Untitled 1 Volume 16 2017 e17068 Original Article 1 DDS, MS, PhD student, Universidade Estadual do Oeste do Paraná – UNIOESTE, School of Dentistry, Department of Restorative Dentistry, Cascavel, Paraná, Brazil 2 DDS, MS, PhD, adjunctive professor, Universidade Estadual do Oeste do Paraná – UNIOESTE, School of Dentistry, Department of Restorative Dentistry, Cascavel, Paraná, Brazil 3 DDS, MS, PhD, Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba – FOP/UNICAMP, School of Dentistry, Department of Dental Materials, Piracicaba, São Paulo, Brazil 4 DDS, MS, PhD, adjunctive professor, Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba – FOP/ UNICAMP, School of Dentistry, Piracicaba, São Paulo, Brazil Corresponding author: Bianca Medeiros Maran, Engenharia 464 – Universitário – Cascavel, PR, zip code: 85819-190 – Brazil. Phone: +55 45 99968-3042 Email: medeiros.bianca@hotmail.com Received: June 25, 2017 Accepted: September 20, 2017 Biological and mechanical degradation affecting the surface properties of aesthetic restorative Bianca Medeiros Maran1, Fabiana Scarparo Naufel2, Andréia Bolzan de Paula3, Giovana Spagnolo Albamonte Araújo3, Regina Maria Puppin-Rontani4 Aim: To evaluate the roughness (Ra), Knoop hardness (KHN) and change of color (∆E) of esthetic restorative materials (Filtek Z350-composite nanoparticle; Empress Direct-composite nanohybrid and IPS e.Max-ceramic) subjected to contact with the Streptococcus mutans biofilm (biological degradation) associated with abrasion generated by tooth brushing (mechanical degradation). Methods: Ten specimens of each material were prepared, and the surface properties initial were evaluated. All specimens were exposed to Streptococcus mutans inoculum; after 7 days, surface properties were evaluated. The specimens were submitted to a 30,000 toothbrushing cycles, using a toothpaste slurry, then, surface properties were evaluated again. Data were analyzed by Proc-Mixed, One-way ANOVA, Tukey-Kramer and Tukey’s tests (α = 0.05). Results: At the baseline, ceramic showed the highest Ra and KHN values; after the biological degradation the composites showed increased Ra, but KHN did not change; after the mechanical degradation, Empress showed decreased Ra and Z350 showed similar Ra, the KHN increased to both composites, and all materials had increased lightness after the mechanical degradation. Conclusions: The results suggest that, when exposed to Streptococcus mutans biofilm and toothbrush abrasion, the ceramics undergoes minimal degradation and the composites exhibited variable degradation, depending on the composition of the material. Keywords: Biofilms. Surface properties. Dental Materials. http://dx.doi.org/10.20396/bjos.v16i0.8651058 2 Maran et al. INTRODUCTION All restorative materials are susceptible to degradation. The degradation of restorative materials can be caused by low pH because of the cariogenic biofilm, consumption of acidic drinks or foodstuffs, and toothbrushes and the water present on saliva as well other components of the saliva1. Tooth brushing is the most used and efficient mechanical method to remove dental bio- film from all accessible tooth surfaces2. Published studies have shown that this method may cause tooth and composite abrasion. This degradation process may lead to sev- eral drawbacks, such as an increase in wear and surface roughness, softening and a decrease in the hardness of dental materials3-5. Over time, intraoral degradation also interferes with the fracture strength of the material, culminating in a lower durability of the restoration in the long term6. Surface texture, gloss and color are also included among the important characteristics that determine the aesthetic effect of these com- posite restorations, and they are also influenced by the intraoral surroundings7,8. It is important to know about this process, because the search for dental esthetics has been one of the main reasons why patients seek a dentist. Thus, the need for tooth-colored fillings has increased, decreasing the use of metal restorations and dental amalgam fillings or cast metal, unlike the use of aesthetic materials, such as composite and ceramics, which have been increasingly used9. Ceramics are considered the most inert of all dental materials used for restorations, composed of metal elements (aluminum, calcium, lithium, magnesium, potassium, sodium, lanthanum, tin, titanium and zirconium) and non-metal substances (silicon, boron, fluorine and oxygen), and characterized by two phases: a crystalline phase surrounded by a vitreous layer10. So far, little information about surface degradation by biofilm is available in the literature. Some studies have evaluated the interaction between biofilm and ceramics, but they verified only the biofilm characteristics instead of the biodegradation