X-RAY ANALYSIS OF BONE AND TEETH

BY H. H. ROSEBERRY, A. BAIRD HASTINGS, AND J. K. MORSE

(From the Department of Physics and the Lasker Foundation for Medical
Research and the Department of Medicine of the
University of Chicago, Chicago)

PLATES 1 AND 2

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(Received for publication, November 3, 1930)

IiYTRODUCTION

The chemical composition of bone has been intensively studied

by a succession of investigators dating from Hoppe-Seyler and
lately exhaustively studied by Kramer and his associates (6, 7).
Exact knowledge of the chemical composition of bone is necessary
for an intelligent study of whether or not equilibrium exists
between bone and body fluids. To know simply that Ca, COs,
and PO4 exist in bone, however, is not sufficient as previous
experiments by one of us have illustrated (Hastings, Murray,
and Sendroy (3)). Equilibration of blood serum with CaC03
(calcite) led one group of investigators to the conclusion that
serum might be in equilibrium with at least one bone constituent,
CaC03; equilibration of serum with Ca3(PO& led another group
(4) to conclude that serum was supersaturated with respect to
Ca3(PO&; and equilibration of serum with CaHP04 has resulted
in a third group (6) concluding that serum was undersaturated
with respect to CaHP04. All of these conclusions may be
independently correct and yet have no relation to the situation
existing between serum and bone. It becomes of paramount
importance to know whether or not the calcium salts of bone
exist in crystalline form and what this crystal form is.
An attack on this problem has been made by de Jong (5) who
concluded that a mineral was present in bone belonging to the
apatite series and which can be represented by the formula
395
396 x-Ray Analysis of Bone and Teeth
3Ca3(P0&.CaC03. Gassmann (1) has presented chemical
evidence that the composition of bones and teeth may be repre-
sented by the formula

While our work was in progress Taylor and Sheard (8) published a
study of the refractive index and x-ray spectrograms of bone,

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teeth, various pathological concretions, and certain calcium salts.
They concluded that bone contains a mineral of the apatite
series having the type formula 3Ca3(P0&.CaXz but does not
contain the mineral brushite, CaHP04.2Hz0 in significant
amount.
This paper contains the results of a study of bone and various
calcium salts by means of x-ray spectrograms. In the main,
our result,s confirm those of de Jong (5), and of Taylor and
Sheard (8).
The questions which we attempted to answer were: (1) Does
bone contain definite crystalline salts? (2) With what mineral
does bone correspond? (3) Is CaHP04 present in bone? (4) Is
the substance known chemically as tertiary calcium phosphate,
Ca,(PO&, crystalline and is it present in bone? (5) What
role does calcium carbonate play in the structure of bones?
(6) Does the crystalline character of teeth differ from that of
bone? (7) What is the size of the unit crystal cell?

Techniques and Methods

Powder Photographs-The x-ray spectrograms were secured by
passing a beam of monochromatic x-rays through a mount on
which were placed the two samples, so that a comparison might
be made.
The samples were prepared by powdering finely in an agate
mortar till the powder would pass through a loo-mesh sieve.
The powdered samples were then placed in a mount which
consisted of a piece of x-ray film from which the emulsion had
been removed. The mount had the dimensions of approximately
2 cm. long, 0.5 cm. wide, and 0.025 cm. thick. Two rectangular
holes were cut opposite each other in which the samples were
 Roseberry, Hastings, and Morse 397
placed. The mount was then enclosed with cigarette paper.
The complete mount was then placed in position at the center of a
cassette which formed a quadrant of a circle, and the film was
placed on the circumference.
Monochromatic x-rays from a molybdenum target filtered by a
$rconium filter were used. The incident wave-length was 0.712
A. The x-rays were defined by a narrow slit, and the exposure
was approximately 45 hours.
The resulting photograph contained the x-ray spectrograms of

