1http://dx.doi.org/10.20396/bjos.v19i0.8660181 Volume 19 2020 e200181 Original Article 1 Chair of Dental Materials, Faculty of Dentistry, University of the Republic, Montevideo, Department of Montevideo, Uruguai. 2 Department of Dental Materials, School of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Corresponding author: Fabrício Mezzomo Collares Department of Dental Materials, School of Dentistry, Federal University of Rio Grande do Sul. Ramiro Barcelos Street, 2492, Rio Branco, 90035-003, Porto Alegre, RS, Brazil. fabricio.collares@ufrgs.br Phone number: +55 51 33085198 Received: June 22, 2020 Accepted: September 25, 2020 Surface and mechanical properties of adhesives with calcium phosphates challenged to different storage media Marcelo Matias Mederos Gómez1 , Isadora Martini Garcia2 , Vicente Castelo Branco Leitune2 , Fabrício Mezzomo Collares2,* Aim: To evaluate the behavior of experimental dental adhesives with hydroxyapatite (HAp), alpha-tricalcium phosphate (α-TCP) or octacalcium phosphate (OCP) after storing them in three different media: dry storage, distilled water, or lactic acid. Methods: An experimental adhesive resin was formulated with bisphenol A glycol dimethacrylate, 2-hydroxyethyl methacrylate, and photoiniciator/co-initiator system. HAp (GHAp), α-TCP (Gα-TCP), or OCP (GOCP) were added to the adhesive resin at 2 wt.%, and one group remained without calcium phosphates to be used as a control (GCtrl). The adhesives were evaluated for surface roughness, scanning electron microscopy (SEM), and ultimate tensile strength (UTS) after storing in distilled water (pH=5.8), lactic acid (pH=4) or dry medium. Results: The initial surface roughness was not different among groups (p>0.05). GHAp showed increased values after immersion in water (p<0.05) or lactic acid (p<0.05). SEM analysis showed a surface variation of the filled adhesives, mainly for Gα-TCP and GHAp. GHAp showed the highest UTS in dry medium (p<0.05), and its value decreased after lactic acid storage (p<0.05). Conclusions: The findings of this study showed that HAp, OCP, and α-TCP affected the physical behavior of the experimental adhesive resins in different ways. HAp was the calcium phosphate that most adversely affected the surface roughness and the mechanical property of the material, mainly when exposed to an acid medium. Keywords: Dentin-bonding agents. Calcium phosphates. Acids. Polymers. Tensile strength. http://orcid.org/0000-0002-1561-2283 https://orcid.org/0000-0002-7388-0200 https://orcid.org/0000-0002-5415-1731 https://orcid.org/0000-0002-1382-0150 2 Gómez et al. Introduction Recurrent caries at the tooth-restoration interface is one of the major causes of res- toration replacement over time1. This outcome is related to materials’ hydrolytic and enzymatic degradation when in contact with the oral environment, leading to higher surface roughness, biofilm accumulation at the margin, and caries development2. Moreover, restorative materials may not completely seal the tooth interface. Mainly over time, the sealing ability still is a concern since gaps are prone to caries devel- opment3. Restorative resin-based materials have been modified to decrease polym- erization shrinkage, hydrolysis degradation, and to decrease gaps formation at the interface via a biomimetic remineralization approach4. Bioactive fillers have been added to resins to provide them bioactivity with the ultimate purpose of reducing the incidence of caries around the restoration’s margin. Calcium orthophosphates (CaP) are the most representative fillers able to release calcium and phosphate ions, which are retained in the oral biofilm and induce dental remineralization5-8. CaP present different molecular forms, crystalline structures, and solubility values9,10. Pre- vious studies evaluated CaP as fillers in experimental adhesive resins showing promising results such as increased bond strength11,12 and mineral deposition at the tooth6,8,11. Currently, studies that evaluate ion-releasing bioactive materials, such as those filled with bioglasses and CaP, stored them in water13,14, ethanol13, artificial saliva15 or simulated body fluid5. However, the release of ions may increase materials’ roughness over time16, espe- cially when they are exposed to acid medium, which could occur in the presence of an aci- dogenic biofilm17. The results of a recent in vitro study suggest that low pH increases the surface roughness and alter the superficial topography of resin-based orthodontic adhe- sives18. The change in the roughness may indicate modifications not only in the morphol- ogy of the material but also in its chemical and physical stability19,20. The bioactive material must maintain its mechanical and chemical properties to seal the cavity adequately21. In a previous study, the authors formulated an experimental adhesive resin composed of bisphenol A glycol dimethacrylate, 2-hydroxyethyl methacrylate, and a photoiniti- ator/co-initiator system11. The material was filled with different calcium orthophos- phates at 2 wt.