8
128 Proc. roy. Soc. Med. Volume 68 February 1975
The Effects of Hydrocortisone on the Mandibular Condylar Cartilage of the Rat by N D Priest Bsc (Department of Oral Anatomy, Royal Dental Hospital ofLondon, School of Dental Surgery, Leicester Square, London WC2H 7LJ)
The object of the present investigation is to study the effect of hydrocortisone on the growing mandibular condylar cartilage of the rat. Hydrocortisone acetate suspension was administered to 23-day-old male wistar rats in daily subcutaneous injections over a period of ten consecutive days in doses of 5, 15 and 50 mg kg-1. The condylar cartilages were examined by conventional histological techniques, electron microscopy and tissue section autoradiography using SH-thymidine (1.0 ,uCi g-1), 3H-proline (2.0 ,Ci g-1) and 35S-sulphate (4.0 jCi g-1).
Growth and Cellular Differentiation At 33 days of age the mandibular condylar cartilage of the rat is composed of two morphologically and physiologically distinct elements, an outer fibrous layer which forms the articular covering of the condyle and an inner layer of growth cartilage. The growth cartilage consists of three cell zones namely the proliferative zone, early hypertrophic zone and hypertrophic zone (Fig 1). Studies using 8H-thymidine have shown that cell proliferation is confined mainly to the proliferative zone and to some adjacent cells in the early hypertrophic zone. Measurement of the proliferative activity of these cells may be taken as an indicator of the growth potential of the cartilage (Blackwood 1966). Cellular proliferation: Control and hydrocortisone-treated rats were sacrificed four hours after a single subcutaneous injection of a1Hthymidine. The distribution of labelled cells within the cartilage was plotted by allocating 14 nominal cell positions of which 10 lay within the proliferative zone and 4 in the immediately adjacent early hypertrophic zone. The positions of labelled cells in the articular zonewere recorded separately (Fig 2). Compared with the control animals a reduction of 29% in the proliferative activity, as measured by the reduction in number of labelled cells, was observed in the cartilages of those animals receiving the 5 mg kg-1 dose of hydrocortisone; reductions of 88% and 98% were observed in those animals receiving the 15 and 50 mg kg-1 doses of hydrocortisone respectively. Cell size: The effects of hydrocortisone on cell size were compared by measuring the diameters of the cells or their lacunm in control and hydro-
Fig 1 Photomicrograph of the 33-day-old mandibular condylar cartilage of a rat, showing the articular zone (az), proliferative zone (pz), early hypertrophic zone (ehz), and hypertrophic zone (hz). Araldite section stained with toluidine blue. x 180
cortisone treated cartilages. Owing to tissue shrinkage during processing the cell diameters were not recorded in standard units of linear measurement but in arbitrary units using an eyepiece graticule at standard magnification. In hydrocortisone treated cartilages there was a tendency towards reduction in cell size. This was particularly marked in the cells of the early hypertrophic zone of the animals receiving the 50 mg kg-' dose of steroid. However, the cells of the hypertrophic zone of these animals did not show an equivalent reduction in size and it was assumed that these cells had undergone hypertrophy before hydrocortisone administration had been commenced (Fig 3). Cell glycogen: The periodic acid Schiff reaction with diastase control was used to identify glycogen within the cells of the cartilage. In the control animals glycogen was absent from the cells of the proliferative zone, but appeared as peripheral deposits in the cells of the early hypertrophic zone and as scattered deposits in the cytoplasm of all hypertrophic chondrocytes. Treatment with hydrocorticone reduced the
9
129
Section of Odontology
amount of glycogen in the cells of the animals receiving 5 and 15 mg kg-" doses of the steroid and in animals receiving the 50 mg kg-1 dose glycogen remained only in the hypertrophic chondrocytes. If glycogen is regarded primarily as a cellular store of carbohydrate matrix precursors the disappearance of glycogen from these cells may effect changes in matrix production.
