Oxygen Inhibition of Surface Composites and Its Correlation with Degree of Conversion and Color Stability.

This study investigated the effects of oxygen inhibition and finishing/polishing procedures on the composite resin properties. One bulk-fill and two conventional composite resins (nanoparticle and microhybrid) were evaluated. Specimens were prepared using 4 surface treatments: control, no treatment; Gly, oxygen inhibition with glycerin; FP, finishing and polishing; Gly + FP, glycerin followed by finishing and polishing. The degree of conversion (DC) was measured using Fourier Transformed Infrared Spectroscopy (FTIR) immediately and after 15 days (n=5). Color stability (ΔEab, and ΔE00) and opacity were evaluated using a spectrophotometer after 15 days of immersion in coffee, using the CIELAB system (n=5). Data were analyzed by two-way ANOVA and Tukey tests (α=0.05) and opacity by two-way repeated-measures ANOVA. Glycerin usage increased significantly the DC however had no influence on the ΔEab, ΔE00 and, opacity values. Finishing and polishing reduced ΔEab and ΔE00 values, regardless of composite resins. Microhybrid showed higher opacity, followed by the nanoparticle and bulk fill, regardless of surface treatment. Post-polymerization polishing procedures resulted in lower conversion than using an oxygen inhibitor agent (Gly condition), but similar staining caused by coffee.

Nowadays, microhybrid and nanoparticle composites are considered universal resin-based restorative materials suitable for the restoration of anterior and posterior teeth due to their excellent aesthetic properties. These nanomaterials use submicrometre particles to further enhance the optical and physical properties of the resins (3). Bulk fill composite resin, flowable and higher viscosity, claim to enable the posterior restoration of build-up in thick layers, 4 to 5 mm, reaching adequate polymerization in deeper regions (4,(7)(8)(9). Sufficient depth of cure may be achieved by using specific polymerization modulators, by improving the translucency, or by using more potent initiator systems. Restoring posterior cavities using bulkfill composite resins can result in reduced shrinkage stress and cusp deflection, which might improve the clinical performance of the restoration (10) To the best of our knowledge, the literature is scarce in terms of the ideal moment to perform surface finishing and polishing procedures of composite resins modulated by presence of unpolymerized monomers on the surface. This condition could absorb pigments, especially on the

Introduction
Composite resins are materials widely used in daily practice that need adequate polymerization for good clinical performance (1). These materials are undergoing chemical degradation processes in the oral cavity due to diets that contain staining solutions, as well as acidic foods and drinks (2,3). Degradation of composite resin, including color alteration, may result in additional costs due to early replacement of restorations. The degree of conversion (DC) of monomers is measured by the percentage of double bonds of carbon consumed during the polymerization reaction (1). The DC depends on the emission spectra of light curing units to match the absorption spectra of the photoinitiators used in these materials. Additionally, the light must actually reach all area of the restoration (4).
During composite light-curing, the contact of oxygen produces a surface layer of uncured resin (5,6). The oxygen inhibits the polymerization reaction, resulting in the formation of a polymer chain more prone to staining and wearing (5). The oxygen-inhibited layer thickness for composite resins ranges from 4 µm to 40 μm (5). The thickness of the oxygen-inhibited layer depends on the type of monomer, initiator-activator systems, particle morphology, concentration of free radicals, and the oxygen consumption rate (5,6). Some clinicians have applied glycerin gel or water-soluble gel over the last increment light-curing trough the transparent layer avoiding the oxygen inhibition and finishing/polishing procedures on the degree of conversion (DC) and color stability (ΔE ab , ΔE 00 and opacity) of bulk-fill and conventional composite resins. The null hypothesis was that the use of the glycerin oxygen inhibitor gel or its association with immediate finishing and polishing would not improve the color stability and the degree of cure of bulk-fill or conventional composite resins.

