2.1

Adhesive formulation

Experimental etch-and-rinse, two bottles, adhesive systems containing different concentrations of L-arginine (Sigma-Aldrich, Inc, St Louis, MO, USA) were fabricated. The primer solution consisted of 15% ethoxylated bisphenol-A dimethacrylate (BisEMA; Esstech Inc., Essington, PA, USA), 10% hydroxyethyl methacrylate (HEMA; Sigma-Aldrich), 10% urethane dimethacrylate (UDMA; Sigma-Aldrich), 10% triethylene glycol dimethacrylate (TEGDMA; Sigma-Aldrich), 15% distilled water and 40% ethanol, and at a ratio of 45% monomers and 55% solvents. The adhesive consisted of 40% UDMA, 30% TEGDMA, 17% BisEMA, 10% bisphenol A glycidyl methacrylate (BisGMA, Sigma-Aldrich), 1.5% diphenyliodonium hexafluorophosphate (DPIHP, Sigma-Aldrich) and 0.5% camphorquinone/1% amine (CQ/EDAB, Sigma-Aldrich) in the monomer mixture previously homogenized at a ratio of 97% monomers and 3% photo-initiator agents. L-arginine was added to the adhesive and homogenized at the weight ratio concentrations of 0% (Arg0; control), 5% (Arg5), 7% (Arg7), and 10% (Arg10). Of note, pilot tests were carried out in order to observe how arginine particles would dissolve with different dental adhesives monomers blends. Some properties were estimated such as arginine-dissolution, phase separation, wettability and viscosity. Once the adhesive blend presented clear-uniform appearance, proper viscosity for dental application and absence of phase-separation, arginine was added at concentrations ranging from 2 to 18%. The arginine concentrations of 5%, 7% and 10% were selected because these presented the best saturated mixtures. The adhesive systems were prepared in a dark room under controlled temperature and humidity, and then kept under refrigeration (4 °C). Prior to use, the adhesives were stirred for 15 min. All concentrations are provided here at a weight ratio.

2.2

Ultimate tensile strength (UTS)

Specimens (n = 10) were prepared using silicon molds with an hourglass shape of 10 × 4 mm and sectional area of 1.5 mm ² (Odeme Dental Research, Luzerna, SC, Brazil). Each adhesive was placed into the molds, covered with a clear transparent Mylar-matrix and coverslip, and then light-cured at 1000 mW/cm ² for 20 s (Valo, Ultradent, South Jordan, UT, USA). Specimens were fitted in a testing jig device and submitted to tensile strength test. Load was applied perpendicular to the plane of the cured adhesive in a semi-universal testing machine OM100 (Odeme Dental Research) at 0.75 mm/min. Ultimate tensile strength (UTS) was calculated in MPa using the formula: UTS = F/A , in which, F was the tensile strength (N) and A the transversal cross section area (mm ² ).

2.3

Flexural strength (FS) and flexural modulus ( E )

Bar-shaped specimens (n = 7) were prepared using a silicon mold of 10 × 2 × 2 mm (Odeme Dental Research). Each adhesive was placed into the molds, covered with a clear transparent Mylar matrix and coverslip, and then light-cured as described above. The cross sectional area was approximately 4.0 mm ² . Specimens were stored at 37°C for 24 h and subjected to three-point bending test in a universal testing machine (Instron, Norwood, MA, USA) with 8 mm span between supports and at crosshead speed of 0.5 mm/min. The maximum load for the specimens at fracture was recorded and the FS calculated using the following equation: FS = 3FL/(2bh ² ), where F was the maximum load (N), L the distance (mm) between supports, B the width (mm) and H the height (mm). B and H were measured immediately prior the testing. In the Instron machine, E data was based on the first load-displacement curve of the linear portion of the graphic obtained from the BlueHill 3 software, and used with the standard equation E = L ³ F/4 w H ³ d, in which L is the support span length (mm), F the maximum load (N), w the specimen width (mm), H the specimen height and d is the deflection (mm) at load F.

