Dental discoloration caused by Grey-MTAFlow cement: analysis of its physicochemical, biological and antimicrobial properties

Abstract Tricalcium silicate-based cement are materials used in reparative and regenerative procedures in endodontics. A recently proposed formulation aimed to enhance handling during clinical use with a versatile material applicable by syringe. Although, the use of bismuth oxide as radiopacifier and grey raw powder are drawbacks considering aesthetics. Objectives Evaluate physicochemical, biological, and antimicrobial properties of Grey-MTAFlow (Ultradent) and assess whether the addition of zinc oxide (ZnO) prevents dentinal discoloration caused by bismuth oxide. Methodology Grey-MTAFlow was manipulated in 'thin' consistency for all tests. Luminosity, color change, ion migration to dentine, radiopacity, setting time, ISO 6876:2012 linear flow, volumetric lateral flow and central filling of simulated grooves scanned using micro-computed tomography (μCT), pH, calcium release, volumetric change using μCT, chemical characterisation, cytotoxicity, and antimicrobial activity were assessed. Addition of 5% ZnO to Grey-MTAFlow and a bismuth-containing experimental composition were comparatively tested. Statistical analyses used Shapiro-Wilk, T-test, ANOVA, and Kolmogorov-Smirnov (p<0.05). Results The addition of ZnO to Grey-MTAFlow prevented dentine darkening after 90 days due to bismuth migration reduction, although no statistical difference was found (p=0.863). ZnO addition significantly enhanced Grey-MTAFlow radiopacity without differences in initial setting time. Grey-MTAFlow presented an ISO linear flow of 10.9 mm and a balanced volumetric lateral flow with central filling in μCT evaluation. All compositions presented an alkaline pH after immersion. Grey-MTAFlow had a significantly higher calcium ion release after 28 days in comparison to 24 hours (p=0.011) and volumetric expansion of 0.4±1.8% after immersion. ZnO addition altered the hydrated cement matrix once calcium hydroxide (portlandite) could not be detected in characterisation. Neither of the materials produced inhibition halos nor reduced bacterial turbidity, but all presented cytocompatibility above 100%. Conclusion Grey-MTAFlow expanded after immersion and exhibited higher luminosity values after the evaluation period when ZnO was added, but chemical modifications after this addition occurred.


Introduction
Tricalcium silicate-based cement has been widely used for endodontic therapy since the 1990s, when mineral trioxide aggregate (MTA) was introduced. 1 These types of cement are essential to the range of endodontic materials due to its repair-inductive biological properties. 2 One of the shortcomings with the use of MTA is the susceptibility to tooth discoloration, which is caused by the interaction between the bismuth oxide contained in its formulation with dental hard structures 3 and sodium hypochlorite used during root canal therapy. 4,5 The radiopacifier bismuth oxide present in the MTA composition was shown to interact with dentine and other components, resulting in the migration of ions into the dentine and tooth staining. 3,4,6 Bismuth oxide is beneficial regarding radiopacity due to its high molecular weight (465.96 g/mol), which requires small amounts for an ideal level of radiopacity (i.e., 3 mm of Al -ISO 6876:2012) compared to alternative radiopacifiers, such as calcium tungstate (287.92 g/ mol), and zirconium oxide (123.21 g/mol). 7 Grey-MTAFlow cement (Ultradent Products Inc., South Jordan, UT, USA) containing tricalcium and dicalcium silicate and bismuth oxide has recently been introduced in the market. This material is available in powder and water-based gel and can be prepared at different consistencies, namely: thin, thick, and putty. Its thin consistency allows it to be delivered with syringe and needle, which facilitates its insertion clinically. Grey-MTAFlow cement has an alkalinizing capability, low solubility, adequate radiopacity, biocompatibility, and induction of mineralisation. 8,9 However, no studies have yet evaluated its color stability. This material is grey, and the presence of bismuth oxide in its composition is a factor that may cause discoloration after contact with the dentine. 3 Aluminum fluoride 10 and zinc oxide (ZnO) 11 were added in small amounts to bismuth-containing formulations and tested in order to keep bismuth oxide as radiopacifier, but avoiding color changes. The addition of ZnO to MTA Angelus in small amounts (5%) prevented dental discoloration caused by bismuth oxide. 11 The addition of ZnO to a different composition material such as Grey-MTAFlow cement might prevent an expected darkening of the dental structures.
This study aimed to investigate the physicochemical, biological, and antimicrobial properties of Grey-MTAFlow in comparison to the 5% ZnO in-weight added to this commercial formulation and an experimental cement composed of white tricalcium silicate, bismuth oxide and similar addition of 5% ZnO. The hypothesis tested is that Grey-MTAFlow cement containing bismuth oxide causes dental discoloration, which can be inhibited by adding 5% ZnO without significant Both Grey-MTAFlow compositions were mixed in 'thin' consistency (1 big-end plus 1 small-end spoon [0.19 g] to 3 drops) following the manufacturers' recommended powder to liquid ratio in order to obtain the best fluidity of this material. ZnO was added to the Grey-MTAFlow cement's original formula in-weight at a percentage of 5% by using an electronic analytical scale (Gehaka AND-GR-202, Tokyo, Japan) with 10 -3 precision. The experimental cement was mixed in a constant ratio of 1 g of powder to 0.25 mL of liquid.

