Auks w-al8101.c’ol Xl. pp. 317 to 325. Pergamon Press 1975. Prmted m Great Brltam

STRUCTURAL FEATURES OF HUMAN DENTAL ENAMEL AS REVEALED BY ISOTHERMAL WATER VAPOUR SORPTION R. T.

ZAHRADNIK

and E. C. Mo~t~o

Forsyth Dental Center, Boston, Massachusetts 02115. U.S.A. Summary-Water sorption isotherms for intact human enamel samples are reported at 25 and 50°C. The isotherms display pronounced hysteresis consistent with the presence of constricted pores, some of which extend to molecular dimensions. The total water holding capacity (apparent porosity) of surface enamel was in the range of 0.4.50.62 per cent (w/w) for the various tooth types studied at 25°C and it increased by 20 to 35 per cent at 50°C. which is indicative of an activated diffusion. Within a tooth type, the water sorption capacity decreases with the age of the tooth and, within a single tooth, it increases from the enamel surface toward the dentinoenamel junction. The pore volume distribution functions show a bimodal pore system with smaller hydraulic radii (0.7 and 1.3 nm) for canine enamel than for enamel from molars and incisors (I.0 and 2.5 nm). The pore volume distribution did not change after cold-ashing of the enamel, but the hystereses in the sorption isotherms are drastically reduced indicating that the pore constrictions are associated with the presence of organic matter.

INTRODUCTION

Several investigators (Gray, Francis and Griebstein, 1962; Holly and Gray, 1968; Higuchi et al., 1969; Moreno and Zahradnik, 1974) have postulated diffuSiOml models to explain the process of demineralization which occurs within human enamel in uivo. Attempts have been made, moreover, directly to measure transport rates in oitro by using diffusional techniques (Holly and Gray, 1968; Braden, Duckworth and Joyston-Bechal, 1971; Burke and Moreno, 1974). Important as these studies may be in predicting values for mass fluxes through enamel, any reasonable interpretation of diffusional observations remains incomplete without a clear understanding of the structural features of the enamel which control these fluxes, such as total porosity and the size and geometry of the pores, Approaches involving electron and polarizing microscopy, and vapour uptake, have been used to characterize the pore system of enamel (Darling et al., 1961; Poole, 1971; Gustafson and Gustafson, 1967). The results obtained with polarizing microscopy suggest that the enamel behaves as a molecular sieve because of the presence of extremely small pores. Recently, it has been reported (Moreno and Zahradnik, 1973) that dental enamel does not contain micropores and that the sieve behaviour is probably related to the presence of pore constrictions which in turn are associated with the organic matter content. The organic matter in enamel only amounts to 0.5 1 per cent by weight (Brudevold, Steadman and Smith, 1960) but it has been shown preferentially to occupy the interprismatic diffusional pathways in enamel (Ronnholm, 1962; Meckel, Griebstein and Neal, 1965; Linden, 1968) and so it is of paramount importance in sorption and diffusional studies. In fact, the lack of complete rehydration of dental enamel which has been exposed to temperatures above 60°C 317

(Burnett and Zenewitz, 1958) may relate to denaturation, shown to begin generally at temperatures near 60°C for proteins (Bull, 1944; Drost-Hansen. 1971). Thus, measurements of physical properties (specific surface area, porosity, etc.) of anorganic enamel do not necessarily reflect the properties of the intact tissue. In addition, the various methods proposed for isolating the inorganic fraction (Williams and Irvine, 1954; Peckham, Losee and Ettleman, 1956: Quigley and Zwarych, 1963; Skinner, Kempher and Pak, 1972) involve high-temperature ashing (> 500°C) or wet chemistry extractions, both of which can cause undesirable alterations in the inorganic phase of enamel (Neuman and Mulryan, 1949; Peckham et ul., 1956). The purpose of the present investigation was to determine porosities, specific surface areas, and pore volume distributions of intact dental enamel samples using water vapour sorption isotherms.

