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J Dent Res 85(9):804-808, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Remineralization of Enamel Caries Can Decrease Optical Reflectivity

R.S. Jones, and D. Fried

Department of Preventive and Restorative Dental Sciences, Box 0758, 707 Parnassus Ave., University of California, San Francisco, San Francisco, CA 94143-0758, USA; dfried{at}itsa.ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The remineralization of enamel caries can lead to distinct optical changes within a lesion. We hypothesized that the restoration of mineral volume would result in a measurable decrease in the depth-resolved reflectivity of polarized light from the lesion. To test this hypothesis, we measured optical changes in artificial caries undergoing remineralization as a function of depth, using Polarization-sensitive Optical Coherence Tomography (PS-OCT). Lesions were imaged non-destructively before and after exposure to a remineralization regimen. After imaging, microradiographs of histological thin sections indicated that the significant reflectivity reduction measured by PS-OCT accurately represented the increase in mineral content within a larger repaired surface zone. Mineral volume changes arising from remineralization can be measured on the basis of the optical reflectivity of the lesion.

KEY WORDS: artificial caries • early caries • diagnostic systems • polarization • optical coherence tomography • remineralization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Enamel caries has the potential to be arrested when the causal biofilm is removed, and the lesion can be restored with new mineral, especially with the aid of topical fluorides. Several studies have demonstrated that the process of remineralization restores mineral to a greater extent near the surface than in the underlying lesion body (ten Cate and Arends, 1977, 1980; ten Cate et al., 1981; Iijima et al., 1999). It has been well-documented that caries lesions can possess an intact surface zone with a histological appearance similar to that of sound enamel (Silverstone, 1973). When the surface zone is thick enough, it is considered to be characteristic of an "arrested" lesion, since the lower permeability may protect the underlying lesion from further progression and cavitation (Holmen et al., 1985). The difficulty with assessing depth-resolved changes within a remineralized lesion based on mineral volume, histology, or even mechanical properties is that it requires the destruction of the tooth and cannot be applied to an understanding of the process of remineralization in vivo. Clinical assessment of remineralized lesions, such as surface texture and appearance, are limited to the lesion surface. In contrast, direct measurement of changes in the optical reflectivity within different layers of a remineralized lesion may prove to be valuable for clinical evaluation of lesion structure and severity. In this study, we hypothesized that the restoration of mineral volume would result in a measurable decrease in the depth-resolved reflectivity of polarized light from the lesion. The significance of this work is that optical reflectivity may provide a non-destructive means of quantifying the extent of enamel remineralization and, potentially, lesion activity.

To test our hypothesis, we measured optical changes in artificial caries undergoing remineralization as a function of depth, using Polarization-sensitive Optical Coherence Tomography (PS-OCT). Two-dimensional images of the reflectivity of polarized light vs. depth from the enamel surface were measured with a depth (axial) resolution of 11 µm. PS-OCT and conventional OCT have found broad applications in the non-destructive imaging of biological structures (Huang et al., 1991; Hee et al., 1992; Fercher et al., 1993; Tearney et al., 1997; Fujimoto et al., 1999), including dental hard and soft tissue (Colston et al., 1998; Matheny et al., 2004). Dental OCT systems can utilize near-IR light, notably 1310 nm, since dental enamel has been shown to be nearly transparent in the near-IR spectrum (Fried et al., 1995; Jones et al., 2003). Early dental PS-OCT work identified enamel demineralization from sound tissue through an increase in reflectivity and changes in enamel birefringence (Baumgartner et al., 2000). PS-OCT can utilize linearly polarized incident light and measure the reflected light in 2 orthogonal polarization axes that are parallel and perpendicular to the incident beam. Optical reflectivity can be used to quantify caries when the reflectivity in the perpendicular axis to the incident polarized beam is measured from each layer, and the cumulative intensities are integrated (Fried et al., 2002; Jones et al., 2004, 2006a). Since pores within each layer of enamel caries highly scatter and depolarize incident polarized light, the PS-OCT perpendicular axis scans can resolve changes in the reflectivity of both the surface and subsurface enamel without interference from the strong surface reflection.