Color change in dentine
A total of 25 stain-free bovine teeth were selected for this study, according to a previously reported methodology 3 . Samples were sectioned to obtain enamel-dentine blocks measuring 10×10 mm and thickness standardised in 3.5±0.1 mm. A cavity of 5.0 mm in diameter and 1.5 mm in depth was prepared at the centre of the dentine surface using a high-speed number 4054 bur (Medical Burs Sorensen, São Paulo, SP, Brazil). The specimens were then immersed in 1% sodium hypochlorite for 30 minutes, washed, immersed in 20% EDTA (pH 7.7) for 2 minutes, irrigated with a final flush of distilled water, and dried with gauze.
Dental discoloration caused by Grey-MTAFlow cement: analysis of its physicochemical, biological and antimicrobial properties 2020;28:e20200269 3/15 Only the edges of the cavities were conditioned with 37% phosphoric acid for 30 seconds, and a layer of adhesive (Adper Single Bond 2, 3M ESPE, Sumaré, SP, Brazil) was applied only to the conditioned edge of the cavity and then light-cured (Optilight LD Max, Gnatus, Ribeirão Preto, SP, Brazil) for 20 seconds. In sequence, the tested compositions were inserted into the cavities at a depth of 1.5 mm. After the initial setting time, the cavities were sealed with a natural flow resin (B2, Nova DFL, Rio de Janeiro, RJ, Brazil).
The polymerization was performed with a LED curing light unit (Optilight LD Max) for 60 seconds, and the specimens were stored in separate flasks containing tap water at 37 o C throughout the test period. Triple antibiotic paste (metronidazole, ciprofloxacin, and minocycline), and unfilled samples served as positive and negative controls, respectively. Luminosity (L) assessment was performed before filling, 24 hours, 28 days, and 90 days after filling.
A spectrophotometer (Vita EasyShade V, VITA Zahnfabrik, Bad Sackingen, Germany) was used to obtain the values of the International Commission on Illumination (CIE) 'L,' 'a' and 'b' in a light-controlled room. The values were recorded and the color change (ΔE) between 90 days and before the filling was calculated by using the following formula: ΔE = [(L 1 -L 0 ) 2 + (a 1 -a 0 ) 2 + (b 1 -b 0 ) 2 ] 1/2 where '0' stands for values before filling and '1' the 90-days values.

Radiopacity
Radiopacity was evaluated according to the ISO 6876:2012 standard and a previously established methodology 7 . Three metallic rings per group were used to shape the cement specimens (10×1 mm), which were kept at 37±1°C and relative humidity until the final set. After this period, the specimens were radiographed with digital sensor (Micro Imagem, Indaiatuba, Sao Paulo, Brazil) at 60 kV, 10 mA, 0.3-second exposure, and focus-film distance of 30 cm. Instead of a radiographic film, a digital sensor was used to avoid film processing effects. An aluminum scale was used as a comparative radiographic density, and samples were evaluated in grey-scale values and converted into aluminum equivalent thickness (mm Al) using the Image J software (National Institutes of Health, Bethesda, MD, USA).
Setting time ISO 6876:2012 standard and a previously reported methodology 12 were followed for setting time analysis.
Freshly mixed cement was placed into metallic rings of 10 mm internal diameter and 2 mm thickness  (n=3). The specimens were kept at a temperature of 37±1°C and humidity of 95±5% during the test and periodically subjected to vertical pressure by using Gilmore needles (according to ASTM-266/2008) in controlled room temperature at 20±1°C, in which a needle weighing 113.4 g was used for initial setting time and one weighing 453.6 g for final setting time.
The setting time (in minutes) was determined from the mixing to the moment that it was no longer possible to observe the marking of each needle on the surface of the specimens. Central cavity filling was obtained considering the volume of material present in this region (in mm 3 ).

pH and calcium ion release in solution
Fifteen acrylic teeth (n=5) with standardised root-end cavity with 3 x 1±0.1 mm (depth versus width) were used for assessment. 12 The cavities were filled with the tested cement and immersed in individual flasks containing 10 mL of deionized water.
Experimental periods of 3 hours, 24 hours and 28 days were used (this was due to the expected completion of the setting reaction of tricalcium silicate-based materials 14 ). For analysis of pH, a calibrated pH-meter (371; Micronal, Sao Paulo, Brazil) was used, and measurements performed in each period. Standard calcium solutions were used as a reference for calcium ion release and evaluated by using atomic absorption spectroscopy (AA6800; Schimadzu, Tokyo, Japan) equipped with a calcium-specific hollow cathode lamp, the calcium ion release results were expressed in mg/L.