MATERIALS Enamel

AND METHODS

sect;or1s

A group of thirty clinically sound teeth. which included incisors, canines, and molars, were selected for this study and stored in a moist environment at 4’C. These teeth were classified as to the age and sex of the donor. Thin enamel slices (lot--300 pm in thickness and 5-20 mg in weight) were cut parallel to the labial surfaces using a water-cooled thin sectioning device and a diamond blade. It was possible to obtain multiple samples from various locations within a single tooth by using this sectioning system. Individual enamel slices were suspended from one arm of a recording micro-electrobalance (sensitivity +O.l pg) fixed inside a vacuum chamber (see Fig. 1). The total pressure within the chamber was kept at one atmosphere except for some preliminary sorption experiments done under vacuum conditions. The

R. T. Zahradnik and E. C. Moreno

318

Thermostated

ible changes in the sorption characteristics were observed. For all the weights recorded at 25 and 50°C the accuracy was kO.4pg and the precision was better than + 1.Opg.

aw both

Pow volume distribution and suvfhce area analysis

Fig. 1. Schematic representation of the gravimetric water vapour sorption apparatus (see text for details). entire set-up was enclosed trolled air box (+OOl’C).

within a temperature

con-

Sorption isotherms The raw sorption data consisted of a series of values for the weight changes of the enamel sample as a function of the relative water vapour pressure of the surrounding atmosphere. The relative humidity of the environment was changed by steps between zero and 100 per cent by using a series of saturated 1960). Weight electrolyte solutions (Rockland, changes were detected by the recording microbalance and they were converted to percentages of the sample weight at zero relative humidity for plotting purposes. Complete sorption isotherms were generated by first increasing the relative humidity from a dry atmosphere to vapour saturation and then following a stepwise decrease back to dryness. Steady state was assumed when the enamel sample weight did not change for at least 15 hr. The adsorption and desorption branches of the isotherms for non-ashed samples were not coincident, thus producing a hysteresis loop. A procedure referred to as “scanning the hysteresis loop” was employed (Rao, 1941; Katz, 1949); i.e. the direction being followed along the relative pressure axis was reversed before either end point of the isotherm was reached, and the new direction was followed until the scanning curve intersected one of the main isotherm curves. Cycling within the region of hysteresis yields information on the nature of the pores in enamel responsible for the hysteresis effect. Adsorption capacities (sample weight increase when the relative humidity is changed from 0 to 100 per cent) were determined for thirty enamel sections at 25°C and complete sorption isotherms were obtained for 12 of these samples at the same temperature. Adsorption capacities were determined for six of these samples at 50°C; complete sorption isotherms were obtained for the remaining six samples at this temperature. These latter six samples were then low-temperature ashed (as explained below) and their adsorption capacities were redetermined; complete isotherms after cold-ashing were established for the two thinnest (z 1OOpm) sections at 25 and 50°C. The highest temperature (50°C) used in these experiments was below the temperature (60°C) at which irrevers-