2.4

Knoop hardness (KHN)

Disk shaped specimens (n = 5) were prepared by placing each adhesive into a rubber mold of 5 × 1 mm (Odeme Dental Research). A Mylar strip and a coverslip were placed over the adhesive/mold and light-cured as described above. Specimens were kept stored for 24 h in dry conditions at 37°C. Next, the top surfaces were polished under water with a 1200 grit SiC sandpaper to obtain a polished surface. KHN test was carried out in a microindenter HMV-2 (Shimadzu, Tokyo, Japan) with a load of 50 g and dwell-time of 15 s in order to obtain five measurements from each specimen. The mean KHN value was obtained by averaging the five indentations.

2.5

Degree of conversion (DC)

Disk shaped specimens (n = 5) were prepared in the same manner as for KHN and evaluated immediately after light-activation. DC was determined by a Fourier Transform Infrared spectrometer (Tensor 27, Bruker Optics GmbH, Ettlingen, Germany), coupled to an attenuated total reflectance (ATR). Absorbance spectra included 16 scans at a resolution of 1 cm ⁻¹ . Unpolymerized blends were scanned after been placed into a Teflon mold (Φ = 5 mm, 1 mm thick) and taken to the ATR. The adhesive blends were light-cured through a polyester strip using a light-curing unit (Valo, Ultradent, USA) for 20 s at 1,000m/Wcm ² . The polymerized samples were then scanned, and unconverted carbon double bonds were quantified by calculating the ratio derived from the aliphatic C C (vinyl) absorption (1638 cm ⁻¹ ) to the aromatic C C absorption (1608 cm ⁻¹ ) peaks for both polymerized and unpolymerized samples. The DC for each resin was calculated according to the follow equation: DC(%) = {1-(X _a /Y _a )/(X _b /Y _b )} × 100, where, X _a (polymerized) and X _b (unpolymerized) represent the bands of the polymerizable aliphatic double bonds, and Ya (polymerized) and Yb (unpolymerized) represent the bands of the aromatic double bonds.

2.6

Microtensile bond strength (μTBS)

Under a protocol approved by the University of Florida Institutional Review Board, 30 intact non-carious and non-restored human third molars were collected from the Department of Oral Surgery, College of Dentistry University of Florida. Teeth were stored in 5% chloramine-T solution at 4°C and used within 3 months following extraction. The roots were cut 3 mm below the cementum-enamel-junction (CEJ) using an orthodontic trimmer, and glued to a phenolic ring pre-filled with acrylic resin. With the aid of a diamond saw, the middle dentin of the teeth was exposed and a standard smear layer created by grinding the dentin on 600-grit silicon carbide paper under running water . The exposed coronal dentin surface of each tooth was cut into four proportional quarters , and each quarter was randomly assigned to the following groups (n = 30): Arg0, Arg5, Arg7 and Arg10. While a quarter was being treated with the respective adhesive, all other quarters were protected by a thick Teflon tape. Accordingly, phosphoric acid was applied to the dentin surface for 15 s, rinsed for 15 s, and blot dried. The experimental primer was applied with a rubbing motion for 10 s, followed by a very gentle air dry. A layer of the adhesive was then uniformly applied with a microbrush for 10 s. Next, the excess was removed with absorbent paper sheets and gently air dried to evaporate the solvent, and then light-cured for 10 s. Incremental layers (1.7 mm) of resin-based composite resin (Filtek Z250, 3 M ESPE, St. Paul, USA) were layered over the adhesive and light cured individually for 20 s, creating a composite core of approximately 5 mm. Sections of 0.9 mm on X and Y directions generated composite-dentin beams. The bonded interface area of each beam was individually measured with a digital caliper (Mitutoyo Corporation, Tokyo, Japan), and then actively gripped onto a Geraldeli’s device-V2 with cyanoacrylate glue and tested in a universal testing machine (Odeme OM100, Odeme Dental Research) at 0.75 mm/min . Final values was expressed in MPa considering the equation: μTBS = F/A. The microtensile bond strength (μTBS) of each quarter was defined as the average of the tested beams. Failure mode was determined by evaluating each beam with stereomicroscope (50×, Nikon, model SMZ-1B, Japan) . The failure mode was classified in adhesive (A), cohesive within dentin (CD), cohesive within resin composite (CR) and mixed (M), which involves dentin, composite and adhesive.

