Antibacterial and remineralizing orthodontic adhesive containing quaternary ammonium resin monomer and amorphous calcium phosphate nanoparticles

Graphical abstract

Abstract

Objectives

To evaluate the bonding performance, antibacterial activity, and remineralization effect on enamel of the orthodontic adhesive containing MAE-DB and NACP.

Methods

Eighty non-carious human premolars were divided into 3 groups: Transbond XT (TB), PEHB + 5% MAE-DB (PD), and PEHB + 40% NACP + 5% MAE-DB (PND). Premolars were bonded with orthodontic brackets, the first subgroup (n = 10) and the second subgroup (n = 10) were subjected to shear bond strength testing after immersed in water for 1 day and in demineralization solution for 28 days respectively and then tested surface roughness, while the third subgroup (n = 6) was used for microhardness evaluation after aged in demineralization solution for 28 days. For each adhesive, fifty disk samples were prepared for antibacterial study. Specimens measuring 12 mm × 2 mm × 2 mm were fabricated for ion release test.

Results

Bond strengths were in the order TB = PND > PND = PD for “1-day in water”, and in the order TB = PND > PD for “28-days in pH 4 solution”. No significant difference in the ARI scores for the three adhesive. Numerous bacteria adhered to TB surface, while PD and PND had minimal bacterial growth and activity. PND showed high levels of Ca and P ions release and enamel hardness. The surface roughness of enamel in PND was much lower than TB and PD and showed no significant difference with the sound, control enamel.

Conclusion

PND adhesive with 5% MAE-DB and 40% NACP exhibits antibacterial and remineralizing capabilities, and did not adversely affect bond strength compared to commercial adhesive.

Clinical significance

Novel adhesive containing quaternary ammonium monomer and nano-amorphous calcium phosphate represents a promising candidate in combating enamel white spot lesions and even dental caries.

Introduction

Formation of enamel white spot lesions around bonded orthodontic brackets is a serious and common complication during orthodontic treatment [ ]. The prevalence of white spot lesions in patients with fixed appliance therapy has been reported to be 73–95% [ ]. Previous studies have shown that the rate of enamel demineralization in orthodontic patients is higher than those without orthodontic treatment [ ]. Placement of fixed appliances increases plaque accumulation around the brackets and colonization of cariogenic bacteria [ ]. Organic acids produced by those bacteria such as lactic, propionic and butyric acids result in surface and subsurface demineralization of the tooth enamel [ ].

Because of the esthetic concerns associated with peri-bracket white spot lesions, continuing efforts have been made to manage these lesions by preventing enamel demineralization or promoting enamel remineralization [ ]. Topical fluoride or fluoride-releasing adhesives and cements have been used to prevent enamel demineralization around orthodontic brackets [ ]. However, fluoride application to enamel prior to the placement of orthodontic brackets renders the enamel more resistant to phosphoric acid-etching. This reduces bonding efficacy and leads to premature bond failure [ ]. Prescription of topical fluoride destined for home-use is usually inefficacious due to low patient compliance [ ]. An alternative preventive approach is based on calcium phosphate (CaP) remineralization [ ]. Calcium (Ca) and phosphate (P) ions released from CaP biomaterials create an ionic reservoir in dental plaques. This reservoir of supersaturated Ca and P ions helps to prevent demineralization and facilitates enamel remineralization [ ].

Resin-based materials containing nanoparticles of amorphous calcium phosphate (NACP) have been developed recently. The NACP-containing materials are capable of inducing enamel remineralization by releasing high levels of Ca and P ions [ ]. Because of the small particle size and high surface area of these nanoparticles, resins that contain NACP have much higher Ca and P ion release than conventional CaP-filled resins [ ]. Compared with a fluoride-releasing composite, NACP-containing resin composite possesses a 4-fold capacity for remineralization of incipient enamel lesions [ ]. The NACP nanocomposite rapidly neutralizes a pH 4 demineralization solution and raises its pH to 5.5 and higher, thereby reducing the rate of enamel demineralization [ ]. Incorporation of up to 40% NACP in a bonding agent has no adverse effect on bond strength [ ]. However, the Ca and P ions release of the conventional CaP and NACP containing resin materials lasted for weeks to months, and then the release would decrease over time [ ]. In our previous studies, novel dental resins containing NACP and PMGDM/EBPADMA were developed with the high capabilities of substantial recharge and sustained long-term release of Ca and P ions [ ]. The rechargeable NACP orthodontic adhesive showed high bracket shear bond strength and Ca and P ion release capability. However, whether the new NACP orthodontic adhesive can inhibit the enamel demineralization as expection is still unknown and need to be studied.

Polymerizable quaternary ammonium salt (QAS) possesses remarkable antimicrobial activities against a wide range of bacteria, fungi and viruses, and is a broad spectrum cationic biocide [ ]. Cationic QAS disrupts the integrity of bacteria by attracting and puncturing negatively-charged bacterial cell membranes [ ]. 2-methacryloxylethyl dodecyl methyl ammonium bromide (MAE-DB) is an example of a polymerizable QAS. The MAE-DB molecule contains two polymerizable methacrylate groups that enables it to co-polymerize with additional MAE-DB monomers and other resin monomers to produce a resin matrix with stable and long-lasting bactericidal effect [ ]. Incorporation of this QAS resin monomer into a resin comonomer blend does not significantly compromise the biocompatibility, mechanical and bonding properties of the polymerized resin matrix [ ].

