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Resin Infiltration of Natural Caries LesionsDept. of Operative Dentistry and Periodontology, University School of Dental Medicine, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Assmannshauserstr. 4-6, 14197 Berlin, Germany Correspondence: * corresponding author, hendrik.meyer-lueckel{at}charite.de
Infiltration of non-cavitated caries lesions with light-curing resins could lead to an arrest of lesion progression. The aim of this study was to evaluate the penetration of a conventional adhesive into natural enamel caries after pre-treatment with two different etching gels in vitro. Extracted human molars and premolars showing proximal white-spot lesions were cut across the lesions perpendicular to the surface. Corresponding lesion halves were etched for 120 sec with either 37% phosphoric acid gel (H3PO4) or 15% hydrochloric acid gel (HCl), and subsequently infiltrated with an adhesive. Specimens were observed by confocal microscopy. Mean penetration depths (SD) in the HCl group [58 (37) µm] were significantly increased compared with those of the H3PO4 group [18 (11) µm] (p < 0.001; Wilcoxon). It can be concluded that etching with 15% hydrochloric acid gel is more suitable than 37% phosphoric acid gel as a pre-treatment for caries lesions intended to be infiltrated.
Key Words: caries • resin infiltration • etching • acid gel
During the development of subsurface caries lesions, mineral is dissolved out of the enamel, resulting in increased porosities that appear clinically as the so-called ’white-spot’ lesions (Ten Cate et al., 2003). Today, these lesions are commonly treated by enhancing remineralization, e.g., by improving the individual’s oral hygiene or fluoridation. However, in non-compliant individuals with cavitated proximal lesions and greater lesion extension, this strategy has considerable limitations. A promising alternative therapy for the arrest of caries lesions might be the infiltration of subsurface lesions with low-viscous light-curing resins. Since porosities of enamel caries act as diffusion pathways for acids and dissolved minerals, infiltration of these lesions with resins might occlude the pathways, thus leading to an arrest of caries progression. Several studies have demonstrated that artificial caries lesions can be infiltrated by commercially available adhesives and fissure sealants (Davila et al., 1975; Robinson et al., 2001; Schmidlin et al., 2004; Meyer-Lueckel et al., 2006). Moreover, it has been shown that infiltrated artificial lesions do not progress in a cariogenic environment (Mueller et al., 2006; Paris et al., 2006). Thus far, only two in vitro studies have addressed the infiltration of natural lesions. However, these early reports were mainly descriptive (Davila et al., 1975), or used materials which were not clinically applicable due to their unsanitary nature (Robinson et al., 1976). Since there are substantial structural differences between both lesion types, it is not applicable to transfer findings from artificial to natural lesions. The surface layer of enamel caries lesions has a lower pore volume compared with that of the lesion body underneath (Bergman and Lind, 1966; Silverstone, 1973). Since the infiltration of enamel caries with light-curing resins is mainly driven by capillary forces, the pore diameter and volume influence the penetration speed (Paris et al., 2007). Therefore, the surface layer forms a barrier, which might hamper the infiltration of the lesion body. From this follows that removing or perforating the surface layer might be essential for a successful infiltration of the lesion body. In artificial lesions, brief etching with 37% phosphoric acid enhanced resin penetration (Gray and Shellis, 2002). With thicker and more mineralized surface layers in natural lesions (Bergman and Lind, 1966), it was assumed that this etching procedure would not be effective in eroding the surface layer (Meyer-Lueckel et al., 2007). The latter study confirmed that etching with 15% hydrochloric acid gel leads to a more effective erosion of the surface layer compared with 37% phosphoric acid gel, but did not focus on the subsequent infiltration of resins into the lesions. Therefore, the aim of the present study was to evaluate the penetration of a commercial adhesive into natural proximal caries lesions, without pre-treatment and with prior conditioning by two different etching gels in vitro. The working hypotheses were: (Hypothesis 1) The surface layer of natural uncavitated caries is a diffusion barrier, which hampers the penetration of resin. Therefore, no resin penetration occurs without prior etching; and (Hypothesis 2) pre-treatment for 120 sec with 15% hydrochloric acid gel leads to a more pronounced resin penetration compared with 37% phosphoric acid gel.