%: hydroxyapatite (HAp; Ca5(PO4)3(OH)), alpha-tricalcium phosphate (α-TCP; Ca3(PO4)2), or octacalcium phosphate (OCP; Ca8H2(PO4)65H2O) 11. The filled adhesives were compared to the base resin without CaP (control group), and they showed a higher degree of conversion. Furthermore, the α-TCP group showed a high microshear bond strength compared with the other groups. The α-TCP and HAp groups induced mineral deposition at the tooth-resin interface, suggesting that these fillers could be an alternative to formulate bioactive dental resins. Despite these findings, the effect of different storage media on the behavior of adhe- sives composed of α-TCP, HAp, or OCP was not investigated so far. The aim of this study was to evaluate the behavior of experimental adhesive resins with HAp, α-TCP, or OCP after storing them in three different storage media. The null hypotheses to be tested are: (1) there are no differences among the adhesives formulated with different CaP regarding their surface and mechanical properties; (2) different storage media do not influence the surface and mechanical properties of the adhesives. 3 Gómez et al. Materials and Methods In this study, the dependent variables analyzed are surface roughness, surface mor- phology, and ultimate tensile strength. Two independent variables were analyzed: (1) the variable “addition of CaP”, with four different adhesives composed solely by the base resin or with 2 wt.% of hydroxyapatite (HAp), alpha-tricalcium phosphate (α-TCP), or octacalcium phosphate (OCP); (2) the variable “storage medium”, in which the adhesives were immersed: dry storage, distilled water, or acidic solution (Figure 1). OCP Ca8H2(PO4)65H2O α-TCP Ca3(PO4)2 HAp Ca5(PO4)3(OH) Bis-GMA HEMA Photoinitiator system Dry (no treatment) Distilled water (pH 5.8) Lactic Acid (pH 4.0) Adhesive resin Storage media Surface roughness Scanning Electron Microscopy Ultimate Tensile Strenght Figure 1. Representative illustration that summarizes the materials and methods of the study. Experimental Adhesive Resin Formulation The experimental adhesive resin was formulated mixing 66.66 wt.% bisphenol A gly- col dimethacrylate and 33.33 wt.% 2-hydroxyethyl methacrylate. Camphorquinone and ethyl 4-(dimethylamino) benzoate were added as a photoinitiator system at 1 mol%. Butylated hydroxytoluene was added at 0.01 wt.% as polymerization inhibitor. These reagents were purchased from Aldrich Chemical, St Louis, MO, USA. Three CaP were previously synthesized, and they were added in this base resin at 2 wt.%: α-TCP (Gα-TCP, 6.03 µm) 22, OCP (GOCP, 4.94 nm) 23 and HAp (GHAp, 26.7 nm) 24. A group without CaP was used as control (GCtrl), totaling four groups. The mixture (resin/particles) was hand-mixed for 5 min, sonicated for 180 s, and hand-mixed again for 5 min. Surface Roughness (Ra) Five samples per group were prepared (10 mm diameter X 1 mm thickness) using a polyvinylsiloxane mold. The uncured adhesive resins were inserted in the molds between two transparent Mylar strips. The samples were light-cured for 30 s on each side (Radii Cal, SDI; Bayswater, Victoria, Australia, 1200 mW/cm2). The top of each 4 Gómez et al. sample was marked and divided in half. Four measurements of surface roughness were performed immediately on one half using a profilometer (Mitutoyo, Surftest SJ–201P, Chicago, USA) with a tracing length of 2 mm and 0.25 mm cut-off. Then, each sample was submerged and stored in individual-hermetic containers for 3 days at 37 °C17, in 10 mL25,26 of different media as distilled water or lactic acid with pH at 5.8 and 417, respectively. The samples were placed vertically so that the surfaces to be tested were kept exposed to the different media. The pH of these media was evaluated