In this study, optical reflectivity changes during remineralization were measured on artificial lesions that were expected to have a porous surface zone. A previous study by our group determined that the remineralization of artificial lesions created by pH cycling did not repair the lesion surface zone, which suggested that the deposited mineral within demineralized pores during the pH cycling may have limited ion diffusion, restoration of partially demineralized crystals, and the growth of remaining crystals during the fluoride remineralization treatment (Jones et al., 2006b).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Artificial Smooth-surface Caries
Sound human posterior teeth (n = 20), which were collected after approval from the UCSF Committee on Human Research and informed patient consent, were mounted on 12-mm3 acrylic blocks after root resection. An acid-resistant varnish was applied to the teeth on all areas outside the 3x2-mm smooth-surface test region, which was scanned by means of the PS-OCT system. Artificial lesions were formed by exposure of the teeth for 9 days to a 40-mL aliquot of acetate buffer solution containing 2.0 mmol/L calcium, 2.0 mmol/L phosphate, and 0.075 mol/L acetate maintained at pH 4.9 and a temperature of 37°C. After the lesions were created, half the samples (n = 10) were exposed for 20 days to a 20-mL remineralizing solution of 1.5 mmol/L calcium, 0.9 mmol/L phosphate, 150 mmol/L KCl, and 20 mmol/L cacodylate buffer maintained at pH 7.0 and 37°C. To enhance the remineralization effect, we added 2 ppm F–, in the form of NaF, to the solution.

Polarization-sensitive Optical Coherence Tomographic Imaging
The fundamentals of PS-OCT have been detailed in numerous publications (Schmitt, 1999; Fujimoto, 2002), and the system used in this study has been described previously (Fried et al., 2002). PS-OCT measures the intensity and polarization state of back-scattered light as a function of depth at a specific lateral position. In this study, the PS-OCT system consisted of an all-fiber-based Optical Coherence Domain Reflectometer (OCDR) (Optiphase Inc., Van Nuys, CA, USA), which measured the reflected signal in the parallel and perpendicular axes to the polarized incident beam, defined as an a-scan, with a computer-controlled high-speed XY-scanning stage (Newport Corp., Irving, CA, USA), to produce a two-dimensional optical tomographic image, or b-scan (Appendix Fig.).

The PS-OCT system used a 20-mW broadband 1310-nm superluminescent diode (SLD; COVEGA, Jessup, MD, USA). The SLD source possessed a spectral bandwidth (FWHM) of 50 nm that produced an axial resolution when imaging enamel of 11 µm, and the system optics produced a 30-µm lateral resolution.

In less than 1 min, we obtained a two-dimensional OCT b-scan by laterally scanning the beam across the mounted wet tooth and collecting a series of depth-resolved signals. The b-scan images (n = 20) were acquired at day 0 and day 9. Demineralized artificial caries lesions (n = 10) were saved for histological evaluation, and the remaining samples were scanned with PS-OCT following 20 days of remineralization. In total, 8 serial b-scan images were acquired for each tooth at 400-µm intervals, which encompassed a 2.8 mm x 12 mm area consisting of the exposed lesion and the bordering sound enamel. A series of line profile integrations in the perpendicular axis over the depth of each lesion ({Delta}R, decibels [dB] x µm) was averaged and used for the assessment of overall lesion severity. Using a single line profile of the perpendicular-axis image, we determined the real lesion depth and surface zone by dividing the measured optical depth by the refractive index of the enamel (n = 1.63).

