2.1

Development of bioactive bonding agents

The parent primer contained pyromellitic glycerol dimethacrylate (PMGDM) (Esstech, Essington, PA) and 2-hydroxyethyl methacrylate (HEMA) (Esstech) at a mass ratio 3.3/1, with 50% acetone solvent (all mass fractions) . This primer is referred to as “PM primer”. The adhesive consisted of 44.5% of PMGDM, 39.5% of ethoxylated bisphenol A dimethacrylate (EBPADMA) (Sigma-Aldrich, St, Louis, MO), 10% of HEMA and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (Esstech) . 1% of phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide (Sigma–Aldrich) was added to enable light cure . PMGDM and EBPADMA were used because they had cytotoxicity similar to other dental dimethacrylates, but significantly less than the cytotoxicity of BisGMA . In addition, PMGDM is an acidic adhesive monomer , and can chelate with calcium ions from the recharging solution to render the resin rechargeable. HEMA was added to improve the flowablity and hydrophilicity, following a previous study . BisGMA was added because it could improve the cross-linkage of monomers and the bonding properties of the adhesive . This parent adhesive is referred to as “PEHB”.

DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that there action products are generated at virtually quantitative amounts and require minimal purification. Briefly, 10 mmol of 2-(dimethylamino) ethyl methacrylate (DMAEMA, Sigma-Aldrich) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR) were combined with 3 g of ethanolin a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h. The solvent was then removed via evaporation, yielding DMAHDM as a clear, colorless, and viscous liquid . To make the primer antibacterial, DMAHDM was mixed at DMAHDM/(PM primer + DMAHDM) = 5% by mass. The 5% was selected following a previous study . Similarly, to make the adhesive antibacterial, DMAHDM was mixed into adhesive at DMAHDM/(PEHB + DMAHDM) = 5%.

MPC was obtained commercially (Sigma-Aldrich) which was synthesized as previously described . The PM primer was first mixed with DMAHDM as described above. Then 5% by mass of MPC was mixed with the PM-DMAHDM primer. Higher MPC mass fractions were not used due to a decrease in dentin bond strength when combined with DMAHDM in preliminary study. Similarly, 5% MPC was incorporated into the PEHB-DMAHDM adhesive.

NACP [Ca ₃ (PO ₄ ) ₂ ] were synthesized via a spry-drying technique as previously described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution. The concentrations of Ca and P ions were 8 mmol/L and 5.333 mmol/L, respectively. The solution was sprayed into a heated chamber to evaporate the water and volatile acid. The dried NACP powders were collected using an electrostatic precipitator. This yielded NACP with a mean particle size of 116 nm . NACP were incorporated into the adhesive at 0%, 20%, 30% and 40% filler mass fractions, following previous studies . NACP levels greater than 40% were not used due to a decrease in dentin bond strength in preliminary study.

A commercial bonding agent Prime & Bond NT (Dentsply, Milford, DE) served as a comparative control (denoted “commercial control”). According to the manufacturer, NT was a total-etching one-bottle bonding system and contained 30% typical methacrylates, <10% methyl methacrylate, and 60% acetone. NT was combined with a self-cure activator (SCA) at 1:1 ratio to enable dual-cure. Six groups were tested:

• (1)

  Commercial Prime & Bond NT (denoted “commercial control”).

• (2)

  PM primer and PEHB adhesive, no MPC, no DMAHDM, no NACP (denoted “Experimental control”).

• (3)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, no NACP (denoted “MPC+DMAHDM”).

• (4)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 20% NACP (denoted “MPC+DMAHDM+20NACP”).

• (5)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 30% NACP (denoted “MPC+DMAHDM+30NACP”).

• (6)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 40% NACP (denoted “MPC+DMAHDM+40NACP”).