The modelless method (Brunauer, Mikhail and Bodor, 1967) was used to determine the dimensions of the pores within enamel. For these calculations, the equilibrium values from the adsorption branch of the isotherms obtained at 25°C were used. This decision was based on results which indicate that enamel contains a constricted pore system (Moreno and Zahradnik, 1973). With such pore systems the adsorption branch is more representative of the true equilibrium state since, along the desorption branch, the constricted cavities retain water in metastable equilibrium. To apply the modelless method, the adsorption curve is first divided into increments. As the relative pressure is lowered through one of these increments, a corresponding group of pores empty by capillary evaporation. A multilayer of adsorbed film still remains on the walls of the pores, however, so that the term “core” is used to refer to that part of the pore space which is actually emptied. The total volume desorbed through this interval is not the volume of the cores of the group of pores alone. but includes the amount desorbed from the multilayers existing on the walls of the pores already emptied in the previous intervals. A corrected volume, therefore, needs to be determined. The correction term for multilayer evaporation is calculated using t-curves, which are plots of the statistical thickness of an adsorbed film against the relative pressure for a nonporous adsorbent. Curves for water vapour are available (Hagymassy, Brunauer and Mikhail, 1969). The film thickness at a particular relative pressure depends, however, on the heat of adsorption of the adsorbent. Therefore the choice of the proper r-curve for water vapour on enamel was made using the C constant of the BETequation which is proportional to the heat of adsorption. This procedure then corrects the core volume for any contribution due to multilayer evaporation. The surface of the core can be determined by a method based on general thermodynamic principles for capillary evaporation (Kiseler, 1945). The corrected core volume divided by the core surface gives the core hydraulic radius of this particular group of pores. In this way an idealized geometrical shape need not be assumed for the pores. From the result calculated for each pore group, a cumulative core volume curve is constructed. This involves a plot of V(r) vs r, where V(r) is the core volume of all pores that have hydraulic radii equal to or greater than r. To obtain the core volume distribution curve. then, AV(r)/Ar is plotted against r. where AV(r)/Ar is the slope of the cumulative curve between two values of the hydraulic radii and r is the average hydraulic radius in this range. If desired, it is a simple procedure to convert a core‘volume distribution into a pore volume distribution (Brunauer et a/.. 1967). For a given cylindrical pore. the radius of the cylinder is equal to twice the hydraulic radius of the core plus a value for the thickness of the adsorbed layer on the pore walls (obtained

Structural

features

of human

from the appropriate t-curve). For a parallel plate pore. the separation between the plates is equal to twice the hydraulic radius of the core plus twice the value for the thickness of the adsorbed layer. An analysis for the presence of micropores in enamel (pores with Kelvin radii less than about 1.6 nm) was performed using the method outlined by Mikhail. Brunauer and Bodor. 1968. This approach also relies on the so-called r-curves. A plot is constructed of the water adsorbed vs the thickness of the adsorbed layers to be expected at the various relative humidities. The thicknesses are obtained from the t-curve corresponding to a C value very close to that obtained in the BETanalysis for dental enamel. The plot should be linear as long as simple multilayer adsorption occurs, with a slope proportional to the surface area of enamel. However. if micropores exist. then the plot begins to deviate downward as these pores fill up and the available surface area decreases. The downward deviations. if present, are used for the determination of the pore volume distribution of the existing micropores. This kind of deviation was not found in the plots constructed with the present data. The specific surface area of enamel was determined using the BETmethod of analysis (Brunauer, Emmett and Teller, 1938). Data from the low relative pressure region of the adsorption curve (P/Ps < 0.3) were used to determine the monolayer capacity for water in grams per gram of enamel and the values of the C constants. The specific surface area was then calculated from the monolayer capacity using a value of 0. t oh nm’ for the molecular cross-sectional area of an adsorbed water molecule (Livingston, 1944). Low-trmprrc~turr

ushiry

Six of the enamel sections, varying in thickness from 95 to 325 pm, were introduced into a low temperature ashing device after their complete isotherms had been generated. This ashing procedure essentially involved passing a stream of radio-frequency-activated oxygen (13.56MHz) over the enamel sections at low total pressure (1 torr) and at temperatures not exceeding 6O’C for 48 hr. This procedure was tested with samples of powdered enamel and it was found that, on the basis of U.V. adsorption (280 nm) of chromatographed soluble fractions and analyses for primary amines (fluorscamine method), from 60 to 70 per cent of the organic matter was altered. The maximum changes in sorption characteristics of enamel sections induced by the low-temperature ashing were observed when the section thickness did not exceed 13 pm. At the low temperature used, no changes in the inorganic phase are anticipated; thus, the modihcation in sorption behaviour is assumed to be due to the alteration of the organic matter.

dental

enamel

0.6,-

P/ P,

Fig. 2. Water vapour sorption isotherm obtained at 25°C using a surface enamel section (240pm in thickness and 10.9 mg in weight) from a canine tooth (25-yr-old female). The ordinate is the relative weight change of the section (with reference to the section weight W at P/P, = 0) and the abscissa is the relative water vapour pressure. Open circles, adsorption branch; closed circles, desorption branch. Scanning of the hysteresis loop indicated by squares.

