FT-Raman Surface Mapping of Remineralized Artificial Dental Caries

Transcription

1 FT-Raman Surface Mapping of Remineralized Artificial Dental Caries I. Stangel, 1 R. Rubinovitz, 2 G.D. Arndt, 3 D. Byerly, 3 C. Theriot, 3 J.E. Kerr, 4 1 BioMat Sciences, Bethesda, MD; 2 Thermo Fisher Scientific, Lanham, MD; 3 Johnson Space Center, Houston, TX; 4 Notre Dame of Maryland Univ, Baltimore, MD

2 Overview Purpose: The objective of the present work was to test the hypothesis that microwave- irradiated caries preferentially remineralize (compared to controls) when exposed to a mineralization environment. Methods: FT-Raman spectroscopy was used to track mineral concentration of enamel for microwave-irradiated and control samples by determining the area under the 1 PO4 3- band (960 cm -1 ) at baseline and after a remineralization protocol. The percent area under the phosphate band at each of 12 points by sample was calculated (relative to baseline values) to determine the degree of remineralization, and hence, the effect of microwave irradiation. Results: The mean post-remineralization hydroxyapatite concentration (relative to baseline) of the microwave-irradiated enamel samples was 100.6% (SD=43.6) while that of the controls was 39.6% (SD=8.1). An independent t-test revealed that the differences in means were significant at p< Introduction Dental caries, a microbial disease of teeth that infects nearly all Americans during their lifetime, is typically treated by surgical ( drill and fill ) approaches. Here, we investigate a novel non-invasive method of treating caries using microwave energy (ME). The utility of ME is based on the finding that prolonged colonization of enamel surfaces by cariogenic bacteria results in a ph decrease to below 5.5, with this decrease subsequently leading to demineralization. By killing susceptible cariogenic bacteria with ME, we hypothesize that ph can be modulated to above 5.5, which then promotes spontaneous remineralization of enamel from exogenous calcium and phosphate ions in saliva. Human enamel is primarily comprised of hydroxyapatite (Ca10(PO4)6(OH)2). Here, we follow the demineralization/remineralization cycle of enamel with FT-Raman spectroscopy. Raman spectroscopy is a powerful technique for studying the structure of enamel, requiring no sample preparation, and is sensitive to small changes in its crystalline structure. The PO4 3- of hydroxyapatite exhibits a strong band at 960 cm -1, and changes in enamel are easily followed by Raman shifts or area under the phosphate peak. Methods Sample Preparation Enamel fragments obtained from human teeth were mounted in a resin mold, surface abraded and then exposed to a Streptococcus mutans (the predominant bacteria implicated in caries) biofilm to promote artificial caries. Half the samples (n=4) were irradiated by ME and the other half remained as controls. All samples were subjected to a remineralization protocol for 8 days. FT-Raman spectra were obtained at after mounting (baseline) and after the remineralization protocol. FT-Raman Spectroscopy FT-Raman spectra were collected using a Thermo Scientific Nicolet is 50 FT-IR spectrometer, configured with a CaF 2 beamsplitter, InGaAs detector and FT-Raman accessory. The laser wavelength was 1064 nm having a spot size of approximately 50 microns. Spectra were collected at 8 cm -1 resolution and 32 scans per measurement at each of 12 representative points in the enamel slab at baseline. Spectra were again collected for the same points after the remineralization protocol. Spectra collection was automated and processed using Thermo Scientific OMNIC and Thermo Scientific Atlµs software. Data Analysis Mineral concentration, as determined by area under the phosphate peak, was collected for all data points. The mineral concentration at the remineralization endpoint was expressed as a percentage of the area under the phosphate peak relative to its baseline value for a particular data point. Descriptive statistics and data analysis were made using SPSS software (IBM Corporation, Armonk, NY). Statistical comparisons of the mean areas under the phosphate peak at baseline were made using a one-way analysis of variance, while comparisons of the percent mineral concentration of the experimental and control groups relative to baseline were made using a Mann-Whitney U-test. 2 FT-Raman Surface Mapping of Remineralized Artificial Dental Caries