2.2

Dentin shear bond strength testing

Extracted human third molars were stored in 0.01% thymol solution at 4 °C. Each tooth was cut perpendicularly to the long axis of the tooth to expose the mid-coronal dentin using a low speed diamond saw (Isomet, Buehler, Lake Bluff, IL). The dentin surface was polished with 600-grit SiC paper. Then the dentin surface was etched with 37% phosphoric acid gel for 15 s and rinsed with water. A primer was applied with a brush-tipped applicator for 15 s, and the dentin was gently blown with air for 5 s to remove the solvent. An adhesive was then applied and light-cured for 10 s with an Optilux curing unit (VCL 401, Demeron Kerr, Danbury, CT). A stainless-steel cylindrical mold (inner diameter = 4 mm, thickness = 1.5 mm) was placed on the adhesive-treated dentin surface. A composite (TPH, Caulk/Dentsply, Milford, DE) was filled into the mold and light-cured for 60 s. The bonded specimens were stored in distilled water at 37 °C for 24 h. A chisel on a Universal Testing Machine (MTS, Eden Prairie, MN) was aligned to be parallel to the composite-dentin interface . Load was applied at a cross-head speed of 0.5 mm/min until the bond failed. Dentin shear bond strength = 4P/(πd ² ), where P is the load at failure, and d is the diameter of the composite . Ten teeth were tested for each group.

2.3

Measurement of protein adsorption onto resin surface

The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used as molds to fabricate resin disks following a previous study . Briefly, 10 μL of a primer was placed in the bottom of each dent of the 96-well plate. After drying with a stream of air, 20 μL of adhesive was applied to the dent and photo-polymerized for 30 s (Optilux), using a Mylar strip covering to obtain a disk of approximately 8 mm in diameter and 0.5 mm in thickness. The cured resin disks were immersed in 200 mL of distilled water and magnetically stirred with a bar at a speed of 100 rpm for 1 h to remove any uncured monomers . The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC) and de-gassed for 3 days .

Protein adsorption on resin disks was determined using a micro bicinchoninic acid (BCA) method . Each disk was immersed in phosphate buffered saline (PBS) for 2 h, and then immersed in 4.5 g/L bovine serum albumin (BSA, Sigma-Aldrich) solution at 37 °C for 2 h . The disks were rinsed with fresh PBS by stirring at a speed of 300 rpm for 5 min, and then immersed in a solution of 1% sodium dodecylsulfate (SDS) in PBS and sonicated for 20 min to detach the BSA adsorbed on the disk . A protein analysis kit (micro BCA protein assay kit, Fisher Scientific, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. Briefly, 25 μL of the SDS solution and 200 μL of the BCA reagent were mixed into the wells of a 96-well plate and incubated at 60 °C for 30 min . The absorbance at 562 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). Standard curves were prepared using the BSA standard .

2.4

Saliva collection and dental plaque microcosm biofilm formation

The dental plaque microcosm model was approved by University of Maryland Institutional Review Board. Saliva is ideal for growing microcosm biofilms in vitro , with the advantage of maintaining much of the complexity and heterogeneity of dental plaque in vivo . Saliva was collected from ten healthy donors having natural dentition without active caries, and not having used antibiotics within the past 3 months. The donors did not brush teeth for 24 h and abstained from food and drink intake for 2 h prior to donating saliva . An equal volume of saliva from each of the ten donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C for subsequent use .

The saliva–glycerol stock was added, with 1:50 final dilution, into a McBain artificial saliva growth medium as inoculum . This medium contained mucin (Type II, porcine, gastric) at a concentration of 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl ₂ , 0.2 g/L; cysteine hydrochloride, 0.1 g/L; hemin, 0.001 g/L; vitamin K ₁ , 0.0002 g/L, at pH 7 . 2% sucrose was added to this medium. 1.5 mL of inoculum was added to each well of 24-well plates with a resin disk, and incubated at 37 °C in 5% CO ₂ for 8 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for 16 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for another 24 h. This totaled 48 h of culture, which was previously shown to form relatively mature dental plaque microcosm biofilms on resins .

2.5

Live/dead staining of biofilms

Resin disks with 2-day biofilms were washed with PBS and stained using the BacLight live/dead kit (Molecular Probes, Eugene, OR) . Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. The stained disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).

2.6

MTT assay of metabolic activity of biofilms

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate the metabolic activity of biofilms on the disks . MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Disks with 2-day biofilms were transferred to a new 24-well plate, and 1 mL of MTT dye was added to each well and incubated at 37 °C in 5% CO ₂ for 1 h. Then the disks were transferred to a new 24-well plate, and 1 mL of dimethyl sulfoxide (DMSO) was added to resolve the formazan crystals. The plate was incubated for 20 min with gentle mixing in the dark. Then, 200 mL of the DMSO solution from each well was collected, and its absorbance at 540 nm was measured via a microplate reader (SpectraMax M5). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk .

2.7

Colony-forming unit (CFU) counts

Disks with 2-day biofilms were transferred into tubes with 2 mL of CPW, and the biofilms were harvested by sonication and vortexing . Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine the total microorganisms . Second, mitis salivarius agar (MSA) culture plates containing 15% sucrose were used to determine the total streptococci . This is because MSA contains selective agents including crystal violet, potassium tellurite and trypan blue, which inhibit most Gram-negative bacilli and most Gram-positive bacteria except streptococci, thus enabling the streptococci to grow . Third, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine the mutans streptococci . The bacterial suspensions were serially diluted, spread onto agar plates and incubated at 37 °C in 5% CO ₂ for 24 h. The number of colonies that grew was counted and used, along with the dilution factor, to calculate the CFU on each resin disk .

2.8

Ca and P ion concentration measurement of the biofilm culture medium

The experimental control and commercial control groups were not included in Ca and P ion concentration measurements because they did not release Ca and P ions. The biofilm culture media after 72 h of incubation for the other four groups were collected and centrifuged at 12000 rpm for 5 min (Eppendorf Centrifuge 5415, Brinkmann, Westbury, NY). Then, 1 mL supernatant was used and analyzed for Ca and P concentrations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves .

2.9

pH of biofilm culture medium

Each resin disk was placed in a well and 1.5 mL of inoculum was added to each well of 24-well plates. They were incubated at 37 °C in 5% CO ₂ for 24 h as described above. Then, the disks with adherent biofilms were transferred to new 24-well plates with fresh medium, and the pH measurement was started. The pH of the culture medium was measured from 24 h to 72 h using a pH meter (Accumet Excel XL25, Fisher, Pittsburgh, PA) . The pH measurements were not collected for the initial 0–24 h of culture, because the planktonic bacteria in the medium would interfere with the pH. By placing disks with adherent biofilms in new wells with fresh medium and measuring the pH from 24 h to 72 h, it enabled the measured pH to be related to the biofilm on the resin . The pH data were recorded once every hour from 24 h to 32 h of the incubation. Then no pH measurement was made at night for 16 h. The pH measurement was re-started the next morning every two hours from 48 h to 60 h of the incubation. The last pH measurement was made in the next morning at 72 h of the incubation.

2.10

Statistical analysis

One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison tests was used to compare the data at a p values of 0.05.

2.1

Development of bioactive bonding agents

The parent primer contained pyromellitic glycerol dimethacrylate (PMGDM) (Esstech, Essington, PA) and 2-hydroxyethyl methacrylate (HEMA) (Esstech) at a mass ratio 3.3/1, with 50% acetone solvent (all mass fractions) . This primer is referred to as “PM primer”. The adhesive consisted of 44.5% of PMGDM, 39.5% of ethoxylated bisphenol A dimethacrylate (EBPADMA) (Sigma-Aldrich, St, Louis, MO), 10% of HEMA and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (Esstech) . 1% of phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide (Sigma–Aldrich) was added to enable light cure . PMGDM and EBPADMA were used because they had cytotoxicity similar to other dental dimethacrylates, but significantly less than the cytotoxicity of BisGMA . In addition, PMGDM is an acidic adhesive monomer , and can chelate with calcium ions from the recharging solution to render the resin rechargeable. HEMA was added to improve the flowablity and hydrophilicity, following a previous study . BisGMA was added because it could improve the cross-linkage of monomers and the bonding properties of the adhesive . This parent adhesive is referred to as “PEHB”.

DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that there action products are generated at virtually quantitative amounts and require minimal purification. Briefly, 10 mmol of 2-(dimethylamino) ethyl methacrylate (DMAEMA, Sigma-Aldrich) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR) were combined with 3 g of ethanolin a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h. The solvent was then removed via evaporation, yielding DMAHDM as a clear, colorless, and viscous liquid . To make the primer antibacterial, DMAHDM was mixed at DMAHDM/(PM primer + DMAHDM) = 5% by mass. The 5% was selected following a previous study . Similarly, to make the adhesive antibacterial, DMAHDM was mixed into adhesive at DMAHDM/(PEHB + DMAHDM) = 5%.

MPC was obtained commercially (Sigma-Aldrich) which was synthesized as previously described . The PM primer was first mixed with DMAHDM as described above. Then 5% by mass of MPC was mixed with the PM-DMAHDM primer. Higher MPC mass fractions were not used due to a decrease in dentin bond strength when combined with DMAHDM in preliminary study. Similarly, 5% MPC was incorporated into the PEHB-DMAHDM adhesive.

