A Polysaccharide-Based Antibacterial Coating with Improved

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Biological and Medical Applications of Materials and Interfaces

A Polysaccharide-Based Antibacterial Coating with Improved Durability for Clear Overlay Appliances Sohyeon Park, Hyun-hye Kim, Seok Bin Yang, Ji-Hoi Moon, Hyo-Won Ahn, and Jinkee Hong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04433 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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ACS Applied Materials & Interfaces

Article

A Polysaccharide-Based Antibacterial Coating with Improved Durability for Clear Overlay Appliances Sohyeon Park†, Hyun-hye Kim‡, Seok Bin Yang§, Ji-Hoi Moon § , Hyo-Won Ahn* ⊥ , Jinkee , ||

Hong*† †

Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro,

Seodaemun-gu, Seoul, Republic of Korea ‡

Graduate Student, Department of Dentistry, Graduate School, Kyung Hee University, Seoul,

Korea, Republic of Korea §

Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee

University, Seoul, Republic of Korea ||

Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, Republic of Korea

⊥

Department of Orthodontics, Kyung Hee University School of Dentistry, Seoul, Republic of

Korea

ACS Paragon Plus Environment

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ABSTRACT Clear overlay appliances (COAs) are widely used in orthodontic fields because they offer many advantages, such as cost-effectiveness, good formability, and good optical characteristics. However, it is necessary to frequently replace COAs because the thermoplastic polymers that are used to fabricate COAs have poor abrasion resistance and tend to induce bacterial accumulation. Here, we developed polysaccharide-based antibacterial multilayer films with enhanced durability, intended for COA applications. First, multilayer films composed of carboxymethylcellulose (CMC) and chitosan (CHI) were fabricated on polyethylene terephthalate glycol-modified (PETG), which was preferred material for COA fabrication, via a layer-by-layer (LbL) technique. Next, chemical crosslinking was introduced within the LbLassembled multilayer films. The LbL-assembled CMC/CHI film, which was made porous and rough by the crosslinking, formed a super-hydrophilic surface to prevent the adhesion of bacteria and exhibited a bacterial reduction ratio of approximately 75%. Furthermore, the crosslinking of the multilayer film coated on the PETG also improved the chemical resistance and mechanical stability of the PETG under simulated intraoral conditions with artificial saliva, by increasing the bond strength between the polysaccharide chains. We attempted to accumulate datasets using our experimental design and to develop sophisticated methods to assess nanoscale changes through large-scale measurements. 소현

KEYWORDS: clear overlay appliances, polysaccharide, layer by layer assembly, nano-film, antibacterial coating, durability


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 INTRODUCTION Aesthetic factors are significant in the field of dentistry; this has led to the universalization of invisible removable appliances. For example, clear overlay appliances (COAs), such as clear aligners or retainers, are widely used in daily orthodontic clinics for dynamic orthodontic treatment and maintenance.1-4 The COAs are fabricated by imprinting thermoplastic sheets on the dental cast model through a heating cycle combined with either vacuum or pressure forming. Several thermoplastics, such as polyethylene, polycarbonate, polypropylene, and polyethylene terephthalate glycol-modified (PETG), are currently used to prepare COAs.5-7 However, these materials inevitably induce bacterial accumulation which is strong risk factor for the development of bacteria-related disease including dental caries and periodontal disease, and possess mechanical limitations, such as poor abrasion and corrosion resistance.8-9 Consequently, it is necessary to frequently replace COAs, which causes the patient economic burden.7,

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Thus, to overcome these limitations, several approaches have been attempted.

Among them, increasing attention has recently focused on a coating technique that uses nanofilms because it offers site-specific application and can be used to produce COAs with little increase in thickness. To maintain minimal thickness of COAs is critical for the function of temporomandibular joints and patients’ comfort. Recently, urushiol-based coatings have been developed in our laboratory, to enhance the mechanical strength and antibacterial effects of such materials. In addition, we fabricated organosilicate-based super-hydrophilic nano-films with enhanced durability for dental applications.11-12 However, coatings should ideally be prepared using easily obtainable materials and simple methods without employing a complicated synthesis process. Furthermore, rigorous testing and analysis with regard to the chemical and physical stabilities