Experimental Design
This in vitro investigation was conducted using a 3x4x2 factorial study design to evaluate the factors "composite resin -bulk-fill, conventional nanoparticle and microhybrid", "protocols of surface treatments-no treatment, oxygen inhibition with glycerin, finishing and polishing, and glycerin + finishing and polishing" and "assessment time -immediate and mediate analysis". The main response variables included in this study were [1] degree of conversion and [2] color change.

Specimen Preparation
One bulk-fill (Filtek Posterior Bulk-Fill, 3M ESPE, St, Paul, MN, USA), and two conventional nanoparticle (Filtek Z350 XT, 3M ESPE) and microhybrid (Filtek Z250, 3M ESPE) composite resins were evaluated in this study. The compositions of materials used are described in Table 1. To measure the DC, according to the International Standards Organization (ISO) 4049, the composite resins were inserted into a silicone mold (HydroXtreme, Vigodent, Rio de Janeiro, Rio de Janeiro, Brazil) with internal dimensions of 4 mm in diameter x 2 mm of thickness. Specimens for color measurement (ISO/TR 28642) were built-up using a Teflon mold (8 mm of diameter x 2 mm of depth).
A halogen-light-based curing unit (OptiLux 501, Demetron, Danbury, CT, USA -600 mW/cm 2 ) was fixed in a standard device in order to maintain a fixed distance between the light-curing tip and sample surface. After placing the material into the mold, a polyester strip was pressed over the surface with a glass slab to obtain a flat surface. After the glass slab and the strip were removed and the photoactivation was made for 20 s, perpendicular and directly on the top of the specimens, at the shortest possible standardized position between the tip and the mold. Specimens were prepared according to different surface treatments: Control group (Control): the composite resins were light-cured for an additional 20 s.
Glycerin surface treatment (Gly): glycerin (Biopharma, Uberlândia, MG, Brazil) was applied on the surface of composite resin specimens followed by additional light activation for 20 s.
Finishing and polishing (FP): the composite resin specimens were light-cured for an additional 20 s, followed by finishing and polishing with abrasive disc Sof-Lex Pop-On (3M/ESPE) used sequentially according to the abrasiveness (medium, fine, and extra-fine discs). Ten movements were performed for each disc. The surface of the specimen was cleaned with distilled water and the composite resin surface was polished with a polishing paste Fotoacrill (Dhpro, Paranaguá, Paraná, Brazil) associated with a felt disk Diamond (FGM, Joinville, Santa Catarina, Brazil). The felt disk was wiped on the surface of the sample with alcohol 70% (Itajá, Goianésia, Goiás, Brazil) with friction and dried with an air-stream for 10 s following each procedure.
Glycerin + finishing + polishing (Gly + FP): after glycerin surface application and light activation for 20 s, the finishing and polishing were performed using the previously described protocol.
Five specimens were produced with the same finishing/ polishing instrument. Before DC analysis, 70% alcohol with gauze was used with on the specimens to remove the glycerin and post polishing compounds. The same clean protocol was used for control group.

Degree of Conversion Measurement
The specimens were placed on the ATR crystal directly with standardized pressure by the ATR device. The DC was measured immediately after the finishing and polishing surface procedures and again after 15 days using Fourier

Oxygen inhibition effects of composite resins
Transformed Infrared Spectroscopy (FTIR-Vertex 70, Bruker Optik GmbH, Ettlingen, Germany). For the evaluation after 15 days, specimens were stored in a dry and dark container at 37 °C. The DC was assessed using FTIR with attenuated total reflectance (ATR crystal) sampling, mid-infrared (MIR) and deuterated triglycine sulfate (DTGS) detector elements (Bruker Optik), with a 4 cm -1 resolution and coaddition of 32 scans. All analyses were performed under controlled temperature (25±1 °C) and humidity (60±5%) conditions. The DC was calculated from the equivalent aliphatic (1640 cm -1 ) and aromatic (1610 cm -1 ) of cured (C) and uncured (U) composite resin specimens according to the following equation:

Immersion of Coffee and Color Stability Measurement (N=5)
The color analysis was carried out immediately after specimen preparation. After 24 h of specimens being stored in a dry and dark container at 37 °C, they were individually immersed in 1 mL of coffee solution (Nestlé, São Paulo, SP, Brazil) for 15 days at 37°C (9). The solution was replaced daily. After storing, the excess of the solution was removed, and the specimens were ultrasonically (Ultrasonic Cleaner, Thornton -INPEC, Vinhedo, SP, Brazil) washed in distilled water for 10 min and dried.
The readings assessed at baseline were used to calculate the color changes caused by coffee immersion. The baseline color coordinates were assessed in standard conditions by means of a reflectance spectrophotometer (Ci64UV, Xrite, Chandler, Arizona, USA). The device was adjusted for the D65 light source, with 100% ultraviolet and specular reflection included. The observer angle was set at 10 degrees, and the device was adjusted to a small reading area (SAV), with a total area of 4 mm 2 . The color parameters were measured over white background (L*white 85.6, a*white 1.28, b*white 6.83) while the opacity was directly measured by the device. To measure opacity, samples were made against white, black (L*black 26.32, a*black -38, b*black 0.53), and white backgrounds again (11). The spectrophotometer was adjusted for three consecutive readings, which were later averaged. The opacity was calculated by the contrast ratio from the luminous reflectance (Y) of the specimens with a black (Yb) and a white (Yw) backing. A value of Yb/Yw=0 means that the specimen is completely transparent, and Yb/ Yw=100 implies that the specimen is completely opaque.
The results of the color readings were quantified in terms of the L*, a*, and b* coordinate values established by the Commission Internationale de l'Eclairage (CIELAB system). The color difference of the same specimen was calculated by the use of two different equations. The first one is the CIELAB color difference (ΔE ab ) equation, which was calculated as follows: ΔE ab = (ΔL 2 + Δa 2 + Δb 2 ) 1/2 where ΔL*, Δa*, and Δb* refers to lightness, green-red, and blueyellow differences of baseline and post coffee immersion color measurements. The second is the CIEDE2000 color difference (ΔE 00 ), and it was calculated as follows: ΔE 00 = [(ΔL/KLS + (ΔC/KCSC) 2 + (ΔH/KHSH) 2 + RT (ΔC/KCSC) (ΔH/ KHSH)] ½ .where ΔL, ΔC and ΔH are considered lightness, chroma, and hue differences between color measurements. KL, KC, and KH are the parametric factors for viewing conditions and illuminating conditions influence. RT is the function for the hue and chroma differences interaction in the blue region. SL, SC, and SH are the weighting functions for the color difference adjustment considering the location variation of L*, a*, and b* coordinates (12).
Statistical Analysis DC data was tested by normal distribution (Shapiro-Wilk test) and homoscedasticity (Levine test) followed by twoway ANOVA and Tukey tests (α=0.05). The color alteration data were tested by normal distribution (Shapiro-Wilk test) and homoscedasticity (Levine test) followed by two-way ANOVA and Tukey tests (α=0.05). For opacity data, two-way repeated measures ANOVA were applied for each composite resin (α=0.05). Linear regression for ΔE ab and immediate and mediate composite DC data set were calculated.

Results
The DC (%) mean and standard deviation values of the all tested composite resins are shown in Table 2. Two-way ANOVA showed that the composite resin (p<0.01) and surface treatment (p<0.01) and the interaction between both study factors (p=0.005) were significant for DC values. The moment of analysis had no influence in the DC values. Glycerin usage increased significantly (p<0.001) the DC values for all composite resins.
The mean and standard deviation values of opacity before and after coffee storage, are shown in Table 3. Twoway ANOVA showed that the composite resin (p=0.007), and moment of analysis (p<0.001) influenced significantly the opacity but not the interaction of them (p=0.803). Microhybrid composite resin showed higher opacity, followed by the nanoparticle and bulk-fill composite resins, regardless of surface treatment.
The color changes for L*, a* and b* parameters are shown in Figure 1 and the mean and standard deviation values of ΔE ab and ΔE 00 after coffee storage, are shown in Table 4. Two-way ANOVA showed that the composite resin (ΔE ab :p<0.001/ΔE 00 :p<0.001) and surface treatment factor (ΔE ab :p<0.001/ΔE 00 :p=0.003) were significant; however, the interactions between both factors (ΔE ab :p=0.167/ ΔE 00 :p=0.38) were not significant. In general, glycerin usage had not influence on the surface coffee staining for all composite resins. Finishing and polishing caused lowest ΔE ab and ΔE 00 values, regardless of composite resins. Linear regression showed a very weak correlation between ΔE 00 and DC (R ² =0.00548).

