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Natural Enamel Caries: A Comparative Histological Study on Biochemical Volumes ARTICLE in CARIES RESEARCH · DECEMBER 2012 Impact Factor: 2.28 · DOI: 10.1159/000345378 · Source: PubMed
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Original Paper Caries Res 2013;47:183–192 DOI: 10.1159/000345378
Received: July 16, 2012 Accepted after revision: October 22, 2012 Published online: December 5, 2012
Natural Enamel Caries: A Comparative Histological Study on Biochemical Volumes F. Barbosa de Sousa a, b J. Dias Soares b S. Sampaio Vianna c a
Department of Morphology and b Laboratory of Microscopy and Biological Image, Health Sciences Center, Federal University of Paraiba, João Pessoa, and c Department of Physics, Earth and Exact Sciences, Federal University of Pernambuco, Recife, Brazil
Key Words Enamel caries Histopathology Image analysis Microradiography Remineralization
Abstract This study aimed to test the hypothesis that organic volume is the main variable for explaining the optical properties and predictive degree of diffusion of enamel histological points at zones of natural enamel caries (NEC; surface layer, SL, n = 30, and body of the lesion, BL, n = 58) and normal enamel (NE, n = 131). Molars with either NEC or NE were quantitatively analyzed regarding the mineral, organic and water volumes (considered as effective pore volume), opacity (predicted in 94% of cases by water volume in NEC), and water volume more easily available for diffusion, ␣d (squared water volume divided by the nonmineral volume; related to permeability). NEC presented lower mineral volumes and higher organic volumes, effective pore volume and opacity than NE. External origin of organic volume in NEC was evidenced by an organic gradient decreasing from the surface inward (R2 = –0.7), which was not detected in teeth with NE only; ␣d values of the SL and NE were similar and both were lower (p ! 0.0001) than that of the BL. Comparing the SL from both NEC and artificial enamel caries (AEC; published data;
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n = 71), with similar mineral volumes, against developing enamel (published data), AEC showed more effective pore volume (3 times higher), higher ␣d and opacity than NEC mainly due to differences in organic volumes. Our results reasonably matched widely known features of NEC histological zones, and confirmed the organic volume as the main variable for explaining optical properties and ␣d (related to permeability). Copyright © 2012 S. Karger AG, Basel
Natural enamel caries (NEC) presents a heterogeneous mineral volume profile, with the lowest mineral content frequently located beneath a surface layer (SL) of ⬃30 m [Silverstone, 1973]. Diffusion through water in the enamel pores is the main transport process for inward movement of acid [Shellis and Dibdin, 2000], accounting for the extent of the carious lesion, and outward movement of dissolved mineral ions, the latter argued to create supersaturation of hydroxyapatite and, then, remineralization at the SL [Anderson and Elliott, 1987]. There is evidence indicating that the prisms’ sheaths are the main pathways for diffusion of cariogenic acid during progression of enamel caries and mineral ions during remineralization [Shellis, 1996; Shellis and Dibdin, 2000]. Recently, Frederico Barbosa de Sousa Departamento de Morfologia, Centro de Ciências da Saúde Universidade Federal da Paraíba, Cidade Universitária, S/N CEP 58051-900, João Pessoa, Paraíba (Brazil) E-Mail fredericosousa @ hotmail.com
increased attention has been paid to the infiltration of the pores of enamel caries with a curable resin, aiming at blocking further lesion progression [Meyer-Lueckel and Paris, 2008; Paris and Meyer-Lueckel, 2010]. The amount of organic matter in the SL has been debated in remineralization and infiltration studies [Meyer-Lueckel and Paris, 2008; Cochrane et al., 2010], but no quantitative volumetric data on nonmineral contents have been related to the features of that layer comparatively to the others. The limited quantitative volumetric data available on pore volume has been highlighted as a limitation of current knowledge on resin-infiltrated carious enamel [Robinson, 2011]. Permeability through the enamel pores is related to the water and organic contents [Atkinson, 1947]. Water and organic volumes are also predicted to exert an important effect on light scattering [Seinfeld and Pandis, 2006], polarization [Oldenbourg and Ruiz, 1989], and interference [Slayter and Slayter, 2000], affecting detection and monitoring of enamel caries by optical methods. Such volumes have been reported at histological layers of NEC [Medeiros et al., 2012] but have not been rigorously related to the differences among such layers. Thus, the aim of this study was to characterize the relationship between enamel histological points at certain zones (SL; body of the lesion, BL; normal enamel, NE) and the spatial distribution of biochemical volumes (including those related to pore volume) along prisms’ paths. We hypothesized that variations in organic matter play the key role for explaining features related to the optical properties and water more easily available for diffusion (related to permeability).
