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Demineralization with EDTA by constant replacement.

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Demineralization with EDTA by
Constant Replacement
LEONARD F. BBLANGER,' D. HAROLD COPP
AND MARGARET A. MORTON
Departments of Anatomy and Physiology, Faculty of Medicine, University
of British Columbia, Vancouver, B . C., Canada
A n apparatus for demineralization of hard tissues by constant replaceABSTRACT
ment of a saturated solution of EDTA is described. This method allows for more
rapid demineralization through better Ca chelation. EDTA demineralization preserves
cells and matrix constituents particularly the mucopolysaccharides as demonstrated
by staining, Alpharadiography, phase contrast microscopy and microincineration.
Cold acetone fixation followed by cold EDTA demineralization retains several enzymic
activities.
Organic chelates, particularly the sodium salts of ethelenediamine tetraacetic
acid (EDTA) have been used for demineralization of hard tissues since 1951
(Screebny and Nikiforuk, '51; Hahn and
Reygadas, '51; Birge and Imhoff, '52),
providing better preservation of the tissues
and their staining properties, as compared
to acid decalcifying fluids (Hunter and
Nikiforuk, '54; Mardfin and Jones, '55).
Freiman ('54) ; Schajowicz and Cabrini
('56 and '59); Balogh ('62; '63); Balogh
and Nomura ('64) have shown that this
procedure is compatible with the histochemical identification of a variety of enzymes. However, EDTA demineralization
is generally considered as slow and practically applicable only to small fragments
of bones. In our experience, mechanical
agitation alone did not speed up the procedure as already recognized by Lillie for
acids (Lillie, '54). A procedure involving
mechanical agitation and constant fluid
replacement is presently described.
PROCEDURE
Bones from a large variety of animals
(rats, chicks, rabbits, dogs, horses, sheep,
reindeer) and from various parts of the
skeleton (skull, maxilla, mandible, humerus, tibia, femur, vertebrae) were fixed
for 24 hours or more in buffered aqueous
formaldehyde (Lillie, '54) or in a mixture
of acetic acid-formaldehyde-ethanol ( 1-415 parts, A.F.A.).
In the large animals, slabs of various
sizes but averaging 5-10 mm in thickness,
ANAT REC., 153: 41-48.
were cut with a band saw. The samples of
maxillary and mandibular bones of horses
and reindeer generally included either
molar and premolar teeth. Following fixation and washing, the specimens were
placed in individual loose cheese cloth bags
and transferred to an Erlenmeyer flask
(fig. 1C) containing 2,000 ml of a 10%
(saturated) solution of the disodium salt
of ethylenediamine-tetraacetic acid in distilled or demineralized water (EDTA,
Sequestrene-Nan, Geigy (Canada) Ltd.,
Toronto). Between 30 to 60 specimens
were treated at once in this fashion.
The solution was constantly agitated
and slightly heated (to about 30°C) in the
flask by a magnetic stirrer (fig. 1 D ) . The
solution was also constantly replaced at
the rate of approximately 1 drop per second
from a reservoir (fig. 1A) whose flow was
regulated by a screw clamp (fig. 1 B ) .
Flask C outflowed into the drainage system of the laboratory.2
The specimens have been left in this
constant replacement system until they
could be cut easily with a razor blade.
This operation provided an indication that
the tissue is ready for embedding; it also
allowed trimming of the surfaces which
might have been "burned by the band
saw.
At this stage, the specimens were washed in running water for 30 minutes. They
1 Present address:
Department of Histology and
Embryology, Faculty of Medicine, University of
Ottawa Ottawa, Canada,
ZIt (s important that this outflow be diluted constantly with running water if the drain pipes are
metallic.
41
42
LEONARD F. B ~ L A N G E R , D. HAROLD COPP AND MARGARET A. MORTON
EDTA samples from otherwise treated
bones (table 2 ) were taken and analyzed
for comparisons.
Adjacent specimens of bones were demineralized in 5% nitric acid or formiccitrate as recommended by Davenport
('60 j or in 5% trichloracetic acid as recommended by Langeron ('42).
RESULTS
Decalcification time. With the above
described system, the time required for
demineralization of various types of bones
was as shown on table 1.
TABLE 1
Maximum
Rat
Rabbit
Sheep, horse, reindeer
Sheep, horse, reindeer
Sheep
Sheep, horse, reindeer
Fig. 1 The apparatus for constant replacement demineralization:
A , 20 liters plastic reservoir.
B, Screw clamp.
C, 2 liters Erlenmeyer vacuum filtration flask.
D, magnetic stirrer.
were then routinely embedded in paraffin
or celloidin or readily cut with the freezing microtome for enzyme studies.
Routine stains ( Masson trichrome, toluidine blue, Wright stain, Von Kossa) were
applied to sections of C a 5-20 u. Other
sections were examined unstained by phase
contrast microscopy; others were submitted to microincineration or alpharadiography ( BClanger and Bklanger, '59).
