2.1

Specimen preparation

Four commercially available RBCs, with different types of filler particles, were used in the present study, as described in Table 1 . Ten syringes of each RBC (shade A3) were used to prepare a total of 108 discs by packing an excess of uncured composite into a custom-made polytetrafluoroethylene (PTFE) mold with a diameter of 6.0 mm and a thickness of 1.5 mm. After that, the material was covered with a Mylar strip on the top and bottom surfaces of the PTFE mold and condensed against a glass plate. The specimens were then irradiated for 80 s by placing the tip of a hand-held light-curing unit (Spectrum 800; Dentsply International Inc., York, PA, USA) into direct contact with the Mylar strip. The specimens were then randomly divided into 6 groups (Group 1–Group 6). Specimens belonging to Group 1 (n = 18) were prepared as described above but they were light-cured for 80 s by placing the tip of the light-curing unit at 1 mm distance from the surface after removing the Mylar strips, to simulate open-air polymerization.

The following F/P procedures were tested for each RBC on the remaining specimens:

• •

  Group 2 (n = 18) no finishing (Mylar strip)

• •

  Group 3 (n = 18) Al ₂ O ₃ discs (Sof-Lex, 3 M, Maplewood, Minnesota, USA)

• •

  Group 4 (n = 18) one-step rubber points (Opti1Step, Kerr Corp., Orange, California, USA)

• •

  Group 5 (n = 18) diamond bur 8862.314.012 followed by 862EF.314.012 (Komet, Gebr. Brasseler GmbH & Co. KG, Lemgo, Germany)

• •

  Group 6 (n = 18) multi-blade carbide bur H48L.Q.314.012 followed by H48L.UF.314.012 (Komet, Gebr. Brasseler GmbH & Co. KG, Lemgo, Germany).

Group 2 specimens were not furtherly processed. Group 3 specimens were finished and polished with a sequence of three Al ₂ O ₃ discs (medium, fine, superfine) for 30 s each at 20,000 rpm under water irrigation. After each step the specimens were washed with distilled water and air dried for 10 s. After every three specimens, discs were changed with new ones to obtain surfaces with homogeneous characteristics. Group 4–6 specimens were treated for 30 s at 20,000 rpm under water irrigation and a new point/bur was used every three specimens.

After that, all specimens were placed into the wells of a 48-well plate (Nunc, Roskilde, Denmark) and stored under light-proof conditions in distilled water for 6 days at 37 ± 1 °C. To reduce the amount of residual monomer that may leak during the subsequent incubation, each specimen was rinsed twice a day with 1 ml of distilled water. Specimens were subsequently carefully cleaned using an applicator brush tip (3 M, Maplewood, Minnesota, USA) soaked in ethanol (70%) prior to any further processing.

2.2

Roughness measurements

The SR of the specimens (n = 5/group) was measured using a profilometer (Sutronic 3+; Taylor Hobson, Leicester, UK). A distance of 1.75 mm was measured in three line scans perpendicular to the expected grinding grooves for each specimen, using a standard diamond tip (tip radius 2 μm, tip angle 90°) and a cut-off level of 0.25. Data were expressed as Ra.

2.3

Gloss measurements

Gloss measurements (n = 5/group), expressed as gloss units (GU), were performed using a small-area glossmeter (MG6-SA; KSJ, Quanzhou, China) with a square measurement area of 2 × 2 mm and a 60° geometry. A black opaque plastic mold was placed over the specimen during measurements to avoid the influence of the ambient light and to maintain the exact position of the specimen. Three measurements were performed for each specimen.

2.4

SEM and EDS analysis

SEM and EDS analysis were performed on test specimens (n = 3/group) using a TM3030Plus Tabletop scanning electron microscope (Hitachi, Schaumburg, IL, USA) equipped with an EDS probe (SwiftED3000 Oxford Instruments Analytical Ltd., Abingdon, Oxfordshire, UK). Three randomly selected fields were acquired for each specimen at 5000 x magnification to display the influence of the F/P procedures on the surfaces of the tested RBCs. For the EDS analysis, three randomly selected 300 × 300 μm fields were analyzed for each specimen in full-frame mode using an acquisition time of 150 s at 5 and 15 KV accelerating voltage. Acquired data represent the elemental composition of 1 μm superficial layer from which electrons were extracted by the accelerated beam. SEM and EDS analyses were repeated on the same specimens following the microbiological procedures described in paragraph 2.6. Specimens were sonicated (Sonifier Model B-15; Branson, Danbury, CT, USA operating at 40 W energy output for 10 min) and carefully cleaned using a microbrush to remove any biofilm.

