Abstract

This study evaluated physicochemical properties, cytotoxicity and bioactivity of MTA Angelus (MTA), calcium silicate-based cement (CSC) and CSC with 30% Ytterbium oxide (CSC/Yb₂O₃). Setting time was evaluated using Gilmore needles. Compressive strength was evaluated in a mechanical machine. Radiopacity was evaluated using radiographs of materials and an aluminum scale. Solubility was evaluated after immersion in water. Cell viability was evaluated by means of MTT assay and neutral red staining, and the mineralization activity by using alkaline phosphatase activity and Alizarin Red staining. The data were submitted to ANOVA, Tukey and Bonferroni tests (5% significance). The bioactive potential was evaluated by scanning electron microscopy. The materials presented similar setting time. MTA showed the lowest compressive strength. MTA and CSC/Yb₂O₃ presented similar radiopacity. CSC/Yb₂O₃ showed low solubility. Saos-2 cell viability tests showed no cytotoxic effect, except to 1:1 dilution in NR assay which had lower cell viability when compared to the control. ALP at 1 and 7 days was similar to the control. MTA and CSC had greater ALP activity at 3 days when compared to control. All the materials present higher mineralized nodules when compared with the control. SEM analysis showed structures suggesting the presence of calcium phosphate on the surface of materials demonstrating bioactivity. Ytterbium oxide proved to be a properly radiopacifying agent for calcium silicate-based cement since it did not affected the physicochemical and biological properties besides preserving the bioactive potential of this material.

Key Words:
ytterbium; physicochemical analysis; silicate cement; endodontic; cell survival

Resumo

Este estudo avaliou as propriedades físico-químicas, citotoxicidade e bioatividade do MTA Angelus (MTA), cimento à base de silicato de cálcio (CSC) e CSC com 30% de óxido de itérbio (CSC/Yb₂O₃). O tempo de presa foi avaliado usando agulhas Gilmore. A resistência à compressão foi avaliada em uma máquina mecânica. A radiopacidade foi avaliada utilizando radiografias dos materiais e uma escala de alumínio. A solubilidade foi avaliada após imersão em água. A viabilidade celular foi avaliada por meio do ensaio MTT e coloração de vermelho neutro (NR), e a atividade de mineralização por meio da atividade da fosfatase alcalina e a coloração com Vermelho de Alizarina. Os dados foram submetidos aos testes ANOVA, Tukey e Bonferroni (5% de significância). O potencial bioativo foi avaliado por microscopia eletrônica de varredura. Os materiais apresentaram tempo de presa semelhante. O MTA mostrou menor resistência à compressão. MTA e CSC/Yb₂O₃ apresentaram radiopacidade semelhante. CSC/Yb₂O₃ apresentou menores valores de solubilidade. A viabilidade celular realizada pelos ensaios de MTT e NR não revelaram efeitos ctotóxicos em todas as diluições, exceto na diluição 1:1 no NR, o qual mostrou baixa viabilidade celular (p<0.05) em todos materiais testados quando comparado ao controle. A atividade de ALP em 1 e 7 dias foi similar ao controle (p>0.05). MTA e CSC tiveram significante aumento na atividade de ALP aos 3 dias quando comarados ao controle (p>0.05). Todos os materiais apresentaram grande produção de nódulos mineralizados quando comparados ao controle (P<0.05). A análise da SEM mostrou estruturas que sugerem a presença de depósitos de fosfato de cálcio na superfície dos materiais demonstrando bioatividade. O Yb₂O₃ mostrou ser um agente radiopacificador adequado em cimentos à base de silicato de cálcio uma vez que não afetou as propriedades físico-químicas e biológicas e ainda preservou o potencial bioativo desse material.

