Novel protein-repellent and biofilm-repellent orthodontic cement containing 2-methacryloyloxyethyl phosphorylcholine Ning Zhang,1,2 Ke Zhang,1,2 Mary Anne S. Melo,1 Chen Chen,1,3 Ashraf F. Fouad,1 Yuxing Bai,2 Hockin H. K. Xu1,4,5,6 1
Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, Maryland 21201 2 Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China 3 State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China 4 Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201 5 Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201 6 Department of Mechanical Engineering, University of Maryland, Baltimore County, Maryland 21250 Received 26 December 2014; revised 3 March 2015; accepted 9 April 2015 Published online 13 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33444 Abstract: The objectives of this study were to develop the first protein-repellent resin-modified glass ionomer cement (RMGI) by incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC) for orthodontic applications, and to investigate the MPC effects on protein adsorption, biofilm growth, and enamel bond strength. MPC was incorporated into RMGI at 0% (control), 1.5%, 3%, and 5% by mass. Specimens were stored in water at 378C for 1 and 30 days. Enamel shear bond strength (SBS) was measured, and the adhesive remnant index (ARI) scores were assessed. Protein adsorption onto the specimens was determined by a micro bicinchoninic acid method. A dental plaque microcosm biofilm model with human saliva as inoculum was used. The results showed that adding 3% of MPC into RMGI did not significantly reduce the SBS (p > 0.1). There was no significant loss in SBS for RMGI containing 3% MPC after water-aging for 30 days, as compared to 1 day (p > 0.1). RMGI with 3% MPC had protein
adsorption that was 1/10 that of control. RMGI with 3% MPC greatly reduced the bacterial adhesion, and lactic acid production and colony-forming units of biofilms, while substantially increasing the medium solution pH containing biofilms. The protein-repellent and biofilm-repellent effects were not decreased after water-aging for 30 days. In conclusion, the MPC-containing RMGI is promising to reduce biofilms and white spot lesions without compromising orthodontic bracket-enamel bond strength. The novel protein-repellent method may have applicability to other orthodontic cements, dental composites, adhesives, sealants, and cements to repel C 2015 Wiley Periodicals, Inc. J Biomed proteins and biofilms. V Mater Res Part B: Appl Biomater, 104B: 949–959, 2016.
Key Words: protein repellent, bacteria repellent, orthodontic cement, shear bond strength, human saliva microcosm biofilm, white spot lesions
How to cite this article: Zhang N, Zhang K, Melo MAS, Chen C, Fouad AF, Bai Y, Xu HHK. 2016. Novel protein-repellent and biofilm-repellent orthodontic cement containing 2-methacryloyloxyethyl phosphorylcholine. J Biomed Mater Res Part B 2016:104B:949–959.
INTRODUCTION
The requirement for orthodontic therapy is increasing as more people pursue orthodontic treatments around the world.1 While orthodontic materials and therapy methods have advanced, the placement of fixed appliances tend to accumulate dental plaque biofilms and make oral hygiene more difficult.2,3 The fixed appliances can lead to specific changes in the oral environment, such as the greater accumulation of microorganisms, the formation of biofilms, and local acidic pH.3,4 The level of Streptococcus mutans (S. mutans) and Lactobacilli in the oral cavity can be elevated due to the
increased biofilm formation.4,5 The resulting lower pH of the biofilms on enamel surfaces adjacent to fixed appliances results in demineralization. Indeed, enamel demineralization and white spot lesions were observed around orthodontic appliances.6 Although, several approaches were attempted to prevent white spot lesions,7 recent studies indicated that 50– 70% of patients had white spot lesions after fixed orthodontic therapy,8,9 and white spot lesions were the primary undesired side-effect of fixed orthodontic treatments.6–9 Oral hygiene, fluoride regimens, and dietary control have been generally practiced for preventing white spot lesions
Correspondence to: H. Xu; e-mail: [email protected] or Y. Bai; e-mail: [email protected] Contract grant sponsor: School of Stomatology at the Capital Medical University in China (NZ); contract grant number: NIH R01 DE17974 Contract grant sponsor: Seed Grant, the University of Maryland School of Dentistry
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during fixed orthodontic treatments.6,7 However, these preventive methods mainly depend on patient compliance, and hence, are not reliable.8,9 Preventive strategies which do not require patient compliance might be more effective. Recently, resin-modified glass ionomer cements (RMGIs) have been used as orthodontic bracket-bonding adhesives due to their fluoride-releasing ability and clinically acceptable bond strengths.6–11 RMGIs possess stronger bond strengths than conventional glass ionomer cements, and RMGIs were observed to be more effective than other orthodontic adhesives for preventing white spot lesions around orthodontic brackets.6–11 However, the use of RMGIs as orthodontic cements has yielded conflicting results. It was reported that RMGIs remaining around the brackets could be a great predisposing factor for white spot lesions, because the rough surfaces could provide suitable environments for bacterial adhesion.5 It was found that S. mutans adhered significantly more to RMGIs than to resin composites.5 In addition, gaps of 10 lm wide were found at the adhesive-enamel junctions around the bracket base, which could harbor bacteria and facilitate biofilm formation.12 Furthermore, previous studies reported that RMGIs had little demineralization-inhibiting effect, as the low-pH environment hinders the remineralization process.13,14 For these reasons, it was suggested that incorporation of antimicrobials into RMGIs could enhance the antibacterial effect.14,15 Various agents such as nanosilver,16 bioactive glass,17 and nanohydroxyapatite18 were incorporated into orthodontic adhesives. A key step in white spot lesion formation during orthodontic treatments is the initial bacterial adhesion around the brackets.19 The subsequent growth of these bacteria may form dental plaque biofilms with organic acids to cause enamel demineralization.7,8,20,21 Various factors are considered to affect bacterial adhesion to material surfaces.21 An initial salivary protein coating is first needed for subsequent bacterial22 and fungal23 adherence to teeth and to materials in the oral cavity. The adsorption of salivary proteins onto teeth and material surfaces can produce a biologic layer, which is termed the acquired pellicle.21–23 This salivary pellicle provides the essential receptors allowing the adherence of bacteria.21–23 Hence, the adsorption of salivary proteins is a prerequisite for bacterial attachment and biofilm formation. Indeed, several studies showed that salivary proteins allowed the adhesion of bacteria, including S. mutans, Porphyromonas gingivalis, and Actinomyces species, in both in vivo and in vitro situations.22–24 Therefore, it would be highly desirable to develop a protein-repellent RMGI, to inhibit protein adsorption and thereby to inhibit bacterial adhesion at the bracket-adhesive-enamel junction, thus reducing or eliminating white spot lesions around the brackets. However, to date, there has been no report on protein-repellent RMGIs. It has been demonstrated that hydrophilic material surfaces are usually more resistant to protein adsorption than hydrophobic surfaces.21 2-methacryloyloxyethyl phosphorylcholine (MPC) is a methacrylate with a phospholipid polar group in the side chain, and is one of the most common
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biocompatible and hydrophilic biomedical polymers.25,26 Previous studies found that the MPC has excellent ability to reduce protein adsorption and bacterial adhesion.26,27 Several medical devices with MPC have been developed and used clinically, such as artificial blood vessels, implantable artificial hearts, and artificial lungs.27,28 Recently, MPC was incorporated into a dentin bonding agent, achieving a strong proteinrepellent ability.29 However, the incorporation of MPC into RMGIs remains to be investigated. Accordingly, the objectives of this study were to develop novel protein-repellent and fluoride-releasing orthodontic cement by incorporating MPC into a RMGI, and to investigate the effects of MPC mass fraction in RMGI on enamel bond strength, protein adsorption, and biofilm activity for the first time. It was hypothesized that: (1) incorporating MPC into RMGI would not compromise the enamel bond strength; (2) MPC-containing RMGI would have much less protein adsorption than that of commercial control; and (3) MPC-containing RMGI would greatly reduce biofilm growth than commercial control.
