Abstract
Objectives
To explore the feasibility of a 3D-microleakage protocol for the evaluation of various configurations of adhesive–tooth interfaces.
Methods
Three different kinds of specimens were prepared: (1) a Class-I composite restoration placed without any bonding to maximize gap formation at the interface; (2) a glass-fiber post cemented with a self-adhesive composite cement into a prepared root canal; and (3) inlay MOD composite restorations placed with either a 1-step self-adhesive or a 2-step etch-and-rinse composite cement. After silver-nitrate (AgNO 3 ) infiltration, the specimens were scanned using a Skyscan 1172 X-ray microtomograph (μCT; Skyscan Bruker) at 100 kV, 100 μA and 7.8–9.5 μm resolution. Projection images were reconstructed, after which maximum-intensity projections (MIPs) and 3D-volumetric renderings were produced. For the inlays, an additional conventional stereomicroscopic (SM) microleakage evaluation was accomplished after specimen sectioning.
Results
MIPs and 3D-renderings from specimens (1) and (2) revealed strongly varying microleakage patterns along the marginal gap/interface. For the specimens of setup (3), the high radiopacity of the 2-step composite cement hindered evaluation of the MIPs. MIP-microleakage patterns along the enamel margin of the restoration cemented with the 1-step composite cement corresponded well to the stereomicroscopic images.
Significance
The reported μCT-protocol revealed good sensitivity to detect AgNO 3 infiltration at the adhesive–tooth interface when considerable microleakage was present. When microleakage was less apparent and spread in a more diffuse pattern, evaluation with μCT was less sensitive compared to stereomicroscopic evaluation.
1
Introduction
Microleakage is defined as the clinically undetectable penetration of fluid, bacteria, molecules and ions between the cavity wall and the restoration. It is known to cause post-restorative sensitivity, marginal defects and staining, and may eventually even lead to secondary caries and pulp irritation . Acting independently or synergistically, these factors will compromise the longevity of composite restorations in vivo .
Marginal leakage is attributed to different factors, including imperfect bonding at the adhesive-dentin/enamel interface resulting from composite-polymerization shrinkage, mismatch in the coefficients of thermal expansion between composite and tooth, or fatigue resulting from cyclic loading, to which the tooth-restoration complex is exposed during oral function .
The traditional method to detect microleakage is to evaluate the penetration of a specific tracer microscopically after sectioning of the sample . Organic dyes ( e.g. basic fuchsin, methylene blue, rhodamine B) and silver nitrate (AgNO 3 ) are the most commonly used agents for this purpose. AgNO 3 is also electron dense and radiopaque, which allows its use with correlated microscopy techniques, such as scanning or transmission electron microscopy . More recently, AgNO 3 has been applied in conjunction with X-ray micro-computed tomography, commonly known as micro-CT or μCT .
Criticism about microleakage tests encompasses mainly the lack of correlation with clinical data . This can be attributed to several reasons. First, proper standardization of the methodology between studies is lacking, together with the use of different kinds of tracers, concentrations and immersion times. Secondly, variability regarding the quantification of the tracer infiltration, ranging from a semi-quantitative approach based on different scoring systems to more quantitative techniques, and based on different numbers of inspected sites, hinders proper comparison between studies and increases the inter-study variability . However, the main disadvantage of a conventional microleakage test is that a tri-dimensional (3D) microleakage phenomenon is assessed two-dimensionally (2D), often even based on a single longitudinal or transverse section . Moreover, it results in irreversible loss of information because the specimen is partially destructed by sectioning . Only a limited number of locations can be evaluated, which may lead to an underestimation or overestimation of the total leakage and to ignoring detailed information about the distribution pattern.
Several techniques have been proposed to overcome shortcomings associated with a single two-dimensional surface observation in microleakage studies, such as spectrophotometric dye-recovery methods , serial grinding and imaging methods and the ‘whole wall technique’, which involves removal of the restoration and analysis of the entire gingival floor for possible leakage . All these techniques still share unfortunately the same limitation, as they are of destructive nature.
Micro-CT, on the other hand, is a non-destructive technique, originated from further development of conventional computer tomography. It can reach a potential resolution in the submicron range, depending on the computer hardware capabilities and X-ray source characteristics . In dental materials research, desktop μCT has been used for various approaches, including the evaluation of polymerization shrinkage of dental composites , quantification of interfacial void fraction in composite restorations , and presence of marginal/internal gaps in ceramic restorations .
The aim of this study was to explore the feasibility of 3D visualization/quantification of silver-nitrate penetration (microleakage evaluation) in various configurations of adhesive–tooth interfaces using a desktop μCT apparatus.
