Influence of atmospheric pressure plasma treatment on mechanical proprieties of enamel and sealant bond strength Hellen S. Teixeira,1 Paulo G. Coelho,1 Simone Duarte,2 Malvin N. Janal,3 Nelson Silva,4 Van P. Thompson5 1

Department of Biomaterials and Biomimetics, New York University College of Dentistry, New York, New York Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, New York 3 Department of Epidemiology and Health Promotion, New York University College of Dentistry, New York, New York 4 Department of Prosthodontics, Federal University of Minas Gerais, Belo Horizonte School of dentistry, Belo Horizonte, MG, Brazil 5 Department of Biomaterials, Biomimetic & Biophotonics King’s College London Dental Institute, London, UK 2

Received 8 May 2014; revised 29 July 2014; accepted 19 August 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33284 Abstract: Objectives: To define the effect of APP treatments on the mechanical properties of enamel and on its ability to promote sealant bonding to unetched enamel. Methods: Human molar teeth were sectioned exposing flat enamel regions at the buccal and lingual surfaces. The specimens were divided into two substrate groups (etched and unetched) and distributed over three surface treatments (i) 5 slm Argon APP treatment, NaOH surface treatment, and (iii) compressed air application (control). The Enamel surfaces were characterized by SEM, IFM, and Goniometer instruments. For the mechanical tests nanoindentation and microshear bond strength were employed. Initial data evaluation comprised normality verification (SPS S software) and variance checking and the appropriated statistical analysis model employed. For all statistical inferences, significance was set at 0.05. Results: SE was significantly higher for the etched and unetched group treated with Plasma relative to the NaOH

and control groups. Nanoindentation testing determined that Rank hardness was significantly higher in the control and Plasma group relative to NaOH for the etched group. Rank Elastic Modulus was significantly higher on Control groups relative to NaOH and Plasma groups for the etched substrate. No difference was detected between treatments for the unetched group. For the mSBS test, we observed that APP treatment on etched and unetched enamel increased bonds significantly (p < 0.001). Conclusions: This study demonstrated that APP increased SE, surface wettability and bond strength between enamel and sealants potentially serving as a substitute for conventional acid etching procedures or as an adjuvant for self-etch C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: sealants. V Appl Biomater 00B: 000–000, 2014.

Key Words: atmospheric pressure plasma, enamel, bonding, pit and fissures, sealants

How to cite this article: Teixeira H, Coelho P, Duarte S, Janal M, Silva N, Thompson V. 2014. Influence of atmospheric pressure plasma treatment on mechanical proprieties of enamel and sealant bond strength. J Biomed Mater Res Part B 2014:00B:000–000.

INTRODUCTION

Pit-and-fissure sealants are considered an outstanding adjunct to preventive strategies because these sealants decrease the onset and progression of occlusal caries.1 The high susceptibility of pits and fissures to carious attack and the rapid onset of the disease at these sites soon after tooth eruption are reported by several studies.2 In this context, treating caries-susceptible pits and fissures with resin sealants has been considered an adjunctive strategy to decrease occlusal caries initiation and/or progression.2 The preventive benefits of such treatment rely on the ability of the sealing material to thoroughly fill pits, fissures, and/or anatomical defects, and to remain completely intact and bonded to the enamel surface for a lifetime.3–5 However, while the efficiency of pit-and-fissure sealants has been reported in

the literature, its lower than expected utilization has been attributed to lack of confidence in their bonding to enamel and to the difficulty of achieving adequate dry field isolation6 and consequently appropriate bonds to the surface. Also etched surface is high energy and readily adsorbs water leading to reduced bond strength and sealant loss.7 Thus, the development of surface treatment conditions that could either eliminate enamel etching or keep it to the lowest levels while maintaining fully etched bond strength levels is highly desirable. Currently, NaOH treatment prior to sealant application has been performed8 in a tentative to increase surface energy, wettability, and consequently bond strength between the internal surface of the zirconia framework and resin cements. NaOH requires further characterization along with

Correspondence to: H.S. Teixeira; e-mail: [email protected]