Extracted human molars and premolars showing proximal white-spot lesions were used in this study. The study protocol conformed to the principles outlined in the Central German Ethics Committee’s statement (2003) focusing on the use of human body material in medical research. After being carefully cleaned of soft tissues, teeth were stored in 20% ethanol solution until used. Teeth were examined by 20 stereo microscopy (Stemi SV 11; Carl Zeiss, Oberkochen, Germany), and cavitated as well as damaged lesions were excluded. For radiographic examination, teeth were positioned in a silicone base with the buccal aspects facing a radiographic tube (Heliodent MD; Siemens, Bensheim, Germany). To simulate cheek scatter, we placed a 15-mm wall of clear Perspex between the tube and the teeth. Standardized radiographs (0.12 sec, 60 kV, 7.5 mA) were taken of each tooth (Ektaspeed; Kodak, Stuttgart, Germany) and developed in an automatic processor (XR 24-II; Dürr Dental, Bietigheim-Bissingen, Germany). The radiographic lesion depths were independently assessed by two examiners and scored (Marthaler and Germann, 1970): no translucency (R0), translucency confined to the outer half on enamel (R1), translucency confined to the inner half of enamel (R2), translucency confined to the outer half of dentin (R3), or translucency confined to the inner half of dentin (R4). In case of disagreement in an assessment of radiographic lesion depth, a consensus rank was reached.
The roots of the teeth were removed, and the crowns were cut across the caries lesions perpendicular to the surface (Band Saw; Exakt Apparatebau, Norderstedt, Germany), providing two halves of each lesion (Figs. 1a, 1b
Subsequently, the cut surfaces were covered with nail varnish. In the treatment (TRT) group, corresponding lesion halves were etched either with 37% phosphoric acid gel (H3PO4; total etch; IvoclarVivadent, Schaan, Liechtenstein), or with an experimental 15% hydrochloric acid gel (HCl). The HCl gel contained hydrochloric acid 15%, glycerol 19%, highly dispersed silicon dioxide 8%, and methylene blue 0.01% in aqueous solution. After 120 sec, the gels were rinsed thoroughly with water spray for 30 sec. In the control (CTR) group, no acid etching was performed. Lesions were immersed in pure ethanol for 30 sec and subsequently dried for 60 sec with oil-free compressed air. A dental adhesive (Excite; IvoclarVivadent, Schaan, Liechtenstein) labeled with 0.1% tetramethylrhodamine isothiocyanate (TRITC; Sigma Aldrich, Steinheim, Germany) was applied to the lesion surfaces. The resin was allowed to penetrate the lesions for 5 min. Subsequently, excess material was removed by means of cotton pellets, and the resin was light-cured for 30 sec (Translux CL; Heraeus Kulzer, Hanau, Germany) at 400 mW/cm2. The nail varnish was carefully removed, and specimen halves were fixed on object holders parallel to the cut surface and polished (Exakt Mikroschleifsystem, Abrasive Paper 2400, 4000; Exakt Apparatebau, Norderstedt, Germany). To stain remaining pores, we immersed the specimens in 50% ethanol solution containing 100 µM/L sodium fluorescein (Sigma Aldrich) for 3 hrs. Specimens were observed by confocal laser scanning microscopy (CLSM Leica TCS NT; Leica, Heidelberg, Germany) in dual-fluorescence mode and with a 10x objective. The excitation light had two wavelength maxima, at 488 and 568 nm. The emitted light was split by a 580-nm reflection short-pass filter and passed through a 525/50-nm band-pass filter for FITC and a 590-nm long-pass filter for RITC detection. Images with a lateral dimension of 1000 x 1000 µm2 and a resolution of 1024 x 1024 pixels were recorded and analyzed by AxioVision LE software (Zeiss, Oberkochen, Germany). Penetration depths and thicknesses of the (residual) surface layer for the lesion halves were measured at up to 10 defined points (depending on the lesion size; indicated by a 100-µm grit), and mean values were calculated. Additionally to CLSM analysis, acid-etched as well as infiltrated lesion surfaces were observed by scanning electron microscopy (APPENDIX). Statistical analysis was performed with SPSS software (SPSS for Windows 11.5.1; SPSS, Chicago, IL, USA). Data were checked for normal distribution by the Kolmogorov-Smirnov test. To analyze differences in penetration depth between lesion halves/acid gels, we used the Wilcoxon test for paired samples. For comparison between unpaired groups, we performed Mann-Whitney and Kruskal-Wallis tests. Penetration depths were analyzed with regard to possible differences between various histological lesion extensions (C1-C3) and radiological grades (R0-R3). The level of significance was set at 5%.