INTRODUCTION

Mineral Trioxide Aggregate (MTA) is a biomaterial composed of Portland cement (PC) ¹1 Kulan P, Karabiyik O, Kose GT, Kargul B. Biocompatibility of Accelerated Mineral Trioxide Aggregate on Stem Cells Derived from Human Dental Pulp. J Endod 2016;42:276-279. and bismuth oxide as radiopacifier ²2 Hwang YC, Lee SH, Hwang IN, Kang IC, Kim MS, Kim SH, et al.. Chemical composition, radiopacity, and biocompatibility of Portland cement with bismuth oxide. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:96-102.. PC is calcium silicate-based cement (CSC) that contains other chemical compounds such as tricalcium aluminate and presents physicochemical, mechanical and biological properties similar to MTA ²2 Hwang YC, Lee SH, Hwang IN, Kang IC, Kim MS, Kim SH, et al.. Chemical composition, radiopacity, and biocompatibility of Portland cement with bismuth oxide. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:96-102.^,33 Islam I, Chng HK, Yap AU. Comparison of the physical and mechanical properties of MTA and portland cement. J Endod 2006;32:193-197.. Calcium silicate cements might be suitable for biomedical applications as biomaterial for bone/dental repair ⁽⁴4 de Aza PN, Zuleta F, Velasquez P, Vicente-Salar N, Reig JA. (a’(H))-Dicalcium silicate bone cement doped with tricalcium phosphate: characterization, bioactivity and biocompatibility. J Mater Sci Mater Med 2014;25:445-452.. However, CSC do not have radiopacity to be distinguished from the surrounding anatomic structures ²2 Hwang YC, Lee SH, Hwang IN, Kang IC, Kim MS, Kim SH, et al.. Chemical composition, radiopacity, and biocompatibility of Portland cement with bismuth oxide. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:96-102.^,33 Islam I, Chng HK, Yap AU. Comparison of the physical and mechanical properties of MTA and portland cement. J Endod 2006;32:193-197..

Bismuth oxide (Bi₂O₃) reduces calcium hydroxide release, increases solubility, and causes deterioration in dimensional stability of MTA ⁵5 Cutajar A, Mallia B, Abela S, Camilleri J. Replacement of radiopacifier in mineral trioxide aggregate; characterization and determination of physical properties. Dent Mater 2011;27:879-891.. Furthermore, lower cell viability was demonstrated for CSC with Bi₂O₃ in comparison with zirconium oxide and calcium tungstate ⁽⁶6 Gomes Cornelio AL, Salles LP, Campos da Paz M, Cirelli JA, Guerreiro-Tanomaru JM, Tanomaru Filho M. Cytotoxicity of Portland cement with different radiopacifying agents: a cell death study. J Endod 2011;37:203-210.. New radiopacifiers have been studied as alternatives to Bi₂O_3.

Ytterbium (Yb) is a very ductile metal that slowly reacts with water and are used in imaging exams ⁷7 Xing H, Bu W, Ren Q, Zheng X, Li M, Zhang S, et al. A NaYbF4: Tm3+ nanoprobe for CT and NIR-to-NIR fluorescent bimodal imaging. Biomaterials 2012;33:5384-5393.. The incorporation of Ytterbium trifluoride (YbF₃) into glass ionomer cement promoted an increase in hardness of the material, and reduction in the setting and working times ⁽⁸8 Prentice LH, Tyas MJ, Burrow MF. The effect of ytterbium fluoride and barium sulphate nanoparticles on the reactivity and strength of a glass-ionomer cement. Dent Mater 2006;22:746-751.. The use of YbF₃ in the concentration of 30% was sufficient to promote proper radiopacity, without changing the properties of experimental cement ⁹9 Collares FM, Ogliari FA, Lima GS, Fontanella VR, Piva E, Samuel SM. Ytterbium trifluoride as a radiopaque agent for dental cements. Int Endod J 2010;43:792-797.. The association of PC with YbF₃ demonstrated radiopacity higher than 4 mm/Al and an increase in the compressive strength of the material ¹⁰10 Antonijevic D, Jeschke A, Colovic B, Milovanovic P, Jevremovic D, Kisic D, et al.. Addition of a Fluoride-containing Radiopacifier Improves Micromechanical and Biological Characteristics of Modified Calcium Silicate Cements. J Endod2015;41:2050-2057..

