Journal of Dental Research

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Changes in Bovine Dentin Mineral with Sodium Hypochlorite Treatment T. Sakae, H. Mishima and Y. Kozawa J DENT RES 1988 67: 1229 DOI: 10.1177/00220345880670091601 The online version of this article can be found at: http://jdr.sagepub.com/content/67/9/1229

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Changes in Bovine Dentin Mineral with Sodium Hypochlorite Treatment T. SAKAE, H. MISHIMA, and Y. KOZAWA Department of Anatomy, Nihon University, School of Dentistry at Matsudo, Chiba 271, Japan Dentin powders from bovine incisors were treated with 10% NaCIO solution. Differential thermal analysis (DTA) indicated the removal of organic material from the dentin sample following the treatment, since the exothermic reaction at 320'C had disappeared. X-ray diffraction studies revealed a change in the crystallinity of the dentin crystals and the formation of calcite after the treatment. Infrared absorption analysis showed that the band due to carbonate ions was weakened after the treatment, while atomic absorption spectroscopic analysis showed that magnesium ions had been dissolved from the dentin sample. The a-axis lengths of treated and heated dentin samples differed from those of untreated and heated samples. Whitlockite was always found in the untreated/heated samples, whereas it was absent in the treated/heated samples. The unit cell dimensions of the whitlockite indicated the partial substitution of magnesium for calcium. Magnesium ions seemed to be more effective than carbonate ions in forming whitlockite. These results showed that some magnesium and carbonate ions were removed from the dentin crystal structure upon NaCIO treatment, while at the same time organic materials were removed from the dentin sample. It was suggested that crystals in the NaCIO-treated dentin were similar to enamel crystals from a crystallographic viewpoint. J Dent Res 67(9):1229-1234, September, 1988

Introduction. Dentin crystals have a more variable morphology, chemical

composition, and crystalline nature than do enamel crystals because the former are embedded in an abundant organic matrix. In order for the mineral phase of dentin to be separated from its organic milieu, the following methods are in common use (Rowles, 1967; Kato and Ogura, 1975): (i) dry-ashing in a muffle furnace at temperatures in excess of 500TC, (ii) KOHglycol-ashing at temperatures greater than 200TC, (iii) KOHethylene-glycol-ashing at 205'C, (iv) ethylene-diamine-ashing at 118TC, (v) hydrazine decomposition at 930C, and (vi) lowtemperature-ashing with the use of plasma treatment. Rowles (1967) reviewed the effects of methods ii-v on the mineral phase, and concluded that structural or compositional changes in, or loss of, inorganic material occurred. Tochon-Danguy et al. (1978) showed that low-temperature ashing also altered the biological crystals by trapping gas in the crystal structure. We have found that heating at temperatures of more than 500'C resulted in the recrystallization of dentin apatite. All of the abovementioned methods resulted in alteration of the mineral phase. Another de-proteinization process, treatment with NaClO solution at room temperature (Boyde and Hobdell, 1969), has also been applied to dentin (Shellis, 1983). This method has an advantage of low temperature, and may not affect the dentin crystals drastically, although the precise effects are not clear. The aim of the present study was to describe the changes in dentin crystals induced by NaClO treatment and to obtain some information on the nature of the dentin crystals themReceived for publication February 10, 1988 Accepted for publication April 20, 1988 This investigation was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 62771441).

selves by means of thermogravimetry (TG), differential thermal analysis (DTA), infrared absorption spectroscopy (IR), and x-ray diffraction analysis.

