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The age factor in the response of bone tissue to alizarin dyes and the mechanism of dye fixation.

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THE AGE FACTOR I N T H E RESPONSE OF BONE
TISSUE TO ALIZARIN DYES AND T H E
MECHANISM O F DYE FIXATION
N. ERCOLI AND M. N. LEWIS
Research Laboratories, Hoffmaiut-La Roche, Inc., Nutley, New Jersey
INTRODUCTION
The coloring of growing bone with madder, first experimentally studied by Belchier (1736), came into general use in ossification studies after the work of Du Hamel (1739). Within the
last two centuries numerous authors have studied the mechanism of dye fixation in bone first using madder (Bazano, 1738 ;
Haller, 1747 ; MacDdnald, 1799 ; Paolini, 1841 ; Serrhs and
Doyhre, 1842; Brull6 and Hugueny, 1845), or, later, one of its
coloring principals, alizarin (Flourens, 1847 ; Lieberkuhn,
1864), and, finally, a synthetic soluble derivative, sodium
alizarin sulfonate (Gottlieb, ’14 ; Proell, ’26 ; Cameron, ’30 ;
Proell and Diener, ’33; Schour, ’41). The conclusion drawn
from the early studies was that the dye becomes fixed in the
calcium salts which are deposited during treatment. This
hypothesis was presented for the first time by Rutherford,
before 1798. Some authors, Paolini, Serr6s-Doyhre, and
Flourens, considered the coloration due to the staining of calcium phosphate. Coloration of the inorganic substrate by
madder during the calcification process was accepted by all
authors, with the exception of Strelzoff (1873) and Katschenko
(1882), who considered the fixation as taking place in the organic matrix of bone.
Most of the recent authors ascribe the fixation of alizarin to
its affinity for “newly formed’’ calcium salts or “free calcium”. Under this assumption, the extent of coloration served
67
68
N . ERCOLI A N D M. N. LEWIS
to explain the process of calcification. According to Gottlieb
(’14)the variation in coloration intensity in different parts
of bone is due to progressive chemical stages of calcification.
According to Macklin ( ’17) the areas more deeply stained are
composed of freshly deposited calcium salts. According to
von Mollendorf (’20) the coloration is due to the formation
of calcium alizarate in bone. Proell ( ’26) considered alizarin
as an indicator of “free” calcium in bone. Cameron (’30)
suggested that “the nature of the arrangement of the calcium
complexes” may be different in young and old bone. Proell
and Diener (’33) affirmed that the colored zone consists of
freshly deposited calcium alizarate. Schour ( ’41) interprets
the coloration as being due to a selective staining response of
the calcifying or calcio-receptive zone of the collagenous
matrix.
Previous in vitro studies have shown that the fixation of
alizarin, or its synthetic analogues, is a process of adsorption
taking place between bone calcium phosphate and dye. The
dye anion is adsorbed, while PO1 anion passes into solution
(Ercoli, ’38).
I n vivo the reaction is considered to take place with the
calcium phosphate previously laid down and it may be assumed that the reaction will occur not only in growing but
also in adult bone, irrespective of age. It was the purpose of
this study to investigate the conditions of fixation of osteotropic substances with relation to the age of the animal and
to analyze the mechanism of dye fixation in bone.
EXPERIMENTAT,
Material and methods
Albino mice of different strains were used in the experiment.
Adult animals were more than 7 months of age and weighed
from 24 to 31.5 gm. These animals had not increased in weight
for a period of 10 days preceding the treatment with dye.
Growing animals were chosen from a group of mice 1P21
days old and weighing 8 to 11gm.
OSTEOTROPISM
69
Alizarin Red S (sodium 1.2.dihydroxyanthraquinone-3-sulfonate) was used as the osteotropic dye. The concentrations
were so adjusted that the dosage could be administered in
0.3-0.4 cc. per mouse. Injections were made into the tail vein
except in those groups which received subcutaneous injections
(the latter are tabulated separately). The experiments on
the young and adult mice were done simultaneously. Fortyeight to 72 hours after injection, when elimination of the dye
was complete, the animals were sacrificed by jugular bleeding.
