Vol. 47, No. 1
Printed in U.S.A.
T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Copyright © 1967 by The Williams & Wilkins Co.
AN
IMPROVED
STAIN
FOR
FRESH,
MINERALIZED
BONE
SECTIONS
USEFUL IN THE DIAGNOSIS OF CERTAIN METABOLIC DISEASES OF THE BONE
A. R. VILLANUEVA, B.S.
Orthopaedic Research Division, Henry Ford Hospital, Detroit, Michigan
Until recently, little attention was given
to the development of staining methods for
mineralized {i.e., undecalcified) sections of
bone. This may be partially the result of
the small volume of work done with these
sections, and partially the result of the
tendency to embed them in nitrocellulose or
other plastics before sectioning. As a result,
little light microscopic histologic work has
been done with undecalcified tissues.
This paper reports on Osteochrome stain,*
intended for use with fresh, unfixed, unembedded, mineralized bone sections. The stain
is easy to use, and it is available as a single,
prebuffered dye powder that is dissolved in
70% reagent methanol to make a single
solution (pH 7.3). The tinctorial patterns of
the stain are specific, reproducible, and
relatively stable. Its attributes make it
suitable for evaluation of metabolic bone
disease in human biopsy material. The
Osteochrome stain is basically the tetrachrome stain17 previously described, but
modified with respect to preparation and
pH to avoid difficulties that were encountered in the standardization of preparation
and use by other laboratories. In our laboratory, at least, the results of the old and new
methods are the same.
MATERIALS AND METHODS
For the best results, the stain should be
used on fresh, unfixed, hydrated, mineralized
bone sections. If processing of the material
is not done immediately, specimens may be
placed in 95 % ethyl alcohol and temporarily
stored at 4 C. In most instances, satisfactory
results are obtained with tissue preserved in
Received, March 21, 1966.
This investigation was based on work supported
by Grants No. 293, Henry Ford Hospital, and
AM-04186, National Institutes of Health, "United
States Public Health Service.
* Available as Osteochrome from Harleco,
Harlan-Leddon Co., Philadelphia, Pennsylvania.
the refrigerator for several days, although
the histologic features may not be as sharp
as they are with fresh tissue. Fixation in
80 % ethyl alcohol or equal parts of acetone
and ethyl alcohol is permissible. Fixing
agents such as formalin, acids, salts of heavy
metals, or mixtures of these are not recommended.
If the specimen is to be shipped for study
elsewhere, it should be placed in a bottle of
80 % ethyl alcohol.
Composition of the Osteochrome Stain
1. Fast green FCF (C.I. No. 42053;
Certificate No. 10, Matheson, Coleman &
Bell, Norwood, Ohio) 0.25 Gm.; Orange G
(C.I. No. 16230; Cert. No. 13; Harleco,
Hartman Leddon Co., Philadelphia, Pennsylvania) 0.75 Gm.; distilled water, 50.00
ml.; glacial acetic acid (analytic reagent;
F.W. 60.054), 0.60 ml.
2. Methylene blue MX 9S5 (C.I. No.
52015; Cert. No. 40; Matheson, Coleman &
Bell) 0.25 Gm.; sodium bicarbonate (analytic reagent, F.W. S4.01), 0.25 Gm.; distilled water, 40.00 ml.
3. Basic fuchsin (C.I. 43500; Cert. No.
10; Matheson Coleman & Bell), 1.00 Gm.;
distilled water, hot, 50.00 ml.
4. Sodium acetate, crystals (analytic
reagent; F.W. 136.085), 2.72 Gm.; distilled
water, 10.00 ml.
Working solution. Prepare 0.25 C. solution
of the Osteochrome in 70 % reagent methanol.
When most of the crystals are dissolved,
filter the solution; it is then ready to use.
Store in a tightly stoppered, gas-tight, brown
container.