Because bacteria-derived acids generate an acidic environment that hampers enamel remineralization, MAE-DB was used in conjunction with NACP in the present study to develop an orthodontic adhesive for exerting antibacterial activity and promote remineralization simultaneously, without compromising the quality and longevity of the bonded interface. Accordingly, the objectives of the study were to test the null hypotheses that: 1) there is no difference in resin-enamel bond strength between the experimental MAE-DB and NACP-containing orthodontic adhesive and a commercial orthodontic adhesive that does not contain these components, and 2) the experimental MAE-DB and NACP-containing orthodontic adhesive does not possess significantly better bactericidal properties against Streptococcus mutans biofilms, or significantly better enamel remineralization potential compared with a commercial orthodontic adhesive that does not contain these components.

Materials and methods

Synthesis of NACP

A spray-drying technique was used to fabricate NACP, as previously described [ ]. Calcium carbonate (CaCO 3 ) and dicalcium phosphate anhydrous (CaHPO 4 ) were dissolved in an acetic acid solution to obtain final Ca and PO 4 ionic concentrations of 8 and 5.333 mmol/L, respectively. Formation of NACP was initiated by atomizing a suspension of CaP micro-droplets followed by drying in a heated chamber to generate solid particles. The resulted NACP has a mean particle size of 116 nm, as reported previously [ ].

Orthodontic adhesives

Two experimental orthodontic adhesives were formulated. The first consists of 44.5 wt percent (wt%) pyromellitic glycerol dimethacrylate (PMGDM) (International Laboratory USA, South San Francisco, CA, USA) and 39.5 wt% ethoxylated bisphenol A dimethacrylate (EBPADMA; MilliporeSigma, St. Louis, MO, USA; Table 1 ). Being an acidic adhesive monomer [ ], PMGDM possesses recharging capability by chelating calcium ions from a recharging solution [ ]. Ten wt% 2-hydroxyethyl methacrylate (HEMA; MilliporeSigma) and 5 wt% bisphenol A glycidyl dimethacrylate (BisGMA) (MilliporeSigma) were added to the PMGDM-EBPADMA mixture. These weight fractions were selected because previous studies showed that addition of 10% HEMA and 5% BisGMA into the PMGDM-EBPADMA resin comonomer blend increased its bond strength to dentin [ ]. Benzoyl peroxide (BPO, 0.8 wt%; Alfa Aesar, Heysham, Lancashire, United Kingdom) was added to enable chemical-cure, and camphorquinone (CQ, 0.2 wt%; MilliporeSigma) was added as photoinitiator to provide the capacity for the resin blend to be light-cured. The resin was designated as PEHB. The polymerizable QAS, MAE-DB ( Fig. 1 ), was added to the PEHB at a weight fraction of 5% ( Table 1 ) according to the previous study [ ]. For the second experimental orthodontic adhesive, NACP fillers were added to PEHB at a weight fraction of 40% [ ]. The MAE-DB resin monomer was subsequently added to the filled adhesive mixture at a weight fraction of 5% ( Table 1 ). According to the results of preliminary experiment, NACP filler level > 40% and MAE-DB filler level >5% were not used to avoid the adverse effect on bracket-enamel bond strength.

Table 1
Composition (in wt%) of the experimental adhesives used in the present study.
Adhesives PMGDM EBPADMA HEMA Bis-GMA BPO CQ NACP MAE-DB
44.5 39.5 10 5 0.8 0.2
(100%)
PD 95 0 5
PND 55 40 5
PMGDM: pyromellitic glycerol dimethacrylate; EBPADMA: ethoxylated bisphenol A dimethacrylate; Bis-GMA: bisphenol A glycidyl dimethacrylate; HEMA: 2-hydroxyethyl methacrylate; BPO: benzoyl peroxide; CQ: camphorquinone; NACP: amorphous calcium phosphate nanoparticles; MAE-DB: 2-methacryloxylethyl dodecyl methyl ammonium bromide.

Fig. 1
Structure of 2-methacryloxylethyl dodecyl methyl ammonium bromide (MAE-DB).

Transbond XT Light Cure Adhesive (TB; 3 M Unitek, Monrovia, CA, USA) was used as the control. The adhesive/cement was used in accordance with the manufacturer’s instructions. According to the manufacturer, TB contains silane-treated quartz (70–80 wt%), bisphenol A diglycidyl ether dimethacrylate (10–20 wt%), bisphenol-A-bis (2-hydroxyethyl) dimethacrylate (5–10 wt%), silane-treated silica (< 2 wt%) and diphenyliodonium hexafluorophosphate (< 0.2 wt%).

The three experimental groups were: (1) TB; (2) PEHB + 5% MAE-DB [designated as PD]; and (3) PEHB + 5% MAE-DB + 40% NACP [designated as PND].

