2.1

MPC incorporation into bonding agent

Scotchbond multi-purpose (3M, St. Paul, MN), referred to as “SBMP”, was used as the parent bonding system. According to the manufacturer, SBMP etchant contained 35% phosphoric acid. SBMP primer single bottle contained 35–45% 2-hydroxyethylmethacrylate (HEMA), 10–20% copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contained 60–70% BisGMA and 30–40% HEMA.

MPC (Sigma-Aldrich, St. Louis, MO) was synthesized via a method reported by Ishihara et al. . The MPC powder was mixed with SBMP primer at MPC/(SBMP primer + MPC) mass fraction of 7.5%. This mass fraction was selected from previous study showing that 7.5% MPC yielded the strongest protein-repellent property without compromising the dentin bond strength . MPC was added into primer and magnetically stirred with a bar at a speed of 150 rpm (Bellco Glass, Vineland, NJ) for 24 h to completely dissolve MPC in primer . Similarly, 7.5% of MPC was also mixed into the SBMP adhesive.

2.2

DMAHDM incorporation into bonding agent

DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine was reacted with an organo-halide . A benefit of this reaction is that the reaction 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 ethanol in 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 . The SBMP primer was first mixed with MPC as described above. Then, DMAHDM was mixed into the SBMP-MPC primer, at DMAHDM/(SBMP primer + DMAHDM) mass fraction of 5%. DMAHDM mass fractions of 7.5% or higher were not used due to a decrease in dentin bond strength when combined with MPC in preliminary study. Similarly, 5% DMAHDM was incorporated into the SBMP-MPC adhesive.

2.3

NACP incorporation into bonding agent

A spray-drying technique was used to synthesize NACP (Ca ₃ [PO ₄ ] ₂ ) as described previously . The NACP had a mean particle size of 116 nm . NACP were incorporated into the SBMP adhesive, but not into the primer, because preliminary study showed that adding NACP into primer decreased the dentin bond strength . NACP was mixed into the adhesive at NACP/(SBMP adhesive + NACP) = 0, 20, 30, and 40% by mass. NACP mass fraction of less than 20% was not used due to the need for sufficient ion release. NACP of more than 40% was not used due to a decrease in dentin bond strength .

2.4

Dentin shear bond strength testing

As listed in Table 1 , five groups were used for dentin shear bond strength testing. The results showed that group 5 had significantly lower dentin bond strength, while all other groups had dentin bond strengths similar to SBMP control. Therefore, group 5 was not included in subsequent experiments. Groups 1–4 were used in SEM examination, and protein adsorption and biofilm assays.

Extracted caries-free human molars were used. The tips of the crowns were cut off using a diamond saw (Isomet, Buehler, Lake Bluff, IL). The cut surface of the tooth was ground using 320 grit SiC paper until occlusal enamel was completely removed . After etching for 15 s and rinsing with water , a primer was applied, and the solvent was removed with air. An adhesive was applied and air-blown for 5 s to produce a thin adhesive layer , and then light-cured for 10 s (Optilux-VCL401, Demetron, Danbury, CT). A stainless-steel iris with a central opening (diameter = 4 mm, thickness = 1.5 mm) was held against the adhesive-treated dentin surface. The opening was filled with a composite (TPH, Caulk/Dentsply, Milford, DE) and light-cured for 60 s . The bonded specimens were stored in distilled water at 37 °C for 24 h . The dentin shear bond strength, S _D , was measured as previously described . A chisel was held parallel to the composite–dentin interface and loaded via a Universal Testing Machine (MTS, Eden Prairie, MN) at 0.5 mm/min until the composite–dentin bond failed. S _D = 4 P /( πd ² ), where P is the load at failure and d is the diameter of the composite . Ten teeth were tested for each group.

2.5

Fabrication of root dentin slabs

Root dentin slabs were fabricated using bovine instead of human root dentin, since the development and inhibition rates of caries are similar in both tissues , and bovine teeth are easier to obtain. The specimen preparation procedure followed previous studies . Extracted bovine incisors with intact roots were used. The root was separated from the crown at 1–2 mm below the cemento-enamel junction using a water-cooled diamond saw (Isomet) . Then, root dentin slabs were prepared from buccal and lingual root surfaces with a water-cooled diamond saw . The slabs were mounted with sticky wax on plexiglas blocks to facilitate handling, and were serially polished to remove the cementum and flatten the surfaces . The polishing procedure was performed with wet SiC papers up to 4000 grit. The polished slabs were examined microscopically (20×), to ensure that a smooth dentin surface was obtained . The root slabs were cleaned by sonication for 10 min in distilled water . The final dimensions of the root dentin slabs were approximately 5 mm in length, 3 mm in width, and 2 mm in thickness . One dentin surface of 5 mm × 3 mm faced the cementum, while the other 5 mm × 3 mm surface faced the pulp. Primer and adhesive were applied to the dentin surface that faced the cementum with open dentinal tubules, in order to simulate the clinical application of using an adhesive to cover roots with missing cementum and open tubules .