Histological Analysis
We used high-resolution (2.15 µm) digital transverse microradiography (TMR) of 150-µm-thick sections to measure the mineral loss or gain from the lesions before (n = 6) and after (n = 6) the remineralization treatment. From the TMR technique, we obtained the quantitative mineral loss profiles (Angmar et al., 1963) taken normal to the outer enamel surface. The relative mineral loss (vol% x µm), {Delta}Z, was calculated as the difference between the sound and lesion profiles on the same sample. We examined the hydrated tooth sections under a polarized light microscope (Series 7, Westover Scientific, Seattle, WA, USA; x400, NA = 0.25) to measure the thickness of the restored surface layer and overall lesion depth.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PS-OCT b-scan images, in both the parallel and perpendicular axes, of the 9-day-demineralized caries lesions (Figs. 1C, 1DGo) are in contrast to images of the bordering sound enamel that was protected from dissolution (Figs. 1A, 1BGo). The parallel-axis image of the artificial caries lesion (Fig. 1CGo) shows the intense reflectivity within each layer of the lesion, compared with the reflectivity within regions of the sound enamel (Fig. 1AGo). In the perpendicular axis (Fig. 1DGo), each distinct layer of the lesion depolarized the incident polarized light upon scattering. The integrated reflectivity in the perpendicular axis ({Delta}R) within the lesion was markedly greater than that of the sound enamel (Fig. 1BGo) caused by native birefringence. After exposure to the fluoride remineralizing solution for 20 days, layers near the surface of the lesion had a reflectivity (Figs. 1E, 1FGo) that was similar to that of sound enamel. Since the perpendicular axis (Fig. 1FGo) was less affected than the parallel axis (Fig. 1EGo) by the confounding surface reflection of the incident polarized light, we used the reflectivity in that polarization state to quantify the properties of the enamel layers near the surface and to determine the thickness of this surface zone. Beneath the surface zone, higher reflectivity regions within the lesion body remained after remineralization.


Figure 1
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  		Figure 1. PS-OCT b-scan images of bordering sound enamel in the parallel (A) and perpendicular (B) axes. Near the enamel surface (arrows), the signal in the perpendicular axis is minimal compared with the moderate back-scattered intensity in the parallel axis. Parallel-axis images of the nine-day artificial caries lesion (C) and the lesion after fluoride remineralization treatment (E) show the decrease in overall reflectivity with the existence of a surface zone of lower scattering after treatment. All of the parallel-axis images possess intense surface reflection that confounds analysis of the subsurface enamel and surface zone. Perpendicular-axis images of the artificial caries lesion (D) and the remineralized lesion (F) are shown. The perpendicular-axis image clearly resolves a surface zone, with optical properties similar to those of the sound enamel (B), and illustrates that the overall reflectivity of the lesion decreased after remineralization. Images are displayed in a false-color scale (bottom), and the blue scale bar (A) is 200 µm in real depth.

 
The {Delta}R, relative mineral loss ({Delta}Z), lesion depth, and surface zone width for the artificial lesions before and after exposure to the remineralization regimen are summarized (Tables 1Go, 2Go). The {Delta}R of the lesion decreased after the treatment (paired t test, p < 0.05). This decrease was caused from the minimal reflectivity of the surface zone layers. There was no measurable reduction in the reflectivity of the underlying lesion body from that of the same region in the artificial caries lesion. As a control in this experiment, there was no measurable difference in the {Delta}R of the bordering sound enamel before (21 ± 28 dB x µm) and after (19 ± 20 dB x µm) remineralization. Comparison of the TMR images of the 2 lesion groups (Figs. 2C, 2DGo) shows that the {Delta}Z was significantly less for the lesions after remineralization. The remineralization treatment restored mineral volume within the lesion. PS-OCT and PLM image analysis confirmed that the overall lesion depth did not substantially change after remineralization (Table 2Go). PLM revealed that the demineralized lesion samples possessed an intact enamel surface zone of 10 ± 4 µm (Fig. 2AGo). The thickness of the surface zone increased to 33 ± 5 µm for the lesion samples exposed to the fluoride remineralization solution (Fig. 2BGo). Except for subtle differences in birefringence, the optical properties of the surface zone were restored to those of the sound enamel. According to the relative mineral loss of the most superficial 40-µm region of the lesion, the TMR images indicated that mineral volume near the surface of the lesion was substantially restored after remineralization (t test, p < 0.01). The PS-OCT perpendicular-axis images did not identify a surface zone from the demineralized lesion, but did indicate a 35 ± 7 µm enamel surface zone after exposure to the remineralization solution. This surface zone possessed significantly less reflectivity in the perpendicular axis image than did the artificial lesion (paired t test, p < 0.001).