2.5

Saliva collection

Paraffin-stimulated whole saliva was collected from three healthy volunteer donors in accordance with the protocol published by Guggenheim et al. . Briefly, saliva was collected in chilled test tubes, pooled, heated to 60 °C for 30 min to inactivate endogenous enzymes and was then centrifuged (12,000 rpm for 15 min at 4 °C). The supernatant was transferred into sterile tubes, stored at −20 °C, and thawed at 37 °C for 1 h prior to the experiments.

2.6

Microbiological procedures

Culture media were obtained from Becton-Dickinson (BD Diagnostics-Difco, Franklin Lakes, NJ, U.SA) and reagents were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, U.S.A.). Mitis Salivarius Bacitracin agar (MSB agar) plates were inoculated with S. mutans (ATCC 35668) and incubated at 37 °C for 48 h in a 5% CO ₂ -supplemented environment. A pure culture of the microorganism in Brain Heart Infusion broth (BHI) was obtained from these plates after incubation at 37 °C for 12 h in a 5% CO ₂ -supplemented environment. Cells were harvested by centrifugation (2200 rpm, 19 °C, 5 min), washed twice with sterile phosphate-buffered saline (PBS) and resuspended in the same buffer. The cell suspension was subsequently subjected to sonication (Sonifier model B-150; Branson, Danbury, CT, USA; operating at 7W energy output for 30 s) to disperse bacterial chains. Finally, the suspension was adjusted to a McFarland scale 1.0 optical density, corresponding to a concentration of approximately 6.0 × 10 ⁸ cells/mL.

2.7

MDFR procedures

The drip flow reactor (MDFR) used in the study was a modification of a commercially available Drip Flow Reactor (DFR 110; BioSurface Technologies, Bozeman, MT, USA). The modified design allowed the placement of customized specimen trays on the bottom of the flow cells and the complete immersion of the RBC surfaces into the surrounding flowing medium . All specimens (n = 18/group) were placed in a PTFE tray on the bottom of the flow cell and exposed to the surrounding medium. All tubing, specimens, and the specimen-containing trays were sterilized before incubation using a chemiclave (Sterrad; ASP, Irvine, CA, USA). By limiting the maximum temperature to 45 °C, heat-related damage of the RBC specimens was avoided. The whole MDFR was then assembled inside a sterile hood and transferred into an incubator at 37 °C.

A total of 10 ml of thawed sterile saliva was placed into each flow cell and the MDFR was then incubated at 37 °C for 24 h to allow the formation of a salivary pellicle on the surface of the specimens. After this incubation, saliva was removed by gentle aspiration and each flow cell was inoculated with 10 ml of S. mutans suspension to allow bacterial colonization of the RBCs surfaces. After 4 h, a multichannel, computer-controlled peristaltic pump (RP-1; Rainin, Emeryville, CA, USA) was turned on to provide a constant flow of nutrient medium through the flow cells. Sterile culture medium including 2.5 g/L mucin (type II, porcine gastric), 2.0 g/L bacteriological peptone, 2.0 g/L tryptone, 1.0 g/L yeast extract, 0.35 g/L NaCl, 0.2 g/L KCl, 0.2 g/L CaCl2, 0.1 g/L cysteine hydrochloride, 0.001 g/L hemin, and 0.0002 g/L vitamin K1, supplemented with 1% sucrose was used. The flow rate was set to 9.6 ml/h . Viable biomass assessment (n = 16/group) and CLSM microscopy (n = 2/group) were performed after 24 h of incubation.

2.8

Viable biomass assay

A tetrazolium salt stock solution was prepared by dissolving 5 mg/mL of 3-(4,5)-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) in sterile PBS. A phenazinium salt stock solution was prepared by dissolving 0.3 mg/mL of N -methylphenazinium methyl sulfate (PMS) in sterile PBS. The solutions were stored at 2 °C in light-proof vials until the day of the experiment, when a fresh measurement solution (FMS) was prepared by mixing 1 ml of MTT stock solution, 1 ml of PMS stock solution, and 8 ml of sterile PBS. A lysing solution (LS) was prepared by dissolving 10% v/v of sodium dodecyl sulfate and 50% v/v dimethylformamide in distilled water. It was stored at 2 °C until the day of the experiment and warmed at 37 °C for 2 h before use.