Discussion
In the present study, the DC of bulk-fill and conventional composite resins was affected by the glycerin usage and the color stability was influenced by the finishing and polishing procedures. Therefore, the null hypothesis of this study was rejected.
DC is the proportion of single carbon-carbon bonds in a polymer matrix to double carbon bonds between monomers (1). It has been shown that the clinical performance (13) of dental composites can be affected by mechanical properties that are influenced mainly by the DC (14), filler content and type of matrix (13,15). Low DC values might have negative influence on fracture resistance, wear resistance, compressive strength and can lead to early replacement of long-term restorations caused by detachment or discoloration around the adhesive interfaces (16). Low DC values can also increase the release of toxic monomers and initiators in the oral environment (5,17). In this study, a higher DC was observed for groups with oxygen inhibition surface treatment by glycerin, regardless of composite resins tested. During polymerization, oxygen reacts rapidly with free oxidized radicals and its presence slows the reaction. Oxygen inhibition improved polymerization of the surface layer (6). This aspect could also have impact on the surface hardness when the matrix band for making a direct restoration is removed after the light curing procedure (5,6).
Low DC was observed in the control group and when composite materials were submitted only to finishing 39.0 ± 9.5Ba Means followed by the different letters (uppercase for comparing the "surface treatment within assessment time for each composite resin -in columns"; lowercase for comparing the "resins within each assessment-in rows") indicate significant difference at Tukey`s test (p<0.05). Gly: glycerin; FP: finishing and polishing.