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  		Table 1. Lesion Severity
 

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  		Table 2. Lesion Depth and Surface Zone Width
 

Figure 2
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  		Figure 2. Polarized light microscopy (400x) of transverse sections of hydrated artificial enamel caries samples (A) and samples exposed to 20 days of fluoride remineralization solution (B). The artificial caries samples in (A) had an intact surface zone with a dark depolarizing lesion body. After remineralization, a zone of repaired enamel was evident over the remaining depolarizing lesion body. High-resolution digital transverse microradiography (TMR) revealed extensive mineral loss, with a higher mineral volume surface zone, after the nine-day exposure to the demineralization solution (C). After exposure to the remineralization solution, the mineral volume increased in the lesion body and was completely restored near the surface zone of the artificial lesion (D). The blue scale bars are 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An increase in mineral volume from the fluoride-enhanced remineralization significantly decreased the optical reflectivity of artificial lesions within an enlarged surface zone. However, the reflectivity did not decrease significantly in the body of the underlying lesion after remineralization. It is important to consider that the lesion body did not remineralize to the same level as the surface zone—that is, remineralization increased the center of the lesion body from 29 ± 7 to 48 ± 6% mineral volume. Optical property experiments of enamel caries have shown that the change in scattering intensity depends on the mineral volume range (Darling et al., 2006). There was a dramatic reduction in scattering between regions of 70–86% mineral volume, which was similar to the changes seen near the surface zone. In contrast, demineralized enamel between 30 and 70% mineral volume did not have significantly different scattering intensities, thus explaining the similar reflectivity within the lesion body after treatment. A possible mechanism is that the pore volume may have decreased in the lesion body by a reduction in the overall pore size, but the number of pores remained high. Also, the lesion body may not have been repaired by an organized crystal arrangement, because of the substantial volume loss from the demineralization. Similar results were observed with histological thin sections examined with polarized light microscopy, for which only the surface zone of enamel appeared to be restored and repaired to a similar orientation and degree as that of sound enamel. These results suggest that optical reflectivity depends on both the total volume of mineral and the directional nature of the repair.

The integrated reflectivity in the perpendicular axis ({Delta}R) measured by PS-OCT provided valuable information for identification and quantification of the surface zone and lesion body of the remineralized lesion. Since each layer of the repaired surface zone had a significantly reduced reflectivity, the surface zone thickness could be calculated, and corresponded with the thickness measured by histology. Likewise, the overall lesion depth did not change substantially after the remineralization regimen. The reduced lesion severity from the surface zone remineralization and restoration was quantified by the measured reduction in the {Delta}R. Although the depth and severity of the lesions produced in this study were similar to the demineralization and remineralization regimens used in other studies (e.g., Larsen and Fejerskov, 1989; Iijima et al., 1999), the demineralization and remineralization regimens used in this study should be treated as an artificial model that may not precisely simulate the complex process that occurs in vivo. The surface zone of the demineralized lesion may be accurately measured in the future with only a slight improvement to the current 11-µm axial resolution with a wider source bandwidth.

The ability to quantify the remineralized surface zone thickness and to identify the underlying lesion body, through differences in optical reflectivity, could be invaluable in lesion assessment and for caries diagnosis. Depth-resolved changes in optical reflectivity can be non-destructively measured by PS-OCT, and topography does not mitigate the imaging ability (Fried et al., 2002); therefore, the remineralization of occlusal caries can also potentially be assessed. Optical reflectivity measurements can be made by PS-OCT in imaging and processing times comparable with those of conventional radiography.

Future work will focus on the use of PS-OCT to monitor the remineralization of natural caries lesions both ex vivo and in vivo. The potential of PS-OCT for nondestructive measurement of the surface zone thickness of caries lesions in vivo is likely to provide a unique tool for the assessment of the outcome of non-surgical therapy and increase our overall understanding of the remineralization of enamel caries.


   ACKNOWLEDGMENTS
 
This work was supported by NIH/NIDCR R01 DE14698/T32 DE07306. The authors gratefully acknowledge the contributions of Cynthia Darling and John Featherstone.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received December 1, 2005; Last revision May 21, 2006; Accepted June 5, 2006


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 ABSTRACT
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angmar B, Carlstrom D, Glas JE (1963). Studies on the ultrastructure of dental enamel. IV. The mineralization of normal human enamel. J Ultrastruct Res 8:12–23.[Medline]

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