2.7

Morphology of the resin-dentin interface

A tooth for each study group was restored following the same method as described for μTBS. They were sectioned into slices of 2 mm thickness using a diamond saw and included in epoxy resin (Buehler epoxycure, Illinois, USA). Next, the slices were worn out with silicon carbide sandpaper in ascending granulations (#600-2500, Norton Saint-Gobain, Guarulhos, SP, Brazil) and polished by felt pads with diamond grinding polishing pastes (6 μm-0.25 μm, Ted-Pella Inc., Redding, CA, USA) followed by a conditioned with phosphoric acid 50%, rinsed and air-dried overnight. All samples were sputter-coated with gold-palladium for 60 s at 45 mA in a vacuum-metalizing chamber (MED 010; Balzers, Liechtenstein). Representative images of the resin-dentin interfaces were obtained using a scanning electron microscope (LEO 435 VP; Carl Zeiss, Jena, Germany), operated at less than 20 kV.

2.8

Arginine release and recharge

For arginine release, specimens with a diameter of 10 mm and a thickness of 1.2 mm and containing either Arg0 or Arg7 were light-cured on both sides for 20 s each. Arg7 specimens were chosen for these assays due to the positive results obtained during the mechanical tests performed for this group. Specimens (n = 3) were immersed in 2 mL high-purity water at 37 °C as previously described with some modifications. A 2 mL quantity of equilibrated water was taken at 0, 2, 4, 8, and then every 24 h up to 10 days, and finally at 30 days. The concentration of arginine released in the solution was analyzed by Liquid Chromatography Mass Spectrometry (LC–MS/MS). The same specimens used for arginine release were later used for the arginine recharge experiment. For arginine recharge, specimens were rinsed with high-purity water, dried and then submitted to 2 recharging cycles per day for 3 days. During each recharging cycle, the specimens were imersed in 2 mL of 1.5% arginine aqueous solution for 1 min (% of marketed arginine-toothpastes), rinsed for 10 s, dried, and then immersed in 2 mL high-purity water at 37 °C for 12 h (collection solution). The concentration of arginine was measured in each collected solution by LC–MS/MS.

2.9

Antibacterial activity

The effect of the adhesive Arg7 on bacterial growth and biofilm formation was evaluated as previously described with some modifications. Planktonic growth of Streptococcus mutans UA159 and Streptococcus gordonii DL1 was tested using the Bioscreen CTM (Oy Growth Curves AB Ltd, Helsinki, Finland). These strains were selected based on the facts that S. mutans is a well-recognized pathogen and S. gordonii is an ADS-positive species. Ring specimens (n = 3; inner diameter: 3.1. mm, outer diameter: 5.2 mm, thickness: 1.1 mm) were made placing the adhesive into a Teflon ring mold and light-curing for 20 s. After ultraviolet (UV) sterilization, the specimens were laid at the bottoms of 100-well honeycomb microtiter plates of Bioscreen CTM. Overnight cultures of the tested strains in Brain Heart Infusion (BHI: 3.7% containing a final concentration of 2 g of glucose/L; Difco Laboratories, Detroit, MI, USA) broth were transferred to fresh media and grown until mid-exponential phase (OD ₆₀₀ = 0.5) in a 5% CO ₂ aerobic atmosphere at 37 °C. The cultures were diluted 1:100 into fresh BHI broth at pH 7.0 or 5.7, and aliquots (250 μL) were applied to the honeycomb plates. Bacterial growth was monitored every half-hour and continuously for 24 h using the Bioscreen CTM with moderate shaking for 10 s prior to OD measurements .