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the two substances to be compared. The distance of each line
from the zero line was measured in cm. and tabulated. Also the
spacings of the spectral lines in ingstrijm units were recorded
with a General Electric ruler.
Laue Photograph-A beam of x-rays was passed through a
section of the enamel of teeth. The sections were cut from the
enamel normal to the axis of growth, and normal to the vertical
surface. These sections were mounted on a glass slide over a
small hole bored in the glass, so that the beam of x-rays would
pass through only the section of the tooth. The section was then
ground to a thickness of approximately 0.40 cm., the grinding
being done on a ground glass surface with wet emery.
The sections were subjected to both beams of white radiation
and monochromatic x-rays. The white radiation was secured
from a tungsten Coolidge tube of universal type, operated at
approximately 71,000 peak volts. The time of exposure was
approximately 20 hours. Data from the photographs so secured
were not valuable for computations but they did show the
presence of crystal planes in the enamel, and furnished informa-
tion concerning their orientation.
Monochromatic x-rays were secured from the same machine
from which the powder photographs were made. The rings
secured by passing a beam of monochromatic x-rays through a
section of enamel gave definite informat.ion concerning the
symmetry of the crystals inside the enamel.

Results
An example of an x-ray spectrogram of bone and the mineral
dahlite is given in Fig. 1. The bone used in this experiment
was a specimen of young bone supplied by Dr. Charles B. Hug-
398 x-Ray Analysis of Bone and Teeth
gins. It had been prepared by his method osteogenesis and
was not more than 30 days old. It is apparent by inspection
that a close similarity exists between lines present in the bone
spectrogram and the dahlite spectrogram. Spectrograms of the
tooth enamel and of a sample of tertiary calcium phosphate
are also included in Fig. 1.
A more exact comparison may be made, however, by plotting
the data obtained from such films (Tables I to III) in the manner
shown in T:xt-fig. 1. Here the spacings between the planes are

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plotted in Angstrom units along the abscissas and the estimated
intensity of the lines, referred to the strongest line as 100, are
plotted as ordinates. The first point of significance is that bone
presents characteristic and reproducible x-ray spectrograms.
This may be interpreted as indicating that bone is built up of
minute crystals which are oriented at random. The second
point of interest is that tht strongest line of the bone, correspond-
ing to a spacing of 2.72 Angstrom units, is present in old and
young bone, whether fresh or ashed, in enamel or dentine, in
fluoroapatite, chloroapatite, the carbonate apatite (dahlite), and
in the substance known as tertiary calcium phosphate. The
third point of significance is the fact that most of the lines found
in an x-ray spectrogram of bone and enamel are found in the
spectrograms of the mineral dahlite and correspond as to spacings
and approximately as to intensities. The fourth point of sig-
nificance is that the prominent lines characteristic of secondary
calcium phosphate are absent from the spectrograms of bone
and enamel. For example, the strongest line found in the
tpectrogram of CaHP04 corresponding to a spacing of 2.91
Angstrom units, is entirely absent from the spectrogram of bone.
By mixing bone and CaHP04 in the ratio 9 : 1 characteristic lines of
CaHP04 were visible. It may therefore be stated that CaHP04,
if present, is there in an amount less than 10 per cent of the total
weight. It is perhaps of importance that young bone, prepared
by Huggins’ technique, the oldest portion of which was not more
than 30 days old yielded a spectrogram in which no CaHPO4
lines were present. It is of further importance that no lines
corresponding to those found in the form of calcium carbonate,
known as calcite, are present in the bone spectrograms. These
two points make it apparent that studies such as one of us has
 Roseberry, Hastings, and Morse 399
TABLE I
Compa: ris on of Spacings in &gstrdm Units Obtained from x-Ray
ectro zms of Bone, Teeth, and Various Apatites
- - -
BOlX3
FlUOK I- ( :hlor Dah-
, d Ether
extrm
cortex
.shed at
apatits e I w&i1 lite high
cortex #E2Ilper-
ature
-_ --
4.05
3.85 3.85 3.82 3.85