CONTROL
2121 1
Matrix Production The intercellular matrix of cartilage consists of carbohydrate and protein components. 8Hproline and 35S-sulphate are commonly employed to study the production of these components and in the mandibular condylar cartilage such studies have shown that matrix is produced mostly in the early hypertrophic zone (Oberg 1964, Oberg et al. 1969). These labelled matrix precursors have been used in the present investigation to study the changes in matrix formation caused by the administration of
924 2
10.81
~ ~ ~ 17-17 5mg Kg-'
1722 197
15 mg Kg-'
23 96
hydrocortisone. CONTROL 4hrs.
]
50
6b6523
5-83
738
a.z. p.z.
eh.z.
h.z.
50mgKg'
0/0
INHIBITION 193
0
5mg Kg-' HYDROCORTISONE
50
I
Ul
O
n,-l-ft 15mg
500
o17 5
Kg-'HYDROCORTISONIE
0 L V
50mg Kg-' HYDROCORTISONIIE
50 -
o
0I
1
..-
Articular'
Proliferative
zone
zone
I
Hypertrophic zones
Fig 2 Histograms showing the number and distribution of 8H-thymidine labelled cells in control and hydracortisone treated cartilages
c.h.z.
Fig 3 Histograms showing the mean diameter of cells in the articular zone (az), proliferative zone (pz) early hypertrophic zone (ehz), hypertrophic zone (hz) and calcified hypertrophic zone (chz) ofcontrol and hydrocortisone treated cartilages. The measurements on the vertical axis are quoted in arbitrary units using an eye-piece graticule at standard magnification
Uptake of H-proline: Autoradiographs of the condylar cartilages were prepared from control and hydrocortisone treated animals which had been given a single injection of 3H-proline four hours before sacrifice. In the control animals collagen synthesis as indicated by the uptake of 3H-proline was most marked in the articular, proliferative and early hypertrophic zones of the cartilage. With increasing dosage of hydrocortisone labelling was progressively reduced. In the animals receiving the 50 mg kg-1 dose of hydrocortisone labelling was conspicuous only in the early hypertrophic zone, although some activity was present in other zones. Uptake of 35S-sulphate: In the control animals most 35S-sulphate labelling appeared in the early hypertrophic zone of the cartilage. Labelling of the articular, proliferative and hypertrophic zones was also present but to a lesser extent. In the steroid treated animals there was a general reduction in the labelling observed as the higher dose levels of hydrocortisone were approached. Comparison of 35S-sulphate autoradiographs with the 3H-proline labelled sections suggested
130 Proc. roy. Soc. Med. Volume 68 February 1975 a greater inhibition of 35S-sulphate labelling due to hydrocortisone administration than of the 3H-proline. This would indicate that the hormone causes greater inhibition of sulphated carbohydrate production than of collagen production.
Ultrastructure Hydrocortisone produced little change in the ultrastructure of the cells in the proliferative zone. In the cells of the early hypertrophic zone some of the normal ultrastructural features such as the peripheral glycogen deposits and swollen endoplasmic -reticulum were absent. Cells in cartilages treated with the highest dose of steroid possessed collapsed endoplasmic reticulum and a -reduced volume of both Golgi saccules and vesicles. These changes are consistent with the reduction in the synthetic activity of these cells as demonstrated with 3H-proline and 35Ssulphate autoradiography. In animals receiving 50 mg kg-" of hydrocortisone two different types of cell were observed in the hypertrophic zone. One type was found in the uppermost layer of this zone and was small with few cytoplasmic organelles; the other resembled the normal hypertrophic chondrocyte except that it did not exhibit the degree of degeneration usually associated with such cells. These hypertrophic chondrocytes were surrounded by densely mineralized perilacunar matrix in which up to seven concentric electron dense rings were present. It would appear likely that these rings represent periodic changes in matrix mineralization, possibly related to the individual daily injections of hydrocortisone. It is possible that the larger hypertrophic chondrocytes had differentiated before steroid administration was commenced and have retained their normal ultrastructure. This is explained by the extremely low growth rate of the cartilage in the animals receiving the high doses of hydrocortisone (Fig 2).