along with the study via a digital pH meter (DM-22, Digimed, São Paulo, SP, Brazil). After 3 days of storage, four new measurements were performed on the other half on the top of each sample. The initial roughness (Ra1), final roughness (Ra2), and roughness variation (ΔRa) were recorded for each group. Surface morphology via scanning electron microscopy (SEM) The surface morphology of three samples per group used in the roughness assess- ment (stored in distilled water and lactic acid) was evaluated via SEM. Other three samples per group were prepared and stored in a dry environment inside a desiccator with silicon dioxide at 37 °C for 3 days to be also analyzed via SEM. The samples were placed on metallic stubs and gold-sputter coated (15–25 nm) (SDC 050, Baltec, Vaduz, Liechtenstein). SEM analysis (SEM, JSM 6060, JEOL, Tokyo, Japan) was per- formed under 7 kV, at 5,000´ and 8,000´ magnification. Ultimate Tensile Strength (UTS) Thirty samples per group were prepared in a metallic matrix with an hourglass shape (8 mm long, 2 mm wide, 1 mm thickness, and 1 mm2 at constriction area) after photo- activation for 30 s on each side27. After photoactivation, the samples were measured with a digital caliper (Mitutoyo, Kawasaki, Kanagawa, Japan; accuracy of 10 µm) to obtain the constriction area of each one. Then, the thirty samples from each group were divided and submerged into 1 mL of the three media of storage (n = 10, dry envi- ronment in a desiccator, distilled water, or lactic acid) to be sat for 3 days at 37 °C. The specimens were fixed in metallic jigs with cyanoacrylate resin to be tested for tensile strength. The tests were performed in a universal testing machine (EZ Test EZ-SX, Shimadzu, Japan) at a crosshead speed of 1 mm/min. The values were obtained in newtons, and the final UTS was expressed in megapascals (MPa) using the constric- tion area of each sample. Statistical Analysis The data were analyzed using the software SigmaPlot®, version 12.0 (Systat Software, Inc., San Jose, CA, USA). Data distribution was evaluated using the Shapiro–Wilk test. One-way analysis of variance (ANOVA) was used to compare groups for initial surface roughness. Paired t test was used for each group to evaluate the difference between immediate and final surface roughness. Krus- kal-Wallis was used to compare ΔRa among groups in both media, and Dunn’s was used as post-hoc after immersion in lactic acid. Two-way ANOVA was used to compare groups for UTS dry medium, distilled water or lactic acid. A signifi- cance level of 0.05 was considered. 5 Gómez et al. Results The results of surface roughness before (Ra1) and after (Ra2) immersion of the exper- imental adhesive resins, as well as the ΔRa, in distilled water and in lactic acid are shown in Table 1. The authors did not perform other statistical analysis such as three- way ANOVA, split-plot two-way ANOVA, or two-way ANOVA with repeated measures to analyze this data because there is a dependency within the same group (the same group was tested before and after the storage in the liquids). Moreover, the samples tested for immersion in water or lactic acid are not the same since this is a destructive method and the same sample could not be immersed in both liquids one after the other. Statistical analysis with repeated measures considering different immersions (in water or lactic acid) should not be applied. In this context, the one-way ANOVA revealed no statistically significant differences among groups for Ra1 (p>0.05). After immersion in distilled water, GHAp roughness increased (p<0.05), while the other groups showed no statistically significant differences (p>0.05). Moreover, there was no differ- ence among groups for ΔRa after immersion in water (p>0.05). On the other hand, the materials presented different behavior after immersion in lactic acid solution. While there was no difference among groups for Ra1 (p>0.05), the roughness of Gα-TCP and GHAp increased after immersion in lactic acid (p<0.05) and GCtrl and GOCP had no differ- ences between Ra1 and Ra2 (p>0.05). When comparing ΔRa after immersion in lactic acid, GHAp showed the highest variation among groups, with statistical difference in comparison to GCtrl (p<0.05). Table 1. Results of surface roughness of the experimental adhesive resins with different calcium phosphates before and after the immersion in distilled water or