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Pergamon Press 1977. Punted m Great Bratam
PROGRESSIVE DEMINERALIZATION
STAGES OF SUBSURFACE OF HUMAN TOOTH ENAMEL
R. T. ZAHRADNIKand E. C. MORENO Forsyth Dental Center, 140 Fenway. Boston, Massachusetts 02115, U.S.A. Summary-Samples of intact human dental enamel were exposed to a lactate buffer known to produce subsurface demineralization. After exposure times of 0, 18, 36 and 72 h, the water vapour sorption isotherms were determined and the corresponding pore-volume distributions and specific surface areas calculated. Three distinct demineralization stages were recognizable. In the first stage, only’the larger peak in the bimodal distribution for intact enamel was affected; the second stage was marked by the beginning enlargement of the smaller accessible pores, finally, there was a dramatic increase in accessible pore volume and specific surface area with increase in the frequency but not in the size of the smaller pores preponderant in intact enamel. The last stage appears to be related to the removal of constrictions that hinder transport into the cores of the prisms. It is suggested that the constrictions are associated with organic matter which is thus intimately involved in incipient enamel demineralization.
INTRODUCTION Human dental enamel consists of prisms radiating from the dentine-enamel (DE) junction toward the outer surface (Meckel, Griebstein and Neal, 1965; Gustafson and Gustafson, 1967). Each prism consists of large numbers of small hydroxyapatite crystals mainly oriented axially along the prism; this parallel orientation does not extend to the boundary zones between adjacent prisms which are the regions with the highest organic content (Ronnholm, 1962; Meckel et al., 1965; Linden, 1968). The crystal arrangement of enamel gives rise to two main categories of pores corresponding to the spaces between single crystals in the prism cores and those between crystals in the region between prisms. Using isothermal water vapour sorption techniques, we established that intact human enamel has a bimodal pore-volume distribution (Moreno and Zahradnik, 1973; Zahradnik and Moreno, 1975); the larger pores are presumably related to spaces in the interprismatic regions and the smaller are probably associated with the spaces within the prisms themselves. There is little information on how the enamel pore structure changes with the onset of caries. Such information would be useful in determining acid-entry pathways and in establishing the enamel regions most susceptible to acid attack. Dibdin (1969) published preliminary results on porosity changes in ground enamel resulting from exposure to acid buffers. Using krypton gas sorption techniques, he found that the pore size distribution after acid exposure was bimodal with a new peak within the smallest calculated pore size range (Kelvin radii < 1.4nm). Significant increases in the specific surface area and porosity of the enamel powder were observed; an especially marked increase in the specific surface area of ground enamel occurred between one and two weeks of exksure to an acidified gel. No explanation of these observations was advanced. Several investigators (Nichol, Judd and Ansell, 1973; Simmelink. Nygaard and Scott, 1974) using
scanning and transmission electron microscopy (SEM and TEM) to study the effect of acid etching upon enamel structure, have shown that the crystals within the core of the enamel prisms are preferentially dissolved while the peripheral regions of the prisms remain relatively unaffected; this results in a honeycomb appearance of the enamel surface. There must be some differences in solubility which permit the structure to be brought out in relief. Nevertheless, incipient carious lesions do not develop by such surface etching but instead develop as a region of demineralization underlying a smooth surface layer which shows little or no damage (Darling, 1956; Gustafson, 1957), suggesting that other factors besides simple surface solubility differences must be involved. Studies of early carious enamel employing SEM (Kerckaert, 1973; Scott, Simmelink and Nygaard, 1974) show that the initial attack appears to occur along the prism boundaries. Enamel from more advanced lesions exhibits the classic demineralized prism cores with a significantly less affected prism boundary region. However, no information is available concerning the sequential changes that must occur in the properties of the pore structure of the enamel with a progressing carious lesion. Such a study, based on natural caries, would be extremely difficult, given the multifactorial nature of caries and the impossibility of controlling the rate of demineralization under in-Go conditions. The use of in-vitro models seems appropriate. .We have advanced a hypothesis (Moreno and Zahradnik. 1974) in terms of which the conditions of acidity can be predicted under which enamel subsurface demineralization and not surface dissolution should occur. This hypothesis was developed solely on the basis of the chemistry of calcium phosphates. A model was tested experimentally by exposing extracted teeth to acid buffers of predetermined composition; the results were in close agreement with our hypothesis, provided that appropriate consideration was given to transport rates of dissolved mineral away from the tooth surface. Furthermore, the struc585
R. T. Zahradnik and E. C. Moreno
586
ture of the demineralized enamel was consistent with the morphology we have referred to for natural incipient carious lesions. Our purpose here was to measure the sequential changes in the properties of the pore structure of dental enamel when the tissue is demineralized. MATERIALS
AND METHODS
Four human teeth with clinically sound enamel were thin-sectioned parallel to their labial surfaces using a water-cooled sectioning device and a diamond disc. The enamel slices (approximately 250 m thick) were suspended from the arm of a recording microbalance (sensitivity = 0.1 pg) which was mounted in a thermostat-controlled chamber at 30°C. The partial water-vapour pressure (P/P,) was varied by using a series of saturated electrolyte solutions (Rockland. 1960). The initial enamel weight for P/p, = 0 was determined by the use of P,05 in the chamber and weight at saturation (P/PS = 1) was obtained by the use of distilled, deionized water. A value of 31.82nm Hg was used for the vapour pressure of water, P,. at 30°C (Weast, 1973). The change in sample weight was determined as a function of the relative water vapour pressure within the chamber: both the adsorption and desorption branches of the isotherms were determined. Detailed information about the technique was given previously (Zahradnik and Moreno, 1975). The sorption parameters used to characterize the pore structure of enamel included the following: (a) total porosity, (b) specihc surface area, (c) pore volume distribution. The porosity was obtained directly by determining the adsorption capacity or weight increase of each slice as the relative humidity was changed from 0 to 100 per cent. We have pointed out (Zahradnik and Moreno, 1975) that this porosity represents a minimum value because a fraction of the enamel water is retained by the tissue even at the lowest value of P/P,; the relative measurements reported here, however, are not hampered by this situation. The specific surface area of enamel was determined using the method of analysis of Brunauer, Emmett and Teller (1938) and the data from the low relative pressure region of the adsorption curve (P/P, < 0.3). The initial calculation yielded a value for the monolayer capacity of enamel (for water) in terms of grams per gram of enamel. The value for the specific surface area was then calculated from the monolayer capacity using a value of 0.106nm’ for the molecular cross-sectional area of an adsorbed water molecule (Livingston, 1944). The dimensions of the pores within enamel were determined using the model-less method (Brunauer, M&hail and Bodor. 1967; Zahradnik and Moreno, 1975). With this approach, an idealized geometrical shape does not have to be assumed. Instead, dimensions are given in terms of hydraulic radii which are determined by dividing a core-volume parameter by a core-surface parameter. The pores are described in terms of their core size. or that portion of the pore which ‘is emptied by capillary evaporation (it excludes the portion occupied by the multilayer of adsorbed water). For our calculations, the equilibrium values from the adsorption branch rather than from the desorption branch of the isotherms were used to provide a more mean-