After a 24 h of incubation, the medium flow was stopped and the flow cells were opened. The trays containing the specimens were placed into Petri plates containing sterile PBS at 37 °C. Specimens were gently removed from the trays, washed three times with sterile PBS to remove non-adhered cells and finally placed in 48-well plates. The MTT assay was performed as follows : 300 μl of FMS were placed in each well, then the plates were incubated for 3 h in light-proof conditions at 37 °C. Electron transport across the microbial plasmatic membrane and, to a lesser extent, microbial redox systems, converted the yellow MTT salt to insoluble purple formazan during the incubation. The conversion at the microbial membrane level was facilitated by the intermediate electron acceptor (PMS). The FMS was then gently removed by aspiration and the intracellular formazan crystals were dissolved by adding 300 μl of lysing solution to each well. Plates were then incubated for 1 h at room temperature in lightproof conditions. The optical density (550 nm) of 100 μl of the suspension in each well was measured with a spectrophotometer (Genesys 10-S; Thermo Spectronic, Rochester, NY, USA). Results were recorded as OD units and were proportional to the amount of viable and metabolically active cells adherent to the sample surface .

2.9

CLSM morphological analysis

Two specimens for each group were analyzed using CLSM. After a 24 h incubation, the biofilm growing on the specimens was gently washed three times with PBS to remove non-adherent cells and stained using the FilmTracer live/dead Biofilm Viability Kit (Invitrogen, Carlsbad, California, USA). Biofilm was observed using a confocal laser scanning microscope (Leica TCS SP2; Leica Microsystems, Wetzlar, Germany). Three randomly selected image stack sections were recorded for each specimen. Confocal images were obtained using a dry 20 × (NA 0.7) objective and digitalized using Leica Application Suite Advanced Fluorescence (LAS AF; Leica microsystems, Wetzlar, Germany), at a resolution of 1024 × 1024 pixels, with a zoom factor of 1.0. For each image stack section, 3D-rendering reconstructions were obtained using Drishti 3D software .

2.10

Statistical analysis

Data were analyzed using statistical software (JMP 12 Pro; SAS, Cary, NC, USA) with a significance level of 5%. The variables gloss and viable biomass showed normal (Shapiro-Wilk’s test) and homogeneous (Levene test) distributions. The two variables were submitted to 2-way ANOVA considering RBC and F/P procedures as fixed factors, followed by the Tukey test. SR data were log-transformed to approach a normal distribution, then were submitted to 2-way ANOVA, followed by the Tukey test.

Regression analyses were performed to determine any possible correlation between gloss and SR parameters.

2.1

Specimen preparation

Four commercially available RBCs, with different types of filler particles, were used in the present study, as described in Table 1 . Ten syringes of each RBC (shade A3) were used to prepare a total of 108 discs by packing an excess of uncured composite into a custom-made polytetrafluoroethylene (PTFE) mold with a diameter of 6.0 mm and a thickness of 1.5 mm. After that, the material was covered with a Mylar strip on the top and bottom surfaces of the PTFE mold and condensed against a glass plate. The specimens were then irradiated for 80 s by placing the tip of a hand-held light-curing unit (Spectrum 800; Dentsply International Inc., York, PA, USA) into direct contact with the Mylar strip. The specimens were then randomly divided into 6 groups (Group 1–Group 6). Specimens belonging to Group 1 (n = 18) were prepared as described above but they were light-cured for 80 s by placing the tip of the light-curing unit at 1 mm distance from the surface after removing the Mylar strips, to simulate open-air polymerization.

The following F/P procedures were tested for each RBC on the remaining specimens:

• •

  Group 2 (n = 18) no finishing (Mylar strip)

• •

  Group 3 (n = 18) Al ₂ O ₃ discs (Sof-Lex, 3 M, Maplewood, Minnesota, USA)

• •

  Group 4 (n = 18) one-step rubber points (Opti1Step, Kerr Corp., Orange, California, USA)

• •

  Group 5 (n = 18) diamond bur 8862.314.012 followed by 862EF.314.012 (Komet, Gebr. Brasseler GmbH & Co. KG, Lemgo, Germany)

• •

  Group 6 (n = 18) multi-blade carbide bur H48L.Q.314.012 followed by H48L.UF.314.012 (Komet, Gebr. Brasseler GmbH & Co. KG, Lemgo, Germany).

Group 2 specimens were not furtherly processed. Group 3 specimens were finished and polished with a sequence of three Al ₂ O ₃ discs (medium, fine, superfine) for 30 s each at 20,000 rpm under water irrigation. After each step the specimens were washed with distilled water and air dried for 10 s. After every three specimens, discs were changed with new ones to obtain surfaces with homogeneous characteristics. Group 4–6 specimens were treated for 30 s at 20,000 rpm under water irrigation and a new point/bur was used every three specimens.