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3.81
3.75
3.35 3.35 3.35 3.35 3.35 3.3: i 3.35
3.40 3.40
3.15 3.15
3.20
3.02 3.05 3.02 3.02 3.02 3.02 3.02 3.02 3.02 3.05
2.80
2.72 2.72 2.72 2.72 2.72 2.72 2.72 2.72 2.72 2.72
2.60 2.60 2.60 2.60 2.60 2.60
2.51 2.50
2.25 2.26 2.24 2.25 2.25 2.25 2.25 2.25 2.25 2.25
2.13 2.13 2.12 2.13 2.13 2.13
2.05 2.02 2.05 2.05 2.02
1.93 1.93 1.93 1.93 1.93 1.93 1.93 1.93 1.93 1.93
1.88 1.88 1.87 1.88 1.89 1.88
1.83 1.82 1.83 1.83 1.83 1.83 1.83 1.83 1.83 1.83
1.79 1.78
1.75 1.75 1.75 1.75
1.71 1.71 1.71 1.71 1.71 1.71 1.71 1.71 1.71 1.71
1.64 1.64 1.62 1.64 1.64
1.53 1.52 1.51 1.53 1.53 1.53
1.49 1.49 1.49 1.49 1.49 1.49
1.44 1.44 1.44 1.44 1.44 1.43 1.44 1.44 1.44 1.44
1.34 1.32 1.33 1.34 1.34
1.305 1.30 1.30: 1.30! 5 1.30: i 1.30
1.27 1.27 1.27 1.27 1.27 1.27
1.25 1.25 1.25
1.23 1.23 1.23 1.23 1.23 1.23
1.21 1.20 1.21 1.21 1.21 1.21
1.14 1.14 1.14 1.14 1.14 1.14 1.14 1.14 1.14
1.10 1.10 1.11 1.10 1.10 1.10 1.10 1.10 1.10 1.10
1.09 1.09
1.07 1.07 1.06
1.02 L.03 1.02 1.02 1.02 1.02 1.02 1.02 1.02 1.02
0.98
0.97 b.97 I.98 0.98 0.98 0.93 0.97
- - -
A few lines of shorter spacing have been omitted in this table.
400 x-Ray Analysis of Bone and Teeth
TABLE II
Comparison of Intensities in Per Cent Obtained from x-Ray Spectrograms of
Bone, Teeth, and Various Apatites
B0ne
rertiar~ Ether cortex
Fluoro
apatitc
:hloro
.patitc ?iE- xdcium
phos-
Dentine ‘ZFd
bone
?xtract
bone
tshed at
high
phate ;emper-
ature
5
2 5 5 15
5

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10
25 20 25 25 5 20 25 20
15 60
5 15
20
5 15 15 10 10 2 5 10 10 20
85
100 100 100 100 100 100 100 100 100 100
20 15 10 20 20 75
2 5
25 40 20 10 10 3 25 20 20 50
20 15 5 5 5 50
20 20 25 25 20
40 50 25 10 10 5 20 25 25 15
20 20 15 5 20 75
40 50 25 20 40 5 20 25 25 40
5 75
5 20 2 30
15 10 5 15 20 5 20 25 25 40
10 5 5 10 20
15 5 2 15 5 20
10 20 2 2 2 20
15 40 10 15 20 3 10 15 15 25
2 10 2 2 5
10 5 5 5 2 10
10 15 2 5 2 15
5 2 15
5 35 5 5 10 2 5 5 20
5 2 10
5 10 5 5 5 2 5 5 10
5 20 5 5 5 2 5 5 5 20
5 3
2 1 2
2 5 3 2 2 2 5 5
2
2 10 3 2 2
 Roseberry, Hastings, and Morse 401

TABLE III
Comparison of Spacings and Intensities of Bone, CaHP04, and Calcium
Carbonates
T
BOW
-T &HP04
-
Calcite
T -_
p-CaCOs’