MATERIALS AND METHODS
Fabrication of MPC-containing RMGI Vitremer (3M, St. Paul, MN) was used as the parent system to develop a MPC-containing RMGI for protein-repellent properties. Vitremer consisted of fluoroaluminosilicate glass and a light-sensitive, aqueous polyalkenoic acid. According to the manufacturer, 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 following the manufacturer’s instructions. The purpose was to investigate a model system, and then the method of incorporating MPC to achieve protein-repellent capability can be applied to other RMGIs and other types of orthodontic cements. MPC was obtained from Sigma-Aldrich (St. Louis, MO) which was synthesized via a method reported by Ishihara et al.25 The MPC powder was mixed with RMGI at MPC/ (RMGI 1 MPC) mass fractions of 0% (control), 1.5%, 3%, and 5%. MPC mass fractions >5% were not tested due to lower enamel bond strength in preliminary study. As another control, a commercial non-fluoride-releasing composite (Transbond XT, 3M Unitek, Monrovia, CA) was also used. According to the manufacturer, Transbond XT consisted of silane treated quartz (70–80% by weight), bisphenol-Adiglycidyl ether dimethacrylate (10–20%), bisphenol-A-bis (2-hydroxyethyl) dimethacrylate (5–10%), silane-treated silica ( 0.1). Most of the TB control specimens failed at the bracketadhesive interface. The adhesive-enamel interface was the most common site of failure for Groups 2–5. There was no noticeable difference between 1 day and 30 days (p > 0.1). Protein adsorption on the disks is plotted in Figure 2 (mean 6 sd; n 5 6). Adding MPC into VT significantly decreased the protein adsorption. VT with 3% MPC had the least protein adsorption, which was nearly 1/10 that of commercial controls (p < 0.05). Water-aging for 30 d did not reduce the protein-repellent efficacy, compared to 1 day (p > 0.1). TABLE I. ARI Scores of Orthodontic Cements (n 5 10) ARI Scores Group TB control VT control VT 1 1.5% MPC VT 1 3% MPC VT 1 5% MPC TB control VT control VT 1 1.5% MPC VT 1 3% MPC VT 1 5% MPC a
Water-Aging
0
1
2
3
Siga
1 day 1 day 1 day 1 day 1 day 30 days 30 days 30 days 30 days 30 days
0 3 4 4 4 0 4 4 4 5
1 7 6 6 6 1 6 6 6 5
5 0 0 0 0 6 0 0 0 0
4 0 0 0 0 3 0 0 0 0
A B B B B A B B B B
Sig refers to statistical significance, with different letters (A, B) indicating significant differences in the ARI scores (p < 0.05).
FIGURE 2. Protein adsorption onto orthodontic cement disk surfaces (mean 6 sd; n 5 6). Dissimilar letters indicate values that are significantly different from each other (p < 0.05).
Figure 3 plots the pH of culture medium with biofilms on the disks: (A) 1 day, and (B) 30 days (mean 6 sd; n 5 6). In (A), for VT with 3% MPC, the pH remained at around 6.5. For all other groups, the pH decreased with time, reaching 5.1 for VT with 1.5% MPC, 4.7 for VT control, and 4.2 for TB control at 48 h. Comparing (A) with (B) showed a similar trend and similar end pH, indicating that the ability to repel bacteria and reduce acid production for VT with 3% MPC was maintained, with no significant decrease from 1 day to 30 days (p > 0.1). The 2-day biofilm properties on the disks are plotted in Figure 4: (A) metabolic activity, and (B) lactic acid production (mean 6 sd; n 5 6). Biofilms on TB control had the highest metabolic activity and lactic acid production. Incorporation of MPC into VT greatly decreased the metabolic activity and lactic acid, as compared to controls (p < 0.05). VT with 3% MPC had the least metabolic activity and lactic acid that was about 40% that of TB control (p < 0.05). There was no significant difference between 1 day and 30 days (p > 0.1). Figure 5 plots the 2-day biofilm CFU counts for: (A) total microorganisms, (B) total streptococci, and (C) mutans streptococci (mean 6 sd; n 5 6). Incorporation of MPC into VT greatly reduced all three CFU counts, compared to commercial controls (p < 0.05). All three CFU counts of biofilms on VT with 3% MPC were an order of magnitude lower than those of TB control (p < 0.05). There was no significant difference in CFU before and after 30 days of water-aging for each group (p > 0.1). Typical live/dead staining images of the 2-day biofilms on the disks are shown in Figure 6. In (A), VT was fully covered by a layer of biofilm consisting of primarily live bacteria. TB control (not shown) was also completely covered with live biofilms similar to (A). In (B), VT with 3% MPC
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FIGURE 4. Quantitative viability of 2-day biofilms on orthodontic cement disks: (A) MTT metabolic activity, and (B) lactic acid production of biofilms (mean 6 sd; n 5 6). In each plot, dissimilar letters indicate significantly different values (p < 0.05).
