the characterization of enamel surface demineralization, remineralization, and associated hardness...

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Arch oral Biol. Vol. 14 :pp. 1407-1417. 1969. Pergamon Press. Printed in Great Britain. THE CHARACTERIZATION OF ENAMEL SURFACE DEMINERALIZATION, REMINERALIZATION, AND ASSOCIATED HARDNESS CHANGES IN HUMAN AND BOVINE MATERIAL F. FEAGIN, T. KOULOURIDES and W. PIGMAN University of Alabama in Birmingham Institute for Dental Research and Department of Physiology and Biophysics, and New York Medical College, New York, N.Y. 10029, U.S.A. Smnmary-Enamel demineralization and remineralization were investigated in relation to change of surface microhardness. Within the investigated range of 120 hardness units from the initial, mineral loss or gain were reflected in parallel, linear changes of microhardness. Changes of one hardness unit corresponded to about0*04 pmoles of Ca’+/cm’of test surface. The molar Ca/P ratio was l-65 for both the demineralization as well as the remineralization process; this result indicated a stoichiometric dissolution and redeposition of hydroxyapatite in the enamel. On the basis of calculations of indenter penetration, softened enamel was shown to have gradients of mineral density from the surface inwards depending upon the demineralization method. The softening and rehardening were shown to take place in the outer 5 p of enamel surface. The acid resistance of the rehardened enamel was similar to that of the original. INTRODUCTION HEAD (1912) proposed that the hardness of enamel is a function of the degree of mineralization. He showed that enamel hardness decreases with weak acid treatments and increases after its exposure to saliva. Later reports indicate that tests of surface microhardness provide a sensitive measure of mineral loss (CALDWELL et al., 1958). Using the same method KOULOURIDES, CUETO and PIGMAN (1961); PIGMAN, CWTO and BAUGH(1964) ; KOULOURIDES, FEAGINand PIGMAN (1965) described conditions affecting the rehardening of softened enamel surfaces. An indication of parallelism between calcium loss and decrease in hardness during weak acid softening of enamel surfaces has been reported (KOULOURIDES and REED, 1964). By morphological evi- dence, MUHLEMANN, LENZ and ROSSINSKY (1964) attempted to relate hardness and electron microscopic evidence of demineralization and remineralization of enamel. Using different conditions for both demineralization and remineralization, they found that demineralization produced distinct subsurface intercrystalline spaces. After remineralization these spaces were filled with an amorphous precipitate unlike the original crystals of enamel. Recent papers have reviewed the problems and presented concepts of enamel remineralization (KOULOURIDES, 1968 ; LENZ, 1967; FRANK, 1967). Apparently, demineralization of enamel by weak acids causes a reduction in crystal size with result- ing increases in the volume of microspaces within the enamel (LENZ, 1967; DARLING 1407

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Arch oral Biol. Vol. 14 :pp. 1407-1417. 1969. Pergamon Press. Printed in Great Britain.

THE CHARACTERIZATION OF ENAMEL SURFACE DEMINERALIZATION, REMINERALIZATION, AND

ASSOCIATED HARDNESS CHANGES IN HUMAN AND BOVINE MATERIAL

F. FEAGIN, T. KOULOURIDES and W. PIGMAN

University of Alabama in Birmingham Institute for Dental Research and Department of Physiology and Biophysics, and New York Medical College,

New York, N.Y. 10029, U.S.A.

Smnmary-Enamel demineralization and remineralization were investigated in relation to change of surface microhardness. Within the investigated range of 120 hardness units from the initial, mineral loss or gain were reflected in parallel, linear changes of microhardness. Changes of one hardness unit corresponded to about0*04 pmoles of Ca’+/cm’of test surface. The molar Ca/P ratio was l-65 for both the demineralization as well as the remineralization process; this result indicated a stoichiometric dissolution and redeposition of hydroxyapatite in the enamel. On the basis of calculations of indenter penetration, softened enamel was shown to have gradients of mineral density from the surface inwards depending upon the demineralization method. The softening and rehardening were shown to take place in the outer 5 p of enamel surface. The acid resistance of the rehardened enamel was similar to that of the original.