The bones were cleaned as much as possible and placed in
saline for 3-4 hours, then under running tap water for 18-20
hours. After this they were put into a 10% solution of sodium
hydroxide. The adhering tissue was removed by agitating in
alkaline water (pH 8-8.5). After rinsing in tap water, the
bones were placed in 95% alcohol for 10 minutes and then
dried. Since gross observation shows the coloration more
definitely than microscopic examination of sections, the observations here reported were made with the dissecting
microscope.
GROWING ANIMALS
Ilztravenozcs ilzjectiolz
Mice were arranged in several groups and each mouse was
given one intravenous injection of Alizarin Red S. The indiand 80 mg./lcg. respectively.
vidual doses were 20,30,40,50,60,
The results are reported in table 1A. The following zones were
found to be colored in the skull: the condyloid process of the
mandible; the crest between the condyli; the zygomatic
process ;the alveolar part of the maxillary ; the premaxillary ;
the laminae of the posterior part of the ethmoid bone (there
was no coloration in the cribrosa) ; in-the pelvis, the crest of
the ilium, the tuberosity of the ischium, the descending ramus
of the pubis ;also the articular processes of the vertebrae (the
bodies showed only diffuse coloration) ; the sternal part of the
ribs; the epiphyses of long bones; the deltoid tubercle; the
olecranon ; the linea aspera ; and the junctions of the lamellar
bones.
70
R’. ERCOLI A N D M . N. LEWIS
TABLE 1
A. Fixation of AZizarin Red S in bone of growing mice.
Intravenous injection.
I
NO.
MICE
DOSE MCI./KQ.
( 0 ) control
20
30
40
50
60
80
.
AVERAGE
WEIGHT GM.
NO.
COLORED
NO. FAINTLY
11
9.1
10.2
8.5
9
10.5
10
..
..
4
2
6
6
3
3
2
COLORED
NO.
UNCOLORED
..
4
3
4
1
..
..
..
* .
..
..
..
R. Fixation of Alizarin Red S in bone of adult mice.
Intravenous in,jection.
0
27
27
27
30
25
25
24
28.5
28
20
30
40
50
60
80
120
200
;!
40
60
..
..
1
1
2
2
2
4
1
2*
1
1
1
1
;
..
..
C. Fixation o f Alizarin Red S in bone of growing mice.
Subcutaneous injection.
~
~
I:.*
9.2
10.5
10.5
~
..
4
1
2
1
1
..
..
..
..
______~----
* - One died 2 minutes after injection.
Subcutaneous injection
A number of mice were arranged in different groups and
each mouse received one subcutaneous injection of Alizarin
Red S. The individual doses were 20, 30, 40, and 60 mg/kg
respectively. The same zones were colored as by intravenous
injection. The results are reported in table 1C.
According to the results summarized in tables 1A and C, a
single intravenous or subcutaneous injection of 20-30 mg./kg.
of Alizarin Red S causes coloration of bone in 50% of growing
OSTEOTROPISM
71
mice injected, and 50 mg./kg. gives coloration in all mice. I n
order to ascertain the time of occurrence of coloring after injection, 3 mice were injected with 60 mg./kg. of the dye and
killed 2, 3, and 5 minues, respectively, after injection. The
bones of these mice were found to be intensely colored. It was
concluded that coloring takes place immediately after injection or resorption of the dye administered.
ADULT ANIMALS
Intravenows injection
A single intravenous injection of Alizarin Red S was given
to different groups of mice in doses of 2 4 30, 40, 50, 60, 80,
120, and 200 mg./kg., respectively. The results are reported
in table 1B. 'These results show that the dose causing coloration in 50% of adult mice is 50-60 mg./kg. The same zones
were colored as in the young animals, except that the coloration of the vertebrae was not diffused. The follpwing protocol
is characteristic for an adult mouse injected with 120 mg./kg.
of the dye.
