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

(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

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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, 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

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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 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-

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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 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

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FlUOK apatits

Compa: ris on of Spacings in &gstrdm Units Obtained from x-Ray

3.85 3.85 3.81

3.35 3.75 3.35

3.40 3.15 3.15

3.02 3.05 3.02 3.02

2.72 2.60 2.51 2.25 2.13 2.05 1.93 1.88 1.83 1.79 1.75 1.71 1.64 1.53 1.49 1.44 1.34 1.305 1.27 1.25 1.23 1.21 1.14 1.10 1.09 1.07 1.02 0.98 0.97

2.72 2.72 2.72 2.60 2.60 2.60

2.26 2.13 2.02 1.93 1.88 1.82

2.24 2.12

1.93 1.87 1.83

2.25 2.13 2.05 1.93 1.88 1.83

1.75 1.71 1.64 1.52 1.49 1.44 1.32

1.27

1.23

1.14 1.10

L.03

b.97

1.71 1.62 1.51 1.49 1.44 1.33 1.30 1.27 1.25 1.23 1.20 1.14 1.11 1.09 1.07 1.02

1.75 1.71

1.53 1.49 1.44

1.30: 1.27

1.23

1.14 1.10

1.06 1.02

I.98 0.98

:hlor Dah- w&i1 lite

-

I- ( e I

-_

Roseberry, Hastings, and Morse 399

TABLE I

ectro zms of Bone, Teeth, and Various Apatites

3.82

3.35

3.02

2.72 2.60

2.25 2.13 2.05 1.93 1.89 1.83

1.71 1.64 1.53 1.49 1.44 1.34 1.30! 1.27

1.21 1.14 1.10

1.02

0.98

5

-

--

3.35 3.35

-

d

i 3.3: 3.35

3.02 3.02 3.02 3.02

2.72 2.72 2.72 2.72

2.25 2.25 2.25 2.25

1.93

1.83

1.71

1.43

1.21 1.14 1.10

1.02

0.93

1.93

1.83

1.71

1.44

1.30:

1.21

1.10

1.02

1.93 1.93

1.83 1.83

1.71

1.44

1.14 1.10

1.02

1.71

1.44

i

1.23

1.14 1.10

1.02

- -

-

, Ether extrm

cortex

BOlX3 cortex

.shed at high

#E2Ilper- ature

4.05 3.85

3.40

3.20 3.05 2.80 2.72 2.60 2.50 2.25 2.13 2.02 1.93 1.88 1.83 1.78 1.75 1.71 1.64 1.53 1.49 1.44 1.34 1.30 1.27 1.25 1.23 1.21 1.14 1.10

1.02

0.97

A few lines of shorter spacing have been omitted in this table.

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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

Fluoro :hloro apatitc .patitc

2

15 5

5

100 20

2 25 20 20 40 20 40

5 5

15 10 15 10 15

2 10 10 5 5 5 5 5 5 2 2 2 2

5

25

15

100 15

40 15 20 50 20 50

20 10

5 5

20 40 10

15

35

10 20

3

5

10

?iE-

10 20

15

15

100 10

20 5

25 15 25

5 5 2 2

10 2 5 2 2 5 2 5 5

1 3

3

rertiar~ xdcium phos- phate

5 5

25 25

10 10

100 20

100 20

10 10 5 5

25 25 10 10

5 20 20 40

2 15

15 2

15

5 5

5

5 5

2 2

2

20 10

5 2

20 2 5 2

10

5 5

2

2

Dentine

5

2

100

3

5

5

5

3

2

2 2

20 25 20

5

100

10

100

10

100

25 20 20

20

20

20

10

2

5

5

2

25 25

25 25

25 25

15

5 5

5

15

5

5 5

5

‘ZFd bone

Ether ?xtract bone

15

B0ne cortex

tshed at high

;emper- ature

5

60

20 20 85

100 75

5 50 50 20 15 75 40 75 30 40 20 20 20 25

5 10 15 15 20 10 10 20

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Roseberry, Hastings, and Morse 401

TABLE III

Comparison of Spacings and Intensities of Bone, CaHP04, and Calcium Carbonates

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

BOW -T SPrtC-

ing

A.

3.35 3.02 2.72 2.25 1.93 1.83 1.71 1.44 1.30 1.21 1.10 1.02

hlten- sity

20 5

100 25 20 20 20 10

2 5 5 2

T

&HP04

Spac- ing

8.

3.30 3.10 2.91 2.71 2.60 2.45 2.21 2.14 2.06 1.97 1.90 1.84 1.78 1.71 1.67 1.63 1.60 1.56 1.53 1.50 1.47 1.44 1.40 1.35 1.31 1.26 1.22 1.20 1.18 1.14 1.11 1.09

- hltA?Il-

sity

95 20

100 90

5 15 10

5 5 5

20 25 15 35 20 15

5 5 5 5 5 5 2

20 5 5 5

10 10

5 5 5

Calcite

Spac- ing

il.