Materials and Methods Samples Twenty extracted erupted human third molars were collected from volunteers who signed an informed written consent, according to procedures previously approved by the Ethical Committee on Research in Humans of the Lauro Wanderley Hospital of the Federal University of Paraiba (Brazil). Uncavitated approximal enamel carious lesions (opaque enamel with surface brightness after removal of dental plaque with water spray and dehydration with compressed air for 5 s; carious enamel group, n = 10; one lesion per tooth) and approximal surfaces with NE at the mid-coronal region (NE group, n = 10) were selected under stereomicroscopy by a calibrated examiner (kappa = 0.98) with regard to the diagnosis of noncavitated white-spot enamel caries lesions according to the criteria of Nyvad et al. [1999]. Ground sections (final thickness of ⬃100 m) were prepared as described recently elsewhere [Medeiros et al., 2012].
184
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Quantification of Mineral Volume Samples and an aluminum (purity of 99%) step wedge standard (10 steps with sheets of 20 m, yielding a range of 20– 200 m) were radiographed in high-resolution radiographic plates (AGHD, Microchrome Technology, San Jose, Calif., USA) using a Faxitron X-ray machine (model MX20, Faxitron, Tucson, Ariz., USA; tungsten anode filtered with a 0.25-mm-thick beryllium window) with 20 keV, 0.3 mA, and 90 min of exposure time. The emission peak energy is 8.5 keV [Boone et al., 1997]. Digital photographs of the developed radiographic plates were obtained in transmitted light microscope (10! objective; bright field). Correction of uneven illumination in digital photographs (a prerequisite for quantitative analysis of gray levels in photomicrographs) was performed by image calculations with raw (R), flat field (F; field of view without sample) and dark field (D; camera front lens blocked) images. Corrected image was obtained by (R – D)/(F – D) in an image freeware (ImageJ, NIH, USA). The same software was used for obtaining of a calibration curve between gray levels and aluminum step wedge (R 2 = 0.9999), and measuring gray levels at the histological points. In order to measure sample thickness, and following a procedure described previously [Bergman and Lind, 1966], each sample was sectioned (after all measurements of birefringence) with a stainless steel blade positioned parallel to the direction of the enamel prisms and close (⬃100–150 m) to the points of measurement, and then a digital photomicrograph (resolution of 0.7 m) of the cut edge (positioned perpendicular to the objective’s lens) was obtained in a transmitted polarized light microscope with a 20! objective. Thickness was obtained (mean of ten measurements) at the histological points using ImageJ (NIH, USA) assuming that the thickness at the cut edge was the same as that at the closest histological point (located ⬃100–150 m away from the cut edge) along the transversal. Quantification of mineral volume using Angmar’s equation [Angmar et al., 1963] was performed as described previously [Medeiros et al., 2012]. The linear attenuation coefficients of enamel and aluminum were 205.245 and 109.138, respectively. These values were calculated using the mass attenuation coefficients of the elements of the formula for enamel hydroxyap-atite [density of 2.99 g/cm3; Ca8.856 Mg 0.088 Na 0.292 K 0.010 (PO 4) 5.312 (HPO 4) 0.280 (CO3) 0.407(OH) 0.702 Cl0.078(CO3)0.050] proposed by Elliott [1997], and aluminum (density of 2.7 g/cm3), available in the literature for the X-ray parameters used [NIST, 2004]. Measurements were performed up to a distance of 600 m along a transversal line following prism paths in carious (points at the following distance from the surface: 20 m, 50 m, and then at intervals of 50 m up to reaching NE; in NE intervals were of 100 m; fig. 1) and NE (20, 50, 100 m, and then at intervals of 100 m), hereafter referred to as carious and NE transversals, respectively. Carious transversals were placed where NE (mineral volume 188%) could be detected at the deeper points. Three zones in carious enamel were analyzed: SL (first measured point), BL (points located between the SL and the first point with mineral volume 688%, regardless of the sign of birefringence), and NE. For each lesion, two additional points (equidistants 50 m from the transversal) in the SL were selected, yielding 30. Quantification of Water and Organic Volumes Phase retardance (in nm) in water (24 h of immersion) at histological points was measured (mean of five measurements) in a