Materials and methods. Dentin samples were prepared from six bovine permanent central incisors. The tooth crown was cut off, and the outer cementum-side layer and the inner pulp-side layer were ground off with a dental disk, leaving the root dentin. The root dentin was then pulverized by an agate mortar and ball with a Retsch vibrator. The whole-dentin powder was then passed through a 200-mesh sieve. The dentin powder, 100 mg, was treated with 300 mL of 10% NaCIO reagent-grade solution for 30 min at room temperature with mild vibration. The powder was then filtered through a cellulose membrane filter (type TM-3, TOYO Co. Ltd., Tokyo, Japan) with aspiration, washed with H20, and dried in air. The magnesium content of the treated solution was analyzed with a Perkin-Elmer atomic absorption spectrometer. For the removal of excess sodium ions from the solution, an ion-exchange resin column method (Ooki et al., 1962) was employed. The solution was evaporated over a steam bath, and the residue was dissolved in 50 mL H20. The aqueous solution was poured into a column of Amberlite A.G.IR-120B(H) pretreated with 6 mol/L HCI, and 100 mL H20, 150 mL 0.4 mol/ L HCl, and 150 mL 0.8 mol/L HCI were successively loaded in order to extract P043-, NV-, and Mg2+, respectively. The atomic absorption measurement conditions were as follows: flame, air-acetylene; lamp current, 15 mA; wavelength, 285 nm; slit, 4. Thermogravimetric (TG) and differential thermal analyses (DTA) were carried out on the NaCIO-treated and untreated dentin samples by means of a TAS-100 TG-DTA apparatus (Rigaku Co. Ltd., Tokyo, Japan). The TG-DTA measurement conditions were as follows: sample weight, 10-20 mg; reference material, corundum; DTA sensitivity, 0.1 RV; TG sensitivity, 0.01 mg; heating rate, 50C/min; temperature range, from room temperature to 1000'C; atmosphere, air; thermocouple, Pt-PtRh. The sample was held at 1000'C for 10 min, then cooled with air. Infrared (IR) absorption spectroscopy was carried out on the NaClO-treated and untreated dentin samples with an IRA-2 spectrometer (JASCO Co. Ltd., Tokyo, Japan) using the KBr disk method. An x-ray diffraction study was carried out with an x-ray powder diffractometer (Rigaku Co. Ltd., Tokyo) on the NaClOtreated and untreated samples and also on the samples heated to 1000'C for the thermal analysis. Each sample was mixed with KC1, which was used as an internal standard and also as a sample-holding material. The unit cell dimensions of the crystals were calculated by the least-squares method.

Results.

Fig. 1A shows the DTA pattern common to the untreated dentin samples. The pattern exhibited a weak and broad en1229 Downloaded from jdr.sagepub.com by guest on July 10, 2011 For personal use only. No other uses without permission.