Oxygen inhibition effects of composite resins
and polishing protocol. Also, these data were lower than the literature reports (3,1). In this research, one possible explanation for this phenomenon may be related to the fact that a polyester strip was used to obtain a flat surface but it was removed before photoactivation, allowing contact between the resin and oxygen. Additionally, maybe finishing and the polishing protocol was not able to completely remove the unpolymerized resin layer, resulting in a lower DC in the resin composite surface. However, finishing and polishing (5) surface procedures can reduce color variation (ΔE ab and ΔE 00 ) after immersion in coffee. The esthetic features of restorations should not be defined as a factor indicating some intervention in posterior restorations. However, it is well-known that resin staining remains a cause of re-intervention even in posterior teeth, and more color-stable composites can prevent over-interventions. In case finish and polish procedures are awkward to achieve in posteriors composite restoration, light-curing in the absence of oxygen should be considered, especially when performing composite restoration in esthetic areas or in areas difficult to access such as the proximal surfaces.
Instrumental techniques for measuring color alteration include colorimetry and spectrophotometry, with good reliable performance for dental materials. Spectrophotometry is more accurate than measuring by using colorimeter, which is not influenced by ambient light (2). For the objective color difference measurement in dentistry, the CIELAB color difference formula has been extensively used, allowing for comparison with previous similar studies on dental composites. It assumes the uniformity of CIELAB color space and the equal importance of CIELAB individual parameters (L*, a*, and b*). However, a discrepancy sensitivity on the L*, a*, and b* parameters has been demonstrated concerning visual perceptibility and acceptability thresholds. (12,19). The CIEDE2000 metrics have also been then proposed due better indicative of human visual thresholds, even closer with the adjustment of parametric factor KL, KC, and KH set (2:1:1) (12). Similar results were observed for both color difference parameters in this research.
In the present study, microhybrid composite resin storage for 15 days in coffee had different performance, in terms of color change than conventional nanoparticle or bulk-fill composite resins, regardless of surface treatment. The difference in staining may be attributed to the composition of the materials and the characteristics of the particles. The hydrophilicity and degree of water sorption of a resin matrix could affect the staining susceptibility of resin composites. Although the particle sizes of nanoparticle resin composite smaller than microhybrid, it is expected to show less water sorption, thus less ΔE. The discrepancy Figure 1. Color changes of composite resins after storage in coffee, according surface treatments. Arepresents the change lightness within a specimen and ranges from 0 (black) to 100 (white). B=represents the change of degree of green/red measurement and C represents the degree of blue/yellow color change of composite resins after the storage in coffee.
could be due to resin matrices and additives such as dyes, photosensitizer molecules, and other chemicals in these materials. Moreover, both bulk-fill and nanoparticle (enamel shade) have high translucency and hightranslucent materials had the lowest color stability (19) The aging process was simulated to examine changes in the color alteration of the composite resins over time (14,16,20). In vitro studies evaluating color stability have examined composite specimens immersed in staining solutions over a particular period of time (9,14,16). Some beverages can alter the color of composite resins through the absorption and/or adsorption of colorants during the period of exposure (9,21). In most studies, the specimens have been immersed in coffee, grape juice, wine, and other beverages continuously for long periods of time (hours or days) (3,9). However, the exposure of composite restorations to beverages in vivo occurs through several cycles of a few seconds each. The temperature of a beverage can also increase its staining effect, as volumetric changes caused by heat or cold affect possible specimen defects (22) For most restorative materials, there is a complex process in the oral environment that includes disintegration and dissolution in saliva and other types of physical/chemical degradation, such as wear and erosion caused by food and drinks, chewing, and bacterial activity (9,17). This study demonstrated that bulk-fill composite resin storage for 15 days in coffee had different performance, in terms of opacity, than conventional composite resins, regardless of surface treatment. While there is a less evident change in the chemical composition in bulk-fill and conventional composites, the enhanced depth of cure in several bulk-fill is not a result of an improved refractive index mismatch between resin and filler but seems rather have been carried out by reducing the amount of pigments and enlarging the filler size. The higher level of translucency already characteristic of bulk-fill and nanoparticle resins may be responsible for the lower opacity values than microhybrid resin.
More studies are still necessary, especially clinical trials, with real time follow-up. Additionally, in laboratorial study should perform thermal/mechanical fatigue, wettability, water sorption and solubility, the thickness of the unpolymerized surface layer, biofilm presence for degradation and cumulative deleterious effects, to better respond to the dilemma of long-term maintenance of restorative materials after oxygen inhibition and also the consequence of the finishing and polishing procedures. The degradation can be prolonged by the maintenance of a low pH in the oral cavity (9,23). Associated with stains produced by immersion in drinks such as coffee, this aspect may also contribute to marginal discoloration, which is wrongly defined as the main reason for the replacement of esthetics restorations (18,24,25) In this study, it was simulated the effect of intermittent usage of coffee on the composite resins. The immersion of coffee negatively influenced the physical and chemical properties of the composite resins tested. To maintain the esthetic performance of composite resin restoration, it is suggested to make finishing and polishing surface procedures after conclude the restorative procedure. It is an important indicator to reduce the possibility of changing color parameters and could prevent degradation and darkening of composite resins. Furthermore, patients should be informed about the deleterious effects caused by the abusive use of coffee solutions on composite resin restorations.
The clinical significance of this study is that the degree of conversion of the composite resins surface can be improved by using glycerin to reduce the oxygen presence and the surface staining can be reduced if this previous procedure is associated with immediate finishing and polishing procedures. Within the limits imposed in the experimental design, it is possible to conclude that the glycerin usage increased the degree of conversion and had no effect on the surface coffee staining of tested composite resins and the finishing and polishing surface procedures reduced significantly the color variation (ΔE ab / ΔE 00 ) on the surface of bulk-fill and conventional composite resins.