The response of cartilage and bone of the newborn guinea pig to stimulation by various hormones (anterior hypophyseal extract estrogen thyroxin).код для вставкиСкачать
TTIE RESPOKSE O F C’ARTITAGE AND BONE O F T H E NEWBORN GUIXEA P I G TO STIMULBTIOh’ BY V-4RIOUS HORRIONES (ANTERIOR HYPOPHYSICAL EXTRACT, ESTROGEN, THYROXIN)’ MARTIN STIJBERRERG AND RUTH SILBERBERG of 8eaearc.l~ Putliology, Oscar J o h n s o n Institute, Wnshingtoir F n w c r s i t y School of Merlirinc, S t . Louis, N is s our i Laboratory T W O FIGURES In a previous paper we have shown the age factor to he significant for the mode of reaction of the cartilage of the guinea. pig to the administration of anterior liypophyseal extract of cattle (Silbcrberg and Silberberg, ’39). Tlie g r o ~ t h proiiiotiiig effect of the extract is mo1.e pronounced in immature guinea pigs than in young mature aiiiiiials, and more so in the latter than in old mature ones. On the other hand, the tendency of the cartilage to undergo retrogressive changes under the influeiicc of the extract iiicreases with advancing age of the guinea pig. Consequently, two questions arose : (1) as to whether the growtli-promoting ef’fect of the extract would be still greater in newborn aiiimals ; (2) as to whether R similar role of the age factor in the response of tlie cartilwgc to the action of other hormones could be established. MATERIAL AND METHODS The material for this investigation was secured from experiirieiits of Dr. Leo Loeb, who suggested that we make use of tlie These investigations mere carried out with the aid of grants from t h e Comiriittee on Scientific Research of the American Medical Association, from the Iiiteriiational Cancer Research Foundation, a n d from the Committee on Research i n Endocrinology of the National Resrarch Council. We are indebted to Dr. Erivin Schwreiik of the Schering Corporation for the Pi,ogynoii-B used in these experiments. 549 550 MABTlN SILBBRBERG A N D RUTH SZLBEBBERG bones of animals which had served for other experimental purposes. Twenty-two guinea pigs, approximately 1 week old and were used. Five animals received weighing from 93 to 123 gm., daily intraperitoneal injections of bovine anterior hypophyseal extract, one each €or periods of 15, 19 and 24 days, and two for 28 days, the dose rising from 0.5 cc. to 2 cc. with increasing weight. Four animals were injected with an oily solution of estrogen, two receiving 50 rat units once weekly for 3 and 4 weeks, and two receiving 100 rat units of this substance for periods of 24 and 5 weeks. Five additional guinea pigs rcceived daily subcutaneous injections of 0.05 to 0.5 cc. thyroxin ; two of these animals were examined after 9, and one each after 10,16, and 28 days, respectively. Eight animals of corresportding weights served as normal controls. The upper end of the tibia was used in the microscopic study of the effect of these hormones. Concerning the technical procedure employed, we refer to former publications ( Silberberg and Silberberg, '38). OBSERVATIOKS After irijections of aiiterior hypophyseal extract, the newborn animals gained weight steadily and more quickly than non-injected control guinea pigs and in contradistinction to the older guinea pigs, which lost weight during the first weeks of treatment with the extract. During the first 10 days of administration of estrogen the weight of the animals increased less than that of the controls, but after 4 weeks, no great differences in the weight of the two groups were noted. Treatment with thyroxin caused a loss or lack of gain in weight f o r a period of about 1week; then a satisfactory gain took place, provided the animal survived the first period of loss in weight. MTCROSCOPIC EXAMINATTO?i I . E f e c t s of pituitary extract The epiphyseal disks were enlarged in all cases. The cell count in the epiphyseal cartilage could be as high as eighteen HOILMONES ON CAIlTlLAGE O F GUINEA P I G S 551 columnar and five hypertrophic cells in one row of cartilage cells, instead of the normal figures of ten columnar and four hypertrophic cells. The slight swelling of the cartilaginous iiiatrix seen after 15 days of injections, increased at later stages; but only small areas were thus affected and marked retrogressive changes were lacking. Replacement of such swollen areas by bone could not be found. The so-called restin? cartilage cells were hyperplastic and hypertrophic. The columnar cells were well preserved and had proliferated noticeably by way of mitosis, especially in places, where the adjacent ground substance was loosened. The arrangement of the cells within a single row was regular and the conversion of the columnar cartilage cells into those of the hypertrophic type proceeded regularly. At the periphery of the hypertrophic cartilage cells an increased amount of calcium was laid down, the cells were readily opened by capillaries, and the demarcation of the layer of the hypertrophic cartilage toward the bone marrow was always sharp. A proliferation of the cells of the cartilaginous covering of the joint went hand in hand with the growth processes in the epiphyseal zones. The cells of the sliding., transitional