Preparation of enamel-bracket bonding specimens

Eighty non-carious human premolars were collected from the clinics of the School of Stomatology, Fourth Military Medical University (FMMU), Xi’an, China. The protocol was approved by the FMMU Institutional Review Board. All experiments were performed in accordance with approved guidelines and regulations. The teeth were disinfected in a 0.005% promodyne solution for 4 h and stored at 4 °C in deionized water. The premolars were randomly distributed among three adhesive groups, each consisting of 26 teeth. The addition two teeth were used as control during morphologic examination.

The buccal enamel surface of each tooth was etched with 35% phosphoric acid (Scotchbond, 3 M ESPE, St. Paul, MN, USA) for 30 s, rinsed with water for 30 s, and dried thoroughly using oil-and-moisture-free air. Prior to bracket placement, a thin uniform coat of Transbond XT primer was applied to the etched surfaces and cured for 10 s. Each adhesive was applied to the base of the bracket (Shinye, Hangzhou, China), which was lightly placed on the tooth surface and pressed firmly into the final position. Excess adhesive around the bracket was removed without disturbing the bracket. The adhesive was polymerized from four sides (occlusal, gingival, mesial and distal) of the bracket for 10 s each, using a light-emission diode curing unit (Elipar S10, 3 M ESPE, St. Paul, MN, USA) operated in the standard curing mode. The light-curing unit was kept approximately 1 mm from the bracket. Bonded specimens were stored in water at 37 °C for 24 h. All the brackets were bonded by an experienced clinician who was blinded with respect to the kind of adhesive utilized during bracket placement.

Aging

The bonded premolars in each adhesive group were divided into 3 subgroups. The first subgroup was subjected to shear bond strength testing after the specimens were immersed in deionized water for 24 h (referred to as 1-day in water, n = 10). The second subgroup (n = 10) was subjected to shear bond strength testing after the specimens were aged in demineralization solution (pH 4) for 28 days. The third subgroup (n = 6) was used for microhardness evaluation after the specimens were aged in demineralization solution (pH 4) for 28 days. The demineralization solution consisted of 3.0 mmol/L CaCl 2 , 1.8 mmol/L KH 2 PO 4 , 0.1 mol/L lactic acid, and 1% carboxymethylcellulose, with pH adjusted to 4 with KOH [ ]. This mimicked the biofilm acids that the orthodontic adhesive-enamel bonded areas would encounter in vivo , which could potentially degrade the resin-enamel bond strength [ ]. Aging in demineralization solution was used to simulate cariogenic challenge on the bonded interface, for evaluating the bond durability and remineralising ability of the experimental adhesives [ ]. This was accomplished by placing each bonded tooth flat in a tube with the buccal surface in contact with the bottom of the tube. The bonded interface was completely immersed in 1.5 mL of the demineralization solution. The solution was changed daily so that the pH of the solution was consistently maintained at 4.

Bracket shear bond testing and adhesive remnant index

Shear bond strengths of the orthodontic brackets to enamel were tested as previously described [ ]. Each tooth was embedded vertically in self-curing acrylic resin (Lang Dental Manufacturing, Wheeling, IL, USA). The buccal axis of the clinical crown was oriented so that the labial surface was parallel to the applied force during bond testing. To measure the shear bond strength, a chisel attached to a Universal Testing Machine (AGS-10kNG, Shimadzu, Japan) was positioned over the upper part of the bracket base and parallel to the bonded interface. An occlusogingival load was applied at a cross-head speed of 0.5 mm/min until the bracket detached [ ].

After the bracket was debonded, each tooth surface was observed under a stereomicroscope (SMZ1500, Nikon, Japan) to examine the failure mode. The adhesive remnant index (ARI) was determined based on the presence of remaining adhesive on enamel. Evaluation was performed using the following criteria [ ]: 0 = no adhesive remaining on enamel; 1 = less than half of the adhesive remaining on enamel; 2 = more than half of the adhesive remaining on enamel; 3 = all the adhesive remaining on enamel.

Antibacterial activity

Experimental adhesives and commercial control adhesive were placed individually into disk-shaped glass molds (inner diameter: 10 mm; depth: 1.5 mm) to prepare polymerized resin disks (n = 15). The top and bottom surfaces of each adhesive were covered with a Mylar strip and a microscope slide; the latter was pressed gently to remove excess material. The adhesives were light-polymerized for 60 s on each side with the light-curing light [ ]. After the adhesive disks were removed from the mold, they were immersed in distilled water with agitation for 1 h to remove any uncured monomer [ ]. The disks were subsequently sterilized with ethylene oxide and degassed in a fume hood for 48 h prior to testing [ ].

Streptococcus mutans (UA159; State Key Laboratory of Military Stomatology, School of Stomatology, FMMU, Xi’an, China) was cultured overnight at 37 °C in Brain Heart Infusion (BHI) broth (Difco, Detroit, MI, USA) in an anaerobic atmosphere enriched with 5% CO 2 . The resulting bacterial suspension was adjusted to an optical density of 0.5 at 600 nm and diluted one hundred folds with fresh BHI for further use [ ].

