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Variations in the Crystalline Components of Human Dental Calculus: I. Crystallographic and Spectroscopic Methods of Analysis
Racquel Zapanta Le Geros J DENT RES 1974 53: 45 DOI: 10.1177/00220345740530012801 The online version of this article can be found at: http://jdr.sagepub.com/content/53/1/45

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Variations in the Crystalline Components of Human Dental Calculus: I. Crystallographic and Spectroscopic Methods of Analysis
RA CQUEL ZAPANTA LE GEROS New York University Dental School, New York, New York 10010, USA

The diffractometric method has more advantages than the camera technique for Xray diffraction analysis of the crystalline components of human dental calculus. With the use of this crystallographic method and infrared absorption techniques, it was found that the magnesium content of human dental calculus is the result of partial substitution of magnesium for calcium in whitlockite, and that the carbonate content is the result of substitution in apatite.
The use of X-ray diffraction powder-camera techniques in the analysis of the crystalline components of human dental calculus has been accepted since 1938.1 Brushite, CaHPO4 * 2H20; octacalcium phosphate, Ca8H2 (P04) 6 * 5H20; apatite, Ca10 (P04) 6(OH) 2a; and whitlockite, (Ca, Mg) 3 (P04) 2' have been identified in different amounts in numerous samples of dental calculus.2-6 In some instances, monetite, CaHPo4, and calcite, CaCO3, presumably have been detected.7 The powder-camera method has the advantage that only small amounts of samples (1 to 3 mg) are used, but the method has some major disadvantages. For example, when octacalcium phosphate (OCP) and apatite are present simultaneously, they are differentiated with great difficulty because of the similarity in the diffraction angles of some of their reflections.8 Also, it is difficult to assess the relative intensities of the bands, and hence the relative amounts of the different components on the same sample, without the use of a densitometer and without the tedious stanThis study was supported by USPHS Research Grant DE-00159. Received for publication December 26, 1972. a A simplified formula of biologic apatite that contains other substituents in the apatite structure.

dardization of the development of films. Finally, the actual identification of the components of the mixture is inconclusive. More accurate identification and more meaningful information can be obtained with the use of the X-ray diffractometer and infrared absorption techniques. The purpose of this paper is to present the information obtainable when X-ray diffractometric -and infrared absorption techniques are used to analyze dental calculus. This study shows that magnesium content is related to the whitlockite component and carbonate content is related to the apatite component.

Materials and Methods Thirty-five samples of human dental calculus and two stones from salivary ducts were analyzed by the X-ray diffractometric technique and infrared absorption spectrophotometry. For X-ray diffraction analysis, about 40 to 80 mg of finely powdered washed and dried human dental calculus was required to fill the diffractometer sample holder, which has an adjustabale depth of 1 to 1.5 mm. X-irradiation was produced with 35 kv, 20 ma on a copper target, using a nickel filter, one-degree slit. Scanning speed was oneeighth degree per minute in the ranges 4.0 to 5.20 20, 10 to 130 20 (for the strongest reflections at 4.7 and 11.70 20 of OCP and brushite, respectively), and 20 to 350 20 (for major apatite and whitlockite reflections). The amount of magnesium substituted in the whitlockite structure was approximated from a plot of amount of magnesium in whitlockite structure vs a-axis dimensions (Fig 1) . The a-axis dimensions of whitlockite were calculated from positions of the 220 reflection: a is 4 d220, where d is the d spacing
45

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,20

Z'

a
0

4.0

'15
510

3.0
2.0

.3

C
-,
I

1.0
0 o

-5
10.34

10.32

10.36 10.38 a-axis (A)

10.40

10.42' 1044 pwe TCP

FIC 1.-Amount of magnesium substituting for calcium in whitlockite structure, as exhibited by contraction of a-axis dimensions. Pure whitlockite (fi-TCP), or .8-Cas (PO4),, has a axis of 10.428 -+- 0.005 A.

2000 1300 1100 900 700 400 Frequency (cm-') FIG 3.-Infrared absorption spectra of OCP (A), apatite (B), and magnesium whitlockite, (C), X-ray diffraction patterns of these materials are shown in Figure 2.