Confocal microscopy was used for analysis of the biofilms. As previously done for planktonic assays, mid-exponential phase cultures of UA159 and DL1 were diluted 1:100 into fresh TY (3% tryptone and 0.5% yeast extract; Difco Laboratories) supplemented with 25 mM glucose (TY–25 mM glucose) at pH 7.0 or 5.7, and aliquoted into 8-well iBidi glass slides (vials ibidi GmbH, Munich, Germany) containing the adhesive discs. Biofilms were allowed to form for 48 h in a 5% CO ₂ aerobic atmosphere at 37 °C with media refresh after 24 h. Growth media and planktonic cells were removed after 48 h and the biofilm formed was washed gently with sterile phosphate-buffered saline (PBS). For analysis by confocal laser scanning microscopy, biofilms were stained with a LIVE/DEAD BacLight bacterial viability kit (Thermo Fisher, USA), and the images were acquired using a spinning disk confocal system connected to a Leica DM IRB inverted fluorescence microscope that was equipped with a Photometrics cascade-cooled EMCCD camera. Syto9 fluorescence was detected by excitation at 488 nm, and emission was collected using a 525 nm (+/- 25 nm, green) band-pass filter. Detection of propidium iodide (PI) fluorescence was performed using a 642 nm excitation laser and a 695 nm (+/- 53 nm, red) band-pass filter. All z-sections were collected at 1 μm intervals using a 63/1.40 oil objective lens. Image acquisition and processing were performed using VoxCell (VisiTech International, Sunderland, United Kingdom).

2.10

Statistical analysis

All data management and statistical analyses was performed using SAS procedures (SAS 9.1.3). For descriptive analysis, distribution of percentages and means were calculated when appropriate. One-way ANOVA and Tukeýs tests were used to test the differences of UTS, FS, E , DC, KHN and μ-TBS means, with a significance level of 95% (α = 0.05). For arginine release and recharge and antibacterial activity experiments, T-test was used to test the differences of continuous variables; and chi-square test was used for categorical variables, also with a significance level of 95% (α = 0.05).

2.1

Adhesive formulation

Experimental etch-and-rinse, two bottles, adhesive systems containing different concentrations of L-arginine (Sigma-Aldrich, Inc, St Louis, MO, USA) were fabricated. The primer solution consisted of 15% ethoxylated bisphenol-A dimethacrylate (BisEMA; Esstech Inc., Essington, PA, USA), 10% hydroxyethyl methacrylate (HEMA; Sigma-Aldrich), 10% urethane dimethacrylate (UDMA; Sigma-Aldrich), 10% triethylene glycol dimethacrylate (TEGDMA; Sigma-Aldrich), 15% distilled water and 40% ethanol, and at a ratio of 45% monomers and 55% solvents. The adhesive consisted of 40% UDMA, 30% TEGDMA, 17% BisEMA, 10% bisphenol A glycidyl methacrylate (BisGMA, Sigma-Aldrich), 1.5% diphenyliodonium hexafluorophosphate (DPIHP, Sigma-Aldrich) and 0.5% camphorquinone/1% amine (CQ/EDAB, Sigma-Aldrich) in the monomer mixture previously homogenized at a ratio of 97% monomers and 3% photo-initiator agents. L-arginine was added to the adhesive and homogenized at the weight ratio concentrations of 0% (Arg0; control), 5% (Arg5), 7% (Arg7), and 10% (Arg10). Of note, pilot tests were carried out in order to observe how arginine particles would dissolve with different dental adhesives monomers blends. Some properties were estimated such as arginine-dissolution, phase separation, wettability and viscosity. Once the adhesive blend presented clear-uniform appearance, proper viscosity for dental application and absence of phase-separation, arginine was added at concentrations ranging from 2 to 18%. The arginine concentrations of 5%, 7% and 10% were selected because these presented the best saturated mixtures. The adhesive systems were prepared in a dark room under controlled temperature and humidity, and then kept under refrigeration (4 °C). Prior to use, the adhesives were stirred for 15 min. All concentrations are provided here at a weight ratio.

