White spot lesions often occur in orthodontic treatments. The objective of this study was to develop a novel resin-modified glass ionomer cement (RMGI) as an orthodontic cement with protein-repellent, antibacterial and remineralization capabilities.
Protein-repellent 2-methacryloyloxyethyl phosphorylcholine (MPC), antibacterial dimethylaminohexadecyl methacrylate (DMAHDM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP) were incorporated into a RMGI. Enamel shear bond strength (SBS) was determined. Calcium (Ca) and phosphate (P) ion releases were measured. Protein adsorption onto specimens was determined by a micro bicinchoninic acid method. A dental plaque microcosm biofilm model was tested.
Increasing the NACP filler level increased the Ca and P ion release. Decreasing the solution pH increased the ion release. Incorporating MPC into RMGI reduced protein adsorption, which was an order of magnitude less than that of commercial controls. Adding DMAHDM and NAg into RMGI yielded a strong antibacterial function, greatly reducing biofilm viability and acid production. Biofilm CFU counts on the multifunctional orthodontic cement were 3 orders of magnitude less than that of commercial control ( p < 0.05). These benefits were achieved without compromising the enamel shear bond strength ( p > 0.1).
A novel multifunctional orthodontic cement was developed with strong antibacterial and protein-repellent capabilities for preventing enamel demineralization.
The new cement is promising to prevent white spot lesions in orthodontic treatments. The method of incorporating four bioactive agents may have wide applicability to the development of other bioactive dental materials to inhibit caries.
Orthodontic treatments with fixed appliances often cause white spot lesions in enamel, resulting from local biofilm accumulation and acid demineralization . Fluoride (F) therapy via fluoridated dentifrices, fluoridated oral rinses and topical F application can be a valuable method for preventing white spot lesions . However, these measures depend on patient compliance and therefore are unreliable . Hence, preventive measures that do not depend on patient compliance would be more effective. Various resin-based materials have been used in dentistry in composites, cements and adhesives . Resin-modified glass ionomer cements (RMGIs) possess desirable F-releasing capability and clinically acceptable enamel bond strengths, therefore have been used as orthodontic cements . However, white spot lesions around orthodontic brackets are still common, jeopardizing the health and esthetics of the teeth, and indicating that F alone cannot prevent enamel demineralization . Hence, it is beneficial to incorporate antimicrobial agents into RMGI to enhance the antibacterial ability to reduce biofilm acids .
Quaternary ammonium methacrylates (QAMs) are effective anti-biofilm agents . QAMs have been incorporated into dental materials including composites, bonding agents and RMGI, achieving strong antibacterial functions . The antibacterial potency of QAMs was shown to increase with increasing the alkyl chain length (CL) of the ammonium groups . Recently, dimethylaminohexadecyl methacrylate (DMAHDM) with a CL of 16 was synthesized and incorporated into composites and bonding agents, producing a strong antibacterial activity . However, the antibacterial activity of QAM in an orthodontic cement is limited to the material’s surface due to the “contact-inhibition” mechanism . It is beneficial for the orthodontic cement to kill not only the biofilms on its surface, but also the nearby biofilms away from its surface, because plaque buildup in the vicinity of the bracket can still produce acids to cause white spot lesions. Silver (Ag) is antibacterial against a wide range of microorganisms . Previous studies developed dental resins containing nanoparticles of silver (NAg) with a potent antibacterial activity . NAg resins can inhibit biofilms away from the resin surface due to the release of Ag ions . NAg in a RMGI indeed inhibited biofilms away from its surface, without compromising the color of RMGI because of the low NAg concentration . Therefore, the present study combined DMAHDM with NAg in RMGI for the first time as an orthodontic cement to enhance its antibacterial potency and inhibit adherent biofilms as well as biofilms away from its surface.
Another approach is to develop protein-repellent materials. RMGIs accumulated more bacteria than other orthodontic cements, due to the relatively rough surfaces, high surface-free energy and polarity of RMGIs . Salivary proteins are known to act as receptors for bacterial adhesion . Hence, it would be desirable to develop a RMGI that can repel protein adsorption, thereby reducing bacterial adhesion. 2-methacryloyloxyethyl phosphorylcholine (MPC) is a methacrylate with a phospholipid polar group in the side chain . It was found that MPC could resist protein adsorption and bacterial adhesion . Recently, MPC was incorporated into dental composite, dentin bonding agent and RMGI, achieving a strong protein-repellent ability. 15,16,28
Previous studies showed that RMGI had little demineralization-inhibiting effect, because the low-pH environment due to oral biofilms inhibits the remineralization . Therefore, it is beneficial to incorporate remineralization agents into RMGI to enhance the remineralization capability of RMGI. Nanoparticles of amorphous calcium phosphate (NACP) were synthesized and incorporated into composites and adhesives . These NACP composites and adhesives showed effective remineralization and caries-inhibition . However, there has been no report on the incorporation of NACP into a RMGI. Furthermore, there has been no report on the development of a protein-repellent, antibacterial and remineralizing RMGI as an orthodontic cement for preventing white spot lesions.