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potential treatments for bonding improvement such as the application of APPs. These APPs, differently than the commonly used high temperature and low pressure plasmas utilized for various engineering applications, represent a promising alternative for improving bond strength levels between enamel and adhesive/RBCs systems due to its ability to widely modify and functionalize surfaces. A recent review has pointed that APPs represent a technology that can improve interactions between materials and biological systems.9 As an example, this technology allows the chemical functionalization and sterilization of surfaces as a result of reactions induced by one or more plasmaproduced chemical species. In addition, APP-generated free radicals and ultraviolet light can be utilized for polymerization purposes, providing a unique opportunity for improvements in enamel bonding. This project used APPs to change the enamel surface chemistry in an attempt to enhance physico–chemical interaction with dental sealants, potentially substituting for acid etching procedure, and resulted in stable and reliable bonding of sealant systems on enamel surfaces. MATERIALS AND METHODS

Atmospheric pressure plasma device (APP) The plasma that was utilized in this study (KinPen 09, INP Greifswald, Germany)10—consists of a hand-held unit (170 mm length, 20 mm diameter, weighing 170 g) connected to a high-frequency power supply (frequency 1.1 MHz, 2–6 kV peak-to-peak, 8 W system power) for the generation of a plasma jet at atmospheric pressure. The handheld unit has a pin-type electrode (1 mm diameter) surrounded by a 1.6 mm quartz capillary. An operating gas consisting of Ar at a flow rate of 5 slm was used. The plasma plume emerging at the exit nozzle is about 1.5 mm in diameter and extends into the surrounding air for a distance of up to 15 mm. Sample preparation For the present study, 96 de-identified extracted human molar teeth were collected under a protocol approved by the NYU Medical School Institutional Review Board. A diamond wafer blade was used to separate the crown from root of each tooth. Subsequently, the crowns were sectioned in half in the mesial-distal direction along the tooth long axis. The buccal and lingual sections were mounted in acrylic resin, and a region of interest was prepared in the enamel region of each tooth hemi section . The ROI preparation was performed by grinding the enamel surface to a 600 grit finish with SiC papers until a flat area presenting minimum dimensions of 3 mm in height and 6 mm in length was obtained. All teeth were immersed in water and kept moist until treated. We determined two substrate conditions (etched and unetched) and three treatment groups under each condition (Control, APP, and NaOH). Before any experimental surface treatment is applied, half (n 5 48) of the total number of specimens (n 5 96) were acid etched with 37% phosphoric acid for 30 seconds

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and then washed off with a gentle stream of tap water for 30 seconds and finally dried for 5 seconds using a gentle stream of compressed air. No acid-etching was employed on the other half (n 5 48). The etched and unetched enamel groups (n 5 48/group) were divided in subgroups and received APP treatment for 15 seconds, NaOH tratment for 60 seconds and the control etched and unetched groups received 15 seconds of compressed air application. NaOH group and APP group Prior to mechanical and chemical testing of both etched and unetched samples, the specimens were treated with alkaline solution (NaOH—0.1 mol). This basic solution was applied by eyedropper, onto the enamel surface of each tooth (n 5 15), and allowed to remain undisturbed for 60 seconds. Each drop contained 0.4 mL of the solution and was applied two drops per sample. The solution was then washed off with a gentle stream of tap water for 30 seconds and then dried with compressed air for 5 seconds. Samples were tested immediately after that. Both etched and unetched samples were positioned 3 mm away from, and perpendicular to, the center of the APP handheld tip. Then, plasma jet was applied in a special hood for 15 seconds utilizing Argon (Ar) as the source of gas at a 5 standard liters per minutes (slm) flow rate. Plasma was applied on etched [35% Phosphoric Acid (Ultraetch, Ultradent Corp, Salt Lake City)] and unetched surfaces. Samples were tested immediately after APP treatment.11,12 For the control group both etched and unetched samples were dried by compressed air application for 15 seconds before tested. Physicochemical characterization Scanning electron microscopy and surface roughness assessment. Prior and after the different surface treatments described for the different groups, the texture and surface roughness parameters were determined by scanning electron microscopy (SEM) and optical interferometry (IFM) (Phase View 2.5, Palaiseau, France), respectively. SEM was performed on both etched and unetched tooth sections. (Philips XL 30, Eindhoven, The Netherlands) at various magnifications at an acceleration voltage of 15kV. For IFM, both buccal and lingual ROIs were evaluated (n 5 3 teeth per group, seven measurements from each ROI) and arithmetic average high deviation (Sa) and root mean square (Sq), and the developed surface ratio (Sdr) parameters were collected. A filter size of 100 3 100 mm was utilized under an optical magnification of 50X (Leica MZAPO, IL). Surface wettabillity and energy assessement. Both buccal and lingual ROIs were evaluated (n 5 3 teeth per group, three measurements from each buccal and lingual ROIs). The ROI were horizontally positioned into a contact angle meter (OCA 30; Data Physics Instruments GmbH, Filderstadt, Germany) for contact angle measurements and SE calculations. Contact angle determinations were obtained via Young’s equation:

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gsv 5gsl 1glv cos u; Where u is the measured contact angle and gsv is the SE of the solid-vapor,13 gsl solid–liquid,14 and gIv liquid–vapor15 interface. For surface energy assessment, the Owens–Wendt– Rabel–Kaelble (OWRK) method16 was used for the SE determination by depositing 0.5 mL droplets of purified water, ethylene glycol, and methylene iodide on the surface of each disk with a micropipette (OCA 30; Data Physics Instruments GmbH, Filderstadt, Germany). Images were then captured and analyzed using software (SCA30; version 3.4.6 build 79). The relationship between the contact angle and SE was determined. The SE was calculated by g L 5 g DL 1 g PL, being gL the SE, gDL the disperse component, and gPL the polar component. The disperse component of the SE, characterizes the interaction between the surface and the dispensed liquid in terms of the non-polar interactions between molecules.17 The roughness, unevenness, and the branching level of the surface contribute to this component.18 The polar component of the SE characterizes the polar interaction between the surface of the material and the working fluid. This component is determined by the presence of polar groups, electric charges, and free radicals on the surface such as those obtained by treatment in oxygen-containing plasma.18 Micromechanical testing Fifteen teeth per group were utilized for nanomechanical testing. A total of 20 indents per tooth flange were made by a Berkovitch indenter for hardness and reduced modulus assessment. The enamel was detected by imaging under the light microscope (Hysitron TI 950, Minneapolis, MN) and indentations were performed in the polished samples (Samples will be polished to a 0.5 lm abrasive diamond paste and then surface treated). A nanoindenter (Hysitron, Minneapolis, MN) equipped with a Berkovich diamond three-sided pyramid probe was used. Indentations in the same specimen were performed in enamel with a distance of at least 10 mm from each other so that no interactions between them affected the mechanical results.19 A wax chamber was created above the acrylic plate so that tests were able to be performed in water (Wallace, JM, 2012). A loading profile with a peak load of 300 mN at a rate of 60 mN/s, followed by a holding time of 10 seconds and an unloading time of 2 seconds was utilized. The extended holding period allows tissue relaxation to a more linear response, so that no tissue creep effect was occurring in the unloading portion of the profile (ISO 145774). Therefore, from each indentation, a load–displacement curve was obtained.20 Microshear bond strength (uSBS) analysis For both unetched and etched enamel groups, a sealant sysR plustm). Ten teeth were rantem was used (UltraSeal XTV domly assigned to each surface treatment group. Cylindrical R plustm) were fabricated on columns of RBC (UltraSeal XTV each tooth ROI (buccal and lingual) using a plastic matrix of

2.30 mm in diameter and 2.30 mm in height with the RBC placed and cured in incremental layers according to the manufacturer’s instructions. The specimens, after aging in water for 2 days, were positioned in a universal testing machine and were subjected to a shear load at a strain rate of 1 mm/min. Following mechanical testing, the fracture mode of the samples was evaluated under a stereomicroscope and assigned to one of the three fracture categories: (i) adhesive, (ii) mixed, and (iii) cohesive in composite (Figure 8 ).21 Statistical analysis Initial data evaluation comprised normality verification (SPS S software) and variance checking. Then appropriate transformations were performed when necessary. For the surface roughness analysis, ANOVA was applied considering substrate (etched vs. non-etched) and surface treatment (control, NaOH, and Plasma) as independent variables and Sa, Sq, and Sdr as dependent variables. The surface energy statistical analysis was performed by one-way ANOVA. For the micromechanical testing, rank transformation was performed and a mixed model was utilized (independent variables were substrate and surface treatment and dependent variables were rank modulus and rank hardness). For the microshear bond strength, a mixed model was employed (independent variables were substrate and surface treatment and dependent variable microshear bond strength) following data rank transformation. Statistical analysis of the miscroshear bond strength fracture modes was performed by multiple chi-squared tests. For all statistical inferences, significance was set at 0.05. RESULTS