produced on the material surfaces11,12. Composites are currently the most used material in the field of restorative dentistry. Basi- cally, these materials are composed of three chemically different components: a polymeric matrix of dimethacrylate monomers, filler particles (dispersed phase) and an organosi- lane, which is a coupling agent that bonds the fillers to the polymeric matrix13. In this context, nanotechnology, consisting of nanofillers, has emerged in the dental market14. This technology came with the intention of improving the electrical, chemi- cal, mechanical and optical properties of restorative materials with advantages such as less toothbrush abrasion, greater hardness and better translucency, polish, gloss and opacity options being used for restorations of anterior and posterior teeth; as a result, studies have been done to prove these characteristics15,16. The nanohybrid composite IPS Empress Direct promises similar aesthetics to those made of ceramics in addition to the advantages of easy handling of the composite (Ivoclar Vivadent). Therefore, it becomes interesting to compare the IPS Empress Direct composite with a 100% nanofiller resin such as Z350 (3M ESPE St. Paul, MN, USA), as well as with the 3 Maran et al. ceramics IPS e.Max (IPS Empress; Ivoclar-Vivadent, Schaan, Liechtenstein), consider- ing the material that undergoes minimal degradation. Thus, the aim of this study was to test the hypothesis that aesthetic restorative mate- rials submitted to the Streptococcus mutans biofilm associated with brushing abra- sion would differ in surface stability to degradation, depending on their composition. MATERIALS AND METHODS Specimen Preparation 10 specimens of each material tested (described in Table 1) were fabricated using silicon molds (Express 3M ESPE, St. Paul, Minn, USA) of 8 mm in diameter and 2 mm deep, with the exception of the ceramics. Composite materials were inserted in a mold using incremental technique and covered with polyester strips and a glass slide to obtain a smooth flat surface. All specimens light cured using a LED light unit (Elipar Freelight, 3M ESPE, St. Paul, MN, USA) for 40 s on the top surface. The light intensity of the curing (1000 mW/cm2) device was checked with a curing light meter (Hilux Den- tal Curing Light Meter, Benlioglu Dental Inc., Demetron, Ankara, Turkey). Then, spec- imens were storage for 24 h in 100% relative humidity at 37°C, and then, they were polished with sequential abrasive discs (Soflex Pop-On, 3M ESPE, St. Paul, MN, USA). Table 1. Material, composition, color and batch of the tested materials. Materials Composition Mean Filler Size Color Batch # Composite Filtek Z350 XT (3M ESPE St. Paul, MN, USA) Bis-GMA (1-10 wt%); UDMA (1-10 wt%); TEGDMA (< 5 wt%); Bis-EMA (1-10 wt%); PEGMA (< 5 wt%) Silica, zirconia, zirconia/ silica (78.5 wt%) 0.6-1.4 µm (cluster) 5–20 nm (nanofiller) A3E 1124300109 IPS Empress Direct (Ivoclar- Vivadent, Schaan, Liechtenstein) UDMA (10-<20 wt%); TEGDMA (3-<5 wt%); Bis-GMA (2.5-<3 wt%) Barium glass, ytterbium trifluoride, mixed oxide, silicon dioxide and copolymer (77.5-79 wt%) Additives, catalysts, stabilizers and pigments (<1.0 wt%) 0.4 to 0.7 µm A3E N32078 Ceramic IPS e.Max (Ivoclar- Vivadent, Schaan, Liechtenstein) SiO 2 , Li 2 O, K 2 O, MgO, ZnO, Al 2 O 3 , P 2 O 5 and others oxides -- A3E P82207 4 Maran et al. Ceramic specimens were fabricated with the same dimensions of the composite, in a prosthetic laboratory using the pressing process in an oven (Programat P500–Ivoclar Vivadent, Schaan, Liechtenstein), and they were glazed. Then, all specimens were stored in water at 37° C for 24 h for the evaluation of the baseline properties. Measurements of Surface Roughness The surface roughness (Ra) was measured in a rugosimeter (Surfcorder SE 1700, Kosaka, Tokyo, Japan) at a constant speed of 0.5 mm/s with a load of 0.7 mN. The cut-off value was set at 0.25 mm to maximize the filtration of the surface wavi- ness. The Ra values for each specimen were taken across the diameter over a stan- dard length of 1.25 mm. The mean surface roughness values (μm) of the specimens were obtained from three successive measurements of the center of each disk, in dif- ferent directions (45°). A calibration was done periodically to check the performance of the surface roughness-measuring instrument. Measurements of Hardness Three Knoop hardness (KHN) indentations were made on the surface of the specimen under a load of 50 g for 10 s (HMV-2, Shimadzu, Tokyo, Japan). The KHN for each spec- imen was recorded as the average of the three readings distant 100 µm each other. Measurement of Color The readings were performed using a spectrophotometer (CM-700d, Konica Minolta, Osaka, Japan). Initially, the ambient light was calibrated in a light cabin, (GTI Mini Matcher