The objective of this study was to develop a novel RMGI orthodontic cement with protein-repellent, antibacterial and remineralization capabilities for the first time. The following hypotheses were tested: (1) Incorporation of MPC, DMAHDM, NAg, and NACP into RMGI would not compromise the enamel bond strength; (2) RMGI containing NACP would be “smart” and could increase the calcium and phosphate ion release at cariogenic pH, when these ions would be most needed to combat demineralization; and (3) RMGI containing MPC, DMAHDM, NAg, and NACP would greatly reduce protein adsorption, biofilm growth and lactic acid production, compared to commercial orthodontic cements.
Materials and methods
MPC incorporation into RMGI
A RMGI (Vitremer, 3 M, St. Paul, MN; referred to as VT) consisted of fluoroaluminosilicate glass particles and a light-sensitive, aqueous polyalkenoic acid. Indications include Class III, V and root-caries restoration, Class I and II in primary teeth, and core-buildup. A powder/liquid mass ratio of 2.5/1 was used according to the manufacturer. VT was selected because RMGIs have been used as orthodontic bracket-bonding cements . The purpose was to investigate a model system, and then the method of incorporating MPC, DMAHDM, NAg, and NACP can be applied to other orthodontic cements.
MPC was obtained commercially (Sigma-Aldrich, St. Louis, MO) which was synthesized via a method reported by Ishihara et al. The MPC powder was mixed with VT at MPC/(VT+MPC) mass fraction of 3%. A previous study showed that the incorporation of 3% MPC yielded a strong protein-repellent capability without compromising the enamel bond strength. 28
NAg incorporation into RMGI
Silver 2-ethylhexanoate (Strem, Newburyport, MA) of 0.1 g was dissolved into 0.9 g of 2-( tert -butylamino)ethyl meth-acrylate (TBAEMA, Sigma-Aldrich) . TBAEMA improved the solubility by forming Ag-N bonds with Ag ions to facilitate the Ag salt to dissolve in the resin solution . TBAEMA contains reactive methacrylate groups which can be chemically bonded in the resin upon photo polymerization. The Ag solution was incorporated into VT at a silver 2-ethylhexanoate/(VT + silver 2-ethylhexanoate) mass fraction of 0.1%. This mass fraction was selected based on previous studies showing a strong antibacterial activity without compromising mechanical properties .
DMAHDM incorporation into RMGI
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, St. Louis MO) 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 to yield DMAHDM . The DMAHDM was mixed with VT at a DMAHDM/(VT + DMAHDM) mass fraction of 1.5%. DMAHDM mass fractions ≥ 2% were not used due to a decrease in the enamel bond strength when DMAHDM was combined with 3% MPC and 0.1% NAg in RMGI in preliminary study .
NACP incorporation into RMGI
NACP was synthesized using a spray-drying technique . Briefly, calcium carbonate and dicalcium phosphate were dissolved in acetic acid to produce Ca and P concentrations of 8 mmol/L and 5.333 mmol/L, respectively. The Ca/P molar ratio for the solution was 1.5, the same as that for ACP. The solution was sprayed into a heated chamber of a spray-drying apparatus. The dried particles were collected via an electrostatic precipitator (Air Quality, Minneapolis, MN), yielding NACP with a mean particle size of 116 nm.
To mix NACP into RMGI, a resin was added to RMGI so that the mixed paste was cohesive and not dry. The resin consisted of BisGMA (bisphenol A glycidyl dimethacrylate) and TEGDMA (triethylene glycol dimethacrylate) (Esstech, Essington, PA) at a mass ratio of 1:1, which was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-N,Ndimethylaminobenzoate (mass fractions). This photo-activated BisGMA-TEGDMA resin (referred to as BT) was added into at a BT/(VT + BT) mass fraction of 15%. NACP was incorporated at NACP/(VT + NACP) mass fractions of 10%, 15%, and 20%, respectively. NACP mass fractions > 20% were not used because the cement paste became relatively dry.
