16. A C T I O N S OF H O R M O N E S
599
Perisutti and Brand (1949) and which may also be present in embryo
extract.
Although these results demonstrated that insulin causes abnormal
development of bone rudiments it seemed essential to make certain
that this effect is due to insulin itself and not to the hyperglycaemic
factor (HGF) which may be present even in highly purified insulin
preparations. T o clarify this point, Chen compared the effect of HGFfree insulin with that of HGF itself and found that the HGF-free
insulin produced the typical abnormalities, while rudiments grown with
HGF alone grew and differentiated normally.
Chen's findings were confirmed by Hay (1958) and Zwilling (1959).
Hay investigated the effect of insulin on cultures of femora and tibiae
from 6-8-day chick embryos grown in natural medium without embryo
extract. In contrast to the immediate growth inhibition described
by Chen (1954) the growth in length of the rudiments was stimulated
shortly after the addition of insulin but retarded in the second half of
a culture period, which lasted altogether 8 days. On the second day
of growth the femora were 6-3 % longer than the control rudiments
but on the eighth day they were shorter by 8-8°/o. After 4-6 days
in vitro the treated bones were broader and showed larger epiphyses
than the controls, and some femora and all tibiae became bent and
distorted, a result identical with that obtained by Chen. The surrounding soft tissue was also more abundant than that of the controls.
Periodic acid-Schiff (PAS) staining showed a deposition of glycogen
in the hypertrophic zone ofthe cartilage which was absent in the control
explants and the cells in this region were larger than the cells in comparable areas of the controls. However, the zone of flattened cells was
less well defined and contained less glycogen than in the control bones.
There was a marked increase in wet (28%) and dry weight (17°/o) in
insulin-treated explants over that of their controls. The total nitrogen
content of the treated bones was increased, but if expressed as a percentage of weight there was no significant difference between control
and treated cultures.
Zwilling (1959) studied the effect of the hormone on identical
material in vivo and in vitro. Tibiae from 6-8-day chick embryos injected with insulin on day 5 were explanted in normal medium and
tibiae from normal 6-day embryos were grown in the presence of insulin.
The chief effects of the hormone in vivo were a marked degeneration of
the chondrogenic tissue in the centre of the epiphysis and a disturbance
of the hypertrophic cartilage and of matrix formation in the shaft.
In the bones cultivated in the presence of insulin there was little degeneration at the ends, which were considerably enlarged and filled with
small-celled cartilage.
599
Perisutti and Brand (1949) and which may also be present in embryo
extract.
Although these results demonstrated that insulin causes abnormal
development of bone rudiments it seemed essential to make certain
that this effect is due to insulin itself and not to the hyperglycaemic
factor (HGF) which may be present even in highly purified insulin
preparations. T o clarify this point, Chen compared the effect of HGFfree insulin with that of HGF itself and found that the HGF-free
insulin produced the typical abnormalities, while rudiments grown with
HGF alone grew and differentiated normally.
Chen's findings were confirmed by Hay (1958) and Zwilling (1959).
Hay investigated the effect of insulin on cultures of femora and tibiae
from 6-8-day chick embryos grown in natural medium without embryo
extract. In contrast to the immediate growth inhibition described
by Chen (1954) the growth in length of the rudiments was stimulated
shortly after the addition of insulin but retarded in the second half of
a culture period, which lasted altogether 8 days. On the second day
of growth the femora were 6-3 % longer than the control rudiments
but on the eighth day they were shorter by 8-8°/o. After 4-6 days
in vitro the treated bones were broader and showed larger epiphyses
than the controls, and some femora and all tibiae became bent and
distorted, a result identical with that obtained by Chen. The surrounding soft tissue was also more abundant than that of the controls.
Periodic acid-Schiff (PAS) staining showed a deposition of glycogen
in the hypertrophic zone ofthe cartilage which was absent in the control
explants and the cells in this region were larger than the cells in comparable areas of the controls. However, the zone of flattened cells was
less well defined and contained less glycogen than in the control bones.
There was a marked increase in wet (28%) and dry weight (17°/o) in
insulin-treated explants over that of their controls. The total nitrogen
content of the treated bones was increased, but if expressed as a percentage of weight there was no significant difference between control
and treated cultures.
Zwilling (1959) studied the effect of the hormone on identical
material in vivo and in vitro. Tibiae from 6-8-day chick embryos injected with insulin on day 5 were explanted in normal medium and
tibiae from normal 6-day embryos were grown in the presence of insulin.
The chief effects of the hormone in vivo were a marked degeneration of
the chondrogenic tissue in the centre of the epiphysis and a disturbance
of the hypertrophic cartilage and of matrix formation in the shaft.
In the bones cultivated in the presence of insulin there was little degeneration at the ends, which were considerably enlarged and filled with
small-celled cartilage.