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The demonstration of centers of osteoblastic activity by use of vital dyes of the benzidene series.

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THE DEMONSTRATION OF CENTERS OF OSTEOBLASTIC ACTIVITY BY USE OF VITAL DYES
OF THE BENZIDENE SERIES
P. G. SHIPLEY
Fi
A
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C.
D C . hIhCI<LIS
om the Dcpoi tment of d n a t o n i ? ~.Johns
,
Hopkinc Univei ~ i / y Ilnltim
,
v e , lltl.
The sites of osteoblastic activity have hitherto been studictl in two
ways: by use of microscopic, sections, and by the examination of gros?
specimens in which the areasshowing bone formation have been revealed by rnakirig the surrounding tissues transparent. Of the socalled ‘clearing methods,’ the best known and most used are those of
Schultzc arid Spaltcholz, the first of which depends for its success on the
relativc opacity of accumulations of calcium salts, and the latter on
the post iriortem staining of bone by solutions of alizarin. Therefore
they are deficient since they shed no light on the beginning of the ossifi(~atioriprocess, and they (30 not make visible the areas in which it is
t:tking place until extensive calcification of thc locus has occurred.
If mi zzo dyc (c.g., trypan blue) he atitninisterc4 t o a very young
:ttiitiinl the k)ones are stained quickly arid very intensely with the vital
color. The staining is so rapid and sclective that thc growing osscoiis
system is colored blue black throughout its entire extent, whereas the
organs (the liver, spleen, etc.), which are so deeply staiiied :tfter the
same treatment in the norrnal adult, are in thvse young animals in a
grcat nieasure excliitletl from participation in the storage of the dyc.
The bones, if tlisscctctl out arid cleared, show distinct epiphyses and
apophysrs sharply outlined within their cartilaginous capsules, and
separated by intervening cartilage from the diaphyses. In preparations of this kind, the various cerit,ers of osscous developnicnt may he
readily seen and studied.
The method pursued to attain this end is hiefly as follows: Young
animals are given a single large dose of a sterile 1 per cent solution of
trypan blue. The site of election for the exhibition of the dye is the
peritoneal cavity, because the superficial veins are very tiny, and the
subcutaneous injection of large amounts of fluid favors pressure necro .
sis. That the different stages in the process of ossification may he as
sharply cut as possible, it is best t o administer a large quantity of tryp a n blue solution a t once, rather than t o give the same amount of dye
in dividcd doses. Concentrated solutions should be given to avoid the
mechanical difficulties involved in the administration of large quantities of fluid. The animal is sacrificed forty-eight hours after staining
and the tissues are fixed by injection of a 10 per cent solution of neu597
598
P. G. SHIPLEY AND C . C. MACXLIN
t r d formalin through the hlootl vessels, followed by immersion in t,hc
sanw solution for a period of from tweiity-four to forty-eight hours.
Tlic bones are then washed thoroughly, and hardened in ascending
grades of alcohol, after which the soft parts are dissected away. Thc
boncs are now dehydrated in absolute alcohol, and, after immersion in
henzol for twenty-four h o u d , are cleared in oil of wintergreen according to the method of Spalteholz. Oil of wintergreen is an excellent clenring agent and causes little or no shrinkage in the tissues
even after long immersion in the fluid. These preparations are cotivenicntly studied with the aid of the dissecting or binocular microscope.
Bones which have been tlecalcificd previous t o clearing may be used
for study in the sanic way, and d rward embcddcd in paraffin directly
from oil of wintclrgl ccn and sectioned; or bones previously cleared withOUL dedcification may be retmnrd through absolute to 95 per cent
alcohol, decal(4k.l and redehytliated, embedded and cut.
In hones which have been decalcified before clearing, the osteogciictic foci are as distinct though not as opaque as in bones which have
iiot bceii treated with acid. ?‘he loss in opacity is due t o the cxtractioii
of a sinall aniount of &in which goes hand in hand with the solution
of the calcium salts. I t is sonietiirnes of advantage in these specirnens t o
introduce as :L contrast a very li7ht eosin stain.
The pictures seen in examining these bones are very beautiful. The
cyiphyscal and apophyseal centers, colored deep blue, are seen em1 ccltlctl in their transparent cartilaginous matrices, separated from the
growing end of the diaphysis by a disc of modified cartilage which is
readily recognized by the striatelcl appearance which is given it hy thc
:trrangenient of its cells in parallel columns. The growing ends of the
(liaphysis are much more darklg stained than the remainder of the
]_)one’sshaft; this phenomenon can be readily seen in the advancing
ventral ends of the ribs and in thc ends of the digital diaphyses adjacent
t o the epiphyses.
I t may be well to emphasize here the fact that stained areas in these
hones are not necessarily the so-called primary ossification centers.
The stain is deposited in the sites, where, at the t h e of the exhibition
of the t1yc1, active ossification is in progress, and tliesc loci of osteo11I:istic activity may or niay not be identical with the ossification ccnter
o f thc. tiorics or epiphysis under ohservation. Nowhere is the difference
hetween the primary ossification center and the focus of active growth
I)rouglit so iharply to the attention of the observer as in the membrane
t)oncs of thcslcull. If oneof these bones isrxamined, i t is apparent even
to t h e iinaidrtl eye that the vital stain is deposited in a heavy line along
the rapidly growing edge of the bone, while the primary ossification
wntcrs are sceri t o be alrriost entirely without color.
Thc unique feature of this method is that it demonstrates clearly the
tliffercnt ossification areas even before the process of deposition of calcium salts has occurred in any marked degree. I n centers where, by
the older methods, the new bone is almost or quite invisible (on account
CENTERS O F OSTEOBLASTIC ACTIVITY
599
of the slight amount of calcium salt present) this method brings out the
locus of ossific activity very distinctly.
A large field for the application of this method is suggested by the
appearance of the bones which have been examined. In the skull, for
instance, graphic demonstrations of the growth of membrane bones and
closure of the sutures are afforded. I n the base of the skull the various
anlagen are easily separated through the sharpness of the contrast between the stained new bone and the intervening masses of cartilage. In
the long bones, the epiphyses and apophyses stand out with diagrammatic clearness.
The physiological and histological aspects of the vital staining of
bone with trypan blue are now being considered, and will be published
in the near future.
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dyes, series, demonstration, vita, activity, osteoblastic, use, center, benzidene
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