Volume change
Volume change was evaluated by using a μCT scanner based on a material's amount compatible with a surgical root-end cavity 3 × 1±0.1 mm as previously reported. 12 Cavities were filled with the cement (n=5), and the initial scanning was performed according to the same previously described parameters. After scanning, each specimen was individually immersed in flasks containing 15 mL of deionized water and stored at 37ºC for 28 days. After this period, they were removed from the flasks, dried with filter paper, and re-scanned with the same initial parameters. The values of rescanned volume were compared to the initial ones, representing the percentage volume change.

Agar diffusion test
This analysis was based on a previously described methodology 17 . Enterococcus faecalis (ATCC 29212) and Porphyromonas gingivalis (ATCC 49417) bacterial strains were previously sub-cultured on appropriate medium plates and particular gaseous conditions. E.
Three sterile stainless-steel cylinders (4.0 × 1.0 × 10 mm; inner diameter of 6 mm) filled with freshly-spatulated material were put in contact with the inoculated surface for each microorganism in separate plates. Due to the silicone components in the liquid of the Grey-MTAFlow cement, an additional metallic disc containing only its water-based gel was also tested. A metallic cylinder containing a sterile paper disc impregnated with 2% chlorhexidine gel was used as a negative control. Plates were kept at

Results
Color change in dentine Mean, standard deviation, and statistical difference for L and ΔE are shown in Table 1 The bismuth used as radiopacifier migrated from the materials into the tooth structure, being more intense at the dentine/material interface in the Grey-MTAFlow.
Considering the Grey-MTAFlow + 5% ZnO interface, bismuth ions were more concentrated in the material matrix. The migration of Si into the dentine was also evident in the elemental maps for all tested cement.

Radiopacity
The results for radiopacity are listed in Table 2. All the tested compositions achieved a minimum of 3 mm of Al in a cement thickness of 1 mm as required by the ISO standard. A significant increase in radiopacity was observed for Grey-MTAFlow + 5% ZnO in comparison to pure Grey-MTAFlow (p=0.0004).

Setting time
The results for the initial and final setting times are listed in   cement (a, b), Grey-MTAFlow cement + 5% ZnO (c, d), experimental cement (e, f) and negative control (g, h). Darkening is evident in Grey-MTAFlow cement. Dentine is stained, with grey color visible on the buccal surface. Stereomicroscopic images at 2x magnification. Scanning electron micrographs and energy dispersive analysis with elemental maps of sectioned teeth filled with the tested materials. Grey-MTAFlow cement (i), Grey-MTAFlow cement + 5% ZnO (j) and experimental cement (k). Calcium (Ca), silicon (Si), and phosphorus (P) were found in all specimens. Bismuth (Bi) was found in Grey-MTAFlow cement, Grey-MTAFlow cement + 5% ZnO and experimental cement corresponding to the radiopacifier. Zinc (Zn) was verified in Grey-MTAFlow cement + 5% ZnO and experimental cement corresponding to the additive. The migration of radiopacifier and Si into the dentine was evident in the elemental maps. The molecular weight of Bi was found to be high in the Grey-MTAFlow cement, with a high incidence of these ions at the cement/dentine interface. This was not verified in the Grey-MTAFlow cement + 5% ZnO, whose molecular weight of Bi was lower and ions evenly distributed in the material matrix. 2020;28:e20200269 8/15 are listed in Table 2. ISO flow analysis showed that the experimental cement had a significantly higher flow, followed by Grey-MTAFlow + 5% ZnO, with statistical difference compared to the other materials (p<0.050).
The volumetric analysis revealed that the experimental cement presented the lowest central cavity filling and lateral volumetric flow, probably due to its consistency (p<0.050). On the other hand, Grey-MTAFlow showed the highest volumetric filling in the central cavity, and ZnO addition did not alter the volumetric material filling (p>0.050).

pH and calcium ion release in solution
The mean values of pH and calcium ion release obtained are shown in Table 3. All the tested compositions presented alkaline pH ranging between 8 and 9. ZnO addition significantly increased the pH values of Grey-MTAFlow (p=0.022) after 28 days. No differences for pH values were observed between 24hour and 28-day periods, regardless of the material (p=0.461).