The CSC-based cements are described as bioactive, stable materials, capable of promoting the hydroxyapatite crystal formation ¹¹11 Han L, Okiji T, Okawa S. Morphological and chemical analysis of different precipitates on mineral trioxide aggregate immersed in different fluids. Dent Mater J 2010;29:512-517.. Analysis of the structure and chemical composition of these materials has shown the formation of calcium carbonate and calcium hydroxide when immersed in distilled water, and the formation of calcium phosphate when immersed in PBS ⁽¹¹11 Han L, Okiji T, Okawa S. Morphological and chemical analysis of different precipitates on mineral trioxide aggregate immersed in different fluids. Dent Mater J 2010;29:512-517.. The calcium ions released by MTA react with the phosphate of PBS, resulting in the formation of hydroxyapatite or carbonated apatite, components which are attributed to the mineralization ability of the calcium silicate-based cements ¹²12 Tay FR, Pashley DH, Rueggeberg FA, Loushine RJ, Weller RN. Calcium phosphate phase transformation produced by the interaction of the portland cement component of white mineral trioxide aggregate with a phosphate-containing fluid. J Endod 2007;33:1347-1351.^,1313 Bozeman TB, Lemon RR, Eleazer PD. Elemental analysis of crystal precipitate from gray and white MTA. J Endod 2006;32:425-428..

Yb₂O₃ is a potential radiopacifier for calcium silicate-based cements. Therefore, the aim of this study are to confirm that the association of Yb₂O₃ with calcium silicate-based cement presents physicochemical and biological properties for use as dental repair material. The null hypothesis is that there is no difference in physicochemical, biocompatibility and bioactivity of the calcium silicate-based cement (CSC), CSC with 30% Ytterbium oxide (CSC/Yb₂O₃), an experimental material, and the reference material (MTA) for endodontic purposes.

MATERIAL AND METHODS

The experimental materials and their powder-liquid ratios are in Table 1. CSC was evaluated in pure form or prepared in association with the 30% Yb₂O₃ (% by weight), which was mixed with distilled water using a powder/liquid ratio determined by previous tests to obtain consistency of retrofilling material and proper radiopacity.

The following tests were performed to evaluate the physicochemical and biological properties of the experimental materials.

Setting Time

The setting time was evaluated by dispensing the sealers into stainless steel molds measuring 10 mm in diameter and 1 mm high (n = 6). The materials were kept in an oven at 37 ºC and 95% humidity during the experiment. The setting time test was performed in accordance with ISO 6876 ¹⁴14 British Standard Institution. Dental root canal sealing materials. BS EN ISO 6876; 2012.. A Gilmore needle with a mass of 100 ± 0.5 g and 2 ± 0.1 mm in tip diameter was used. The setting time was determined as the mean time between beginning manipulation and the time elapsed until needle no longer produced marks on the cement surfaces.

Solubility

Circular molds were fabricated using polyvinyl siloxane impression material, based on the measurements recommended by Carvalho-Júnior et al. ⁽¹⁵15 Carvalho-Junior JR, Correr-Sobrinho L, Correr AB, Sinhoreti MA, Consani S, Sousa-Neto MD. Solubility and dimensional change after setting of root canal sealers: a proposal for smaller dimensions of test samples. J Endod 2007;3:1110-1116., with a thickness of 1.5 mm and internal diameter of 7.75 mm. Each mold was placed on a glass slide covered with cellophane paper film and filled with a material to be evaluated (n=6). A nylon thread was included in the mass of the cements. The set was stored in an oven at a temperature of 37°C and 95 % humidity for a period corresponding to three times the setting time of each material. The test specimens were removed from the molds, weighed on a precision balance HM-200 (A & D Engineering, Inc., Bradford, MA, USA) until stabilization of the mass (initial mass) and suspended by means of fixing the nylon threads inside plastic receptacles with lids, containing 7.5 mL of distilled and deionized water. The receptacles remained in the oven at 37°C for 7 days, when the test specimens were removed from the distilled water, dried and placed in a dehumidifying chamber, then to be weighed again until stabilization of the mass, thus obtaining the final mass. The loss of mass was expressed in percent of the original mass. Weighing procedures were repeated three times for each sample.

Compressive Strength

The cements were placed in a cylinder-shaped mold measuring 12 mm high and 6 mm in diameter. The set was placed in the oven. The set was stored at a temperature of 37°C and 95% humidity for a period corresponding to three times the setting time of each material. Then the samples were removed from the mold, and the flat surface of each sample was gently abraded with 600 grit abrasive paper. The samples remained in the oven (37 °C and 95% humidity) up to the time intervals of 24 h and 21 days, when they were submitted to the compression test in a universal testing machine (Emic DL Equipamentos e Sistemas de Ensaio, São José dos Pinhais, PR, Brazil) with a load cell of 5 KN and a speed of 0.5 mm/min. The maximum stress in MPa was calculated, using the maximum force and the diameter of the cylinders (1 MPa = 1N/1m²).