FIGURE 3. Effect of MPC incorporation on the decrease in pH of culture medium with dental plaque microcosm biofilms. (mean 6 sd, n 5 6). (A) pH of medium with biofilms on orthodontic cement disks after being water-aged for 1 day. (B) pH of culture medium with biofilms cultured on the orthodontic cement disks after being water-aged for 30 days.
had much less biofilm coverage. There was little difference between 1 day and 30 days. In (E), VT with 1.5% to 3% MPC had significantly (p < 0.05) less biofilm coverage than the commercial controls (mean 6 sd; n 5 6). DISCUSSION
The present study represents the first report on the development of a protein-repellent RMGI, which could be used as
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an orthodontic cement to repel proteins to reduce biofilm acids and white spot lesions. The new method successfully reduced protein adsorption on VT by an order of magnitude, which in turn greatly reduced oral biofilm formation and lactic acid production, and substantially increased the pH to avoid the cariogenic low pH. The inhibition of bacteria attachment and biofilm growth was achieved without compromising the enamel shear bond strength of the proteinrepellent VT, as compared to the unmodified commercial VT. Biofilms around orthodontic brackets can produce acids and cause white spot lesions.7,8 Bacterial adhesion is the first step of biofilm formation,20,21 because the adsorption of salivary proteins is a prerequisite for bacterial
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FIGURE 5. Colony-forming unit (CFU) counts of 2-day biofilms on orthodontic cement disks: (A) total microorganisms, (B) total streptococci, and (C) mutans streptococci (mean 6 sd; n 5 6). In each plot, dissimilar letters indicate significantly different values (p < 0.05).
adhesion.21–24 Proteins were found to adsorb preferentially to hydrophobic surfaces.21 Highly hydrophilic surface coatings utilizing MPC are well known to reduce protein adsorption and bacterial adhesion.27–29 MPC is one of the most common biocompatible and hydrophilic biomedical polymers.25,26 Regarding the protein-repellent mechanism, it was reported that, in the hydrated MPC polymer, there is an abundance of free water but no bound water.25,26 The bound water would result in protein adsorption.27,50 On the other hand, the large number of free water around the phosphorylcholine group could detach proteins, thereby repelling protein adsorption.27,50 On the basis of this mechanism, it could be assumed that increasing the mass fraction of MPC in RMGI would increase its protein-repellent potency. Indeed, increasing the MPC mass fraction from 0% to 3% in the RMGI significantly decreased the protein adsorption (Figure 2). The results in Figures 3–6 confirmed that VT with 3% MPC greatly reduced bacterial adhesion, CFU counts, metabolic activity and lactic acid production of biofilms. These results demonstrate that the idea of developing a protein-repellent RMGI is a promising approach to combating biofilms and inhibiting white spot lesions in orthodontic treatments. Acidogenic bacteria in biofilms can metabolize carbohydrates to acids and cause a local plaque pH to decrease to 4.5 or even 4 after a sucrose rinse.51 It was reported that, below pH of about 5.5, the demineralization dominates, resulting in a net enamel mineral dissolution.51 Hence, it would be highly desirable for the local pH to maintain greater than 5.5, thereby preventing enamel demineralization around the orthodontic brackets. In the present study, the two commercial controls with biofilms resulted in pH below 5. It should be noted that VT had a beneficially higher pH than that of TB control. The reason for this is likely that the fluoride ion release of VT contributed to reducing the acid production of the bacteria, by inhibiting the metabolic pathways such as the fermentation pathway for lactic acid production.52,53 This result is confirmed by VT having less CFU, and lower metabolic activity and lactic acid production of biofilms, than TB control. Therefore, the additional protein-repellent ability of VT with 3% MPC was beneficial in further reducing the lactic acid production of bacteria, thereby successfully maintaining a safe pH of around 6.5 to avoid demineralization. Previous studies found that at pH 4 in the plaque around orthodontic brackets, the remineralization process was adversely affected and even more fluoride in the oral environment did not inhibit demineralization.13,14 Hence, the incorporation of MPC into RMGI could enhance the fluoride remineralization effect of RMGI. The results of this study indicate that adding 3% MPC into RMGI with microcosm biofilms in the presence of sucrose would be able to maintain the local pH at a safe level to prevent tooth mineral dissolution. Regarding the long-term protein-repellent durability, it was reported that MPC contains reactive methacrylate groups, which can be copolymerized and covalently bonded with the resin matrix upon photo-polymerization.54 A previous study evaluated the durability and protein-repellent
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FIGURE 6. Live/dead images of 2 day biofilms on orthodontic cement disks. Live bacteria were stained green, and dead bacteria were stained red. Live and dead bacteria in the proximity of each other produced yellow/orange colors. (A, B) Representative images of biofilms on disks after bring water-aged for 1 day. (C, D) Biofilms on disks after being water-aged for 30 days. (E) Area fraction of live bacteria on disks (mean 6 sd; n 5 6). Dissimilar letters indicate significantly different values (p < 0.05).
action of acrylic resin containing MPC. The results indicated that MPC was copolymerized with acrylic resin through covalent bonding, and the strong CAC bonding offered durable resistance to protein adsorption.54 In the present study,
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VT consisted of polymerizable resin,10,11 which could copolymerize with MPC. Photo-induced polymerization could immobilize the MPC in the resin matrix through strong covalent bonding, thus providing durable protein-repellent
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ability. Furthermore, another study investigated poly-MPC (PMPC) grafting onto surfaces through covalent bonding, which was resistant to mechanical stresses.50 In the present study, MPC was incorporated into RMGI throughout the volume and not just on the surface. Therefore, the wear and brushing actions around orthodontic brackets in vivo will not remove the protein-repellent capability. Indeed, the protein-repellent and biofilm-repellent effects of MPCcontaining RMGI were not significantly reduced after wateraging for 30 days. Further study is needed to investigate the protein-repellent activity of MPC-containing RMGI for longer than 30 days. Enamel bond strength is an important parameter in orthodontic treatments.32–36 The bond strength should be high enough to avoid failure, but low enough to allow debonding without damaging the enamel when the treatment is completed.36 The optimal bond strengths were about 8–9 MPa.55 The application of phosphoric acid complicates the removal of residual adhesive on enamel and can cause enamel cracks.31,34,56 Additionally, enamel demineralization may be promoted by the acid-etching procedure.6,8 RMGIs can be used as orthodontic adhesive without acid etching due to ionic bonding between the hydroxyapatite in teeth and carboxyl groups of polyalkenoic acid.31,34–36 There are many benefits of bonding the brackets without etching the enamel, such as reducing saliva contamination, protecting the enamel, and saving chair time.10,31 In the present study, Groups 2–5 were applied without acid etching. The SBS of Groups 2–4 were clinically acceptable, based on literature values. The SBS of VT with 5% MPC was lower, likely because 5% MPC may have rendered the VT too hydrophilic, with water adsorption degrading the bond.57,58 In contrast, the SBS of VT with 3% MPC was nearly 8.8 MPa, which is clinically ideal. These results suggest that the use of an optimal MPC amount in RMGIs is important to obtaining the maximal protein-repellent ability and SBS. Three points should be noted regarding SBS. First, while studies in the literature usually measured the bond strength after 1 day or 30 days immersion, it is also important to measure the early bond strength at 15 min after being bonded. This is the critical time for the orthodontist who will be applying forces via arch wires, ligatures, and elastics. At the time of initially applying these forces, the brackets must be well adhered to the enamel surface. Second, while studies in the literature including the present study report bracket adhesion in MPa, another important measurement would be the force. This is because if the force generated by the testing instrument on the bracket to catastrophic debonding is greater than the force applied in the treatment, then the adhesive cement would be sufficient. Third, while the present study tested the bonded specimens after storage in distilled water for 1 day or 30 days, it would be interesting to grow oral biofilms on the bonded teeth for 30 days or longer, and then test the adhesion to determine the biofilm acid effects on possible bond degradation. Further study is needed to investigate these three aspects. After debonding, enamel surface should remain intact with as little residual adhesive on it as possible.31 ARI score