2.6

Scanning electron microscopic (SEM) examinations

Because adhesives can provide a barrier to protect the exposed roots from acid attacks , and a thicker coating is expected to provide a stronger barrier, the coating thickness was measured. Root dentin surface (with cementum removed) was covered with a single coat of an adhesive via the micro-brush supplied in the package, following the manufacturer’s instructions. First, the root dentin slab surface was etched with etchant for 15 s and rinsed with water, and dried with air . A primer was applied to the dentin surface and left for 20 s, and air-blown for 5 s . Then, 20 μL of adhesive was applied using pipette and rubbed in for 10 s with the micro-brush . The adhesive was light-cured for 10 s (Optilux). The different viscosity of the four adhesive systems produced different coating thicknesses.

Cross-sectional cutting would yield information on coating thickness and the resin–dentin interface; however, the adhesive coating on dentin could chip and dislodge in the cutting process. To avoid adhesive coating fracture and to enable the measurement of coating thickness, a composite (TPH) was placed on the top of the adhesive coating and light-cured for 60 s, to facilitate the subsequent cutting and the measurement of the adhesive coating thickness . The samples were cut through the center perpendicularly to the coated surface via a diamond saw (Isomet) with copious water. The cross-section was polished with increasingly finer SiC papers up to 4000 grit . The specimens were air dried and sputter-coated with gold, and examined in SEM (Quanta 200, FEI, Hillsboro, OR). The thickness of adhesive coating on root dentin was measured for six specimens. Four readings were taken at random locations on each specimen, yielding a total of 24 measurements per group.

To examine the interfaces, additional specimens were treated with 50% phosphoric acid for 30 s and 5% NaOCl for 10 min before being prepared for SEM observation . To observe the adhesive surface texture, root dentin surfaces were coated with one of the adhesives in the same manner as described above (without TPH and without cutting). The surfaces were then air-dried and sputter-coated with gold for SEM observations .

2.7

Measurement of protein adsorption onto resin surface

To test the protein-repellent properties of groups 1–4, resin disks were fabricated. The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used as molds 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 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, following a previous study . The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC) and de-gassed for 3 days .

The amount of protein adsorbed on resin disks was determined by the micro bicinchoninic acid (BCA) method . First, the disks were immersed in phosphate buffered saline (PBS) for 2 h. Then they were immersing in 4.5 g/L bovine serum albumin (BSA) (Sigma-Aldrich) solution at 37 °C for 2 h. This solution had a concentration of 4.5 g of BSA per 1 L of PBS, following previous studies . The disks then were rinsed with fresh PBS by stirring at a speed of 300 rpm for 5 min (Bellco Glass, Vineland, NJ), immersed in sodium dodecyl sulfate (SDS) 1 wt% in PBS, and sonicated at room temperature for 20 min to completely detach the BSA adsorbed onto the disk surface. 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 was mixed with 200 μL of the BCA working reagent in a 96-well plate, which was incubated at 60 °C for 30 min . Then the 96-well plate was cooled down to room temperature and 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. From the concentration of protein, the amount of protein adsorbed on the disk surface was calculated . Six disks were evaluated for each group.

2.8

Saliva collection for dental plaque microcosm biofilm model

The dental plaque microcosm model using human saliva as inoculum has been approved by the University of Maryland Baltimore Institutional Review Board (# HP-00050407). Saliva is ideal for growing dental plaque microcosm biofilms in vitro , with the advantage of maintaining much of the complexity and heterogeneity of the dental plaque in vivo . Saliva was collected from 10 healthy adult donors having natural dentition without active caries or periopathology, and without the use of antibiotics within the last 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. Stimulated saliva was collected during paraffin chewing and was kept on ice. An equal volume of saliva from each of the 10 donors was combined. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C .

2.9

Dental plaque microcosm biofilm formation and live/dead assay

The saliva–glycerol stock was added, with 1:50 final dilution, to a growth medium as inoculum . The growth 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 K1, 0.0002 g/L, at pH 7 . Each resin disk was placed into a 24-well plates, 1.5 mL of inoculum was added to each well, 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. After 16 h, the disks were transferred to new 24-well plates with fresh medium and incubated for 24 h. This totaled 2 days of incubation, which was shown to form mature biofilms on resin .

