 4. Pericellufar region from hy~rtrophic chondrocyte of a rachitic animal showing a~~ulations of granules in the pericellular region (7). There is no vesicle or matrix calcitication. Ruthenium red, x 18,000
Distribution
of proteoglycans
Plate
1
381