ingful reflection of the dimensions of the core bodies (Zahradnik and Moreno, 1975). The stepwise conversion of adsorption data into a core volume distribution curve has been described previously (Zahradnik and Moreno, 1975). The final result of this procedure consisted of a plot of the distribution function AV(r)/Ar (the slope of the cumulative core volume curve between two values of the hydraulic radii) against r, the average hydraulic radius. The sorption data obtained initially for each of the enamel slices served as a baseline (intact enamel). The slices were placed, individually at room temperature, into beakers containing 50ml of a lactic acid buffer demonstrated by microradiographic methods (Moreno and Zahradnik, 1974) to produce subsurface demineralization in enamel. The buffer was 0.1 M lactic acid, adjusted to a pH of 4.4 with potassium hydroxide. 0.5 g/liter of a commercial preparation of hydroxyapatite (CYa:P 1.63, specific surface area 68 m’/g) was added to the lactic acid to bring the calcium and phosphate levels in the buffer to values of 5 x toe3 and 3 x 10m3M, respectively; the final pH of this system after HA dissolution was 4.64. The values for Ca and P correspond to one-half of the levels expected for saturation with respect to enamel mineral (Moreno and Zahradnik, 1974). After acid exposure, the enamel slice was placed in distilled water for 24 h to remove traces of the buffer from within the slab and then the adsorption parameters for each slice were redetermined. This procedure was repeated twice again for a total of three consecutive acid-exposures. The total time spent in the lactic acid for the various stages in in-vitro subsurface demineralization were 0, 18, 36 and 72 h. After 72 h in this buffer, it has been shown (Moreno and Zahradnik, 1974) that, for extracted teeth, a well-defined demineralized region of approximately 50 to 75 pm exists below an apparently intact surface layer of approximately 25 pm. As we were interested in measuring the structural changes in enamel during the very initial formation of an artificial lesion, vapour sorption data on enamel exposed to the buffer for periods longer than 72 h were not determined. Altogether, the experimental work involved the determination of 16 sorption isotherms each isotherm branch defined by 11 to 13 experimental points. RESULTS The water vapour sorption isotherms obtained from the four enamel samples before acid demineralization were qualitatively similar. The isotherms had a desorption branch with two distinct plateaus and a hysteresis loop, extending to the lowest P/P, values, indicating a solid with a pore system dominated by narrow constrictions (Moreno and Zahradnik, 1973; Zahradnik and Moreno, 1975). The magnitude of the maximum measured adsorption capacity and the degree of hysteresis changed during acid exposure; the general isotherm shape, however, was maintained. Fig. 1 shows a typical set of sorption isotherms prior to and after acid exposure. Those obtained after 72 h of acid treatment yielded values for the total adsorption capacity more than double the value obtained for the same enamel sections before treatment. In both cases, the adsorption and desorption branches
587
Progression of enamel subsurface deminerahzation
I
I
x
3 \
II
/
/
0.6
04
0.2
-u c
(3.2
0.4
06
08
I.0
P/P,
Fig. I. Water-vapour sorption Isotherms obtained at 30°C using an enamel sectton from an incisor of a 54-yr old woman. Circles, intact enamel; squares, enamel after 72 hr of buffer exposure. The ordinate is the relative weight change of the section (with reference to the section weight Wat P/P, = 0) and the absctssa the relative water vapour pressure Open symbols. adsorption branches; solid sym-
bols, desorption branches.
of the isotherms are not coincident (hysteresis). The hysteresis loop for the acid-exposed sample closed, however, at a partial vapour pressure of approximately 0.30, but the loop before exposure did not close until the very lowest partial water vapour pressure values were reached. There were variations between the 4 samples m both the magnitude of the adsorption capacity and the degree of hysteresis. Table 1 summarizes the results obtained with the four specimens, listing the changes in measured porosity and specific surface area brought about by increasing exposure times to the acid buffer. During a 72 h period of acid exposure, the total porosity increased
substantially m all samples; this increase was shght initially. the major change occurring generally between 36 and 72 h. A similar trend was observed for the calculated specific surface-area values. Sample number 3 1s the obvious exception with both of these changes occurring at an earlier point in the demineralization process (between 18 and 36 h). The reported porosity values determined after acid treatment probably do not represent the total porosity. In the case of 72 h exposure, based on our unpublished microradiographic analysis, the increased porosity would be expected to be much greater than the five per cent found in our present study. The reported values should be considered as a measure of the space occupied by those pores still remaining in the size region of capillary condensation after acid demineralization; for water vapour this would be ag proximately 16 nm in radms or less. The specific surface area calculations do not have this limitation and should reflect valid physical measurements of the total internal surface area available to water vapour at 30°C. The preponderant pore sizes within enamel were determined using the model-less method of Brunauer et al. (1967). Figure 2 shows a series of core-volume distributions. for a single enamel sample as a function of the time spent in the lactate buffer. The distributron for intact enamel, anticipated from previous work (Moreno and Zahradnik, 1973; Zahradnik and Moreno. 1975), is bimodal; i.e.. there are two predominant pore sizes. This observation was consistently made with each intact enamel section studied and the maxima in the four distributions occur at approximately the same values of hydraulic radii. r,,. Changes in pore size during demmeralizatron were expressed in terms of the relative changes observed in the value of v,, with increasing times of buffer exposure (Table 2). It would be possible to express the core hydrauhc radii in more conventional terms if we assume a geometrical model; e.g., if enamel were to contain cylindrical pores. the two peaks for intact enamel would correspond to pore bodies with radii of approximately 2.6 and 5.8 nm. and for a parallel plate model, the plate separations would be about 3.2 and 6.6 nm. However. for the present purposes, hydraulic radii are sufficiently illustrative and are not bound to assumed particular shapes.
Table 1. Changes in the adsorption parameters of enamel wrth increasing times of buffer exposure Time of buffer exposure (h) 72 0 18 36
Sample* 1 (54 yr) Total measured porosity (%)
Specific surface area (m*/g) 2 (65 yr) 3 (28 yr) 4 (54 yr)
* Incisors from females. t Not determined.
1.86 4.2 1.97 4.2 2.51 5.2 2.19 4.4
2.19 4.4 2.58 5.1 2.94 5.5 2.46 4.7
2.28 4.7 2.74 5.3 4.28 6.9 2.76 N.D.t
4.65 7.3 3.81 6.6 4.58 7.1 364 N.D.t
R. T. Zahradnik and E. C. Moreno
588
No acid
exposure
18h acid
exposure
36 h acid
exposure
72 h acid exposure
f 1 IO
I 2.0
I 3.0
, 4.0
rho
nm
I 5.0
% I 70
6.0
I IO
I 2.Q
I 3.0 rh,
1 40
I 5.0
60
I 70
nm
Fig. 2. Core-volume distributions for enamel of a 54-yr old woman obtained on the basis of the isotherms for sample 1 generated after each period of buffer exposure. The core-volume distribution function is plotted vs the hydraulic radius for four acid-buffer exposure times. Upper left, 0 h; lower left, 18 h; upper right, 36 h; lower right, 72 h.