Staining Procedure
Either cut bone sections with a thin
sectioning machine (such as the Gillings
Bronwill semi-automatic, Scientific Products, Evanston, Illinois), or cut them into
slabs with a fine-toothed jeweler's saw.
78
i « 4
I
%
F I G . 1 (upper). Green osteoid seam without peripheral ring. Mineralized cross-section of a human
rib, showing the osteoid seam bracketed by the white bars. Note t h a t the peripheral ring (pr) and the
clear zone (zd) of the osteoid seam are absent. Compare with Figure 2, in which both of these structures
are present. Osteochrome bone stain. X 375.
F I G . 2 (lower). Green osteoid seam with peripheral ring. Fresh, unfixed, hydrated, mineralized,
inicmbedded cross-section of cortex from human rib. The osteoid seam is the light band labeled 0.
Note that there is a clear zone just outside the seam. This is the zone of demarcation (zd). Tetracycline
labeling occurs here. Centrifugal to this zone is a darker band (peripheral ring) which is relatively basophilic (pr), and immediately outside of this is another dark region (IB), which is referred to in this
paper as partially mineralized bone. Observe t h a t some osteocytes are embedded in the osteoid. At the
extreme left are two resorption areas (H). The darkly stained nuclei in the haversian and extrahaversian
bone are osteocytes. Osteochrome bone stain. X 170.
79
so
Vol. 4.7
VILLANTJEVA
Thickness before staining should be 75 to
100 M.2
Wash sections in tap water; then rinse
each in distilled water for several minutes.
Stain in the Osteochrome solution for 90
min. for quick diagnosis or 4S hr. for
complete permeation of the numerous tiny
lacunae usually found in bone. Best results
are obtained after staining for 48 hr.
Regrind both surfaces lightly under running water on used, fine, waterproof sandpaper (grit No. 400) to remove surface
stain. (This step may be omitted if the
sections were ground smoothly at desired
thickness during the first grinding, or if the
thin sectioning machine is used.)
Agitate manually or mechanically for a
few minutes in 0.1 % invert soap (zephiran
chloride) or in 0.01 % of any commercial
household detergent solution, then rinse in
tap water and again thoroughly in distilled
water. (This cleans debris from the surface
of the sections.)
Differentiate with gentle agitation in
0.01 % glacial acetic acid in 95 % alcohol
while periodically observing the color of the
osteoid seams under medium power microscopy. The end of differentiation is marked
by the appearance of a green, blue-green,
or jade green color in some of the seams.
Not all seams turn green; some remain red
or pink. Differentiation requires 3 to 5
min. for the sections stained for 90 min.,
and 20 to 25 min. for those stained for 48
hr. (If differentiation is found to be incomplete in the final mount, return through the
xylol to absolute alcohol, perform the rest of
the procedure, and complete the differentiation.)
Transfer the sections to two changes of
95%
alcohol, for 4 min. each time; then
transfer to absolute alcohol for two changes,
each 3 min. in duration.
Clear for 2 min. in equal parts of absolute
alcohol and xylol; 1 min. in one part of
absolute alcohol and three parts of xylol; 1
min. in one part of absolute alcohol and
nine parts of xylol; and then in two changes
of xylol, for 1 min. in each.
Mount in Harleco's microscopic mounting
medium.
RESULTS
Microscopic Description
Three major types of osteoid seams are '
observed: the green osteoid seams without
peripheral ring; the green osteoid seams
with peripheral ring; and the red osteoid
seams.
Green osteoid seams loithotd peripheral
ring (Fig. 1). The main body of these seams
stains a homogeneous green. They are
observed partially in areas coincident with
resorption, and to some degree are seen
during the late stages of bone mineralization.