INTRODUCTION

HEAD (1912) proposed that the hardness of enamel is a function of the degree of mineralization. He showed that enamel hardness decreases with weak acid treatments and increases after its exposure to saliva. Later reports indicate that tests of surface microhardness provide a sensitive measure of mineral loss (CALDWELL et al., 1958). Using the same method KOULOURIDES, CUETO and PIGMAN (1961); PIGMAN, CWTO and BAUGH (1964) ; KOULOURIDES, FEAGIN and PIGMAN (1965) described conditions affecting the rehardening of softened enamel surfaces. An indication of parallelism between calcium loss and decrease in hardness during weak acid softening of enamel surfaces has been reported (KOULOURIDES and REED, 1964). By morphological evi- dence, MUHLEMANN, LENZ and ROSSINSKY (1964) attempted to relate hardness and electron microscopic evidence of demineralization and remineralization of enamel. Using different conditions for both demineralization and remineralization, they found that demineralization produced distinct subsurface intercrystalline spaces. After remineralization these spaces were filled with an amorphous precipitate unlike the original crystals of enamel.

Recent papers have reviewed the problems and presented concepts of enamel remineralization (KOULOURIDES, 1968 ; LENZ, 1967; FRANK, 1967). Apparently, demineralization of enamel by weak acids causes a reduction in crystal size with result- ing increases in the volume of microspaces within the enamel (LENZ, 1967; DARLING

1407

1408 F. FEAGIN, T. KOULOVRIDES AND W. PIGMAN

et al., 1961; SMITH and SHACKLEFORD, 1968). This porosity of partially demineralized enamel indicates that components of enamel dissolved preferentially (POSEN, 1962; DARLING, 1958; JOHANSEN, 1965; COOLIDGE, 1965).

Conversely, in remineralization the microspaces are filled with minerals derived from equilibrating mineralizing solutions. Since these mineral changes of enamel are assumed to be reflected in changes of microhardness, a quantitative definition of demineralization and remineralization in terms of hardness is desirable. This was the purpose of the present study. Calcium and phosphate changes were measured and the Ca/P ratio used as a criterion of the mineral phase involved.

MATERIALS AND METHODS

Preparation of enamel surfaces and hardness measurements

Parallelogram shaped enamel slabs of precise dimensions were cut from adult human and bovine teeth with a Gillings-Hamco thin sectioning machine. Initially, O-25 cm2 (bovine) surfaces were prepared on the enamel slabs, and the surface hard- ness was measured with a Kentron microhardness tester and Knoop indenter as described by KOULOCIRIDES et al. (1961). Except for the test surface, exposed enamel was sealed with wax.

Average Knoop Hardness Numbers (KHN) of 5 indentations per surface have been reported to represent statistically the hardness of enamel prepared by this method (PICKJXL et al., 1965). In this work 5 indentations were made initially on each enamel surface. After softening and/or rehardening an indentation was made within 60 p of each initial indent. The average of the hardness changes for each surface showed little variability. Average hardness changes of at least 10 enamel surfaces were used for each value presented in this work. Standard deviation (S.D.) of the mean of the hardness changes for each experimental group are presented.

Depths of penetration of the hardness indenter into the enamel surface was calcu- lated from the measured length of the indentations. The Knoop indenter has a pyra- midal shape and the lengths of indents made by vertical drops onto flat surfaces are 30 times the depth of indentation (KNOOP, PETERS and EMERSON, 1939). Depths of indenter penetration were determined for loads of 1,4, 10,25, 50, 100, 300 and 500 g.

Demineralizing and remineralizing procedures

Enamel surfaces were demineralized (softened) in 0.001 M potassium acetate buffer, pH 5 *5, at 37°C (KOULC~~RIDES et al., 1961). Surfaces used for remineralization studies were softened 100-120 KHN in excessive volumes of acetate buffer with stirring. Usually 4-5 hr were required for this amount of softening. When changes of calcium, phosphorus and hardness were measured during enamel softening, individual surfaces were exposed to 8 to 15 ml acetate buffer for 8 to 48 hr without stirring. Groups of individual softening surfaces were exposed to identical volumes of buffer for the same length of time.

Remineralizing solutions contained 1 to 3 mM calcium, Ca/P 1.67, 0.05 mM fluoride, O-15 M sodium chloride, and were adjusted to pH 7 *3 (PIGMAN et al., 1964).