NACP [Ca ₃ (PO ₄ ) ₂ ] were synthesized via a spry-drying technique as previously described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution. The concentrations of Ca and P ions were 8 mmol/L and 5.333 mmol/L, respectively. The solution was sprayed into a heated chamber to evaporate the water and volatile acid. The dried NACP powders were collected using an electrostatic precipitator. This yielded NACP with a mean particle size of 116 nm . NACP were incorporated into the adhesive at 0%, 20%, 30% and 40% filler mass fractions, following previous studies . NACP levels greater than 40% were not used due to a decrease in dentin bond strength in preliminary study.

A commercial bonding agent Prime & Bond NT (Dentsply, Milford, DE) served as a comparative control (denoted “commercial control”). According to the manufacturer, NT was a total-etching one-bottle bonding system and contained 30% typical methacrylates, <10% methyl methacrylate, and 60% acetone. NT was combined with a self-cure activator (SCA) at 1:1 ratio to enable dual-cure. Six groups were tested:

• (1)

  Commercial Prime & Bond NT (denoted “commercial control”).

• (2)

  PM primer and PEHB adhesive, no MPC, no DMAHDM, no NACP (denoted “Experimental control”).

• (3)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, no NACP (denoted “MPC+DMAHDM”).

• (4)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 20% NACP (denoted “MPC+DMAHDM+20NACP”).

• (5)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 30% NACP (denoted “MPC+DMAHDM+30NACP”).

• (6)

  Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 40% NACP (denoted “MPC+DMAHDM+40NACP”).

2.2

Dentin shear bond strength testing

Extracted human third molars were stored in 0.01% thymol solution at 4 °C. Each tooth was cut perpendicularly to the long axis of the tooth to expose the mid-coronal dentin using a low speed diamond saw (Isomet, Buehler, Lake Bluff, IL). The dentin surface was polished with 600-grit SiC paper. Then the dentin surface was etched with 37% phosphoric acid gel for 15 s and rinsed with water. A primer was applied with a brush-tipped applicator for 15 s, and the dentin was gently blown with air for 5 s to remove the solvent. An adhesive was then applied and light-cured for 10 s with an Optilux curing unit (VCL 401, Demeron Kerr, Danbury, CT). A stainless-steel cylindrical mold (inner diameter = 4 mm, thickness = 1.5 mm) was placed on the adhesive-treated dentin surface. A composite (TPH, Caulk/Dentsply, Milford, DE) was filled into the mold and light-cured for 60 s. The bonded specimens were stored in distilled water at 37 °C for 24 h. A chisel on a Universal Testing Machine (MTS, Eden Prairie, MN) was aligned to be parallel to the composite-dentin interface . Load was applied at a cross-head speed of 0.5 mm/min until the bond failed. Dentin shear bond strength = 4P/(πd ² ), where P is the load at failure, and d is the diameter of the composite . Ten teeth were tested for each group.

2.3

Measurement of protein adsorption onto resin surface

The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used as molds to fabricate resin disks following a previous study . Briefly, 10 μL of a primer was placed in the bottom of each dent of the 96-well plate. After drying with a stream of air, 20 μL of adhesive was applied to the dent and photo-polymerized for 30 s (Optilux), using a Mylar strip covering to obtain a disk of approximately 8 mm in diameter and 0.5 mm in thickness. The cured resin disks were immersed in 200 mL of distilled water and magnetically stirred with a bar at a speed of 100 rpm for 1 h to remove any uncured monomers . The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC) and de-gassed for 3 days .

Protein adsorption on resin disks was determined using a micro bicinchoninic acid (BCA) method . Each disk was immersed in phosphate buffered saline (PBS) for 2 h, and then immersed in 4.5 g/L bovine serum albumin (BSA, Sigma-Aldrich) solution at 37 °C for 2 h . The disks were rinsed with fresh PBS by stirring at a speed of 300 rpm for 5 min, and then immersed in a solution of 1% sodium dodecylsulfate (SDS) in PBS and sonicated for 20 min to detach the BSA adsorbed on the disk . A protein analysis kit (micro BCA protein assay kit, Fisher Scientific, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. Briefly, 25 μL of the SDS solution and 200 μL of the BCA reagent were mixed into the wells of a 96-well plate and incubated at 60 °C for 30 min . The absorbance at 562 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). Standard curves were prepared using the BSA standard .