were developed to evaluate the amount of adhesive remaining on enamel after debonding.39 Acid etching caused more adhesive remaining on enamel than that without acid-etching.31 Moreover, the amounts of residual adhesive tend to be greater with a high SBS.34,36 In the present study, TB control showed higher SBS values, and higher ARI scores than other groups. Groups 2–5 had similar ARI scores, which were lower than that of TB control. These results indicate that incorporating MPC into RMGI did not result in significant changes to the bonding mechanism, and the predominant mode of bracket failure of Groups 2–5 was at the adhesive-enamel interface, leaving less than half of the adhesive on enamel. This could be clinically beneficial, because tooth cleaning after debonding is likely to be simpler and quicker with less residual adhesive.31,34–36 Although MPC-containing RMGI can reduce bacterial adhesion, they do not possess a strong antibacterial function and bacteria-killing capability. Further studies are needed to incorporate both antibacterial agents and MPC into RMGI to develop a bioactive orthodontic cement with triple benefits of fluoride release, protein-repellent and antibacterial capabilities, in order to effectively inhibit biofilms and eliminate white spot lesion formation. CONCLUSIONS
The first protein-repellent orthodontic cement was developed by incorporating MPC into a RMGI to repel bacteria attachment and inhibit enamel lesion formation. Adding 3% of MPC into RMGI had enamel bond strength similar to that of commercial unmodified RMGI, both at 1 day and 30 days of water-aging treatment. RMGI containing 3% of MPC greatly reduced protein adsorption, bacterial adhesion, biofilm CFU, and lactic acid production, while being able to raise cariogenic low pH to a safe pH level to avoid demineralization. The protein-repellent and biofilm-repellent effects were not decreased after water-aging for 30 days. Therefore, the MPC-containing RMGI is promising to reduce biofilm formation and the occurrence of enamel white spot lesions during orthodontic treatments. The novel proteinrepellent method may have applicability to other types of orthodontic cements as well as dental composites, adhesives, sealants, and cements to repel proteins and biofilms. ACKNOWLEDGEMENTS
The authors thank Dr. Satoshi Imazato, Dr. Michael D. Weir and Dr. Junling Wu for discussions and experimental help. REFERENCES 1. King G. Access to Orthodontic Services in the US. American Association of Orthodontics website. http://www.aaomembers.org/ mtgs/upload/King-Access-to-Orthodontic-Care-The-Problem-and-SomeSolutions.pdf. May 1, 2012. 2. Uysal T, Amasyali M, Ozcan S, Koyuturk AE, Sagdic D. Effect of antibacterial monomer-containing adhesive on enamel demineralization around orthodontic brackets: An in-vivo study. Am J Orthod Dentofacial Orthop 2011;139:650–656. 3. Santamaria M Jr, Petermann KD, Vedovello SA, Degan V, Lucato A, Franzini CM. Antimicrobial effect of Melaleuca alternifolia dental gel in orthodontic patients. Am J Orthod Dentofacial Orthop 2014;145:198–202.
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27. Ishihara K, Nomura H, Mihara T, Kurita K, Iwasaki Y, Nakabayashi N. Why do phospholipid polymers reduce protein adsorption? J Biomed Mater Res 1998;39:323–330. 28. Sibarani J, Takai M, Ishihara K. Surface modification on microfluidic devices with 2-methacryloyloxyethyl phosphorylcholine polymers for reducing unfavorable protein adsorption. Colloids Surf B Biointerfaces 2007;54:88–93. 29. Zhang N, Melo MA, Bai Y, Xu HH. Novel protein-repellent dental adhesive containing 2-methacryloyloxyethyl phosphorylcholine. J Dent 2014;42:1284–1291. 30. Scougall-Vilchis RJ, Yamamoto S, Kitai N, Hotta M, Yamamoto K. Shear bond strength of a new fluoride-releasing orthodontic adhesive. Dent Mater J 2007;26:45–51. 31. Scougall-Vilchis RJ, Ohashi S, Yamamoto K. Effects of 6 selfetching primers on shear bond strength of orthodontic brackets. Am J Orthod Dentofacial Orthop 2009;135:424.e1–424.e7. 32. Poosti M, Ramazanzadeh B, Zebarjad M, Javadzadeh P, Naderinasab M, Shakeri MT. Shear bond strength and antibacterial effects of orthodontic composite containing TiO2 nanoparticles. Eur J Orthod 2013;35:676–679. 33. Bishara SE, Soliman M, Laffoon J, Warren JJ. Effect of antimicrobial monomer-containing adhesive on shear bond strength of orthodontic brackets. Angle Orthod 2005;75:397–399. 34. Cheng HY, Chen CH, Li CL, Tsai HH, Chou TH, Wang WN. Bond strength of orthodontic light-cured resin-modified glass ionomer cement. Eur J Orthod 2011;33:180–184. 35. Paschos E, Kleinschrodt T, Clementino-Luedemann T, Huth KC, Hickel R, Kunzelmann KH, Rudzki-Janson I. Effect of different bonding agents on prevention of enamel demineralization around orthodontic brackets. Am J Orthod Dentofacial Orthop 2009;135: 603–612. 36. Tuncer C, Tuncer BB, Ulusoy C. Effect of fluoride-releasing lightcured resin on shear bond strength of orthodontic brackets. Am J Orthod Dentofacial Orthop 2009;135:14.e1–e6. 37. Moreau JL, Weir MD, Giuseppetti AA, Chow LC, Antonucci JM, Xu HH. Long-term mechanical durability of dental nanocomposites containing amorphous calcium phosphate nanoparticles. J Biomed Mater Res B 2012;100:1264–1273. 38. Weir MD, Moreau JL, Levine ED, Strassler HE, Chow LC, Xu HH. Nanocomposite containing CaF2 nanoparticles: Thermal cycling, wear and long-term water-aging. Dent Mater 2012;28:642–652. 39. Artun J, Bergland S. Clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. Am J Orthod 1984;85:333–340. 40. Cheng L, Weir MD, Zhang K, Xu SM, Chen Q, Zhou X, Xu HH. Antibacterial nanocomposite with calcium phosphate and quaternary ammonium. J Dent Res 2012;91:460–466. 41. Imazato S, Ehara A, Torii M, Ebisu S. Antibacterial activity of dentine primer containing MDPB after curing. J Dent 1998;26:267– 271. 42. Zhou C, Weir MD, Zhang K, Deng D, Cheng L, Xu HH. Synthesis of new antibacterial quaternary ammonium monomer for incorporation into CaP nanocomposite. Dent Mater 2013;29:859–870. 43. Zhang K, Li F, Imazato S, Cheng L, Liu H, Arola DD, Bai Y, Xu HH. Dual antibacterial agents of nano-silver and 12-methacryloyloxy dodecylpyridinium bromide in dental adhesive to inhibit caries. J Biomed Mater Res B 2013;101:929–938. 44. Moro T, Kawaguchi H, Ishihara K, Kyomoto M, Karita T, Ito H. Wear resistance of artificial hip joints with poly (2-methacryloyloxyethyl phosphorylcholine) grafted polyethylene: comparisons with the effect of polyethylene cross-linking and ceramic femoral heads. Biomaterials 2009;30:2995–3001. 45. McBain AJ. Chapter 4: In vitro biofilm models: an overview. Adv Appl Microbiol 2009;69:99–132. 46. Cheng L, Exterkate RA, Zhou X, Li J, ten Cate JM. Effect of Galla chinensis on growth and metabolism of microcosm biofilms. Caries Res 2011;45:87–92. 47. McBain AJ, Sissons C, Ledder RG, Sreenivasan PK, De Vizio W, Gilbert P. Development and characterization of a simple perfused oral microcosm. J Appl Microbiol 2005;98:624–634. 48. Cheng L, Weir MD, Xu HH, Kraigsley AM, Lin NJ, Lin-Gibson S, Zhou X. Antibacterial and physical properties of calcium–
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49.