was used to interpret the scanning results (Rao, 1941). The fact that the desorption scan intersects the two branches of the isotherm whereas the adsorption scan meets the adsorption branch only at very high relative humidities necessitates the presence of some constricted or ink bottle type pores (Moreno and Zahradnik, 1973). Pore size analyses were performed on the basis of the adsorption data of the isotherms and a typical core volume distribution is shown in Fig. 3. The distribution was determined from the adsorption data of Fig. 2. The distribution is bimodal with values of 0.7 and 1.3 nm for the maxianalysis

-I I

G 4 :

RESULTS

The

water

vapour

adsorption

isotherms

for

the

non-ashed samples were qualitatively similar regardless of the source of the enamel sections. A typical example is shown in Fig. 2. All isotherms exhibited extensive hysteresis throughout the range of relative pressures as well as a general shape characteristic of the type IV isotherm (Brunauer et al., 1938). Representative desorption and adsorption hysteresis scans are included in Fig. 2. Rao’s method of

a

L

0

I

20

I 40

rn’

I 60

I

80

I IO0

H

Fig. 3. Core volume distribution obtained on the basis of the isotherm given in Fig. 2. The core volume distribution function is plotted vs the hydraulic radius. The ratios AVi Ar,, is the slope of a cumulative curve (core volume vs. rh) at rh.

R. T. Zahradnik and E. C. Moreno

320

Table 1. Water sorption capacity of molar surface enamel as a function of age at 25”C* Age of donor (yrs)

Sample dry weight (mg)

Water sorption capacity “/‘,dry weight

12 13 24 28 53 56 65 75

7.1 5.6 11.9 10.2 9.4 5.3 7.7 8.6

0.56 + 0.02t 0.59 IfI 0.03 0.57 * 0.01 0.56 + 0.01 0.46 + 0.01 0.51 f 0.03 0.46 k 0.02 0.45 * 0.02

* Female subjects. t S.E.M. mum of the two distribution peaks. Virtually the same distribution was found for a sample from another canine tooth. These values compare with values of 1.0 + 0.1 nmand 2.5 f 0.3 nm found for the distribution maxima with enamel samples from the incisors and molars used in this investigation. Assuming a cylindrical model, the two peaks in the core body distribution for canine enamel would correspond to pore bodies with radii of 1.9 and 3.3 nm, for molar and incisor enamel, the corresponding radii would be 2.6 and 58 nm. If a parallel plate model is assumed, the plate separations would be 2.4 and 4.0 nm for the pores in canine enamel, and 3.2 and 6.6 nm for the pores in enamel from molars and incisors. The measured water vapour adsorption capacity of enamel at 25°C was observed to depend both on the age of the tooth and upon the location of the enamel sample within the tooth. The two features are illustrated in Tables 1 and 2, respectively. The enamel samples from subjects ranging in age from 12 to 28 yr disp1ayed.a significantly higher sorption capacity than those samples from subjects in the age bracket of 53 75 yr (Table 1). The surface enamel sections used in connection with Tables 1 and 2 were 2OOpm thick and they were cut after the outermost 2(r30 pm layer had been removed by grinding. The figures in Table 2 show that the enamel sorption capacity doubles in going from the surface to the vicinity of the dentineenamel junction. The deep samples were cut as close

Table 2. Water sorption capacity of enamel as a function of depth at 25°C

Sample

Location

Incisor, 18 yr female Surface De-junction Molar, 25 yr male Surface De-junction

Sample weight (mg)