In order to appreciate the calcium chelating efficiency of the above system, samples of the outflow solution were taken at
different time intervals (table 2). From
these, 1.0 ml aliquots were ashed in a n
oven at 800" for eight hours. Demineralized H20, 2.0 ml and N/10 HC1, 1.0 ml
were then added to the ash. A n aliquot of
0 . 2 ml of the dissolved ashes was titrated
according to Copp's method for EDTA titration of calcium (Copp, '63)
tibia
4 days
8 days
talus
8 days
vertebra
skull
(parietal) 16 days
skull
30 days
(frontal)
mandible
(with
30 days
teeth)
tibia and
humerus
(diaphysis) 30 days
By comparison, rat and sheep bones
placed in 2,000 ml of EDTA solution at
room temperature, took at least twice as
long for complete dernineralization.
At 5"C, rat tibiae fixed in acetone, took
six days for complete demineralization
when the solution was changed every two
days. Horse skull and vertebrae under
similar conditions could be sectioned with
the freezing microtome after 14 days but
they still contained some mineral salt.
Slabs of parietal bone from sheep were
demineralized in seven days in 5% nitric
acid and also in 5% trichloracetic acid.
Adjacent specimens in formic-citrate were
still hard after 16 days.
Cu content of EDTA solution. The
groups in table 2 represent various species
and also a different total mass of bones in
each group. However, comparisons within
each group are valid and even some general conclusions are possible.
Demineralization by the continuous replacement process (table 2, group 1) apparently proceeds at a n uneven rate. With
DEMINERALIZATION WITH EDTA
TABLE 2
C a content of EDTA solution
Group 1
Reindeer bones, continuous replacement, 30°C:
mgm C a %
After
1 hour
2 hours
4 hours
8 hours
1 day
2 days
4 days
6 days
8 days
10 days
12 days
14 days
16 days
2.313
3.15
5.19
3.99
4.80
3.99
2.01
2.01
1.17
1.434
0.96
1.278
1.038
Checks
8 days, 1 hour
14 days, 1 hour
0.558
0.318
Group 2
Sheep bones, 2000 ml., changed every seven days,
30°C:
After
mgm %
1 week
2 weeks
3 weeks
4 weeks
3.57
1.63
0.92
0.74
Group 3
Horse bones, 2000 ml., changed every three days,
5OC:
After
4 days
7 days
10 days
14 days
Blank
mgm %
2.40
3.12
1.80
1.11
0.27
Group 4
R u t bones, 2000 ml., changed every two days,
5°C
After
mgm
2 days
4 days
6 days
2.78
3.01
1.94
samples of reindeer parietal bones, the
largest amount of chelation took place during the first two days; the rate of chelation decreased rapidly on days 4 and 6 to
reach a plateau from day 8 to day 16. It
would seem that some calcium was still
being removed even on an hourly rate if
the two checks of group 1 are compared
with the blank in group 3. However the
Von Kossa reaction was negative on sections of specimens demineralized for 16
days.
43
Specimens of sheep parietal bones demineralized at room temperature in two
liters of an EDTA solution renewed at
weekly intervals took twice as long. The
percentage of chelated calcium decreased
rapidly from week to week (table 2 , group
2).
Horse parietal specimens demineralized
under similar circumstances but at 5°C
seemed to yield amounts of chelated calcium comparable to those of group 2 at 7
and 14 days (table 2, group 3 ) .
Rat tibiae were completely demineralized after 6 days at 5"C, as compared to 4
days (table 1 ) for the continuous replacement process at 24°C.
Comparative staining properties of
EDTA and Acid-demineralized sections.
As previously reported (Hunter and Nikiforuk, '54), the cellular constituents of
bone and marrow were remarkably well
preserved even after 30 days in EDTA.
The acids used presently (nitric, trichloracetic) have destroyed or deformed most
of the cells.
Toluidine blue metachromasia (Belanger
and Hartnett, '60) has been remarkably
well preserved in paraffin embedded material following neutral formaldehyde fixation and EDTA demineralization. On the
other hand, specimens demineralized in the
acid solutions presently used, have shown
practically no metachromasia.
The matrix appears to have been disorganized in general after acid demineralization, as compared to that of similar material submitted to EDTA, as judged by the
relative amount and distribution patterns
of the red and green staining material in
the matrix after the Masson trichrome.
The Von Kossa reaction was negative in
all material described above as completely
demineralized (table 1) .
Physical methods. Sections of demineralized bones submitted to alpharadiography (Belanger and Belanger, '59) have
shown the already described pattern of
high peripheral density of trabeculae
(Belanger et al., '63) after EDTA (fig. 2 )
or acid demineralization (fig. 3 ) . The organic material however appeared greatly
disorganized and less dense after the nitric
(fig. 3) or trichloracetic acid treatment.
The midtrabecular areas of osteolytic resorption (B6langer et al.,'63) easily recog-
44
LEONARD F. BSLANGER, D. HAROLD COPP A N D MARGARET A. MORTON
nized after EDTA (fig. 2, R) were difficult
to distinguish from artefacts in the aciddemineralized specimens (fig. 3 ) .