SPrtC- hlten- Spac- hltA?Il- Spac- IJltX3~ SPW Illtell. Spac- :nten-
ing sity ing sity ing sity ing sity ing sity
--
A. 8. il. 2. H.
1 3.35 20 3.30 95 3.75 60 3.80 5 3.59 75

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2 3.02 5 3.10 20 3.39 20 3.25 100 3.29 75
3 2.72 100 2.91 100 2.98 100 2.67 25 2.71 60
4 2.25 25 2.71 90 2.72 15 2.45 15 2.31 30
5 1.93 20 2.60 5 2.45 50 2.32 40 2.05 100
6 1.83 20 2.45 15 2.25 50 2.16 10 1.85 25
7 1.71 20 2.21 10 2.07 50 2.10 10 1.82 25
8 1.44 10 2.14 5 1.90 75 1.96 40 1.64 50
9 1.30 2 2.06 5 1.85 75 1.86 40 1.53 5
10 1.21 5 1.97 5 1.59 45 1.80 30 1.46 5
11 1.10 5 1.90 20 1.50 45 1.72 40 1.36 5
12 1.02 2 1.84 25 1.46 20 1.61 5 1.28 30
13 1.78 15 1.41 30 1.52 10 1.14 5
14 1.71 35 1.33 5 1.49 5 1.10 5
15 1.67 20 1.28 20 1.46 10
16 1.63 15 1.23 20 1.40 15
17 1.60 5 1.17 35 1.35 20
18 1.56 5 1.14 40 1.26 10
19 1.53 5 1.05 2 1.23 35
20 1.50 5 1.03 40 1.20 20
21 1.47 5 1.01 35 1.18 5
22 1.44 5 0.98 30 1.16 15
23 1.40 2 0.96 30 1.12 10
24 1.35 20 0.93 30 1.10 5
25 1.31 5 0.89 10 1.08 5
26 1.26 5 0.85 5 1.04 20
27 1.22 5 0.81 5 1.02 20
28 1.20 10 0.79 5 1.00 20
29 1.18 10 0.78 2 0:97 15
30 1.14 5 0.77 2 0.95 5
31 1.11 5 0.70 2 0.93 5
32 1.09 5 0.68 2 0.90 2

*These data are taken from the work of Gibson, Wychoff, and
Merwin (2).
 x-Ray Analysis of Bone and Teeth

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TEXT-FIQ. 1. The data obtained from x-ray spectrograms plotted with

intensities as ordinates and spacings as abscisss.
 Roseberry, Hastings, and Morse 403

made on equilibria between biological fluids and calcite, CaC03,

or such as others have made on equilibria between biological
fluids and secondary calcium phosphate, CaHP04, are without
biological significance.
It would appear that such equilibrium experiments should be
carried on between biological fluids and dahlite if the conclusions
to be drawn are to be capable of biological interpretation.
Regarding the question of the independent existence of the

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chemical substance Cas(PO& it may be stated that it may exist

L47 COJP&

I ,

R = 20.32 cm

TEXT-FIG. 2. The data obtained from x-ray spectrograms plotted with

intensities as ordinates and spacings as abscissae.

as a definite independent crystal form and further appears to

be the fundamental nucleus of the members of the apatite series.
It may have associated with it CaO, CaC&, CaF2, CaCOE, etc.
so placed within the crystal unit that no difference in the im-
portant planes is detectable. An analogy to such a situation
exists in the case of amphibole, a member of the asbestos family,
in which there may be substitution of a variety of the acidic
and basic atoms without detectable difference in the spectrograms
404 x-Ray Analysis of Bone and Teeth
(Warren (9)). Our conclusion therefore is that a substance of
the chemical analysis Ca3 (PO& may exist, that it belongs to the
apatite series of mineral, but that it probably does not represent
as stable a form of the series as is represented by the generalform
CaX2.nCaa(P0& where X may be Cl, F, 3 0, or $$O,, and n is not
less than 2 or greater than 3.
Through the kindness of Dr. L. Emmet Holt we were supplied
with a variety of specimens of tertiary calcium phosphate which
varied in analysis both below and above the theoretical Ca :P