The sterile resin disks in each adhesive group were equally divided into five subgroups and placed in five 24-well plates containing 2-mL of BHI broth per well. Twenty microliter of the diluted S. mutans suspension was then added to each well. Because of the difference in surface roughness of resin disks prepared from PD with no nanofillers and the nanofiller-containing PND, it was anticipated that the bacterial killing efficacy of the co-polymerized MAE-DB resin in the two adhesives would be masked by the difference in bacteria adherence between the two materials. According, different bacteria culture time-points were used to examine the percentage of live bacteria within biofilms grown on resin disks prepared from the PD and PND groups until no more difference between the two materials could be detected. Accordingly, biofilms were evaluated by confocal laser scanning microscopy after 6, 12, 24, 48, and 72 h of anaerobic culture.

At each designated time-point, the biofilm-coated disks from the 5 subgroups were rinsed with sterile phosphate-buffered saline (PBS) to remove loose bacteria. The resin disks were stained with Live/Dead BacLight Bacterial Viability Kit L13152 (Molecular Probes, Invitrogen, Eugene, OR, USA). With live/dead staining, live bacteria produce green fluorescence upon staining with Syto 9, and bacteria with compromised membranes produce red fluorescence upon staining with propidium iodide. After incubation with the fluorescent dyes, the stained resin disks were rinsed with distilled water and examined with a confocal laser scanning microscope (CLSM; FluoView FV1000, Olympus, Tokyo, Japan). Excitation with a 488-nm laser resulted in green fluorescence emission of live bacteria; excitation with a 543-nm laser resulted in red fluorescence emission of bacteria with damaged membranes. Five 316.8 μm x 316.8 μm images were taken from each disk at random locations (5 images for each adhesive at each culture time-point). The live/dead ratio of biofilm cells was quantified using the Imaris software (Bitplane AG, Zurich, Switzerland).

The remaining 2 resin disks from each subgroup was used to prepared 6-h or 72-h biofilms for scanning electron microscopy. The biofilm-coated disks were gently rinsed with sterile PBS, soaked in 3% glutaraldehyde at 4 °C overnight, washed twice with PBS, dehydrated in a graded series of ethanol solutions, and dried in a critical-point drier. The specimens with sputtered-coated with gold-palladium and examined with a field emission-scanning electron microscope (FE-SEM; S-4800, Hitachi, Tokyo, Japan) at 5 kV.

Ca and P ion release

Sodium chloride solution (133 mmol/L) was buffered to pH 4 with 50 mmol/L lactic acid to measure the ion release, simulating a low pH, cariogenic condition [ ]. Specimens measuring 12 mm × 2 mm × 2 mm were fabricated using the three orthodontic adhesives. Three specimens from each group were immersed in 50 mL of the pH-adjusted NaCl solution to yield a specimen volume/solution of 2.9 mm 3 /mL, based on previous studies [ ]. The concentrations of Ca and P released from the specimens were measured at 1, 3, 5, 7, 14, 21, 28, 35 and 42 days, using a previously-reported protocol [ ]. At each designated time-period, a 0.5 mL aliquot of solution was retrieved from each storage medium and replaced with fresh NaCl solution. The pH of the incubating solutions was monitored and adjusted to pH 4 with 50 mmol/L lactic acid using a combination pH electrode (Orion, Cambridge, MA, USA) [ ]. The retrieved aliquots were analyzed for Ca and P concentrations using an inductively-coupled plasma emission spectrometer (ICP-MS; Model iCAP-6300, Thermo Fisher Scientific, USA) using known standards and calibration curves. Experiments were conducted in triplicates (n = 3).

Microhardness

Microhardness was used an indirect indicator of the extent of enamel demineralization and remineralization. Six teeth from each adhesive group were used for the experiment. Each bonded tooth was placed flat inside a tube with the buccal surface in contact with the bottom of the tube. The bracket-buccal bonded interface was completely immersed in 1.5 mL of demineralization solution (pH 4), while the lingual surface of each bonded tooth was not exposed to the solution. The demineralization solution was changed daily for 28 days prior to retrieval of the specimens for microhardness determination.

An operator who was blinded to the group allocation performed the analysis. Each tooth was hemi-sectioned in a sagittal direction through the center of the bracket into mesial and distal halves, using a low-speed diamond saw (Kejing, Shenyang, China) under water cooling. The sectioned specimens were embedded in self-curing epoxy-resin (EpoKwick, Buehler, Lake Bluff, IL, USA), leaving the cut face exposed. The resin-embedded specimens were polished with 3 grades of abrasive papers (320, 600 and 1200 grit). Final polishing was achieved with 1-μm diameter diamond cream and polishing-cloth disk (Buehler).

Microhardness measurement was conducted using a hardness tester (HXD-1000 TM/LCD, Digital Micro Hardness Tester, Shanghai Taiming Optical Instrument Co. Ltd., Shanghai, China) equipped with a Vickers diamond indenter at a load of 50 gf and a dwell time of 15 s. Four indentations were made in each embedded tooth-half from 4 locations and 2 depths (12 tooth-halves; n = 12). On the buccal surface, indentations were made under the bracket and at the occlusal and cervical edges of the bracket. An additional indentation made at the lingual enamel surface served as the internal control. For each location, two indentations were made at 30 μm and 120 μm from the external enamel surface. The latter was used to represent the part of the tooth enamel that was not affected by surface demineralization.

