Turki A. Bakhsh

Non-invasive quantification of resin-dentin interfacial gaps using optical coherence tomography: validation against confocal microscopy.

OBJECTIVES Regardless of the cause, gap formation at the tooth-restoration interface may result in treatment failure; non-destructive assessment and monitoring of these defects are important. The aim of this in vitro study is to assess the tooth-restoration interface using a non-invasive technique; swept source optical coherence tomography (SS-OCT) and to confirm the findings with confocal laser scanning microscope (CLSM). METHODS Cylindrical class-I cavities (3mm in diameter and 1.5mm in depth) were prepared in the occlusal surface of human premolars. Each cavity was restored using an all-in-one adhesive system (Clearfil Tri-S Bond) and one of the three types of composites placed in bulk; Majesty Posterior, AP-X and Majesty LV (all by Kuraray Medical, Japan). Ten serial cross-sectional images of the whole restored cavity were obtained by SS-OCT at 1319 nm center wave length, to which locations the specimens were later trimmed, polished and observed under CLSM. An image analysis software was used to detect significant peaks in the signal intensity at the resin-dentin interface of the cavity floor. The presence and dimensions of gaps at the interface were also confirmed by CLSM. RESULTS Increased SS-OCT signal intensity along the interface corresponded well to the interfacial gaps detected by CLSM. The actual gap size detected ranged from 26 μm to 1.9 mm in length, and the universal composite APX showed lowest interfacial gaps. CONCLUSION SS-OCT imaging technology can be used to non-invasively detect and quantify micrometer gaps at the bottom of composite restorations, and potentially become a monitoring tool for composite restorations both in the laboratory research, and in the clinics.

Monitoring remineralization of enamel subsurface lesions by optical coherence tomography.

Abstract. Optical coherence tomography (OCT) is a potential clinical tool for enamel lesion monitoring. Swept-source OCT findings were compared with cross-sectional nanohardness findings of enamel. Subsurface bovine enamel lesions in three groups were subjected to (1) deionized water (control), (2) phosphoryl oligosaccharide of calcium (POs-Ca) or (3) POs-Ca with 1 ppm fluoride for 14 days. B-scans images were obtained at 1310-nm center wavelength on sound, demineralized and remineralized areas after 4, 7, and 14 days. The specimens were processed for cross-sectional nanoindentation. Reflectivity from enamel that had increased with demineralization decreased with remineralization. An OCT attenuation coefficient parameter (μt), derived based on the Beer-Lambert law as a function of backscatter signal slope, showed a strong linear regression with integrated nanohardness of all regions (p<0.001, r=−0.97). Sound enamel showed the smallest, while demineralized enamel showed the highest μt. In group three, μt was significantly lower at four days than baseline, but remained constant afterwards. In group two, the changes were rather gradual. There was no significant difference between groups two and three at 14 days in nanohardness or μt POs-Ca with fluoride-enhanced nanohardness of the superficial zone. OCT signal attenuation demonstrated a capability for monitoring changes of enamel lesions during remineralization.

Concurrent evaluation of composite internal adaptation and bond strength in a class-I cavity.

OBJECTIVES This study investigated class-I cavity floor adaptation by swept-source optical coherence tomography (OCT) in combination with microtensile bond strength (MTBS) using different filling methods. METHODS Two adhesive systems; Tokuyama Bond Force and Tri-S Bond Plus were used in conjunction with a universal composite (Estelite Sigma Quick) placed either incrementally (oblique) or in bulk with or without a flowable composite lining (Palfique Estelite LV). Ten serial B-scan images were obtained throughout each cavity by OCT (center wavelength: 1319nm). In order to evaluate adaptation defined as the cavity floor percentage showing no gap, a significant increase in the signal intensity was considered as gap at the bonded interface of the cavity floor. The same specimens were then cut into beams to measure MTBS at the cavity floor. RESULTS Two-way ANOVA demonstrated that the interaction of adhesive systems and filling techniques was significantly affecting both adaptation and MTBS (p<0.05). There was a significant correlation between MTBS and adaptation at cavity floor (p<0.05). Cavity floor adaptation and MTBS were improved when incremental filling technique was applied, while the outcome of lining technique was variable. CONCLUSIONS Quantitative assessment by OCT can non-destructively provide information on the performance and effectiveness of dental composites and restoration techniques. There was a moderate correlation between floor adaptation and bond strength in class-I cavities. Incremental application of composite restoration showed the best performance in terms of bond strength and internal adaptation. CLINICAL SIGNIFICANCE Incremental application of composite restoration was the most advantageous placement technique in terms of bond strength and internal adaptation. The lack of placement pressure with flowable composites may affect their adaptation to all-in-one adhesives; therefore, the outcome of cavity lining by flowable composite was variable.