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ratio of 1.50. x-Ray spectrograms of these salts were taken and
the data of three have been plotted in Text-fig. 2 together with
those obtained from spectrograms of two commercial samples of
tertiary calcium phosphate. The data of the spectrogram of
CaHP04 have been added for reference. It will be seen that the
spectrograms of all of the specimens of tertiary calcium phosphate
are essentially identical except in the case of the Kahlbaum salt.
In this sample the 2.91 and the 1.35 lines, characteristic of
CaHPO+ were present. This is interpreted as indicating the
presence of CaHP04 in this salt.

Conclusions Concerning the Crystal Cell

The Laue photographs furnished interesting information con-

cerning the structure of bone and enamel. An example of a
Laue photograph secured with white radiation is shown in Fig. 2.
Concentric rings were secured for sections cut from any portion
of the enamel. The rings were very narrow and defined in the
case of monochromatic radiation. The fact that concentric
rings were secured makes it evident that the crystals of the enamel
have no definite orientation in regard to histological elements,
the so called rods and prisms. The crystals are packed in the
rods and prisms at random and covered with a sheath which
composes the histological unit.
It was interesting to study the possible crystal structure of the
unit crystal. With the postulate that the enamel crystal was
similar to that of apatite, due to the similarity of patterns and
intensity of the lines in the spectrograms, an effort was made
to account for all the reflecting planes. All the lines were
 Roseberry, Hastings, md Morse 405
accounted for on the basis of a close packed hexagonal lattice
containing 4 molecules which when referred to orthorhombic
axes gave the constants: a0 = 20.8 X 1O-s cm., bo = 12.0 X
1O-8 cm., CO = 8.82 X 1O-8 cm. The degree of correspondence

TABLE IV
Comparison of Theoretical and Observed Spacings of the Unit Crystal
Cell o.f Enamel
Ob- Theo-
Iten- Dis- GE

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tadians Degrees Sine 9 served retical
sity tance SC&
a,ij agij
--
em. d. d. A.
1 2 3.32 0.0811 4” 38’ 3.0808 4.41 4.40 4.41 002
2 5 3.75 0.0916 5” 15’ 3.0915 3.89 3.85 3.92 130
3 25 4.30 0.1051 6” 1’ 3.1048 3.39 3.35 3.36 402
4 10 4.79 0.1170 6” 41’ 3.1164 3.06 3.02 3.02 620
5 100 5.32 0.1299 7” 26’ 3.1294 2.75 2.73 2.75 531
6 20 5.55 0.1356 7” 44’ 3.1346 2.64 2.60 2.60 800
7 10 6.46 0.1577 9” 2’ 3.1570 2.26 2.25 2.25 802
8 5 6.80 0.1661 9” 31’ D.1653 2.15 2.13 2.18 842
9 25 7.10 0.1734 9’56’ D.1725 2.06 2.05 2.04 404
10 20 7.55 0.1844 10” 34’ 0.1834 1.94 1.93 1.96 260
11 20 7.73 0.1889 10” 41’ 0.1854 1.92 1.89 1.92 134
12 40 7.95 0.1942 11” 7’ 0.1928 1.84 1.83 1.80 524
13 20 8.52 0.2081 11” 55’ 0.2065 1.72 1.71 1.73 660
14 10 8.89 0.2172 12” 26’ 0.2153 1.65 1.64 1.66 844
15 5 9.50 0.2320 13” 17’ 0.2298 1.55 1.53 1.55 372
16 2 9.80 0.2395 13” 43’ 0.2371 1.50 1.49 1.50 080
17 20 10.14 0.2477 14” 11’ 0.2450 1.45 1.44 1.44 480
18 2 10.95 0.2674 15” 19’ 0.2641 1.34 1.34 1.36 664
19 5 11.20 0.2736 15” 40’ 0.2700 1.32 1.30, 1.32 046
20 2 11.50 0.2807 16” 4’ 0.2768 1.28 1.27 1.28 806
21 10 12.00 0.2931 16” 47’ 0.2888 1.23 1.21 1.21 484
22 5 12.85 0.3137 17” 57’ 0.3082 1.15 1.15 1.15 302
23 5 13.35 0.3261 18” 41’ 0.3203 1.11 1.10 1.11 702
24 2 14.50 0.3541 20” 18’ 0.3469 1.02 1.02 1.02 448
25 2 15.20 0.3713 21” 17’ 0.3630 0.98 0.98 0.98 520
26 2 15.95 0.3896 22” 18’ 0.3795 0.94 0.93 0.93 668