Radiopacity

The radiopacity test was performed based on ISO 6876 ¹⁴14 British Standard Institution. Dental root canal sealing materials. BS EN ISO 6876; 2012. standards. Teflon rings with an internal diameter of 10 mm and height of 1 mm were used to fabricate 6 test specimens of each material. After manipulation, the materials were placed in the rings, which were kept at 37 °C and 100% humidity for 48 h until the materials set. After this time interval, a disc of each material was placed on an aluminum scale with occlusal film under it, and the samples were thus radiographed. Radiographs were taken with an X-ray appliance model GE 1000 (General Electric, Milwaukee, WI, USA), operating at 50 kV, 10 mA, 18 pulses per second, with a focal distance of 33 cm, on occlusal film (Insight - KodakComp, Rochester, NY, USA). The films were processed in an automatic developer (A/T 2000®XR, Ar Techniques Inc., Hicksville, NY, USA), digitized with a scanner, and evaluated using the UTHSCSA ImageTool for Windows version 3.00 software, in which the areas of each degree of the aluminum scale and the cements were selected, to determine the equivalence of radiopacity of the cements in millimeters of aluminum.

Cell Cultures and Preparation of Extracts of the Materials

The Saos-2 cells (ATCC HTB-85) were cultivated in T-75 flasks (Jetbiofil, Elgin, SR, USA), containing D-MEM medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, Grand Island, NY, USA), penicillin (100 IU/mL) (Sigma-Aldrich), streptomycin (100 mg/mL) (Sigma-Aldrich) in 95% humidified atmosphere, 5% CO₂ and 37°C until confluence was reached.

To prepare the materials, one gram of powder was weighted and manipulated with distilled water, on a sterile glass plate. After preparation, the material was adapted to the bottom of a well of a 12-well plate and hydrated with dampened gauze. The plates were then maintained at 37 °C for 15 h to allow the complete setting reaction of the materials. After this time interval, the plates were exposed to ultraviolet light for 30 min, for disinfection, and 5 mL D-MEM culture medium, without FBS, was put into each well of the plates, and maintained at 37°C, 95% humidity and 5% CO₂ for 24 h. After 24 h, to observe the dose-response of each material, dilutions of the extracts (1:1, 1:2, 1:4 e 1:8) were made in D-MEM without PBS.

MTT Assay

Cell viability was determined by MTT assay. This assay is based on the capacity of the mitochondrial dehydrogenase enzyme to convert yellow tetrazolium 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium (MTT; Sigma-Aldrich) into colored formazan compounds, whose absorbance is proportional to the quantity of live cells. After counting and plating the Saos-2 osteoblast cells (1x10⁵ cells/mL) in 96-well plates with D-MEM culture medium with 10% FBS, the cells were exposed to the cement extracts (200 mL/ well) in dilutions of 1:1; 1:2; 1:4; 1:8 and control (cells not exposed to the extracts) for 24 h. After 24 h, the culture medium was replaced by 100 mL MTT (0.55 mg/mL) with incubation for 3 h. After this, each well was washed with 200 ul of phosphate buffer 1X (PBS 1X, Sigma-Aldrich), and 100 mL of isopropyl alcohol (HCl 0.04N isopropyl alcohol, Sigma-Aldrich) was added to each well to solubilize the formazan. The optical density (OD = 570 nm) was measured using an automated microplate reader (Elx800; Bio-Tek Instruments, Winooski, VT, USA). Three independent experiments were performed (n=12 per group).

