2.1

Incorporation of MPC into RMGI

An RMGI cement (GC Ortho LC, Fuji, Aichi-ken, Japan) consisted of fluoroaluminosilicate glass particles, hydroxyethyl methacrylate (HEMA) monomer and aqueous polyalkenoic acid. A powder/liquid mass ratio of 2.5/1 was used according to the manufacturer. MPC was obtained commercially (Sigma-Aldrich, St. Louis, MO) which was synthesized via an approach by Ishihara et al. . The MPC powder was mixed into RMGI at a MPC/(RMGI + MPC) mass fraction of 3%, following a previous study .

2.2

Incorporation of DMAHDM into RMGI

DMAHDM with an alkyl chain length of 16 was synthesized via a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that the reaction products are generated at 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 and viscous liquid . The DMAHDM was mixed with RMGI at a DMAHDM/(RMGI + DMAHDM) mass fraction of 1.5%, following a previous study .

2.3

Incorporation of NACP into RMGI

NACP were synthesized via a spray-drying technique as described previously . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution to achieve final Ca and P ionic concentrations of 8 mmol/L and 5.333 mmol/L, which led to Ca/P molar mass ratio was 1.5, the same as that for ACP. This solution was sprayed into a heated chamber apparatus, and the dried particles were collected via an electrostatic precipitator (AirQuality, Minneapolis, MN), producing NACP with a mean particle size of 116 nm measured before .

Adding NACP powder into the RMGI paste significantly increased the paste viscosity. Therefore, to maintain a viscosity similar to that of the commercial RMGI, photo-activated resin monomers were added together with NACP into RMGI. The monomers consisted of bisphenol A glycidyl dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) (Esstech, Essington, PA) at a 1:1 mass ratio, with 0.2% camphorquinone and 0.8% ethyl 4-N,Ndimathylaminobenzoate. The BisGMA-TEGDMA resin (referred to as BT) at a BT/(final cement) mass fraction of 15%, as well as NACP at a NACP/(final cement) mass fraction of 20% was mixed to form the BT-NACP paste. Meanwhile, MPC at a MPC/(final cement) mass fraction of 3% and DMAHDM at a DMAHDM/(final cement) mass fraction of 1.5% were mixed into the powder of the RMGI to form the RMGI-MPC-DMAHDM blend. Then, this blend was mixed with the BT-NACP paste to form the final RMGI + MPC + DMAHDM + NACP cement. The percentages of each component were selected following a previous study . This yielded a modified RMGI paste containing BT and NACP that had an acceptable viscosity for the final paste and good enamel bond strength.

2.4

Preparation of orthodontic bracket-enamel bonded samples

Nighty-six freshly extracted, caries-free premolars for orthodontic reasons were collected and randomly assigned to four groups shown in Table 1 (n = 24 per group). The use of extracted human teeth has been approved by the Ethical Committee of Beijing Stomatological Hospital and the Institutional Review Board of University of Maryland in Baltimore. The selection criteria for teeth included intact buccal enamel without WSLs, and without visible cracks or enamel irregularities. Teeth were cleaned and stored in 0.1% thymol solution at 4 °C before use within 1 month. For each group, twenty-four teeth were randomly divided into two groups of 12 teeth each. One group was used for the shear bond strength test, and the other group for the demineralization experiment. The four types of cements are listed below:

• (1)

  Transbond XT control (referred to as TB control);

• (2)

  RMGI (GC Ortho LC, Fuji);

• (3)

  95.5% RMGI + 3% MPC + 1.5% DMAHDM (referred to as “RMGI + MPC + DMAHDM”);

• (4)

  60.5% RMGI + 3% MPC + 1.5% DMAHDM + 20% NACP + 15% BisGMA-TEGDMA (referred to as “RMGI + MPC + DMAHDM + NACP”).

According to the manufacturer, Transbond XT (3 M Unitek, Saint Paul, MS, USA) contained silane-treated quartz, bisphenol-A-diglycidyl ether dimethacrylate, bisphenol-A-bis (2-hydroxyethyl) dimethacrylate, and silane-treated silica. For enamel shear bond test, each tooth was embedded in a self-curing acrylic resin (New Century Dental Materials, Shanghai, China) perpendicularly to the buccal axis of the crown, in order that the buccal surface would be parallel to the force during testing ( Fig. 1 A). Enamel was etched for 30 s with 37% phosphoric acid gel (3 M Unitek). The tooth was dried with air stream until a chalky appearance was observed . Standard straight-wire premolar brackets (OPK-A, Tomy, Fukushima-ken, Japan) were bonded according to manufacturers’ instructions and literature for Transbond control and GC control . The bonding procedure for the two novel groups was the same as that for GC control. To make the cement thickness consistent for each sample, a 3.0 N force was applied vertically on the bracket for 5 s via an FGP-0.5 gauge (SHIMPO, Japan). The samples were light-cured for a total 40 s from mesial and distal sides at a 45° angle and a distance of 2 mm to the enamel surface (Elipar™ S10 LED, 3 M Unitek, Saint Paul, MS, USA).

