Penetration pattern of rhodamine dyes into enamel and dentin: confocal laser microscopy observation

Transcription

1 International Journal of Cosmetic Science, 2012, 34, doi: /j x Penetration pattern of rhodamine dyes into enamel and dentin: confocal laser microscopy observation S. R. Kwon*, P. W. Wertz, Y.Lià and D. C. N. Chan *Department of Restorative Dentistry, Center for Dental Research, Loma Linda University, School of Dentistry, Loma Linda, California, Oral Pathology, Radiology and Medicine, Dows Institute for Dental Research, University of Iowa, College of Dentistry, Iowa City, Iowa, àcenter for Dental Research, Loma Linda University, School of Dentistry, Loma Linda, California and Department of Restorative Dentistry, University of Washington, School of Dentistry, Seattle, Washington, USA Received 15 August 2011, Accepted 19 September 2011 Keywords: confocal laser microscope, dentin, enamel, penetration, Rhodamine B Synopsis Enamel and dentin are susceptible to extrinsic and intrinsic stains. The purposes of this study were to determine the penetration pattern of Rhodamine B and dextran-conjugated Rhodamine B into and to relate it to the penetration pattern of hydrogen peroxide commonly used as an active ingredient in tooth-whitening agents and high-molecular-weight staining molecules. Eighteen recently extracted human maxillary anterior teeth were used. Teeth were cleaned and painted with nail varnish except for the crown area above the cemento-enamel junction (CEJ). The painted teeth were then immersed in Rhodamine B and dextran-conjugated Rhodamine B ( MW) for 4, 7, 10 and 15 days. Teeth were sliced to 3 mm thickness in transverse plane and mounted on a glass slide just prior to observation with confocal laser microscopy. Rhodamine B and dextran-conjugated Rhodamine B readily penetrated into the enamel and dentin when exposed for 4 and 7 days, respectively. Rhodamine B penetrated along the interprismatic spaces of the enamel into the dentin. The penetration was accentuated in sections with existing crack lines in the enamel. Rhodamine B was readily absorbed into the dentinal tubules at the dentino-enamel junction and continued to penetrate through the dentin via the dentinal tubules into the pre-dentin. Within the limitations of this study, it is concluded that Rhodamine B and dextran-conjugated Rhodamine B when applied to the external surface of the tooth readily penetrate into the enamel and dentin via the interprismatic spaces in the enamel and dentinal tubules in the dentin, suggesting that stain molecules and bleaching agents possibly exhibit similar penetration pathways. Résumé L émail et la dentine peuvent montrer des taches de l extérieur et de l intérieur. Le but de cette étude est de déterminer la voie de pénétration dans l émail et la dentine de la Rhodamine B et de la Correspondence: So Ran Kwon, DDS, MS, PhD, MS, Associate Professor, Department of Restorative Dentistry, Center for Dental Research, Loma Linda University, School of Dentistry, Taylor Street, Loma Linda, California 92350, USA. Tel.: ; fax: ; sorankwon@llu.edu Rhodamine B conjuguée au dextran par l observation en microscopie confocale laser et de la transposer à celle du peroxyde d hydrogène communément utilisé comme ingrédient actif dans le blanchiment des dents et aux agents de coloration à haut poids moléculaire. Dix-huit dents antérieures humaines récemment extraites ont été utilisées. Les dents ont été nettoyées et peintes avec du vernis à ongles à l exception du secteur de la couronne situé au dessus de la jonction émail-cément (CEJ). Les dents peintes ont ensuite été immergées dans la rhodamine B et la de Rhodamine B dextran conjuguée ( MW) pendant 4, 7, 10, 15 jours. Les dents ont été coupée en échantillons de 3 mm d épaisseur dans le plan transversal et montée sur une lame de verre juste avant l observation en microscopie confocale. La Rhodamine B et la Rhodamine B dextran conjuguée ont aisément pénétré dans l émail et la dentine respectivement lors des 4 et 7 jours d exposition. La Rhodamine B a pénétré le long des espaces interprismatique de l émail vers la dentine. Le taux de pénétration