2
Materials and methods
Three different types of specimens were prepared on six extracted teeth that were stored in 0.5% aqueous chloramine solution at 4 °C for less than 6 months:
Specimen setup 1 : A 4 mm × 4 mm × 2 mm Class-I cavity ( n = 1) was prepared on a sound premolar using a micro-specimen former (University of Iowa, Iowa City, USA), equipped with a high-speed regular-grit (100 μm) diamond bur (842, Komet, Lemgo, Germany). The cavity was filled with the microhybrid composite Gradia Direct (shade A2; GC, Tokyo, Japan) without any adhesive pre-treatment at the interface to intentionally create a marginal gap. To maximize exposure of the gap to the infiltration medium, the occlusal margins of the restoration were additionally prepared with the bur to remove possible areas of overfilling after curing the restorative material.
Specimen setup 2 : A composite glass-fiber post (Parapost FiberLux, Coltène-Whaledent, Altstätten, Switzerland) ( n = 1) was cemented into the root canal of an extracted lower premolar using a self-adhesive cement (Rely-X UniCem, 3M ESPE, Seefeld, Germany). The root canal was prepared as described in detail elsewhere . Briefly, the tooth was cut at the most apical point of the cement–enamel junction using a low-speed diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). Endodontic treatment was performed following a standardized crown-down technique using the Profile system (Dentsply-Maillefer, Konstanz, Germany) coupled to a X-Smart endodontic motor (Dentsply-Maillefer). The apical third of the root was left filled with gutta-percha to exclude possible leakage through the apex. After preparation of the post-space with a low-speed bur, rinsing with 2.5% NaOCl solution and drying with paper points, the fiber posts were cemented following the manufacturer’s instructions.
Specimen setup 3 : Inlay MOD cavities ( n = 4) with their mesial cervical margin located in enamel and the distal one in dentin were prepared in human sound molars. Indirect composite restorations (Gradia Direct composite, shade A2; GC) were cemented with either the self-adhesive dual-cure composite cement MaxCem (Kerr, Orange, CA, USA) ( n = 2) or the 2-step etch-and-rinse composite cement (Variolink II with Excite, Ivoclar-Vivadent, Schaan, Liechtenstein) ( n = 2).
With the exception of a 1-mm thick area around the restoration margin or the post-cement interface, the specimens where covered with two layers of nail varnish. A 50% ammoniacal AgNO 3 solution was used as a tracer and after 24 h of water storage at 37 °C, all the specimens were infiltrated as described by Tay et al. .
2
Materials and methods
Three different types of specimens were prepared on six extracted teeth that were stored in 0.5% aqueous chloramine solution at 4 °C for less than 6 months:
Specimen setup 1 : A 4 mm × 4 mm × 2 mm Class-I cavity ( n = 1) was prepared on a sound premolar using a micro-specimen former (University of Iowa, Iowa City, USA), equipped with a high-speed regular-grit (100 μm) diamond bur (842, Komet, Lemgo, Germany). The cavity was filled with the microhybrid composite Gradia Direct (shade A2; GC, Tokyo, Japan) without any adhesive pre-treatment at the interface to intentionally create a marginal gap. To maximize exposure of the gap to the infiltration medium, the occlusal margins of the restoration were additionally prepared with the bur to remove possible areas of overfilling after curing the restorative material.
Specimen setup 2 : A composite glass-fiber post (Parapost FiberLux, Coltène-Whaledent, Altstätten, Switzerland) ( n = 1) was cemented into the root canal of an extracted lower premolar using a self-adhesive cement (Rely-X UniCem, 3M ESPE, Seefeld, Germany). The root canal was prepared as described in detail elsewhere . Briefly, the tooth was cut at the most apical point of the cement–enamel junction using a low-speed diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). Endodontic treatment was performed following a standardized crown-down technique using the Profile system (Dentsply-Maillefer, Konstanz, Germany) coupled to a X-Smart endodontic motor (Dentsply-Maillefer). The apical third of the root was left filled with gutta-percha to exclude possible leakage through the apex. After preparation of the post-space with a low-speed bur, rinsing with 2.5% NaOCl solution and drying with paper points, the fiber posts were cemented following the manufacturer’s instructions.
Specimen setup 3 : Inlay MOD cavities ( n = 4) with their mesial cervical margin located in enamel and the distal one in dentin were prepared in human sound molars. Indirect composite restorations (Gradia Direct composite, shade A2; GC) were cemented with either the self-adhesive dual-cure composite cement MaxCem (Kerr, Orange, CA, USA) ( n = 2) or the 2-step etch-and-rinse composite cement (Variolink II with Excite, Ivoclar-Vivadent, Schaan, Liechtenstein) ( n = 2).