SAKAE et aLD J Dent Res

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sample. The calculated organic contents lay within a small range, 18.4 wt% on average (Table 1). Within a temperature range of 600-10000C, the average weight loss was 2.6 wt% for the untreated dentin samples. The weight losses of the NaCIO-treated samples with the same temperature range were a little larger than those of the untreated samples, 4.7 wt%. The IR spectra of the NaCIO-treated and untreated dentin samples are shown in Figs. 2A and 2B, respectively. After the NaClO treatment, the intensity of the band at 1650 cm-' was weakened relative to those of the bands at 1400-1600 cm -. Figs. 2A and 2B also show that the band at 870 cm strengthened after the treatment. The IR spectrum of the treated dentin sample was, on the whole, similar to that for human enamel (Fig. 2C). The x-ray diffraction pattern of the untreated sample is shown in Fig. 3A. The broad peaks apparent in the x-ray pattern indicated that the dentin apatites were poorly crystallized. The peak broadening was measured as the full width at the halfmaximum (FWHM) of the apatite (002) reflection (Tables 2 and 3). The FWHM value for the untreated sample was larger than that for the corresponding treated sample, except for the BD-2 sample. The average of the FWHM for the untreated dentin samples was also larger than that for the treated samples, 0.510 and 0.470, respectively. These results showed that the crystallinity of the dentin mineral generally became more pronounced after the NaCIO treatment. The c-axis length of the dentin apatite was roughly calculated from the d-spacing of the (002) peak in the x-ray diffraction pattern (Tables 2 and 3). The values for the untreated and NaClO-treated dentin samples were 0.689 and 0.690 nm. There was no apparent difference between these values. This result does not mean that there was no difference in the crystal lattice, but rather that it was difficult to reveal any differences by this rough measurement. In the x-ray diffraction pattern of the NaClO-treated dentin sample, calcite appeared (Fig. 3B). Treated samples showed the presence of calcite, but differences in the amount of calcite ( wtit were apparent (Table 3). The BD-2 sample showed the most 0 abundant amount of calcite after the treatment. Fig. 4 shows the x-ray diffraction patterns of the NaCIOtreated and untreated dentin samples after being heated at 1000'C. The poorly crystalline dentin apatite was apparently into a well-crystallized form, while whitlockite re-organized -20 was formed in the untreated/heated dentin sample (Fig. 4A) but not in the treated/heated sample (Fig. 4B). The relative intensity ratios (Wh/Ap) of the whitlockite (217) peak to the apatite (211) peak are listed in Table 2. The ratio varied from -40 0.03 to 0.72. The apatite unit cell dimensions of the samples after being heated at 10000C were calculated by the least-squares method (Tables 2 and 3). The estimated standard deviation for the T1G-DTA t~~~I : B values was 0.0002 nm at maximum. For the untreated dentin samples, the average value was 0.9422 nm for the a-axis and 0.6882 nm for the c-axis length, and for the NaClO-treated samples, 0.9418 nm and 0.6883 nm, respectively. The values TG for the c-axis showed no significant differences. The a-axis lengths of the untreated samples were a little larger than those -20 of the treated samples, and also larger than that of hydroxyapatite, a = 0.94184 nm (Young and Holcomb, 1982). The unit cell dimensions of the whitlockites were also calculated by the least-squares method (Table 2). The estimated -40 standard deviations were within 0.0002 nm for the a-axis val_A 500 6000 7 _ 00 JU 200 00 500 7100 8003 900 A 100 0) ues and 0.002 nm for the c-axis values. The average a-axis Temperature CC) length was 1.0361 nm, and the average c-axis length was 3.715 Fig. 1-DTA and TG curves for untreated (A) and NaClOtreated (B) nm. The unit cell dimensions of the whitlockites were close to dentin samples (No. BD-5). The vertical scales are (left) wt% loss for TG values a = 1.0350 nm and c magnesium-whitlockite, Downloaded from jdr.sagepub.com by guest on July 10, 2011 Forthe personal use only.for No other uses without permission. for DTA. and (right)

dothermic peak at about 600C and a strong exothermic peak at 3200C with a shoulder. It was noticed in this study that the DTA curves leaned slightly toward the endothermic side at temperatures of more than 700'C. In contrast to the DTA pattern of the untreated dentin sample, that of the NaCIO-treated dentin sample (Fig. 1B) showed no exothermic peaks over a temperature range from 200'C to 6000C. A weak endothermic peak below 1000C and a faint endothermic peak at 6600C were observed. It was also noticed that the DTA curves leaned slightly toward the endothermic side at temperatures of more than 7000C. The TG curve for the untreated dentin sample (Fig. lA) showed two apparent steps of weight loss occurring from the starting temperature to ca. 200'C and from ca. 200'C to ca. 6000C, and then showed a gradual decrease in weight at temperatures of more than 6000C. In contrast, the TG curve of the NaCIG-treated dentin sample (Fig. 113) showed that the weight loss at temperatures between 2000C and 6000C was markedly decreased. For convenience, the weight losses of the NaCIO-treated and untreated dentin samples were calculated from the TG curves over three temperature ranges: room temperature (R.T.)-2000C, and 600-10000C, and these are listed in Table 1. The weight losses between R.T. and 200TC occurred over a small range, 10.7 wt% on average for the untreated dentin samples and 11.3 wt% on average for the NaClO-treated dentin samples. The weight loss between 200'C and 600'C was 21.5 wt% on average for the untreated dentin samples (Table 1). However, the amount of weight loss was remarkably decreased to 3.1 wt% on average for the NaCIO-treated samples. The content of organic material in each dentin sample was calculated by subtracting the weight loss between 200'C and 6000C of the treated sample from that of the corresponding untreated

200-6o0C,

l l

DTA

100

200

xV

300'

400

-

Sample No.