and pressure zones underwent hypertrophy and multiplied, partly mitotically (fig. l).2Mitoses were also found very close t o the surface of the joint. The maximum of proliferation was seen during the third week of administration of the hormoric ; but after 4 weeks of treatment, proliferation was still markedly stimulated. As a rule, degeneration of cells did not occur. The osseous border lamella, which delimits the cartilao.' +inom covering from the bone marrow, was enlarged and had partlv replaced the zone of hypertrophic cartilaqc. I n the subchondral tissues of both epiphysis and metaphysis iriaturc bony spiculae were more nunierous than are commonlv found ; they were surrounded by proliferatinq epithelioid cells which acted as osteoblasts, or which coalesced and formed osteoclastic giant cells. However, peritrabecular apposition predominated over the processes of resorption ; thus, the We are indebtrd t o MY. S. J . Hayward for the photomicrographs. 552 MAILTIN SlLBEILBERG bN1) RUTH SILBERRERG spiculae bccaiiie tliickcr than they usually arc. The periosteal bone formation took a corresponding course, the cortex being thicker and smoother than normally. Fig. 1 Section through the joint surface of the upper tibia of a fcmale guinea pig which had been injected daily with anterior hypophyseal extract of cattle for 19 days. The nninial was 6 days old and weighed 107 gm. at tlic beginning of the experiment. The final woiglit was 205 gm. Note a mitosis in each of the transitional and pressure zones of the cartilage. Magnification X 500. Fig. 2 Section through the joiut surfaec of the upper tibia of a female guinea pig which had received daily injcctions of thyroxin for 16 days. The animal was 7 days old and weighed 116 gm. at the beginning of the treatment. The final weight was 190 gm. Note the mitotic division of a cell of thc sliding zone close to the surface. Magilification X 500. II. E f d s of estrogem Administration of estrogen produced after 3 weeks a narrowing, and after 4 weeks and longer, an enlargement of the growth zones. During the stage of greatest narrowing the cell count had fallen to two hypertrophic and fire columnar carti- IIOIlMONES ON CARTILAGE O F GUINEA PIGS 553 lage cells in one row; at the time of the enlargement of the epiphyseal zone the columnar cells could be as numerous as eighteen, while the number of hypertrophic cells was normal (four cells). After 3 weeks of administration of the smaller dose of estrogen, and as early as after 16 days following injections of the larger dose, the cartilaginous matrix was dense and more basophilic than ordinarily; but after 4 and 5 weeks, it was swollen and only slightly basophilic, its fibrils being loosened. At the earlier stages the columnar and resting, as well as the hypertrophic cartilage cells were smaller in size than normally, and this condition, together with the reduction in the number of cells, led to a considerable narrowing of the cartilaginous disk. The conversion of the columnar into hypertrophic cartilage was regular. At later stages, however, the cartilage rows increased in length and became even longer than usual. This was due to a marked proliferation of the columnar cartilage cells which frequently underwent initotic division. The “resting” cartilage cells likewise proliferated. Simultaneous, retrogressive changes in the cartilage cells produced in some places a slight localized disintegration of the cartilage cell columns, but on the whole, the arrangement of the columns was regular. Even as late as after 5 weeks administration of the high dose of estrogen, neither hyalinization nor osseous replacement of the areas of retrogressed cartilage could be observed. Corresponding to the changes observed in the epiphpseal plate, the cartilage cells of the joint decreased at first in size and the intercellular matrix became denser and hyalinized. Decreased absorption led to a thickening of the bony border lamella underneath the articular cartilage. Again, as was the case in the epiphpseal zone, a marked proliferation of the cartilage of the joint followed the first stage, which was characterized by atrophy. While the ground substance softened, the cells of the various zones increased in size and multiplied quite noticeably, as indicated by the appearance of mitotic figures. The hypertrophic cartilage cells also regained 554 MARTIN SILBERBERG AND RUTH SILBERBERC: their normal size and they were readily replaced by bone. The capillary activity was increased as compared with the earlier stage, and so was the absorption of bony substance leading to a relative thiniiing of the osseous border lamella. The subchondral layers of both the epiphysis and metaphysis were narrowed a t first, on account of a decrease in the capillary activity. Small epithelioid cells were present in fairly large numbers, whereas, the production of osteoclastic giant cells was less prominent. There was only a limited tendency of the osteoblasts to multiply and to form bone; the osseous spiculae were thinner than normally. However, during the second stage the subchondral zones were richly vascularized and enlarged. Epithelioid cells