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of such materials are required because the conditions in the oral cavity are very complicated owing to the various enzymes present, its variable pH and physical stimulation. Here, we developed biocompatible polysaccharide-based antibacterial films, optimized for oral conditions. Specifically, we fabricated multilayer films composed of chitosan (CHI) and carboxymethylcellulose (CMC) on PETG, which is the most commonly used material for COAs, using a layer-by-layer (LbL) technique; we also introduced a crosslinking technique to the LbL-assembled multilayer film. Both CHI and CMC are linear polysaccharides, which are highly compatible with each other owing to their similar chemical structures. CHI possesses rich amine groups that exert an antibacterial effect, and each molecule of CMC can form hydrogen bonds with six water molecules.13 Owing to these features, they are considered suitable materials for the production of super-hydrophilic coatings that exert an antibacterial effect. Bacteria often adhere to the surface of in-dwelling medical devices and form a complex microbial community called the "biofilm". Because biofilms have a strong tendency to adhere to surfaces and increase resistance to antibiotics and host immune systems, it is extremely significant to prevent their initial adhesion in medical devices associated with bacterial infection.14-15 The super-hydrophilic coating on medical devices inhibits biofilm adhesion by forming a thin water film which interferes with the interaction between the surface of the medical device and the biofilm.11, 16-19 The LbL technique involves the alternating deposition of oppositely charged polyelectrolytes via electrostatic interaction; it can be applied to various substrates regardless of their shape and size.20-26 In addition, the crosslinking of such LbL-assembled multilayer films not only results in the production of a porous structure with a super-hydrophilic surface but also improves the


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chemical and physical stability of the coating by enhancing the strength of the bonds between the polysaccharide chains.27-28 Therefore, the objective of this study is to investigate the antibacterial effect of the PETG coated with the crosslinked polysaccharide multilayer film, and assess the chemical resistance and mechanical properties of the coating under simulated intraoral conditions with artificial saliva.

 RESULTS AND DISCUSSION Preparation of polysaccharide-based coating via LbL assembly and crosslinking. Both CMC and CHI are linear polysaccharides. Specifically, CMC is the sodium salt of a cellulose derivative, in which carboxymethyl groups (-CH2-COOH) are bound to some of the hydroxyl groups of the glucopyranose monomers that form the cellulose backbone. CMC is negatively charged in aqueous solutions because its -COOH groups are ionized to -COO-. CHI is composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). CHI is positively charged in aqueous solutions because its NH2 groups are ionized to NH3+.28-29 The CMC and CHI solutions were prepared with a pH of 4 because more than 80% of both these polysaccharides are ionized at this pH.13 Specifically, the driving force in this polysaccharide-based LbL assembly is considered to be owed to the electrostatic interaction between these materials. Prior to the LbL assembly, the oxygen plasma-treated substrates (Si wafer and PETG) were incubated in a branched polyethylene imine (BPEI) solution. BPEI is a polymer that is frequently used as a material for surface modification because it has inherently high adhesion and many amine groups. Thus, hydrogen bonding and electrostatic interaction are formed between the hydroxy groups of the oxygen plasma treated substrate and the amine


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groups of BPEI, thereby achieving surface modification for LbL deposition. BPEI also acts as a cushion layer by providing functional groups on both the substrate and the CMC due to the flexibility due to the branched structure.30-32 The surface modification allows the polysaccharide multilayer film to be robustly formed on the substrate surface. 33-34 In the case of the surface-amine modified substrates by BPEI, the LbL assembly commences with the deposition, via electrostatic interaction, of a negatively charged CMC layer, followed by a positively charged CHI layer. To demonstrate the successful assembly of (CMC/CHI)n nanofilms, the thicknesses of the LbL-assembled multilayered films on substrates (n = 5) were measured using a profilometer. Three sites were randomly selected for each substrate and the thickness was measured twice. By repeating these alternating depositions, the polysaccharide multilayer films are obtained. The thickness growth curve in figure 1 shows the successful deposition process of polysaccharides by the result that the multilayer film thickens with increasing the number of deposition. The multilayer films grow relatively linearly without any distinct irregularities, and the final thickness of the (CMC/CHI)20 film was approximately 1,818 nm. According to our previous paper, the (CMC/CHI)10 films also had super-hydrophilic properties, and thus we believed that the (CMC/CHI)10 films also exhibit anti-biofilm adhesion effects. However, we concluded that thicker thickness than 10 bilayer films would be more effective, to improve the mechanical strength for practical use. Moreover, the film with a thickness more than 2μm is not suitable for coating because it can easily be peeled off from the substrate. Therefore, we used the (CMC/CHI)20 film with thickness of 1.5μm - 2μm in this experiment.


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Figure 1. Curve showing the increase in the thickness of the CMC/CHI multilayer film (a); FT-IR spectra of CMC (black), CHI (purple), non-crosslinked (CMC/CHI)20 film (green), and crosslinked (CMC/CHI)20 film (red) (b).