Barbosa de Sousa /Dias Soares / Sampaio Vianna
transmitted polarized light microscope (Axioskop, Carl Zeiss, Germany) with a 0–5 orders Berek compensator and a green interference filter (550 nm, bandwidth of 10 nm). The signal of the observed birefringence (BRobs) was determined using both a Red I interference filter and the Berek compensator. In order to measure water and organic volumes, a theoretical BRobs value equal to the experimental BRobs was computed by [Sousa et al., 2006]: 2
V1V2 1 V1 n12 n22
BRobs 0.0065 0.85 V1 (1) 2 V1n1 V2n2 ¡ 1 V1 n22 V2n12 ¯° ¢ ±
where, on the right-hand side, the first term is the intrinsic birefringence (BR intr) and the second term is the form birefringence (BR form). V1 is the mineral volume fraction measured by microradiography, V2 is the nonmineral volume fraction (V1 + V2 = 1), and n1 (1.62) and n2 are the refractive indexes of the mineral and nonmineral volumes, respectively. The latter was given by Sousa et al. [2006]:
n2 1.33
␣ V2
1.56

(2)
V2
where ␣ is the total water volume fraction (refractive index of 1.33), and  is the organic volume fraction (refractive index of 1.56; V2 =  + ␣). With this approach, there were two unknowns (␣ and ) and two equations: V2 =  + ␣ and theoretical BRobs = experimental BRobs. Theoretical BRobs and experimental BRobs were equalized by changing the values of ␣ and  (precision of 10 –3 for ␣ and  and 10 –5 for BRobs) iteratively. A limitation of such an interpretation is that the required mathematical fitting of experimental and theoretical BRobs can only be achieved to mineral volumes ^95% [Sousa et al., 2006]. Detailed description and examples have been recently reported [Medeiros et al., 2012]. In addition, we introduced a new parameter, the water volume more easily available for diffusion (␣d):
␣ ¬ ␣ V2 ®
␣d
(3)
where ␣ is multiplied by the fraction of V2 filled with water. Here we consider that ␣ is equal to the sum of the firmly (␣1) and loosely bound (␣2) water volumes, and that ␣2 is proportional to the ratio ␣/V2. ␣d ranges from 0 (V2 = ␣ =  = 0) to 1 [mineral volume (V1) = 0 and  = 0], and for a given 0 ! V2 ! 1 the higher the organic volume () the lower ␣d. Moreover, it was assumed that the V2 fraction filled with organic volume [/V2; (/V2 + ␣/V2) = 1] is an important factor restricting water mobility. The following published predicted nonmineral volumes as a function of the mineral volume (V1), derived from normal and developing enamel, were used in agreement tests [Sousa et al., 2009]:
␣1 = 0.1988 – 0.1652 V1
(4a)
␣2 = 0.6987 – 1.2487 V1 + 0.544 (V1)
(4b)
 = 0.086054 + 0.46808 V1 – 0.584 (V1)2
(4c),
2
where the total water volume fraction is equal to ‘␣1 + ␣2’. Source of Data on the SL of Artificial Enamel Caries Data on mineral volume (based on a mineral density of 3.15 g/cm3) and BRobs in water from the SL of artificial enamel
Natural Enamel Caries: Biochemical Volumes
caries (n = 71) were collected from the image of the figure where they were published [Theuns et al., 1993] using a software of image analysis and following the procedures described elsewhere [Sousa et al., 2009]. Published mineral volumes [V1(3.15)] were converted to new values based on a mineral density of 2.99 g/cm3 [V1(2.99)] according to Sousa et al. [2006]: V1 2.99 0.954913 V1 3.15 0.1057567
(5)
Then, the water, organic, and ␣d volumes were obtained from the interpretation of BRobs as performed with the experimental data of the present study.