2.2

Ultimate tensile strength (UTS)

Specimens (n = 10) were prepared using silicon molds with an hourglass shape of 10 × 4 mm and sectional area of 1.5 mm ² (Odeme Dental Research, Luzerna, SC, Brazil). Each adhesive was placed into the molds, covered with a clear transparent Mylar-matrix and coverslip, and then light-cured at 1000 mW/cm ² for 20 s (Valo, Ultradent, South Jordan, UT, USA). Specimens were fitted in a testing jig device and submitted to tensile strength test. Load was applied perpendicular to the plane of the cured adhesive in a semi-universal testing machine OM100 (Odeme Dental Research) at 0.75 mm/min. Ultimate tensile strength (UTS) was calculated in MPa using the formula: UTS = F/A , in which, F was the tensile strength (N) and A the transversal cross section area (mm ² ).

2.3

Flexural strength (FS) and flexural modulus ( E )

Bar-shaped specimens (n = 7) were prepared using a silicon mold of 10 × 2 × 2 mm (Odeme Dental Research). Each adhesive was placed into the molds, covered with a clear transparent Mylar matrix and coverslip, and then light-cured as described above. The cross sectional area was approximately 4.0 mm ² . Specimens were stored at 37°C for 24 h and subjected to three-point bending test in a universal testing machine (Instron, Norwood, MA, USA) with 8 mm span between supports and at crosshead speed of 0.5 mm/min. The maximum load for the specimens at fracture was recorded and the FS calculated using the following equation: FS = 3FL/(2bh ² ), where F was the maximum load (N), L the distance (mm) between supports, B the width (mm) and H the height (mm). B and H were measured immediately prior the testing. In the Instron machine, E data was based on the first load-displacement curve of the linear portion of the graphic obtained from the BlueHill 3 software, and used with the standard equation E = L ³ F/4 w H ³ d, in which L is the support span length (mm), F the maximum load (N), w the specimen width (mm), H the specimen height and d is the deflection (mm) at load F.

2.4

Knoop hardness (KHN)

Disk shaped specimens (n = 5) were prepared by placing each adhesive into a rubber mold of 5 × 1 mm (Odeme Dental Research). A Mylar strip and a coverslip were placed over the adhesive/mold and light-cured as described above. Specimens were kept stored for 24 h in dry conditions at 37°C. Next, the top surfaces were polished under water with a 1200 grit SiC sandpaper to obtain a polished surface. KHN test was carried out in a microindenter HMV-2 (Shimadzu, Tokyo, Japan) with a load of 50 g and dwell-time of 15 s in order to obtain five measurements from each specimen. The mean KHN value was obtained by averaging the five indentations.

2.5

Degree of conversion (DC)

Disk shaped specimens (n = 5) were prepared in the same manner as for KHN and evaluated immediately after light-activation. DC was determined by a Fourier Transform Infrared spectrometer (Tensor 27, Bruker Optics GmbH, Ettlingen, Germany), coupled to an attenuated total reflectance (ATR). Absorbance spectra included 16 scans at a resolution of 1 cm ⁻¹ . Unpolymerized blends were scanned after been placed into a Teflon mold (Φ = 5 mm, 1 mm thick) and taken to the ATR. The adhesive blends were light-cured through a polyester strip using a light-curing unit (Valo, Ultradent, USA) for 20 s at 1,000m/Wcm ² . The polymerized samples were then scanned, and unconverted carbon double bonds were quantified by calculating the ratio derived from the aliphatic C C (vinyl) absorption (1638 cm ⁻¹ ) to the aromatic C C absorption (1608 cm ⁻¹ ) peaks for both polymerized and unpolymerized samples. The DC for each resin was calculated according to the follow equation: DC(%) = {1-(X _a /Y _a )/(X _b /Y _b )} × 100, where, X _a (polymerized) and X _b (unpolymerized) represent the bands of the polymerizable aliphatic double bonds, and Ya (polymerized) and Yb (unpolymerized) represent the bands of the aromatic double bonds.