With the exception of a 1-mm thick area around the restoration margin or the post-cement interface, the specimens where covered with two layers of nail varnish. A 50% ammoniacal AgNO 3 solution was used as a tracer and after 24 h of water storage at 37 °C, all the specimens were infiltrated as described by Tay et al. .
3
Micro-CT analysis
After the AgNO 3 infiltration, the specimens were scanned using a Skyscan 1172 desktop μCT scanner (Skyscan Bruker, Kontich, Belgium) at 100 kV, 100 μA using an aluminum and copper (Al + Cu) filter. The specimens were rotated 180° with a rotation step of 0.87° and an exposure time of 590 ms. For each step, 16 frames were averaged into a shadow projection image to minimize noise. To minimize ring artifacts, a random movement of 30 detector lines was used. The overall scanning time was approximately 60 min per specimen. Attention was paid not to leave any part of the specimens outside the scanning field during the scanning procedure, as incomplete projections from out of field objects may considerably increase streaking or shading artifacts. This limits the maximum obtainable magnification. All specimens could be scanned with a voxel size of 7.8 μm, except for the glass fiber post (setup 2), which was scanned with a voxel size of 9.5 μm.
Projection images were reconstructed into slice views, perpendicular to the specimen rotation axis. The scans were reconstructed using a modified Feldkamp cone-beam reconstruction algorithm (NRecon 1.4.4 software; Skyscan Bruker) . The slices were converted into an 8-bit BMP output format and the values within the dynamic range were mapped into gray levels [0–255]. This output format was suitable for further processing using the open-source ImageJ interface , while 3D renderings were generated using VGStudio Max 2.0 (Volume Graphics, Heidelberg, Germany). To enhance the visualization of the AgNO 3 infiltration, maximum-intensity projections (MIPs) were generated from selected volumes of interest (VOIs) that were centered at the interfaces where microleakage was suspected. A MIP visualizes the voxels with maximum intensity in the rays perpendicular to the projection plane. MIP imaging preserves structures having high attenuation in the final image and displays the continuity of such structures that run obliquely through the selected volume of interest, in this case being the interfacial area.
4
Conventional microleakage evaluation
After micro-CT scanning, the specimens from setup 3 were sectioned in the mesio-distal direction by a 0.3 mm thick diamond cut-off wheel (Struers, Ballerup, Denmark) mounted to a low-speed saw (Isomet, Buehler) under water cooling. For visual observation after sectioning of the AgNO 3 infiltrated specimens by stereomicroscopy, they were immersed in photo-developer solution while exposed to ultraviolet light for 8 h to reduce the silver ions to metallic silver. Next, the degree of microleakage was assessed with a conventional stereomicroscope and scored according to the criteria mentioned in Table 1 . These scores were also applied to the corresponding μCT-based MIPs obtained from the same specimens. For the specimens from setup 3, two coordinate axes were used to generate the MIPs: a Z -axis MIP, corresponding to the stereomicroscopic slice view, and an X -axis MIP, resulting from the slices in the occluso-cervical direction.
| Score | Description |
|---|---|
| 0 | No dye penetration |
| 1 | Silver nitrate penetration extending less than or up to one half of the cervical wall |
| 2 | Penetration greater than one half of the cavity depth but not extending to the axial wall |
| 3 | Penetration into the axial wall |
5
Results
The micro-CT-based results after AgNO 3 infiltration for the direct composite restoration (setup 1) are shown in Fig. 1 . By projecting the maximum gray-value intensity from each Z -axis slice into a single projection, a maximum-intensity projection (MIP) from the VOI selected from Fig. 1 a was obtained, corresponding to the pattern of AgNO 3 infiltration ( Fig. 1 b). The AgNO 3 solution had heterogeneously penetrated the interface, in a branch-like pattern, resulting in variable depths ( Fig. 1 b, red arrows). Silver-nitrate deposits at the marginal gap around the Class-I restoration appeared with sufficient contrast on the μCT slices to allow segmentation and 3D rendering of the AgNO 3 distribution ( Fig. 1 c). Furthermore, as the gray values that correspond with the silver nitrate (225–255) were sufficiently different from the range of gray values that correspond with the tooth and composite restoration (30–185) ( Fig. 1 d), a relative simple threshold could be implemented to quantify the infiltrated AgNO 3 volume. For this direct composite restoration, a total volume of 1.12 mm 3 of AgNO 3 solution had infiltrated the entire tooth–composite interface. Compared to the total restoration volume (5.42 mm 3 ), a 0.21 ratio between the volume of silver-nitrate infiltration and the total restoration volume was obtained.