Neutral Red (NR) Assay

Neutral red is a vital dye that is incorporated though endocytosis and accumulates in the lysosomes of cells that have intact membranes. For this assay, 1x10⁵ cells/mL were plated in 96-well plates with D-MEM medium with 10% FBS, which were incubated for 24 h. After this period, the cells were exposed to the cement extracts (200 mL/ well) for 24 h; the extracts were replaced with 100 mL of 50 mg NR / mL solution (Sigma-Aldrich), and then incubated at 37ºC, 95% humidity and 5% CO₂ for 3 h. Afterwards, 100 mL of the solution (50% ethanol and 1% acetic acid) (Sigma-Aldrich) was used for solubilization. The optical densities were measured in a spectrophotometer (Elx800; Bio-Tek Instruments, Winooski, VT, USA) at a wavelength of 570 nm ¹⁶16 Scelza MZ, Linhares AB, da Silva LE, Granjeiro JM, Alves GG. A multiparametric assay to compare the cytotoxicity of endodontic sealers with primary human osteoblasts. Int Endod J 2012;45:12-18.. Three independent experiments were performed (n=12 per group). According to the results of MTT and neutral red, the cement dilution selected for subsequent analyses was 1:8.

Alkaline Phosphatase Activity

Alkaline phosphatase activity was evaluated by means of the Labtest kit (Labtest, Lagoa Santa, MG, Brazil). The principle of the method is thymolphthalein monophosphate hydrolysis by alkaline phosphatase produced by the cells. After 24 h of plating (1x10⁵ cells / mL in 96-well plates), Saos-2 cells were exposed to the cement extracts in the dilution of 1:8, throughout the time intervals of 1, 3 and 7 days. The eluted materials were renewed every two days throughout all of the 7 days of exposure of the samples. After each experimental time interval, all the cement extracts were removed and the cells were washed with 200 μL PBS 1X, followed by the addition of 200 μL of a lauryl sulfate solution (1% in distilled water, Sigma-Aldrich) to each well. The samples went through a period of rest for 30 min at room temperature. In accordance with the manufacturer’s recommendation, 5 μL of each sample in lauryl sulphate solution was transferred to microtubes (Eppendorf, Hamburg, Germany), containing the enzyme substrate and buffer. The systems were incubated at 37°C for 10 min and then 500 mL of color reagent was added. The optical density was evaluated in an automatic microplate reader (ELx800, Bio-Tek Instruments, Winooski, VT, USA) at 590 nm. The assay was performed in sextuplicate and repeated three times in an independent manner (n=18/group). The data were expressed as ALP activity normalized by the number of viable cells in the respective time interval of culture (OD = 570 nm).

Alizarin Red Staining (ARS)

The alizarin red stain is used to identify calcium deposits in cell cultures. In order to evaluate the calcium deposited by the cells exposed to the different calcium silicate-based materials, Saos-2 cells were counted and plated (1x10⁴ cells/mL) in 12-well pates. For this experiment the culture medium was supplemented with 50 µg/mL of L-ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich). For 21 days, the D-MEM medium with and without the cement extracts (dilution 1:8) were renewed every two days.

After this period the medium was aspirated, the wells were washed with PBS, and the cells fixed with 10% paraformaldehyde (Sigma-Aldrich) at room temperature for 15 min. The monolayers were then washed twice with distilled water, before adding 1 mL of alizarin red staining (2% - pH 4.1 from Sigma-Aldrich), and thus maintained at room temperature for 20 min. After this period, the dye was aspirated and the wells were washed 4 times with distilled water (1 mL). The plates were then slightly inclined to favor aspiration, and left for 2 min to facilitate excess water removal, and were immediately photographed (Canon EOS-1D, Canon Inc, Tokyo, Japan) and analyzed with the ImageTool program (Dental School of the University of Texas Health Science Center, San Antonio, TX, USA). Three independent experiments were performed (n=12 per group).

Bioactivity

The bioactivity test was performed based on Bosso-Martelo et al. ¹⁷17 Bosso-Martelo R, Guerreiro-Tanomaru JM, Viapiana R, Berbert FL, Basso Bernardi MI, Tanomaru-Filho M. Calcium Silicate-Based Cements Associated with Micro- and Nanoparticle Radiopacifiers: Physicochemical Properties and Bioactivity. Int Sch Res Notices 2015;23,2015:874283.. The materials were manipulated and adapted in cylindrical molds measuring 1 mm high X 7.5 mm in diameter, and remained in an oven at 95% humidity and 37 °C. After this period of setting, the samples were completely immersed in 5 mL a PBS solution (for 500 mL distilled water; anhydrous NaCl - 90 g; Na₂HPO₄ - 13.65 g; Na₂H₂PO₂H₂O - 2.42 g). The test specimens remained immersed in PBS and kept in an incubator at 37 °C for 28 days. The PBS solution was not changed during the analysis period. After this period, silica gel and lime soda were placed in a receptacle, close to the samples, and maintained in the oven for 12 h to remove excess humidity from the materials, which were afterwards coated with carbon so that there would be electrical conductivity. Microstructural evaluation of the cement surfaces was performed by means of visualization in a scanning electron microscope SEM (JEOL, JSM-6610LV Scanning Electron Microscope, USA) in the secondary electron mode.