2.6

Microtensile bond strength (μTBS)

Under a protocol approved by the University of Florida Institutional Review Board, 30 intact non-carious and non-restored human third molars were collected from the Department of Oral Surgery, College of Dentistry University of Florida. Teeth were stored in 5% chloramine-T solution at 4°C and used within 3 months following extraction. The roots were cut 3 mm below the cementum-enamel-junction (CEJ) using an orthodontic trimmer, and glued to a phenolic ring pre-filled with acrylic resin. With the aid of a diamond saw, the middle dentin of the teeth was exposed and a standard smear layer created by grinding the dentin on 600-grit silicon carbide paper under running water . The exposed coronal dentin surface of each tooth was cut into four proportional quarters , and each quarter was randomly assigned to the following groups (n = 30): Arg0, Arg5, Arg7 and Arg10. While a quarter was being treated with the respective adhesive, all other quarters were protected by a thick Teflon tape. Accordingly, phosphoric acid was applied to the dentin surface for 15 s, rinsed for 15 s, and blot dried. The experimental primer was applied with a rubbing motion for 10 s, followed by a very gentle air dry. A layer of the adhesive was then uniformly applied with a microbrush for 10 s. Next, the excess was removed with absorbent paper sheets and gently air dried to evaporate the solvent, and then light-cured for 10 s. Incremental layers (1.7 mm) of resin-based composite resin (Filtek Z250, 3 M ESPE, St. Paul, USA) were layered over the adhesive and light cured individually for 20 s, creating a composite core of approximately 5 mm. Sections of 0.9 mm on X and Y directions generated composite-dentin beams. The bonded interface area of each beam was individually measured with a digital caliper (Mitutoyo Corporation, Tokyo, Japan), and then actively gripped onto a Geraldeli’s device-V2 with cyanoacrylate glue and tested in a universal testing machine (Odeme OM100, Odeme Dental Research) at 0.75 mm/min . Final values was expressed in MPa considering the equation: μTBS = F/A. The microtensile bond strength (μTBS) of each quarter was defined as the average of the tested beams. Failure mode was determined by evaluating each beam with stereomicroscope (50×, Nikon, model SMZ-1B, Japan) . The failure mode was classified in adhesive (A), cohesive within dentin (CD), cohesive within resin composite (CR) and mixed (M), which involves dentin, composite and adhesive.

2.7

Morphology of the resin-dentin interface

A tooth for each study group was restored following the same method as described for μTBS. They were sectioned into slices of 2 mm thickness using a diamond saw and included in epoxy resin (Buehler epoxycure, Illinois, USA). Next, the slices were worn out with silicon carbide sandpaper in ascending granulations (#600-2500, Norton Saint-Gobain, Guarulhos, SP, Brazil) and polished by felt pads with diamond grinding polishing pastes (6 μm-0.25 μm, Ted-Pella Inc., Redding, CA, USA) followed by a conditioned with phosphoric acid 50%, rinsed and air-dried overnight. All samples were sputter-coated with gold-palladium for 60 s at 45 mA in a vacuum-metalizing chamber (MED 010; Balzers, Liechtenstein). Representative images of the resin-dentin interfaces were obtained using a scanning electron microscope (LEO 435 VP; Carl Zeiss, Jena, Germany), operated at less than 20 kV.

2.8

Arginine release and recharge

For arginine release, specimens with a diameter of 10 mm and a thickness of 1.2 mm and containing either Arg0 or Arg7 were light-cured on both sides for 20 s each. Arg7 specimens were chosen for these assays due to the positive results obtained during the mechanical tests performed for this group. Specimens (n = 3) were immersed in 2 mL high-purity water at 37 °C as previously described with some modifications. A 2 mL quantity of equilibrated water was taken at 0, 2, 4, 8, and then every 24 h up to 10 days, and finally at 30 days. The concentration of arginine released in the solution was analyzed by Liquid Chromatography Mass Spectrometry (LC–MS/MS). The same specimens used for arginine release were later used for the arginine recharge experiment. For arginine recharge, specimens were rinsed with high-purity water, dried and then submitted to 2 recharging cycles per day for 3 days. During each recharging cycle, the specimens were imersed in 2 mL of 1.5% arginine aqueous solution for 1 min (% of marketed arginine-toothpastes), rinsed for 10 s, dried, and then immersed in 2 mL high-purity water at 37 °C for 12 h (collection solution). The concentration of arginine was measured in each collected solution by LC–MS/MS.