During the earliest stages of acid attack, as indicated by the core volume distribution for 18 h (Fig. 2) there appears to be a substantial enlargement of the pores associated with the distribution peak having a larger hydraulic radius, while the first peak (smaller pore system) remains relatively unchanged. With continued acid exposure (36 h), the hydraulic radius of the second peak continued to enlarge to a point no longer detectable with this technique, simply because pore size increased to dimensions where capillary condensation was ‘no longer possible. However, the smaller pore system also undergoes a change ret&ted by a reduction in frequency as well as the appearance of a new peak with a larger radius than that of the original smaller pores. If the acid attack is left to continue, the last core volume distribution (Fig. 2) is obtained. This figure is remarkable that, instead of a continued decrease in frequency or enlargement of the size of the smaller pores, there is a tremendous
Table 2. Core volume distribution
changes with increasing times of buffer exposure Time of buffer exposure (h) 18 36
0 1 Predominant peaks (rmaX,nm) Frequency (AV/Ar, ml/g.cm) x 10e3 2 3 4
1.0 15 0.9 26 0.9 15 0.9 12
increase in their frequency without any significant change in the hydraulic radius. Table 2 lists the values for the hydraulic radii at which maxima in the core volume distribution curves occur, rmax. The values for the distribution functions of these peaks are included to indicate changes in the contribution of the various pore sixes to the total measured porosity. The exact location of the maximum for the peak with the larger r,, is uncertain because of the limited number of experimental points. Thus, the rmar values in Table 2 correspond to the experimental measurement rather than to the true radius for this peak. Close inspection of the table shows that all specimens underwent changes in their core volume distributions that were similar to those just described. However, sample 3 deviated slightly from the norm, with the dramatic increase in the volume contribution of the smaller pores occurring between 18 and 36 h. The core-volume distributions do not, of course,
2.4 6 2.1 8 2.4 10 2.4 6
1.2 15 1.1 27 1.1 10 0.9 12
* Produced by splitting of the original peak with lowest rmax.
5.4 3 2.6 16 1.5* 9 5.5 4
1.1 12 0.9 16 0.9 30 1.1 9
1.9* 8 1.3* 13 1.6* 10 1.6* 7
72 1.1 28 0.9 34 1.0 15 1.0 26
1.7* 8 2.6* 9
Progression of enamel subsurface demineralization
589
creases in total pore volume and. in the time sequence, the increase in both of these parameters coincides with the dramatic increase in the pore volume occupied by pores with hydraulic radii in the order of 0.9 to l.Onm (Fig. 2 and Table 2) which is the radius for the smaller size-pore preponderant in intact enamel. Thus, although probably reprecipitation of solid phases occurs (Gray and Francis, 1963 ; Moreno and Zahradnik 1974) this reprecipitation could not be the most plausible explanation for the observed increase in specific surface area. A more probable explanation is that the observed increase in specific surface area corresponds to an increase pore DISCUSSION space available for the water vapour molecules as acid Our results for intact enamel agree with those attack progresses; i.e., at some stage in the deminerareported previously (Moreno and Zahradnik, 1973; lization process, pores that were not available for free Zahradnik and Moreno, 1975). The isotherms display transport of water become permeable. It is pertinent marked hysteresis between the adsorption and then to interpret the present results in the light of desorption branches. Hysteresis in the intermediate this explanation and our present knowledge of the relative pressure region of the type reported is not enamel structure. uncommon for porous solids and has been interpreted Our investigation, taken in toto, suggests the fol(deBoer, 1958) as resulting from irregularly shaped lowing stages for the process of an incipient subsurpores having zones or necks which are narrower than face demineralization: the initial demineralization the other regions of the pores (e.g., ink-bottle type occurs mostly along the interprismatic spaces; thus, pores). However, the low relative pressure hysteresis the larger pore-size in the enamel is shifted towards implies that these constrictions extend down to very larger hydraulic radii. whereas the frequency and the small dimensions, i.e. molecular ones. This interpreradius of the smaller pores are unaffected (Fig. 2 and tation is supported (Zahradnik and Moreno, 1975) Table 2). In the second stage, the enlargement of the by vapour sorption measurements at several temperainterprismatic spaces continues to the point that tures where a phenomenon of activated diffusion was capillary condensation does not occur any more in observed; that is, the adsorption capacity (measured the enlarged pores; a modest demineralization also porosity) of enamel increases rather than decreases occurs which affects a small fraction of the accessible with rising temperature. The four enamel slices also smaller pores resulting in a new peak in the region display the typical bimodal pore volume distribution below 2 nm in the pore-volume distribution function: for Intact enamel. Our present findings do not allow probably, at this stage, some demineralization occurs us to assign a specific location for the two pore-cateat the periphery of the enamel prisms but the bulk gories. It is known (Lyon and Darling, 1957; Poole of the pores in the prism core have still not been and Brooks, 1961) however. that the enamel crystals affected. The isotherms corresponding to this second lie parallel to the long axis of the prisms in the core stage (36 h) still show hysteresis extending to the lowregion. but deviate considerably in the peripheral est relative vapour pressures, indicating that there are zones of the prisms. Thus, closer packing (smaller very small constrictions still present in the system. pores) would be expected in the core than in the inter- Finally, with prolonged acid attack (72 h). the pore prismatic regions. Consequently, we assume here volume in the cores of the prisms becomes accessible these two general locations for the two dominant pore with a corresponding increase in specific surface area sizes observed. Measurements of spaces between en- and total porosity (Table 1). The hysteresis in the sorption isotherms after 72 hr amel crystals (Orams et al., 1974) by electron microscopy have yielded values covering the range from of buffer exposure decreased markedly and closed at 1 to IOnm, in presumably random observations. The P/ps values in the order of 0.3, i.e., the microconstrictwo peaks we found for the pore volume distributions tions are no longer present. Previously (Zahradnik are within this range. and Moreno, 1975) we associated the microconstricExposure of enamel to the acid buffers, however, tions in enamel with the presence of organic matter. Our present findmgsindicate that the absence of microbrought about unexpected changes. If the incipient demineralization of enamel is visualized as a simple constrictions, presumably resulting from alteration dissolution of crystal, it is reasonable to expect that of the organic matter, coincides with an increased acthe volume occupied by pores with apparent radii cessibility of the smaller pores in the prisms. Thereof 16 nm and less should increase with increasing fore, organic matter, present in small amounts (0.5 times of exposure to the acid buffers. Table 1 seems to 1.0 per cent by weight; Brudevold, Steadman and to fulfill this expectation, but the internal surface area Smith, 1960), cannot be ignored in formulating also increased on exposure to the buffers, whereas, mechanistic models for caries. The closing of the hysfor a simple dissolution process, this parameter teresis in the isotherms at P/Ps values of about 0.3 should have decreased. Reprecipitation of solid was also observed (Zahradnik and Moreno, 1975) phases in a fine state of subdivision might be associ- when enamel slices were subjected to cold-ashing ated with the increase in specific surface area. Our (activated oxygen), a procedure mild enough to alter results, however. make this explanation improbable. only the organic phase of the enamel. The present Indeed. the marked increase in the internal surface results suggest that the microconstrictions in intact area appears concomitantly with the marked in- enamel are located in the periphery of the prisms: include pores with radii greater than 16nm. Nevertheless, for those pores that are included in the measured range, the information on the distributions of buffer-exposed enamel reflects distinct changes from the pore network of intact enamel. More specifically, two points become clear: (a) demineralization appears to follow a stepwise mechanism, initially affecting only the larger diffusional pathways, (b) at a certain point in demineralization, there is an increase in the number of very small pores to which the water molecule has access.