Statistical Analysis

Normality and equal variances tests were conducted prior to One-way ANOVA statistical analysis to guarantee that the data followed normal distribution and presented homoscedasticity. The physicochemical tests were submitted to Tukey´s comparison post-hoc test. Comparisons of cell viability and ALP activity were analyzed by two-way ANOVA with the Bonferroni post-test. Comparisons of alizarin red were performed by one-way ANOVA followed by the Tukey test. In all cases, the results were considered for a p value of less than 0.05.

RESULTS

Table 2 presents the mean values of setting time, solubility, compressive strength and radiopacity tests.

Setting time of CSC/Yb₂O_3, pure CSC and MTA were similar (p>0.05). All the materials presented low solubility, and MTA and CSC/Yb₂O₃ were similar (p>0.05). The compressive strength of the association CSC/Yb₂O₃ and pure CSC were similar (p>0.05) (24 h and 21 days). Radiopacity of CSC/Yb₂O₃ and MTA was similar (p>0.05).

The cell viability using MTT assay showed that MTA, CSC and CSC/Yb₂O₃ presented no cytotoxic effect, since the cell viability was higher for MTA and CSC/Yb₂O₃ (p<0.05) in all dilutions (Fig. 1A). Therefore, the 1:8 dilution was selected for the bioactivity experiments. The NR assay complemented the results of the MTT assay, and also showed that MTA, CSC and CSC/Yb₂O₃ presented no cytotoxic effect, except for the dilution1:1, in which there was lower cell viability for of the materials in comparison with the control group. In the 1:8 dilution, cell proliferation was significantly higher for CSC/Yb₂O₃ (p<0.05), (Fig. 1B).

Figure 1
Viability of human osteoblast cells (Saos-2) evaluated by MTT (A) and NR (B) assays after 24 h of exposure to different dilutions of the cements (1:1, 1:2; 1:4 and 1:8) CSC; CSC/Yb₂O₃ and MTA, to culture medium without FBS (control). Bars with different letters represent statistically significant difference between extracts of the cements and control group. ANOVA and Bonferroni (p<0.05). CSC = calcium silicate-based cement; calcium silicate-based cement associated with 30% ytterbium oxide radiopacifier; and MTA = Mineral Trioxide Aggregate; and C = control.

Figure 2 shows the structures that suggest the presence of calcium phosphate deposits on the surface of materials, demonstrating their bioactive potential.

Figure 2
Spherical structures, suggesting bioactivity of the material (calcium phosphate).

The lowest cell viability and alkaline phosphatase activity were observed after one day of exposure (Fig. 3A and Fig. 3B). ALP activity increased after this time (Fig. 3B). On the third day of exposure, a higher ALP activity value was observed for the MTA and CSC groups when compared with control (p>0.05). On the seventh day of exposure, all the groups were similar to the control as regards alkaline phosphatase activity (p>0.05).

Figure 3
(A) Cell viability of cell lineage Saos-2; and (B) Alkaline Phosphatase Activity (ALP) evaluated after exposure to extracts of the cements CSC; CSC/Yb₂O₃ and MTA in the dilution of 1:8, and culture medium (control) in time intervals of 1, 3 and 7 days. (C) Alizarin red staining (ARS). (D) Images of mineralized area with 2% Alizarin red staining. Bars with different letters represent significant differences between cement extracts and negative control group in each period. CSC = calcium silicate-based cement; calcium silicate-based cement associated with 30% ytterbium oxide radiopacifier; and MTA = Mineral Trioxide Aggregate; and C = control.

All materials induced a greater production of mineralized nodules when compared with the control (p<0.05). MTA and CSC produced more mineralized nodules than CSC/Yb₂O₃ (p<0.05) (Fig. 3C and Fig. 3D).