Swept source optical coherence tomography for quantitative and qualitative assessment of dental composite restorations

The aim of this work was to explore the utility of swept-source optical coherence tomography (SS-OCT) for quantitative evaluation of dental composite restorations. The system (Santec, Japan) with a center wavelength of around 1300 nm and axial resolution of 12 μm was used to record data during and after placement of light-cured composites. The Fresnel phenomenon at the interfacial defects resulted in brighter areas indicating gaps as small as a few micrometers. The gap extension at the interface was quantified and compared to the observation by confocal laser scanning microscope after trimming the specimen to the same cross-section. Also, video imaging of the composite during polymerization could provide information about real-time kinetics of contraction stress and resulting gaps, distinguishing them from those gaps resulting from poor adaptation of composite to the cavity prior to polymerization. Some samples were also subjected to a high resolution microfocus X-ray computed tomography (μCT) assessment; it was found that differentiation of smaller gaps from the radiolucent bonding layer was difficult with 3D μCT. Finally, a clinical imaging example using a newly developed dental SS-OCT system with an intra-oral scanning probe (Panasonic Healthcare, Japan) is presented. SS-OCT is a unique tool for clinical assessment and laboratory research on resin-based dental restorations. Supported by GCOE at TMDU and NCGG.

Optical coherence tomography for evaluation of enamel and protective coatings

Optical coherence tomography (OCT) is an interferometric imaging technique. This study aimed to employ OCT to evaluate four different resin-based materials including a coating containing glass-ionomer filler and calcium, a giomer, and two fluoride-releasing self-etch resins. The coating and its underlying and adjacent enamel were monitored using swept-source OCT (center wavelength: 1330 nm) at baseline, after 5,000 thermal cycles, and after 1, 4 and 7 days of demineralization (pH 4.5). The coatings showed different thicknesses (60-250 micrometers) and various levels of structural and interfacial integrity. OCT could detect a demineralization inhibition zone adjacent to the edge of the fluoride- and calcium-releasing material. Localized demineralization was occasionally observed under thinner coatings. Protection of susceptible enamel surfaces by thin resin-based bioactive coatings provides protection from demineralization. OCT can be used to non-destructively monitor the integrity of such coatings, as well as enamel changes beneath and adjacent to them.

Focused ion beam processing for transmission electron microscopy of composite/adhesive interfaces

Although most transmission electron microscope (TEM) investigations were carried out using conventional ultramicrotomy, they were limited to the tooth/adhesive resin interface and were difficult to accomplish for the resin-composite interface. Some of these limitations have been overcome with the introduction of focused ion beam (FIB) milling. Therefore, the objective of the study was to compare different composites/adhesive interfaces using FIB/TEM technique. Cylindrical cavities were prepared in extracted human molar teeth. The restored cavities were divided into four groups: (1) One-step self-etch Scotchbond Universal (SBU; 3M ESPE, USA), (2) all-in-one Xeno-V+ adhesive (X5P; DENTSPLY, Germany), (3) two-step etch-and-rinse Prime and Bond NT (PNT; DENTSPLY, Germany) that were restored with Filtek Supreme Ultra Universal composite (3M ESPE, USA), and (4) teeth restored with the two-step self-etch Filtek Silorane adhesive (SSA; 3M ESPE, USA) that were restored with its corresponding Filtek Silorane composite (3M ESPE, USA). All specimens were cross-sectioned and subjected to FIB preparation followed by composite/adhesive interfacial TEM examination. The TEM findings were variable and ranged from concentrated clusters of nanoparticles to phase separation of adhesive components and large osmotic blisters. The osmotic blisters in all-in-one adhesives appeared to be influenced by the presence of water and the solvent’s vapor pressure in the cavity. In conclusion, FIB/TEM is a powerful tool allowing the study of biomaterials interaction in situ. The absence of residual water in the adhesives may reduce osmotic blistering that, in turn, may improve the reactivity of the resin monomers, as well as interfacial bonding.

Ultra-structural characterization of enamel–resin interface using FIB-TEM technology

This study evaluated the enamel–resin interface of three adhesive resins (ARs) by a transmission electron microscope (TEM) after milling with focused ion beam (FIB). Simple Class-I cavities (0.5 mm within enamel depth, 4 mm length, and 2 mm width) were prepared on human caries-free molars. The enamel of two groups was conditioned by one-step self-etch bonding system (Scotchbond™/Single Bond Universal [SBU], and Xeno V®+ [XV+]), while the enamel of the third group was etched by phosphoric acid, and then treated by two-step self-priming etch-rinse system (Prime&Bond® NT [PBNT]). The application of the adhesive systems was carried out according to their respective manufacturer’s instructions. The cavities were restored by a nanofill resin composite (Filtek™ Z350 XT Universal; 3 M ESPE) in one bulk-fill, and cured for 40 s at 550 mW/cm2 by a halogen light (Optilux 501, Demetron/Kerr, Danbury, USA). The specimens were milled by FIB into 100 nm thickness slices, and then observed under TEM. The transmission electron micrographs showed an adequate adhesion of both two-step etch-rinse (PBNT) and one-step self-etch (SBU) to enamel surface. The deeply etched enamel prisms were impregnated by the etch-rinse PBNT adhesive. A relatively inadequate adhesion associated with some areas of bond degradation underneath the hybrid layer and within the adhesive was noted for the XV+ AR. Apart from the mild acidic adhesives of one-step self-etch and two-step etch-rinse investigated, the highly hydrophilic and acidic water-based one-step self-etch adhesive (XV+) proved to be less effective enamel bond by ultra-structural characterization technique using FIB-TEM.