1231

CHANGES IN DENTIN MINERAL WITH NaClO

Mol. 69 N~o. 7

TABLE 1 THE PERCENTAGE WEIGHT LOSSES OF DENTIN SAMPLES MEASURED FROM THE TG CURVES Temperature Range (0C) Untreated NaCIO-treated R.T. -200 200-600 600-1000 200-600 600-1000 R.T. -200

(B)

(A) BD-1 21.3 11.4 10.8 21.6 BD-5 22.3 BD-2 11.1 9.8 21.3 BD-3 BD-4 9.4 20.5 11.9 21.8 BD-6 MEAN 10.7 21.5 was means calculated by of the equation *Organic content

Organic content*

2.0 3.4 2.9 2.7 2.6 1.7 2.6 (C) = (A)

-

(C) 4.9 3.6 3.3 4.5 5.3 3.5 4.7

3.8 2.9 2.4 2.5 3.3 3.7 3.1

11.5 11.3 11.0 10.6 11.0 12.6 11.3

17.5 18.7 19.9 18.8 17.2 18.1 18.4

(B).

A

_-*i' i \< 1 , @* IIIIIfff1f1I 111111 35 40 25 30

20

45

1.1

50

1515 5

*

B

*

*

20

cm1 Fig. 2-Infrared absorption spectra for untreated (A) and NaClO-treated (B) dentin samples (No. BD-3). C, Infrared absorption spectrum of human enamel. = 3.7085 nm (Gopal et al., 1974), rather than to that of calcium-whitlockite, a = 1.0439 nm and c = 3.7375 nm (Dickens et al., 1974). This result indicated that the whitlockites contained some magnesium ions in their crystal structures. The removal of magnesium ions from the dentin sample by NaClO treatment was checked by atomic absorption spectroscopy. Magnesium contents of the solutions after treatment varied between 0.03 ppm and 0.20 ppm (Table 3). The NaClO reagent solution contains about 0.01 ppm magnesium. Therefore, it was clear that magnesium ions were removed from the dentin sample by the NaClO treatment.

I I

25

1

30

I II 35

40

I II 45

50

I

55

Diffraction Angle (CuKc,2e) Fig. 3-X-ray diffraction patterns for untreated (A) and NaCIO-treated (B) dentin samples (No. BD-2). Peaks marked by asterisks are for the internal standard, KCI. Arrowed peaks are those of calcite.

Discussion. The DTA pattern of the untreated dentin samples was basically identical to the DTA curves of the dentin reported by Brauer et al. (1970), Lezovic et al. (1975), and Aoki et al. (1977). The average weight loss between R.T. and 200TC was 10.7 wt% for the untreated dentin samples. This value is similar to the moisture content of 10.0 wt% for the whole dentin (Burnett and Zenewitz, 1958) and also to the value of 9.7 wt%, which is the weight loss of the dentin between 20TC and 228TC


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TABLE 2 X-RAY DIFFRACTION DATA FOR UNTREATED DENTIN SAMPLES Unheated Heated at 10000C whitlockite apatite apatite c-axis a-axis c-axis c-axis a-axis (002)

Sample No. FWHM* (nmt) 0.690 BD-1 0.51 0.689 BD-5 0.54 BD-2 0.43 0.689 0.689 BD-3 0.50 0.42 0.690 BD-4 0.689 BD-6 0.63 0.51 0.689 MEAN HA: Ca-TCP$ Mg-TCP§ *Full width at the-half-maximum of the (002) peak. tCalculated from the (002) peak position. tHydroxyapatite; Young and Holcomb (1982). ,l-Ca3 (PO4)2; Dickens et a!. (1974). *Mg-whitlockite; Gopal et al. (1974).