a été accentué dans les sections présentant des lignes de fissure au sein de l émail. La Rhodamine B est facilement absorbée par les tubules de la dentine à la jonction émail-dentine et continue à pénétrer à travers la dentine par l intermédiaire des tubules dans la prédentine. Dans les limites de cette étude, il est conclu que la Rhodamine B et la Rhodamine B dextran conjuguée, une fois appliquées à la surface externe de la dent, pénètrent facilement dans l émail et la dentine par les espaces interprismatique de l émail et les tubules de la dentine, ce qui suggère que les molécules à l origine des taches et les agents de blanchiment utilisent éventuellement des voies de pénétration similaire. Introduction Tooth whitening is a non-invasive and highly effective method to whiten discoloured teeth. It is a treatment option to enhance the aesthetics of the teeth that has been practiced in dentistry for over 100 years. Thus, the safety and efficacy of this procedure have been well established [1]. However, the basic mechanism of tooth whitening with hydrogen peroxide as the main active ingredient has not been fully explained. It has been demonstrated that hydrogen peroxide readily penetrates into the pulp cavity within 5 15 min when applied to the external surface of the tooth [2, 3]. The penetration is facilitated by the use of higher peroxide concentration, heat, light and ICS ª 2011 Society of Cosmetic Scientists and the Société Française de Cosmétologie 97

2 younger age of the tooth [2, 4 6]. It has been proposed that during the course of penetration, the oxidative free radicals generated from hydrogen peroxide interact with organic stain molecules, breaking them down into smaller molecules or breaking double bonds of pigment molecules to alter their configurations and consequently optical properties, resulting in the perception of a lighter colour. This conversion of stain molecules seems to change the light reflection of the tooth making it appear lighter [7]. To elucidate the mechanism, it seems essential to better understand the interaction chemistry of peroxides and stain molecules in the tooth and the penetration pathway of peroxides within the enamel and dentin. The aetiology and type of discolouration is an essential component of proper diagnosis and treatment planning for tooth whitening and usually dictates the prognosis of the whitening outcome. Discolouration has been traditionally classified as extrinsic, intrinsic or a combination of both [8]. Unlike extrinsic discolourations that occur on tooth surfaces, intrinsic discolourations are attributable to the presence of stain molecules within the enamel and dentin, incorporated either during tooth formation or after eruption [9]. It is noteworthy that in some cases, extrinsic stains like nicotine stain can eventually become intrinsic [10]. However, how readily extrinsic stain molecules can penetrate the tooth surface and what type of penetration pattern they will exhibit have not been well demonstrated. Rhodamine B is a water-soluble molecule with a molar mass of 479 g mol )1. It is often used as a tracer dye within water to determine the direction of flow and transport. It was hypothesized that Rhodamine B as a water soluble and low-molecular-weight substance would penetrate the enamel and dentin in a similar pattern as hydrogen peroxide (Molar mass: 34 g mol )1 ) used for whitening teeth. Dextrans are hydrophilic polysaccharides characterized by their moderate to high molecular weight, good water solubility and low toxicity. Dextran-conjugated Rhodamine B of molecular weight was used to simulate the penetration pattern of high-molecular-weight stain molecules. A common stain molecule tannin that is found in coffee, chocolate and orange-coloured juices can have molecular weights ranging from 500 to over 3000 and up to [11]. The purposes of this study were to determine the penetration pattern of Rhodamine B and dextran-conjugated Rhodamine B into and to relate the results to the penetration pattern of hydrogen peroxide and other stain molecules. Materials and methods Recently extracted human maxillary anterior teeth without