Coloration was found in the following zones : the premaxillaries ; the malar process and around the alveoli of the maxillary (the alveolar wall was more intensely colored) ; the ethmoid; the bulla tympanica; the condyloid proces of the mandible (intensely colored) the mandibular notch; the mandibular symphysis ;the alveolar part of the incisors ;the occipital
condyles ; the crests and marginal parts of the parietal, interparietal, and squamous ; the vertebrae - the bodies and the
posterior articular, transverse and tubercular processes ; the
distal part of the caudal vertebrae ; the coxal bone - the iliac
crest, the inferior ventral spine, the articular surface of the
sacrum, the acetabulum, and the ascending ramus pubis on
the medial margin ;the scapula - the vertebral border and the
coracoid process (less intensively) ; the femur - the linea
aspera, the third trochanter and the epiphyses around the condyle ;the humerus - the epiphyses and the deltoid tuberosity;
the ulna - the olecranon and the epiphyses ; the epiphyses on
72
N . ERCOLI AND M.
N. LEWIS
the tibia, fibula and radius; and the ribs-the
sternal
third part.
The pattern of coloration most frequently found corresponds to this description.
Subcutaneous injectiorz
Six adult mice have been injected with doses of 80-120
mg./kg. Three showed slight coloration of the zones indicated.
Toxicological observations
During these experiments the possible toxic effects of
Alizarin Red S (i.v.) were also watched. The reactions (dyspnea, jumping convulsions) were definitely not the same as the
tetanic symptoms produced on injection of oxalate, which is
known to reduce the concentration of circulating calcium ions.
Three adult mice injected i.p. with doses of 500 mg./kg. of
Alizarin Red S gave no tetany. They died 8-10 hours after
injection, showing muscular weakness and dyspnea. It is concluded that Alizarin Red S does not bind circulatory calcium.
DISCUSSION AND CONCLUSIONS
The experimental results reported here demonstrate that
bones of adult animals take up the alizarin dye if the injected
dose, i.e., the concentration reached in the blood, is sufficiently
high. Fixation can be considered to follow the absorption immediately since it occurs in less than 2 minutes after intravenous injection. To obtain 50% coloration adult mice require
two to three times as great a dose as growing mice. Further,
it was found that a dose of 80-120 mg./kg. given to adult mice
by subcutaneous injection has only a slight coloring effect,
but produces definite coloration by intravenous injection.
Thus, in order to obtain visible fixation in adult bone, the intravenous administration is more adequate since it results in
a higher concentration of the dye in the blood stream.
In order to obtain coloration of adult bone a higher dye
concentration is required, presumably since in the older ani-
OSTEOTROPISM
73
ma1 certain conditions would tend to diminish the quantity of
dye which could reach the inorganic material ; viz. :
1. Lower vascularization -According to Strelzoff, in pigeons, the diameter of the Haversian canals diminishes from
0.114-0.027 mm. in the 2-day-old animal, to 0.015-0.003 mm.
in the adult. According to Lexer ( '03) upon aging the vessels
of bone become relatively thinner and their number diminish.
2. Lower permeability of adult bone-The increase of the
relative mineral content at the expense of organic material
and water (Weidenreich, '30) evidently results in more compactness and less premeability.
The percentage solubility of calcium alizarate (0.02% ),
higher than that of calcium carbonate (0.0013%), speaks
against the assumption that the coloring may be due to calcium
alizarate formed by reaction between dye and circulating calcium. An anion forming a more soluble calcium salt than carbonate could not possibly precipitate the calcium present in
serum. I n fact, the toxicological data show that Alizarin Red
S, unlike oxalate, does not reduce the concentration of Ca ions
in circulation.
Previously reported experiments and the results of this
study suggest the following hypothesis concerning the mechanism of fixation of alizarin dye in bone. The circulating dye
in contact with calcium phosphate is adsorbed with liberation
of phosphate ion. Consequently, the coloration is localized
mainly around the Haversian canals (Strelzoff, 1873) where
contact occurs between inorganic material and dye. The onsorption reaction is irreversible ; therefore, once fixed, the dye
remains in bone tissue until the latter is resorbed (Flourens,
1847). The higher vascularity and permeability of growing
bone leads to an uptake of the alizarin dye with lower doses
than is the case in the adult. The zones where the coloration
is maximum are the same in young and adult animals. I n
these zones, vascularization is greatest and metabolic processes, no doubt, are most intense. Emphasis is placed on the
consideration that the higher fixation of alizarin in young
74
N. ERCOLI AND M . N . LEWIS
bone is due to the anatomical setup and not to any specifio
chemical condition of the calcium.
