2.1

Specimen preparation

Twenty intact human molars with no visible evidence of caries or cracks were collected after obtaining the informed consent according to a protocol approved by the Institutional Review Board of Tokyo Medical and Dental University (approval no. 725). The molars were kept in a preserving solution (NaN ₃ , 0.2%; NaCl, 0.8%; KH ₂ PO ₄ , 0.6%; NaOH added to adjust pH to 7.4) at 4 °C until use. The teeth were cut mid-region to separate the buccal from the palatal or lingual surfaces using a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA). Forty tooth blocks including the cervical region were made by reducing both sides to the size of 5 mm × 5 mm × 3 mm. A class V cavity (surface diameter: 2.5 mm, maximum depth: 2.0 mm) was prepared in the cervical part of each tooth block using a diamond bur (FG 102R; Shofu, Kyoto, Japan) attached to an air turbine headpiece under copious cooling water. The occlusal region of the preparation margins was located in the enamel and the cervical region was located in the dentin ( Fig. 1 ). Twenty specimens were treated by Clearfil Tri-S Bond ND Quick (TS; Kuraray Noritake Dental, Tokyo, Japan) and another twenty by Clearfil SE Bond (SE; Kuraray Noritake Dental, Tokyo, Japan). Then, they were irradiated with a halogen light curing unit with 600 mW/cm ² output (Optilux 501; Kerr, Orange, CA, USA) for 10 s. All specimens were restored with a flowable resin composite (Estelite Flow Quick, A2; Tokuyama Dental, Tokyo, Japan) following the manufacturer’s instruction and light cured for 20 s. After restoring, the surfaces of all specimens were polished with two kinds of silicone polishers (Brownie Mini Point FG, Greenie Mini Point FG; Shofu, Kyoto, Japan). They were examined using SS-OCT (IVS-2000; Santec, Komaki, Japan) to confirm that no overhangs existed. Then, four edges of all specimen surfaces were covered with a thin layer of acid-resistant varnish (Shiseido, Tokyo, Japan), approximately 0.5 mm from the restored cavity margin. In this way, 40 restored specimens were prepared and they were separated into the demineralized group (SE: n = 10, TS: n = 10) and the control group (SE: n = 10, TS: n = 10). The schematic diagram of the study design and methodology was shown in Fig. 1 . The composition and application of the materials was shown in Table 1 .

Schematic diagram of the study design and methodology. After the preparation of class V cavities, the specimens were either treated with one-step adhesive (TS) or two-step adhesive (SE), restored with a flowable resin composite (Estelite Flow Quick). The demineralized group was incubated for 2 weeks after S. mutans biofilm formation in an oral biofilm reactor (OBR), while the control group was incubated for 2 weeks without biofilms. The specimens were subjected to SS-OCT observation at baseline and after incubation. CLSM was used for confirmation of SS-OCT findings after cutting the specimens. TS: Clearfil Tri-S Bond ND Quick; SE: Clearfil SE Bond.

2.2

Biofilm-induced demineralization

2.2.1

Bacteria suspensions and nutrient broth

In the demineralized group, artificial biofilms were formed on restored surfaces using a laboratory strain of oral cariogenic bacteria S. mutans MT8148. A suspension of S. mutans at an optical density of 2.5 at 500 nm (OD ₅₀₀ = 2.5) was prepared from a 16-h fresh culture in Brain Heart Infusion (BHI; Becton Dickinson, Sparks, MD, USA) broth. The suspension was washed three times with phosphate buffered saline (PBS) and stored at 4 °C with gentle stirring. For growing biofilms, a solution of Heart Infusion (HI; Becton Dickinson, Sparks, MD, USA) broth with sucrose (at 1–2% final concentration) was used.

2.2.2

Specimen assembly and biofilm formation in the oral biofilm reactor

The artificial biofilms were formed on the surfaces in an oral biofilm reactor (OBR) according to the previously reported method . In brief, OBR is equipped with two chambers, with each chamber containing a warm water jacket to maintain a constant interior temperature, to grow biofilms under anaerobic conditions. A flat-bulb pH electrode is used to monitor the pH beneath the biofilm continuously.