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51. 52. 53.
phosphate and calcium–fluoride nanocomposites with chlorhexidine. Dent Mater 2012;28:573–583. Lima JP, Sampaio de Melo MA, Borges FM, Teixeira AH, SteinerOliveira C, Nobre Dos Santos M, Rodrigues LK, Zanin IC. Evaluation of the antimicrobial effect of photodynamic antimicrobial therapy in an in situ model of dentine caries. Eur J Oral Sci 2009; 117:568–574. Goda T, Konno T, Takai M, Ishihara K. Photoinduced phospholipid polymer grafting on Parylene film: Advanced lubrication and antibiofouling properties. Colloids Surf B Biointerfaces 2007;54: 67–73. Dawes C. What is the critical pH and why does a tooth dissolve in acid? J Can Dent Assoc 2003;69:722–724. Rawls HR. Fluoride-releasing acrylics. J Biomater Appl 1987;1: 382–405. Shinohara MS, De Goes MF, Schneider LF, Ferracane JL, Pereira lito V, Nikaido T. Fluoride-containing adhesive: DuraPN, Di Hipo bility on dentin bonding. Dent Mater 2009;25:1383–1391.
54. Takahashi N, Iwasa F, Inoue Y, Morisaki H, Ishihara K, Baba K. Evaluation of the durability and antiadhesive action of 2methacryloyloxyethyl phosphorylcholine grafting on an acrylic resin denture base material. J Prosthet Dent 2014;112:194–203. 55. Reynolds IR. A review of direct orthodontic bonding. Br J Orthod 1975;2:171–178. 56. Correr GM, Alonso RC, Consani S, Puppin-Rontani RM, Ferracane JL. In vitro wear of primary and permanent enamel. Simultaneous erosion and abrasion. Am J Dent 2007;20:394–399. 57. Park JG, Ye Q, Topp EM, Misra A, Spencer P. Water sorption and dynamic mechanical properties of dentin adhesives with a urethane-based multifunctional methacrylate monomer. Dent Mater 2009;25:1569–1575. 58. Singh V, Misra A, Marangos O, Park J, Ye Q, Kieweg SL, Spencer P. Viscoelastic and fatigue properties of model methacrylate-based dentin adhesives. J Biomed Mater Res B 2010;95:283–290.
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Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, Maryland 21201 2 Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China 3 State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China 4 Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201 5 Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201 6 Department of Mechanical Engineering, University of Maryland, Baltimore County, Maryland 21250 Received 26 December 2014; revised 3 March 2015; accepted 9 April 2015 Published online 13 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33444 Abstract: The objectives of this study were to develop the first protein-repellent resin-modified glass ionomer cement (RMGI) by incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC) for orthodontic applications, and to investigate the MPC effects on protein adsorption, biofilm growth, and enamel bond strength. MPC was incorporated into RMGI at 0% (control), 1.5%, 3%, and 5% by mass. Specimens were stored in water at 378C for 1 and 30 days. Enamel shear bond strength (SBS) was measured, and the adhesive remnant index (ARI) scores were assessed. Protein adsorption onto the specimens was determined by a micro bicinchoninic acid method. A dental plaque microcosm biofilm model with human saliva as inoculum was used. The results showed that adding 3% of MPC into RMGI did not significantly reduce the SBS (p > 0.1). There was no significant loss in SBS for RMGI containing 3% MPC after water-aging for 30 days, as compared to 1 day (p > 0.1). RMGI with 3% MPC had protein
adsorption that was 1/10 that of control. RMGI with 3% MPC greatly reduced the bacterial adhesion, and lactic acid production and colony-forming units of biofilms, while substantially increasing the medium solution pH containing biofilms. The protein-repellent and biofilm-repellent effects were not decreased after water-aging for 30 days. In conclusion, the MPC-containing RMGI is promising to reduce biofilms and white spot lesions without compromising orthodontic bracket-enamel bond strength. The novel protein-repellent method may have applicability to other orthodontic cements, dental composites, adhesives, sealants, and cements to repel C 2015 Wiley Periodicals, Inc. J Biomed proteins and biofilms. V Mater Res Part B: Appl Biomater, 104B: 949–959, 2016.
Key Words: protein repellent, bacteria repellent, orthodontic cement, shear bond strength, human saliva microcosm biofilm, white spot lesions
How to cite this article: Zhang N, Zhang K, Melo MAS, Chen C, Fouad AF, Bai Y, Xu HHK. 2016. Novel protein-repellent and biofilm-repellent orthodontic cement containing 2-methacryloyloxyethyl phosphorylcholine. J Biomed Mater Res Part B 2016:104B:949–959.