Schematic diagram illustrating the workflow of this study. A, test of shear bond strength; B, bracket bonding and varnish covering of premolar buccal surface; C, in-vitro dental plaque microcosm model for a 14-day demineralization; D, buccolingually cut of a demineralized sample; E, principle of lesion depth measurements of Group A slices using polarized-light microscopy (PLM). F, cross-sectional micro-hardness (CSMH) analysis and scanning electron microscopy (SEM) of Group B.

For demineralization test, the roots were cut off via a water-cooled diamond bur. Coronal pulp was removed, then the pulp cavities were sealed by wax. To prevent the surrounding enamel from being etched, a piece of tape with a rectangular cut-out window of the size of the bracket base was used. The etching and bracket bonding procedures were the same as the samples in the shear bond test. Then, each bracket-enamel bonded sample was painted with an acid-resistant nail varnish around the bracket, leaving a 1 mm space of exposed enamel ( Fig. 1 B). All the samples were disinfected with ethylene oxide and de-gassed for 3 d (Anprolene AN 74i, Andersen, Haw River, NC).

2.5

Enamel shear bond strength (SBS) test

The bracket-bonded samples of each group (n = 12) were stored in distilled water at 37 °C for 24 h, and then they were used for shear bond testing. A chisel shear tester was connected to a computer-controlled Universal Testing Machine (AG-IS 500N, SHIMADZU Corporation, Japan). Each embedded sample was fixed on the working stage, and the chisel tip was carefully adjusted and positioned on the upper part of the bracket base ( Fig. 1 A). An occlusal-gingival load was applied at a displacement rate of 0.5 mm/min until the bracket bond failed. The enamel shear bond strength (SBS) = the debonding force/bracket contact surface area, following previous studies .

2.6

Dental plaque microcosm biofilm model for enamel demineralization

Human saliva and sucrose were used to establish an in-vitro dental plaque microcosm biofilm model . This model was confirmed to have the capability to produce acidic pH due to polymicrobial biofilm growth and acid production . Saliva was collected from ten healthy adult donors having natural dentition without active caries or periopathology, and without the application of antibiotics within the last 3 months. The donors did not carry out any procedures of oral hygiene for 24 h and abstained from food and drink intake for 2 h prior to donating saliva. An identical volume of saliva from each donor was blended to form the saliva sample. The saliva was then diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C before use .

The culture 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. In each well of 24-well flat-bottomed plates, 1.5 mL of medium with the saliva-glycerol mixture (1:50 of volume) and 0.2% sucrose was added as inoculum. To ensure biofilm standardized growth, the inoculum was first incubated anaerobically at 37 °C for 8 h, then refreshed and incubated for another 16 h, for a total of 24 h. A previous study showed that an acidic environment could be obtained by biofilm metabolism after 24 h incubation . Then, each prepared crown sample was placed into a well with medium for continuous 14 d demineralization, while the medium was changed every 24 h ( Fig. 1 C).

2.7

Polarized-light microscopy (PLM)

After the aforementioned biofilm acid challenge for 14 days, the demineralized samples were taken out and rinsed with deionized water. Each sample was buccolingually sectioned into two equivalent parts using a diamond rim saw ( Fig. 1 D). They were randomly divided into two groups. Group A was used for PLM. Group B was used for Section 2.8 . Samples in group A were dehydrated by gradient ethanol solutions (at 90%, 95% and 100%), and the varnish was rinsed off with acetone. Each sample was embedded and then segmented parallel to the buccal-lingual axis using a hard-tissue cutting system (E300CP, EXAKT, Germany) into a cross-sectional slice and polished with 800- to 4000-grit abrasive papers (Hermes, Hamburg, Germany) using a micro-grinding system (E400CS, EXAKT, Germany). Finally, an 80 to 100 μm thickness slice was derived for lesion depth evaluation using a polarized-light microscope (Axio Scope A1, Zeiss, Germany). Regions of demineralization at 25× magnification were centered and photographed ( Fig. 1 E). For each slice, the lesion depth was measured at three distance ranges, from 50 to 150 μm, 150 to 250 μm, and 250 to 350 μm, respectively, to the margin of the adhesive. The three ranges were marked with the digital segment template lines of isometric widths by Image Pro Plus 6.0 (Media Cybernetic, Silver Spring, MD) software. The average lesion depth for each range was measured as the area of the body of the lesion divided by the width of the template line.

2.8

Cross-sectional hardness testing

Each sample of group B was stick to a glass slide via a thin layer of self-curing resin (Technovit 4000, Heraeus Kulzer GmbH, Germany) using a parallel carrier sheet pressing device (E401, EXAKT, Germany). The cross-sectional surfaces were serially polished to 4000-grit. This process was to ensure that the tested surface was parallel to the platform of the hardness tester (FM-700, Future-Tech, Kawasaki, Japan). A Knoop indenter was used to measure the hardness KHN at a force of 25 g and a dwell time of 5 s. On each sample, three lanes of 24 indentations were made on three equal interval distances, at 100, 200, and 300 μm away from the adhesive margin. Eight indentations for each lane from the external side to the internal side were made at 25, 50, 75, 100, 125, 150, 175, and 200 μm depth ( Fig. 1 F). Another lane of indentations was made on sound enamel beneath the varnish-covered region as the hardness baseline. Integrated demineralization (ΔS) of each sample was calculated by the following steps. First, all the lanes of KHN values in the experimental region and the baseline were obtained. Second, these data were transformed into mineral content percent values that were linearly proportional to the square root of KHN, according to the relation between hardness and transverse microradiography (TMR) . Then, the mineral content percentage was plotted against the depths to obtain the integrated hardness profile. ΔS was computed based on the profile area of the demineralized enamel and subtracted from the area of sound enamel, following a previous study .