Unmodified VT served as a control. Another orthodontic cement (Transbond XT, 3 M, Monrovia, CA) served as another control (referred to as TB control). According to the manufacturer, TB consisted of silane-treated quartz (70–80% by weight), bisphenol-A-diglycidyl ether dimethacrylate (10–20%), bisphenol-A-bis (2-hydroxyethyl) dimethacrylate (5–10%), silane-treated silica (< 2%) and diphenyliodonium hexafluorophosphate (<0.2%). TB was used to provide a higher end of the enamel bond strength range in orthodontic applications . VT was used as a control with a medium and acceptable bond strength for bonding orthodontic brackets to enamel. MPC, DMAHDM, NAg and NACP were incorporated into VT, but not into TB, for the purpose of formulating a new orthodontic cement with both fluoride release as well as protein-repellent, antibacterial and remineralizing capabilities. The following six groups were tested:
Transbond XT control (referred to as TB control);
Vitremer control (referred to as VT control);
95.4% Vitremer + 3% MPC + 1.5% DMAHDM + 0.1% NAg (referred to as “VT + MPC + DMAHDM + NAg”).
70.4% Vitremer + 3% MPC + 1.5% DMAHDM + 0.1% NAg + 10% NACP + 15% BisGMA-TEGDMA (referred to as “VT + MPC + DMAHDM + NAg + 10NACP”);
65.4% Vitremer + 3% MPC + 1.5% DMAHDM + 0.1% NAg + 15% NACP + 15% BisGMA-TEGDMA (referred to as “VT + MPC + DMAHDM + NAg + 15NACP”);
60.4% Vitremer + 3% MPC + 1.5% DMAHDM + 0.1% NAg + 20% NACP + 15% BisGMA-TEGDMA (referred to as “VT + MPC + DMAHDM + NAg + 20NACP”).
Enamel shear bond strength (SBS) and adhesive remnant index (ARI)
One hundred and twenty extracted human maxillary first premolars were randomly divided into 6 groups of 20 teeth each. The protocol of human teeth collection was approved by the University of Maryland Baltimore Institutional Review Board. The criteria for tooth selection included intact buccal enamel that had no visible cracks and no enamel irregularities . The teeth were cleaned and polished with a fluoride-free pumice slurry and rubber cups for 10 s, thoroughly washed, and then dried with an oil-free air stream . Each tooth was embedded vertically in a self-curing acrylic resin (Lang Dental, Wheeling, IL) taking into account the buccal axis of the clinical crown, so that the labial surface would be parallel to the force during the shear bond testing. Premolar metal orthodontic brackets (Ormco 2000, Sybron Dental, Orange, CA) were used. Group 1 samples were bonded following the manufacturers’ recommendations. Enamel was etched for 30 s with 37% phosphoric acid (Scotchbond, 3M ESPE, St. Paul, MN) and then rinsed for 10 s. The tooth was dried with a stream of air until a chalky white appearance was observed . Transbond XT primer was applied to the etched surface. Then, Transbond XT light-cured adhesive paste was applied to the bracket base and pushed against the enamel surface. A bracket placement plier was used to hold and keep the bracket in position on the center of the enamel surface. A 300-g force was applied vertically on the bracket for 5 s using a force gauge (Correx, Bern, Switzerland) to ensure a uniform bonding pressure and adhesive thickness . Excess adhesive around the bracket base was removed with a clinical probe. Then the specimen was photo-cured (VCL 401, Demetron, CA) for a total of 40 s. The curing light was held against the bracket and the tooth on the mesial aspect for 20 s, followed by 20 s against the distal aspect at a distance of 3 mm and a 45° angle to the enamel surface .
For groups 2–6, RMGI can be used for bonding brackets without acid etching, according to the manufacturer as well as literature . Hence, the bonding procedures consisted of pumicing the enamel surface for 10 s with flour pumice, followed by a rinse of 10 s with water. Each tooth was then wiped with a moist cotton roll to ensure that the bonding surface was not desiccated, and excess water was removed . RMGI paste was applied to the bracket base and the bracket was positioned and bonded to the enamel. The bracket was then light-cured for a total of 40 s as described above.
Samples of each group were randomly divided into 2 subgroups of 10 samples each. They were stored in distilled water at 37 °C for 1 d or 30 d, respectively. Then they were used for shear bond testing. A chisel was connected with a computer-controlled Universal Testing Machine (MTS, Eden Prairie, MN) and the chisel tip was positioned on the upper part of the bracket base. An occlusal-gingival load was applied to the bracket at a displacement rate of 0.5 mm/min until the bond failed . The enamel shear bond strength SBS = the debonding force/bracket contact surface area, following previous studies .