After that, all specimens were placed into the wells of a 48-well plate (Nunc, Roskilde, Denmark) and stored under light-proof conditions in distilled water for 6 days at 37 ± 1 °C. To reduce the amount of residual monomer that may leak during the subsequent incubation, each specimen was rinsed twice a day with 1 ml of distilled water. Specimens were subsequently carefully cleaned using an applicator brush tip (3 M, Maplewood, Minnesota, USA) soaked in ethanol (70%) prior to any further processing.

2.2

Roughness measurements

The SR of the specimens (n = 5/group) was measured using a profilometer (Sutronic 3+; Taylor Hobson, Leicester, UK). A distance of 1.75 mm was measured in three line scans perpendicular to the expected grinding grooves for each specimen, using a standard diamond tip (tip radius 2 μm, tip angle 90°) and a cut-off level of 0.25. Data were expressed as Ra.

2.3

Gloss measurements

Gloss measurements (n = 5/group), expressed as gloss units (GU), were performed using a small-area glossmeter (MG6-SA; KSJ, Quanzhou, China) with a square measurement area of 2 × 2 mm and a 60° geometry. A black opaque plastic mold was placed over the specimen during measurements to avoid the influence of the ambient light and to maintain the exact position of the specimen. Three measurements were performed for each specimen.

2.4

SEM and EDS analysis

SEM and EDS analysis were performed on test specimens (n = 3/group) using a TM3030Plus Tabletop scanning electron microscope (Hitachi, Schaumburg, IL, USA) equipped with an EDS probe (SwiftED3000 Oxford Instruments Analytical Ltd., Abingdon, Oxfordshire, UK). Three randomly selected fields were acquired for each specimen at 5000 x magnification to display the influence of the F/P procedures on the surfaces of the tested RBCs. For the EDS analysis, three randomly selected 300 × 300 μm fields were analyzed for each specimen in full-frame mode using an acquisition time of 150 s at 5 and 15 KV accelerating voltage. Acquired data represent the elemental composition of 1 μm superficial layer from which electrons were extracted by the accelerated beam. SEM and EDS analyses were repeated on the same specimens following the microbiological procedures described in paragraph 2.6. Specimens were sonicated (Sonifier Model B-15; Branson, Danbury, CT, USA operating at 40 W energy output for 10 min) and carefully cleaned using a microbrush to remove any biofilm.

2.5

Saliva collection

Paraffin-stimulated whole saliva was collected from three healthy volunteer donors in accordance with the protocol published by Guggenheim et al. . Briefly, saliva was collected in chilled test tubes, pooled, heated to 60 °C for 30 min to inactivate endogenous enzymes and was then centrifuged (12,000 rpm for 15 min at 4 °C). The supernatant was transferred into sterile tubes, stored at −20 °C, and thawed at 37 °C for 1 h prior to the experiments.

2.6

Microbiological procedures

Culture media were obtained from Becton-Dickinson (BD Diagnostics-Difco, Franklin Lakes, NJ, U.SA) and reagents were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, U.S.A.). Mitis Salivarius Bacitracin agar (MSB agar) plates were inoculated with S. mutans (ATCC 35668) and incubated at 37 °C for 48 h in a 5% CO ₂ -supplemented environment. A pure culture of the microorganism in Brain Heart Infusion broth (BHI) was obtained from these plates after incubation at 37 °C for 12 h in a 5% CO ₂ -supplemented environment. Cells were harvested by centrifugation (2200 rpm, 19 °C, 5 min), washed twice with sterile phosphate-buffered saline (PBS) and resuspended in the same buffer. The cell suspension was subsequently subjected to sonication (Sonifier model B-150; Branson, Danbury, CT, USA; operating at 7W energy output for 30 s) to disperse bacterial chains. Finally, the suspension was adjusted to a McFarland scale 1.0 optical density, corresponding to a concentration of approximately 6.0 × 10 ⁸ cells/mL.

2.7

MDFR procedures

The drip flow reactor (MDFR) used in the study was a modification of a commercially available Drip Flow Reactor (DFR 110; BioSurface Technologies, Bozeman, MT, USA). The modified design allowed the placement of customized specimen trays on the bottom of the flow cells and the complete immersion of the RBC surfaces into the surrounding flowing medium . All specimens (n = 18/group) were placed in a PTFE tray on the bottom of the flow cell and exposed to the surrounding medium. All tubing, specimens, and the specimen-containing trays were sterilized before incubation using a chemiclave (Sterrad; ASP, Irvine, CA, USA). By limiting the maximum temperature to 45 °C, heat-related damage of the RBC specimens was avoided. The whole MDFR was then assembled inside a sterile hood and transferred into an incubator at 37 °C.