Water sorption capacity % dry weight

13.0 25,3

0.55 f 0.01* 1.18 + 0.01

10.7 4.1

0.57 f 0.02 1.25 k O-04

* S.E.M

as possible to the junction without contaminating the sample with dentine, which was verified by microscopic examination. The internal specific surface area of outer enamel, as determined by the BET method (Brunauer et al., 1938) for 12 enamel sections are given in Table 3. The corresponding C values for enamel are of the order of 30 f 7. It is apparent that the specific surface areas are similar for all the teeth regardless 01 the tooth type or the age of the subject. The actual values in Table 3 (3.5-4.5 mz g- ‘) are considerably higher than those reported using krypton as the molecular probe (Dibdin, 1969). However, other investigators using nitrogen (Huget, Brauer, and Loebenstein, 1968) reported values comparable to those reported here. Difficulties arise in surface area determinations if the system contains micropores. However, using a separate micropore analysis (Mikhail et al., 1968) it was possible to establish that there is an absence of a detectable number of micropores within the pore network of enamel. Six of the enamel sections were used to generate isotherms at 50°C in addition to 25°C. The effect of temperature on the water vapour sorption of enamel is illustrated by the representative plot in Fig. 4. The two sorption isotherms clearly show that enamel’s apparent water sorption capacity increases with increasing temperature. In Table 4 are given the water sorption capacities for the six enamel sections tested at the two temperatures. All enamel sections exhibited an increased water sorption capacity with tempera-

Table 3. Surface area of human surface enamel

Tooth type Molar

Premolar Incisor Canine

Age (yrs)

Sex

Thickness (pm)

Sample dry weight (mg)

Specific surface area m”/g

18 22 27 37 54 65 12 13 18 63 25 48

M F F M M F M F F F F M

240 265 300 270 125 205 210 240 210 265 240 235

Il.9 11.4 12.2 14.6 9.1 7.7 9-2 12.7 13.0 16.4 10.9 5.6

4.3 4.5 3.9 44 4.3 3.8 4.2 4.3 45 3.9 3.5 3.7

Structural features of human dental enamel

,”

0

m

I 0.2

I I O-4 0.6 P/P,

I 0.8

I I.0

I

I

I

02

04

Ocs

I OR

I 10

P/P,

Fig. 4. Effect of temperature on the water vapour sorption isotherm of a surface enamel section (I 25 pm in thickness and 9.7 mg in weight) from a molar tooth (54 year-old male). Left, at 25 C’: right at 50°C. ture. Sorption isotherms at 25°C obtained with samples previously used in experiments at 50°C coincided with those obtained initially at the former temperature. thus verifying the reversibility of the sorption characteristics. It was not possible to determine sorption capacities for temperatures above 50°C because irreversible changes did occur in such samples; thus, the sorption capacities determined at 25°C could not be reproduced if the sections had been heated at 60°C. For this reason no upper limit on total water sorption capacities could be established. It is, however, clear that the increase in measureable sorption capacity is due to an increased access to the water already contained or trapped within enamel rather than simply an increase in enamel’s capacity to adsorb at higher temperatures. This conclusion results from the observation that the total sample weight at 100 per cent relative humidity actually decreases slightly (z 0.1 per cent) in going from 25 to 50°C. The possible role of the organic matter on sorption characteristics was investigated with the two thinnest (94 and 125 pm) of the six sections that had been Table 4. Water sorption

Tooth

type

Incisor* Canine Molar*

* Enamel t S.E.M.

sections

capacity

Thickness

tested at the two temperatures. These two sections were low-temperature ashed and their isotherms were redetermined at 25°C. The results obtained. illustrated in Fig. 5 for one of the samples, were quite similar for both sections. The water sorption capacity was somewhat reduced ( : 15 per cent) after ashing. The striking difference, however, is the drastic reduction in hysteresis, especially in the low relative pressure region. Sorption studies done at a higher temperature (50°C) revealed that the ashed enamel sections exhibited a reduction (220 per cent) in adsorbing capacity with the increase in temperature. This is opposite to what was observed before ashing and is more in line with sorption by a porous system free of constrictions. The sorption results reported in this paper were obtained at a total pressure of one atmosphere. Preliminary water vapour sorption experiments using vacuum, however. yielded isotherms that were identical to those obtained in air. The sorption of water, therefore, is not affected by diluting it with air which apparently cannot compete with the water for the internal surface of enamel.

of human

enamel at two temperatures

Age (yrs)

Sex

Olm)

Sample dry weight (mg)

18 54 25 25 27 54

F F F M F M

210 235 240 95 300 125

25.3 16.3 10.9 4.1 12.2 9.7

from the vicinity of the dentine-enamel

junction.