Surface roughness

After shear bond strength testing, specimens that had been immersed in demineralization solution (pH 4) for 28 days prior to testing were examined by FE-SEM and atomic force microscopy (AFM, Series 5500, Agilent, Santa Clara, CA, USA). Four debonded specimens from the TB, PD and PND groups with less than half of the bonded enamel adhesive covered by adhesive (i.e. ARI score 1) were used for examination. Two additional non-carious premolars were used as control to record the original enamel surface morphology. Each tooth was cut with a low-speed diamond saw into two halves along the mesiodistal plane. The buccal half specimens were ultrasonicated for 15 min to remove debris and dried with nitrogen gas [ ]. Two buccal halves were sputter-coated with gold/palladium for SEM, using the S-4800 FE-SEM at 5 kV. The remaining 2 specimens were used for AFM examination and surface roughness examination. Surface profiling was performed using a tapping model etched silicon probe (Keysight, Böblingen, Germany). Four AFM images were collected from each location of a tooth-half. Surface roughness was determined from the statistical data derived from those images (n = 4 × 2 tooth-halves = 8) and expressed as root mean square height (Sq, in μm).

Statistical analyses

Interval data generated from the experiments were evaluated for their normality (Shapiro-Wilk test) and equal variance assumptions (modified Levene test) prior to the adoption of parametric statistical methods. Statistical significance for all analyses was pre-set set at α = 0.05.

Data derived from bonding of orthodontic brackets to enamel (n = 10) were analyzed with two-factor analysis of variance (ANOVA) to examine the effect of “adhesive” and “aging”, and the interaction of these two factors on shear bond strength. Post-hoc pairwise comparisons were performed using the Holm-Šidák statistic. For analysis of the ARI results (n = 10), the Kruskal-Wallis H test was used to evaluate the scores among the three adhesive subgroups at each time-point; the Wilcoxon signed-rank test was used to compare the scores of each adhesive between the two time-points.

The percentages of live bacteria in biofilms taken with CLSM (n = 5) were analyzed with a two-factor ANOVA design to examine the effect of adhesive (TB, PD or PND) and culture time (6, 12, 24, 48 or 72 h), and the interaction of those 2 factors on antibacterial efficacy. Post-hoc pairwise comparisons were conducted using the Holm-Šidák statistic.

For microhardness evaluation, a 3-factor ANOVA design was originally planned for examining the effect of adhesive (TB, PB or PND), indentation location (occlusal, under bracket, cervical or lingual), the depth from which indentation was made (30 or 120 μm from enamel surface), and the interaction of those 3 factors on microhardness. However, the normality and equal variance assumptions of the data sets were not fulfilled despite different forms of non-linear transformation of the data sets. Accordingly, data derived from the 24 groups (n = 12) were analyzed using Kruskal-Wallis H test and Dunn’s multiple comparisons test.

For surface roughness evaluation, data from the control, TB PB and PND groups (n = 8) were logarithmically-transformed to satisfy the normality and homoscedasticity assumptions for the use of parametric statistical methods. The transformed data sets were analyzed with one-factor ANOVA and the Holm-Šidák method for pairwise comparisons.

Materials and methods

Synthesis of NACP

A spray-drying technique was used to fabricate NACP, as previously described [ ]. Calcium carbonate (CaCO 3 ) and dicalcium phosphate anhydrous (CaHPO 4 ) were dissolved in an acetic acid solution to obtain final Ca and PO 4 ionic concentrations of 8 and 5.333 mmol/L, respectively. Formation of NACP was initiated by atomizing a suspension of CaP micro-droplets followed by drying in a heated chamber to generate solid particles. The resulted NACP has a mean particle size of 116 nm, as reported previously [ ].

Orthodontic adhesives

Two experimental orthodontic adhesives were formulated. The first consists of 44.5 wt percent (wt%) pyromellitic glycerol dimethacrylate (PMGDM) (International Laboratory USA, South San Francisco, CA, USA) and 39.5 wt% ethoxylated bisphenol A dimethacrylate (EBPADMA; MilliporeSigma, St. Louis, MO, USA; Table 1 ). Being an acidic adhesive monomer [ ], PMGDM possesses recharging capability by chelating calcium ions from a recharging solution [ ]. Ten wt% 2-hydroxyethyl methacrylate (HEMA; MilliporeSigma) and 5 wt% bisphenol A glycidyl dimethacrylate (BisGMA) (MilliporeSigma) were added to the PMGDM-EBPADMA mixture. These weight fractions were selected because previous studies showed that addition of 10% HEMA and 5% BisGMA into the PMGDM-EBPADMA resin comonomer blend increased its bond strength to dentin [ ]. Benzoyl peroxide (BPO, 0.8 wt%; Alfa Aesar, Heysham, Lancashire, United Kingdom) was added to enable chemical-cure, and camphorquinone (CQ, 0.2 wt%; MilliporeSigma) was added as photoinitiator to provide the capacity for the resin blend to be light-cured. The resin was designated as PEHB. The polymerizable QAS, MAE-DB ( Fig. 1 ), was added to the PEHB at a weight fraction of 5% ( Table 1 ) according to the previous study [ ]. For the second experimental orthodontic adhesive, NACP fillers were added to PEHB at a weight fraction of 40% [ ]. The MAE-DB resin monomer was subsequently added to the filled adhesive mixture at a weight fraction of 5% ( Table 1 ). According to the results of preliminary experiment, NACP filler level > 40% and MAE-DB filler level >5% were not used to avoid the adverse effect on bracket-enamel bond strength.