2.4

Saliva collection and dental plaque microcosm biofilm formation

The dental plaque microcosm model was approved by University of Maryland Institutional Review Board. Saliva is ideal for growing microcosm biofilms in vitro , with the advantage of maintaining much of the complexity and heterogeneity of dental plaque in vivo . Saliva was collected from ten healthy donors having natural dentition without active caries, and not having used antibiotics within the past 3 months. The donors did not brush teeth for 24 h and abstained from food and drink intake for 2 h prior to donating saliva . An equal volume of saliva from each of the ten donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C for subsequent use .

The saliva–glycerol stock was added, with 1:50 final dilution, into a McBain artificial saliva growth medium as inoculum . This medium contained mucin (Type II, porcine, gastric) at a concentration of 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl ₂ , 0.2 g/L; cysteine hydrochloride, 0.1 g/L; hemin, 0.001 g/L; vitamin K ₁ , 0.0002 g/L, at pH 7 . 2% sucrose was added to this medium. 1.5 mL of inoculum was added to each well of 24-well plates with a resin disk, and incubated at 37 °C in 5% CO ₂ for 8 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for 16 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for another 24 h. This totaled 48 h of culture, which was previously shown to form relatively mature dental plaque microcosm biofilms on resins .

2.5

Live/dead staining of biofilms

Resin disks with 2-day biofilms were washed with PBS and stained using the BacLight live/dead kit (Molecular Probes, Eugene, OR) . Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. The stained disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).

2.6

MTT assay of metabolic activity of biofilms

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate the metabolic activity of biofilms on the disks . MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Disks with 2-day biofilms were transferred to a new 24-well plate, and 1 mL of MTT dye was added to each well and incubated at 37 °C in 5% CO ₂ for 1 h. Then the disks were transferred to a new 24-well plate, and 1 mL of dimethyl sulfoxide (DMSO) was added to resolve the formazan crystals. The plate was incubated for 20 min with gentle mixing in the dark. Then, 200 mL of the DMSO solution from each well was collected, and its absorbance at 540 nm was measured via a microplate reader (SpectraMax M5). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk .

2.7

Colony-forming unit (CFU) counts

Disks with 2-day biofilms were transferred into tubes with 2 mL of CPW, and the biofilms were harvested by sonication and vortexing . Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine the total microorganisms . Second, mitis salivarius agar (MSA) culture plates containing 15% sucrose were used to determine the total streptococci . This is because MSA contains selective agents including crystal violet, potassium tellurite and trypan blue, which inhibit most Gram-negative bacilli and most Gram-positive bacteria except streptococci, thus enabling the streptococci to grow . Third, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine the mutans streptococci . The bacterial suspensions were serially diluted, spread onto agar plates and incubated at 37 °C in 5% CO ₂ for 24 h. The number of colonies that grew was counted and used, along with the dilution factor, to calculate the CFU on each resin disk .

2.8

Ca and P ion concentration measurement of the biofilm culture medium

The experimental control and commercial control groups were not included in Ca and P ion concentration measurements because they did not release Ca and P ions. The biofilm culture media after 72 h of incubation for the other four groups were collected and centrifuged at 12000 rpm for 5 min (Eppendorf Centrifuge 5415, Brinkmann, Westbury, NY). Then, 1 mL supernatant was used and analyzed for Ca and P concentrations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves .

2.9

pH of biofilm culture medium

Each resin disk was placed in a well and 1.5 mL of inoculum was added to each well of 24-well plates. They were incubated at 37 °C in 5% CO ₂ for 24 h as described above. Then, the disks with adherent biofilms were transferred to new 24-well plates with fresh medium, and the pH measurement was started. The pH of the culture medium was measured from 24 h to 72 h using a pH meter (Accumet Excel XL25, Fisher, Pittsburgh, PA) . The pH measurements were not collected for the initial 0–24 h of culture, because the planktonic bacteria in the medium would interfere with the pH. By placing disks with adherent biofilms in new wells with fresh medium and measuring the pH from 24 h to 72 h, it enabled the measured pH to be related to the biofilm on the resin . The pH data were recorded once every hour from 24 h to 32 h of the incubation. Then no pH measurement was made at night for 16 h. The pH measurement was re-started the next morning every two hours from 48 h to 60 h of the incubation. The last pH measurement was made in the next morning at 72 h of the incubation.

2.10

Statistical analysis

One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison tests was used to compare the data at a p values of 0.05.