lactic acid. Group Storage in distilled water Storage in lactic acid Roughness before immersion Roughness after immersion ΔRa (%) Roughness before immersion Roughness after immersion ΔRa (%) (Ra1, μm) (Ra2, μm) (Ra1, μm) (Ra2, μm) GCtrl 0.09 (±0.01) Aa 0.12 (±0.05) a 47.93 (±70.28) A 0.11 (±0.04) Aa 0.13 (±0.03) a 24.74 (±49.88) B GOCP 0.10 (±0.03) Aa 0.15 (±0.07) a 59.35 (±76.50) A 0.10 (±0.04) Aa 0.16 (±0.10) a 59.07 (±75.10) AB Gα-TCP 0.08 (±0.03) Aa 0.13 (±0.03) a 78.85 (±97.40) A 0.10 (±0.03) Aa 0.21 (±0.06) b 116.94 (±83.58) AB GHAp 0.07 (±0.01) Aa 0.21 (±0.08) b 173.91 (±96.28) A 0.08 (±0.02) Aa 0.30 (±0.06) b 285.64 (±110.34) A Different capital letters indicate statistically significant difference in the same column (p<0.05). Different small letters indicate statistically significant difference in the same row within the same medium of storage (distilled water or lactic acid) (p<0.05). The images from SEM analyses corroborate the findings of surface roughness mea- surement. Few differences can be observed in the surface of GCtrl between dry stor- age and distilled water storage (Figure 2 A–D). Higher irregularities are identified for GCtrl when it was stored in lactic acid (Figure 2 E, F). As well as observed for GCtrl, almost no differences are observed among images of dry storage and distilled water for GOCP (Figure 3 A–D). When exposed to lactic acid, more irregularities are observed (Figure 3 E, F). Compared with GCtrl, GOCP showed higher defects when exposed to 6 Gómez et al. lactic acid. The surface of Gα-TCP stored in distilled water, and lactic acid (Figure 4 C–F) showed larger irregularities than GCtrl. GHAp showed the highest difference on the sur- face between dry storage and distilled water storage (Figure 5 A–D) compared with GCtrl, GOCP, and Gα-TCP. In addition, after storing in lactic acid (Figure 5 E, F), GHAp presented higher irregularities, with larger cracks and cavities with irregular borders distributed on an irregular surface. Figure 2. Scanning electron microscopy of GCtrl at dry (no treatment), distilled water, or lactic acid storage. Few differences are observed for this group without calcium phosphates addition when the surface is exposed to dry storage (A and B) compared to that after water storage (C and D). After the exposition to lactic acid, GCtrl presents higher irregularities (E and F). N o tr ea tm en t Control group A ft er 3 -d ay s to ra ge in di st ill ed w at er ( pH =5 .8 ) A ft er 3 -d ay s to ra ge in la ct ic a ci d (p H =4 .0 ) A C E B D F 7kV 7kV 7kV 7kV 7kV 7kV 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 7 Gómez et al. Figure 3. Scanning electron microscopy of GOCP at dry (no treatment), distilled water, or lactic acid storage. Few differences are observed for GOCP when the surface is exposed to dry storage (A and B) compared to that after water storage (C and D). After the exposition to lactic acid, this group showed higher irregularities (E and F). N o tr ea tm en t Octacalcium phosphate group A ft er 3 -d ay s to ra ge in di st ill ed w at er ( pH =5 .8 ) A ft er 3 -d ay s to ra ge in la ct ic a ci d (p H =4 .0 ) A C E B D F 7kV 7kV 7kV 7kV 7kV 7kV 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 8 Gómez et al. Figure 4. Scanning electron microscopy of Gα-TCP at dry (no treatment), distilled water, or lactic acid storage. High differences are observed within this group when “no treatment” (A and B) is compared to the surfaces after water (C and D) or lactic acid (E and F) exposition. Observe that Gα-TCP shows a much more irregular surface after water or lactic acid storage in comparison to GCtrl and GOCP. N o tr ea tm en t Alpha-tricalcium phosphate group A ft er 3 -d ay s to ra ge in di st ill ed w at er ( pH =5 .8 ) A ft er 3 -d ay s to ra ge in la ct ic a ci d (p H =4 .0 ) A C E B D F 7kV 7kV 7kV 7kV 7kV 7kV 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 9 Gómez et al. The results of the UTS of the experimental adhesive resins exposed to different media are shown in Table 2. After dry storage, the values ranged from 28.69 (±10.93) MPa for GCtrl to 46.34 (±10.72) MPa for GHAp, with a statistically significant difference between GCtrl and GHAp (p<0.05). The values of UTS after distilled water storage ranged from 30.53 (±6.07) MPa for GOCP to 39.26 (±9.44) MPa for GCtrl, without differences among groups (p>0.05). After lactic acid storage, the