Statistical Analysis The organic volumes along both transversals were normalized separately and correlated to the normalized distance from the enamel surface. Regarding the carious transversals, the first point at NE was considered as the deepest point. The normality of data was tested (Kolmogorov-Smirnov test). Statistics for each biochemical volume at each zone (SL, BL, and NE) were calculated. Groups were compared regarding the water and ␣d volumes by Mann-Whitney statistical test (significance level of 0.05) using ranked data (ranking as 1, 2, 3 …). Agreements between experimental [current data and data from the SL of artificial enamel caries published by Theuns et al., 1993] and predicted [Sousa et al., 2009] nonmineral volumes were tested using Bland and Altman [1986] plots. Correlations between positive BRobs and the biochemical volumes from NEC were analyzed using Pearson correlation.
Results
A total of 145 points (30 from the SL, 58 from the BL, and 57 from NE) from the carious transversals and 90 points from the NE transversals were analyzed (table 1). Organic and water volumes were not obtained at 16 histological sites (8 out of 90 sites from the NE transversals, and 8 out of 57 NE sites from carious enamel transversals) because mineral volume exceeded 95% [Sousa et al., 2006]. Statistics on biochemical volumes (including mineral volume) were calculated for the fitted points (n = 219) only (table 1). Lesion depth ranged from 250 to 500 m (mean of 340 8 77.5 m). The typical profiles from the carious transversals showed that the organic volume decreased from the SL inward (fig. 2a, c; both from lesions shown in fig. 1); both water volume and ␣d presented the highest values at the center of the BL, but with the difference that water volume values in the SL were higher than that of NE while ␣d values in the SL were similar to that in NE (fig. 2b, d). Two mineral volume profiles were obtained, with the mineral volume of the SL either lower (fig. 2a, related to lesion shown in fig. 1a) or similar to that of the BL (fig. 2c, Caries Res 2013;47:183–192
185
Color version available online
Fig. 1. a, b PLM images of two NEC lesions with the transversals and points located at 100, 200, 300, 400, 500, and 600 m from the enamel surface. c, d Microradiographs of lesions shown in a and b, respectively. Corresponding biochemical volumes are shown in figure 2. Bars = 100 m.
a
b
c
d
Table 1. Statistics (sample size, mean, SD, median, and Kolmogorov-Smirnov significance) for the biochemical volumes in NE, SL, and BL
Volume
n
Mean, %
SD
Median, Statistical % significance
Mineral Water Organic ␣d
131 131 131 131
92.37 5.56 2.07 4.15
1.61 0.72 1.26 0.81
92.68 5.57 1.81 4.02
0.009 0.000 0.069 0.015
Mineral Water Organic ␣d BL (all points) Mineral Water Organic ␣d
30 30 30 30
70.92 11.26 17.65 4.52
6.95 3.18 5.28 1.91
72.71 10.30 15.78 3.76
0.004 0.040 0.038 0.004
58 58 58 58
76.68 12.59 10.73 6.97
8.26 4.26 4.90 2.72
76.94 11.92 9.47 7.21
0.068 0.200 0.002 0.200
NE
SL
Total
219
related to lesion shown in fig. 1b), each type found in 50% of the carious transversals. NE transversals presented approximately flat profiles (fig. 2e, f). A negatively birefringent SL was detected in all but one lesion. Many sites with negative BRobs were included in carious enamel (fig. 2). From carious transversals, the normalized organic volume was linearly correlated 186
Caries Res 2013;47:183–192