Furthermore, Fourier transform-infrared (FT-IR) spectra shown in Figure 1b exhibited clear CMC/CHI film formation and crosslinking between multilayers. The spectra of CMC and CHI clearly reflected the inherent peaks of polysaccharides, but CMC/CHI films exhibited relatively monotonous peaks due to the electrostatic interaction between CMC and CHI, rather than their respective singlets. Meanwhile, crosslinking of LbL assembled CMC/CHI film was performed using

1-Ethyl-3-(3-Dimethylaminopropyl)

Carbodiimide

hydrochloride

(EDC)/N-

hydroxysulfosuccinimide (NHS) chemistry. First, EDC reacts with the carboxylate molecule, CMC, to form an unstable reactive o-acrylisourea ester. The formed ester reacts with NHS, and converts into a semi-stable amine-reactive NHS ester. Finally, the NHS ester promotes crosslinking between the polysaccharide chains by forming stable amide bonds with the amine groups of CHI. Glutaraldehyde was also used as a second crosslink agent, providing additional chemical bonds between the polysaccharide chains. The reactive end groups of glutaraldehyde allow the formation of covalent bonds between two polysaccharides containing hydroxyl groups or primary amine groups.35-38 In the FT-IR spectra of the CMC/CHI film, there are several notable peaks, namely overlapping O–H and N–H peaks at around 3400 cm-1, a C–H bond peak at around 2800 cm-1, as well as C–O–H, C–O–C, and C–N peaks corresponding to the polysaccharide structure at around 1600 cm-1 and 1150 cm-1. CMC, CHI and CMC/CHI films formed almost similar spectra by showing all the peaks represented by polysaccharides.27


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In the FT-IR spectra of the crosslinked CMC/CHI film, two distinctly separated peaks attributed to O=C, at around 1680 cm-1 and a N–H peak, at around 1645 cm-1 due to the amide bond formation, were observed.39-40 It is also possible to observe that the intensity of the C–N peak at around 1150 cm-1 was reduced. This corresponds to the formation of the C=N peak at around 1700–1600 cm-1, which is owed to crosslinking reaction of the glutaraldehyde.41-43 We also demonstrated successful chemical crosslinking using X-ray photoelectron spectroscopy (XPS) in our previous work.44

Film morphology and wettability analysis. The crosslinking induces additional bonds and reassembly between the polysaccharide chains that form the multilayer film, transforming the dense structure of the multilayer film into a porous structure. The effect of crosslinking on the morphologies of the multilayer thin films was investigated via field-emission scanning electron microscope (FE-SEM) and atomic force microscopy (AFM) observation. First, the roughness of the films was measured by AFM. According to the results of AFM measurement in figure 2, the Rq values of non-crosslinked (CMC/CHI)20 film and crosslinked (CMC/CHI film)20 were 20.32 nm and 57.71 nm, respectively. The SEM images in Figure 3 also show the increased roughness and porosity of the multilayer-film surface following crosslinking.


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Figure 2. AFM images of the non-crosslinked (CMC/CHI)20 film (a) and the crosslinked (CMC/CHI)20 film (b), and roughness of the films measured using AFM. The Brunauer−Emmett−Teller (BET) analysis results included in our previous study, demonstrate that the total porosity increased after crosslinking.44 This surface morphology change is the result of the effect of PBS used in post-crosslinking washes as well as additional bond formation due to crosslinking. The salts of PBS used for washing of the EDC/NHS crosslinking agent were sandwiched between the multilayered films which have already been swollen by the wet cross-linking, thereby ensuring the area where the pores can be formed. A porous structure was then formed in the area of the PBS salt removed after several washes with distilled water. Such a change in the morphology also influences the wettability of the multilayer film. According to the Wenzel and Cassie-Baxter theory, rough surfaces with a high surface energy are super-hydrophilic, while rough surfaces with a low surface energy are superhydrophobic.45-47 To compare the wettability of the films with changes in the surface morphology prior to and following crosslinking, statics water contact angles of non-crosslinked (CMC/CHI)10 film and crosslinked (CMC/CHI film)10 were measured. As expected, prior to crosslinking, the surface of the multilayer film exhibited hydrophilicity, with a contact angle of 35°; this was owed to the hydrophilic functional groups, namely –COOH and –NH2 of the CMC and CHI. However, following crosslinking, the multilayer film exhibited superhydrophilicity, with a contact angle of less than 5° (Figure 3). It is unlikely that the surface energy has increased rapidly because of the crosslinking, since surface energy is a materialspecific property. Therefore, the change in the contact angle signifies the increased surface roughness, which is owed to crosslinking.47


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Figure 3. Images showing the changes in the surface properties. Cross-sectional SEM images of the (CMC/CHI)10 films (a) and crosslinked (CMC/CHI)10 films (b). Static water-contactangle images of the (CMC/CHI)10 films (c), and crosslinked (CMC/CHI)10 films (d).