Physicochemical characterization Scanning electron microscopy and surface roughness. The scanning electron micrographs as well as the 3D reconstructions obtained by optical interferometry are presented in Figures 1 and 2 for the etched and non-etched groups, respectively. The Sa, Sq, Sdr, and Sds parameters for the different groups are presented in Tables I and II. Relative to the etched substrates, the non-etched substrates presented smoother profiles regardless of subsequent treatment (Figures 1 and 2). For the etched control group (no subsequent surface treatment), a amorphous phase layer was present and prevented the exposure of the enamel microstructure of enamel rods. On the other hand, both the etched NaOH and etched Plasma treated surfaces did not present with a amorphous layer covering the surface and the enamel rod microstructure was evident. However, while the enamel rods were selectively etched by the NaOH treatment, their microstructure was preserved by the Plasma treatment [Figure 1(E)]. The statistical summary (mean 6 standard error) for the different surface roughness parameters evaluated by ANOVA is presented in Tables I and II for the etched and non-etched groups. Overall, considering etched and non-etched substrates and the different surface conditions, a significant effect of

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FIGURE 1. (left) Scanning electron micrographs and (right) three-dimensional surface reconstruction for the three different etched substrates and subsequent surface treatments. (A,B) control group, (C,D) NaOH treated group, and (E,F) Plasma treated group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

substrate (p < 0.001) was detected whereas no effect of surface was observed (p > 0.68) for the Sa parameter, No effect was detected for the interaction between substrate and surface treatment (p > 0.75). The Sa values were 0.83 6 0.06 lm and 0.09 6 0.06 lm for the etched and non-etched groups, respectively. Among the etched and non-etched groups, no significant differences in Sa were observed between the control, NaOH, and Plasma groups. When comparing etched and non-etched results for the control, NaOH, and Plasma, significantly higher Sa values were observed for the etched substrates. In contrast to the etched groups (Figure 1) all nonetched groups presented a smooth surface where no enamel rod miscrostructure was observed (Figure 2). No qualitative differences were detected between the non-etched Control, NaOH, and Plasma groups (Figure 2). In agreement with the Sa statistical findings, statistical analysis of the Sq parameter showed a significant effect of substrate (p < 0.001) and no effect of surface was observed (p > 0.75). No effect was detected for the interaction between substrate and surface treatment was detected (p > 0.79). The Sq values were 1.01 6 0.07 lm and 0.11 6 0.07 lm for the etched and non-etched groups,

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respectively. Among the etched and non-etched groups, no significant differences in Sq were observed between the control, NaOH, and Plasma groups. When comparing etched and non-etched results for the control, NaOH, and Plasma, significantly higher Sa values were observed for the etched substrates. Concerning the developed surface ratio (Sdr), a significant substrate effect was detected (p < 0.001) and a borderline statistical significance for the surface treatment was detected (p 5 0.0526). The Sdr values were 21.01 6 2.64 % and 5.25 6 2.64 lm for the etched and non-etched groups, respectively. Among the etched samples, the highest Sdr value was observed for the NaOH group and the lowest for the Plasma group with the Control group presenting intermediate values. While no differences were detected between the control and the plasma samples a significant difference was observed between the NaOH and the Plasma groups. No significant difference was observed between the NaOH and Control samples. Regarding the non-etched surfaces no differences between surface treatments were detected. When comparing etched and non-etched results for the control, NaOH, and Plasma, significantly higher Sdr values were observed for the etched substrates.