THE CHARAcTERIzAnoN OF ENAMEL SURFACE DE MINERALIZATTON 1409

A ratio of enamel surface area to volume of remineralizing solutions of 0 -25 cm2 to 1.0 ml was maintained for solutions used for chemical analysis and hardness change. The system shown in Fig. 1, with two enamel surfaces exposed to 2 -0 ml of l-5 mM calcium solution, was used for remineralization at 37°C with gentle shaking for periods of l-6 hr. When remineralization was determined by hardness change alone, the sur- face to volume ratio was about 1 cm*/500 ml (PIGMAN et al., 1964).

Methods of calcium and phosphorus analysis

Changes of the concentration of calcium in the demineralization and remineraliza- tion solutions were measured with the calorimetric o-cresolphthalein complexone method (SARKAR and CHAUHAN, 1967). Amounts of calcium from 2 to 6 pg were linearly related to optical density between O-2 to O-6 units. Phosphorus concentrations were measured by the calorimetric method of CIXEN, TORIBARA, and WARNER (1956) over the range of 3-12 pg. The calcium and phosphorus methods were accurate within 1 per cent. The accuracy of the analyses was checked during each experiment by analysis of a standard synthetic apatite solution. The Ca/P ratio range for a number of such solutions varied from 1 a64 to 1.70 and averaged 1.67.

Enamel uptake of calcium and phosphorus during rehardening was determined by calculating the differences of concentrations between the averages for control solutions and experimental solutions. Control solutions were identical to experimentals without

of tube -6mm

Rubber stopper Icm

FIG. 1. System used for remineralization of enamel surfaces with the maximum surface to volume ratio. Two enamel slabs inserted into the test tube containing the mineralizing

solution.

1410 F. mAGIN, T. KOULOURD~B AND W. PraMAN

enamel surfaces in the system (Fig. 1). The Ca/P ratio of mineral gain during reharden- ing was calculated for the residual solution after rehardening and by mineral uptake.

RESULTS

Calcium, phosphate, and hardness changes during demineralization and remineralization of enamel surfaces

Table 1 presents a summary of demineralization studies on enamel surfaces from bovine incisors. Each surface was exposed to 8, 12 or 15 ml acetate buffer from 8 to 48 hr. The surfaces of each group were demineralized for the same volume and time.

TARLE 1. CALCIUM, PHOSPHORUS, AND HARDNESS LOSS DURlNG DEMlNERAUZA-IlON OF GROUPS OF

ENAMEL SURFACES

No. of enamel KHN surfaces decrease

25 33f 5

Z 51* 66f 4 5 39 86f 6 14 109 f 10 10 126 f 11

Micromoles loss/cm2 from enamel surface Calcium Phosphorus

1.31 f 0.33 O-77 f O-16 2.30 f O-30 l-42 + 0.20 2.77 f 0.40 1.73 f o-25 3.83 f O-24 2.29 f O-22 4-80 f O-60 2.99 & O-40 5.56 f 0.70 3-46 & 0.45

Molar Ca/P

l-70 f o-03 l-61 f 0.03 l-60 f o-04 l-67 f O-19 1.61 f O-05 1.61 f O-10

Individual enamel surfaces (O-25 cm2, bovine) demineralized in 8 to 15 ml acetate buffer, pH 5.5, 37”C, without stirring, for 848 hr. Enamel surfaces of each group were demineralized in the same volume for the same length of time.

The time of exposure to the acetate buffer was determined by hardness testing of con- trol surfaces until the enamel surfaces softened the intended amount. The KHN decreases were found to be linearly related to the calcium and phosphate losses from the enamel surfaces under these conditions. The molar Ca/P ratio for the mineral loss from groups of enamel surfaces varied from l-60 to 1.70 and averaged 1.64. A 100 KHN decrease corresponds to a calcium loss of approximately 4 pmoles/cm* enamel surface. Calcium losses of enamel surfaces from human teeth gave similar results (not presented).