Conclusion The foregoing experiments indicate that in the mandibular condylar cartilage of the rat hydrocortisone: reduces cell proliferation, interrupts cell hypertrophy, inhibits cell degeneration, inhibits glycogen accumulation and reduces the production of both the carbohydrate and protein portions of the intercellular matrix. It can be concluded that hydrocortisone inhibits cartilage growth by interference with the growth mechanisms of cell proliferation, cell hypertrophy and matrix production.
10 REFERENCES Blackwood H J J (1966) Archives ofOral Biology 11, 493 Oberg T (1964) Transactions of the Royal Schools of Dentistry (Stockhobn and Omea) 10, 1 Oberg T, Frajers C-M, Friburg U & Lohmander S (1969) Acta odontologica Scandinavica 27,425
Growth of the Cartilages of the Cranial Base: Preliminary Studies on Rattus norvegicus by Graham J Roberts BDS (Department ofChildren's Dentistry, Royal Dental Hospital ofLondon School of Dental Surgery, Leicester Square, London WC2H7LJ) The series of experiments reported here have been carried out as an attempt to elucidate the mechanism of growth of cartilage, and in particular, the synchondroses of the cranial base. For this purpose X-ray cephalometry and histological techniques supported by radio-isotope labelling of the cartilage cells and matrix have been used. Assessment ofPhysiological Age Six methods are used to assess physiological age, namely, chronological age, morphological age, skeletal age, dental age, secondary sex character age, and neurological age (Tanner 1962). As humans exhibit a marked degree of polymorphism the most reliable indicator is a combination of chronological and skeletal age, whereas in the
laboratoryrat, which exhibits little polymorphism, a combination of chronological and morphological age is satisfactory. With laboratory animals such as the rat further standardization can be achieved by limiting genetic and environmental influences. 350
-
300 /,Z ridifction
250
200
.150100
smaLl Litter males
large litter males
50 --
large litter
females
0 10 20 30 40 50 60 70 Acknowledgments: I am grateful to Dr M TIME IN OAYS Meredith Smith for providing the electron micrographs and to Professor H J J Blackwood Fig 1 Graph showing growth rate in relation to litter for supervising this work. size and sex ofRattus norvegicus
80
128 Proc. roy. Soc. Med. Volume 68 February 1975
The Effects of Hydrocortisone on the Mandibular Condylar Cartilage of the Rat by N D Priest Bsc (Department of Oral Anatomy, Royal Dental Hospital ofLondon, School of Dental Surgery, Leicester Square, London WC2H 7LJ)
The object of the present investigation is to study the effect of hydrocortisone on the growing mandibular condylar cartilage of the rat. Hydrocortisone acetate suspension was administered to 23-day-old male wistar rats in daily subcutaneous injections over a period of ten consecutive days in doses of 5, 15 and 50 mg kg-1. The condylar cartilages were examined by conventional histological techniques, electron microscopy and tissue section autoradiography using SH-thymidine (1.0 ,uCi g-1), 3H-proline (2.0 ,Ci g-1) and 35S-sulphate (4.0 jCi g-1).