with the normalized distance from the surface (fig. 2h; R 2 = –0.702 and p ! 0.0001), while no correlation was found from NE transversals (fig. 2g; R 2 = –0.000036, p = 0.687). Experimental and predicted [Sousa et al., 2009] (Eqs. 4a–c) water and organic volumes were plotted as a function of the mineral volume (fig. 3a, b). Good agreement was obtained between experimental and predicted organic volumes of NE (fig. 3c), while organic volumes from NEC were higher than predicted (fig. 3d). Carious enamel as a whole had water volumes lower than predicted (fig. 4b), and this difference was a bit higher when only the SL was considered (fig. 4c). When data from the SL of artificial caries [Theuns et al., 1993; data related to the same range of mineral volume as that of the SL of NEC; n = 71] were considered, lower differences were detected (fig. 4d). Approximately 3 times higher water volumes were observed in artificial caries compared to natural lesions. Because positively birefringent enamel is opaque [a common feature of carious enamel; Darling, 1958], we calculated the correlation between the biochemical volumes and BRobs. Water volume had the highest correlation (R 2 = 0.94, and p ! 0.0001; the higher water volume the higher the positive BRobs), closely followed by ␣d (R 2 = 0.92, and p ! 0.0001), and significantly higher than the mineral (R 2 = –0.35, and p ! 0.0001) and organic (R 2 = 0.033, and p = 0.09) volumes. The water volumes of the SL and BL did not differ (mean ranks: 40.03 for SL and 47.53 for BL, p = 0.196), but both were higher than that of NE (mean ranks for paired Barbosa de Sousa /Dias Soares / Sampaio Vianna
Volume (%)
a
Mineral Organic
Lesion 1
18
Water ␣d 9
0 0
100
200
90 80 70 60 50 40 30 20 10 0
c
Volume (%)
Lesion 1
300
400
500
600
Lesion 2 Mineral Organic
0
100
200
300
400
500
600
b
0
Volume (%)
Volume (%)
90 80 70 60 50 40 30 20 10 0
100
200
22 20 18 16 14 12 10 8 6 4 2 0
d
600
Water
␣d
0
60
Mineral Organic
40 20
Volume (%)
Volume (%)
500
100
200
300
400
500
600
Normal enamel Normal enamel
80
0
6 Water
␣d
4
2 0
e
100
200 300 400 Distance from surface (μm)
500
600
1.0 0.8 0.6 0.4 0.2
0
f Normalized organic volume
Normalized organic volume
400
Lesion 2
100
R2 = –0.000036 0
0.2 0.4 0.6 0.8 Normalized distance from surface
100
200 300 400 Distance from surface (μm)
1.0
Fig. 2. a–f Plots of typical distributions of biochemical volumes along the transverses from two NEC, one with arch-shaped (a, b) and the other with ‘linear’ (c, d) distribution of mineral volumes within carious enamel, and one NE (e, f). Organic, water, and ␣d
volumes present arch-shaped distributions in both lesions, while they are shown as flat profiles in NE (e, f). Areas with diagonal
500
600
1.0 0.8 0.6 0.4 0.2 R2 = 0.702
0
0
g
300
0
h
0.2 0.4 0.6 0.8 Normalized distance from surface
1.0
lines: sites with positive BRobs or pseudoisotropy. g, h Plots of the normalized distance from enamel surface against the normalized organic volumes for NE (g) and NEC (h), and corresponding linear fits showing no correlation for NE (R 2 = –0.000036) and a negative correlation for NEC (R 2 = –0.702).
comparisons: 146.43 for SL and 66.02 for NE, p ! 0.000001; and 158.44 for BL and 67.15 for NE, p ! 0.000001). A comparison made between the water volumes of the SL and the points of the BL presenting positive BRobs only revealed, however, a statistically significantly lower water volume at the SL (mean ranks: 21.57 for SL and 42.8 for BL, p ! 0.00001). Regarding ␣d, no statistical difference was observed between the SL and NE (mean ranks: 73.60 for SL and 75.97 for NE, p = 0.789), but both had ␣d values lower than those of the BL (points with both positive and
negative BRobs; mean ranks for paired comparisons: 28.70 for SL and 53.29 for BL, p ! 0.0001; and 71.67 for NE and 127.29 for BL, p ! 0.00001).
