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The response of cartilage and bone of the newborn guinea pig to stimulation by various hormones (anterior hypophyseal extract estrogen thyroxin).

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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.
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