Addition of zirconium oxide to Biodentine increases radiopacity and does not alter its physicochemical and biological properties

Abstract Objectives: To evaluate the radiopacity of Biodentine (BD) and BD associated with 15% calcium tungstate (BDCaWO4) or zirconium oxide (BDZrO2), by using conventional and digital radiography systems, and their physicochemical and biological properties. Materials and Methods: Radiopacity was evaluated by taking radiographs of cement specimens (n=8) using occlusal film, photostimulable phosphor plates or digital sensors. Solubility, setting time, pH, cytocompatibility and osteogenic potential were also evaluated. Data were analyzed using one-way ANOVA and Tukey post-test or two-way ANOVA and Bonferroni post-test (α=0.05). Results: BD radiopacity was lower than 3 mm Al, while BD ZrO2 and BD CaWO4 radiopacity was higher than 3 mm Al in all radiography systems. The cements showed low solubility, except for BDCaWO4. All cements showed alkaline pH and setting time lower than 34 minutes. MTT and NR assays revealed that cements had greater or similar cytocompatibility in comparison with control. The ALP activity in all groups was similar or greater than the control. All cements induced greater production of mineralized nodules than control. Conclusions: Addition of 15% ZrO2 or CaWO4 was sufficient to increase the radiopacity of BD to values higher than 3 mm Al. BD associated with radiopacifiers showed suitable properties of setting time, pH and solubility, except for BDCaWO4, which showed the highest solubility. All cements had cytocompatibility and potential to induce mineralization in Saos-2 cells. The results showed that adding 15% ZrO2 increases the radiopacity of BD, allowing its radiography detection without altering its physicochemical and biological properties.


Introduction
Mineral trioxide aggregate (MTA) is an endodontic repair material used for root perforation, root-end filling and vital pulp therapy, due to its sealing capability, biocompatibility and ability to induce mineralization [1][2][3] .
It is composed of Portland cement clinker (80%) and bismuth oxide (20%) as a radiopacifier 4 . However, MTA is difficult to manipulate and insert into cavities, has a long setting time 5 and causes tooth discoloration due to the chemical reaction of Bi 2 O 3 with the dentin matrix 6 .
In an attempt to improve the drawbacks of the MTA, tricalcium silicate-based cements have been developed. One such formulation is Biodentine ® (BD) (Septodont; Saint-Maur-des-Fossés, France) which was developed for use as a bioactive dentin substitute, and has been indicated for coronal and radicular restorations, pulp capping, pulpotomy, root repair and root-end filling [7][8][9] . BD powder contains 80% tricalcium silicate, 15% calcium carbonate and 5% zirconium oxide (ZrO 2 ), which is used as a radiopacifier agent. The mixing liquid is composed of water, calcium chloride and a hydrosoluble polymer 7 . Studies have shown that this cement has better handling conditions 10 and lower setting time in comparison with MTA 11 . Moreover, BD has biocompatibility, bioactivity and ability to induce mineralized tissue formation 9,12 .
Despite of the good properties of BD, researchers who have used it as root-end filling have reported that its primary clinical limitation is low radiopacity, which hinders radiographic assessment of treatment and follow-up 8,13 . Considering the appropriate properties of tricalcium silicate-based cements associated with the radiopacifiers ZrO 2 or calcium tungstate (CaWO 4 ) [14][15][16][17] , an option to improve the radiopacity of BD is to associate it with these radiopacifiers.
On the other hand, there is no consensus among in vitro studies that evaluated the radiopacity of BD.
Some studies showed radiopacity lower 18,19 and others higher 16,20 than that recommended by the International Standards Organization 21 , which is at least 3 mm Al.
ISO 6876:2012 21 recommends that radiopacity of endodontic materials must be evaluated in conventional radiographic films. However, nowadays, the radiopacity of these materials has been evaluated using digital images 20,22 . Nonetheless, there is no consensus on how digital radiography influences the radiopacity of materials. Rasimick, et al. 23 (2007) reported that barium-containing materials tend to be 13% more radiopaque in radiographs obtained by digital sensor than on conventional film. On the other hand, other endodontic materials appeared to be from 7% to 20% less radiopaque on radiographs obtained by photostimulable phosphor plates than on the conventional type 24 . However, the only studies that showed radiopacity of BD higher than 3 mm Al used photostimulable phosphor plates 16,20 . Therefore, it is important to evaluate the radiopacity of BD and BD associated with CaWO 4 or ZrO 2 , using conventional and digital radiography systems, and, in addition, to evaluate the effects on the physicochemical and biological properties of BD when these radiopacifiers are added.
The aim of this study was to evaluate (1) the radiopacity of BD and BD associated with CaWO 4 or ZrO 2 using conventional and digital radiography systems, and (2) the physicochemical properties of setting time, pH and solubility, and biological properties of cytocompatibility and osteogenic potential of these cements. The null hypothesis was that there would be no difference in the radiopacity values of BD using conventional or digital radiography systems, and that CaWO 4 or ZrO 2 associated with BD would not change its radiopacity nor its biological and physicochemical properties.

