12]
tinuoug
| iterative activity of chondrocytes (percent labeled
proliferative zone cells)—as well as to the inability
Studies
fradiat, §
1., and
blood-
ot the tissue to clacify and undergo bony replacement.
However, the autoradiographic studies with 7HTdR
-.;] to delineate any specific cellular mechanism
TABLE 56.
Group
Division or Rats py Group
“ich, in the absence of deficient calcification, might
. plain why rickets occurs. With time, the proliferative
1
Purina Chow, ad lub.
Purina Chow, pair-fed with
cer 40, # the competent cells to synthesize DNA seems normal.
3
Rachitogenic diet fortified with
phosphate and vitamin D: ad
lib.
Rachitogenic diet, 21 days, ad
lib.
Rachitogenic diet (14 days) and
phosphate (7-10 days)
Rachitogenic diet (14 days) and
vitamin D,» (7-10 days)
Rachitogenic diet (14 days) and
phosphate plus vitamin D,
(7-10 days)
posure, fF
vupacity of chondrocytes decreases, but the ability of
ogy of
ntane. §
340),
Rachitogenic diets enriched with phosphate were more
effective than vitamin D alonein reversing the even-
). Thee tual decline in proliferative potential of rachitic carti-
iduced E
7, 269-5
ity of &
!
jae.
The present
autoradiographic
investigation
was
wanlertaken to determine whether the abnormalities
vtserved during the development of rickets in rats
reflected an altered ability of cartilage and bone cells
4 and§ tu produce the extracellular collagenous components
itional F
vision F
4
5
6
7
| of their respective matrices. We have used *H-pro-
2 heta
.
line as a tracer based upon evidence that it is a selec-
tive marker for collagen formation.{-) The inBone
triecllular pathway involving the Golgi apparatus
F ty which protocollagen is synthesized in chondrocytes
'4< been established by electron microscopic auto-
idiography.4% 1 The export of radioproline from
‘hondroeytes and osteoblasts and its incorporation
into collagen with time has been effectively demon-
‘ular I
lism §
gly- §
ritic fk
ysis f
die- F
~trated by light microscopic autoradiography ,“@!
and by experiments conducted in vitro.¢% This study
Iso permitted a further investigation of the capac-
itv of dietary supplements of phosphate and/or vitanun Des to heal rickets, and the opportunity to
‘valuate the results
from several
other labora-
ones 17 which suggest that collagen formation is
accelerated in rickets.
tter §
MATERIALS AND METHODS
ita- §
tal to that employed previously by Kunin and his
coworkers. 7 8) Rickets was produced in 6 male
weanling Sprague-Dawley rats (40-50 g) utilizing a
high caleium (1.2%), low phosphate (0.1%), vitamin
')-free diet™®) for 21-23 days. For comparison, litterinates were fed either Purina Laboratory Chow (cal‘ium, 1.42%; phosphorus, 0.96%) ad kbitum, or pair-
In
;
rity F
age FE
ely
ate
arral §
The experimental protocol was essentially identi-
ied with this commercial preparation in amounts
consumed daily by the rats on the rachitogenic diet.
‘\ third group was sustained on the basal rachitogenic
‘het which was supplemented with NaH»PQ, (calcium to phosphorus ratio = 1.4:1), and vitamin De
‘10 IU. per gram diet) ad libitum. Free access to de-
“nized distilled water was permitted, and the colony
«a8 maintained under shielded incandescent lighting
‘l constant temperature rooms at 22°C. In order to
Numberof rats
.
.
Dietary regimen
group 4
sacrificed at
21 days
23 days
3
3
3
3
3
3
3
3
3
3
3
3
3
3
-
study the ability of inorganic phosphate and vitamin
D. to heal rickets, 3 groups of 6 rats each were
raised on the basal rachitogenic ration for 14 days.
Subsequently, these animals were maintained for an
additional 7-10 days on diets which were selectively
enriched with either NaHsPO,, vitamin De, or both,
as outlined above. The division of rats by group is
listed in Table 56.
On the 21st day of the experiment, all the animals
were injected intraperitoneally with 2.0 »Ci *H-pro-
line* per gram body weight in 0.1-0.3 ml 0.01 N HCl.
Three rats from each group were sacrificed by cervical
dislocation at 4 hr and 3 days after isotope injection.
These time periods were chosen so that both the
initial pattern of isotope uptake by chondrocytes
and bone cells could be followed, as well as the
subsequent rates of endochondral ossification and for-
mation of new bone by osteoblasts. The tibias re-
moved from the rats at autopsy were fixed in 10%
neutral formalin, decalcified in 10% EDTA (pH 7.47.6), embedded in paraffin and sectioned with the
bones oriented in their long axes on a rotary microtome at 4 «. Slides bearing deparaffinized sections were
autoradiographed by dipping in liquid Kodak NTB-2
emulsion. After a 9-week exposure period in a freezer,
the slides were developed for 5 min in Kodak D-19
{20° C), fixed in acid fixer, washed thoroughly, and
stained through the emulsion with hematoxylin and
eosin.
Autoradiographic Studies
The pattern of silver grains developed in the emul-
sion was examined to determine which cell types in
* L-proline-3,4-"H, New England Nuclear Corporation, Lot
No. 343-207, Specific Activity 5.86 Ci/mM.