MM1e, GTI Graphic Technology Inc., Newburgh, NY, USA), and the specimens were positioned in a sample carrier for the baseline readings. The parameters L*, a* and b* from the color space, referred to as CIELAB (L*, a*, b*), were recorded. The L* indi- cates lightness (L* + = lightness and L* - = darkness), the a* coordinate represents the red/green range (a* + = redness and a* - = greenness) and the b* coordinate represents the yellow/blue range (b* + = yellowness and b* - = blueness). The L*a*b* system allows the numeric definition of a color, as well as the difference between two colors using the following formula: ΔE= [(ΔL)2 + (Δa)2 + (Δb)2]1/2. The data acquisition was performed by a microcomputer using On Color QC Lite software (Konica Minolta, Osaka, Japan). Biofilm Growth – Biological degradation After the measurements of surface roughness, hardness and color, the all the spec- imens were sterilized for 4 h in an ethylene oxide chamber (Ferlex, São Paulo, SP, Brazil). A Streptococcus mutans (UA 159) strain was obtained from the culture of the Department of Microbiology and Immunology, Piracicaba Dental School, University State of Campinas. To prepare the inoculums, the Streptococcus mutans was first grown on mitis salivarius agar plates (Difco Laboratories, Sparks, MI, USA) at 37°C for 24 h in an environment supplemented with 5% CO2. Subsequently, single colonies were inoculated into 5 mL of brain-heart infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA) and incubated at 37º C for 24 h. The spec- 5 Maran et al. imens were exposed under static conditions to 25 μL of Streptococcus mutans inocu- lums adjusted to an optical density of 0.6 at 550 nm (approximately 8x1011 CFU/mL), and after 2 hours at room temperature the non-adhering cells were removed by wash- ing twice with 0.9% NaCl solution (saline). A single material disk was inserted in each well of 48-well polystyrene plates (Nunc mul- tidish, Sigma, St. Louis, MO, USA) with 2 mL of sterile, fresh BHI broth with the addition of 1% sucrose (wt/vol). The bacterial accumulation occurred at 37ºC in an environment supplemented with 5% CO2, developing 7-day-old biofilms. The medium was renewed every 48 h. At the end of the experimental period, the specimens were ultrasonically (UNIQUE 1400, Indaiatuba, SP, Brazil) washed for 10 minutes, and soon after the mea- surements were repeated. Three-body abrasion test – Mechanical degradation. Three-body Abrasion Test – Mechanical degradation After biological degradation, the tooth brushing test was conducted at 250 cycles/min for 30,000 cycles with a 200 gF load. The Oral B Pró Saúde toothpaste (Procter & Gamble, São Paulo, SP, Brazil) was diluted in distilled water (1:2) and used as an abra- sive third body. Specimens were washed in an ultrasonic bath for 10 min and gently dried with absorbent paper. Then, three final surface roughness readings were taken from each specimen in the opposite direction to that of the tooth brushing movement; Knoop hardness and color were also evaluated as previously reported after mechani- cal degradation using the same pattern described above. Statistical Analysis For Ra, KNH and L*, after the exploratory data analysis and selection of the best cova- riance structure, data were analyzed by means of mixed models (Proc-Mixed) and Tukey-Kramer test (α = 0.05). The data of hardness suffered logarithmic transforma- tion to meet the assumptions of a parametric analysis. For ΔE, after the exploratory analysis the data were analyzed by One-way ANOVA and Tukey’s test (α = 0.05). RESULTS There was a significant difference among the materials studied (p < 0.0001 – for all the variables analyzed) and between the degradation methods (baseline/biological biodeg- radation/mechanical degradation) (for Ra and KHN: p < 0.0001; for L*: p = 001; for initial ΔE: p = 0005); in addition, there was significant difference for the interaction between the three studied factors (for Ra: p < 0.0001; for KHN: p = 0.0327; for L*: p = 0.05). Table 2 shows the Ra averages found by different materials and different degradation processes. At baseline, e.Max showed the highest roughness and the Empress and Z350 composite showed very low and similar roughness. After biological degradation, the Ra values of e.Max increased still remaining statistical similarity to the baseline, but it was showed a significant increase for Z350 and Empress composite, being that Z350 showed the lowest roughness. However, after mechanical degradation, the roughness of Z350 remained similar to the biological degradation and higher than baseline values; for Empress and e.Max, Ra was smaller than biological values; being statistically similar to the baseline values for e.Max, but for Empress it was lower than those obtained after biological degradation but higher than baseline values (Table 2). 