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R. T. Zahradnik and E. C. Moreno
upon prolonged acid attack, the organic matter associated with these constrictions is either dissolved or sufficiently modified to allow freer access into deeper regions of the prisms. Although the results do not give direct evidence concerning the location of the microconstrictions, it is reasonable to assume that they are located towards the cortex of the prisms where independent studies (Ronnholm, 1962; Meckel et al., 1975; Linden, 1968) have shown a higher concentration of organic matter than in the rest of enamel. The three demineralization stages suggested by the vapour sorption measurements probably correspond to the three stages described by Darling (1963) based on histology. He concluded that the attack occurs first along the interprismatic region, then extends to cross-striations penetrating the prisms and finally progresses along the prism cores leaving the cortices of prisms relatively unaffected. With our techniques, it is not possible to ascertain the equivalence of the various stages. However, if the second stages are equivalent. it would be necessary to conclude that the most vulnerable sites of the prism are the intersections of its cortex with the cross striations. According to Darling (1963), there is a soluble fraction in enamel matrix; it is tempting to speculate that soluble organic matrix may exist at the points of greater vulnerability. The three sequential stages of enamel demineralization are also consistent with electron microscopy of natural lesions. Scott (1974) and Kerckaert (1973), using SEM and TEM, concluded that initial demineralization takes place in the interprismatic spaces; at a more advanced stage, demineralization within the prism becomes apparent and more accentuated with respect to the prism periphery. Probably these two stages correspond to the first and third stages described here; the second stage, initial changes in the smaller predominant pore size of intact enamel, cannot be observed directly with electron microscopy as it appears to depend on properties of the organic matter. The apparent contradiction between our findings and those of Dibdin (1969) referred to earlier is probably due to the use of krypton at low temperatures for the isothermal sorption measurements which yields much lower porosity values. Evidently, the pore accessibility for krypton is smaller than for water vapour. Indeed Dibdin found, when powdered enamel was exposed to an acid acetate buffer, a bimodal pore volume distributions did appear Thus, the acid treatment removed the initial restrictions to the flow of the krypton molecules. There was an increase in the smallest pore size measurable with his method which would be difficult to explain unless accessibility to that pore size was actually increased by the acidbuffer treatment. Dibdin also found significant increases in the pore volume and the specific surface area of enamel treatment with an acidified gel, and by heating to 36Y’C. Such observations agree with our findings and explanations concerning the role of organic matter. In early studies on th- composition of enamel protein, Bibby (1952), based on the resistance of isolated enamel protein to bacterial destruction, suggested that the organic matter may provide a passive defense
against caries. Apostolopoulos and Buonocore (1966) found little difference in acid dissolution rates between intact and anorganic enamel. However, enamel dissolution experiments do not proceed in the same fashion as with the buffers we used or in the mouth, where a subsurface demineralization marks the onset. Simmelink et al. (1974) believe that mineralization is not affected by the small organic matter content of enamel. However, Silness, Hegdahl and Gustavsen (1973) have shown that, although the organic matter content is low by weight, there is an extensive organic-inorganic interface in enamel and thus a small amount of organic matter may have a large influence. Its distribution is more important than its amount. Weatherell (1974) suggested that the protein in the enamel interior might encourage reprecipitation of calcium phosphate during the caries process. Apostolopoulos and Buonocore (1966) suggested that properties of the organic matrix, e.g. degree of swelling and net electrical charge, may retard diffusion in dentine and bone, particularly under low pH conditions; such effects may be responsible for the reduced solubility rates observed for the intact as opposed to the anorganic tissues. It is conceivable that in enamel a barrier to ionic transport exists at the organic-inorganic interface, and that this barrier becomes more effective under acidic conditions favouring subsurface demineralization. Proteinaceous pellicles of salivary origin developed on tooth surfaces display permeability selectivity which Zahradnik, Moreno and Burke (1976) showed affect the rates of demineralization. Finally, acid etching of enamel results in a preferential dissolution of the prism cores (Nichol et al., 1973; Simmelink et al., 1974). Thus, the crystals in cores would be the most susceptible to acid attack, and therefore involved in the initial stages of subsurface demineralization. However, electron microscopy shows that the initial loss of mineral in early natural caries is from the interprismatic region, so more is needed than a simple model involving only the solubility characteristics of the mineral phase. We believe that our present results and previous ones (Moreno and Zahradnik, 1973; Zahradnik and Moreno, 1975) strongly suggest that the organic matter in dental enamel is important in the incipient carious lesion. It is possible that the second stage of demineralization may mark the point where advancing caries cannot be reversed by simple deposition of mineral.
Acknowledgement-This
investigation
was supported in
part by NIDR Grant DE 03187. REFERENCES Apostolopoulos A. X. and Buonocore M. G. 1966. Comparative dissolution rates of enamel, dentin, and bone. I. Effect of the organic matter. J. dent. Res. 45, 10931100. Bibby B. G. 1952. The organic structure of dental enamel as a passive defense against caries. J. dent. Res. 12, 99-116. Brudevold F., Steadman L. T. and Smith F. A. 1960. Inorganic and organic components of tooth structure. Ann. N.Y Acad. Sci. 8s, 110-132.
Progression of enamel subsurface demineralizatlon Brunauer S., Emmett P. H. and Teller E. 1938. Adsorption of gases m multimolecular layers. J. Am. them. Sot. 6u, 309-319. Brunauer S., Mikhail R. SH. and Boder E. E. 1967. Pore structure analysis without a pore shape model. J. Colloid interface Sci. 24, 451463. Darling A. I. 1956. Studies on the early lesions of enamel caries with transmitted light, polarized light and radiography. Br. dent. J. 101, 289-297 and 329-341. Darling A. I. 1963. Microstructural channes in earlv dental caries. In: Mechanisms of Hard &sue Destruction (Edited by Sognnaes R. F.). pp. 171-186. American Assoclation for the Advancement of Science, Washington. deBoer J. H. 1958. The shape of capillaries. In: Structure and Properties of Porous Materials (Edited by Everett D. H. and Stone F. S.), pp. 68-94. Butterworth, London. Dibdin G. H. 1969. The internal surface and pore structure of enamel. J. dent. Res. 48, 771-776. Gray J. A. and Francis M. D. 1963. Physical chemistry of enamel dissolution. In: Mechanisms of Hard Tissue Destruction (Edited by Sognnaes R. F.), pp. 213-260. American Association- for ihe Advancemeni of Science, Washinnton. Gustafson”G. 1957. The histopathology of caries of human dental enamel: with special reference to the division of the carious lesion into zones. Acta odont. stand. 15, 13-55.