4000 3000

w
AA

A, W
(I)

WA

AW
I
A.0
W

A~~~~~~~~~~
OJ1 Nw

I-'0 A J1

AO,
0 A0 9

O,W.A
U

(II)

Wt

~~~WA

I I
I,

,l

2'I
B
,,

(111)
I

(IV)

1
-8~~~~

X,

//1,
(V)

-I-I

4 5

26

30
Diff ra cti on

340 2.
Angle

26
Di

.30
FFRACTION ANGLE

340 20

FIG 2.-X-ray diffraction patterns of pure compounds prepared at 37 C (A and B) and 60 C (C) by diffusion of ions in gel systems. A, apatite; B, OCP (dotted line indicates reduced intensity of 100 reflection from x 0.25 to x 1.0.); C, magnesium-substituted whitlockite.

FIG 4.-X-ray diffraction patterns of some samples of human dental calculus. Different crystalline components are apatite (A), octacalcium phosphate (0), and whitlockite (W). IV and V are the side and outside layers, respectively, of one calculus sample.

46
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V~ol 53 No. 1

VARIATIONS IN CALCULUS COMPONENTS

47

FIG 5.-Layered structure of produced at 67%).

calculus sample viewed under microscope (orig

mag

100;

re-

that corresponds
and
a

to

the 20 diffraction angle

is the a-axis dimension in angstroms.9

For example, when the 220 reflection is at 34.76 20, the corresponding d is 2.581 and the calculated a axis is 10.322 -+- 0.005 A, and the amount of magnesium in the whitlockite is about 3.8 wt%. The values in Figure 1 were derived from synthetic whitlockite with known amounts of magnesium substituted for some of the calcium in the structure.10,11 For the infrared absorption analyses, 0.6 to 1.0 mg of finely powdered samples was mixed with 300 potassium bromide (infrared grade) and pressed into a pellet by use of 20,000 psi. The scanning speed was about eight minutes for each range, 4000 to 1300 cm-1 and 1300 to 400 cm-1. For a more critical study of the spectra, the scanning speed may be reduced to 30 minutes for each range. To better demonstrate the C03 vibration bands, 2 to 3 mg samples may be used. X-ray diffraction patterns (Fig 2) and infrared absorption spectra (Fig 3) of pure materials were used for comparison. These materials were prepared at 37 C for brushite, OCP, and whitlockite, and at 60 C for apatite in gel systems.'2-14
Results

sample to sample. Variations within the sample, that is, between the inside and outside layers of the sample (Fig 4, IV, V) also were observed. The layered appearance of the calculus sample, which indicates discontinuous mineralization, is shown in Figure 5. When the OCP component is present simultaneously with the apatite component, it can be differentiated accurately and without difficulty by the presence of the strongest OCP

Enamel

|C-alculu| Calculus
-I

I --or-

I-- -

r -

X-RAY

DIFFRACTION ANALYSES.

X-ray dif-

raction analyses of 35 samples using diffractometric techniques demonstrated variations in the crystalline components of human dental calculus. For example, from the diffraction patterns of the five samples shown in Figure 4, it can be observed that the relative amounts of the components OCP (0), apatite (A), and whitlockite (W) vary from

Dentin
|

26

30

3420

FiG 6.-Comparative crystallinity of powdered

enamel, dentin, and calculus sample. Calculus sample was mainly apatite.

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48

LE GEROS
A
0-

J Dent Res January-February 1974

C3
P04

reflection (200) at 4.74 20 (d = 18.64 A). Brushite was identified in only 3 of 35 samples, although it had been reported as the major component in young calculus (3 days old) by Schroeder.8 Only whitlockite was present in the calculus found in the parotid gland. The crystallinity (reflecting crystallite size) of the apatite component of most of the dental calculus samples was between that of dentin and enamel (Fig 6). A semiquantitative determination of the relative amounts of each component in a sample can be made by obtaining the ratio of the heights of the major reflections of the component in the sample to that of the pure material. Whitlockite appeared as a major component in many of the samples. The amount of magnesium substitution in the whitlockite structure can be approximated from the shifts of the 220 reflections to lower diffraction angles, as compared with the positions of pure whitlockite (Fig 7). The shift of these reflections indicates a reduction in the a-axis dimensions because of the partial substitution of the smaller magnesium ions (ionic radius, 0.67 A) for the calcium ions (ionic radius, 0.99 A) in the whitlockite structure, Ca3 (P04) 2.10-16 The amount of magnesium substitution in the whitlockite component