A summary of data for a series of remineralization experiments is presented in Table 2. The enamel surfaces from bovine teeth were demineralized in an excess of acetate buffer with stirring (KWLOURIDES et al., 1961) until hardness testing showed an average hardness decrease of about 100 KHN (4-5 hr). KHN increases of enamel surfaces, two surfaces per sample, were measured and related to calcium and phospho- rus losses from remineralizing solutions of 1.5 mM calcium ion concentration (Ca/P 1.67). The extent of remineralization of the groups of enamel surfaces was controlled by the time of exposure to the solutions as indicated in Fig. 3. Hardness increases correspond linearly to calcium gains until about 80 per cent of complete rehardening (line 5, Table 2). Slight surface crusting was seen microscopically as the rehardening

THECHARACIgWAnONOFENAMELSURFACEDEMINERALIZAnON 1411

TBLB 2. CALCIUM AND PHOSPHORUS C&UN AND HARDNESS INCREASE OF QROUPS OF R-zED

ENAMELSURFACES

No. of samples Hr

Enamel uptake pmoles/cm’

Calcium Phosphorus

After rehardening remaining solution

Ca/P

13 1 21* 3 0.666 *o-o5 - - 7 2 41* 5 1.51 f 0.17 0.95 f 0.10 1.68 f 0.03

24 : 72 f 10 2.66 & 0.09 1.64 f 0.16 1.69 * 0.03 8 80& 5 2.90 f0.14 1.59 f 0.14 l-54 f o-05

11 6 91* 9 3.86 & l-9 2.38 f O-32 1*66&0*04

Enamel surfaces mesoftened 100-120 KHN with excessive volumes of 0.001 M acetate buffer, pH 5-5, with stirring (4-5 hr).

Mineralizing solution: l-5 mM calcium (Ca/P l-67); O-05 mM fluoride; O-15 M sodium chloride; pH 7-3; 37°C. Each value is the average for 2 enamel surfaces (O-25 cm’, bovine) remineralized in 2 ml solution l-6 hr.

approached 100 per cent. The Ca/P molar ratios of the calcifying solutions after reminer- alization were close to 1.67, except for the solution indicated in line 4 (Ca/P l-54) which may suggest an error in the phosphorus analysis for this group.

The calcium and phosphate gained by the enamel was calculated by taking the differences between calcium and phosphorus contents of an equal number of control solutions, systems without enamel surfaces, and solutions after remineralization. As the errors of the analyses are compounded by subtractions, only the Ca/P ratios of the solutions after rehardening are presented.

A comparison of demineralization properties of previously untreated bovine enamel surfaces (A) with those that had been demineralized and remineralized (B) is shown in Table 3. The enamel surfaces of group (B) were initially demineralized 117 KHN and remineralized 87 KHN (75 per cent recovery) by the methods described

TABLE 3. COMPOSITIONOFMINERALREMOVED DIJRINGDEMINERALIZATIONOFREHARDENED

ENAMEL SURFACES

Knoop hardness change

Initial After Subsequent Enamel mineral loss No. demineral- remineral- demineral- (p moles/cm2 Molar

samples ization ization ization Calcium Phosphorus Ca/P

(A) 20 - - -61 f 9 2.63 & 0.30 1.60 f 0.17 1.64 + 0.06

(B) 20 -117 f 16 +87 f 10 -52f5 2.26 50-15 l-39 f0.10 1.63 ~kO.05

Initial demineralization of enamel surfam (0.25 cm*, bovine) in excessive volumes of acetate buffer, pH 5-5 with stirring (4-5 hr). Subsequent demineralization in 0.001 M acetate buffer, 8-O ml/enamel for 12 hr without stirring.

Remineralization solution: 1.5 mM Ca*+ (Ca/P l-67); O-05 mM F-; O-15 M NaCl; pH 7.3. Enamel surfaces remineralized 4 hr with 5 surfaces per 500 ml solution.

1412 F. FEAGIN, T. KOUL~URIDES AND W. RGMAN

by PIGMAN et al. (1964). Each enamel surface of groups (A) and (B) was then demineral- ized in 8 -0 ml acetate buffer for 12 hr. The hardness of the untreated enamel surfaces (A) decreased a small amount more than the remineralized surfaces (B). This difference in hardness loss was reflected by a greater loss in calcium and phosphate from the un- treated enamel (A). The molar Ca/P ratios of the dissolved mineral for the two groups were the same, 1.64. The presence of O-05 mM fluoride ions in the mineralizing solution accounts for a small difference in the demineralizing characteristics (FEAGIN, KOULOURIDES and PIGMAN, 1969).