2.9

Scanning electron microscopy (SEM)

Samples after hardness testing were removed from glass slides. They were sputter-coated with gold and examined via a scanning electron microscope (SEM, EVO MA25, Zeiss, Oberkochen, Germany). SEM images were obtained at 100 to 300 μm from the adhesive margin.

2.10

Statistical analysis

SBS, PLM and hardness results were statistically examined with SPSS 17.0 for Windows (SPSS Inc. Chicago, IL, USA). The normal distribution assumption and Levene’s homogeneity test were confirmed for the dependent variables. For SBS and lesion depths of PLM, one-way ANOVA was performed. For ΔS and hardness, ANOVA with Tukey post-hoc tests were used. The significance level of p was set at 0.05.

2.1

Incorporation of MPC into RMGI

An RMGI cement (GC Ortho LC, Fuji, Aichi-ken, Japan) consisted of fluoroaluminosilicate glass particles, hydroxyethyl methacrylate (HEMA) monomer and aqueous polyalkenoic acid. A powder/liquid mass ratio of 2.5/1 was used according to the manufacturer. MPC was obtained commercially (Sigma-Aldrich, St. Louis, MO) which was synthesized via an approach by Ishihara et al. . The MPC powder was mixed into RMGI at a MPC/(RMGI + MPC) mass fraction of 3%, following a previous study .

2.2

Incorporation of DMAHDM into RMGI

DMAHDM with an alkyl chain length of 16 was synthesized via a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that the reaction products are generated at 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 and viscous liquid . The DMAHDM was mixed with RMGI at a DMAHDM/(RMGI + DMAHDM) mass fraction of 1.5%, following a previous study .

2.3

Incorporation of NACP into RMGI

NACP were synthesized via a spray-drying technique as described previously . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution to achieve final Ca and P ionic concentrations of 8 mmol/L and 5.333 mmol/L, which led to Ca/P molar mass ratio was 1.5, the same as that for ACP. This solution was sprayed into a heated chamber apparatus, and the dried particles were collected via an electrostatic precipitator (AirQuality, Minneapolis, MN), producing NACP with a mean particle size of 116 nm measured before .

Adding NACP powder into the RMGI paste significantly increased the paste viscosity. Therefore, to maintain a viscosity similar to that of the commercial RMGI, photo-activated resin monomers were added together with NACP into RMGI. The monomers consisted of bisphenol A glycidyl dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) (Esstech, Essington, PA) at a 1:1 mass ratio, with 0.2% camphorquinone and 0.8% ethyl 4-N,Ndimathylaminobenzoate. The BisGMA-TEGDMA resin (referred to as BT) at a BT/(final cement) mass fraction of 15%, as well as NACP at a NACP/(final cement) mass fraction of 20% was mixed to form the BT-NACP paste. Meanwhile, MPC at a MPC/(final cement) mass fraction of 3% and DMAHDM at a DMAHDM/(final cement) mass fraction of 1.5% were mixed into the powder of the RMGI to form the RMGI-MPC-DMAHDM blend. Then, this blend was mixed with the BT-NACP paste to form the final RMGI + MPC + DMAHDM + NACP cement. The percentages of each component were selected following a previous study . This yielded a modified RMGI paste containing BT and NACP that had an acceptable viscosity for the final paste and good enamel bond strength.

2.4

Preparation of orthodontic bracket-enamel bonded samples

Nighty-six freshly extracted, caries-free premolars for orthodontic reasons were collected and randomly assigned to four groups shown in Table 1 (n = 24 per group). The use of extracted human teeth has been approved by the Ethical Committee of Beijing Stomatological Hospital and the Institutional Review Board of University of Maryland in Baltimore. The selection criteria for teeth included intact buccal enamel without WSLs, and without visible cracks or enamel irregularities. Teeth were cleaned and stored in 0.1% thymol solution at 4 °C before use within 1 month. For each group, twenty-four teeth were randomly divided into two groups of 12 teeth each. One group was used for the shear bond strength test, and the other group for the demineralization experiment. The four types of cements are listed below:

• (1)

  Transbond XT control (referred to as TB control);

• (2)

  RMGI (GC Ortho LC, Fuji);

• (3)

  95.5% RMGI + 3% MPC + 1.5% DMAHDM (referred to as “RMGI + MPC + DMAHDM”);

• (4)

  60.5% RMGI + 3% MPC + 1.5% DMAHDM + 20% NACP + 15% BisGMA-TEGDMA (referred to as “RMGI + MPC + DMAHDM + NACP”).

Table 1

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