proliferated freely and deposited more bone. Thus the trabeculae became thicker as compared with the earlier stage. However, even as late as after 5 weeks of injections of 100 rat units of estrogen, the thickness of the trabeculae did not exceed that of trabeculac of nornial guinea pigs of corresponding age. H I . Effects of thyroain I n three cases the epiphyseal zone was narrowed. After 9 and 10 days the cell count in a single cartilage row could be as low as one hypertrophic and six columnar cells. I n two cases, after 16 and 28 days of administration of thyroxin, the number of cartilage cells was normal or slightly above normal, namely, four to five hypertrophic and twelve columnar cells. The columnar arrangement as such was always preserved. I n those animals in which the weight had remained low, the ground substance was dense and contained small o r moderate amounts of calcium, whereas, the matrix was less basophilic and softer and the fibrils loosened if the animals had shown a greater gain in weight. I n the former, the rows of cartilage cells were short and the cells were inore numerous than ordinarily, and the columnar cells proliferated, here and there, by way of mitosis, their conversion into hypertrophic cartilage being greatly accelerated. Retrogressive changes were only rarely found. Simultaneously with the stimulation of growth HORMONES O N CARTILAGE OF GUINEA PIGS 555 processes, the breakdown of the hypertrophic cells and their replacement by bone were intensified, as evidenced by the narrowing of the epiphyseal line, which occurred in spite of the increased proliferation. The hypertrophic cartilage cell layer was sharply demarcated toward the bone marrow. The cells of the sliding, transitional and pressure zones of the articular cartilage were more numerous and not infrequently multiplied mitotically (fig. 2). They enlarged and finally resembled typically hypertrophic cartilage cells, although they were smaller than the latter. The original zone of hypertrophic cartilage was narrowed, ossificd for the greater part and incorporated in the bony border lamella. Thus ossification of the cartilage was accelerated, but simultaneously, increased absorption took place from below by advancing capillaries. Around the trabeculae resorption of bone was likewise increased, owing to the activity of many osteoclastic giant cells which originated from coalescing epithelioid cells. On the other hand, the numerous proliferating epitlielioid cells arranged themselves along the well preserved chondroid processes, remnants of the cartilaginous matrix, and deposited bone around them, building up in this way a great number of osscous trabeculae. Nevertheless, the absorption of bone was so accentuated that in spite of the increased apposition the trabeculae were thinner than normally. Around the bony shaft, increased lacunar and capillary absorption led likewise to a diminution of the osseous tissue. COMMENT I n newborn guinea pigs, injections of pituitary extract of cattle f o r periods of from 35 to 28 days, promote the proliferation and hypertrophy of the cartilage of the epiphyseal growth zones and the joints, but do not cause any appreciable retrogressive changes. The amount of newly produced endochondral and periosteal bone is increased, apposition of osseous tissue being more pronounced than absorption. During the first 3 weeks, treatment with estrogen causes a suppression 556 MAItTIW SILBERBEKG A N D FLUTH SILBERBERG of the growth of the cartilage of the opiphyseal disks and the joints, which is followed, at a later stage, by a strong proliferation of the cartilage cells. Retrogressive changes are insignificant. Trabecular and periosteal bone production is diminished during the first phase, and as compared with t.his phase, it is increased during the second stage. However, deposition of bone exceeding the normal does not occur during 5 weeks of administration of estrogen. Under the influence of thyroxin administered up to 4 weeks, growth and differentiation of the cartilage of the epiphyseal lines and joints are accelerated, and also the endwhondral ossification is enhanced. Resorptive processes acting 011 the bony trabeculae and on the shaft predominate over appositional processes. A comparison between the changes in the skeletal tissues of the newborn guinea pig caused by these various hormones, with those obtained previously in siniilar experiments in older animals, reveals definite differences as to the response of the cartilage in these groups. The newborn guinea pigs treated with pituitary extract showed a greater stimulation of proliferation than did any group of older guinea pigs (Silberberg and Silberberg, '35 and '39) which had received a corresponding dose of this extract. Xoreover, the tendency of the cartilage to undergo degeneration and ossification under the influence of the pituitary extract became more pronounced with advancing age of the animals, whereas, this tendency was practically absent in guinea pigs which were injected with pituitary extract very shortly after birth. I n the guinea pigs which had received estrogen from a very early age, a twofold reaction