A high degree of diffusibility, electronegativity, and the
irreversibility of the adsorption product have been reported
as necessary for the fixation of a substance in the inorganic
material of bone (Ercoli, '38, '41). In the light of the aforementioned hypothesis, the role of these factors becomes
obvious :
a. Diffusibility facilitates the penetration of the dye into
the compact material of bone.
b. Electronegativity allows its adsorption, since bone calcium phosphate behaves as an electro-positive adsorbent.
c. Irreversibility of the (dye) -Ca-phosphate adsorption
product makes the fisstion permanent. Trypan blue, an electronegative dye which has been found to produce temporary
coloring of growing bone (Blotevogel, '26), forms a reversible
adsorption product.
The small solubility of the calcium salt of alizarin led to
the belief that the coloration is due to the formation of Ca salt.
The relative insolubility of the Ca salt, however, is an indirect
factor since only anions giving slightly soluble calcium salts
arc irreversibly adsorbed by calcium phosphate.
The following experimental data confirm the hypothesis that
coloring of bone takes place by polar adsorption between preexisting calcium phosphate and circulating dye, rather than
by formation of calcium alizarate or any reaction with Ca ions :
1. I n vitro the reaction between inorganic bone material
and alizarin is a polar adsorption process, closely following
the isotherm of Freundlich (Ercoli, '38) ;
2. As reported in the experimental part, adult bone may be
stained with the dye ;
3. The fast rate at which coloring sets in;
4.The absence of toxic reactions, such as would be indicative
of the disappearance of circulating Ca after administration of
alizarin sulfonate, shows that no precipitation of Ca ions
occurs;
5. The disappearance of coloration after decalcification
proves that the dye is attached to the inorganic material ; while
OSTEOTROPISM
75
6. The permanence of coloration after treatment with alkali
and destruction of the organic matrix also shows that the dye
is fixed in the inorganic material.
I n conclusion, the fixation of alizarin in bone in the form of
a product of adsorption with pre-existing calcium phosphate,
precludes its use as an indicator of the age of the deposited
calcium salt or of the biochemical conditions underlying calcification. Nevertheless, the value of alizarin for studies of ossification remains unchanged, if its use is limited to determining
the addition of new substance to that existing at the moment
of the injection, or to the measurement of zones lying between
those colored by dye administered at intervals.
SUMMARY
The influence of age on the fixation of Alizarin Red S was
studied.
It was found that a single intravenous injection of 20-30
mg./kg. of dye caused bone coloration in growing mice. Adult
mice required two to three times this amount to produce coloration. Coloration was produced in bone immediately after
intravenous injection.
The anatomical zones in which coloration takes place have
been described.
It is concluded that fixation occurs by a process of adsorption between existing calcium phosphate and dye anions, rather
than by the supposed formation of calcium alizarate or a reaction with free calcium ions.
The higher dosage required for coloration in adult mice
depends on the anatomical and physical, not on the chemical,
Conditions of bone.
I n adult mice, subcutaneous injection produced less coloring
than intravenous injection.
LITERATURE CITED
BAZANO,M. 1738 De eoloratis animalium quorundam vivokum ossibus. De
Bononiensi Seientiarum ot Artium Insitutio atque Academia Commentari., 1701. I, 1st part, p. 129 (1745).
BELCHIER,
J. 1736 An account of the bones of animals being changed to a red
colour by aliment only. Philosophieal transactions, vol. 39, p. 287.
