Streptococcus mutans suspension was applied to form a cariogenic biofilm on composite restoration.
Swept-source optical coherence tomography (SS-OCT) was used to observe the carious demineralization at the cavosurface margins.
Demineralized enamel around the restorations was observed as intensified brightness in SS-OCT image.
Cariogenic demineralization took place with the gap formation at the enamel–resin interface.
SS-OCT appears to be a promising modality for the detection of secondary caries at early stage.
The aim of this study was to evaluate enamel demineralization at composite restoration margins caused by cariogenic biofilm using swept-source optical coherence tomography (SS-OCT).
Sixty round-shaped cavities were prepared on the mid-buccal enamel surface of extracted human molars. The cavities were restored with Estelite Flow Quick flowable composite using either Clearfil SE Bond or Clearfil Tri-S Bond ND bonding agents. Streptococcus mutans suspension was applied to form a cariogenic biofilm on the surface. After 1, 2, or 3 weeks of incubation ( n = 10), the biofilm was removed to observe the carious demineralization at the cavosurface margins using SS-OCT. The gap along the enamel–composite interface was recorded on each adhesive system. Confirmatory direct observation was accomplished at the same location using confocal laser scanning microscope.
The demineralized enamel around the restorations was observed as a zone of intensified brightness in SS-OCT. The demineralized lesion on the cervical enamel was significantly deeper than that on the occlusal enamel ( p < 0.05). However, the extension of enamel demineralization at the enamel–composite interface was significantly deeper at the occlusal wall than the cervical wall ( p < 0.05). The extension in Tri-S Bond ND group was significantly deeper than in SE Bond group ( p < 0.05). A significant increase in gap formation was found after the extension of demineralization compared with the baseline.
The carious demineralization around composite restorations were observed as a bright zone in SS-OCT during the process of bacterial demineralization. SS-OCT appears to be a promising modality for the detection of caries adjacent to an existing restoration.
Caries adjacent to existing restoration is the term describing lesions at the tooth-restoration interface, often cited as the most frequent reason for restoration replacement . Despite recent advancements in adhesive dentistry, composite restorations may still result in the onset of the so-called secondary caries due to the inherent property of polymerization shrinkage and long-term bond degradation . Nearly 70% of composite restorations are replacements for failed restorations, of which the primary reason for the failure is caries adjacent to restoration , followed by fractures . The integrity of interface between resin composite and tooth structure is the most significant factor that determines the long-tern clinical success of restoration .
The process underlying caries formation involves cariogenic bacteria in a biofilm capable of fermenting carbohydrates to produce acid. This acid leads to the demineralization of tooth structures . Streptococcus mutans is regarded as a primary cause of caries . S. mutans is able to penetrate into marginal gaps of composite restorations and contribute to the development of caries adjacent to restoration .
The caries lesions forming between the enamel and resin composite can occasionally present a diagnostic challenge because these lesions cannot be observed until they have significantly progressed and reached an advanced stage . Accurate characterization of the existing composite margins and early detection of caries adjacent to restoration can help in decision making over preventive strategies, maintenance, minimally invasive repairs or replacements. The traditional diagnostic assessment of margin quality, such as marginal color change and tactile probing, appear to have a low sensitivity in diagnosing caries . In the radiographic assessment which frequently used by clinicians, a translucent zone detected on a radiograph can be associated with either the presence of a radiolucent restorative material, a thick adhesive layer, an open margin or caries adjacent to restoration. The radiographic methods can only detect advanced caries under composite restorations .
Optical coherence tomography (OCT) is a noninvasive cross-sectional imaging method that uses low-coherence interferometry. This technique allows the visualization of the microstructure of tissues and biomaterials in real-time without the requirement of tissue sectioning or specimen preparation. Optical properties of the tissues including absorption and scattering have an influence on their appearance in OCT images. Cross-sectional images are created by linear scanning of the tissue by an optical beam. By measuring the echo time delay and intensity of backscattered light from within the scattering medium, depth information of the structure can be resolved. Using the obtained backscatter signal as an input, a computer can be used to reconstruct the visual image .