In the CLSM images, the penetrated resin showed a red fluorescence, whereas remaining pores within the lesion, as well as dentin, appeared green (Figs. 1c–1e  ). Solid material, such as sound enamel or the surface layer, was displayed black.
Penetration depths varied considerably. For lesion halves etched with HCl gel, the mean penetration depth (standard deviation) [58 (37) µm] was significantly higher compared with that of those lesions treated with H3PO4 gel [18 (11) µm] (p < 0.001; Wilcoxon) (Fig. 2
For radiological grading of lesion extensions, good inter-observer agreement could be found (  = 0.804). Similar to histological lesion extension (C1–C3), no significant differences in penetration depth could be observed among different radiological grades (R0–R3) (Table ).
For those lesions where the surface layer was completely removed (CTR, n = 0; H3PO4, n = 2; HCl, n = 8), significantly higher (p < 0.01; Mann-Whitney) penetration depths [65 (35) µm] could be found compared with those lesions where residues of the surface layer remained after etching [33 (31) µm]. Surface layer thickness was significantly reduced after HCl etching [20 (18) µm], compared with that in the lesions etched with phosphoric acid [37 (25) µm] and with the non-etched CTR group [42 (23) µm] (p > 0.05; Mann-Whitney).
In previous studies where confocal microscopy was used, resin penetration was visualized by labeling of the resin with fluorescent dyes (Schmidlin et al., 2004). In another approach, the remaining (not infiltrated) pores were marked with a dye-labeled polymer (Meyer-Lueckel et al., 2006; Paris et al., 2006), or by imbibition in a fluorescent solution (González-Cabezas et al., 1998). In the present study, these two methods were modified and combined. The penetration of the resin was visualized by the red fluorescence of TRITC. The remaining lesion pores were indicated by the green fluorescence of sodium fluorescein. Hard tissues with small pore volume including the surface layer or sound enamel were neither infiltrated by the resin nor stained by the green solution and appeared black. Thus, the dual-fluorescence technique used in the present study allowed for the simultaneous observation of the porous lesion structure, the penetrated resin, as well as structures with small pore volume. Caries infiltration might be a promising approach for the treatment of uncavitated caries lesions. In contrast to fissure sealing, where the diffusion barrier is placed on top of the (lesion) surface, the infiltration technique aims to create the diffusion barrier inside the lesion, replacing lost mineral with resin. Therefore, the infiltration treatment should be differentiated from sealing techniques, where a resin layer (Goepferd and Olberding, 1989; García-Godoy et al., 1997; Tantbirojn et al., 2000) was established on the caries lesions. A recent clinical trial, where proximal enamel lesions were superficially sealed with an adhesive, found a significantly reduced but still relatively high (43.5%) lesion progression over an 18-month observation period (Martignon et al., 2006). Another clinical investigation did not find any significant differences in lesion progression between the sealed and the control group (Gomez et al., 2005). The latter authors speculated that treatment failures might be due to incomplete sealing or sealant disintegration over time. Moreover, laboratory studies confirmed the inferior resistance of unfilled resins to mechanical and chemical stress (Schmidlin et al., 2002, 2006). Therefore, it is questionable whether superficial smooth-surface sealing with unfilled resins is, as yet, generally applicable in daily practice. Compared with the latter concepts, the infiltration treatment might bear several advantages. With the infiltration technique, excessive resin is removed from the tooth surface before light-curing, whereby clinical application is greatly simplified. With application strips coated on one side, proximal lesions can be infiltrated without special protection for the adjacent tooth, and after only minimal tooth separation, e.g., by means of a wooden wedge or an orthodontic rubber band (unpublished results). Moreover, with this