Characterization of sound, softened and remineralized enamel surfaces by the depth of penetration of the Snoop hardness tester

Figure 2 is a plot of the logarithm of the weight on the Knoop indenter and the penetration, in microns, into the test surface of sound softened and remineralized enamel surfaces (bovine and human). The differences between the depths of penetra- tion for each load on the indenter were almost constant over the range of weights used, i.e., about l-7 p between the untreated enamel surface and the softened surface, and 0 * 3 p between the untreated and the remineralized surface. The primary region of surface change during hardening and softening was in the outermost 2 TV of the enamel. This showed that the resistance of enamel to the penetration of the indenter progressively increased with the depth from the test surface. The outermost 1.7 p of the demineralized surface offered little resistance to the indenter. For the enamel sur- faces demineralized by bacterial action, the outer surface layers were softened to a less extent than the underlying regions, an indication of subsurface demineralization.

I

6- o

Artificial mouth demineralized

Sound enamel

Remineralized enamel

Demineralized enamel

0 I.00 200

Log of indenter load, g

FIG. 2. The relation of the load on the Knoop indenter to the depth of penetration into enamel surfaces.

lXECHARAiXERlZATIONOPENAMELSuRFACEDEMlNERALlZA~ON 1413

Remineralization of ertamel surfaces softened in different ways

Table 4 shows the results of rehardening of enamel surfaces from human teeth softened by bacterial action (CALDWELL et al., 1958), bacterial action and acetate buffer, and acetate buffer alone. Remineralization of enamel surfaces softened by bacterial action was much less than for acetate buffer softened enamel, and remineraliz- ation stopped at 4 hr. Lowering the calcium concentration of the calcifying solutions or lowering the pH removed the block to rehardening and caused the rehardening process to continue over the time of the study. Additional softening of the bacterial softened enamel with acetate buffer caused the surface to continue rehardening in 3 .O mM calcium solutions.

CaZ + No. of

(mM/l) teeth

Hardness

decrease

Hardnessincrease(KHN)at:

2 hr 4 hr 6 hr 8 hr

O-5 12 125 10 + 6 20 5 12 36&12 - 1.0 11 130 12 f 8 285 8 48& 6 56 i 16 1.5 8 113 32 + 4 6Oi 4 96&12 - 3.0 12 135 20 rt 8 36 f 12 36 f 12 - 3*0* 8 128 36 f 8 525 8 60&12 - 3*0t 8 111;144 38 f 8 72 i 12 96 f 12 120& 8 3.0: 15 120 74 f 8 92*10 - -

Calcifying solution: Ca/P 1.67; F-, 0.05 mM/l; NaCI, 150 mM/l; pH 7.3; temp. 37°C; solutions changed every 2 hr.

* Calcifying solution, pH 6.8.

t Enamel surfaces additionally demineralized with 0.001 M acetic acid, pH 5.5 (1 hr), excessive volumes/surfaces and stirring.

!: Control enamel surfaces demineralized with 0.001 M acetic acid, pH 5.5 (4 hr), excessive volumes/surface and stirring.

Figure 3 shows the results obtained from the use of mineralizing solutions of different calcium concentrations (Ca/P 1.67) for the rehardening of enamel softened with acetate buffers. Each point presents the hardness increase of 10 bovine and 10 human enamel surfaces exposed to 500 ml of remineralizing solution. Unlike the entire linear relationship of the 1 *O mM calcium solution, the 1.5 and 3 a0 mM calcium solutions were linear to about 75 per cent of complete hardness recovery. The results suggest that too rapid rehardening causes remineralization of the outermost enamel surface and limits diffusion into the interior of the softened structure. The remineraliza- tion characteristics of human and bovine enamel were the same.

1414 F. FEAGIN, T. K~~LO~RIDES AND W. PIGMAN

A I.0 mM Ca2+

x 1.5mM Ca2+ o 3.OmM Ca2+

Rehardening time, hr

Solution: Co/P 1.67M; 0.05 mMF_0~15M NaCl ; pH 7.3; 37T

Enamel surfaces presoftened 100 to 120 KHN

FIG. 3. Rate of rehardening of enamel surfaces in calcifying solutions at different calcium concentrations.

