AMONG the many factore that control skeletal growth, hormones are known to

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1 Q. Jl exp. Physiol. (1969) 54, THE EFFECTS OF CORTISONE AND SOMATOTROPHIN ON BONE GROWTrH IN RATS. By A. RAMAN. From the Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. (Received for publication 23rd April 1968) The effects of cortisone and growth hormone on bone growth in rats were determined by the use of the tetracycline staining technique. The results obtained showed that both cortisone and growth hormone in a dosage of 2 mg./kg. body weight significantly affected appositional growth rate as well as total bone formation rate (p < -1). Growth hormone produced increased bone formation, but cortisone depressed bone formation. AMONG the many factore that control skeletal growth, hormones are known to play a very important part by influencing the process of bone formation and resorption. Most of our knowledge concerning the effects of hormones on bone growth has been derived from clinical observations and histological studies. However, these studies have been to a large extent qualitative, and until it can be shown quantitatively what precisely the hormones do, there will always be disagreement about the role of hormones on bone growth. The observation that the tetracycline antibiotics are permanently fixed in mineralizing new bones in vivo [Milch et al., 1958] has given us a new and powerful tool in bone growth studies. So in recent years a large amount of quantitative work has been published on various aspects of skeletal physiology using this technique [Frost, 1963; Lee, 1964; Marotti, 1963]. The tetracycline labelling technique has many advantages over other methods used in skeletal studies, such as uptake of radioactive isotopes and radiography. In the dosage given, the drug is harmless and does not affect the growth process. Unlike the radioactive isotopes, tetracycline does not diffuse into the surrounding tissue, and the technique is simple and does not entail elaborate equipment. In view of the scanty information available with regard to quantitative measurements on bone formation and growth under the influence of hormones, it was decided to determine the effects of cortisone and growth hormone on bone formation rates in rats. This paper describes such a study. MATERIALS AND METHODS Male Wistar rats, 6 weeks old and with an average weight of 1 g., were chosen and grouped into three groups of 1 animals each. They were fed rat pellets and water ad libitum. The multiband tetracycline staining technique [Frost et al., 1961] was used to label the bones. Briefly the technique is as follows: on the first day of the experiment, the animals were given drinking water containing 2 g./litre of Terramycin (Pfizer). The water was changed exactly twenty four hours later. Any new bone mineralized during this period would be labelled by the drug. From days 2 to 11 inclusive, no Terramycin was given. This was the control period and during 11

2 12 Raman this period, the control band of new bone was being formed on top of the first labelled band. On the 12th day, Terramycin was again given in the drinking water in the same dosage as before and the drug was removed exactly twenty four hours later. This would produce a second tetracycline label. From 13 to 22 inclusive, the animals in Group B were given daily intraperitoneal injections of cortisone acetate, 2 mg./kg. body weight, while the animals in Group C had intraperitoneal injections of porcine somatotrophin (Sigma), 2 mg./kg. body weight. The animals in Group A which acted as controls were given daily intraperitoneal injections of 1 ml. normal saline ( 9 per cent NaCI). This constituted the experimental period, and during this time the experimental band of bone would be deposited. On the 23rd day, Terramycin was again given in the drinking water for exactly twenty four hours as before. The animals were killed on the 26th day and the femora and tibiae removed, embedded, sectioned and mounted as described elsewhere (Raman, 1966 a and b). The sections were examined under a fluorescent microscope (Leitz). Three rings of tetracycline labelling (fig. 1) were seen in the sections on the periosteal aspect. The distance between the innermost and middle rings is the appositional growth for the control period and the distance between the middle and outermost rings is the appositional growth for the experimental period. Similarly, the areas of bones between the labels would give the area of new bone formed during the control and experimental period respectively. For determining the appositional growth rate, an Okner screw micrometer eyepiece (Leitz) was used. The distances between the midpoints of the tetracycline labels on the periosteal side were measured to the nearest micron. Eight random readings were taken for each section at different points and averaged and the process repeated for all sections from each bone and the mean appositional growth rate (microns per day) calculated as: Distance between tetracycline markers in microns Number of days between markers For measuring area of total new bone formation, photomicrographs of the sections were made and after suitable enlargement these were printed on to glossy paper, the prints were traced on to transparent square millimetre paper and the area of new bone formation measured by counting the squares between the tetracycline labels. According to Mainland [1929], this method is superior to cutting and weighing or planimetry. Each section was measured twice and the process repeated for all sections from each bone and averaged. The mean total new bone formation rate was then calculated as: Area of bone between markers Total area of bone x number of days between markers X RESULTS The results obtained are shown in Tables I and II. DISCUSSION The present study deals with quantitative measurements of appositional growth rate and new bone formation rate in the diaphyses of the femur and tibia on the periosteal aspect only. It has been shown that bone formation on the endosteal aspect and in the metaphyseal regions need not necessarily be governed to the same extent by the factors that affect periosteal growth [Frost et al., 1961]. These areas are therefore not included.