6 Maran et al. Table 3 shows the KHN values obtained for different materials and degradation mod- els. At baseline, Empress and Z350 composite showed the lowest and similar KHN val- ues. After the S mutans degradation, it can be observed that all materials experienced a decreasing on KHN values. However, in a descending significant order it can be observed that e.Max showed the highest KHN values followed by Empress and Z350. After the mechanical degradation, the hardness increased for Z350 and remained similar to the biological degradation values for Empress. In all conditions, baseline, biodegradation and mechanical degradation exposition, e.Max showed the highest KHN values (Table 3). Table 4 shows the ΔE values for different materials submitted to different degrada- tion models. After the biological biodegradation, the Empress composite showed the lowest ΔE, similar to e.Max values. There was no statistical difference between all materials studied after the mechanical degradation (Table 4). Table 5 shows the values obtained for lightness of the materials submitted to differ- ent degradation models. All materials studied showed the same performance when submitted to different deg- radation methods. At baseline, all materials showed similar lightness pattern. How- ever, the exposition to S mutans biofilm provided the lowest lightness for Empress Table 2. Means (standard deviations) of surface roughness (Ra) (µm) for the different experimental conditions. Materials Baseline Biological biodegradation Mechanical degradation Z350 0.26 (0.09) Bb 1.51 (1.08) Ab 1.48 (0.70) Aab Empress 0.24 (0.07) Cb 2.71 (0.43) Aa 0.86 (0.34) Bb e.Max 2.60 (0.71) ABa 3.26 (0.98) Aa 2.20 (0.79) Ba Means followed by different capital letters in the same line and small letters in the same column were significantly different (p < 0.05). Table 3. Means (standard deviations) of the Knoop hardness (KHN) for the different experimental conditions. Materials Baseline Biological biodegradation Mechanical degradation Z350 62.1 (24.0) Bb 51.6 (15.38) Bc 82.38 (17.8) Ab Empress 82.2 (15.8) Ab 80.4 (13.5) Ab 106.2 (16.7) Ab e.Max 811.7 (139.9) Aa 656.8 (105.6) Aa 757.8 (151.1) Aa Means followed by different capital letters in the same line and small letters in the same column were significantly different (p < 0.05). Table 4. Means (standard deviations) of color change (ΔE) for the different experimental conditions. Materials Biological biodegradation Mechanical degradation Z350 2.8 (1.0) a 1.9 (0.5) a Empress 2.1 (0.5) b 1.2 (0.4) a e.Max 3.0 (0.6) a 1.7 (1.1) a Groups denoted by a different letter represent significant difference (p<0.05). 7 Maran et al. when compared with e.Max and Z350. A significant increase on lightness can be observed for all materials studied after mechanical degradation, although the highest values were observed for Z350 and the lowest for Empress. e.Max showed interme- diary lightness (Table 5). DISCUSSION Aesthetic restorative materials are prone to a gradual degradation process in the oral cavity because of pH changes (chemical or bacterial action), temperature, chewing and brushing, depending on the composition of the restorative material15,17,18. This study revealed that the composites showed similar average roughness after polishing. After biological degradation, the composites show different variations of roughness, which may depend on the hydrolytic stability of the polymer matrix15,16. According to Sarkar19 (2000), these changes are due to the absorption and diffusion of water and organic acids from the bacterial metabolism, internal resin matrix, inter- faces between the inorganic particles, pores and other defects. The greater increase in the roughness of the composite Empress compared to Z350 can be attributed to the fact that the second one is a composite which has only nanoparticles particles as fillers, with less interstitial spacing of the matrix, which decreases its hydrolysis; in addition, we should mention the presence of Bis-EMA, a hydrophobic monomer, which favors the hydrolytic stability13,16,20,21. At baseline, the ceramics showed higher roughness than the polished composites, which is due to the irregularities in the surface from the resulting glazing process. After the biological biodegradation, there was no significant variation in the sur- face roughness of the ceramics, which may be due to the stability of the material, as it is considered the most inert dental material10. These results are in agreement with the study of Padovani et al.1. The final roughness of the ceramics was com- parable to the original, which is in agreement with studies evaluating resistance to toothbrush abrasion8,22. The surface hardness of Z350 remained statistically similar after biological degra- dation; the presence