3.75 3.39 2.98 2.72 2.45 2.25 2.07 1.90 1.85 1.59 1.50 1.46 1.41 1.33 1.28 1.23 1.17 1.14 1.05 1.03 1.01 0.98 0.96 0.93 0.89 0.85 0.81 0.79 0.78 0.77 0.70 0.68

IJltX3~ sity

60 20

100 15 50 50 50 75 75 45 45 20 30

5 20 20 35 40

2 40 35 30 30 30 10

5 5 5 2 2 2 2

T SPW

ing

2.

3.80 3.25 2.67 2.45 2.32 2.16 2.10 1.96 1.86 1.80 1.72 1.61 1.52 1.49 1.46 1.40 1.35 1.26 1.23 1.20 1.18 1.16 1.12 1.10 1.08 1.04 1.02 1.00 0:97 0.95 0.93 0.90

Illtell. sity

5 100 25 15 40 10 10 40 40 30 40

5 10

5 10 15 20 10 35 20

5 15 10

5 5

20 20 20 15

5 5 2

p-CaCOs’ -_

-- Spac-

ing

H.

3.59 3.29 2.71 2.31 2.05 1.85 1.82 1.64 1.53 1.46 1.36 1.28 1.14 1.10

:nten- sity

75 75 60 30

100 25 25 50

5 5 5

30 5 5

*These data are taken from the work of Gibson, Wychoff, and Merwin (2).

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x-Ray Analysis of Bone and Teeth

TEXT-FIQ. 1. The data obtained from x-ray spectrograms plotted with intensities as ordinates and spacings as abscisss.

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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 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

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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 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

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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

1 2 2 5 3 25 4 10 5 100 6 20 7 10 8 5 9 25

10 20 11 20 12 40 13 20 14 10 15 5 16 2 17 20 18 2 19 5 20 2 21 10 22 5 23 5 24 2 25 2 26 2

Iten- sity

Dis- tance

em.

3.32 3.75 4.30 4.79 5.32 5.55 6.46 6.80 7.10 7.55 7.73 7.95 8.52 8.89 9.50 9.80

10.14 10.95 11.20 11.50 12.00 12.85 13.35 14.50 15.20 15.95

Cell o.f Enamel

tadians Degrees

0.0811 4” 38’ 0.0916 5” 15’ 0.1051 6” 1’ 0.1170 6” 41’ 0.1299 7” 26’ 0.1356 7” 44’ 0.1577 9” 2’ 0.1661 9” 31’ 0.1734 9’56’ 0.1844 10” 34’ 0.1889 10” 41’ 0.1942 11” 7’ 0.2081 11” 55’ 0.2172 12” 26’ 0.2320 13” 17’ 0.2395 13” 43’ 0.2477 14” 11’ 0.2674 15” 19’ 0.2736 15” 40’ 0.2807 16” 4’ 0.2931 16” 47’ 0.3137 17” 57’ 0.3261 18” 41’ 0.3541 20” 18’ 0.3713 21” 17’ 0.3896 22” 18’

Ob- Sine 9 served

a,ij --

d.

3.0808 4.41 3.0915 3.89 3.1048 3.39 3.1164 3.06 3.1294 2.75 3.1346 2.64 3.1570 2.26 D.1653 2.15 D.1725 2.06 0.1834 1.94 0.1854 1.92 0.1928 1.84 0.2065 1.72 0.2153 1.65 0.2298 1.55 0.2371 1.50 0.2450 1.45 0.2641 1.34 0.2700 1.32 0.2768 1.28 0.2888 1.23 0.3082 1.15 0.3203 1.11 0.3469 1.02 0.3630 0.98 0.3795 0.94

GE SC&

d.

4.40 3.85 3.35 3.02 2.73 2.60 2.25 2.13 2.05 1.93 1.89 1.83 1.71 1.64 1.53 1.49 1.44 1.34 1.30, 1.27 1.21 1.15 1.10 1.02 0.98 0.93

Theo- retical

agij

A. 4.41 3.92 3.36 3.02 2.75 2.60 2.25 2.18 2.04 1.96 1.92 1.80 1.73 1.66 1.55 1.50 1.44 1.36 1.32 1.28 1.21 1.15 1.11 1.02 0.98 0.93

002 130 402 620 531 800 802 842 404 260 134 524 660 844 372 080 480 664 046 806 484 302 702 448 520 668

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.

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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- 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).

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Roseberry, Hastings, and Morse 407

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.

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THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XC PLATE 1

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

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THE JOURNAL OF BIOLOGICAL CHEMISTRY. VOL. XC PLATE 2

FIG. 2

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

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MorseH. H. Roseberry, A. Baird Hastings and J. K.

TEETHX-RAY ANALYSIS OF BONE AND

1931, 90:395-407.J. Biol. Chem. 

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