INTRODUCTION
The requirement for orthodontic therapy is increasing as more people pursue orthodontic treatments around the world.1 While orthodontic materials and therapy methods have advanced, the placement of fixed appliances tend to accumulate dental plaque biofilms and make oral hygiene more difficult.2,3 The fixed appliances can lead to specific changes in the oral environment, such as the greater accumulation of microorganisms, the formation of biofilms, and local acidic pH.3,4 The level of Streptococcus mutans (S. mutans) and Lactobacilli in the oral cavity can be elevated due to the
increased biofilm formation.4,5 The resulting lower pH of the biofilms on enamel surfaces adjacent to fixed appliances results in demineralization. Indeed, enamel demineralization and white spot lesions were observed around orthodontic appliances.6 Although, several approaches were attempted to prevent white spot lesions,7 recent studies indicated that 50– 70% of patients had white spot lesions after fixed orthodontic therapy,8,9 and white spot lesions were the primary undesired side-effect of fixed orthodontic treatments.6–9 Oral hygiene, fluoride regimens, and dietary control have been generally practiced for preventing white spot lesions
Correspondence to: H. Xu; e-mail: [email protected] or Y. Bai; e-mail: [email protected] Contract grant sponsor: School of Stomatology at the Capital Medical University in China (NZ); contract grant number: NIH R01 DE17974 Contract grant sponsor: Seed Grant, the University of Maryland School of Dentistry
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during fixed orthodontic treatments.6,7 However, these preventive methods mainly depend on patient compliance, and hence, are not reliable.8,9 Preventive strategies which do not require patient compliance might be more effective. Recently, resin-modified glass ionomer cements (RMGIs) have been used as orthodontic bracket-bonding adhesives due to their fluoride-releasing ability and clinically acceptable bond strengths.6–11 RMGIs possess stronger bond strengths than conventional glass ionomer cements, and RMGIs were observed to be more effective than other orthodontic adhesives for preventing white spot lesions around orthodontic brackets.6–11 However, the use of RMGIs as orthodontic cements has yielded conflicting results. It was reported that RMGIs remaining around the brackets could be a great predisposing factor for white spot lesions, because the rough surfaces could provide suitable environments for bacterial adhesion.5 It was found that S. mutans adhered significantly more to RMGIs than to resin composites.5 In addition, gaps of 10 lm wide were found at the adhesive-enamel junctions around the bracket base, which could harbor bacteria and facilitate biofilm formation.12 Furthermore, previous studies reported that RMGIs had little demineralization-inhibiting effect, as the low-pH environment hinders the remineralization process.13,14 For these reasons, it was suggested that incorporation of antimicrobials into RMGIs could enhance the antibacterial effect.14,15 Various agents such as nanosilver,16 bioactive glass,17 and nanohydroxyapatite18 were incorporated into orthodontic adhesives. A key step in white spot lesion formation during orthodontic treatments is the initial bacterial adhesion around the brackets.19 The subsequent growth of these bacteria may form dental plaque biofilms with organic acids to cause enamel demineralization.7,8,20,21 Various factors are considered to affect bacterial adhesion to material surfaces.21 An initial salivary protein coating is first needed for subsequent bacterial22 and fungal23 adherence to teeth and to materials in the oral cavity. The adsorption of salivary proteins onto teeth and material surfaces can produce a biologic layer, which is termed the acquired pellicle.21–23 This salivary pellicle provides the essential receptors allowing the adherence of bacteria.21–23 Hence, the adsorption of salivary proteins is a prerequisite for bacterial attachment and biofilm formation. Indeed, several studies showed that salivary proteins allowed the adhesion of bacteria, including S. mutans, Porphyromonas gingivalis, and Actinomyces species, in both in vivo and in vitro situations.22–24 Therefore, it would be highly desirable to develop a protein-repellent RMGI, to inhibit protein adsorption and thereby to inhibit bacterial adhesion at the bracket-adhesive-enamel junction, thus reducing or eliminating white spot lesions around the brackets. However, to date, there has been no report on protein-repellent RMGIs. It has been demonstrated that hydrophilic material surfaces are usually more resistant to protein adsorption than hydrophobic surfaces.21 2-methacryloyloxyethyl phosphorylcholine (MPC) is a methacrylate with a phospholipid polar group in the side chain, and is one of the most common
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biocompatible and hydrophilic biomedical polymers.25,26 Previous studies found that the MPC has excellent ability to reduce protein adsorption and bacterial adhesion.26,27 Several medical devices with MPC have been developed and used clinically, such as artificial blood vessels, implantable artificial hearts, and artificial lungs.27,28 Recently, MPC was incorporated into a dentin bonding agent, achieving a strong proteinrepellent ability.29 However, the incorporation of MPC into RMGIs remains to be investigated. Accordingly, the objectives of this study were to develop novel protein-repellent and fluoride-releasing orthodontic cement by incorporating MPC into a RMGI, and to investigate the effects of MPC mass fraction in RMGI on enamel bond strength, protein adsorption, and biofilm activity for the first time. It was hypothesized that: (1) incorporating MPC into RMGI would not compromise the enamel bond strength; (2) MPC-containing RMGI would have much less protein adsorption than that of commercial control; and (3) MPC-containing RMGI would greatly reduce biofilm growth than commercial control.
MATERIALS AND METHODS
Fabrication of MPC-containing RMGI Vitremer (3M, St. Paul, MN) was used as the parent system to develop a MPC-containing RMGI for protein-repellent properties. Vitremer consisted of fluoroaluminosilicate glass and a light-sensitive, aqueous polyalkenoic acid. According to the manufacturer, 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 following the manufacturer’s instructions. The purpose was to investigate a model system, and then the method of incorporating MPC to achieve protein-repellent capability can be applied to other RMGIs and other types of orthodontic cements. MPC was obtained from Sigma-Aldrich (St. Louis, MO) which was synthesized via a method reported by Ishihara et al.25 The MPC powder was mixed with RMGI at MPC/ (RMGI 1 MPC) mass fractions of 0% (control), 1.5%, 3%, and 5%. MPC mass fractions >5% were not tested due to lower enamel bond strength in preliminary study. As another control, a commercial non-fluoride-releasing composite (Transbond XT, 3M Unitek, Monrovia, CA) was also used. According to the manufacturer, Transbond XT consisted of silane treated quartz (70–80% by weight), bisphenol-Adiglycidyl ether dimethacrylate (10–20%), bisphenol-A-bis (2-hydroxyethyl) dimethacrylate (5–10%), silane-treated silica ( 0.1). Most of the TB control specimens failed at the bracketadhesive interface. The adhesive-enamel interface was the most common site of failure for Groups 2–5. There was no noticeable difference between 1 day and 30 days (p > 0.1). Protein adsorption on the disks is plotted in Figure 2 (mean 6 sd; n 5 6). Adding MPC into VT significantly decreased the protein adsorption. VT with 3% MPC had the least protein adsorption, which was nearly 1/10 that of commercial controls (p < 0.05). Water-aging for 30 d did not reduce the protein-repellent efficacy, compared to 1 day (p > 0.1). TABLE I. ARI Scores of Orthodontic Cements (n 5 10) ARI Scores Group TB control VT control VT 1 1.5% MPC VT 1 3% MPC VT 1 5% MPC TB control VT control VT 1 1.5% MPC VT 1 3% MPC VT 1 5% MPC a
Water-Aging
0
1
2
3
Siga
1 day 1 day 1 day 1 day 1 day 30 days 30 days 30 days 30 days 30 days
0 3 4 4 4 0 4 4 4 5
1 7 6 6 6 1 6 6 6 5
5 0 0 0 0 6 0 0 0 0
4 0 0 0 0 3 0 0 0 0
A B B B B A B B B B
Sig refers to statistical significance, with different letters (A, B) indicating significant differences in the ARI scores (p < 0.05).