of the TEGDMA monomer is justified in both composites, which increases the degree of conversion, reducing leaching and softening10,21.Hardness becomes an important parameter to measure the performance of materials in the oral environment, being correlated with the resistance to compression and abrasion, and it indirectly reflects the rate of polymerization of the material5. Materials with decreased hardness have reduced longevity and may require early replacement of the restoration5,23. Table 5. Means (standard deviation) of lightness (L*) for the different experimental conditions. Materials Baseline Biological biodegradation Mechanical degradation Z350 72.96 (0.23) Ba 72.78 (0.69) Ba 73.50 (0.29) Aa Empress 68.59 (0.53) Ba 71.81 (0.71) Bb 72.50 (0.51) Ab e.Max 72.60 (0.53) Ba 73.18 (0.55) Ba 73.30 (0.51) Aab Means followed by different capital letters in the same line and small letters in the same column were significantly different (p < 0.05). 8 Maran et al. The hardness of the Z350 composite was lower than the Empress after the biological biodegradation, and this can be attributed to differences in size and distribution of the fillers on these materials4,24; beyond the aforementioned factor, it can be speculated that this is the association of the consequent hydrolysis of the polymeric matrix with the inorganic framework differences of the studied composites24. The ceramic, showed higher hardness, in all degradation methods than the compos- ites studied, which is due to their glass character, as there is the coalescence of the particles and higher solid density in the sintering process1,10. The nanofilled com- posite may be prone to absorbing liquids because of the greater contact area-load matrix, and this interface is more susceptible to fluid accumulation in the bacterial biofilm, or alternatively, the spaces resulting from the presence of the imperfect engagement of charged particles in the polymeric matrix. Spaces or “microvoids” in the polymeric matrix can increase the retention of acids and thereby increase the degradation of Z35024. In the oral cavity, the deleterious effects of the biodegradation are generally associ- ated with toothbrush abrasion, as the abrasiveness of the toothpaste along with the toothbrush may promote the displacement of charged particles, which is directly pro- portional to the size of these effects25. The hardness of the composite Z350 increased, this can be attributed to the process of maturation or late polymerization of the com- posite26, and that the nano-sized loads have greater contact surface with the organic phase, improving the hardness of the material27. The Empress composite showed decreased roughness after the mechanical deg- radation, but not returning to equivalent values to the baseline; this may be due to the losses of larger particles, which weakens the softened matrix and enhances the abraded mass of the polymer, removing the softened layer13,20. The roughness of Z350 remained similar to that observed after the biological degradation, and greater than at the baseline, which can be attribute to the effect of the bristles of the toothbrush atop the smaller interstitial space in the polymeric matrix, which could result in a higher abrasion resistance16. The lightness and stability of the color, important properties of aesthetic restor- ative materials, are influenced by various factors such as the composition of the inorganic portion, diet, habits or even the organic matrix. The sensitivity of the human eye to detect color variation translates to ΔE> 3.3; thus, the color changes were imperceptible to human sensitivity22,28,29. However, analysis of the CIELAB color scale coordinates (L *, a *, and b *) showed significant changes in the val- ues of L*. The lightness is the ability of the material to reflect direct light and is closely related to the surface characteristics of the material, ranging from light (100) to dark (0)8,30. After the mechanical degradation, there were increases in the lightness for all materi- als studied, probably the optical changes that occurred reflect physical and chemical reactions: i) internal – such as hydrolysis – or ii) in the surface – such as increased roughness –, as these affect the lightness through changes in the refractive index and reflection, respectively28, since the specimens were not exposed to any coloring agent and there was standardization of the thickness of the specimens. 9 Maran et al. The biodegradation provided on composite materials as Ra, was recovered after mechanical degradation for all materials. However, for hardness, only Empress Direct has recovered that after bio and mechanical degradation. Color was significantly affect after mechanical degradation for all materials studied. 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