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FIGURE 2. (left) Scanning electron micrographs and (right) three dimensional surface reconstruction for the three different non-etched substrates and subsequent surface treatments. (A,B) control group, (C,D) NaOH treated group, and (E,F) Plasma treated group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Surface wettability The surface wettability results for the different groups are presented in Figures 3 and 4 (surface energy). With the exception of the Plasma treated surfaces, the etched substrates presented significantly higher (p < 0.02) surface energy values relative to

their non-etched counterparts (Figure 3). The overall increase in surface energy values for the etched control and NaOH groups relative to the non-etched primarily arose from increases in the polar component even though a subtle increase in the disperse components were also detected.

TABLE I. Statistical Summary (Mean 6 Standard Error) for the Different Surface Roughness Parameters by ANOVA (Etched Groups)

TABLE II. Statistical Summary (Mean 6 Standard Error) for the Different Surface Roughness Parameters by ANOVA (Non-Etched Groups)

Etched

Unetched Control

Control

Sa (lm)

Sq(lm)

Average Std Error NaOH

0.92 0.1 Sa(lm)

1.09 0.11 Sq(lm)

Average Std Error PLASMA

0.81 0.1 Sa (lm)

1 0.11 Sq(lm)

Average Std Error

0.75 0.1

0.92 0.11

Sds (ummts/mm) 53.67 8.57 Sds (ummts/mm) 67.96 8.57 Sds (ummts/mm) 21.21 8.57

Sdr(%) 12.27 4.57 Sdr(%)

Average Std Error NaOH

33.54 4.57 Sdr(%)

Average Std Error PLASMA

12.22 4.57

Average Std Error

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Sa (lm) Sq(lm)

Sds Sdr(%) (ummts/mm) 0.09 0.11 67.32 5 0.1 0.11 8.57 4.5 Sa (lm) Sq(lm) Sds Sdr(%) (ummts/mm) 0.1 0.12 71.85 5.7 0.1 0.11 8.57 4.5 Sa (lm) Sq(lm) Sds Sdr(%) (ummts/mm) 0.08 0.09 61.7 5 0.1 0.11 8.57 4.5

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FIGURE 3. Surface energy calculated by the OWRK method for the different groups (mean 6 standard deviation). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

When surface energy comparisons are made between etched samples, significant differences were detected between the three surface groups, when the Plasma, NaOH, and control presented the highest, intermediate, and lowest values, respectively (Figure 3). On the other hand, while the non-etched plasma presented the highest and statistically higher values relative to the non-etched NaOH and control groups, no significant difference was detected between these groups (Figure 3). Micromechanical testing Since significant differences in variance between groups were detected for both hardness and elastic modulus, data ranking was performed for appropriate comparisons between groups. The mixed model analysis detected a significant effect of substrate on both rank elastic modulus (p < 0.001) and rank hardness (p < 0.001), where significantly higher values were observed for the non-etched relative to the etched substrate (Figure 5 and 6). When considering rank elastic modulus for the etched substrates, these presented significantly lower values relative to their non-etched counterparts (Figure 5). Within the etched substrates, the control group exhibited significantly higher rank elastic modulus values relative to

FIGURE 5. Ranks elastic modulus as a function of substrate and surface treatment (presented as estimated mean 6 95% confidence interval). The number of asterisks depicts statistically homogeneous groups within each substrate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

the NaOH and Plasma surface treatments. On the other hand, no differences were detected between non-etched substrates subjected to different surface treatments (Figure 5). When considering rank hardness for the etched substrates, these presented significantly lower values relative to their non-etched counterparts (Figure 6). Within the etched substrates, the control group exhibited significantly higher rank hardness values relative to the NaOH surface treatment but not when compared to the Plasma surface treatment. On the other hand, no differences were detected between rank hardness of non-etched substrates subjected to different surface treatments (Figure 6). Microshear bond strength The rank microshear bond strength mixed model analysis showed a significant effect of substrate (p < 0.001) and surface treatment (p < 0.001), where significantly higher values were observed for the etched relative to the non-etched substrate and significantly higher values were observed for the Plasma surface treatment relative to the control and NaOH surface treatments on both substrate conditions (etched and unetched) (Figure 7). When the rank microshear bond strength was evaluated, significantly higher values were observed for the etched samples of each surface treatment group when compared to their non-etched counterparts (Figure 7). For the etched

FIGURE 4. A water drop on Acid-Etched-Plasma group (left) and Acid-Etched-Control group (right). Note the significant reduction of the water contact angle on enamel-etched surface after plasma exposure (left).