After debonding, each enamel surface was observed via a stereomicroscope (Zoom 2000, Leica, Wetzlar, Germany). The ARI scores were assessed as previously defined. . These scores quantified the remnants of resin on enamel . The following scores were used. 0 = no adhesive remained on enamel. 1 = less than half of the bonding area on enamel was covered with adhesive. 2 = more than half of the bonding area on enamel was covered with adhesive. 3 = the entire bonding area on enamel was covered with adhesive.
Calcium (Ca) and phosphate (P) ion release measurement
Groups 4–6 contained NACP and were tested for Ca and P ion release vs . NACP filler level. Each cement paste of was placed into a rectangular mold of 2 × 2 × 12 mm . The specimen was photo-cured for 1 min on each open side of the mold and then stored at 37 °C for 1 d. A sodium chloride (NaCl) solution (133 mmol/L) was buffered to three different pH: pH 4 with 50 mmol/L lactic acid, pH 5.5 with 50 mmol/L acetic acid, and pH 7 with 50 mmol/L HEPES . Three specimens were immersed in 50 mL of solution at each pH, yielding a specimen volume/solution of 2.9 mm 3 /mL, following previous studies . This was similar to a specimen volume per solution of nearly 3.0 mm 3 /mL in a previous study . For each solution, the concentrations of Ca and P ions released from the specimens were measured at 1, 3, 7, 14, 21, and 28 d . At each time, aliquots of 0.5 mL were removed and replaced by fresh solution. The aliquots were analyzed for Ca and P ion concentrations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves following previous studies .
Measurement of protein adsorption
All six groups were tested for protein adsorption and biofilm growth. Each paste was placed into a disk mold of 9 mm in diameter and 2 mm in thickness and light-cured for 40 s on each open side of the mold . The 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 previous studies . The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC) and de-gassed for 3 d .
Protein adsorption onto the disks was determined via a micro bicinchoninic acid (BCA) method . Each disk was immersed in phosphate buffered saline (PBS) for 2 h. The disks then were immersed in a bovine serum albumin (BSA) (Sigma-Aldrich) solution at 37 °C for 2 h, which contained BSA at a concentration of 4.5 g/L following a previous study . 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) at 1 wt% in PBS, and sonicated at room temperature for 20 min to completely detach the BSA from disk surfaces. A protein analysis kit (micro BCA protein assay kit, Fisher Scientific, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. From the protein concentration, the amount of protein adsorbed onto the cement disk was calculated . Six disks were evaluated for each group.
Saliva collection and dental plaque microcosm biofilm model
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 ten healthy adult donors having natural dentition without active caries or periopathology, and without the use of antibiotics within the last 3 months, following previous studies . 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 parafilm chewing and was kept on ice. 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 .
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 . An inoculum of 1.5 mL was added to each well of 24-well plates containing a cement 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. At 16 h, the disks were transferred to new 24-well plates with fresh medium and incubated for 24 h. This totals 48 h of incubation, which was adequate to form microcosm biofilms as shown previously .
Live/dead staining of biofilms
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, and 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). Six specimens were evaluated for each group. Three randomly-chosen fields of view were photographed for each disk, yielding a total of 18 images for each group.
MTT assay of metabolic activity
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. A total of 1 mL of MTT was added to each well and incubated for 1 h. 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 DMSO solution was collected from each well, and its absorbance at 540 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk .
Lactic acid production
Disks with 2-day biofilms were rinsed with cysteine peptone water (CPW) to remove loose bacteria and placed in a new 24-well plate . An aliquot of 1.5 mL of buffered peptone water (BPW) supplemented with 0.2% sucrose was added to each well. Samples were incubated at 37 °C in 5% CO 2 for 3 h to allow the bacteria to produce acid. Lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method . The microplate reader was used to measure the absorbance at 340 nm for the collected BPW solutions. Standard curves were prepared using a lactic acid standard (Supelco Analytical, Bellefonte, PA) .
Colony-forming unit (CFU) counts
Disks with 2-day biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing (Fisher, Pittsburgh, PA) . Three types of agar plates were used to measure the CFU counts. First, tryptic soy blood agar culture plates were used to determine total microorganisms . Second, mitis salivarius agar (MSA) culture plates containing 15% sucrose were used to determine total streptococci . Third, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine mutans streptococci . The bacterial suspensions were serially diluted and spread onto agar plates for CFU analysis .
One-way analysis of variance (ANOVA) was performed to detect the significant effects of the different orthodontic cements on protein-repellent and antibacterial properties. Two-way ANOVA was performed to detect the significant effects of different orthodontic cements and water-aging time on SBS, as well as the effects of NACP filler level and pH value on Ca and P ion release. Tukey’s multiple comparison test was used to compare the data at a p value of 0.05. The chi-square test was used to evaluate the ARI scores.