values of UTS ranged from 29.56 (±6.43) MPa for GHAp to 33.87 (±11.93) MPa for GCtrl, also without differences among groups (p>0.05). GHAp was the only group that presented a statistically significant difference among the different storage media, with lower UTS values after lactic acid storage (29.56 ± 6.43 MPa) than dry medium (46.34 ± 10.72 MPa) (p<0.05). Figure 5. Scanning electron microscopy of GHAp at dry (no treatment), distilled water, or lactic acid storage. High differences are observed within this group when “no treatment” (A and B) is compared to the surfaces after water (C and D) or lactic acid (E and F) exposition. Observe that, mainly after the lactic acid exposition, GHAp shows more irregularities than the other adhesive resins, areas with large cracks and cavities surrounded by irregular borders. N o tr ea tm en t Hydroxiapatite group A ft er 3 -d ay s to ra ge in di st ill ed w at er ( pH =5 .8 ) A ft er 3 -d ay s to ra ge in la ct ic a ci d (p H =4 .0 ) A C E B D F 7kV 7kV 7kV 7kV 7kV 7kV 5μm 5μm 5μm 2μm 2μm 2μm x5,000 x5,000 x5,000 x8,000 x8,000 x8,000 10 Gómez et al. Table 2. Results of ultimate tensile strength of the experimental adhesive resins with different calcium phosphates after their storage in different media: dry, distilled water or lactic acid. Group Dry (MPa) Water (MPa) Lactic Acid (MPa) GCtrl 28.69 (±10.93) Ba 39.26 (±9.44) Aa 33.87 (±11.93) Aa GOCP 35.96 (±17.19) ABa 30.53 (±6.07) Aa 32.61 (±6.93) Aa Gα-TCP 34.43 (±11.48) ABa 38.14 (±7.88) Aa 30.36 (±13.22) Aa GHAp 46.34 (±10.72) Aa 34.60 (±9.89) Aab 29.56 (±6.43) Ab Different capital letters indicate statistically significant difference in the same column (p<0.05). Different small letters indicate statistically significant difference in the same row (p<0.05). Discussion Bioactive materials with ion-releasing fillers such as CaP have been investigated to induce the remineralization process of dental tissues7. Studying the behavior of bioactive materials when exposed to different media could assist in understanding their physical properties. In this study, adhesive resins with HAp, α-TCP, or OCP were tested regard- ing their physical properties after storing in distilled water, lactic acid, or dry medium. There were significant differences among the adhesives with different CaP, leading to the rejection of the first null hypothesis. Furthermore, the storage media influenced the behavior of the adhesives, which led us also to reject the second null hypothesis. Dental materials are susceptible to suffering chemical and physical modifications in the oral environment due to hydrolysis and to bacterial enzymes, leading to their deg- radation over time28. High surface roughness contributes to the attachment of micro- organisms and biofilm development28, besides making it more difficult to maintain hygiene28. In 1990, an in vivo study using fluorethylenepropylene or cellulose acetate strips suggested that the surface roughness of Ra = 0.2 μm was a threshold value for bacterial retention in intraoral surfaces29. Moreover, it is suggested that when the val- ues are lower than 0.2 μm, the materials’ chemical properties may be more important for biofilm formation than the surface roughness. Currently, lower values up to 0.1 µm are recommended for polishing resins with inor- ganic particles to reduce biofilm accumulation2. Gα-TCP and GHAp presented Ra higher than 0.2 µm after immersing in lactic acid, and GHAp showed values above 0.2 µm even after distilled water storage. In addition to inducing remineralization, ion-releas- ing materials have been suggested to inhibit biofilm formation by increasing the pH around them and delaying bacterial colonization30. However, the exposed CaP on the materials’ surface, accompanied by the increase of surface roughness, was shown not to decrease bacterial adhesion17. In this study, as well as in the previous report17, the samples were not subject to pH cycles, which could lead to different results and, maybe, lower surface roughness differences. However, this method is a way to evalu- ate the material over an extreme situation. Besides the surface roughness measurement, the surface morphology of the experi- mental adhesive resins was evaluated via SEM, which supported the results observed for Ra. In distilled water, Gα-TCP and GHAp showed larger holes interspersed with small prominences on an irregular surface