(nm) 0.9420 0.9424 0.9421 0.9422

(nm) 0.6877 0.6882 0.6881 0.6885 0.6884 0.6880 0.6882 0.68800

0.9420 0.9424 0.9422 0.94184

(nm) 1.0356 1.0358 1.0364 1.0366

(nm)

1.0363 1.0358 1.0361

3.715 3.715 3.717 3.711 3.716 3.715 3.715

1.0439 1.0350

3.7375 3.7085

September 1988

Wh/Ap peak ratio 0.72 0.68 0.59 0.48 0.39 0.30 0.53

TABLE 3

MAGNESIUM CONTENTS IN THE NaClO SOLUTION AND X-RAY DIFFRACTION DATA FROM NaCIO-TREATED DENTIN SAMPLES Heated at 1000°C Unheated Solution apatite magnesium apatite c-axis a-axis calcite c-axis content Sample (002) FWHM* No. (ppm) (nmt) formationt (nm) (nm) ++ BD-1 0.20 0.46 0.690 0.9420 0.6885 0.690 + 0.15 0.45 BD-5 0.9419 0.6883 BD-2 0.48 +++ 0.03 0.689 0.9420 0.6882 ++ 0.05 0.45 BD-3 0.690 0.9417 0.6887 0.690 ++ 0.9417 BD-4 0.04 0.40 0.6883 0.690 0.9417 + 0.03 0.57 BD-6 0.6880 0.47 MEAN 0.08 0.690 0.9418 0.6883 HA' 0.94184 0.68800 *Full width at the half-maximum of the (002) peak. tCalculated from the (002) peak position. tIncreasing order: +, + +, + + +. ¶Hydroxyapatite: Young and Holcomb (1982).

due to dehydration of the adsorbed water (Holager, 1970). Therefore, the weight losses between R.T. and 200NC and the first endothermic peak were attributable to dehydration of water adsorbed on the samples. The slight increase, rather than decrease, in the weight loss over this temperature range after the NaClO treatment suggested an increase in the surface areas of the dentin crystals for adsorption of water molecules, because the organic material, which acted as an apparent reservoir of adsorbed water, was lost. The DTA study showed that a strong exothermic peak was observed at 320°C for the untreated dentin samples, whereas it was absent in the NaCIO-treated dentin samples. Brauer et al. (1970) assigned the exotherm in the DTA curve of dentin at 400-5000C to the decomposition of organic constituents, since their anorganic whole-tooth sample showed no peaks over this temperature range. From the results of chemical analysis, Lezovic et al. (1975) attributed the thermal reaction of dentin over this temperature range mainly to the decomposition of organic materials. Therefore, the exothermic peak at 320°C in this study can be attributed to the burn-off of organic materials in the dentin sample. The DTA results showed that the organic materials in the dentin samples were completely removed by the NaClO treatment.

The weight loss between 200°C and 600°C was 21.5 wt% on average for the untreated dentin samples. This value was similar to the weight loss of 21.3 wt% between 228°C and 548°C for dentin, reported by Holager (1970). The amount of weight loss was remarkably decreased to 3.1 wt% on average for the NaCIO-treated samples. Therefore, the organic content was calculated by subtraction and found to be 18.4 wt% on average for the dentin samples. The calculated values were similar to that for a representative organic content of 20 wt%

(Eastoe, 1967).The results of IR analysis indicated a decrease in the relative intensity of the 1650 cm-' band after NaCIO treatment, in agreement with similar results obtained by Emerson and Fischer (1962) for dentin deproteinized with ethylene diamine. The weakening of the band at 1650 cm-' after NaClO treatment suggested that the band was due to certain organic ions. The band at 1650 cm-' can be assigned to the band of amide I (C = 0 stretching). However, the 1650-cm - 1 band survived as a weak band after NaClO treatment. The IR spectrum of the treated dentin sample was similar to that of enamel. As to the IR spectrum of enamel, LeGeros et al. (1969) assigned a band at 1630 cm-1 to the vibration of OH and the bands at 1400-1600 cm -1 to the vi-


CHANGES IN DENTIN MINERAL WITH NaCIO

Vol. 69 No. 7 *

A

*

*

20

30

25

B

35

40

-1

45

50

55

*

1233

diffraction showed the presence of calcite in the treated samples. Calcite shows a strong endothermic peak at 800'C accompanied by a larger weight loss. TG-DTA examination of a mixed sample of calcite and synthetic hydroxyapatite has also shown a strong endotherm at 800'C due to the decomposition of calcite. Therefore, the endothermic reaction of calcite formed by NaClO treatment was presumably hidden in the weak endotherms of more than 7000C, and the endothermic reactions at 660'C and over 700'C in this study were not due to the reaction of calcite or to that of a mixture of calcite and hydroxyapatite. From the TG and differential TG analysis of human dentin, Lim and Liboff (1972) assigned the peak of weight loss between 700'C and 800'C to the transformation of hydroxyapatite to P-tricalcium phosphate. However, our x-ray diffraction study showed that whitlockite was present at the untreated/ heated dentin samples but not at the treated/heated dentin samples. Therefore, the endotherms over 600'C were not directly attributed to the transformation of hydroxyapatite to whitlockite.