P04

OH

CO,

I3

B C

VX~~~~~~~~~~~~
HPO4

D
E

4000

3000

2000

1200

1000

800

600

400 cm-

FREQUENCY

FIG 8.-Infrared absorption spectra of some samples of human dental calculus. Corresponding X-ray diffraction patterns are shown in Figure 4.

CM
M

F4m

ri

DIFFRACTION

ANGLE

FIG 7.-X-ray diffraction pattern of a 1:1 mixture of pure and magnesium-substituted whitlockite (TCP/TCMP). Magnesium substitution in TCMP component is maximum, that is, 16 atomic%. As a consequence of partial substitution, reflections are shifted to higher diffraction angles, which denote contraction of a axis.

ranges from 2 to 3 wt%. When the amount of magnesium in the whole sample is approximated, and when the relative amounts of whitlockite in the calculus samples (either mixed with OCP or with apatite) are considered, the magnesium content in the samples can range between 0.5 to 1.5 wtO. These values agree with the chemical analyses made by Gron et a12 on whole samples of calculus. INFRARED ABSORPTION ANALYSES.-Infrared spectra of several samples of human dental calculus are shown in Figure 8 (see spectra of pure samples, Figure 3). If the spectrum of the sample is superimposed on that of the pure material, the bands that correspond to each of the components can be identified. The CO3 bands in the apatite component of the calculus (Fig 8, B) are similar to the CO3 bands in the human enamel apatite and in synthetic apatite that contains CO3 prepared from aqueous systems at 37C (Fig 9). In previous crystallographic and spectroscopic studies, it was established that the CO3 in enamel apatite and in precipitated apatites that contain carbonate are integral parts of the apatite structure.1718 For qualitative analyses, the presence of an apatite component can be ascertained by the presence of the CO3 vibration bands at

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Vol 53 No. I

VARIATIONS IN CALCULUS COMPONENTS

49

30

250

20

30
04o
~.40

30

FIG 9.-Infrared absorption spectra of dental calculus composed mainly of apatite (A), human enamel (B), and apatite that contains carbonate (C), prepared at 37 C from solutions that contained CO3 and P04 in equal amounts.

1465, 1475, 861, and 869 cm-1. If CaCO3 is present, the CO3 vibration bands in the spectrum occur at different wave numbers than the CO3 bands in the spectrum of the apatite that contains C03.18 Discussion The diffractometric techniques that were used in the preliminary'9 and present investigations of dental calculus have the following advantages compared with the powder camera techniques used by previous investigators'-8: relative accuracy in the determination of the components of the mixtures of the different types of calcium phosphates present in a calculus sample (for example, OCP can be differentiated easily from apatite even when both are present simultaneously); rapidity of analysis (1.5 hours for diffractometric techniques and 9 hours for the powder-camera technique); and convenience (film development and film standardization for accurate determination of intensities of reflections are not necessary for the diffractometric method) . The conditions in the human oral environment are not conducive to the formation of monetite and calcite.17,20 Therefore, the reported occurrence of these two compounds

in human dental calculus7 could have been caused by the limitations of the powdercamera methods. The shifting of the whitlockite reflections in comparison with pure whitlockite, fl-Ca3(P04) 21 is caused by the partial substitution of the smaller magnesium ion for calcium in the structure. Thus, the amount of magnesium being incorporated in the whitlockite component can be approximated from the contraction of the a axis. The vibration bands of C03 in the spectrum of a sample of dental calculus indicate that the C03 group is in a crystal field or environment similar to that of apatite that contains carbonate, in which C03 is a substituent for P04. This indicates that the C03 is an integral part of the apatite component.