In the present experiment, biofilms were formed on restored specimens inside the two identical water jacket-encircled chambers of the OBR as illustrated in Fig. 1 . Specimens were placed on a Teflon holder around a flat-bulb pH electrode of the OBR by using red utility wax (GC Co., Tokyo, Japan) and in such a way that only the restored surface remained open for biofilm attachment. The open surfaces were kept horizontal at the level of the electrode surface. The Teflon holder bearing the specimens was set through the bottom opening of the chamber by a silicon plug. The chamber, encircled by the water jacket, was sealed with another silicon plug fitted with four stainless steel tubes (21-gauge). In this manner, the chamber itself served as an incubator with a 37 °C inner temperature. The other ends of the four stainless steel tubes were connected to four silicon tubes passing through peristaltic pumps regulated by a computer-operated controller (EYELA EPC-2000, Tokyo Rika, Tokyo, Japan). One tube was used to collect the suspension of S. mutans , two to collect sucrose-HI and the other one to collect PBS from the prepared stock as described above. All of these liquids were pumped into the chambers at 6 ml/hr per tube so that they dropped continuously onto the center of the specimen holder. The mixture of these liquids formed water domes which were mixed by the force of gravity exerted from the falling liquid drops on the holder and which were diffusely distributed over all of the specimens. Both chambers operated simultaneously and the pH on the flat-bulb electrode was recorded continuously. Reduction in pH was similar in all the experiments in this study: the pH began to fall from 7.35 within 2 h and was reduced to below 4.0 by 20 h.

The specimens with undisturbed biofilms were further inoculated for 14 days to produce relatively larger-sized secondary caries lesions. Twenty specimens of the demineralized group were kept in separate wells of a 24-well culture plate (Corning Inc., NY, USA) at 37 °C and sucrose-HI was supplemented on alternate days.

Another twenty specimens of the control group, without biofilms formation, were cleaned by 99% ethanol for 30 min and washed three times with PBS. Then they were kept in separate wells of a 24-well culture plate at 37 °C and sucrose-HI was supplemented on alternate days.

2.3

Swept-source optical coherence tomography (SS-OCT)

The SS-OCT system (IVS-2000, Santec, Komaki, Japan) was used to examine the specimens before and after 2-week incubation. This system is a frequency-domain OCT technique that interprets the magnitude and coherence of the light reflected from the subject into the depth-profile of the subject. The system incorporates a high-speed frequency, swept external-cavity laser. The wavelength ranged from 1260 nm to 1360 nm centered at 1310 nm at 20 kHz sweep rate. The optical resolution was 17 μm laterally and 11 μm axially in the air, which corresponds to 7 μm in tissues assuming a refractive index around 1.5.

During the preparation of specimens, two points (the upper-margin center and lower-margin center) were marked on the surface of each specimen. SS-OCT examination was performed along the plane between these two points and central cross-section images were obtained. After 2-week incubation, the specimens of the demineralized group were cleaned using 0.25 M NaOH and washed with distilled water to remove biofilms. The specimens of the control group were cleaned with distilled water. Then, SS-OCT images were obtained from the location along the two marked points, where the SS-OCT scanning had been performed before incubation. The demineralized specimens were cross-sectionally scanned with OCT after gentle blot-drying of the surface leaving it moist without any visible water-droplets. To ensure the reproducibility of the scan, the specimens were placed at the same orientation as accurately as possible. The specimens were tilted at 3–5 degrees to avoid mirror reflections from the surface of specimen on the SS-OCT image.

A custom code in the image analysis software (Image J, version 1.48; National Institutes of Health, Bethesda, MD, USA) was used to read the raw data of SS-OCT. The obtained SS-OCT image was rotated to compensate for the tilting during the scan to reach a horizontal surface. A noise-reducing median filter (size 2) was applied to the raw data ( Fig. 2 b’). A binarization process was applied when measuring the interfacial gap ( Fig. 2 b”). Lesion progression around restorations and interfacial gap were measured as the lesion depth (LD), the interfacial gap depth (GD) and the interfacial gap length (GL) ( Fig. 2 b’ and b”).

2.4

Confocal laser scanning microscope (CLSM)

After the SS-OCT observation, all specimens were fixed in epoxy resin (EpoxiCure; Buehler, Lake Bluff, IL, USA). After 8 h, a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) was used to cut every specimen in half and obtain discs with thickness of approximately 2.5 mm. Then, the slices were trimmed off using wet 2000-grit silicon carbide papers and further polished with diamond paste down to 0.25 μm under running water. The cross-sectional slice examined using the nondestructive SS-OCT imaging was physically separated and examined under CLSM (1LM21H/W; Lasertec Co., Yokohama, Japan) at magnifications of 125× to 500×. The linear measurements of lesion depth, interfacial gap depth and interfacial gap length obtained from CLSM were analyzed in the same manner as OCT images by Image J ( Fig. 2 d and e).

2.5

Statistical analysis

The normal distribution and homogeneity of variance were checked for LD, GD and GL by Shapiro–Wilk and Levene tests, respectively. LD showed a normal distribution and homogeneity in intergroup variance. GD and GL showed a normal distribution, but did not show homogeneity in all intergroup variance. Therefore, LD, GD and GL obtained from SS-OCT and CLSM were compared using Pearson’s correlation. The mean value in LD, GD and GL of four pairs of groups (enamel in SE vs enamel in TS, dentin in SE vs dentin in TS, enamel in SE vs dentin in SE, enamel in TS vs dentin in TS) were compared. LD was analyzed by Independent-samples t -test. GD and GL were analyzed by Welch t -test. The frequency of specimens with/without interfacial gap after incubation was analyzed in Fisher’s exact test. All statistical analyses were performed with 95% level of confidence using the Statistical Package for Medical Science (ver.16.0 for Windows; SPSS, Chicago, IL, USA).