Gustafson G. and Gustafson A. G. 1967. Microanatomy and histochemistry of enamel. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.), Vol. 2. Chap 14, pp. 75-134. Academic Press, New York. Kerckaert G.A. 1
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Pergamon Press 1977. Punted m Great Bratam
PROGRESSIVE DEMINERALIZATION
STAGES OF SUBSURFACE OF HUMAN TOOTH ENAMEL
R. T. ZAHRADNIKand E. C. MORENO Forsyth Dental Center, 140 Fenway. Boston, Massachusetts 02115, U.S.A. Summary-Samples of intact human dental enamel were exposed to a lactate buffer known to produce subsurface demineralization. After exposure times of 0, 18, 36 and 72 h, the water vapour sorption isotherms were determined and the corresponding pore-volume distributions and specific surface areas calculated. Three distinct demineralization stages were recognizable. In the first stage, only’the larger peak in the bimodal distribution for intact enamel was affected; the second stage was marked by the beginning enlargement of the smaller accessible pores, finally, there was a dramatic increase in accessible pore volume and specific surface area with increase in the frequency but not in the size of the smaller pores preponderant in intact enamel. The last stage appears to be related to the removal of constrictions that hinder transport into the cores of the prisms. It is suggested that the constrictions are associated with organic matter which is thus intimately involved in incipient enamel demineralization.
INTRODUCTION Human dental enamel consists of prisms radiating from the dentine-enamel (DE) junction toward the outer surface (Meckel, Griebstein and Neal, 1965; Gustafson and Gustafson, 1967). Each prism consists of large numbers of small hydroxyapatite crystals mainly oriented axially along the prism; this parallel orientation does not extend to the boundary zones between adjacent prisms which are the regions with the highest organic content (Ronnholm, 1962; Meckel et al., 1965; Linden, 1968). The crystal arrangement of enamel gives rise to two main categories of pores corresponding to the spaces between single crystals in the prism cores and those between crystals in the region between prisms. Using isothermal water vapour sorption techniques, we established that intact human enamel has a bimodal pore-volume distribution (Moreno and Zahradnik, 1973; Zahradnik and Moreno, 1975); the larger pores are presumably related to spaces in the interprismatic regions and the smaller are probably associated with the spaces within the prisms themselves. There is little information on how the enamel pore structure changes with the onset of caries. Such information would be useful in determining acid-entry pathways and in establishing the enamel regions most susceptible to acid attack. Dibdin (1969) published preliminary results on porosity changes in ground enamel resulting from exposure to acid buffers. Using krypton gas sorption techniques, he found that the pore size distribution after acid exposure was bimodal with a new peak within the smallest calculated pore size range (Kelvin radii < 1.4nm). Significant increases in the specific surface area and porosity of the enamel powder were observed; an especially marked increase in the specific surface area of ground enamel occurred between one and two weeks of exksure to an acidified gel. No explanation of these observations was advanced. Several investigators (Nichol, Judd and Ansell, 1973; Simmelink. Nygaard and Scott, 1974) using
scanning and transmission electron microscopy (SEM and TEM) to study the effect of acid etching upon enamel structure, have shown that the crystals within the core of the enamel prisms are preferentially dissolved while the peripheral regions of the prisms remain relatively unaffected; this results in a honeycomb appearance of the enamel surface. There must be some differences in solubility which permit the structure to be brought out in relief. Nevertheless, incipient carious lesions do not develop by such surface etching but instead develop as a region of demineralization underlying a smooth surface layer which shows little or no damage (Darling, 1956; Gustafson, 1957), suggesting that other factors besides simple surface solubility differences must be involved. Studies of early carious enamel employing SEM (Kerckaert, 1973; Scott, Simmelink and Nygaard, 1974) show that the initial attack appears to occur along the prism boundaries. Enamel from more advanced lesions exhibits the classic demineralized prism cores with a significantly less affected prism boundary region. However, no information is available concerning the sequential changes that must occur in the properties of the pore structure of the enamel with a progressing carious lesion. Such a study, based on natural caries, would be extremely difficult, given the multifactorial nature of caries and the impossibility of controlling the rate of demineralization under in-Go conditions. The use of in-vitro models seems appropriate. .We have advanced a hypothesis (Moreno and Zahradnik. 1974) in terms of which the conditions of acidity can be predicted under which enamel subsurface demineralization and not surface dissolution should occur. This hypothesis was developed solely on the basis of the chemistry of calcium phosphates. A model was tested experimentally by exposing extracted teeth to acid buffers of predetermined composition; the results were in close agreement with our hypothesis, provided that appropriate consideration was given to transport rates of dissolved mineral away from the tooth surface. Furthermore, the struc585
R. T. Zahradnik and E. C. Moreno
586
ture of the demineralized enamel was consistent with the morphology we have referred to for natural incipient carious lesions. Our purpose here was to measure the sequential changes in the properties of the pore structure of dental enamel when the tissue is demineralized. MATERIALS
AND METHODS
Four human teeth with clinically sound enamel were thin-sectioned parallel to their labial surfaces using a water-cooled sectioning device and a diamond disc. The enamel slices (approximately 250 m thick) were suspended from the arm of a recording microbalance (sensitivity = 0.1 pg) which was mounted in a thermostat-controlled chamber at 30°C. The partial water-vapour pressure (P/P,) was varied by using a series of saturated electrolyte solutions (Rockland. 1960). The initial enamel weight for P/p, = 0 was determined by the use of P,05 in the chamber and weight at saturation (P/PS = 1) was obtained by the use of distilled, deionized water. A value of 31.82nm Hg was used for the vapour pressure of water, P,. at 30°C (Weast, 1973). The change in sample weight was determined as a function of the relative water vapour pressure within the chamber: both the adsorption and desorption branches of the isotherms were determined. Detailed information about the technique was given previously (Zahradnik and Moreno, 1975). The sorption parameters used to characterize the pore structure of enamel included the following: (a) total porosity, (b) specihc surface area, (c) pore volume distribution. The porosity was obtained directly by determining the adsorption capacity or weight increase of each slice as the relative humidity was changed from 0 to 100 per cent. We have pointed out (Zahradnik and Moreno, 1975) that this porosity represents a minimum value because a fraction of the enamel water is retained by the tissue even at the lowest value of P/P,; the relative measurements reported here, however, are not hampered by this situation. The specific surface area of enamel was determined using the method of analysis of Brunauer, Emmett and Teller (1938) and the data from the low relative pressure region of the adsorption curve (P/P, < 0.3). The initial calculation yielded a value for the monolayer capacity of enamel (for water) in terms of grams per gram of enamel. The value for the specific surface area was then calculated from the monolayer capacity using a value of 0.106nm’ for the molecular cross-sectional area of an adsorbed water molecule (Livingston, 1944). The dimensions of the pores within enamel were determined using the model-less method (Brunauer, M&hail and Bodor. 1967; Zahradnik and Moreno, 1975). With this approach, an idealized geometrical shape does not have to be assumed. Instead, dimensions are given in terms of hydraulic radii which are determined by dividing a core-volume parameter by a core-surface parameter. The pores are described in terms of their core size. or that portion of the pore which ‘is emptied by capillary evaporation (it excludes the portion occupied by the multilayer of adsorbed water). For our calculations, the equilibrium values from the adsorption branch rather than from the desorption branch of the isotherms were used to provide a more mean-