any identifiers were obtained from the University of Iowa, and approval of an exempt status from IRB submission and review was received prior to initiating the study. Sample selection and preparation Eighteen recently extracted human maxillary anterior teeth were collected 1 month prior to the study and stored in 0.2% thymol (Sigma-Aldrich, St. Louis, MO, USA) in distilled water at 4 C. Thymol was used only to retard bacterial growth and minimize any effect on the tooth surface that might alter the response with in vivo conditions. Calculus was removed with a sickle scaler (Hu-Friedy, Chicago, IL, USA), and any surface debris was cleaned with plain pumice (PreppiesÔ; Whip Mix Corporation, Louisville, KY, USA) and purple prophy cups (Young Dental, Earth City, MO, USA). All teeth were observed for the absence of developmental anomalies, caries, existing restorations, deep crack lines or severe attrition. All teeth were painted with nail varnish except for the crown area 1 mm above the cemento-enamel junction (CEJ). Dye immersion The painted teeth were then randomly divided into two groups according to the dye solution used. Group 1 was immersed in Figure 2 Sectioning of tooth mounted on a precision cutting machine (Isomet 1000; Buehler Ltda.). Figure 1 Immersion of teeth in dye solution. The bottle was wrapped with a tinfoil (Buffalo Dental MFG) to prevent photo-bleaching of the dye solution. Figure 3 Observation of tooth sample with a LSM 710 confocal laser microscope (Carl Zeiss Microimaging GmbH). 98 ICS ª 2011 Society of Cosmetic Scientists and the Société FrançaisedeCosmétologie International Journal of Cosmetic Science, 34,

3 Figure 4 Control tooth with no dye immersion observed at the enamel. The aprismatic fluoride-rich outer layer exhibits high fluorescence. Figure 6 Tooth immersed in Rhodamine B for 4 days, exhibiting full penetration into the enamel along the interprismatic spaces. Figure 5 Control tooth with no dye immersion observed at the DEJ. The DEJ shows moderate fluorescence. 100 lm Rhodamine B (Sigma-Aldrich) for 4, 10 and 15 days, and group 2 was immersed in 100 lm dextran-conjugated Rhodamine B ( MW, neutral, Invitrogen, Eugene, OR, USA) for 7 and 15 days. The dye solution was replaced every 3 days with a freshly prepared dye solution. The bottle was wrapped with a tinfoil (Buffalo Dental MFG, Syosset, NY, USA) to prevent photo-bleaching of the dye solution (Fig. 1). Figure 7 Tooth immersed in Rhodamine B for 15 days, presenting tree-like branching of the dentinal tubules at the DEJ. Rhodamine B is readily uptaken into the dentinal tubules. Tooth sectioning and mounting After dye immersion of the whole tooth, the root portion was trimmed off with an orthodontic trimmer (3/4 HP Wet Model Trimmer; Whip Mix Corporation). The trimmed tooth was mounted in a precision cutting machine (Isomet 1000; Buehler Ltda., Lake Bluff, IL, USA) and sectioned in transverse plane of the tooth at the cervical height of contour (Fig. 2). The surface of the sectioned tooth was further polished with special silicon carbide grinding papers (Microcut, grit 600/1200, grit P2400; Buehler Ltda.). The 3-mmthick tooth section was mounted on a glass slide and observed with a LSM 710 confocal laser microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) (Fig. 3). All images were further processed with image J software (NIH, USA). Results Rhodamine B and dextran-conjugated Rhodamine B readily penetrated into the enamel and dentin when exposed to the dye solution for 4 and 7 days, respectively, as evaluated with the confocal laser microscope. The human tooth exhibits varying degrees of nat- ICS ª 2011 Society of Cosmetic Scientists and the Société Française de Cosmétologie International Journal of Cosmetic Science, 34,