Table 1
Composition (in wt%) of the experimental adhesives used in the present study.
Adhesives PMGDM EBPADMA HEMA Bis-GMA BPO CQ NACP MAE-DB
44.5 39.5 10 5 0.8 0.2
(100%)
PD 95 0 5
PND 55 40 5
PMGDM: pyromellitic glycerol dimethacrylate; EBPADMA: ethoxylated bisphenol A dimethacrylate; Bis-GMA: bisphenol A glycidyl dimethacrylate; HEMA: 2-hydroxyethyl methacrylate; BPO: benzoyl peroxide; CQ: camphorquinone; NACP: amorphous calcium phosphate nanoparticles; MAE-DB: 2-methacryloxylethyl dodecyl methyl ammonium bromide.

Fig. 1
Structure of 2-methacryloxylethyl dodecyl methyl ammonium bromide (MAE-DB).

Transbond XT Light Cure Adhesive (TB; 3 M Unitek, Monrovia, CA, USA) was used as the control. The adhesive/cement was used in accordance with the manufacturer’s instructions. According to the manufacturer, TB contains silane-treated quartz (70–80 wt%), bisphenol A diglycidyl ether dimethacrylate (10–20 wt%), bisphenol-A-bis (2-hydroxyethyl) dimethacrylate (5–10 wt%), silane-treated silica (< 2 wt%) and diphenyliodonium hexafluorophosphate (< 0.2 wt%).

The three experimental groups were: (1) TB; (2) PEHB + 5% MAE-DB [designated as PD]; and (3) PEHB + 5% MAE-DB + 40% NACP [designated as PND].

Preparation of enamel-bracket bonding specimens

Eighty non-carious human premolars were collected from the clinics of the School of Stomatology, Fourth Military Medical University (FMMU), Xi’an, China. The protocol was approved by the FMMU Institutional Review Board. All experiments were performed in accordance with approved guidelines and regulations. The teeth were disinfected in a 0.005% promodyne solution for 4 h and stored at 4 °C in deionized water. The premolars were randomly distributed among three adhesive groups, each consisting of 26 teeth. The addition two teeth were used as control during morphologic examination.

The buccal enamel surface of each tooth was etched with 35% phosphoric acid (Scotchbond, 3 M ESPE, St. Paul, MN, USA) for 30 s, rinsed with water for 30 s, and dried thoroughly using oil-and-moisture-free air. Prior to bracket placement, a thin uniform coat of Transbond XT primer was applied to the etched surfaces and cured for 10 s. Each adhesive was applied to the base of the bracket (Shinye, Hangzhou, China), which was lightly placed on the tooth surface and pressed firmly into the final position. Excess adhesive around the bracket was removed without disturbing the bracket. The adhesive was polymerized from four sides (occlusal, gingival, mesial and distal) of the bracket for 10 s each, using a light-emission diode curing unit (Elipar S10, 3 M ESPE, St. Paul, MN, USA) operated in the standard curing mode. The light-curing unit was kept approximately 1 mm from the bracket. Bonded specimens were stored in water at 37 °C for 24 h. All the brackets were bonded by an experienced clinician who was blinded with respect to the kind of adhesive utilized during bracket placement.

Aging

The bonded premolars in each adhesive group were divided into 3 subgroups. The first subgroup was subjected to shear bond strength testing after the specimens were immersed in deionized water for 24 h (referred to as 1-day in water, n = 10). The second subgroup (n = 10) was subjected to shear bond strength testing after the specimens were aged in demineralization solution (pH 4) for 28 days. The third subgroup (n = 6) was used for microhardness evaluation after the specimens were aged in demineralization solution (pH 4) for 28 days. The demineralization solution consisted of 3.0 mmol/L CaCl 2 , 1.8 mmol/L KH 2 PO 4 , 0.1 mol/L lactic acid, and 1% carboxymethylcellulose, with pH adjusted to 4 with KOH [ ]. This mimicked the biofilm acids that the orthodontic adhesive-enamel bonded areas would encounter in vivo , which could potentially degrade the resin-enamel bond strength [ ]. Aging in demineralization solution was used to simulate cariogenic challenge on the bonded interface, for evaluating the bond durability and remineralising ability of the experimental adhesives [ ]. This was accomplished by placing each bonded tooth flat in a tube with the buccal surface in contact with the bottom of the tube. The bonded interface was completely immersed in 1.5 mL of the demineralization solution. The solution was changed daily so that the pH of the solution was consistently maintained at 4.

Bracket shear bond testing and adhesive remnant index

Shear bond strengths of the orthodontic brackets to enamel were tested as previously described [ ]. Each tooth was embedded vertically in self-curing acrylic resin (Lang Dental Manufacturing, Wheeling, IL, USA). The buccal axis of the clinical crown was oriented so that the labial surface was parallel to the applied force during bond testing. To measure the shear bond strength, a chisel attached to a Universal Testing Machine (AGS-10kNG, Shimadzu, Japan) was positioned over the upper part of the bracket base and parallel to the bonded interface. An occlusogingival load was applied at a cross-head speed of 0.5 mm/min until the bracket detached [ ].