Natural Enamel Caries: Biochemical Volumes
Caries Res 2013;47:183–192
Discussion
This study shows that the nonmineral profile of NEC sharply differs from that of NE along prism paths, which is important for histopathology and diffusion. Carious 187
36 32 32 28
24
Water volume (%)
Organic volume (%)
28
20 16 12 8
16 12 8
0
4 50
60
2.0
70 80 90 Mineral volume (%)
100
50
b
NE
60
70 80 90 Mineral volume (%)
100
NEC 15
1.5 Mean +1.96 SD 1.18
0.5 Mean +0.07
0
Mean +1.96 SD 9.30
10 Differences
1.0 Differences
20
4
a
–0.5
5 Mean +2.47 0
Mean –1.96 SD –1.04
–1.0
0
1
2 3 4 Mean organic volume (%)
Mean –1.96 SD –4.37
–5
–1.5
c
24
5
5
d
10 15 20 Mean organic volume (%)
25
Fig. 3. a, b Experimental (scattered data) and predicted (lines) organic (a) and water (b) volumes as a function of mineral volume. Experimental and predicted nonmineral volumes reasonably match for NE (mineral volume 688%), but not for NEC
(mineral volume !88%). c, d Bland and Altman plots of the mean of experimental and predicted organic volumes against their differences for normal (c, data from all sections) and carious enamel (d).
enamel presented lower mineral volume, and higher water and organic volumes (table 1; fig. 3). The mineral volume profiles at the carious transversals presented values in the SL much lower than in NE and many data points in the BL with values higher than that in the SL as the profile approached NE (fig. 1a, b), being consistent with previous reports on in vivo/natural [Arends and Christoffersen, 1986; Medeiros et al., 2012] and in vitro caries lesions [Huang et al., 2011]. This is why the mean mineral volume in the SL was higher than in the BL (table 1). It has been shown that variations in mineral volume at the histological layers of enamel caries might be influenced by the random motion of dissolved ions in the pores [Anderson and Elliott, 1992]. As pore volume (which limits the water and organic volumes) is influ-
enced by mineral volume, randomness can influence pore volume in enamel caries. This is why histological points were treated independently. The water volume can be considered as the effective pore volume available for incorporation of new solids (including those created by remineralization and resinbased infiltrants). Based on the strong correlation (R 2 = 0.94) between the water volume and BRobs, and considering that positively birefringent enamel is opaque [Darling, 1958], water volume can also be regarded as a good predictor of optical properties (opacity) under water immersion. ␣d was computed using Eq. 3 in order to provide a measure related to permeability. This idea is based on: (i) the fact that permeability coefficient is the product of the diffusion and solubility coefficients, and all factors
188
Caries Res 2013;47:183–192
Barbosa de Sousa /Dias Soares / Sampaio Vianna
Normal enamel
Natural enamel caries
Mean +1.96 SD 1.11
1.0 0.5
Mean –0.017
0 –0.5 –1.0
Mean –1.96 SD –1.15
–1.5
Mean +1.96 SD 4.31
5 Water volume differences (%)
Water volume differences (%)
1.5
0 Mean –2.67 –5 Mean –1.96 SD –9.64
–10
–15
–2.0 4.0
4.5
5.0
a
5.5 6.0 6.5 7.0 Mean water volume (%)
7.5
10
12 14 16 18 20 Mean water volume (%)
22
–2 –4 Mean –5.83
–6 –8 –10 –12
Mean –1.96 SD –14.27
–14
26
Mean +1.96 SD 5.02
5
0
24
Artificial carious enamel (surface layer)
Mean +1.96 SD 2.61 Water volume differences (%)
Water volume differences (%)
8
b
Natural enamel caries (surface layer) 2
6
8.0
0
Mean –2.00
–5 Mean –1.96 SD –9.03
–10
–15
–16 0
c
10
12 14 16 18 20 Mean water volume (%)
22
24
0
d
10
12
14 16 18 20 22 Mean water volume (%)
24
26
28
Fig. 4. Bland and Altman plots of the mean of experimental and predicted water volumes against their differences for NE (a, data from all sections), NEC (b), SL from NEC (c), and SL from artificial enamel caries (d) [Theuns et al., 1993] (n = 56; all points with
mineral volume data based on a mineral density of 2.99 g/cm3 and within the same mineral volume range as those from the SL of NEC shown in b).