Green osteoid seams ivith peripheral ring
(Fig. 2). The main body of these seams
shows two concentric layers, the inner and
outer, that are stained lighter and darker
green, respectively. Adjacent and peripheral
to the outer layer is a pale blue-green layer
(clear zone) which seems to coincide with
the zone of demarcation, 3 ' " • 1 2 , 1 4 , 1 5 > 1 8 or
the transition zone of Loe.13 Bordering the
clear zone is a bright red-orange peripheral
ring (apparently the sudanophilic zone of
Irving, 9 mineralization front of Thomas,16
and phosphate ridge of Johnson10). These
seams are also observed as part of a quadrant where resorption is in progress.
Red osteoid seams (Fig. 3). These are a
homogeneous red. Occasionally, there is
diffuse red staining at the boundary of these
seams, which are basophilic. Some of them
are much thinner than the average normal
FIG. 3 (upper). Red osteoid seam. Cross-section of human rib showing the osteoid seam lying in the
near center of this illustration. It is indicated by the dark circular band and is bracketed by while bars.
Note the diffuse staining at the periphery of the osteoid and no observable clear zone (zd) like that
seen in the green seams of Figure 1. The lower left corner of the figure is a resorption space, with its
Howship's lacuna (H). Osteochrome bone stain. X 210.
FIG. 4 (lower). Longitudinal sections of a human rib, showing the osteoid seam running horizontally
through the middle portion of the field. Note the difference in the depth of printing of the osteoid seam
in this condition. The dark bands (marked li to li') correspond with the red seam; the light bands (marked
R' to (?) correspond with the green seam. These colors alternate along the length of an actively forming
new osteone (haversian system), the distance between transitions varying in a seemingly random manner
from 0.1 to 1.0 mm. Osteoblasts may also be seen lining the inner wall of the osteoid seam, while osteocytes are arranged randomly along the haversian lamella. Osteochrome bone stain. X 175.
Jan. 1967
AN IMPROVED BONE STAIN
I*
*^ii^^*^
" •
^ ' *** ** ^.^-
SI
S2
VILLANUEVA
seam thickness of 7.5 n, and their surface
areas and circumferences are often also
considerably decreased in comparison with
the normal mean.4
Bone of medium mineralization density
stains orange, whereas that of low density
stains red. Periosteum stains blue-green,
and periosteal cells stain red. Sharpey's
fibers are usually red (if poorly mineralized)
or unstained (if highly mineralized). Osteocytes and their canaliculae take the red or
purple stain. Nuclei of osteoblasts and osteoclasts are red or purple and their cytoplasm
is usually faint green, but occasionally pink.
Mast cell granules are red and eosinophile
granules are blue-green. Nuclei of both cells
are stained red. Red blood cells are yellow
to red. The hematopoietic cells in the marrow and fat cells are also clearly demonstrated. Cartilage cells are stained dark
purple to red.
Effect of Age on the Osteochrome Solution
The intensity of color of the green staining osteoid seams decreases in tissue fixed in
alcohol for longer than 1 year. The working
Osteochrome solution should be replaced
after 1 month, not because the stain constituents deteriorate, but because its pH
changes. The stock solution appears to be
chemically and tinctorially stable.
DISCUSSION
In bones from metabolically normal
people,* and from animals such as monkeys,
dogs, cats, rats, and steers, Osteochromestained osteoid seams appear in two major
colors: red and green. When longitudinal
sections are examined microscopically, it is
seen that the red and green colors alternate
along the length of an osteoid seam, the
distance between this transition ranging
from 0.1 mm. to 1.0 mm. (Fig. 4).
The consistency of the tinctorial patterns
of staining in Osteochrome-treated sections
suggests that there are corresponding and
consistent differences in the chemical prop* Representative sample of individuals w i t h o u t
metabolic disease of the bone, chronic illness or
infection, hepatic disease, edema, diabetes
mellitus, congestive heart failure, metastatic
malignancy, x-ray therapy, or t r e a t m e n t with
cytotoxic or hormonal agents.