After the bracket was debonded, each tooth surface was observed under a stereomicroscope (SMZ1500, Nikon, Japan) to examine the failure mode. The adhesive remnant index (ARI) was determined based on the presence of remaining adhesive on enamel. Evaluation was performed using the following criteria [ ]: 0 = no adhesive remaining on enamel; 1 = less than half of the adhesive remaining on enamel; 2 = more than half of the adhesive remaining on enamel; 3 = all the adhesive remaining on enamel.

Antibacterial activity

Experimental adhesives and commercial control adhesive were placed individually into disk-shaped glass molds (inner diameter: 10 mm; depth: 1.5 mm) to prepare polymerized resin disks (n = 15). The top and bottom surfaces of each adhesive were covered with a Mylar strip and a microscope slide; the latter was pressed gently to remove excess material. The adhesives were light-polymerized for 60 s on each side with the light-curing light [ ]. After the adhesive disks were removed from the mold, they were immersed in distilled water with agitation for 1 h to remove any uncured monomer [ ]. The disks were subsequently sterilized with ethylene oxide and degassed in a fume hood for 48 h prior to testing [ ].

Streptococcus mutans (UA159; State Key Laboratory of Military Stomatology, School of Stomatology, FMMU, Xi’an, China) was cultured overnight at 37 °C in Brain Heart Infusion (BHI) broth (Difco, Detroit, MI, USA) in an anaerobic atmosphere enriched with 5% CO 2 . The resulting bacterial suspension was adjusted to an optical density of 0.5 at 600 nm and diluted one hundred folds with fresh BHI for further use [ ].

The sterile resin disks in each adhesive group were equally divided into five subgroups and placed in five 24-well plates containing 2-mL of BHI broth per well. Twenty microliter of the diluted S. mutans suspension was then added to each well. Because of the difference in surface roughness of resin disks prepared from PD with no nanofillers and the nanofiller-containing PND, it was anticipated that the bacterial killing efficacy of the co-polymerized MAE-DB resin in the two adhesives would be masked by the difference in bacteria adherence between the two materials. According, different bacteria culture time-points were used to examine the percentage of live bacteria within biofilms grown on resin disks prepared from the PD and PND groups until no more difference between the two materials could be detected. Accordingly, biofilms were evaluated by confocal laser scanning microscopy after 6, 12, 24, 48, and 72 h of anaerobic culture.

At each designated time-point, the biofilm-coated disks from the 5 subgroups were rinsed with sterile phosphate-buffered saline (PBS) to remove loose bacteria. The resin disks were stained with Live/Dead BacLight Bacterial Viability Kit L13152 (Molecular Probes, Invitrogen, Eugene, OR, USA). With live/dead staining, live bacteria produce green fluorescence upon staining with Syto 9, and bacteria with compromised membranes produce red fluorescence upon staining with propidium iodide. After incubation with the fluorescent dyes, the stained resin disks were rinsed with distilled water and examined with a confocal laser scanning microscope (CLSM; FluoView FV1000, Olympus, Tokyo, Japan). Excitation with a 488-nm laser resulted in green fluorescence emission of live bacteria; excitation with a 543-nm laser resulted in red fluorescence emission of bacteria with damaged membranes. Five 316.8 μm x 316.8 μm images were taken from each disk at random locations (5 images for each adhesive at each culture time-point). The live/dead ratio of biofilm cells was quantified using the Imaris software (Bitplane AG, Zurich, Switzerland).

The remaining 2 resin disks from each subgroup was used to prepared 6-h or 72-h biofilms for scanning electron microscopy. The biofilm-coated disks were gently rinsed with sterile PBS, soaked in 3% glutaraldehyde at 4 °C overnight, washed twice with PBS, dehydrated in a graded series of ethanol solutions, and dried in a critical-point drier. The specimens with sputtered-coated with gold-palladium and examined with a field emission-scanning electron microscope (FE-SEM; S-4800, Hitachi, Tokyo, Japan) at 5 kV.

Ca and P ion release

Sodium chloride solution (133 mmol/L) was buffered to pH 4 with 50 mmol/L lactic acid to measure the ion release, simulating a low pH, cariogenic condition [ ]. Specimens measuring 12 mm × 2 mm × 2 mm were fabricated using the three orthodontic adhesives. Three specimens from each group were immersed in 50 mL of the pH-adjusted NaCl solution to yield a specimen volume/solution of 2.9 mm 3 /mL, based on previous studies [ ]. The concentrations of Ca and P released from the specimens were measured at 1, 3, 5, 7, 14, 21, 28, 35 and 42 days, using a previously-reported protocol [ ]. At each designated time-period, a 0.5 mL aliquot of solution was retrieved from each storage medium and replaced with fresh NaCl solution. The pH of the incubating solutions was monitored and adjusted to pH 4 with 50 mmol/L lactic acid using a combination pH electrode (Orion, Cambridge, MA, USA) [ ]. The retrieved aliquots were analyzed for Ca and P concentrations using an inductively-coupled plasma emission spectrometer (ICP-MS; Model iCAP-6300, Thermo Fisher Scientific, USA) using known standards and calibration curves. Experiments were conducted in triplicates (n = 3).