Material and methods
The materials evaluated were BD and BD associated with radiopacifiers ZrO 2 or CaWO 4 , in a proportion of 85% BD and 15% ZrO 2 (BD ZrO 2 ) or 15% CaWO 4 (BD CaWO 4 ) by weight. The composition, manufacturer, and proportion used for the materials are shown in

Radiopacity
Eight specimens measuring 10 mm in diameter by 1 mm wide were made for each material, according to the ISO 6876:2012 21 specification. The specimens were stored at 37°C and 95% humidity for 24 hours and, subsequently, they were radiographed using conventional or digital radiography systems.
-Conventional radiography: The specimens were placed on occlusal radiographic E-speed films (Kodak; Rochester, NY, USA) along with an 8-step   Powder: BD (Sepdodont); calcium tungstate (Sigma-Aldrich) Liquid: solution of BD (Sepdodont) 0.7 g BD + 0.12 g CaWO 4 /6 drops The plate was kept at 37°C and 95% humidity for 48 hours. After this time, the cements were exposed to ultraviolet light (UV) under laminar flow for 30 minutes to prevent contamination. Five mL of serum-free DMEM were added in each well of the plate in which the material was accommodated. For 24 h, the plate was maintained at 37°C, 95% humidity and 5% CO 2 to create the extract of each cement 28 . DMEM was used as negative control and 20% dimethyl sulfoxide (DMSO) as positive control.

Cell viability assays
Cell viability was evaluated through methyl-thiazoltetrazolium (MTT) and neutral red (NR) assays. Saos-2 cells were seeded at a density of 1x10 5 cells/mL in a 96-well plate containing DMEM with FBS 10% for 24 hours. After, the cells were exposed to the cement extracts at 1:1, 1:2, 1:4, 1:8 and 1:12 dilutions (v:v) in serum-free DMEM for 24 hours 2,29 . Additionally, the viability of cells exposed to the cement extracts at 1: 8 dilution for 1, 3 and 7 days was assessed by MTT, renewing the extracts every two days.
MTT assay was performed by replacing the cement extracts with 100 µL of a 5 mg/mL MTT solution (Sigma-Aldrich) and the cells were incubated at 37°C, 95% humidity and 5% CO 2 for 3 h. After this period, the well content was removed and 100 µL of acidified isopropyl alcohol (HCl 0.04 N and isopropyl alcohol) was added to the extract. The absorbance of the solutions was measured in a spectrophotometer at wavelength of 570 nm. NR assay was performed by replacing the cement extracts with 100 µL DMEM containing 50 µg NR/mL of solution (Sigma-Aldrich). The cells were incubated at 37°C, 95% humidity and 5% CO 2 for 3 h, and the well content was removed to proceed with solubilization of the colorimetric product in 100 µL of an ethanol solution mixture (50% ethanol and 1% acetic acid).
The absorbance of the solutions was measured in a spectrophotometer at wavelength of 570 nm. In both assays, the absorbance readings were normalized with cells exposed to the culture medium. Three independent experiments were performed for both assays.

Statistical analysis
The results were analyzed using one-way analysis

Radiopacity
According to Table 1, in all the digital and convectional radiography systems used, the BD radiopacity did not reach 3 mm Al, as specified by ISO 6876:2012 21 . BD associated with radiopacifiers ZrO 2 or CaWO 4 had radiopacity higher than 3 mm Al shown in all radiographic systems. The radiopacity of the materials obtained with the use of a Kodak digital sensor was higher than the values obtained through the other systems (p<0.05). The cements associated to the radiopacifiers showed statistically higher radiopacity than the BD (p<0.05). There was no statistically significant difference between BD ZrO 2 and BD CaWO 4 (p>0.05), except for the occlusal film scanned, in which BD CaWO 4 had higher radiopacity than BD ZrO 2 (p<0.05). Radiography images of the materials obtained with different systems are shown in Figure 2.
pH According to Table 2, the deionized water containing the materials had alkaline pH in all time intervals. No differences were found between groups (p>0.05), except for the control one (deionized water), which had significantly lower pH values than the other groups in all time intervals (p<0.05).

Solubility and setting time
According to       (Figure 3a and 3b). Considering the MTT results, the 1:8 dilution was chosen for ALP activity and ARS assays.