Phase contrast observations of adjacent
unstained sections have c o n k e d the decrease and disorganization of fibrillar material in the trabeculae after acid demineralization.
Microincineration of demineralized sections has also brought further evidence
towards this. It has also shown more
abundant ash deposits at the border of the
trabeculae correponding to the patterns
of alpha density reported above (figs. 2
and 3). This procedure has also revealed
that no salt remained in the tissues which
were described above as totally demineralized (table 1).
Enzyme histochemistry. EDTA demineralization at 5°C for periods of up to 14
days following cold acetone fixation preserves several enzymic mechanisms such
as described by Frieman ('54) and Schajowicz and Cabrini ('56) for alkaline phosphatase, and various other enzymes as described by Balogh ( ' 6 3 ) , Balogh and
Nomura ('64) and also by Belanger et al.
('65).
ACKNOWLEDGMENTS
The authors are indebted to Mrs. L. F.
B6langer for skilled technique, to the Medical Research Council of Canada and to
the Canada Department of Agriculture for
financial support. This project was executed under Contract no. AT( 30-1)-2779
with the United States Atomic Energy
Commission, Division of Biology and Medicine.
LITERATURE CITED
Balogh, K. 1962 Decalcification with Versene
for histochemical studies of enzyme systems.
J. Histochem. and Cytochem., 10: 232-233.
1963 Histochemical study of oxidative enzyme systems i n teeth and peridontal
tissues. J. Dent. Res., 4 2 : 1457-1466.
Balogh, K., and Y. Nomura 1964 A technique
for the demonstration of acetylcholinesterase
activity in the inner ear after decalcification
with EDTA. J. Histochem. and Cytochem., 12:
931-933.
BClanger, L. F., and C. BClanger 1959 Alpharadiography: A simple method for determination
of mass concentration in cells and tissues. J.
Biophys. and Biochem. Cytol., 6: 197-202.
BClanger, L. F., and A. Hartnett 1960 Persistent toluidine blue metachromasia. J. Histochem. and Cytochem., 8: 75.
Belanger, L. F., I. Clark, I,. Krook and C. Gries
1965 Persistent protease activity in bone following acetone fixation and EDTA demineralization. J. Histochem. and Cytochem., 13: 404.
Belanger, L. F., J. Robichon, B. B. Migicovsky,
D. H. Copp and J. Vincent 1963 Resorption
without Osteoclasts (Osteolysis). In: Mechanism of Hard Tissue Dsetruction, A.A.A.S.,
Washington, D. C., 531-556.
Birge, E. A., and C. E. Imhoff 1952 Versenate
as a decalcifying agent for bone. Am. J. Clin.
Pathol., 22: 192-193.
Copp, D. H. 1963 Simple and precise micromethod for EDTA titration of calcium. J. Lab.
Clin. Med., 61: 1029-1037.
Davenport, H. A. 1960 Histological and Histochemical Technics. W. B. Saunders Co..
Philadelphia.
Freiman, D. G. 1954 Organic chelating agent
in the demineralization of bone for histochemical study of Alkaline phosphatase. Am. J.
Clin. Pathol., 24: 227-231.
Hahn, F. L., and F. Reygadas 1951 Demineralization of hard tissues. Science, 114: 461-463.
Hunter, H. A., and G. Nikiforuk 1954 Staining reactions following demineralization of
hard tissues by chelating and other dacalcifying agents. J. Dent. Res., 33: 1 3 6 1 4 4 .
Langeron, M. 1942 Precis de Microscopie.
Ed., Masson et Cie. (Paris).
Lillie, R. D. 1954 Histopathologic Technic and
Practical Histochemistry. McGraw-Hill Co.,
New York.
Mardfin, D. H., and V. E. Jones 1955 Comparison of effects of acid decalcification and
chelation at a neutral pH upon histochemical
staining. J. Dent. Res., (abst.), 34: 711.
Nikiforuk, G., and L. Screebny 1953 Demineralization of hard tissues by organic chelating
agents at neutral pH J. Dent. Res., 32: 859867.
Schajowicz, F., and R. L. Cabrini 1956 Chelating agents or histological and histochemical
decalcifiers. Stain Technology, 31: 129-133.
1959 Histochemical demonstration of
acid phosphatase i n hard tissues. Stain Technology, 34: 59-64.
Screebny, L. M., and G. Nikiforuk 1951 Demineralization of hard tissues by organic chelating
agents. Science, 113: 560.
~
~~
PLATE
PLATE 1
EXPLANATION O F FIGURES
46
2
Alpharadiograph of a 7 p section from an EDTA demineralized portion of the parietal skull of a sheep. X 55. R, area of ojteolytic
resorption.
3
Alpharadiograph of a section adjacent to above but from a specimen
demineralized i n 5% nitric acid. X 55.
DEMINERALIZATION WITH EDTA
Leonard F. Bklanger, D. Harold Copp and Margaret A. Morton
PLATE 1
47
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