compared with α-TCP or HAp in a dry medium. A uniform pattern over the entire surface of Gα-TCP and GHAp was observed, probably due to a slight hydrolytic effect on the resin matrix31. After lactic acid storage, the variation 11 Gómez et al. of surface integrity was more pronounced for CaP groups than for GCtrl. This result corroborated the values found for ΔRa, mainly for GHAp, which should statistically sig- nificant difference for GCtrl after immersion in acid. We could also observe that the GOCP showed small grooves and holes scattered on the surface after lactic acid storage, while Gα-TCP presented a similar pattern to Gα-TCP immersed in distilled water but with cracks in greater quantity. The group containing HAp showed larger cavities with irregular limits distributed over a slightly smooth surface. It is possible that these cavities were created due to the release of HAp agglomerates because low values of surface area were found for HAp previously synthesized by the same method24. Nanoparticles are prone to agglomer- ation due to their high surface energy. In composite resins, agglomerates of nanopar- ticles presented lower adhesion to the organic matrix compared with microparticles, detaching over time32. These agglomerates jeopardize the composite resins com- pared to microparticles, making the material more susceptible to mechanical failure and surface wear33. This process could occur with HAp in the experimental adhesive resin because the small molecules of lactic acid could diffuse through pores among HAp agglomerates and produce faster dissolution34. In the mechanical analysis, the immediate UTS increased with HAp incorporation compared with GCtrl, without differences for Gα-TCP and GOCP. In distilled water, there were no differences in UTS, neither among groups nor between the same group comparing dry storage and distilled water storage. On the other hand, the mechan- ical performance was different after immersion in lactic acid solution, with GHAp showing reduced UTS compared with GHAp in dry storage. This group also presented the highest surface roughness variation after exposure to the lactic acid solution. These results suggest that, even without statistically significant differences among Gα-TCP, GHAp, and GOCP after storing in lactic acid, the UTS could be jeopardized for GHAp over time in acid conditions. The pH of the medium and the type of filler determine the release rate of the ions7, altering materials’ mechanical properties. HAp is soluble in acid solutions9, insoluble in alkaline solutions, and distilled water, while α-TCP and OCP are more soluble than HAp at neutral pH9. Even so, there were no differences for GCtrl, GOCP, and Gα-TCP, depend- ing on the storage media. The rationale for that may be a better distribution of OCP and α-TCP within the polymer, leading to lower CaP–resin interfaces to be exposed and to react with lactic acid. Another important factor related to the solubility of CaP is the size of the particles, in which the decrease to a nanoscale level can increase their dissolution35. Furthermore, CaP stability decreases with the increase of impuri- ties’ presence9 and the method used to synthesize the HAp24 leads to the presence of carbonates in the final powder, which may have favored its dissolution9. Here we observed the different behavior of bioactive resin-based restorative mate- rials depending on the type of CaP incorporated into them. Interestingly, the physi- cal response of the materials when facing various storing media depended on the CaP added. Therefore, further evaluations are encouraged in situ and in vivo to deeply understand the biological effects of these bioactive materials in patients with different risks of caries. 12 Gómez et al. In conclusion, the findings of this study showed that HAp, OCP, and α-TCP affected the physical behavior of the experimental adhesive resins in different ways. HAp was the CaP that most adversely affected the surface roughness and the mechanical property of the material, mainly when exposed to an acid medium. Acknowledgments The authors gratefully acknowledge Microscopy and Microanalysis Center (Federal Uni- versity of Rio Grande do Sul) for the transmission electron microscopy analysis. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 – scholarship. Conflicts of Interest: none. 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