X-ray diffraction studies showed that the reaction occurring

20

11111111111111111111111111111 1111111 25 30 35 40 45 50 55 Diffraction Angle (CuKo(,20)

Fig. 4-X-ray diffraction patterns of dentin samples after being heated at 1000TC. A, Untreated sample. B, NaCLO-treated sample (No. BD-1). Peaks marked by asterisks are for the internal standard, KCl. Arrowheads

upon NaClO treatment involved the crystallinity of the dentin apatite and suggested that recrystallization took place. It is assumed that the dentin crystals undergo substitution of certain ions in the crystal lattice upon the NaClO treatment which changes their thermal behavior. X-ray diffraction also revealed that calcite was formed after NaClO treatment. In IR analyses, calcite generally shows a sharp band at 872 cm - and a strong and broad band at 1420 cm-' (White, 1974). Therefore, the bands at 870 cm-1 and part of the broad band at 1400-1480 cm -in the IR spectrum of the treated dentin sample (Fig. 2B) could be assigned to

those of calcite. The source of the calcium and carbonate ions in the formation of calcite seemed to be the dentin apatite. Crowell et al. (1934) proposed a tentative chemical formula of brations of carbonate ions in the apatite crystal structure. These assignments can also be applied to the IR bands of the dentin 3Ca3(PO4)2.CaCO3.Ca(OH)2 for the inorganic residue left after the deproteinization of dental tissues by the KOH-glycol method. samples in this study. The present study, however, did not show a constant amount It has been shown that the carbonate ions in enamel apatite of calcite. The amount of magnesium ions removed also difoccupy two sites in the apatite structure, the OH site and the fered among the samples. These results indicated a variety of P04 site (Elliott, 1964; LeGeros et al., 1969). The present IR chemical compositions among the dentin samples. Driessens method was not sufficiently sensitive to clarify the sites of and Verbeeck (1985) postulated from thermodynamic considoccupancy of the carbonate ions, but from the similarity between the IR spectra of dentin and enamel it is suggested that, erations that a few percent of dolomite, CaMg(CO3)2, may be in dentin apatite, carbonate ions also occupy the same two present in tooth enamel. By analogy with the situation in enamel, dentin may also contain dolomite. However, it remains to be sites in the apatite structure. TG analysis of the NaCIO-treated dentin samples showed proved. In this study, there was no evidence for the presence of any that weight losses occurred at temperatures of more than 200TC. other form of calcium carbonates apart from calcite. This obThis result indicated a removal of ions from the dentin crystals. servation agreed with the thermodynamic consideration that TG analysis of enamel has consistently shown some percentage calcite is the most stable phase among the calcium carbonate loss of weight at temperatures above 200'C (Little and Caspolymorphs under conditions of 0-500C at 1 atm (Usdowski, ciani, 1966; Holager, 1970, 1972; Corcia and Moody, 1974; Davidson and Arends, 1977; LeGeros et al., 1978; Holcomb 1982). However, it is also known that a solution containing and Young, 1980; Sakae et al., 1984). LeGeros et al. (1978) magnesium favors the formation of aragonite rather than calcite and Holcomb and Young (1980) suggested that lattice water (Mackenzie et al., 1983). The NaClO solution was enriched and carbonate ions evolved from the crystals at temperatures with magnesium ions after the treatment. Therefore, the formation of calcite could be explained by the segregation or above 200'C. By analogy with the situation in enamel crystals, we attribute the weight losses that occurred at temperatures of decomposition of dentin crystals in which the calcium, carmore than 200'C to the removal of carbonate ions and of lattice bonate, and magnesium ion constituents were removed from the crystals, leaving insufficient time for the magnesium ions water from the dentin crystals. to affect the polytypism of the calcium carbonate. However, a difference was observed in the amount of weight The carbonate ions in the dentin apatite or calcite did not loss between the untreated and NaCIO-treated dentin samples seem to play an important role in the formation of whitlockite, over a temperature range of 600-1000'C. Within this temperbecause in this study the NaClO-treated dentin sample which ature range, the DTA curves of both the NaClO-treated and contained the segregated calcite did not form whitlockite after untreated dentin samples showed an inclination toward the endothermic side at temperatures of more than 700'C and a weak being heated at 10000C, while the untreated sample, presumendothermic peak at 660'C for Downloaded the treated ably containing magnesium ions, did so. An x-ray examination samples. The x-ray from jdr.sagepub.com by guest on July 10, 2011 For personal use only. No other uses without permission. indicate the peaks for whitlockite.