ingful reflection of the dimensions of the core bodies (Zahradnik and Moreno, 1975). The stepwise conversion of adsorption data into a core volume distribution curve has been described previously (Zahradnik and Moreno, 1975). The final result of this procedure consisted of a plot of the distribution function AV(r)/Ar (the slope of the cumulative core volume curve between two values of the hydraulic radii) against r, the average hydraulic radius. The sorption data obtained initially for each of the enamel slices served as a baseline (intact enamel). The slices were placed, individually at room temperature, into beakers containing 50ml of a lactic acid buffer demonstrated by microradiographic methods (Moreno and Zahradnik, 1974) to produce subsurface demineralization in enamel. The buffer was 0.1 M lactic acid, adjusted to a pH of 4.4 with potassium hydroxide. 0.5 g/liter of a commercial preparation of hydroxyapatite (CYa:P 1.63, specific surface area 68 m’/g) was added to the lactic acid to bring the calcium and phosphate levels in the buffer to values of 5 x toe3 and 3 x 10m3M, respectively; the final pH of this system after HA dissolution was 4.64. The values for Ca and P correspond to one-half of the levels expected for saturation with respect to enamel mineral (Moreno and Zahradnik, 1974). After acid exposure, the enamel slice was placed in distilled water for 24 h to remove traces of the buffer from within the slab and then the adsorption parameters for each slice were redetermined. This procedure was repeated twice again for a total of three consecutive acid-exposures. The total time spent in the lactic acid for the various stages in in-vitro subsurface demineralization were 0, 18, 36 and 72 h. After 72 h in this buffer, it has been shown (Moreno and Zahradnik, 1974) that, for extracted teeth, a well-defined demineralized region of approximately 50 to 75 pm exists below an apparently intact surface layer of approximately 25 pm. As we were interested in measuring the structural changes in enamel during the very initial formation of an artificial lesion, vapour sorption data on enamel exposed to the buffer for periods longer than 72 h were not determined. Altogether, the experimental work involved the determination of 16 sorption isotherms each isotherm branch defined by 11 to 13 experimental points. RESULTS The water vapour sorption isotherms obtained from the four enamel samples before acid demineralization were qualitatively similar. The isotherms had a desorption branch with two distinct plateaus and a hysteresis loop, extending to the lowest P/P, values, indicating a solid with a pore system dominated by narrow constrictions (Moreno and Zahradnik, 1973; Zahradnik and Moreno, 1975). The magnitude of the maximum measured adsorption capacity and the degree of hysteresis changed during acid exposure; the general isotherm shape, however, was maintained. Fig. 1 shows a typical set of sorption isotherms prior to and after acid exposure. Those obtained after 72 h of acid treatment yielded values for the total adsorption capacity more than double the value obtained for the same enamel sections before treatment. In both cases, the adsorption and desorption branches
587
Progression of enamel subsurface deminerahzation
I
I
x
3 \
II
/
/
0.6
04
0.2
-u c
(3.2
0.4
06
08
I.0
P/P,
Fig. I. Water-vapour sorption Isotherms obtained at 30°C using an enamel sectton from an incisor of a 54-yr old woman. Circles, intact enamel; squares, enamel after 72 hr of buffer exposure. The ordinate is the relative weight change of the section (with reference to the section weight Wat P/P, = 0) and the absctssa the relative water vapour pressure Open symbols. adsorption branches; solid sym-
bols, desorption branches.
of the isotherms are not coincident (hysteresis). The hysteresis loop for the acid-exposed sample closed, however, at a partial vapour pressure of approximately 0.30, but the loop before exposure did not close until the very lowest partial water vapour pressure values were reached. There were variations between the 4 samples m both the magnitude of the adsorption capacity and the degree of hysteresis. Table 1 summarizes the results obtained with the four specimens, listing the changes in measured porosity and specific surface area brought about by increasing exposure times to the acid buffer. During a 72 h period of acid exposure, the total porosity increased
substantially m all samples; this increase was shght initially. the major change occurring generally between 36 and 72 h. A similar trend was observed for the calculated specific surface-area values. Sample number 3 1s the obvious exception with both of these changes occurring at an earlier point in the demineralization process (between 18 and 36 h). The reported porosity values determined after acid treatment probably do not represent the total porosity. In the case of 72 h exposure, based on our unpublished microradiographic analysis, the increased porosity would be expected to be much greater than the five per cent found in our present study. The reported values should be considered as a measure of the space occupied by those pores still remaining in the size region of capillary condensation after acid demineralization; for water vapour this would be ag proximately 16 nm in radms or less. The specific surface area calculations do not have this limitation and should reflect valid physical measurements of the total internal surface area available to water vapour at 30°C. The preponderant pore sizes within enamel were determined using the model-less method of Brunauer et al. (1967). Figure 2 shows a series of core-volume distributions. for a single enamel sample as a function of the time spent in the lactate buffer. The distributron for intact enamel, anticipated from previous work (Moreno and Zahradnik, 1973; Zahradnik and Moreno. 1975), is bimodal; i.e.. there are two predominant pore sizes. This observation was consistently made with each intact enamel section studied and the maxima in the four distributions occur at approximately the same values of hydraulic radii. r,,. Changes in pore size during demmeralizatron were expressed in terms of the relative changes observed in the value of v,, with increasing times of buffer exposure (Table 2). It would be possible to express the core hydrauhc radii in more conventional terms if we assume a geometrical model; e.g., if enamel were to contain cylindrical pores. the two peaks for intact enamel would correspond to pore bodies with radii of approximately 2.6 and 5.8 nm. and for a parallel plate model, the plate separations would be about 3.2 and 6.6 nm. However. for the present purposes, hydraulic radii are sufficiently illustrative and are not bound to assumed particular shapes.
Table 1. Changes in the adsorption parameters of enamel wrth increasing times of buffer exposure Time of buffer exposure (h) 72 0 18 36
Sample* 1 (54 yr) Total measured porosity (%)
Specific surface area (m*/g) 2 (65 yr) 3 (28 yr) 4 (54 yr)
* Incisors from females. t Not determined.
1.86 4.2 1.97 4.2 2.51 5.2 2.19 4.4
2.19 4.4 2.58 5.1 2.94 5.5 2.46 4.7
2.28 4.7 2.74 5.3 4.28 6.9 2.76 N.D.t
4.65 7.3 3.81 6.6 4.58 7.1 364 N.D.t
R. T. Zahradnik and E. C. Moreno
588
No acid
exposure
18h acid
exposure
36 h acid
exposure
72 h acid exposure
f 1 IO
I 2.0
I 3.0
, 4.0
rho
nm
I 5.0
% I 70
6.0
I IO
I 2.Q
I 3.0 rh,
1 40
I 5.0
60
I 70
nm
Fig. 2. Core-volume distributions for enamel of a 54-yr old woman obtained on the basis of the isotherms for sample 1 generated after each period of buffer exposure. The core-volume distribution function is plotted vs the hydraulic radius for four acid-buffer exposure times. Upper left, 0 h; lower left, 18 h; upper right, 36 h; lower right, 72 h.