4 Figure 8 Tooth immersed in Rhodamine B for 10 days, showing cross-sectioned dentinal tubules with circular or elliptical orifices with high content of Rhodamine B. Figure 10 Tooth immersed in dextran-conjugated Rhodamine B for 15 days, presenting uptake of dextran-conjugated Rhodamine B from the enamel into the terminal branches of the dentinal tubules at the DEJ. Figure 9 Accumulation of Rhodamine B at the outer boundary of the pulp space in a tooth immersed in Rhodamine B for 17 days. ural fluorescence depending on the mineral content and uptake of other trace molecules. Thus, control teeth not immersed in the fluorescent dye solution were observed under the confocal laser microscope using constant laser parameters to establish the baseline fluorescence of the human dentin and enamel (Figs 4 & 5). The enamel is primarily composed of hydroxyapatites making up the rod and interrod enamel. In the enamel, Rhodamine B penetrated along the interprismatic spaces, so that the longitudinal orientation of the rods was clearly visible. Striae of Retzius generally ascribed to a weekly rhythm in enamel production and appearing as a series of concentric dark lines could be observed (Fig. 6). Rhodamine B was readily absorbed from the enamel into the dentinal tubules at the dentino-enamel junction via the terminal Figure 11 Tooth immersed in dextran-conjugated Rhodamine B for 15 days, showing penetration into the dentinal tubules. branches (Fig. 7). The dentinal tubules are tapered structures with larger diameters and greater density per area in the pulpal region that could be observed in the micrographs by moving from the pulpal region towards the dentino-enamel junction (DEJ). Depending on the viewed area and the angle of sectioning, the dentinal tubules could be observed either longitudinally or cross-sectioned showing the openings (Fig. 8). Rhodamine B penetrated into the dentinal tubules more readily than into the intertubular dentin that was marked and highlighted by greater fluorescence in the dentinal tubules (Fig. 9). Pre-dentin is the first deposited unmineralized matrix at the pulpal boundary that varies in thickness from 10 to 50 lm and consists mainly of collagen [12]. Heavy accumulation of Rhodamine B was observed in the pre-dentin area demonstrating 100 ICS ª 2011 Society of Cosmetic Scientists and the Société FrançaisedeCosmétologie International Journal of Cosmetic Science, 34,

5 full penetration of Rhodamine B from the outer enamel surface into the pulp cavity (Fig. 9). Interestingly, the high-molecular-weight dextran-conjugated Rhodamine B (MW ) penetrated from the enamel surface to the pulp cavity exhibiting the same penetration pattern as the lowmolecular-weight Rhodamine B, suggesting that teeth are susceptible even to high-molecular-weight stain molecules when exposed for up to 7 and 15 days (Figs 10 & 11). Discussion The purposes of this study were to determine the penetration pattern of Rhodamine B and dextran-conjugated Rhodamine B into and to relate it to the penetration pattern of hydrogen peroxide and other stain molecules. This in vitro study has shown that water soluble, low-molecularweight Rhodamine B and even the high-molecular-weight dextranconjugated Rhodamine B readily penetrated into the enamel and dentin through interprismatic spaces in the enamel and via dentinal tubules in the dentin. This confirms that the dental hard tissues are significantly permeable to fluids, and the greatest fluid flow in the enamel is in the interprismatic spaces [13]. The permeability of dentin has been evaluated extensively, and it has been shown that it is dependent on the configuration of intertubular dentin, that is the product of the number and diameter of the dentinal tubules, dentin thickness, temperature and the distance from the pulp chamber [14 16]. In the case of dye penetration, it is also dependent on solubility, concentration and particle size of the dye. Other factors are capillary forces, osmotic gradients and concentration gradients [14, 15, 17]. According to the results of this study, the fluorescent dye molecules are taken up from the interprismatic spaces into the dentinal tubules, which seem to act as channels to lead the dye molecules into the pulp cavity. This is an important aspect, because the human tooth is constantly exposed to the accumulation of stain molecules in food and drugs and still retains the basic tooth colour for a long time. It would be interesting to extend the immersion time of the tooth in the dye solution and