affect permeability and diffusion equally when solubility is constant [Pauly, 1999] (a common situation in enamel caries research, as is the case when tissue variations in a single immersion medium/infiltrant are considered); (ii) evidence indicating that deproteinization of the SL enhances the permeability of NEC to calcium [Robinson et al., 1990]; (iii) evidence indicating that removal of organic matter improves infiltration of aqueous solutions with high refractive indexes into enamel [Houwink, 1971], which can be interpreted as a result of an increase in the loosely bound water volume [Sousa et al., 2006]; (iv) evidence indicating that removal of organic content
accelerates rehydration of enamel by diffusion [Zahradnik and Moreno, 1975]; (v) the assumption that spaces for water are smaller in the organic framework than outside it; and (vi) the Einstein equation for diffusion, which predicts that the diffusion of molecules decreases with increasing frictional resistance of its environment [Dusenbury, 2009] (␣/V2 in Eq. 3 is expected to be inversely proportional to frictional resistance). The parameter ␣d is reported for the first time in the literature. It is known that the water volume can be divided into loosely and firmly bound water volumes, with the former requiring less energy to be removed, and, thus, being
Natural Enamel Caries: Biochemical Volumes
Caries Res 2013;47:183–192
189
more easily available for diffusion [Carlstrom and Glas, 1963; Shellis and Dibdin, 2000]. Dehydration of carious enamel at room temperature (required for measuring water volumes) frequently results in loss of birefringence in both the BL and SL [Medeiros et al., 2012], making it impossible to measure different types of water. In order to circumvent this limitation, we computed the water volume more easily available for diffusion using Eq. 3. Based on what was explained above, enamel histological zones can be compared regarding effective pore volume, ␣d (related to permeability), and opacity under water immersion. Although the lower mineral volumes were found at points of BL with positive BRobs, the organic volume profile presented a decreasing gradient from the surface inward (fig. 2h), suggesting an external origin. Organic and water volumes of NE are consistent with previous reports [Sousa et al., 2006; Medeiros et al., 2012]. The high organic volumes in carious enamel are consistent with previous reports on high amounts of organic matter in undemineralized sections of NEC evidenced by scanning electron microscopy [see fig. 7, 12, and 13 of Frank et al., 1964] and silver nitrate staining showing strong staining in carious enamel decreasing smoothly towards NE [see fig. 60 of Williams, 1923]. It is also consistent with the report of increased penetration of quinoline in the BL of NEC after removal of organic matter [Shellis et al., 2002]. These studies did not report data on the location of the highest organic content in NEC, while our data reports, for the first time with statistics, that the highest organic volume of this gradient is found at the surface layer. As erupted enamel lacks cellular processes capable of removing organic matter, such high organic volume yields lower effective pore volume, so that complete remineralization/resin infiltration is unlikely in the cases reported here. The SL presented effective pore volume significantly higher than that of NE but similar to that of the BL. Compared to the BL, the SL presented: (i) effective pore volume lower than that at the points with positive BRobs, but similar to those at the BL when all points are included no matter the sign of birefringence; (ii) lower ␣d; and (iii) lower opacity and positive BRobs than the points with positive BRobs. Relative to NE, higher effective pore volume and optical properties, and similar ␣d (due to the effect of organic volume in Eq. 3) were measured. The highest values of effective pore volume, ␣d, and opacity/ positive BRobs in NEC can be found in the BL. These comparisons reasonably match with the histopathological features of NEC reported in the literature [Silverstone, 1973; Medeiros et al., 2012]. The higher water volume at 190
Caries Res 2013;47:183–192