Vol. 47
erties of the bone matrix in the osteoid
seams. If simple mixing or chance competition (or both) for the dye molecules represents the cause of the biphasic coloration of
seams, then sections stained long enough
should be found to be homogeneously
stained with respect to color. This, however,
is not the case (time range evaluated: 1 hr.
to 7 days). It is consistently observed that
the tissues maintain their initial color
regardless of the length of staining. Some of
the osteoid seams never destain completely,
which indicates considerable stability of the
bone tissue-stain bond. The Osteochrome
tinctorial differences do not seem to correlate with metachromasia with toluidine blue.
At controlled pH, when fresh, unfixed
preparations are used (pH 6.0 to 8.0, range
tested), all seams {both the red and green
staining seams of Osteochrome preparations)
are metachromatic to toluidine blue. The
metachromasia disappears after immersion
of the section in absolute alcohol.
The actual staining mechanism of osteoid
seams by the Osteochrome method is not
certain. Presumably, the staining mechanism is some combination of four general
aspects of the procedure: a differential
stain extraction during the differentiation
step due to dependence of reversibility of
the dye-matrix combination on pH; a differential stain extraction during the differentiation step due to dependence of solubility
of the dyes on the polar nature of dye and
solvent; a differential stain extraction in
those parts of the sections that are mineralized on the basis of relative diffusibility,
in turn related to the molecular weight,
specific ion effects, and hydrated ionic size
of the dyes; and differing isoelectric points
of the various tissue elements, which should
affect their staining with the dyes.
The stain is useful in the diagnosis of
certain types of osteomalacia. 18 Several
sections made from biopsies from patients
with severe osteomalacia have been diagnosed with this stain. In these sections,
there were no diagnostic abnormalities in
routine demineralized, fixed, hematoxylin
and eosin sections made from the same
biopsy specimen.7 The apparent reason for
this is that the lamellar osteoid seam does
not necessarily exhibit unusual staining
Jan. 1967
AN IMPROVED BONE STAIN
FJG. 5. Doublet osteoid seam. Mineralized cross-section of biopsy of a human 11th rib from a case
of vitamin D-resistant rickets, showing the inner, green osteoid seams, marked 0 and 0', and the outer
red seam, marked R at the periphery. The significance of this finding is difficult to interpret, although it
may mean that bone formation is moving unusually slowly, and that these osteoid seams failed to ossify
completely before laying down another osteoid. Note 0', and compare with zd (zone of demarcation)
in Figure 2. The core of the latter has already mineralized and thus appears unstained or very lightly
stained in the illustration, whereas the former is still a wide layer of unmineralized organic matrix that
stains green. Also note R and compare with IB in Figure 2. The only observed difference is that here R
is definitely an osteoid seam that stains red, whereas that of IB denotes partially mineralized bone
that stains pink. At the extreme lower right corner is another red osteoid seam, marked R. Osteochrome
bone stain. X 250.
properties in demineralized, routinely processed bone material. In other types of osteomalacia, and especially in vitamin D-resistant rickets, a large, perilacunar red stain
5
SUITOUIKIS many of the osteocytes.
Its
origin is not completely understood, but
Frost 0 has suggested that a disturbance in
the metabolism of the osteocyte is responsible. Another interesting oddity associated
with these bone disorders is the presence of
a "doublet" osteoid seam (Fig. 5). I t is not
clear just how this seam is formed.
Finally, the idea of a "peripheral ring"
and "clear zone" of the green osteoid seam
is especially interesting because there is a
strong suggestion that these may represent
one of the initial stages in the transformation of the osteoid matrix to bone. In Osteo-
chrome-stained sections from metabolically
normal people, and from 50 people with
known metabolic bone disorders (such as
osteoporosis, osteogenesis imperfecta, thyrotoxicosis, hyperparathyroidism, acromegaly, osteopetrosis, Morquio's disease, and
Paget's disease), these features are prominent; in osteomalacia and vitamin D-resistant rickets, however, they are absent. I t
has not been possible to devise a hypothesis
that would account for these observations.