2.9

Antibacterial activity

The effect of the adhesive Arg7 on bacterial growth and biofilm formation was evaluated as previously described with some modifications. Planktonic growth of Streptococcus mutans UA159 and Streptococcus gordonii DL1 was tested using the Bioscreen CTM (Oy Growth Curves AB Ltd, Helsinki, Finland). These strains were selected based on the facts that S. mutans is a well-recognized pathogen and S. gordonii is an ADS-positive species. Ring specimens (n = 3; inner diameter: 3.1. mm, outer diameter: 5.2 mm, thickness: 1.1 mm) were made placing the adhesive into a Teflon ring mold and light-curing for 20 s. After ultraviolet (UV) sterilization, the specimens were laid at the bottoms of 100-well honeycomb microtiter plates of Bioscreen CTM. Overnight cultures of the tested strains in Brain Heart Infusion (BHI: 3.7% containing a final concentration of 2 g of glucose/L; Difco Laboratories, Detroit, MI, USA) broth were transferred to fresh media and grown until mid-exponential phase (OD ₆₀₀ = 0.5) in a 5% CO ₂ aerobic atmosphere at 37 °C. The cultures were diluted 1:100 into fresh BHI broth at pH 7.0 or 5.7, and aliquots (250 μL) were applied to the honeycomb plates. Bacterial growth was monitored every half-hour and continuously for 24 h using the Bioscreen CTM with moderate shaking for 10 s prior to OD measurements .

Confocal microscopy was used for analysis of the biofilms. As previously done for planktonic assays, mid-exponential phase cultures of UA159 and DL1 were diluted 1:100 into fresh TY (3% tryptone and 0.5% yeast extract; Difco Laboratories) supplemented with 25 mM glucose (TY–25 mM glucose) at pH 7.0 or 5.7, and aliquoted into 8-well iBidi glass slides (vials ibidi GmbH, Munich, Germany) containing the adhesive discs. Biofilms were allowed to form for 48 h in a 5% CO ₂ aerobic atmosphere at 37 °C with media refresh after 24 h. Growth media and planktonic cells were removed after 48 h and the biofilm formed was washed gently with sterile phosphate-buffered saline (PBS). For analysis by confocal laser scanning microscopy, biofilms were stained with a LIVE/DEAD BacLight bacterial viability kit (Thermo Fisher, USA), and the images were acquired using a spinning disk confocal system connected to a Leica DM IRB inverted fluorescence microscope that was equipped with a Photometrics cascade-cooled EMCCD camera. Syto9 fluorescence was detected by excitation at 488 nm, and emission was collected using a 525 nm (+/- 25 nm, green) band-pass filter. Detection of propidium iodide (PI) fluorescence was performed using a 642 nm excitation laser and a 695 nm (+/- 53 nm, red) band-pass filter. All z-sections were collected at 1 μm intervals using a 63/1.40 oil objective lens. Image acquisition and processing were performed using VoxCell (VisiTech International, Sunderland, United Kingdom).

2.10

Statistical analysis

All data management and statistical analyses was performed using SAS procedures (SAS 9.1.3). For descriptive analysis, distribution of percentages and means were calculated when appropriate. One-way ANOVA and Tukeýs tests were used to test the differences of UTS, FS, E , DC, KHN and μ-TBS means, with a significance level of 95% (α = 0.05). For arginine release and recharge and antibacterial activity experiments, T-test was used to test the differences of continuous variables; and chi-square test was used for categorical variables, also with a significance level of 95% (α = 0.05).