between the theoretical and observed spacings is shown in the

seventh and ninth columns of Table IV. This correspondence
would lead to the conchrsion that the possible crystal structure
of enamel is hexagonal.
406 x-Ray Analysis of Bone and Teeth

SUMMARY

1. x-Ray spectrograms of bone indicate that it has a crystalline

structure.
2. Estimations of the spacing between the planes and the
intensities of the lines indicate that bone has a crystal structure
fundamentally the same as that of other members of the apatite
series.
3. Since chemical analysis indicates that the chemical composi-

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tion of untreated bone is similar to that of the mineral dahlite
and since x-ray spectrograms indicate the similarity in the
crystal structure of bone enamel and dahlite, it is concluded
that the calcium salts of bone and enamel may be represented
by the formula: CaC03+nCaa(PO&, where n is not less than
2 nor greater than 3.
4. No evidence is found that CaHP04 or CaC03 exists in bone
or teeth as such.
5. Cas(POd) is crystalline and seems to belong to the apatite
series.
6. The unit crystal cells of enamel have a random distribution
irrespective of the histological elements, the so called rods and
prisms.
7. The diffraction lines can possibly be accounted for on the
basis of a close packed hexagonal lattice which when referred to
orthorhombic axes has the lattice constants: a0 = 20.8 X 10es
cm., bo = 12.0 X 10s8 cm., co = 8.82 X 10ms cm.

BIBLIOGRAPHY

1. Gassmann, T., 2. physiol. Chem., 178, 62 (1928).

2. Gibson, R. E., Wychoff, R. W. G., and Merwin, H. E., Am. J. Xc., 10,
325 (1925).
3. Hastings, A. B., Murray, C. D., and Sendroy, J., Jr., J. Biol. Chem.,
‘71, 723 (1926-27).
4. Holt, L. E., Jr., La Mer, V. K., and Chown, H. B., J. Biol. Chem., 64,
509 (1925).
5. de Jong, W. F., Rec. trav. chim. Pays-Bus, 45,445 (1926).
6. Kramer, B., and Shear, M. J., J. Biol. Chem., 79,147 (1928).
7. Shear, M. J., Washburn, M., and Kramer, B., J. BioZ. Chem., 83, 697
(1929).
8. Taylor, N. W., and Sheard, C., J. BioZ. Chem., 81,479 (1929).
9. Warren, B. E., 2. Krist., 72,493 (1930).
 Roseberry, Hastings, and Morse 407

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EXPLANATION OF PLATES

PLATE 1
FIG. 1. A reproduction of x-ray spectrograms of (1) tooth enamel, (2)
dahlite, (3) ashed bone, (4) tertiary calcium phosphate.

PLATE 2
FIG. 2. A Laue photograph of tooth enamel.
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XC PLATE 1

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(Roseberry, Hastings, md Morse: x-Ray analysis of bone and teeth)

THE JOURNAL OF BIOLOGICAL CHEMISTRY. VOL. XC PLATE 2

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FIG. 2

(Roseberry, Hastings, and Morse: x-Ray analysis of bone and teeth)