Disks with biofilms grown for 2 days were rinsed with phosphate buffered saline (PBS) and live/dead stained using the BacLight live/dead bacterial viability kit (Molecular Probes, Eugene, OR) . Live bacteria were stained with Syto 9 to produce a green fluorescence. Dead bacteria were stained with propidium iodide to produce a red fluorescence. Disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY). Six specimens were evaluated for each group. Three randomly chosen fields of view were photographed from each disk, yielding a total of 18 images for each group.

2.10

MTT assay of metabolic activity

Resin disks with 2-day biofilms were transferred to a new 24-well plate for the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay . MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. One milliliter of MTT dye (0.5 mg/mL MTT in PBS) was added to each well and incubated at 37 °C in 5% CO ₂ for 1 h. Metabolically active bacteria reduced the MTT to purple formazan. Disks were transferred to a new 24-well plate, and 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The plate was incubated for 20 min with gentle mixing at room temperature in the dark. Then, 200 μL 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 .

2.11

Lactic acid production and colony-forming unit (CFU) counts of biofilms

Disks with 2-day biofilms were rinsed with cysteine peptone water (CPW) to remove loose bacteria . Disks were transferred to 24-well plates containing buffered peptone water (BPW) plus 0.2% sucrose. The disks were incubated in 5% CO ₂ at 37 °C for 3 h to allow biofilms to produce acid . The BPW solutions were collected for lactate analysis using an enzymatic method. The 340-nm absorbance was measured with the microplate reader. Standard curves were prepared using a standard lactic acid (Supelco, Bellefonte, PA).

Disks with biofilms were transferred into tubes with 2 mL CPW, and biofilms were harvested by sonication and vortexing (Fisher, Pittsburgh, PA) . Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine total microorganisms . Second, mitis salivarius agar (MSA) plates with 15% sucrose were used to determine 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, cariogenic mutans streptococci are known to be resistant to bacitracin, and this property is used to isolate mutans streptococci from the highly heterogeneous oral microflora . Therefore, MSA agar culture plates plus 0.2 units of bacitracin per milliliter was used to determine mutans streptococci . The bacterial suspensions were serially diluted and spread onto agar plates for CFU analysis.

2.12

Statistical analysis

All data were first checked for normal distribution with the Kolmogorov–Smirnov test. One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison test was used to compare the data at a p -value of 0.05.

2.1

MPC incorporation into bonding agent

Scotchbond multi-purpose (3M, St. Paul, MN), referred to as “SBMP”, was used as the parent bonding system. According to the manufacturer, SBMP etchant contained 35% phosphoric acid. SBMP primer single bottle contained 35–45% 2-hydroxyethylmethacrylate (HEMA), 10–20% copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contained 60–70% BisGMA and 30–40% HEMA.

MPC (Sigma-Aldrich, St. Louis, MO) was synthesized via a method reported by Ishihara et al. . The MPC powder was mixed with SBMP primer at MPC/(SBMP primer + MPC) mass fraction of 7.5%. This mass fraction was selected from previous study showing that 7.5% MPC yielded the strongest protein-repellent property without compromising the dentin bond strength . MPC was added into primer and magnetically stirred with a bar at a speed of 150 rpm (Bellco Glass, Vineland, NJ) for 24 h to completely dissolve MPC in primer . Similarly, 7.5% of MPC was also mixed into the SBMP adhesive.

2.2

DMAHDM incorporation into bonding agent

DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine was reacted with an organo-halide . A benefit of this reaction is that the reaction 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 ethanol in 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 . The SBMP primer was first mixed with MPC as described above. Then, DMAHDM was mixed into the SBMP-MPC primer, at DMAHDM/(SBMP primer + DMAHDM) mass fraction of 5%. DMAHDM mass fractions of 7.5% or higher were not used due to a decrease in dentin bond strength when combined with MPC in preliminary study. Similarly, 5% DMAHDM was incorporated into the SBMP-MPC adhesive.

2.3

NACP incorporation into bonding agent

A spray-drying technique was used to synthesize NACP (Ca ₃ [PO ₄ ] ₂ ) as described previously . The NACP had a mean particle size of 116 nm . NACP were incorporated into the SBMP adhesive, but not into the primer, because preliminary study showed that adding NACP into primer decreased the dentin bond strength . NACP was mixed into the adhesive at NACP/(SBMP adhesive + NACP) = 0, 20, 30, and 40% by mass. NACP mass fraction of less than 20% was not used due to the need for sufficient ion release. NACP of more than 40% was not used due to a decrease in dentin bond strength .

2.4

Dentin shear bond strength testing

As listed in Table 1 , five groups were used for dentin shear bond strength testing. The results showed that group 5 had significantly lower dentin bond strength, while all other groups had dentin bond strengths similar to SBMP control. Therefore, group 5 was not included in subsequent experiments. Groups 1–4 were used in SEM examination, and protein adsorption and biofilm assays.

Table 1

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