took place similar to that seen in older immature guinea pigs under the influence of this substance (Silberberg and Silberberg, '39). I n both instances the first response consisted of a suppression of growth, the second one in an enlargement of the epiphyseal zones, even beyond the normal width. In the older immature animals this enlargement was followed by a second narrowing of the epiphpseal zones, caused by a decrease in the number of the various cartilage cells and an increase in the sclerosis and ossification HORMOKES ON CARTILAGE OF GCIREA PIGS 55'7 of the matrix, changes which we interpret as premature ageing of the cartilage. I n the newborn guinea pig, on the other hand, the epiphyseal zone showed a regular configuration and no signs of degeneration, sclerosis or increased ossification, even if estrogen had been administered over periods of t h e and in doses which would have suficed to call forth the above processes in older immature animals. It is probable that a second narrowing of the growth zones and the alterations connected with it would eventually have occurred also in the newborn guinea pigs if the dose of estrogen had been increased or the duration of the treatment extended. Further investigations will have to test the correctness of this assumption and to clarify, in particular, the interaction of hormonal influences and local factors. One of these latter factors may be the inherent growth capacity of the epiphyseal cartilage which decreases with advancing age. Such a decrease of the growth capacity might call forth a still different response of the cartilage if estrogen should be given to mature animals. Thyroxin causes in the newborn guinea pig a stimulation of cartilage growth, differentiation and ageing, inasmuch as the formation of cartilage cells from the resting cartilage, as well as their conversion into hypertrophic cartilage and bone, is accelerated. This is an effect similar t o that exerted by thyroid feeding in older immature guinea pigs (Silberberg and Silberberg, '38). However, proliferation of cartilage and resorption of bony substance were more marked in the younger animals. This may be due to the fact that the dose of thyroid hormone given t o the younger animals was relatively much larger than that given to the older ones. Therefore, at present, the significance of the age factor in the reaction of the cartilage to thyroid hormone remains uncertain. The occurrence of mitotic proliferation even in cells of the uppermost, the sliding zone, of the articular cartilage, which takes place under the influence of certain hormones, may allow some conclusions as to the physiological growth processes in this tissue. Harris ('33) has contended that the cartilage cells of the joint proliferate only in the deeper layers ; subsequently 558 MARTIN SILBERBERG AND R,UTH SILBERBERG they are gradually flattened, pushed toward the surface, and finally detached and dissolved, processes which he compares with those taking place in the epidermis. According to this interpretation one should expect thyroxin, which accelerates the ageing of cartilage, to cause an accelerated flattening and an intensified desquamation of cartilage cells. This, however, could not be observed. On the contrary, we found that the very same cells which, according to Harris, should have, under the stimulation of thyroxin, undergone increased senescence and degeneration, showed a marked proliferative activity, and even mitotic division. It may therefore be concluded that the flat cells of the sliding zone do not represent a senescent type of cells, but that they have the potency t o proliferate, even under conditions which otlierwise accelerate age changes. SUMMARY In newborn guinea pigs, the cartilage has the tendency to respond to various hormonal stiniuli with more proliferative and less retrogressive changes than the cartilage of older animals. This is further evidence in favor of the opinion that the age of the animal on which the hormones act, plays an important role in helping to determine the mode of reaction of the cartilage to the administration of various hormones. LITERATURE CITED HARRIS, 11. A . 1933 Bone growth in health and disease. Oxford University Press, London. SILBERBERG, M. 1935 Effects of extract of cattle anterior pituitary gland on eiidoaliondral ossificatjon in young guinea pigs. Procweedings SOC.Exp. Riol. and Med., vol. 32, p. 1423. SILBERBEBQ, M., AND R. SILBERECERO 1938 Effects of anterior pituitary implants and extracts on epiphyses and joints of immature female guinea pigs. Arch. Path., vol. 26, p. 1208. -_____ 1938 The effects of thyroid feeding on growth processes and retro gressire changes in bone and cartilage of the immature guinea pig. Growth, vol. 2, p. 327. -1939 Action of estrogen on skeletal tissues of immature guinea pigs, Arch. Path., vol. 28, p. 345. -___ 1939 A comparison of the effects of anterior pituitary hormone on skeletal tissues and joints of young and mature guinea pigs. Am. J . Path., vol. 15, p. 542. -__ 1940 Effects of prolonged injections of cattle anterior pituitary extract on bone and cartilage of guinea pigs. Arch. Path., vol. 29, p. 355.