Microhardness

Microhardness was used an indirect indicator of the extent of enamel demineralization and remineralization. Six teeth from each adhesive group were used for the experiment. Each bonded tooth was placed flat inside a tube with the buccal surface in contact with the bottom of the tube. The bracket-buccal bonded interface was completely immersed in 1.5 mL of demineralization solution (pH 4), while the lingual surface of each bonded tooth was not exposed to the solution. The demineralization solution was changed daily for 28 days prior to retrieval of the specimens for microhardness determination.

An operator who was blinded to the group allocation performed the analysis. Each tooth was hemi-sectioned in a sagittal direction through the center of the bracket into mesial and distal halves, using a low-speed diamond saw (Kejing, Shenyang, China) under water cooling. The sectioned specimens were embedded in self-curing epoxy-resin (EpoKwick, Buehler, Lake Bluff, IL, USA), leaving the cut face exposed. The resin-embedded specimens were polished with 3 grades of abrasive papers (320, 600 and 1200 grit). Final polishing was achieved with 1-μm diameter diamond cream and polishing-cloth disk (Buehler).

Microhardness measurement was conducted using a hardness tester (HXD-1000 TM/LCD, Digital Micro Hardness Tester, Shanghai Taiming Optical Instrument Co. Ltd., Shanghai, China) equipped with a Vickers diamond indenter at a load of 50 gf and a dwell time of 15 s. Four indentations were made in each embedded tooth-half from 4 locations and 2 depths (12 tooth-halves; n = 12). On the buccal surface, indentations were made under the bracket and at the occlusal and cervical edges of the bracket. An additional indentation made at the lingual enamel surface served as the internal control. For each location, two indentations were made at 30 μm and 120 μm from the external enamel surface. The latter was used to represent the part of the tooth enamel that was not affected by surface demineralization.

Surface roughness

After shear bond strength testing, specimens that had been immersed in demineralization solution (pH 4) for 28 days prior to testing were examined by FE-SEM and atomic force microscopy (AFM, Series 5500, Agilent, Santa Clara, CA, USA). Four debonded specimens from the TB, PD and PND groups with less than half of the bonded enamel adhesive covered by adhesive (i.e. ARI score 1) were used for examination. Two additional non-carious premolars were used as control to record the original enamel surface morphology. Each tooth was cut with a low-speed diamond saw into two halves along the mesiodistal plane. The buccal half specimens were ultrasonicated for 15 min to remove debris and dried with nitrogen gas [ ]. Two buccal halves were sputter-coated with gold/palladium for SEM, using the S-4800 FE-SEM at 5 kV. The remaining 2 specimens were used for AFM examination and surface roughness examination. Surface profiling was performed using a tapping model etched silicon probe (Keysight, Böblingen, Germany). Four AFM images were collected from each location of a tooth-half. Surface roughness was determined from the statistical data derived from those images (n = 4 × 2 tooth-halves = 8) and expressed as root mean square height (Sq, in μm).

Statistical analyses

Interval data generated from the experiments were evaluated for their normality (Shapiro-Wilk test) and equal variance assumptions (modified Levene test) prior to the adoption of parametric statistical methods. Statistical significance for all analyses was pre-set set at α = 0.05.

Data derived from bonding of orthodontic brackets to enamel (n = 10) were analyzed with two-factor analysis of variance (ANOVA) to examine the effect of “adhesive” and “aging”, and the interaction of these two factors on shear bond strength. Post-hoc pairwise comparisons were performed using the Holm-Šidák statistic. For analysis of the ARI results (n = 10), the Kruskal-Wallis H test was used to evaluate the scores among the three adhesive subgroups at each time-point; the Wilcoxon signed-rank test was used to compare the scores of each adhesive between the two time-points.

The percentages of live bacteria in biofilms taken with CLSM (n = 5) were analyzed with a two-factor ANOVA design to examine the effect of adhesive (TB, PD or PND) and culture time (6, 12, 24, 48 or 72 h), and the interaction of those 2 factors on antibacterial efficacy. Post-hoc pairwise comparisons were conducted using the Holm-Šidák statistic.

For microhardness evaluation, a 3-factor ANOVA design was originally planned for examining the effect of adhesive (TB, PB or PND), indentation location (occlusal, under bracket, cervical or lingual), the depth from which indentation was made (30 or 120 μm from enamel surface), and the interaction of those 3 factors on microhardness. However, the normality and equal variance assumptions of the data sets were not fulfilled despite different forms of non-linear transformation of the data sets. Accordingly, data derived from the 24 groups (n = 12) were analyzed using Kruskal-Wallis H test and Dunn’s multiple comparisons test.

For surface roughness evaluation, data from the control, TB PB and PND groups (n = 8) were logarithmically-transformed to satisfy the normality and homoscedasticity assumptions for the use of parametric statistical methods. The transformed data sets were analyzed with one-factor ANOVA and the Holm-Šidák method for pairwise comparisons.

Only gold members can continue reading. Log In or Register to continue

Jun 17, 2018 | Posted by in General Dentistry | Comments Off on Antibacterial and remineralizing orthodontic adhesive containing quaternary ammonium resin monomer and amorphous calcium phosphate nanoparticles
Premium Wordpress Themes by UFO Themes