observe any differences in terms of accumulation of dye molecules into the intertubular spaces of dentin where the stain molecules may not be readily washed away. This accumulation might have a more pronounced effect on the tooth colour. The main limitation of this study was the absence of dentinal fluid flow produced by intrapulpal pressure in vital teeth that might have affected the results as shown by Vongsavan and Matthews [18] who evaluated the diffusion of Evans blue applied on dentine surface and observed that where as the dye diffused readily into dentine in extracted teeth, a lower dye penetration was observed when it was applied in vivo. Further studies on the penetration pattern of stain molecules and hydrogen peroxide into the tooth structure and their interaction with enamel and dentin will assist in elucidating the mechanism of tooth whitening that will ultimately assist in further development of whitening agents that will work more effectively and faster. Conclusions Within the limitations of this study, it is concluded that Rhodamine B and dextran-conjugated Rhodamine B when applied to the external surface of the tooth readily penetrate into the enamel and dentin via the interprismatic spaces in the enamel and dentinal tubules, suggesting that stain molecules and bleaching agents probably exhibit similar penetration pathways. Acknowledgements This study was not a funded research project. Special thanks to Katherine Walters, Jean Ross and Chantal Allamargot from the Central Microscopy Research Facility at the University of Iowa for sharing their expertise. References 1. Haywood, V.B. History, safety, and effectiveness of current bleaching techniques and applications of the nightguard vital bleaching technique. Quintessence Int. 23, (1992). 2. Bowles, W.H. and Ugwuneri, Z. Pulp chamber penetration by hydrogen peroxide following vital bleaching procedures. J. Endod. 13, (1987). 3. Cooper, J.S., Bokmeyer, T.J. and Bowles, W.H. Penetration of the pulp chamber by carbamide peroxide bleaching agents. J. Endod. 18, (1992). 4. Benetti, A.R., Valera, M.C., Mancini, M.N., Miranda, C.B. and Balducci, I. In vitro penetration of bleaching agents into the pulp chamber. Int. Endod. J. 37, (2004). 5. Camargo, S.E., Cardoso, P.E., Valera, M.C., de Araujo, M.A. and Kojima, A.N. Penetration of 35% hydrogen peroxide into the pulp chamber in bovine teeth after LED or Nd:YAG laser activation. Eur. J. Esthet. Dent. 4, (2009). 6. Camps, J., de Franceschi, H., Idir, F., Roland, C. and About, I. Time-course diffusion of hydrogen peroxide through human dentin: clinical significance for young tooth internal bleaching. J. Endod. 33, (2007). 7. Albers, H. Lightening natural teeth. ADEPT Report 2, 1 24 (1991). 8. Watts, A. and Addy, M. Tooth discoloration and staining: a review of the literature. Br. Dent. J. 190, (2001). 9. Dahl, J.E. and Pallesen, U. Tooth bleaching a critical review of the biological aspects. Crit. Rev. Oral Biol. Med. 14, (2003). 10. Haywood, V.B. Tooth Whitening: Indications and Outcomes of Nightguard Vital Bleaching, pp Quintessence Pub Co, Inc, Illinois (2007). 11. Florkin, M. and Mason, H.S. Comparative Biochemistry, pp Academic Press, New York (1962). 12. Nanci, A. Ten Cate s Oral Histology, pp Mosby/Elsevier, Missouri (2008). 13. Ake-Linden, L. Microscopic observations of fluid flow through enamel in vitro. Odontol. Revy 19, (1968). 14. Pashley, D.H., Livingston, M.J. and Whitford, G.M. The effect of molecular size on reflection coefficients in human dentine. Arch. Oral Biol. 24, (1979). 15. Oliver, C.M. and Abbott, P.V. Entrapped air and its effects on dye penetration of voids. Endod. Dent. Traumatol. 7, (1991). 16. Pissiotis, E. and Spangberg, L.S. Dentin permeability to bacterial proteins in vitro. J. Endod. 20, (1994). 17. Vasiliadis, L., Darling, A.I. and Levers, B.G. The histology of sclerotic human root dentine. Arch. Oral Biol. 28, (1983). 18. Vongsavan, N. and Matthews, B. Fluid flow through cat dentine in vivo. Arch. Oral Biol. 37, (1992). ICS ª 2011 Society of Cosmetic Scientists and the Société Française de Cosmétologie International Journal of Cosmetic Science, 34,