ALP activity
According to Figure 4a, Saos-2 cells exposed to cement extracts had viability similar to (p>0.05) or greater (p<0.05) than the control group (culture medium) at 1, 3 and 7 days. The lowest cell viability was detected on the first day of cell exposure to the cement extracts, increasing over the time intervals of 3 and 7 days. At 7 days, Groups BD ZrO 2 and BD CaWO 4 showed higher cell viability values than BD and the control group (p<0.05), however, there was no significant difference between BD and the control group (p>0.05). The ALP activity (Figure 4b) of cement groups at 1, 3 and 7 days was similar to (p>0.05) or greater (p<0.05) than that of the control group.
At 7 days, the highest ALP activity was detected for BD, followed by BD ZrO 2 (p<0.05) and BD CaWO 4 (p<0.05). There was no significant difference between Figure 4-Saos-2 cell viability evaluated by methyl-thiazol-tetrazolium (MTT) assay (a) and alkaline phosphatase (ALP) activity (b) evaluated after exposure to BD, BD ZrO 2 and BD CaOW 4 at 1:8 dilution and culture medium (negative control) for 1, 3 and 7 days. The ALP activity of cement groups at 1, 3 and 7 days was similar to or greater than the control group. At 7 days, the highest ALP activity was detected for Group BD followed by Groups BD ZrO 2 and BD CaWO 4 . There was no significant difference between Group BD CaWO 4 and the control group. Bars with different letters represent significant differences between groups in each period. BD=Biodentine; BD ZrO 2 =BD with addition of 15% zirconium oxide; BD CaWO 4 =BD with addition of 15% calcium tungstate; NC=negative control J Appl Oral Sci.

Alizarin red staining
As observed in Figure 5, all materials induced a greater production of mineralized nodules when compared to the negative control group (p<0.05) after 21 days of cell exposure to the cement extracts.
There was no significant difference among the cement groups (p>0.05).

Discussion
The first aim of this study was to evaluate the radiopacity of BD and BD associated with 15% CaWO 4 or ZrO 2 . For the manipulation of BD and BD associated with radiopacifiers, 6 drops of liquid were used, instead An alkaline medium contributes to osteogenic potential and antibacterial activity of a material 31 .
The addition of radiopacifiers did not change the pH of BD CaWO 4 or BD ZrO 2 when compared with BD; all cements had alkaline pH in all time intervals. Alkaline pH of BD has been studied in literature 12,19 and it results from the hydration reaction of tricalcium silicate, which forms calcium hydroxide that dissociates, thereby alkalinizing the medium 16   All cements induced a greater mineralized nodule production when compared with the negative control group. Bars with different letters represent significant differences between groups. BD=Biodentine; BD ZrO 2 =BD with addition of 15% zirconium oxide; BD CaWO 4 =BD with addition of 15% calcium tungstate; NC=negative control 3 and 10 days of immersion in water, respectively 33 .
Opposite to our results, some researchers reported solubility of BD higher than 3% evaluated in periods from 24 hours to 7 days of immersion in water 11,34 .
The addition of ZrO 2 did not change the solubility of BD, and the addition of CaWO 4 increased it to 3.63%. ALP activity and ARS assays were performed to evaluate the osteogenic potential of the cements. ALP plays a critical role in mineralization 38 . After 7 days of Saos-2 cell exposure to the cement extracts, ALP activity increased, especially for BD and BD ZrO 2 , when compared with untreated cells. These results were in line with those of a previous study that showed that BD and tricalcium silicate-based cement associated with 30% ZrO 2 showed potential to induce mineralization 27 . ARS is a test used to evaluate calcium deposits in cell culture. All materials induced greater production of mineralized nodules when compared with the control group. These results agreed with studies which revealed that BD induced similar or greater production of mineralized nodules than unexposed cells 27 . In summary, BD, BD associated with ZrO 2 or CaWO 4 had cytocompatibility, induced ALP activity and production of mineralized nodules necessary to promote endodontic repair.

Conclusions
BD radiopacity was lower than 3 mm Al, as shown by the conventional and digital radiography systems, and adding 15% ZrO 2 or CaWO4 was sufficient to increase it to values higher than the minimum specified by ISO 6876:2012 21 (>3 mm Al). BD associated with radiopacifiers showed suitable properties of setting time, pH and solubility, except for BD CaWO 4 , which exhibited higher solubility than BD and BD ZrO 2 . All cements evaluated had cytocompatibility and potential to induce mineralization in Saos-2 cells. The results showed that the addition of 15% ZrO 2 increases the radiopacity of BD allowing its radiography detection, without altering its physicochemical and biological properties.