Recently, variations of OCT such as swept-source OCT (SS-OCT) have been developed. SS-OCT can construct images by ultra-high-speed scanning of the generated near-infrared laser wavelength and allows for the non-invasive construction of tomographic images of teeth superficial zones and restorations within a short space of time . Dental composites and hard tissues cause optical scattering; therefore, they are suitable substrates for SS-OCT imaging. The tooth-restoration interface under direct composite restorations has been investigated using this technique .
Previous studies reported that SS-OCT had a high degree of sensitivity and specificity for detecting caries and gaps around restorations . SS-OCT is therefore a promising tool for the observation of demineralization at the tooth-restoration interface. SS-OCT can additionally provide a quantitative measure of the lesion severity and show the lesion depth . It is likely that SS-OCT images of restoration margins would be of great value in determining whether caries adjacent to restoration warrants replacement of the restoration. Previous works have shown the utility of SS-OCT to assess demineralized lesions formed by acidic solution ; however, there are few reports on the evaluation of lesions around composite restoration caused by a cariogenic biofilm.
Several studies have indicated that artificial biofilms can be used for research on caries formation adjacent to restorations. Artificial mouth models can be used in vitro to study development of caries associated with an insufficient marginal adaptation of restorations . An oral biofilm reactor (OBR) has been used to study oral biofilms in vitro , thereby simulating the human oral environment .
The aim of the current study was to investigate enamel demineralization caused by a cariogenic biofilm around a composite restoration using SS-OCT. In addition, the findings were compared with those obtained with a confocal scanning laser microscope (CLSM).
Materials and methods
The SS-OCT (Prototype 2, Panasonic Healthcare Co. Ltd. Ehime, Japan) system used in the current study is a frequency (Fourier) domain technique with a tunable light source. This SS-OCT incorporates a handheld probe with a power of less than 10.0 mW, which is within the safety limits defined by the American National Standards Institute. The spectral bandwidth of the laser is over 100 nm centered at 1330 nm at a 30-kHz sweep rate. The axial resolution of the system is 12 μm in air, which corresponds to 8 μm in tissues, assuming refractive indices of approximately 1.5. The lateral resolution of 20 μm is determined by the objective lens of the probe. The light beam from the laser source is projected onto the tooth surfaces and scans the area of interest using the handheld probe.
Sixty caries-free sound human third molars were chosen. The extracted teeth were used for the study, according to a protocol approved by the Human Research Ethics Committee, Tokyo Medical and Dental University (No.725). The teeth were stored in refrigeration before the study. Cubical specimens (5 mm × 3 mm × 5 mm) were prepared from the mid-buccal portion of the tooth ( Fig. 1 ). The buccal enamel surface was slightly polished with a 1500-grit silicon carbide paper until a flat area was obtained. This was performed to eliminate any possible superficial enamel cracks and to create a flat surface for standard cavities. Round-shaped cavities (2 mm diameter × 1 mm depth) were prepared on the mid buccal enamel of the specimens. A round diamond bur (diameter 2 mm, M265R, JM STERI DIA, Morita, Japan) attached to a high-speed handpiece was used to prepare the cavities under water coolant. The bur was replaced after every preparation to maintain the cutting efficiency. The cavities were randomly divided into two groups of 30 cavities each, according to the materials used: the two-step self-etch adhesive group (Clearfil SE Bond, Kuraray Noritake Dental, Tokyo, Japan) and the one-step self-etch adhesive group (Clearfil Tri-S Bond ND, Kuraray Noritake Dental, Tokyo, Japan) restored with a flowable resin composite (Estelite Flow Quick, Tokuyama Dental, Tokyo, Japan) according to the instructions of the manufacturer ( Table 1 ). After polymerization, the restored surface was slightly polished with 1500-grit silicon carbide paper to remove the excess resin and standardize the surface.