Effects of coating materials on nanoindentation hardness of enamel and adjacent areas

OBJECTIVES Materials that can be applied as thin coatings and actively release fluoride or other bioavailable ions for reinforcing dental hard tissue deserve further investigation. In this study we assessed the potential of resin coating materials in protection of underlying and adjacent enamel against demineralization challenge using nanoindentation. METHODS Enamel was coated using Giomer (PRG Barrier Coat, PBC), resin-modified glass-ionomer (Clinpro XT Varnish, CXT), two-step self-etch adhesive (Clearfil SE Protect, SEP) or no coating (control). After 5000 thermal cycles and one-week demineralization challenge, Martens hardness of enamel beneath the coating, uncoated area and intermediate areas was measured using a Berkovich tip under 2mN load up to 200μm depth. Integrated hardness and 10-μm surface zone hardness were compared among groups. RESULTS Nanoindentation and scanning electron microscopy suggested that all materials effectively prevented demineralization in coated area. Uncoated areas presented different hardness trends; PBC showed a remarkable peak at the surface zone before reaching as low as the control, while CXT showed relatively high hardness values at all depths. SIGNIFICANCE Ion-release from coating materials affects different layers of enamel. Coatings with fluoride-releasing glass fillers contributed to reinforcement of adjacent enamel. Surface prereacted glass filler-containing PBC superficially protected neighboring enamel against demineralization, while resin-modified glass-ionomer with calcium (CXT) improved in-depth protection. Cross-sectional hardness mapping of enamel on a wide range of locations revealed minute differences in its structure.

In situ characterization of resin-dentin interfaces using conventional vs. cryofocused ion-beam milling.

OBJECTIVE The introduction of focused ion beam (FIB) milling has facilitated preparation of hard tissue samples for transmission electron microscope (TEM). However, this technique generates high temperature that may alter or damage morphological features in biological tissue. Therefore, the aim of this study was to determine the effects of cryogenic cooling on the morphological features of dentin interfaces with dental restorative materials in samples prepared by FIB for TEM examination. METHODS After preparation of a cylindrical-shaped cavities in extracted, non-carious premolar teeth, the specimens were restored with dental adhesive/composite and categorized into two restorative materials groups; (PB) a combination of Clearfil Protect Bond (Kuraray Noritake Dental, Japan)/Estelite Sigma Quick composite (Tokuyama Dental, Japan), and (SB) Filtek Silorane restorative system (3M ESPE, USA). The specimens were subjected to interfacial cross-sectioning, followed by observation and area selection using confocal laser microscopy. Later, ultrathin sections were prepared using FIB with cryogenic cooling (PB-C) and (SB-C), or without cooling (PB-NC) and (SB-NC) that all were examined under TEM. RESULTS Resulting TEM images of the ultra-morphological features at the resin-dentin nano-interaction zone were improved when FIB preparation was conducted in the cryogenic condition and no sign of artifacts were detected. SIGNIFICANCE Conducting ion beam milling with cryogenic cooling was advantageous in minimizing the elevation in specimen temperature. This led to preservation of dentin microstructures that revealed additional information about substrates that are necessary for advanced characterization of tooth-biomaterial interactions.

Ultrastructural features of dentinoenamel junction revealed by focused gallium ion beam milling

To take full advantage of focused ion beam (FIB) in preparation of ultrathin sections of biological tissues, we have used a cryo‐milling process. In this study, extracted human teeth were scanned by optical coherence tomography to inspect the samples for intactness and to determine the area of interest. Then, the selected area of interest was cross‐sectioned for examination under a confocal laser scanning microscope to determine the target location of the dentinoenamel junction (DEJ) that was later milled by cryo‐FIB at preset parameters, followed by transmission electron microscope examination of the final sliced specimens for ultrastructural characterization. The proposed technique was able to outline the DEJ and to identify the different tooth layers in a single section, without artefacts or tissue damage. The DEJ was outlined as fine longitudinal projections intermingling between the solid electron‐dense enamel and intricate electron‐lucent hollow dentin. In conclusion, this study has shown the great potential of cryo‐FIB in handling different biological tissues having different physical properties, with great precision and accuracy and minimum artefacts.