A total of 10 ml of thawed sterile saliva was placed into each flow cell and the MDFR was then incubated at 37 °C for 24 h to allow the formation of a salivary pellicle on the surface of the specimens. After this incubation, saliva was removed by gentle aspiration and each flow cell was inoculated with 10 ml of S. mutans suspension to allow bacterial colonization of the RBCs surfaces. After 4 h, a multichannel, computer-controlled peristaltic pump (RP-1; Rainin, Emeryville, CA, USA) was turned on to provide a constant flow of nutrient medium through the flow cells. Sterile culture medium including 2.5 g/L mucin (type II, porcine gastric), 2.0 g/L bacteriological peptone, 2.0 g/L tryptone, 1.0 g/L yeast extract, 0.35 g/L NaCl, 0.2 g/L KCl, 0.2 g/L CaCl2, 0.1 g/L cysteine hydrochloride, 0.001 g/L hemin, and 0.0002 g/L vitamin K1, supplemented with 1% sucrose was used. The flow rate was set to 9.6 ml/h . Viable biomass assessment (n = 16/group) and CLSM microscopy (n = 2/group) were performed after 24 h of incubation.

2.8

Viable biomass assay

A tetrazolium salt stock solution was prepared by dissolving 5 mg/mL of 3-(4,5)-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) in sterile PBS. A phenazinium salt stock solution was prepared by dissolving 0.3 mg/mL of N -methylphenazinium methyl sulfate (PMS) in sterile PBS. The solutions were stored at 2 °C in light-proof vials until the day of the experiment, when a fresh measurement solution (FMS) was prepared by mixing 1 ml of MTT stock solution, 1 ml of PMS stock solution, and 8 ml of sterile PBS. A lysing solution (LS) was prepared by dissolving 10% v/v of sodium dodecyl sulfate and 50% v/v dimethylformamide in distilled water. It was stored at 2 °C until the day of the experiment and warmed at 37 °C for 2 h before use.

After a 24 h of incubation, the medium flow was stopped and the flow cells were opened. The trays containing the specimens were placed into Petri plates containing sterile PBS at 37 °C. Specimens were gently removed from the trays, washed three times with sterile PBS to remove non-adhered cells and finally placed in 48-well plates. The MTT assay was performed as follows : 300 μl of FMS were placed in each well, then the plates were incubated for 3 h in light-proof conditions at 37 °C. Electron transport across the microbial plasmatic membrane and, to a lesser extent, microbial redox systems, converted the yellow MTT salt to insoluble purple formazan during the incubation. The conversion at the microbial membrane level was facilitated by the intermediate electron acceptor (PMS). The FMS was then gently removed by aspiration and the intracellular formazan crystals were dissolved by adding 300 μl of lysing solution to each well. Plates were then incubated for 1 h at room temperature in lightproof conditions. The optical density (550 nm) of 100 μl of the suspension in each well was measured with a spectrophotometer (Genesys 10-S; Thermo Spectronic, Rochester, NY, USA). Results were recorded as OD units and were proportional to the amount of viable and metabolically active cells adherent to the sample surface .

2.9

CLSM morphological analysis

Two specimens for each group were analyzed using CLSM. After a 24 h incubation, the biofilm growing on the specimens was gently washed three times with PBS to remove non-adherent cells and stained using the FilmTracer live/dead Biofilm Viability Kit (Invitrogen, Carlsbad, California, USA). Biofilm was observed using a confocal laser scanning microscope (Leica TCS SP2; Leica Microsystems, Wetzlar, Germany). Three randomly selected image stack sections were recorded for each specimen. Confocal images were obtained using a dry 20 × (NA 0.7) objective and digitalized using Leica Application Suite Advanced Fluorescence (LAS AF; Leica microsystems, Wetzlar, Germany), at a resolution of 1024 × 1024 pixels, with a zoom factor of 1.0. For each image stack section, 3D-rendering reconstructions were obtained using Drishti 3D software .

2.10

Statistical analysis

Data were analyzed using statistical software (JMP 12 Pro; SAS, Cary, NC, USA) with a significance level of 5%. The variables gloss and viable biomass showed normal (Shapiro-Wilk’s test) and homogeneous (Levene test) distributions. The two variables were submitted to 2-way ANOVA considering RBC and F/P procedures as fixed factors, followed by the Tukey test. SR data were log-transformed to approach a normal distribution, then were submitted to 2-way ANOVA, followed by the Tukey test.

Regression analyses were performed to determine any possible correlation between gloss and SR parameters.