During the earliest stages of acid attack, as indicated by the core volume distribution for 18 h (Fig. 2) there appears to be a substantial enlargement of the pores associated with the distribution peak having a larger hydraulic radius, while the first peak (smaller pore system) remains relatively unchanged. With continued acid exposure (36 h), the hydraulic radius of the second peak continued to enlarge to a point no longer detectable with this technique, simply because pore size increased to dimensions where capillary condensation was ‘no longer possible. However, the smaller pore system also undergoes a change ret&ted by a reduction in frequency as well as the appearance of a new peak with a larger radius than that of the original smaller pores. If the acid attack is left to continue, the last core volume distribution (Fig. 2) is obtained. This figure is remarkable that, instead of a continued decrease in frequency or enlargement of the size of the smaller pores, there is a tremendous
Table 2. Core volume distribution
changes with increasing times of buffer exposure Time of buffer exposure (h) 18 36
0 1 Predominant peaks (rmaX,nm) Frequency (AV/Ar, ml/g.cm) x 10e3 2 3 4
1.0 15 0.9 26 0.9 15 0.9 12
increase in their frequency without any significant change in the hydraulic radius. Table 2 lists the values for the hydraulic radii at which maxima in the core volume distribution curves occur, rmax. The values for the distribution functions of these peaks are included to indicate changes in the contribution of the various pore sixes to the total measured porosity. The exact location of the maximum for the peak with the larger r,, is uncertain because of the limited number of experimental points. Thus, the rmar values in Table 2 correspond to the experimental measurement rather than to the true radius for this peak. Close inspection of the table shows that all specimens underwent changes in their core volume distributions that were similar to those just described. However, sample 3 deviated slightly from the norm, with the dramatic increase in the volume contribution of the smaller pores occurring between 18 and 36 h. The core-volume distributions do not, of course,
2.4 6 2.1 8 2.4 10 2.4 6
1.2 15 1.1 27 1.1 10 0.9 12
* Produced by splitting of the original peak with lowest rmax.
5.4 3 2.6 16 1.5* 9 5.5 4
1.1 12 0.9 16 0.9 30 1.1 9
1.9* 8 1.3* 13 1.6* 10 1.6* 7
72 1.1 28 0.9 34 1.0 15 1.0 26
1.7* 8 2.6* 9
Progression of enamel subsurface demineralization
589
creases in total pore volume and. in the time sequence, the increase in both of these parameters coincides with the dramatic increase in the pore volume occupied by pores with hydraulic radii in the order of 0.9 to l.Onm (Fig. 2 and Table 2) which is the radius for the smaller size-pore preponderant in intact enamel. Thus, although probably reprecipitation of solid phases occurs (Gray and Francis, 1963 ; Moreno and Zahradnik 1974) this reprecipitation could not be the most plausible explanation for the observed increase in specific surface area. A more probable explanation is that the observed increase in specific surface area corresponds to an increase pore DISCUSSION space available for the water vapour molecules as acid Our results for intact enamel agree with those attack progresses; i.e., at some stage in the deminerareported previously (Moreno and Zahradnik, 1973; lization process, pores that were not available for free Zahradnik and Moreno, 1975). The isotherms display transport of water become permeable. It is pertinent marked hysteresis between the adsorption and then to interpret the present results in the light of desorption branches. Hysteresis in the intermediate this explanation and our present knowledge of the relative pressure region of the type reported is not enamel structure. uncommon for porous solids and has been interpreted Our investigation, taken in toto, suggests the fol(deBoer, 1958) as resulting from irregularly shaped lowing stages for the process of an incipient subsurpores having zones or necks which are narrower than face demineralization: the initial demineralization the other regions of the pores (e.g., ink-bottle type occurs mostly along the interprismatic spaces; thus, pores). However, the low relative pressure hysteresis the larger pore-size in the enamel is shifted towards implies that these constrictions extend down to very larger hydraulic radii. whereas the frequency and the small dimensions, i.e. molecular ones. This interpreradius of the smaller pores are unaffected (Fig. 2 and tation is supported (Zahradnik and Moreno, 1975) Table 2). In the second stage, the enlargement of the by vapour sorption measurements at several temperainterprismatic spaces continues to the point that tures where a phenomenon of activated diffusion was capillary condensation does not occur any more in observed; that is, the adsorption capacity (measured the enlarged pores; a modest demineralization also porosity) of enamel increases rather than decreases occurs which affects a small fraction of the accessible with rising temperature. The four enamel slices also smaller pores resulting in a new peak in the region display the typical bimodal pore volume distribution below 2 nm in the pore-volume distribution function: for Intact enamel. Our present findings do not allow probably, at this stage, some demineralization occurs us to assign a specific location for the two pore-cateat the periphery of the enamel prisms but the bulk gories. It is known (Lyon and Darling, 1957; Poole of the pores in the prism core have still not been and Brooks, 1961) however. that the enamel crystals affected. The isotherms corresponding to this second lie parallel to the long axis of the prisms in the core stage (36 h) still show hysteresis extending to the lowregion. but deviate considerably in the peripheral est relative vapour pressures, indicating that there are zones of the prisms. Thus, closer packing (smaller very small constrictions still present in the system. pores) would be expected in the core than in the inter- Finally, with prolonged acid attack (72 h). the pore prismatic regions. Consequently, we assume here volume in the cores of the prisms becomes accessible these two general locations for the two dominant pore with a corresponding increase in specific surface area sizes observed. Measurements of spaces between en- and total porosity (Table 1). The hysteresis in the sorption isotherms after 72 hr amel crystals (Orams et al., 1974) by electron microscopy have yielded values covering the range from of buffer exposure decreased markedly and closed at 1 to IOnm, in presumably random observations. The P/ps values in the order of 0.3, i.e., the microconstrictwo peaks we found for the pore volume distributions tions are no longer present. Previously (Zahradnik are within this range. and Moreno, 1975) we associated the microconstricExposure of enamel to the acid buffers, however, tions in enamel with the presence of organic matter. Our present findmgsindicate that the absence of microbrought about unexpected changes. If the incipient demineralization of enamel is visualized as a simple constrictions, presumably resulting from alteration dissolution of crystal, it is reasonable to expect that of the organic matter, coincides with an increased acthe volume occupied by pores with apparent radii cessibility of the smaller pores in the prisms. Thereof 16 nm and less should increase with increasing fore, organic matter, present in small amounts (0.5 times of exposure to the acid buffers. Table 1 seems to 1.0 per cent by weight; Brudevold, Steadman and to fulfill this expectation, but the internal surface area Smith, 1960), cannot be ignored in formulating also increased on exposure to the buffers, whereas, mechanistic models for caries. The closing of the hysfor a simple dissolution process, this parameter teresis in the isotherms at P/Ps values of about 0.3 should have decreased. Reprecipitation of solid was also observed (Zahradnik and Moreno, 1975) phases in a fine state of subdivision might be associ- when enamel slices were subjected to cold-ashing ated with the increase in specific surface area. Our (activated oxygen), a procedure mild enough to alter results, however. make this explanation improbable. only the organic phase of the enamel. The present Indeed. the marked increase in the internal surface results suggest that the microconstrictions in intact area appears concomitantly with the marked in- enamel are located in the periphery of the prisms: include pores with radii greater than 16nm. Nevertheless, for those pores that are included in the measured range, the information on the distributions of buffer-exposed enamel reflects distinct changes from the pore network of intact enamel. More specifically, two points become clear: (a) demineralization appears to follow a stepwise mechanism, initially affecting only the larger diffusional pathways, (b) at a certain point in demineralization, there is an increase in the number of very small pores to which the water molecule has access.