Cell viability and antibacterial function test. Using the polysaccharides as coating material contributed to high cell viability of the coating. Figure 4 shows the viability of the C2C12 cells for the coating, which was normalized to the control cell group. The coating composed of biocompatible polysaccharides exhibited 98% cell viability and this result demonstrated the biocompatibility of the coating. Meanwhile, the antibacterial effect of the coating against S. mutans is shown in Figure 5. S. mutans is the most prominent acidogenic oral bacteria, which can cause white spot lesions (WSLs) or serious demineralization of the tooth enamel.15 The SEM images show that there are enormous biofilms in the bare PETG, whereas there are few biofilms in the coated PETG. In addition, the ATP luminescent signal of the coated PETG is approximately four times lower than that of the bare PETG, which indicates that the coated PETG exhibits low bacterial viability. The antibacterial effect of the coating is owed to its super-hydrophilic properties. Under


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physiological conditions, super-hydrophilic surface forms a thin water film, which interferes with the interaction between the PETG surface and biofilm, preventing the attachment of bacteria.48-49

Figure 4. Cell viability graph with regard to the crosslinked (CMC/CHI)20 films; data was obtained via MTT-assays performed in duplicate using C2C12.

Figure 5. Adhesion of S. Mutans on the PETG surface (a,a‘) and crosslinked CMC/CHI filmcoated PETG surface (b,b’). Quantification of adhered bacteria on the uncoated PETG and crosslinked CMC/CHI film-coated PETG (C).\


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Analysis of the chemical stability of the coatings. Figure 6 shows the normalized-thickness graphs of crosslinked (CMC/CHI)20 films (Group 1) and non-crosslinked (CMC/CHI)20 films (Group 2) under each condition. The thickness of the crosslinked (CMC/CHI)20 films (Group 1) was almost maintained, without significant change, under the artificial saliva, regardless of the pH level and enzymes present. In contrast, the thickness of the non-crosslinked (CMC/CHI)20 films (Group 2) significantly reduced with time. Besides, the stability of the coatings under the artificial saliva with the enzymes could not be measured after 96 h because the group 2 peeled off (Figure S3). Table 1 exhibits the changes in the film thickness for Groups 1 and 2 owing to crosslinking by repeated measures ANOVA for each artificial saliva condition. Significant differences were observed between Groups 1 and 2, except for the case involving artificial saliva at a pH of 4.7 (Table 1). In the case of both Group 1 and 2 under the artificial saliva with a pH of 4.7, there was no significant reduction in thickness. This was because the pH 4.7 was similar to that of the environment in which the multilayer film was prepared. However, it could be observed that the non-crosslinked film (Group 2) degrades more severely under conditions where the pH is higher than 4.7. This is owed to the aggregation of CHI, which occurs as the pH increases, and the poor stability of the multilayer film prior to crosslinking.50 Table 2 shows the results of examining the effects of artificial saliva conditions on each group according to the cross-linking. The change in thickness was not significant in Group 1 with crosslinks in both time, acid, enzyme, or combined conditions. These results demonstrate that crosslinking of the films improved chemical stability under the simulated intraoral conditions. A Bonferroni post hoc test was performed for Group 2, which revealed significant differences in the film thickness changes according to the pH conditions (Table 3). The non-crosslinked (CMC/CHI)20 films (Group 2) were relatively stable at an artificial saliva pH of 4.7, and the film thickness decreased significantly as the pH increased (Table 1, Figures 6a to c).


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Figure 6. Chemical resistance test results obtained for the nanofilms under the simulated intraoral conditions with artificial saliva. The normalized thickness (%) of the crosslinked (CMC/CHI)20 films (red line, group 1) and that of the non-crosslinked films (black line, group 2) are indicated in each graph. (a) Artificial saliva with a pH of 4.7. (b) Artificial saliva with a pH of 6.7. (c) Artificial saliva with a pH of 8.7. (d) Artificial saliva with a pH 6.7, containing α-amylase. (e) Artificial saliva with a pH of 6.7, containing lysozyme. (* indicates a P-value of < 0.05, which represents statistical significance in a repeated measures ANOVA at the last stage.)


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Table 1. Change in the film thickness for Groups 1 and 2, with respect to the crosslinking, under each artificial saliva condition

pH

Time

Crosslinking

Time * crosslinking

pH 4.7

0.1049

0.4958

0.2374

pH 6.7

0.0002***

0.0005***

0.0012**

pH 8.7

A Polysaccharide-Based Antibacterial Coating with Improved

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