76
N . ERCOLI AND M. N. LEWIS
BLOTEVOGEL,
W. 1924 Der vitale Farbstofftransport wahrend der Zahnentwicklung. Ztschr. f . Zellen u. Gewebelehre, vol. 1, p. 601.
BRULLE,AND HUGUENY1845 Sur le developpement des 0s dans les mammifares
et les oiseaux. Ann. Science. Nat., Se'ries 111, vol. 4, p. 287.
CAMERON,
G. E. 1930 The staining of calcium. J. Bact. & Path., vol. 33, p. 929.
DU HAMEL 1739 Sur une racine qui a la facult6 de teindre en Rouge les 08 des
Animaux vivants. Histoire de l'Academie Royale des Sciences - Memoires de M a t h h a t i q u e s et de Physique, p. 1.
ERCOLI,N. 1938 L'osteotropismo dei farmaci, 111, Ricerche in vitro con
l'alizarin 3. sulfonato d i sodio. Boll. SOC.It. Biol. Sperim., vol. 13, p.
757. I fattori chimico-fisici dell 'osteotropismo farmacologico. Proc.
XVI Internat. Meeting of Physiol., Zurich (Switzerland), 11,p. 20.
1941 A chemotherapeutic agent with osteot'ropie properties. Proc.
SOC.Exp. Biol. Med., vol. 48, p. 672.
FLOURENS,
P. 1847 Theorie experimentelle de la formation des 0s: ChAz Baill i h e , Paris.
GOTTLIEB,B. 1914 Die vitale Farbung der kalkhaltigen Gewebe. Anat. Anz.,
vol. 46, p. 179.
HALLER,
A. 1747 Opera anatomica minora., vol. 11, p. 460, Lausanne.
N. 1882 Uber die Erappfarbung der Froschgewebe. Arch f. mikr.
KATSCHENKO,
Anat., vol. 21, p. 357.
LEXER,
E. 1903 Die Entstehung entziindlicher Knochenerde und ihre Beziehung
zu den Arterienverzweigungen der Knochen. Arch f . Klin. Chirurgie,
vol. 71, p. 1.
LIEBERKUHN,
N. 1864 Uber Knochenwachstum. Arch. f. Anat. Physiol. u. Wiss.
Med., p. 598.
MACDONALD,1799 Disputatio Inaguralis. De necrosi ac callo. Edinburgh.
MACKLIN,C. C. 1917 Studies in calcification by the use of vital dyes. J. Med.
Research, vol. 31, p. 493.
vON MOLLENDORF,
W. 1920 Vitale Farbungen a n tierischen Zell. Cap. IX.
Knochen. Erg. Physiol., vol. 18, p. 297.
PAOLINI,
M. 1841 Quorundam experimentorum de vi Rubixe ad ossa, ovorumque
Gallinarum putamina calcaria coloranda. Novi Commentilri Instituti
Bononiensi, vol. 6, p. 469.
PROEL,
F. 1926 Beitrage zur vitalen Knochenfarbung. Ztschr. f. Zellforschung
u. mikr. Anat., rol. 3, p. 461.
PROELL,
F., AND DIENER,A. 1933 Model1 u. Tierversuche zum Problem der Verkalkung von Knochen. Ztschr. f . Zellforschung u. mikr. Anatomie,
vol. 18, p. 244.
RUTHERFORD,
D. before 1798 reported by R. Blake, An essay on the structure
and formation of the teeth in man, Dublin, William Porter, 1801, p. 154.
SCHOUR,
I., AND M. M. HOFFMANN;
B. G . SARNAT,
AND M. B. ENGEL 1941 Vital
staining of growing bones and teeth with Alizarine Red S., J. of Dent.
Res., vol. 20, p. 411.
SERRES, AND DOYERE 1842 Expos6 de quelque faits relatifs a la coloration des 08
chez les animaux soumis au regime de la garance. Comptes Rendus de
1'Acad6mie des Sciences.
STRELZOFF,
1873 Uber Krappfiitterung. Ztrlbl. f . d. Med. Wissensch., p. 737.
WEIDENREICH,
F. 1930 Dxs Knochengewebe, Hdb. d. mikr. Anat., vol. 2 (part 2 ) .
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