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upon prolonged acid attack, the organic matter associated with these constrictions is either dissolved or sufficiently modified to allow freer access into deeper regions of the prisms. Although the results do not give direct evidence concerning the location of the microconstrictions, it is reasonable to assume that they are located towards the cortex of the prisms where independent studies (Ronnholm, 1962; Meckel et al., 1975; Linden, 1968) have shown a higher concentration of organic matter than in the rest of enamel. The three demineralization stages suggested by the vapour sorption measurements probably correspond to the three stages described by Darling (1963) based on histology. He concluded that the attack occurs first along the interprismatic region, then extends to cross-striations penetrating the prisms and finally progresses along the prism cores leaving the cortices of prisms relatively unaffected. With our techniques, it is not possible to ascertain the equivalence of the various stages. However, if the second stages are equivalent. it would be necessary to conclude that the most vulnerable sites of the prism are the intersections of its cortex with the cross striations. According to Darling (1963), there is a soluble fraction in enamel matrix; it is tempting to speculate that soluble organic matrix may exist at the points of greater vulnerability. The three sequential stages of enamel demineralization are also consistent with electron microscopy of natural lesions. Scott (1974) and Kerckaert (1973), using SEM and TEM, concluded that initial demineralization takes place in the interprismatic spaces; at a more advanced stage, demineralization within the prism becomes apparent and more accentuated with respect to the prism periphery. Probably these two stages correspond to the first and third stages described here; the second stage, initial changes in the smaller predominant pore size of intact enamel, cannot be observed directly with electron microscopy as it appears to depend on properties of the organic matter. The apparent contradiction between our findings and those of Dibdin (1969) referred to earlier is probably due to the use of krypton at low temperatures for the isothermal sorption measurements which yields much lower porosity values. Evidently, the pore accessibility for krypton is smaller than for water vapour. Indeed Dibdin found, when powdered enamel was exposed to an acid acetate buffer, a bimodal pore volume distributions did appear Thus, the acid treatment removed the initial restrictions to the flow of the krypton molecules. There was an increase in the smallest pore size measurable with his method which would be difficult to explain unless accessibility to that pore size was actually increased by the acidbuffer treatment. Dibdin also found significant increases in the pore volume and the specific surface area of enamel treatment with an acidified gel, and by heating to 36Y’C. Such observations agree with our findings and explanations concerning the role of organic matter. In early studies on th- composition of enamel protein, Bibby (1952), based on the resistance of isolated enamel protein to bacterial destruction, suggested that the organic matter may provide a passive defense
against caries. Apostolopoulos and Buonocore (1966) found little difference in acid dissolution rates between intact and anorganic enamel. However, enamel dissolution experiments do not proceed in the same fashion as with the buffers we used or in the mouth, where a subsurface demineralization marks the onset. Simmelink et al. (1974) believe that mineralization is not affected by the small organic matter content of enamel. However, Silness, Hegdahl and Gustavsen (1973) have shown that, although the organic matter content is low by weight, there is an extensive organic-inorganic interface in enamel and thus a small amount of organic matter may have a large influence. Its distribution is more important than its amount. Weatherell (1974) suggested that the protein in the enamel interior might encourage reprecipitation of calcium phosphate during the caries process. Apostolopoulos and Buonocore (1966) suggested that properties of the organic matrix, e.g. degree of swelling and net electrical charge, may retard diffusion in dentine and bone, particularly under low pH conditions; such effects may be responsible for the reduced solubility rates observed for the intact as opposed to the anorganic tissues. It is conceivable that in enamel a barrier to ionic transport exists at the organic-inorganic interface, and that this barrier becomes more effective under acidic conditions favouring subsurface demineralization. Proteinaceous pellicles of salivary origin developed on tooth surfaces display permeability selectivity which Zahradnik, Moreno and Burke (1976) showed affect the rates of demineralization. Finally, acid etching of enamel results in a preferential dissolution of the prism cores (Nichol et al., 1973; Simmelink et al., 1974). Thus, the crystals in cores would be the most susceptible to acid attack, and therefore involved in the initial stages of subsurface demineralization. However, electron microscopy shows that the initial loss of mineral in early natural caries is from the interprismatic region, so more is needed than a simple model involving only the solubility characteristics of the mineral phase. We believe that our present results and previous ones (Moreno and Zahradnik, 1973; Zahradnik and Moreno, 1975) strongly suggest that the organic matter in dental enamel is important in the incipient carious lesion. It is possible that the second stage of demineralization may mark the point where advancing caries cannot be reversed by simple deposition of mineral.
Acknowledgement-This
investigation
was supported in
part by NIDR Grant DE 03187. REFERENCES Apostolopoulos A. X. and Buonocore M. G. 1966. Comparative dissolution rates of enamel, dentin, and bone. I. Effect of the organic matter. J. dent. Res. 45, 10931100. Bibby B. G. 1952. The organic structure of dental enamel as a passive defense against caries. J. dent. Res. 12, 99-116. Brudevold F., Steadman L. T. and Smith F. A. 1960. Inorganic and organic components of tooth structure. Ann. N.Y Acad. Sci. 8s, 110-132.
Progression of enamel subsurface demineralizatlon Brunauer S., Emmett P. H. and Teller E. 1938. Adsorption of gases m multimolecular layers. J. Am. them. Sot. 6u, 309-319. Brunauer S., Mikhail R. SH. and Boder E. E. 1967. Pore structure analysis without a pore shape model. J. Colloid interface Sci. 24, 451463. Darling A. I. 1956. Studies on the early lesions of enamel caries with transmitted light, polarized light and radiography. Br. dent. J. 101, 289-297 and 329-341. Darling A. I. 1963. Microstructural channes in earlv dental caries. In: Mechanisms of Hard &sue Destruction (Edited by Sognnaes R. F.). pp. 171-186. American Assoclation for the Advancement of Science, Washington. deBoer J. H. 1958. The shape of capillaries. In: Structure and Properties of Porous Materials (Edited by Everett D. H. and Stone F. S.), pp. 68-94. Butterworth, London. Dibdin G. H. 1969. The internal surface and pore structure of enamel. J. dent. Res. 48, 771-776. Gray J. A. and Francis M. D. 1963. Physical chemistry of enamel dissolution. In: Mechanisms of Hard Tissue Destruction (Edited by Sognnaes R. F.), pp. 213-260. American Association- for ihe Advancemeni of Science, Washinnton. Gustafson”G. 1957. The histopathology of caries of human dental enamel: with special reference to the division of the carious lesion into zones. Acta odont. stand. 15, 13-55.
Gustafson G. and Gustafson A. G. 1967. Microanatomy and histochemistry of enamel. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.), Vol. 2. Chap 14, pp. 75-134. Academic Press, New York. Kerckaert G.A. 1