Water sorption capacity “1 dry weight 50 C 25-C l-18 0.62 0.57 1.25 0.60 0.55

* f * _t + +

0.01t 0.01 0.02 0.04 0.01 0.02

I.34 0.77 0.77 1.4x 0.72 0.69

i_ 0~01 * O~OI _t 0.01 + 004 IfI 0.01 f 0.02

R. T. Zahradnik

322

and E. C. Moreno

I.2 r

P/P,

P/P,

Fig. 5. ElTect of cold-ashing on the water vapour sorption isotherm (at 25°C) of an enamel section (95 pm in thickness and 4.1 mg in weight) taken from near the dentino-enamel junction of a molar tooth (25 year-old male). Left. before cold-ashing; right, after cold-ashing.

DlSCUSSlON

The results of the present investigation have confirmed the observations made on a limited number of enamel samples and previously reported (Moreno and Zahradnik, 1973). The shape of the sorption isotherms, the presence of hysteresis down to very low relative vapour pressures (see Fig. 2) and the hysteresis scans are all consistent with the presence of constrictions in the pore network of enamel. Alternative explanations for the hysteresis are not completely satisfactory. For example, the concept that hysteresis reflects a delay in the meniscus formation in the pore bodies during the adsorption process (Foster, 1952) may be acceptable only for the mesopore (1.6nm < Kelvin radii < 16.0 nm) region but it could not be applied to the low vapour pressure (P/ P, < 0.35) region, simply because in this region there is no capillary condensation but rather multilayer formation. Possible abnormalities brought about by micropores (Kelvin radii < 1.6 nm) should be discarded because the micropore analyses did not support their presence in enamel; in addition micropore systems display a reversible behaviour in their sorption isotherms (Gregg and Sing, 1967). It must be pointed out, however, that the presence of hysteresis at very low vapour pressure implies that the constrictions should extend down to molecular dimensions (core hydraulic radii < 0.45 nm). Evidently, the surface area of the walls of these constrictions is negligibly small compared to the total specific surface area of the enamel; otherwise, their contribution to the total pore volume could have been ascertained in the micropore analyses. The sorption behaviour, judged from the shape and hysteresis of the isotherms, was similar for the enamel of the various tooth types investigated. Significant differences, however, were observed in the pore

volume distribution analyses. In the cases of enamel samples from canine teeth (Fig. 3) the distribution was bimodal as it was for the rest of the teeth, but a conspicuous reduction in the hydraulic radii of the two predominant pore sizes (0.7 and 1.3 nm) were observed in relation to the predominant sizes of incisors and molars (1.0 and 2.5 nm). These results are consistent with those obtained in diffusion studies of water through dental enamel (Burke and Moreno, 1974). It was reported that water had lower transport fluxes and higher activation energies for self-diffusion in enamel sections from canine teeth than from other tooth types. A reasonable assumption is that smaller constrictions should be associated with the smaller pore sizes of the canine enamel, hence a higher activation energy should be expected for the transport of water through these constrictions. Evidence that constrictions control the fluxes of matter through enamel is deduced from the comparison of apparent porosities obtained by vapour sorption with those calculated from diffusion studies. The sorption capacities found in the present study (see Tables 1 and 4) are in the order of 0.6per cent by weight; assuming a value of 3 g cmm3 for the density of dental enamel, the apparent porosity is about 1.8 per cent. The latter value is two orders of magnitude higher than the porosities derived from measurements of water diffusion, which implies that the pore constrictions restrict the mass transport to only a fraction of the existing pores; the remaining pores, presumably with very small constrictions, behave as blind pores for diffusional purposes. The pore volume distributions reported here were calculated on the basis of the adsorption branches of the sorption isotherms. It could be argued that different results would have been obtained if the calculations had been made on the basis of the desorption branch. However. because of the existence of