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FIGURE 6. Ranks hardness as a function of substrate and surface treatment (presented as estimated mean 6 95% confidence interval). The number of asterisks depicts statistically homogeneous groups within each substrate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

substrate, significantly higher values were observed for both Plasma and Control relative to the NaOH surface treatment (Plasma significantly higher than the Control). When considering the surface treatments on non-etched substrates, both the Plasma and the NaOH surface treatments significantly outperformed the Control. It should be noted that the non-etched Plasma group and the etched Control group results were not significantly different (Figure 7). Following miscroshear bond strength testing, three different types of fracture modes were observed: adhesive, mixed, and cohesive on composite were observed (Figure 8). The cross tabulation of the fracture modes (adhesive, mixed, and cohesive) as a function of substrate and surface treatment is presented in Table III and Figure 9. When chisquared tests were run considering adhesive versus mixed/ cohesive failure modes, no significant differences were observed for both etched and non-etched Control and NaOH groups’ fracture distribution (p 5 0.21) even though etching of the substrate shifted failure modes from adhesive to mixed/cohesive (p 5 0.21). When considering the etched and non-etched plasma versus control groups, etching the substrate significantly changed the failure mode of the control group from adhesive to mixed/cohesive, and less of a shift from adhesive to mixed/cohesive was observed for the Plasma when going from non-etched to etched substrates, therefore resulting in a significant difference between both groups’ fracture mode distribution (p 5 0.03). No differences in fracture mode distribution between the Control and Plasma groups were detected since all fractures for both groups were adhesive under unetched condition. Finally, when chi-squared tests were ran to evaluate fracture mode distributions between the Plasma and NaOH groups, no significant differences were observed in both etched (p 5 0.65) and non-etched (p 5 0.21) conditions. DISCUSSION

While enamel bonding far exceeds the performance of dentin bonding, improving adhesive dental procedures to enamel is

highly desirable for preventive, orthodontic, and restorative procedures.22 In preventive dentistry, higher bond strength between sealants and enamel has the potential to decrease caries development on pits and fissures. Pit and fissure caries known to be a major epidemiologic and economic burden in developing and developed countries.23,24 Improvements in bonding to enamel would also benefit orthodontic bracket performance, as well as it would improve the performance of anterior and posterior teeth adhesive restorative procedures by decreasing the likelihood of marginal infiltration and secondary caries development.23 Most adhesive restorative procedures require selective removal of tooth structure in order to increase the surface area available for bonding and mechanical interlocking between the infiltrating resin and tooth structure. Ideally, high bond strength between non etched enamel is desirable, a challenging scenario not only due to the lack of mechanical interlocking between enamel and resin but also the limited surface area relative to the etched counterpart.25 The rationale for employing APP technology is to improve enamel bonding is multifaceted and includes the fact that high temperature chemistry may be delivered at ambient temperature resulting in surface energy increases allowing for a more intimate interaction between substrate and adhesive in both etched and non-etched enamel. It also presents potential for enamel bonding improvements as chemical radicals may be deliberately incorporated in the surface prior to the bonding procedure, resulting in primary chemical bonding to both etched and non-etched enamel.26 Finally, while both previous surface energy and chemical functionalization enhancements are highly desirable, APP sources have decreased in dimension and currently available commercial units are as large as dental light curing units and thereby can be used in private dental offices.26 Thus, the present study is one of the first attempts to employ APPs to modify etched and nonetched enamel surfaces to enhance physico– chemical interaction with dental sealants seeking to achieve more stable and reliable bonding of sealant systems.

FIGURE 7. Rank microshear bond strength as a function of substrate and surface treatment (presented as estimated mean 6 95% confidence interval). The number of asterisks depicts statistically homogeneous groups within each substrate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 8. Three fracture patterns were detected following microshear bond strength testing. (i) adhesive, (ii) mixed, and (iii) cohesive in composite.