the SL compared to NE is consistent with data showing that after dehydration at room temperature changes in birefringence in the SL are stronger than in NE [Medeiros et al., 2012]. Our data suggest that incorporation of new solids (remineralization and infiltrants), permeability (when a single immersion medium/infiltrant is considered), and opacity are expected to be lower in NEC than in developing enamel [data from Sousa et al., 2009]. The water volume of the SL of artificial enamel caries (⬃3 times greater than in NEC, fig. 4) suggests that remineralization/infiltration, permeability, and opacity can be expected to be higher than at the SL of NEC and NE, in spite of the fact SL from NEC and AEC had similar mineral volumes (p 1 0.05). This is consistent with reports showing that removal of the SL in NEC is an indispensable step for proper penetration of resin-based infiltrants, and that the latter have been shown to diffuse deeper in artificial enamel caries compared to natural lesions [Meyer-Lueckel and Paris, 2008]. The artificial lesions analyzed here were similar to NEC in polarized light microscopy and microradiography [Theuns et al., 1993]. Our data show how artificial and natural lesions might differ from variables other than mineral content and qualitative BRobs. The variables described here can be useful to get deeper insights into remineralization and infiltration in enamel caries lesions (all types). Based on evidence that SL deproteinization enhances access of calcium into the BL of NEC [Robinson et al., 1990] (which is consistent with our results), it has been suggested to remove organic matter to enhance remineralization in NEC [Cochrane et al., 2010]. Because organic matter exerts an osmotic pressure in enamel [Atkinson, 1947], complete replacement of water by infiltrant monomer is unlikely. The remaining water volume located between the polymerized resin and the organic network might be able to provide diffusion pathways for cariogenic acids. This might explain the reported (low) progression of demineralization in resin-infiltrated enamel carious lesions [Paris and Meyer-Lueckel, 2010]. The Rayleigh-Debye-Gans approximation [Seinfeld and Pandis, 2006] describes light scattering in materials and predicts that scattering is directly proportional to: (i) the ratio of particle’s (hydroxyapatite crystals in the case of enamel) refractive index to the surrounding medium’s refractive index (organic matter and water in enamel), which can be directly related to ␣d; and (ii) pore sizes (directly related to the effective pore volume). Such a ratio is also expected to affect polarization and interference [Oldenbourg and Ruiz, 1989; Slayter and Slayter, Barbosa de Sousa /Dias Soares / Sampaio Vianna
2000]. In addition, fluorescence from enamel caries has been shown to be increased by both dehydration at room temperature (expected to be directly proportional to ␣d) [Mendes et al., 2004] and organic content [Lussi et al., 2004]. Thus, the biochemical volumes described here influence most of the optical methods currently applied to identify enamel caries, so that the optical properties of enamel cannot be reasonably explained if the effect of organic matter is ignored. The possibility must be considered that a reduced optical contrast between NEC and NE after a baseline severity results from the incorporation of organic matter instead of remineralization, and that a low ␣d caused by a high organic volume might cause falsenegative diagnosis of NEC. However, conclusions derived from data related only to the mineral content are a common feature of the current efforts on the histopathology of enamel caries, which, deprived of information on organic and water contents, potentially tend to lead to the misinterpretation that most of the increased pore volume is always occupied by water. Confirming our hypothesis, either when differences in mineral volumes existed (SL vs. NE in NEC) or not (SL of NEC vs. SL of AEC/developing enamel), our data show that the organic volume is the main variable for explaining differences between enamel histological zones regarding features related to optical properties and diffusion. The advantage of the methodology applied here can
be estimated based on the importance of spatially resolved data on pore volumes, optical properties, and permeability for the pathology, diagnosis, prevention, and treatment of enamel caries. For the future, deeper insights can be obtained by testing the predictive value of ␣d on the volume of solution (immersion medium or infiltrant) penetrated in carious enamel. Another interesting topic to be explored is the effect of organic volume and or ␣d on changes in optical properties and/or remineralization after treatment. In conclusion, NEC presented lower mineral volumes and higher water and organic volumes than NE. Differences among histological layers could be reasonably explained in terms of effective pore volume, ␣d, and opacity/ positive birefringence, which are greatly influenced by the decreasing-gradient profile and degree of organic volume.
Acknowledgments Financial support from CNPq (Brazilian Ministry of Science, Innovation and Technology) is greatly acknowledged.
Disclosure Statement The authors declare that there are no conflicts of interest.
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Barbosa de Sousa /Dias Soares / Sampaio Vianna