Conclusions These studies demonstrate that the diffractometric method is better than the camera technique for the X-ray diffraction analysis of the crystalline components of human dental calculus, and indicate the additional information made available by the application of infrared absorption spectroscopic techniques.

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SO

LE GEROS

J Dent Res January-February 1974

9. FANG, J.H., and BLoss, F.D.: X-ray Diffraction Tables. Southern Illinois Press, 1966. 10. TRAuTz, O.R., and FESSENDEN, E.: Formation and Stability- offs Whitlockite and Octacalcium Phosphate, Two Components of Salivary Calculi, J Dent Res 37: 78, 1958. 11. TRAUrz, O.R., and ZAPANTA-LEGERos, R.: Effect of Magnesium on Various Calcium Phosphates, abstracted, IADR Program and Abstracts of Papers, No. 101, 1962. 12. LEGmtos, R.Z.; REICH, L.; LEGEROs, J.P.; and TRAUTz, O.R.: Calcium Phosphate Crystals Grown in Silica Gel II., abstracted, IADR Program and Abstracts of Papers, No. 678, 1971. 13. LEGEmos, R.Z., and LEGEmos, J.P.: Brushite Crystals Grown by Diffusion in Solution and in Gel, J Cryst Growth 13/14: 476, 1972. 14. LFGEPoz, R. 7.. and MORALES, P.: Renal Stone Crystals Grown in Gel Systems, J Invest Urol, in press. 15. TRAuTZ, O.R., and ZAPANTA-LEGEROS, R.: Effect of Magnesium on Various Calcium Phosphates II., J Dent Res 43: 751, 1964. 16. FRONDEL, C.: Whitlockite, a New Calcium Phosphate, Ca3 (P04) 2, Am Mineral 26: 145, 1941. 17. LEGEROS, RZ.: Crystallographic Studies on the Carbonate Substitution in the Apatite Structure, PhD thesis, New York University,

More significantly, these studies show that the magnesium content of human dental calculus is related to the whitlockite component, and substitutes for some of the calcium in the Ca3 (PO4) 2 crystal structure. The carbonate content is related to the apatite component; CO3 is an integral part of the apatite structure and partially substitutes for the P04 groups. Experimental evidence on some of the effects of magnesium21 and carbonate on the synthetic apatite-whitlockite systems will be presented in subsequent papers.
The authors thank G. M. Domingo, C. Carter, and G. Quirolgico for technical assistance.

References
1.

GLOcK, G.E., and MuRuAy, M.M.: Chemical

Investigations of Salivary Calculus, J Dent Res 17: 257, 1938. 2. GRON, P.; VAN CAMPEN, G.J.; and LINDSTROM, I.: Human and Dental Calculus. Inorganic and Crystallographic Composition, Arch Oral Biol 12: 829, 1967. 3. JENSEN, A.T., and DANO, M.: Crystallography of Dental Calculus and the Precipitation of Certain Calcium Phosphates, J Dent Res 33: 741, 1954. 4. JENsEN, A.T., and HANSEN, H.G.: Tetracalcium Hydrogen Triphosphate, Trihydrate, a Constituent of Dental Calculus, Experientia 13: 31, 1957. 5. JENSEN, A.T., and RowLEs, S.L.: Magnesian Whitlockite, a Major Constituent of Dental Calculus, Acta Odontol Scand 16: 121, 1957. 6. SCHROEDER, H.E., and BAUMBAUER, H.U.: Stages of Calcium Phosphate Crystallization During Calculus Formation, Arch Oral Biol 11: 1, 1966. 7. WEsTERDEN, E.M., and LITTLE, K.: Some Observations on the Composition of Calculus, J Dent Res 37: 749, 1958. 8. SCHROEDER, H.E.: Formation and Inhibition of Dental Calculus, Vienna: Hans Berne Publishers, 1969.

1967.
18. LEGEROS, R.Z.; TRAuTZ, O.R.; KLEIN, E.; and LEGERos, J.P.: Spectral Properties of CO. in C03-containing Apatites, Dev Appl Spec 7B: 3, 1970. 19. LEGERos, R.Z.; TRAurz, O.R.; and KLEIN, E.: Variations in the Crystalline Components of Human Dental Calculus. Effect of Mg and C03, abstracted, IADR Program and Abstracts of Papers, No. 677, 1971. 20. NEWESELY, H.: Changes in Crystal Types of Low Solubility Calcium Phosphates in the Presence of Accompanying Ions, Arch Oral Biol 6:174, 1961. 21. LEGEROS, RZ.; ALFREY, A.C.; and CONTIGUGLA, R.: Pathological Calcification Associated with Uremia. Two Types of Calcium Phosphate Deposits, Cak Tiss Res, in press.

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