Growth and Cellular Differentiation At 33 days of age the mandibular condylar cartilage of the rat is composed of two morphologically and physiologically distinct elements, an outer fibrous layer which forms the articular covering of the condyle and an inner layer of growth cartilage. The growth cartilage consists of three cell zones namely the proliferative zone, early hypertrophic zone and hypertrophic zone (Fig 1). Studies using 8H-thymidine have shown that cell proliferation is confined mainly to the proliferative zone and to some adjacent cells in the early hypertrophic zone. Measurement of the proliferative activity of these cells may be taken as an indicator of the growth potential of the cartilage (Blackwood 1966). Cellular proliferation: Control and hydrocortisone-treated rats were sacrificed four hours after a single subcutaneous injection of a1Hthymidine. The distribution of labelled cells within the cartilage was plotted by allocating 14 nominal cell positions of which 10 lay within the proliferative zone and 4 in the immediately adjacent early hypertrophic zone. The positions of labelled cells in the articular zonewere recorded separately (Fig 2). Compared with the control animals a reduction of 29% in the proliferative activity, as measured by the reduction in number of labelled cells, was observed in the cartilages of those animals receiving the 5 mg kg-1 dose of hydrocortisone; reductions of 88% and 98% were observed in those animals receiving the 15 and 50 mg kg-1 doses of hydrocortisone respectively. Cell size: The effects of hydrocortisone on cell size were compared by measuring the diameters of the cells or their lacunm in control and hydro-
Fig 1 Photomicrograph of the 33-day-old mandibular condylar cartilage of a rat, showing the articular zone (az), proliferative zone (pz), early hypertrophic zone (ehz), and hypertrophic zone (hz). Araldite section stained with toluidine blue. x 180
cortisone treated cartilages. Owing to tissue shrinkage during processing the cell diameters were not recorded in standard units of linear measurement but in arbitrary units using an eyepiece graticule at standard magnification. In hydrocortisone treated cartilages there was a tendency towards reduction in cell size. This was particularly marked in the cells of the early hypertrophic zone of the animals receiving the 50 mg kg-' dose of steroid. However, the cells of the hypertrophic zone of these animals did not show an equivalent reduction in size and it was assumed that these cells had undergone hypertrophy before hydrocortisone administration had been commenced (Fig 3). Cell glycogen: The periodic acid Schiff reaction with diastase control was used to identify glycogen within the cells of the cartilage. In the control animals glycogen was absent from the cells of the proliferative zone, but appeared as peripheral deposits in the cells of the early hypertrophic zone and as scattered deposits in the cytoplasm of all hypertrophic chondrocytes. Treatment with hydrocorticone reduced the
9
129
Section of Odontology
amount of glycogen in the cells of the animals receiving 5 and 15 mg kg-" doses of the steroid and in animals receiving the 50 mg kg-1 dose glycogen remained only in the hypertrophic chondrocytes. If glycogen is regarded primarily as a cellular store of carbohydrate matrix precursors the disappearance of glycogen from these cells may effect changes in matrix production.
CONTROL
2121 1
Matrix Production The intercellular matrix of cartilage consists of carbohydrate and protein components. 8Hproline and 35S-sulphate are commonly employed to study the production of these components and in the mandibular condylar cartilage such studies have shown that matrix is produced mostly in the early hypertrophic zone (Oberg 1964, Oberg et al. 1969). These labelled matrix precursors have been used in the present investigation to study the changes in matrix formation caused by the administration of
924 2
10.81
~ ~ ~ 17-17 5mg Kg-'
1722 197
15 mg Kg-'
23 96
hydrocortisone. CONTROL 4hrs.
]
50
6b6523
5-83
738
a.z. p.z.
eh.z.
h.z.
50mgKg'
0/0
INHIBITION 193
0
5mg Kg-' HYDROCORTISONE
50
I
Ul
O
n,-l-ft 15mg
500
o17 5
Kg-'HYDROCORTISONIE
0 L V
50mg Kg-' HYDROCORTISONIIE
50 -
o
0I
1
..-
Articular'
Proliferative
zone
zone
I
Hypertrophic zones
Fig 2 Histograms showing the number and distribution of 8H-thymidine labelled cells in control and hydracortisone treated cartilages
c.h.z.
Fig 3 Histograms showing the mean diameter of cells in the articular zone (az), proliferative zone (pz) early hypertrophic zone (ehz), hypertrophic zone (hz) and calcified hypertrophic zone (chz) ofcontrol and hydrocortisone treated cartilages. The measurements on the vertical axis are quoted in arbitrary units using an eye-piece graticule at standard magnification
Uptake of H-proline: Autoradiographs of the condylar cartilages were prepared from control and hydrocortisone treated animals which had been given a single injection of 3H-proline four hours before sacrifice. In the control animals collagen synthesis as indicated by the uptake of 3H-proline was most marked in the articular, proliferative and early hypertrophic zones of the cartilage. With increasing dosage of hydrocortisone labelling was progressively reduced. In the animals receiving the 50 mg kg-1 dose of hydrocortisone labelling was conspicuous only in the early hypertrophic zone, although some activity was present in other zones. Uptake of 35S-sulphate: In the control animals most 35S-sulphate labelling appeared in the early hypertrophic zone of the cartilage. Labelling of the articular, proliferative and hypertrophic zones was also present but to a lesser extent. In the steroid treated animals there was a general reduction in the labelling observed as the higher dose levels of hydrocortisone were approached. Comparison of 35S-sulphate autoradiographs with the 3H-proline labelled sections suggested
130 Proc. roy. Soc. Med. Volume 68 February 1975 a greater inhibition of 35S-sulphate labelling due to hydrocortisone administration than of the 3H-proline. This would indicate that the hormone causes greater inhibition of sulphated carbohydrate production than of collagen production.