Structural

features of human dental enamel

constricted pores, the desorption curve reflects the properties of the constrictions and not those of the pore bodies. Thus, for the system as a whole, a point on the desorption branch at a given relative vapour pressure represents a metastable equilibrium with respect to the corresponding point on the sorption curve. Furthermore, when the apparent cause for the hysteresis is greatly reduced by cold ashing, it was observed that the desorption branch was the one greatly affected whereas the adsorption branch remained practically unchanged (see Fig. 5). For these reasons we believe that the correct procedure to calculate pore volume distributions in the enamel is through the use of adsorption data. The same reasons given in the foregoing paragraph are valid for the selection of the lower vapour pressure region (P/P,) < 03) of the absorption branch for the calculation of specific surface areas. The values given in Table 3 (: 4 m2 g-l) are substantially higher than those reported ( 2 @4 m2 g- ‘) using Krypton as the adsorbate (Dibdin, 1969). Explanations for these discrepancies have been given in a previous publication (Moreno and Zahradnik. 1973). The values presently reported are somewhat smaller than those published by other investigators who used powdered enamel and water as the adsorbate (Poole, 1971; Loebenstein, 1973). This is not surprising since significant changes in specific surface areas are often found when a porous material is ground (Gammage and Gregg, 1972; Odler ct ul.. 1972). It should be emphasized that the surface areas reported in Table 3 (calculated from adsorption data at 25 ‘C) do not constitute absolute values because, as shown in the Results section. the vapour sorption behaviour of enamel changes in an atypical fashion as a function of temperature. Thus, if the surface area calculations are made on the basis of the isotherms obtained at 5O’C, an increase of about 50 per cent is obtained in relation to the values given in Table 3. We believe. however. that specific surface area determinations made at or close to physiological temperatures will yield meaningful results in relation to dynamic processes taking place in the oral environment (ionic exchange. diffusion with reaction, etc.). The additional surface area that becomes apparent at higher temperatures is probably not accessible under normal conditions. It is pertinent to point out that there is no explicit relationship between apparent porosity and pore size. In fact it appears that the porosities of canine enamel and that of other tooth types is approximately the same (see Table 4). However, this result should be taken very cautiously because of the limited number of samples with which this comparison can be made. More definitive differences were established in the present study between apparent porosities (as reflected by sorption capacities) and the age of the enamel. The results in Table I clearly show a significant difference, for the same tooth type. between porosities of surface enamel in the age group 12-28 yr (~0.57 per cent) and those in the group 53-75 yr ( -0.47 per cent). At present the reason for this difference is not clear, There are at least two possibilities to be considered; a. very slow post-eruptive mineralization process, or a modification in the nature of the organic matter. An increase in the mineral phase of only 03 per cent by weight would account for the observed difference. Under normal conditions. saliva

i2i

is supersaturated with respect to the enamel mineral; thus, it is not unreasonable to think that a slow diffusion of calcium and phosphorus under small concentration gradients may bring about supersaturation conditions in the liquid phase filling the pores of the enamel with a concomitant precipitation. If this were the case, comprehensive studies on this subject might show a more marked reduction in the porosity of surface enamel than in that of the inner enamel regions in enamel of older subjects. An alternative explanation for the results shown in Table I relates to changes in the water holding capacity of protein in the enamel matrix with age. This latter possibility appears less likely because the reduction in sorption capacities of enamel upon cold ashing does not start approaching the differences reported in Table I for the two age groups. A simple increase in the organic matter content with age (with a consequent displacement of water) would have to be in the order of I5 per cent to be consistent with the present data: such an increase seems improbable. The marked increase in enamel sorption capacity in going from the enamel surface to near the dentin0 enamel junction (see Table 2) is in general agreement with previous reports (Brudevold et al.. 1960; Gwinnett. 1966) based on completely different types of measurements. This result most probably reflects the fact that in the last stages of amelogenesis the calcification proceeds from the surface inward. The determination of sorption capacities and isotherms at a higher temperature than 25