treatment, no sealant margins are produced on the tooth surface that could enhance plaque accumulation and cause periodontal inflammation. Furthermore, infiltration of the porous lesion structures might strengthen the lesion mechanically and prevent cavitation. To infiltrate a caries lesion, the penetrating resin needs access to the porous spaces of the lesion body. It was assumed that the penetration could be hampered by the highly mineralized surface layer, where the pore volume is considerably lower. In fact, in the present investigation, no penetration was found without prior acid-etching. Moreover, it could be demonstrated that penetration was significantly increased in those parts of the lesion where the surface layer was completely removed after acid-etching. Therefore, hypothesis 1 could be corroborated. Recently, 15% hydrochloric acid gel proved to erode the surface layer more effectively than 37% phosphoric acid gel (Meyer-Lueckel et al., 2007). In the present investigation, the effects of etching with these acid gels on resin penetration were compared. Phosphoric acid (37%) is frequently used in restorative dentistry for adhesive purposes. Hydrochloric acid has been previously used for enamel microabrasion (McCloskey, 1984; Mathewson et al., 1987). Although short-term contact of this strong acid with mucosa has been shown to be harmless (Croll et al., 1990), safety precautions, such as a rubber dam, should be used in clinical practice. Since significantly higher penetration depths could be found after etching with hydrochloric acid, hypothesis 2 could be confirmed as well. However, the surface layer could not be eroded completely in 67% of lesions in the HCl group. Thus, longer application times should be considered to achieve complete surface layer erosion. However, resin penetration was not influenced by macroscopic or radiological lesion extension. The penetration depths observed for Excite in natural lesions in the present study were lower compared with those observed in artificial lesions (104 µm/30 sec) in a previous investigation (Meyer-Lueckel et al., 2006), although a ten-fold longer penetration time was chosen in the present study. It might be argued that the incomplete surface layer erosion in natural lesions, even for those etched with HCl, could be responsible for this contradiction. However, in specimens where the surface layer could be totally removed, mean penetration depths were lower as well. In contrast to artificial lesions, the pores of natural caries might be contaminated with organic materials, such as proteins and carbohydrates, that might hamper resin penetration as well. This underlines that findings from artificial lesions cannot necessarily be extrapolated to natural lesions. It can be concluded that the surface layer of a non-cavitated natural caries lesion is a barrier that significantly hampers the penetration of a light-curing resin. Therefore, no substantial resin penetration could be observed without prior acid-conditioning. Etching for 120 sec with hydrochloric acid gel led to deeper resin penetration than etching with phosphoric acid gel, although surface layers could not be removed in all cases.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG; PA 1508/1-1). The authors are indebted to Mrs. Anja Bartels and Mrs. Julia Heinrich (Dept. of Operative Dentistry and Periodontology, CBF, Charité) for their excellent contributions to the experiments, to Dr. Herbert Renz (Dept. of Experimental Dentistry, CBF, Charité) for his assistance with the SEM, and to Prof. Dr. Harald Stein (Institute for Pathology, CBF, Charité) for providing the CLSM. The Charité-Universitätsmedizin Berlin holds US (US10/432,271) and European (EP06021966.4) patent applications for an infiltration technique for dental caries lesions in which the authors of this study are appointed as inventors.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication July 23, 2006. Revision received February 9, 2007. Accepted for publication March 6, 2007.
Journal of Dental Research, Vol. 86, No. 7,
662-666 (2007)
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ABSTRACT
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