FIGURE 2. Protein adsorption onto orthodontic cement disk surfaces (mean 6 sd; n 5 6). Dissimilar letters indicate values that are significantly different from each other (p < 0.05).
Figure 3 plots the pH of culture medium with biofilms on the disks: (A) 1 day, and (B) 30 days (mean 6 sd; n 5 6). In (A), for VT with 3% MPC, the pH remained at around 6.5. For all other groups, the pH decreased with time, reaching 5.1 for VT with 1.5% MPC, 4.7 for VT control, and 4.2 for TB control at 48 h. Comparing (A) with (B) showed a similar trend and similar end pH, indicating that the ability to repel bacteria and reduce acid production for VT with 3% MPC was maintained, with no significant decrease from 1 day to 30 days (p > 0.1). The 2-day biofilm properties on the disks are plotted in Figure 4: (A) metabolic activity, and (B) lactic acid production (mean 6 sd; n 5 6). Biofilms on TB control had the highest metabolic activity and lactic acid production. Incorporation of MPC into VT greatly decreased the metabolic activity and lactic acid, as compared to controls (p < 0.05). VT with 3% MPC had the least metabolic activity and lactic acid that was about 40% that of TB control (p < 0.05). There was no significant difference between 1 day and 30 days (p > 0.1). Figure 5 plots the 2-day biofilm CFU counts for: (A) total microorganisms, (B) total streptococci, and (C) mutans streptococci (mean 6 sd; n 5 6). Incorporation of MPC into VT greatly reduced all three CFU counts, compared to commercial controls (p < 0.05). All three CFU counts of biofilms on VT with 3% MPC were an order of magnitude lower than those of TB control (p < 0.05). There was no significant difference in CFU before and after 30 days of water-aging for each group (p > 0.1). Typical live/dead staining images of the 2-day biofilms on the disks are shown in Figure 6. In (A), VT was fully covered by a layer of biofilm consisting of primarily live bacteria. TB control (not shown) was also completely covered with live biofilms similar to (A). In (B), VT with 3% MPC
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FIGURE 4. Quantitative viability of 2-day biofilms on orthodontic cement disks: (A) MTT metabolic activity, and (B) lactic acid production of biofilms (mean 6 sd; n 5 6). In each plot, dissimilar letters indicate significantly different values (p < 0.05).
FIGURE 3. Effect of MPC incorporation on the decrease in pH of culture medium with dental plaque microcosm biofilms. (mean 6 sd, n 5 6). (A) pH of medium with biofilms on orthodontic cement disks after being water-aged for 1 day. (B) pH of culture medium with biofilms cultured on the orthodontic cement disks after being water-aged for 30 days.
had much less biofilm coverage. There was little difference between 1 day and 30 days. In (E), VT with 1.5% to 3% MPC had significantly (p < 0.05) less biofilm coverage than the commercial controls (mean 6 sd; n 5 6). DISCUSSION
The present study represents the first report on the development of a protein-repellent RMGI, which could be used as
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an orthodontic cement to repel proteins to reduce biofilm acids and white spot lesions. The new method successfully reduced protein adsorption on VT by an order of magnitude, which in turn greatly reduced oral biofilm formation and lactic acid production, and substantially increased the pH to avoid the cariogenic low pH. The inhibition of bacteria attachment and biofilm growth was achieved without compromising the enamel shear bond strength of the proteinrepellent VT, as compared to the unmodified commercial VT. Biofilms around orthodontic brackets can produce acids and cause white spot lesions.7,8 Bacterial adhesion is the first step of biofilm formation,20,21 because the adsorption of salivary proteins is a prerequisite for bacterial
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FIGURE 5. Colony-forming unit (CFU) counts of 2-day biofilms on orthodontic cement disks: (A) total microorganisms, (B) total streptococci, and (C) mutans streptococci (mean 6 sd; n 5 6). In each plot, dissimilar letters indicate significantly different values (p < 0.05).
adhesion.21–24 Proteins were found to adsorb preferentially to hydrophobic surfaces.21 Highly hydrophilic surface coatings utilizing MPC are well known to reduce protein adsorption and bacterial adhesion.27–29 MPC is one of the most common biocompatible and hydrophilic biomedical polymers.25,26 Regarding the protein-repellent mechanism, it was reported that, in the hydrated MPC polymer, there is an abundance of free water but no bound water.25,26 The bound water would result in protein adsorption.27,50 On the other hand, the large number of free water around the phosphorylcholine group could detach proteins, thereby repelling protein adsorption.27,50 On the basis of this mechanism, it could be assumed that increasing the mass fraction of MPC in RMGI would increase its protein-repellent potency. Indeed, increasing the MPC mass fraction from 0% to 3% in the RMGI significantly decreased the protein adsorption (Figure 2). The results in Figures 3–6 confirmed that VT with 3% MPC greatly reduced bacterial adhesion, CFU counts, metabolic activity and lactic acid production of biofilms. These results demonstrate that the idea of developing a protein-repellent RMGI is a promising approach to combating biofilms and inhibiting white spot lesions in orthodontic treatments. Acidogenic bacteria in biofilms can metabolize carbohydrates to acids and cause a local plaque pH to decrease to 4.5 or even 4 after a sucrose rinse.51 It was reported that, below pH of about 5.5, the demineralization dominates, resulting in a net enamel mineral dissolution.51 Hence, it would be highly desirable for the local pH to maintain greater than 5.5, thereby preventing enamel demineralization around the orthodontic brackets. In the present study, the two commercial controls with biofilms resulted in pH below 5. It should be noted that VT had a beneficially higher pH than that of TB control. The reason for this is likely that the fluoride ion release of VT contributed to reducing the acid production of the bacteria, by inhibiting the metabolic pathways such as the fermentation pathway for lactic acid production.52,53 This result is confirmed by VT having less CFU, and lower metabolic activity and lactic acid production of biofilms, than TB control. Therefore, the additional protein-repellent ability of VT with 3% MPC was beneficial in further reducing the lactic acid production of bacteria, thereby successfully maintaining a safe pH of around 6.5 to avoid demineralization. Previous studies found that at pH 4 in the plaque around orthodontic brackets, the remineralization process was adversely affected and even more fluoride in the oral environment did not inhibit demineralization.13,14 Hence, the incorporation of MPC into RMGI could enhance the fluoride remineralization effect of RMGI. The results of this study indicate that adding 3% MPC into RMGI with microcosm biofilms in the presence of sucrose would be able to maintain the local pH at a safe level to prevent tooth mineral dissolution. Regarding the long-term protein-repellent durability, it was reported that MPC contains reactive methacrylate groups, which can be copolymerized and covalently bonded with the resin matrix upon photo-polymerization.54 A previous study evaluated the durability and protein-repellent
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FIGURE 6. Live/dead images of 2 day biofilms on orthodontic cement disks. Live bacteria were stained green, and dead bacteria were stained red. Live and dead bacteria in the proximity of each other produced yellow/orange colors. (A, B) Representative images of biofilms on disks after bring water-aged for 1 day. (C, D) Biofilms on disks after being water-aged for 30 days. (E) Area fraction of live bacteria on disks (mean 6 sd; n 5 6). Dissimilar letters indicate significantly different values (p < 0.05).