MTAFlow showed significantly higher ion release
at 3-hours compared to Grey-MTAFlow + 5% ZnO (p<0.0001), but similar to that of the experimental cement (p=0.112). On the other hand, ZnO addition significantly increased the calcium ion release at the 24-hours period (p=0.043), although no difference was observed between these two compositions at 28 days (p=0.954). Considering the experimental periods, the 28-day period showed significantly higher values regardless of the material analysed (p=0.011).

Volume change
The results for volume change are listed in Table 3

Discussion
Tooth discoloration caused by MTA has been widely demonstrated both in vitro and clinically. This material drawback occurs due to the interaction between the radiopacifier bismuth oxide and dental structures. 3,18,19 Bismuth oxide is present in the composition of the Grey-MTAFlow and, consequently, is expected a color change when in contact with dentine. A previous investigation 11 showed that 5% ZnO used as an additive in a white-powder material (MTA Angelus) inhibited the destabilisation of bismuth oxide and consequent discoloration of the tooth. The present study tested the hypothesis that the addition of 5% ZnO into a grey-powder composition would also prevent tooth discoloration caused by this material.
This hypothesis was partially accepted once L values after 90 days were higher after addition, although no significant difference was observed.
Aesthetics is an important aspect to be observed after dental restorative procedures. 20 Tooth discoloration was evident in Grey-MTAFlow cement after 90 days as the staining of dentine was intense, where bismuth ions were detected. This finding is in accordance with previous studies, 3,5 which found bismuth and silicon ions in dentine. The L values continually decreased after the insertion of Grey-MTAFlow, indicating a tendency of light absorbance.
The addition of 5% ZnO to Grey-MTAFlow inhibited tooth discoloration visually, but the difference was not statistically significant compared to the group using pure Grey-MTAFlow. This fact can be attributed to the period of analysis that had a maximum of 90 days.
The tendency of darkening in the group using pure Grey-MTAFlow would probably continue over time, and thus, a statistical difference could be detected.
Regarding the experimental cement, high values of L were observed for this cement, suggesting that its color was stable and that ZnO prevented discoloration.
In the sectioned specimens, there was an evident area of different color at the dentine/material interface. The use of bovine teeth in the present study aimed to provide a sufficiently flat surface, enabling color assessments, and thus standardized measurements.
Previous investigations did not report differences in dental discoloration pattern when using bovine or human teeth models. 3,5 Radiopacity is a vital material characteristic and should be sufficient to allow distinction from dentin and adjacent anatomical structures in follow-up radiographic and tomographic exams. 7 The results obtained in the present study showed that Grey-

MTAFlow radiopacity was significantly enhanced by
ZnO addition. This result could be explained by the additional radiopacity provided by the ZnO addition.
However, all the tested compositions presented adequate radiopacity (i.e., at least 3 mm of Al), according to the ISO standard. A previous study 8 found similar values of radiopacity for the Grey-MTAFlow. Considering the clinical procedures in which these reparative materials are used, short initial setting time is a critical characteristic. 8,25 ZnO addition slightly prolonged the final setting time compared to that of pure Grey-MTAFlow. The experimental cement had the longest initial and final setting times, which is probably related to the particle size and prolonged hydration reaction as a result. Setting time is directly related to solubility, which was significantly higher in the experimental cement. Grey-MTAFlow and Grey-MTAFlow + 5% ZnO cement showed similar volume changes. However, Grey-MTAFlow cement presented a slight volume increase after immersion, probably due to water absorption during the immersion period.  27 In the present study, all the tested materials presented pH values of above 8 after a 28-day immersion in water. Guimarães, et al. 8 (2017) found higher pH values for Grey-MTAFlow cement, whose 'putty' consistency was possibly associated with higher solubility and calcium ion release in water.

Considering clinical use, facilitated handling
For the experimental cement, calcium release was significantly higher after 28 days, suggesting that a higher volume loss contributed to these increased values. A balance between material-matrix compounds release and stability is vital to provide sealing after long periods since these materials are not expected to be replaced.
The material characterisation was performed for both un-hydrated and hydrated cement after 28 days of immersion in HBSS. This methodology is well documented in the literature and has been used to evaluate the composition of endodontic materials. [27][28][29] In the present study, calcium hydroxide was detected

Conclusion
Grey-MTAFlow containing bismuth oxide as a radiopacifier can potentially cause dentine discoloration, which was influenced by the addition of ZnO considering luminosity. Physical properties were not affected by such addition, although chemical 2020;28:e20200269 13/15 modifications were verified with no detection of calcium hydroxide deposition. Biologically, all the tested materials presented cytocompatibility, but no detectable antimicrobial activity. Besides, the use of an inert alternative radiopacifier would be of great advantage considering the long-term aesthetic outcome.