DISCUSSION

Hardness decrease of enamel surfaces as related to mineral loss

Partial demineralization of enamel has been described as enlargement of micro- spaces within the ultrastructure arising from complete or partial dissolution of crystals (DARLING et al., 1961). In the present study, penetration of the hardness indenter as a function of the load suggests uniform distribution of microspaces in sound enamel unlike the distribution in softened enamel. With enamel demineralized by acetate buffer, the principal change of mineral density occurred in the outermost 2 I_L of the surface with gradients of smaller mineral losses to the depth of unaffected tissue

(5-7 A+ On the basis of enamel density (2.9) and its calcium content (35 per cent) each

micron layer of 1 cm* surface area contains about

(2.9 x 10m4 g/u/cm*) X

(35 g)

(40 g/M) (100 g enamel) = 2.54 x low6 M Ca, or

2.54 p moles of calcium. The loss of 4 p moles of calcium corresponding to about a 100 KHN decrease suggests extensive, but not complete, dissolution of the outer 2 p layer of enamel surface. The outermost 2 p of softened enamel surface, however, main- tains a rigid structure which contains calcium and phosphate ions in the approximate ratio of hydroxyapatite (unpublished data). Since the original volume of the demineral- ized enamel persists, the values showing loss of sufficient calcium to account for the total amount in the outer 2 ,u indicates dissolution below this depth.

THECHARACZERIZATlONOPENAMELSURFACEDEh4INERALUATION 1415

Enamel softening produced by bacterial action presents a dissimilar case. The difference between initial and hnal depths of penetration increases with the heavier loads. The outermost layers of the test surface resist the weight of small loads. However, contribution of this outer surface resistance toward the pressure of 500 g is not evident. This type of surface preservation, as discussed earlier by one of us (KOULOURIDES, 1968), indicates that the density profiles of demineralized enamel can be different, depending on the conditions of demineralization.

The molar Ca/P ratio of the mineral removed from the enamel surfaces during demineralization indicates stoichiometric dissolution of hydroxyapatite, as reported earlier (DARLING, 1967; SMITH er al., 1968). The linear relationship between decrease in enamel hardness and calcium loss or gain shows that hardness measurements can be used as an indication of the degree of enamel mineralization. A necessary condition for these studies is the preservation of the original test surface which can be followed by observing the initial indentations. Loss of surface is reflected in distortion and decrease in size of the initial indentations.

Remineralization

The increases of hardness and the corresponding uptake of calcium and phosphate ions by softened enamel from mineralizing solutions show that rehardening is remineralization. The solid that forms in the mineralizing treatment is a calcium phos- phate with Ca/P ratio of hydroxyapatite (l-67). On subsequent demineralization, remineralized enamel gave about the same results as sound enamel indicating similar composition and crystal size. Electron microscopy of rehardened enamel showed filling of sz&wr$zce microspaces by an amorphous solid (MUHLEMANN et al., 1964). The conditions of these experiments may differ regarding the depths of indenter penetration into the enamel surface (5 CL), areas studied by electron microscopy (to 100 TV depth) and the presence of carbonates in mineralizing solutions. The state of solid crystallinity under the conditions of enamel rehardening is a subject of con- tinuing investigation.

Complete rehardening of softened enamel occurs in solutions containing calcium concentrations of 1 to 3 mM, Ca/P l-67, and O-05 mM fluoride (Fig. 3). Increases of calcium concentrations caused greater initial rates of rehardening. Rates of growth of hydroxyapatite crystals are known to depend on the degree of supersaturation of the solution (POSNER, HARPER and MULLER, 1965b). The presence of 0.05 mM fluoride increases the rate of enamel rehardening (KOULOURIDES et al., 1961) and promotes crystallinity of apatite (POWER, EANES and ZIPKIN, 1965a). The rapid linear rates of rehardening, Ca/P ratio near 1.67 for mineral uptake during rehardening, and dissolu- tion character following rehardening suggests the formation of one mineral phase during the course of remineralization of partially demineralized enamel.

When rehardening was studied with an indenter load of 500 g it was found that the rehardening of the bacterial softened enamel was inhibited when compared to the usual buffer softened enamel under conditions of rapid remineralization. The evidence of subsurface softening by the bacterial action leads to an explanation of this poor rehardening. The outermost surface appears to remineralize and prevent diffusion into

1416 F. FEAGIN, T. KOULOURIDES AND W. PIGMAN

the deeper enamel layers. This explanation is strengthened by the small (1 hr) addi- tional acetate buffer softening of the bacterial softened enamel, which allowed rehardening of similar magnitude as obtained with buffer softened enamel.