Ultrastructure Hydrocortisone produced little change in the ultrastructure of the cells in the proliferative zone. In the cells of the early hypertrophic zone some of the normal ultrastructural features such as the peripheral glycogen deposits and swollen endoplasmic -reticulum were absent. Cells in cartilages treated with the highest dose of steroid possessed collapsed endoplasmic reticulum and a -reduced volume of both Golgi saccules and vesicles. These changes are consistent with the reduction in the synthetic activity of these cells as demonstrated with 3H-proline and 35Ssulphate autoradiography. In animals receiving 50 mg kg-" of hydrocortisone two different types of cell were observed in the hypertrophic zone. One type was found in the uppermost layer of this zone and was small with few cytoplasmic organelles; the other resembled the normal hypertrophic chondrocyte except that it did not exhibit the degree of degeneration usually associated with such cells. These hypertrophic chondrocytes were surrounded by densely mineralized perilacunar matrix in which up to seven concentric electron dense rings were present. It would appear likely that these rings represent periodic changes in matrix mineralization, possibly related to the individual daily injections of hydrocortisone. It is possible that the larger hypertrophic chondrocytes had differentiated before steroid administration was commenced and have retained their normal ultrastructure. This is explained by the extremely low growth rate of the cartilage in the animals receiving the high doses of hydrocortisone (Fig 2).
Conclusion The foregoing experiments indicate that in the mandibular condylar cartilage of the rat hydrocortisone: reduces cell proliferation, interrupts cell hypertrophy, inhibits cell degeneration, inhibits glycogen accumulation and reduces the production of both the carbohydrate and protein portions of the intercellular matrix. It can be concluded that hydrocortisone inhibits cartilage growth by interference with the growth mechanisms of cell proliferation, cell hypertrophy and matrix production.
10 REFERENCES Blackwood H J J (1966) Archives ofOral Biology 11, 493 Oberg T (1964) Transactions of the Royal Schools of Dentistry (Stockhobn and Omea) 10, 1 Oberg T, Frajers C-M, Friburg U & Lohmander S (1969) Acta odontologica Scandinavica 27,425
Growth of the Cartilages of the Cranial Base: Preliminary Studies on Rattus norvegicus by Graham J Roberts BDS (Department ofChildren's Dentistry, Royal Dental Hospital ofLondon School of Dental Surgery, Leicester Square, London WC2H7LJ) The series of experiments reported here have been carried out as an attempt to elucidate the mechanism of growth of cartilage, and in particular, the synchondroses of the cranial base. For this purpose X-ray cephalometry and histological techniques supported by radio-isotope labelling of the cartilage cells and matrix have been used. Assessment ofPhysiological Age Six methods are used to assess physiological age, namely, chronological age, morphological age, skeletal age, dental age, secondary sex character age, and neurological age (Tanner 1962). As humans exhibit a marked degree of polymorphism the most reliable indicator is a combination of chronological and skeletal age, whereas in the
laboratoryrat, which exhibits little polymorphism, a combination of chronological and morphological age is satisfactory. With laboratory animals such as the rat further standardization can be achieved by limiting genetic and environmental influences. 350
-
300 /,Z ridifction
250
200
.150100
smaLl Litter males
large litter males
50 --
large litter
females
0 10 20 30 40 50 60 70 Acknowledgments: I am grateful to Dr M TIME IN OAYS Meredith Smith for providing the electron micrographs and to Professor H J J Blackwood Fig 1 Graph showing growth rate in relation to litter for supervising this work. size and sex ofRattus norvegicus
80