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showed that the calcite/synthetic hydroxyapatite mixture produced calcium oxide and hydroxyapatite on being heated. The unit cell dimensions of the whitlockite crystals indicated that the whitlockites were not pure tricalcium phosphate but contained magnesium substituted for calcium in certain amounts. A thermal study of enamel has suggested that the first whitlockite crystals to appear upon being heated were richer in magnesium ions than those formed after further subsequent heating (Sakae and Hirai, 1984). These results strongly suggest that magnesium ion is one of the most important factors involved in the formation of whitlockite from dentin apatite as well as enamel apatite.

Acknowledgments. The authors thank Prof. G. Hirai, Department of Anatomy, Nihon University School of Dentistry at Matsudo, for his helpful advice, and Dr. A. Fujii, Department of Pharmacology, for his technical advice on operation of the atomic absorption spectrometer. REFERENCES AOKI, H.; BAN, T.; AKAO, M.; KATO, K.; and IWAI, S. (1977):

Thermal Analysis of Calcified Tissues, Rept Inst Med Dent Eng 11:27-31. BOYDE, A. and HOBDELL, M. H. (1969): Scanning Electron Microscopy of Primary Membrane Bone, Z Zellforsch 99:98-108. BRAUER, G.M.; TERMINI, D.J.; and BURNS, C.L. (1970): Characterization of Components of Dental Materials and Components of Tooth Structure by Differential Thermal Analysis, J Dent Res 49:100-110. BURNETT, G.W. and ZENEWITZ, J.A. (1958): Studies of the Composition of Teeth. VII. The Moisture Content of Calcified Tissues, J Dent Res 37:581-589. CORCIA, J.T. and MOODY, W.E. (1974): Thermal Analysis of Human Dental Enamel, J Dent Res 53:571-580. CROWELL, C.D.; HODGE, H.C.; and LINE, W.R. (1934): Chemical Analysis of Tooth Samples Composed of Enamel, Dentine, and Cementum, J Dent Res 14:251-268. DAVIDSON, C.L. and ARENDS, J. (1977): Thermal Analysis Studies on Sound and Artificially Decalcified Tooth Enamel, Caries Res 11:313-320. DICKENS, B.; SCHROEDER, L.W.; and BROWN, W.E. (1974): Crystallographic Studies of the Role of Mg as a Stabilizing Impurity in 3-Ca3(P04)2 I. The Crystal Structure of Pure 1-Ca3(PO4)2, J Solid State Chem 10:232-248. DRIESSENS, F.C.M. and VERBEECK, R.M.H. (1985): Dolomite as a Possible Magnesium-containing Phase in Human Tooth Enamel, Calcif Tissue Ist 37:376-380. EASTOE, J.E. (1967): Chemical Organization of the Organic Matrix of Dentine. In: Structural and Chemical Organization of Teeth, A.E.W. Miles, Ed., New York: Academic Press, pp. 279-316. ELLIOTT, J.C. (1964): The Crystallographic Structure of Dental Enamel and Related Apatites, Ph.D. Thesis, University of Lon-

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