The APP device utilized created desirable surface modifications for bonding to enamel. From a morphologic standpoint, APP application resulted in unnoticeable qualitative changes in the substrate morphologies of both non-etched and etched enamel. From a quantitative perspective and in agreement with the current literature, a strong effect of substrate etching was observed for all parameters of surface roughness (Sa, Sq, Sdr, and Sds) analyzed. More importantly, no significant effects were detected for the interaction between substrate and the various treatments, indicating that the main driver for surface roughness effects was etching the substrate. When non-etched, no significant differences were observed between control, NaOH, and the Plasma

groups. Thus, both surface qualitative and quantitative morphologic demonstrated that the APP application treatment did not substantially change etched and non etched surface morphology as per the instrumentation resolution employed for surface roughness analyzes. When surface energy was evaluated, the control (EA) and NaOH treated etched substrates (EN) presented significantly higher values relative to their non-etched counterparts, accounted for primarily by the increase in the polar component and by both polar and dispersive component for the control and NaOH groups, respectively. The increase in the dispersive component value for the NaOH etched surface energy potentially resulted from subtle alterations in

TABLE III. Cross Tabulation of Fracture Modes as a Function of Substrate and Surface Treatment Fracture Mode

non-etched

condition

ctrl NaOH Plasma

Total etched

condition

ctrl NaOH Plasma

Total Total

condition

ctrl NaOH Plasma

Total

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Count % within Count % within Count % within Count % within Count % within Count % within Count % within Count % within Count % within Count % within Count % within Count % within

condition condition condition condition condition condition condition condition condition condition condition condition

Adhesive

Mixed

10 100.0% 7 70.0% 10 100.0% 27 90.0% 0 0.0% 3 30.0% 5 50.0% 8 26.7% 10 50.0% 10 50.0% 15 75.0% 35 58.3%

0 0.0% 3 30.0% 0 0.0% 3 10.0% 10 100.0% 3 30.0% 5 50.0% 18 60.0% 10 30.0% 6 30.0% 5 25.0% 21 35.0%

Cohesive on Composite

0 0.0% 4 40 0% 0 0.0% 13.3% 0 0.0% 4 20.0% 0 0.0% 4 6.7%

Total 10 100.0% 10 100.0% 10 100.0% 30 100.0% 10 100.0% 10 100.0% 10 100.0% 30 100.0% 20 100.0% 20 100.0% 20 100.0% 60 100.0%

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FIGURE 9. Fracture mode distribution for the different groups. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

surface roughness resulting from selective etching by the strong base. The only group that presented comparable surface energy values for unetched and etched conditions was the Plasma group, where the highest values of surface energy components were observed. The surface energy levels achieved for the unetched plasma were higher than control and NaOH etched groups, a feature highly desirable for improving bonding. While APP treatment did not morphologically change etched and unetched subtrates it increased their respective surface energies to the highest levels observed. AAP effects on the mechanical properties of the enamel substrates were unknown. For this purpose, nanoindentation was conducted and overall data evaluation demonstrated that unetched substrates presented significantly higher hardness and modulus values relative to etched substrates. Such results are due to the selective removal of mineral content from the enamel during the etching procedure resulting in a porous structure.22 In general, both surface treatments (NaOH and APP) decreased both elastic modulus and hardness values, and this observation was especially pronounced in the etched substrates elastic modulus while not as pronounced for hardness. Such result is important as etched substrates treated with NaOH and APP, while being more readily wet by sealants—a desirable property, may present lower biomechanical integrity in the micrometer range after the bonding procedure, which might lower bond stability given sealant polymerization shrinkage and mechanical stresses from mastication. The microshear bond strength testing addressed the different groups’ short term bond performance. As expected due to the increase in surface area, surface energy, and the mechanical interlocking between substrate and resin for the etched group, significantly higher bond strengths resulted. There was an unequivocal contribution of the APP surface treatment on both etched and non etched substrates to bond strength, APP application to non etched surfaces resulted in significantly higher microshear bond strength