3 Methods (schematic) Tooth Map of enamel slabs to be harvested 8 enamel slabs harvested Each enamel slab FT-Raman mapping at 12 points/slab Artificial caries induced by S. mutans biofilm Enamel Mean 960 phosphate peak area calculated for each of 12 points/slab Image showing 12 points interrogated Biofilm FT-Raman remapping of 12 baseline points - mean 960 area by slab calculated Slabs 1-4 Slabs 5-8 ME group Control group Remineralization protocol Results FIGURE 1. Representative baseline spectrum of enamel averaged from individual spectra collected at 12 points in a single sample. Peaks attributed to phosphate are shown in the spectrum v3 PO v4 PO4 430 v2 PO4 v1 PO4 960 Thermo Scientific Poster Note PITTCON PN52683-EN 0315S 3

4 FIGURE 2. The characteristic 1 PO4 3- band of hydroxyapatite at 960 cm -1 is shown. The area under the phosphate peak was calculated for each of the spectra collected at baseline and after the remineralization protocol. FIGURE 3. Box plot showing the area distribution under the 960 cm-1 peak for each of the eight enamel slabs at baseline. Wide variation in area means, as well as distribution between and within slabs can be noted, despite all slabs having been harvested from the same tooth. The difference in means were statistically significant (p<0.01). FIGURE 4. The remineralization patterns of the ME samples varied with respect to the 960 cm -1 peak areas with several showing a net decrease while others showed a net increase. (This variation can be observed in the slopes of the different ME samples, as seen in Figure 7.) Below, the 960 cm -1 peaks for each of 12 points for an ME sample at baseline (blue spectra) and after the remineralization protocol (red spectra) show a net decrease of about 50% in mineral concentration from baseline values. 4 FT-Raman Surface Mapping of Remineralized Artificial Dental Caries

5 FIGURE 5. The remineralization pattern for the ME sample below showed a net increase in mineral concentration from baseline values. Again, the 960 cm -1 peaks for each of 12 points are shown at baseline (blue spectra) and after the remineralization protocol (red spectra) FIGURE 6. The control groups predominantly showed a pattern where the postremineralization area under the 960 peaks was substantially lower than those at baseline. Below is the typical spectral pattern seen in the control samples, where the net loss was approximately 25% of baseline values. The net decrease in the 960 peak areas for the control groups trended well below that of the ME samples. FIGURE 7. Changes in mean area under the 960 cm -1 peak at baseline and postremineralization by ME and control group samples. Thermo Scientific Poster Note PITTCON PN52683-EN 0315S 5

6 FIGURE 8. Means and 95% confidence intervals (CI) for the percent mineral concentration of the ME and control groups relative to baseline. A Mann-Whitney U- test indicated that the mean percent mineralization of the ME group (samples 1-4) was statistically greater than that of the control group (samples 5-8), p< FT-Raman Surface Mapping of Remineralized Artificial Dental Caries

7 Conclusions The null hypothesis is rejected; hence, our hypothesis that microwave exposure of artificial caries promotes statistically greater remineralization compared to controls is supported by these data. A wide variation in the concentration of hydroxyapatite exists within very close regions of enamel harvested from the same tooth and at regions that closely approximate each other. This may be due to the abrasion of the enamel in the resin molds during sample preparation, thus non-uniformly exposing sub-surface enamel where crystallinity tends to vary as a function of depth. There was no significant shifting of the PO4 3- band at 960 cm -1 for nearly all spectra collected, indicating that ME used under the conditions of this experiment has no effect on the crystallinity of enamel. FT-Raman spectroscopy is an excellent tool for systematically determining changes in the mineral content of enamel. In particular, the auto-mapping feature facilitates systematic data collection of large data sets. Acknowledgements This research was supported by grant # from the National Science Foundation. We would further like to acknowledge Dr. Jack Guralnik for his excellent discussions on the data analysis Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. SPSS is a registered trademark of International Business Machines Corporation (IBM). All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientific, San Jose, CA USA is ISO 9001 Certified. Africa Australia Austria Belgium Brazil Canada China (free call domestic) PN52683-EN 0315S Denmark Europe-Other Finland France Germany India Italy Japan Korea Latin America Middle East Netherlands New Zealand Norway Russia/CIS Singapore Sweden Switzerland Taiwan UK/Ireland USA