2.1

Specimen preparation

Twenty intact human molars with no visible evidence of caries or cracks were collected after obtaining the informed consent according to a protocol approved by the Institutional Review Board of Tokyo Medical and Dental University (approval no. 725). The molars were kept in a preserving solution (NaN ₃ , 0.2%; NaCl, 0.8%; KH ₂ PO ₄ , 0.6%; NaOH added to adjust pH to 7.4) at 4 °C until use. The teeth were cut mid-region to separate the buccal from the palatal or lingual surfaces using a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA). Forty tooth blocks including the cervical region were made by reducing both sides to the size of 5 mm × 5 mm × 3 mm. A class V cavity (surface diameter: 2.5 mm, maximum depth: 2.0 mm) was prepared in the cervical part of each tooth block using a diamond bur (FG 102R; Shofu, Kyoto, Japan) attached to an air turbine headpiece under copious cooling water. The occlusal region of the preparation margins was located in the enamel and the cervical region was located in the dentin ( Fig. 1 ). Twenty specimens were treated by Clearfil Tri-S Bond ND Quick (TS; Kuraray Noritake Dental, Tokyo, Japan) and another twenty by Clearfil SE Bond (SE; Kuraray Noritake Dental, Tokyo, Japan). Then, they were irradiated with a halogen light curing unit with 600 mW/cm ² output (Optilux 501; Kerr, Orange, CA, USA) for 10 s. All specimens were restored with a flowable resin composite (Estelite Flow Quick, A2; Tokuyama Dental, Tokyo, Japan) following the manufacturer’s instruction and light cured for 20 s. After restoring, the surfaces of all specimens were polished with two kinds of silicone polishers (Brownie Mini Point FG, Greenie Mini Point FG; Shofu, Kyoto, Japan). They were examined using SS-OCT (IVS-2000; Santec, Komaki, Japan) to confirm that no overhangs existed. Then, four edges of all specimen surfaces were covered with a thin layer of acid-resistant varnish (Shiseido, Tokyo, Japan), approximately 0.5 mm from the restored cavity margin. In this way, 40 restored specimens were prepared and they were separated into the demineralized group (SE: n = 10, TS: n = 10) and the control group (SE: n = 10, TS: n = 10). The schematic diagram of the study design and methodology was shown in Fig. 1 . The composition and application of the materials was shown in Table 1 .

Schematic diagram of the study design and methodology. After the preparation of class V cavities, the specimens were either treated with one-step adhesive (TS) or two-step adhesive (SE), restored with a flowable resin composite (Estelite Flow Quick). The demineralized group was incubated for 2 weeks after S. mutans biofilm formation in an oral biofilm reactor (OBR), while the control group was incubated for 2 weeks without biofilms. The specimens were subjected to SS-OCT observation at baseline and after incubation. CLSM was used for confirmation of SS-OCT findings after cutting the specimens. TS: Clearfil Tri-S Bond ND Quick; SE: Clearfil SE Bond.

Table 1

Materials used in this study.

Material (abbreviation); manufacture; lot no.	Composition	Application instructions
Clearfil Tri-S Bond ND Quick (TS); Kuraray Noritake Dental Inc; AF0001	Bis-GMA, MDP, HEMA, hydrophilic aliphatic dimethacrylate, hydrophobic aliphatic methacrylate, colloidal silica, NaF, CQ, accelerators, initiators, ethanol, water, pH = 2.3.	Apply the bond and strong air blow for 5 s. Light cure for 10 s.
Clearfil SE Bond (SE); Kuraray Noritake Dental Inc; 000014	Primer: MDP, HEMA, hydrophilic aliphatic dimethacrylate, dl-CQ, N,N-diethanol-p-toluidine, water, pH = 2.0. Bond: MDP, bis-GMA, HEMA, hydrophobic aliphatic dimethacrylate, dl-CQ, N,N-diethanol-p-toluidine, colloidal silica.	Apply the primer for 20 s and mild air blow. Apply the bond and air blow gently. Light cure for 10 s.
Composite Estelite Flow Quick; Tokuyama Dental; 063043P	Bis-MPEPP, TEGDMA, UDMA, silica-zirconia filler, silica-titania fillers (53% filler by volume, 0.04–0.6 μm particle size), CQ.	Dispense in layers up to 2 mm in thickness. Light cure for 20 s.

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