action of acrylic resin containing MPC. The results indicated that MPC was copolymerized with acrylic resin through covalent bonding, and the strong CAC bonding offered durable resistance to protein adsorption.54 In the present study,
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VT consisted of polymerizable resin,10,11 which could copolymerize with MPC. Photo-induced polymerization could immobilize the MPC in the resin matrix through strong covalent bonding, thus providing durable protein-repellent
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ability. Furthermore, another study investigated poly-MPC (PMPC) grafting onto surfaces through covalent bonding, which was resistant to mechanical stresses.50 In the present study, MPC was incorporated into RMGI throughout the volume and not just on the surface. Therefore, the wear and brushing actions around orthodontic brackets in vivo will not remove the protein-repellent capability. Indeed, the protein-repellent and biofilm-repellent effects of MPCcontaining RMGI were not significantly reduced after wateraging for 30 days. Further study is needed to investigate the protein-repellent activity of MPC-containing RMGI for longer than 30 days. Enamel bond strength is an important parameter in orthodontic treatments.32–36 The bond strength should be high enough to avoid failure, but low enough to allow debonding without damaging the enamel when the treatment is completed.36 The optimal bond strengths were about 8–9 MPa.55 The application of phosphoric acid complicates the removal of residual adhesive on enamel and can cause enamel cracks.31,34,56 Additionally, enamel demineralization may be promoted by the acid-etching procedure.6,8 RMGIs can be used as orthodontic adhesive without acid etching due to ionic bonding between the hydroxyapatite in teeth and carboxyl groups of polyalkenoic acid.31,34–36 There are many benefits of bonding the brackets without etching the enamel, such as reducing saliva contamination, protecting the enamel, and saving chair time.10,31 In the present study, Groups 2–5 were applied without acid etching. The SBS of Groups 2–4 were clinically acceptable, based on literature values. The SBS of VT with 5% MPC was lower, likely because 5% MPC may have rendered the VT too hydrophilic, with water adsorption degrading the bond.57,58 In contrast, the SBS of VT with 3% MPC was nearly 8.8 MPa, which is clinically ideal. These results suggest that the use of an optimal MPC amount in RMGIs is important to obtaining the maximal protein-repellent ability and SBS. Three points should be noted regarding SBS. First, while studies in the literature usually measured the bond strength after 1 day or 30 days immersion, it is also important to measure the early bond strength at 15 min after being bonded. This is the critical time for the orthodontist who will be applying forces via arch wires, ligatures, and elastics. At the time of initially applying these forces, the brackets must be well adhered to the enamel surface. Second, while studies in the literature including the present study report bracket adhesion in MPa, another important measurement would be the force. This is because if the force generated by the testing instrument on the bracket to catastrophic debonding is greater than the force applied in the treatment, then the adhesive cement would be sufficient. Third, while the present study tested the bonded specimens after storage in distilled water for 1 day or 30 days, it would be interesting to grow oral biofilms on the bonded teeth for 30 days or longer, and then test the adhesion to determine the biofilm acid effects on possible bond degradation. Further study is needed to investigate these three aspects. After debonding, enamel surface should remain intact with as little residual adhesive on it as possible.31 ARI score
were developed to evaluate the amount of adhesive remaining on enamel after debonding.39 Acid etching caused more adhesive remaining on enamel than that without acid-etching.31 Moreover, the amounts of residual adhesive tend to be greater with a high SBS.34,36 In the present study, TB control showed higher SBS values, and higher ARI scores than other groups. Groups 2–5 had similar ARI scores, which were lower than that of TB control. These results indicate that incorporating MPC into RMGI did not result in significant changes to the bonding mechanism, and the predominant mode of bracket failure of Groups 2–5 was at the adhesive-enamel interface, leaving less than half of the adhesive on enamel. This could be clinically beneficial, because tooth cleaning after debonding is likely to be simpler and quicker with less residual adhesive.31,34–36 Although MPC-containing RMGI can reduce bacterial adhesion, they do not possess a strong antibacterial function and bacteria-killing capability. Further studies are needed to incorporate both antibacterial agents and MPC into RMGI to develop a bioactive orthodontic cement with triple benefits of fluoride release, protein-repellent and antibacterial capabilities, in order to effectively inhibit biofilms and eliminate white spot lesion formation. CONCLUSIONS
The first protein-repellent orthodontic cement was developed by incorporating MPC into a RMGI to repel bacteria attachment and inhibit enamel lesion formation. Adding 3% of MPC into RMGI had enamel bond strength similar to that of commercial unmodified RMGI, both at 1 day and 30 days of water-aging treatment. RMGI containing 3% of MPC greatly reduced protein adsorption, bacterial adhesion, biofilm CFU, and lactic acid production, while being able to raise cariogenic low pH to a safe pH level to avoid demineralization. The protein-repellent and biofilm-repellent effects were not decreased after water-aging for 30 days. Therefore, the MPC-containing RMGI is promising to reduce biofilm formation and the occurrence of enamel white spot lesions during orthodontic treatments. The novel proteinrepellent method may have applicability to other types of orthodontic cements as well as dental composites, adhesives, sealants, and cements to repel proteins and biofilms. ACKNOWLEDGEMENTS
The authors thank Dr. Satoshi Imazato, Dr. Michael D. Weir and Dr. Junling Wu for discussions and experimental help. REFERENCES 1. King G. Access to Orthodontic Services in the US. American Association of Orthodontics website. http://www.aaomembers.org/ mtgs/upload/King-Access-to-Orthodontic-Care-The-Problem-and-SomeSolutions.pdf. May 1, 2012. 2. Uysal T, Amasyali M, Ozcan S, Koyuturk AE, Sagdic D. Effect of antibacterial monomer-containing adhesive on enamel demineralization around orthodontic brackets: An in-vivo study. Am J Orthod Dentofacial Orthop 2011;139:650–656. 3. Santamaria M Jr, Petermann KD, Vedovello SA, Degan V, Lucato A, Franzini CM. Antimicrobial effect of Melaleuca alternifolia dental gel in orthodontic patients. Am J Orthod Dentofacial Orthop 2014;145:198–202.
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