values relative to the non etched control, slightly higher values relative to unetched NaOH group, but more importantly, to comparable bond strength levels to etched controls. Such unprecedented improvement in bond strength to non etched substrates demonstrates the effectiveness of APP treatment in increasing the surface energy and bonding ability of the system without compromising the surface morphology and substrate mechanical properties, highly desirable features for enamel bonding. While surface energy was increased by the NaOH and APP treatment of etched substrates, these treatments resulted in decreases in the micromechanical properties of the etched substrates. Despite the potential decrease in bond strength reliability due to the detrimental effect of APP application on the mechanical properties of etched surfaces, microshear bond strength values for that group were significantly higher than all other groups evaluated. On the other hand, the increase in surface energy value observed for the etched NaOH relative to etched control appeared to be counteracted by the decrease in the substrate mechanical properties; resulting in microshear bond strength values significantly lower than the control and AAP groups. According to the microshear results, the application of NaOH was only significantly beneficial to unetched surfaces. As expected due to the mechanical interlocking between sealant and enamel, etching the surface resulted in failure modes that more often occurred in a mixed/cohesive mode than adhesively. Surprisingly, the EP group had a higher number of adhesive failures than the E and EN groups yet significantly higher mean bond strength. The selection choose of argon gas for the plasma instead of oxygen lies on the fact that oxygen species and in particular ozone inhibits resin based composites (RBC) polymerization which may impact on clinical performance of sealants.25 In light of the increased popularity of (ultra-) mild selfetch adhesives, adhesion to enamel requires more attention regarding surface-preparation methods than more traditional etch and rinse adhesives.27 Milder-Self-Etch sealants are less technique sensitive (less contamination sensitive), however they will not etch enamel well enough to ensure long-term sealant survival. APP modified the enamel surface increasing bond strength of sealants on non-etched enamel surfaces offering a potential alternative for improving longterm performance of milder self-etch sealants. APP treatment on dental enamel improved surface free energy partially by enhancing surface polarity, transforming the surface into a more hydrophilic state.28 As the hydrophilic adhesives improve retention of sealants even when enamel is contaminated23 and APP is known to improve surface energy and polarity of the substrate,28 the combination of APP treatment followed by sealant placement can be a good alternative for the long-term sealant survival. The increase in polarity for Plasma group may account for the increased mSBS values. The greater the polarity the smaller is the contact angle between the sealant and enamel surface. This increased wettability of the substrate, and may reduce void spaces in the enamel–sealant interphase

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improving the long term performance of sealants,23 This increased wettability may result in a better marginal seal which is extremely important in fissure sealant therapy.24 Additionally, contamination of etched enamel with saliva has been shown to result in sealant failure, techniques that could either eliminate enamel etching or keep it to the lowest levels while maintaining great sealant infiltration are highly desirable.29 Incomplete infiltration of the resin components into the tooth substrate might reduce the bond strength as well as the durability of the restoration.25 An advantage of hydrophilic adhesives is that the infiltration of the resin monomer occurs simultaneously with the water displacement, reducing the risk of discrepancies between the depth of demineralization and monomer infiltration25 and potentially improving the degree of polymerization. Improvement on the hydrophilicity of the enamel surface by APP treatment may lead to a greater graded interface between brittle enamel and low modulus adhesive which can play a role in improved mechanical performance and is important in maintaining the integrity of tooth structure.29 Finally, improvement in the hydrophilicity of the enamel surface caused by APP application, may have a positive effect on the retention of sealants and reduce margin leakage. However, studies on sealant microleakage after plasma treatment are necessary.24

8.

9.

10. 11.

12.

13.

14.

15.

16. 17.

CONCLUSION

The results presented that depicted increased SE, surface wettability and bond strength between sealants and enamel for the APP treated group. However, in vivo studies and clinical evaluation including APP are warranted before the method being considered as a clinically valid substitute for conventional acid etching procedure or as an adjuvant for self-etch sealants. ACKNOWLEDGMENTS

The authors would like to acknowledge the support of Department of Biomaterials and Biomimetics, New York University College of Dentistry, New York, NY, USA.

18. 19.

20.

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22.

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ATMOSPHERIC PLASMA EFFECTS ON SEALANT AND ENAMEL