The demineralization methods used in this study apparently involve dissolution of the more accessible ultrastructural enamel constituents as suggested by JOHNSON

(1966) and FOSDICK and HUTCHINSON (1965). After partial demineralization the residual structure preserves the volume of the original surface and serves as a calcifi- able matrice. Complete remineralization restores the density close to the original values, as shown by hardness recoveries.

Ackrtowledgements-This work has been supported by grants from the National Institutes for Dental Research, No. DE02537, and the American Chicle Company, Division of Warner-Lambert Pharmaceutical Company.

R&urn&La demineralisation et la remineralisation de l’dmail sont Btudiees en fonction des changements de micro-durete de la surface. Dans la gamme de 120 unites de durete ttudiee, la perte ou le gain mineral se traduit par des changements paralleles et lineaires de microdurete. Un changement d’une unite de microdurete correspond a environ 0,04 p moles de Ca2/cm2 de surface testee. Le rapport molaire Ca/P est de 1,65 pour le deminer- alisation et la remineralisation. Ce resultat traduit une dissolution et un depot stoechio- metrique de l’hydroxyleapatite. En se basant sur des calculs de penetration du diamant, Pernail ramolli presente des gradients de densite minerale, a partir de la surface vers l’interieur, en fonction de la methode de demineralisation. Le ramollissement et le redurcissement de la surface de l’email se produisent dans une couche de 5 p d’epaisseur. La resistance aux acides de l’email redurci est identique a celle dun Cmail normal.

Zusannnenfassnng-Schmelzdemineralisation und -remineralisation wurden in Bezie- hung zur Verlnderung der Oberflachen-Mikrohlrte untersucht. Innerhalb des untersuch- ten Bereichs von 120 Hlrteeinheiten spiegelte sich der Mineralverlust oder -gewinn in parallelen und linearen h;nderungen der Mikrohlrte wider. Anderungen im AusmaB einer Harteeinheit entsorachen etwa 0.04 Mmol Caz/cm2 der Testoberlllche. Das molare Ca/P-Verhaltnis- betrug sowohl ‘fur die Demmeralisation als such fur den Remineralisationsprozess 1,65. Dieses Ergebnis deutet auf eine stiichiometrische Auflosung und Wiederanlagerung von Hydroxylapatit im Schmelz hin. Nach Berech- nungen der Eindrticke zeigte der erweichte Schmelz, von der Demineralisationsmethode abhlngend, von der Oberhache zur Tiefe hin Unterschiede in der Mineraldichte. Es wurde gezeigt, da13 die Erweichung und Wiedererhlrtung in den lugeren 5 TV der Schmelzoberflache stattfand. Die Saureresistenz des wiedererharteten Schmelzes war der des ursprtinglichen Schmelzes Ihnlich.

REFERENCES

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CHEN. P. S.. JR.. TORIBARA. T. Y. and WARNER, H. 1956. Microdetermination of phosphorus. Analyt. dhem. ‘28, i756-1758.’

COLLIDGE, T. B. 1965. Biochemistry of the sheath spaces in caries. Ann. N. Y. Acad. Sci. 131,884-892. DARLING. A. I. 1958. Studies of the early lesion of enamel caries, its nature, points of entry, modes of

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DARLING, A. I., MORTIMER, K. Y., POOLE, D. F. C. and OLLIS, W. D. 1961. Molecular sieve behaviour of normal and carious dental enamel. Archs oral Biol. 5,251-273.

THE CHARACTERlzATION OF ENAMEL SURFACE DEMrNERALlzATION 1417

FEAGM, F., Kouroua~~ss T. and PIGMAN, W. 1969. Formation of hydroxyapatite under various condi- tions of remineralization of dental enamel. Archs oral Biol, in press.

FOSDICK, L. S. and HUTC~NSON, A. C. W. 1965. The mechanism of caries of dental enamel. Ann. N. Y. Acad. Sci. 131,758-770.

FRANK, R. M. 1967. The ultrastructure of the tooth from the point of view of mineralization, de mineralization, and remineralization. Znt. dent. J. 17, 661-683.

HEAD, J. H. 1912. A study of saliva and its action on tooth enamel in reference to its hardening and softening. J. Am. med. Ass. 59,2118-2122.

JOHANSEN, E. 1965. Comparison of the ultrastructure and chemical composition of sound and carious enamel from human permanent teeth. In: Tooth Enamel (edited by STACK, M. V. and FEARNHEAD, R. W.) pp. 177-182. John Wright, Bristol.

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