3 FIG. 1. Cross SectioIn of Rat FemrntIF ( x 46) shiow'ing tihe three tetracycline labels. Teil rmy(-cin ad(litmnistered oin three occasions. No hormtionet' treatmvient. [To face page 12

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5 14 Raman With reference to Table I, both cortisone and growth hormone affect skeletal growth considerably. We know from clinical experience that lamellar bone formation is increased in gigantism and acromegaly, while hypofunction of the pituitary leads to dwarfism and diminished bone formation. Bell and Cuthbertson [1941] have shown that administration of anterior pituitary extracts produces heavier and stronger bones in growing TABLE II. AVERAGE BODY WEIGHTS (G.) OVER EXPERIMENTAL PERIOD. Average Weight Average Weight Average Weight at the end of at beginning at the end of Experimental of Experiment Control Period Period Control animals * Cortisone treated animals *6 Somatotrophin treated animals animals. Comparison of the growth hormone treated animals with the control group shows that there is a significant increase in bone formation and appositional growth rates in both tibia and femur (p <1). The results are in agreement with those of Tapp [1966] who showed that bone growth is increased under the influence of growth hormone. Although the growth hormone used in this study was from porcine sources, Papkoff and Li [1962] have stated that the rat is responsive to this growth hormone. With respect to cortisone, it is evident that there is a significant depression of lamellar bone formation (p < 1). While it is generally accepted that adrenal corticoids suppress lamellar bone formation, there is a lot of disagreement as to whether the hormones cause increased bone resorption as well. Working with human material, Frost et al., [1962] and Klein et al. [1965] have shown that the adrenal corticoids regularly inhibit bone resorption. The results obtained in the present study seem to agree with this concept. Labelling at the start of the experiment is not affected at all by the cortisone but remains completely intact. Unlike man, the rat does not develop osteoporosis when cortisone is administered. Nevertheless, if one assumes that in a steady state, the resorption of old bone and formation of new bone are equal in extent, it would follow that osteoporosis would appear if the rate of bone formation is depressed while resorption is also inhibited, although to a lesser degree. The adrenal corticoids probably act in this way on bone growth. The results obtained here are in general agreement with those of Stanisavljevic et al. [1962] and Hulth and Oleurd [1963]. Earlier studies on the effects of hormones on bone growth have been mainly qualitative, and it is felt that by using the tetracycline staining technique, it is possible to compare the growth of different animals under different conditions.

6 Cortisone and Somatotrophin and Bone Growth 15 ACKNOWLEDGMENTS My thanks are due to Professor G. H. Bell of the University of Dundee, Scotland, for suggesting this study and advice, and to the Department of Medical Illustrations, University of Malaya, for the excellent photographic work. REFERENCES BELL, G. H. and CUTHBERTSON, D. P. (1943). J. Endocrinol. 3, FROST, H. M. (1963). Canad. J. Biochem. Physiol. 41, FROST, H. M., ROTH, H., VILLANUEVA, A. R., and STANISAVLJEVIc, S. (1961). Henry Ford Hosp. Bull. 9, FROST, H. M., STANISAVLJEVIC, S., VITINUEVA, A. R., and ROTH, H. (1962). Henry Ford Hosp. Bull. 1, HuLTH, A. and OLERUD, S. (1963). Brit. J. exp. Path. 44, KLEIN, M., VI.LANUEVA, A. R., and FROST, H. M. (1965). Acta orthop. Scand. XXXV, LEE, W. R. (1964). J. Anat. (London) 98, MAINLAND, D. (1929). J. Anat. (London) 63, MARoTTI, G. (1963). Acta Anat. 52, MLcH, R. A., RAT, D. P., and TOBIE, J. E. (1958). J. Bone Jt. Surg. 4A, PAPKOFF, H., and Li, C. H. (1962). In Methods in Hormone Research (R. Dorfman, ed.) vol. II, N. York: Academic Press. RAmA, A. (1966 a). Far East Med. J. 2, RAMAN, A. (1966 b). Acta orthop. Scand. 37, STANISALVJEVIC, S., ROTH, H., VILLANu-EVA, A. R. and FROST, H. M. (1962). Henry Ford Hosp. Bull. 1, TArP, E. (1966). J. Bone Jt. Surg. 48B,