|Materials; Lot no||Manufacture||Composition||Application instruction|
|Clearfil SE Bond
|Kuraray Noritake Dental||Primer: MDP, HEMA, hydrophilic dimethacrylate, photoinitiator, water Adhesive: MDP, Bis-GMA, HEMA, hydrophobic dimethacrylate, camphorquinone, silanated colloidal silica||Primer: Apply 20 s. Air blow
Adhesive: Apply. Air blow. Light cure 10 s
|Clearfil Tri-S Bond ND; 00008A||Kuraray Noritake Dental||MDP, bis-GMA, HEMA, photo-initiators, ethanol, water, silanted colloidal silica||Apply 20 s, Air blow 5 s, Light cure 10 s|
|Estelite Flow Quick (A2); J047||Tokuyama Dental||Bis-MPEPP, TEGDMA, UDMA, silica-zirconia filler, silica-titania fillers, CQ||Light cure 20 s|
The bonded cavities were then subjected to the demineralization protocol using a cariogenic biofilm. To ensure the repeatability of the SS-OCT scans for the same specimen, two small holes were drilled at the occlusal and the cervical sides of the specimen to facilitate SS-OCT scans along the center of the cavity. Before the demineralization, all specimens were scanned under SS-OCT for bonding defects. The specimens showing the formation of an apparent defective margin, void inclusion, or any other defect were excluded from the study and replaced.
Specimen assembly and biofilm formation in the oral biofilm reactor
In the current study, cariogenic biofilms were formed on the resin composite restoration surfaces using S. mutans MT8148 ( S. mutans ). A suspension of S. mutans in phosphate-buffered saline (PBS) was prepared from a 16 h fresh culture in brain heart infusion (BHI, Becton Dickinson, Sparks, MD, USA) broth after washing three times with PBS and stored at 4 °C with gentle stirring. For growth of the biofilms, a solution of heart infusion (HI, Becton Dickinson, Sparks, MD, USA) broth with sucrose (to obtain a 1% final concentration; HI-sucrose) was used.
The OBR used in the current study was equipped with two chambers containing a warm water jacket to maintain a constant interior temperature of 37 °C, as described previously ( Fig. 1 ). Biofilms were formed on the specimens inside the water jacket-encircled chamber of the OBR for 20 h. A flat-bulb pH electrode was used to continuously monitor the pH values beneath the biofilm. The suspension of S. mutans , HI-sucrose, and PBS were pumped into the chambers to continuously drop onto the center of the specimen holder.
After 20 h, the specimens with the undisturbed biofilms were further inoculated. Each group of specimens was again divided into three subgroups ( n = 10) that were incubated for 1, 2, and 3 weeks. All specimens were kept in separate wells at 37 °C in a 24-well culture plate (Corning Inc., NY, USA), and HI-sucrose was supplemented by changes of solution on alternate days. After the incubation periods, the specimens were rinsed with NaOH (0.25 mol) solution and fixed in 4% paraformaldehyde with 1% glutaraldehyde in PBS for a day. The specimens were then rinsed with PBS and preserved for observation.
Observation of demineralization at the enamel–composite interface
After the incubation for each experimental period, the occlusal and cervical cavosurface margins of composite restorations were subjected to SS-OCT observation to assess the enamel demineralization and interfacial gap at the cavity wall. To facilitate data analysis, each SS-OCT image was digitally analyzed using ImageJ (ver. 1.42q, National Institute of Health, Bethesda, MD, USA). A custom computer code that had been developed as a plugin for ImageJ based on a binarization process was used to facilitate the image analysis procedure and to distinguish the pixel clusters of a higher brightness, which indicate the demineralized area or gap .
The depth of demineralization was obtained from two depth locations, namely along the enamel–composite interface and 50 μm away from the margin ( Fig. 2 ). The gap length was measured from the high brightness line along the cavity wall. The depth of demineralization at each point and the gap length were measured as the distance from the surface level of composite restorations. The depth of demineralization or gap length was calculated as the percentage of the distance from surface to the cavity bottom (cavity depth) on the occlusal side and cervical side individually, according to the following equation:
Depth of demineralization ( % ) = depth of demineralization distance from the surface to cavity bottom × 100 , % Gap length ( % ) = gap length distance from the surface to cavity bottom × 100 , %