Further studies are necessary to determine
the nature of this phenomenon.
SUMMARY
An improved bone stain called the Osteochrome has been developed for fresh, unfixed,
unembedded, mineralized bone sections. It is
S4
VILLANUEVA
available as a single prebuffered powder
with a pH comparable with that of blood.
Osteoid seams are differentially stained
and cytologic details are clearly demonstrated. This staining method has proved
to be specific, stable, reliable, and reproducible. It is a valuable aid in the diagnosis
of certain metabolic bone diseases, especially
the osteomalacias.
Acknowledgments. Dr. C. L. Mitchel, Dr. G.
Fine, Dr. D. C. Mitchel, and the late Dr. J. L.
Fleming provided human biopsy material. Dr.
J. R. R.amser, Dr. IT. Duncan, and Mr. Leon
Ilnicki made pertinent suggestions. Dr. IT. M.
Frost helped extensively and made constructive
criticisms.
REFERENCES
1. Duncan, H.: Personal communication.
2. Frost, H. M.: Preparation of thin, undecalciy
fied bone sections by rapid manual method.
Stain Technol., 33: 273-277, 1958.
3. Frost, H. M.: Tetracycline labelling of the
zone of demarcation of osteoid seams.
Canad. J. Biochem. Physiol., 40: 485-489,
1962.
4. Frost, H. M.: Thickness of osteoid seams in
human beings. Clin. Orthop., 25: 175-179,
1962.
5. Frost, H. M.: A unique histological feature of
vitamin D-resistant rickets observed in
four cases. Acta Orthop. Scandinav., 33:
220-226, 1963.
6. Frost, H. M.: Some observations on bone
mineral in a case of vitamin D-resistant
rickets. Henry Ford Hosp. M. Bull., 6:
300-310, 1958.
Vol. 4-7
7. Frost, H. M.: Unpublished observations.
8. Hurxthal, L. M., Dotter, W. E., Clerkin, E. P.,.
and Baylink, D. J.: Two new methods for
the stud}' of osteoporosis and other metabolic bone disease. Lahey Clin. Bull., 13:
155-166, 1964.
9. Irving, J. T.: Histochemioal changes in theearly stages of calcification. Clin. Orthop.,.
17: 92-102, 1960.
10. Johnson, L.: In Frost, H. M.: Bone Biodynamics, Boston: Little, Brown & Company,
1964, pp. 543-654.
11. Lacroix, P., and Ponlot, R.: Remarque sur
1'histopathologic de l'osteoporose posttraumatique. Acta Med. Belg., Suppl.
2: 149-164, 1956.
12. Leblond, C. P., Lacroix, P., Ponlot, R., and
Dhem, A.: Les stades initiaux de l'ostGogenese: nouvelles donniSes histochimiqueet autoradiographiques. Bull. Acad. Roy.
Med. Belg., 24: 421^143, 1959.
13. Loe, H.: Bone tissue formation—a morphological and histochemioal study. Acta Odontol.
Scandinav.. 17: 311-427, 1959.
14. Meyer, P. C : Histological identification of
osteoid tissue. J. Path. Bact., 71: 325-333,
1956.
15. Ponlot, R.: L'interfit du noir Sudan B en
histologic des os. Bull. Micros. Appl., Sr
125-127, 1958.
16. Thomas, W. C.: Comparative studies on bonematrix and osteoid by histochemioal techniques. J. Bone Joint Surg., 43-A: 4KM27,
1961.
17. Villanueva, A. R.; Hattner, R., and Frost r
H. M.: A tetrachrome stain for fresh, mineralized bone sections, useful in the diagnosis of bone diseases. Stain Technol.,
39: 87-94, 1964.
18. Vincent, J.: Recherches sur la constitution du
tissu osseux compact. Arch. Biol. Belg.,
65: 531-569, 1954.