DISCUSSION

The use of the CSC associated with new radiopacifiers has been studied as an alternative to MTA ¹⁷17 Bosso-Martelo R, Guerreiro-Tanomaru JM, Viapiana R, Berbert FL, Basso Bernardi MI, Tanomaru-Filho M. Calcium Silicate-Based Cements Associated with Micro- and Nanoparticle Radiopacifiers: Physicochemical Properties and Bioactivity. Int Sch Res Notices 2015;23,2015:874283.^,1818 Bosso-Martelo R, Guerreiro-Tanomaru JM, Viapiana R, Berbert FL, Duarte MA, Tanomaru-Filho M. Physicochemical properties of calcium silicate cements associated with microparticulate and nanoparticulate radiopacifiers. Clin Oral Investig 2016;20:83-90.^,1919 Mestieri LB, Gomes-Cornélio AL, Rodrigues EM, Faria G, Guerreiro-Tanomaru JM, Tanomaru-Filho M. Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines. Braz Dent J 2017;28:65-71.. A calcium silicate-based cement with ytterbium trifluoride (YbF₃) demonstrated interesting physical-chemical properties for use as biomaterial ¹⁰10 Antonijevic D, Jeschke A, Colovic B, Milovanovic P, Jevremovic D, Kisic D, et al.. Addition of a Fluoride-containing Radiopacifier Improves Micromechanical and Biological Characteristics of Modified Calcium Silicate Cements. J Endod2015;41:2050-2057.. The investigation of materials physicochemical properties is important to predict their clinical application ²⁰20 Lucas CP, Viapiana R, Bosso-Martelo R, Guerreiro-Tanomaru JM, Camilleri J, Tanomaru-Filho M. Physicochemical Properties and Dentin Bond Strength of a Tricalcium Silicate-Based Retrograde Material. Braz Dent J 2017;28:51-56.. The incorporation of radiopacifiers to CSC may increase the material’s setting time ¹⁰10 Antonijevic D, Jeschke A, Colovic B, Milovanovic P, Jevremovic D, Kisic D, et al.. Addition of a Fluoride-containing Radiopacifier Improves Micromechanical and Biological Characteristics of Modified Calcium Silicate Cements. J Endod2015;41:2050-2057.^,1818 Bosso-Martelo R, Guerreiro-Tanomaru JM, Viapiana R, Berbert FL, Duarte MA, Tanomaru-Filho M. Physicochemical properties of calcium silicate cements associated with microparticulate and nanoparticulate radiopacifiers. Clin Oral Investig 2016;20:83-90.. Antonijevic et al. ¹⁰10 Antonijevic D, Jeschke A, Colovic B, Milovanovic P, Jevremovic D, Kisic D, et al.. Addition of a Fluoride-containing Radiopacifier Improves Micromechanical and Biological Characteristics of Modified Calcium Silicate Cements. J Endod2015;41:2050-2057. observed a short setting time for the association of different calcium silicate-based cement with YbF₃ 30% (6, 14 and 20 min). However, the present study using Yb₂O₃ showed that setting time was similar for CSC and CSC/Yb₂O₃. This result suggests that this radiopacifier does not interfere in the hydration process of the CSC.

All the materials presented solubility lower than 3%, in accordance with specifications ISO 6876 ¹⁴14 British Standard Institution. Dental root canal sealing materials. BS EN ISO 6876; 2012.. Low solubility is a favorable property for the biomaterial, and may promote long-term bacterial sealing ability ²¹21 Espir CG, Guerreiro-Tanomaru JM, Spin-Neto R, Chávez-Andrade GM, Berbert FL, Tanomaru-Filho M. Solubility and bacterial sealing ability of MTA and root-end filling materials. J Appl Oral Sci 2016;24:121-125..

The presence of a radiopacifying agent is essential to enable differentiation of the material from the adjacent areas. The results of this study showed that except for pure CSC, the association of CSC/Yb₂O₃ and MTA had radiopacity higher than 3 mm/Al, in accordance with the recommendation of the ISO 6876 ¹⁴14 British Standard Institution. Dental root canal sealing materials. BS EN ISO 6876; 2012.. In the present study, 30% of Yb2O3, by percentage of mass, was added to the CSC (CSC/Yb₂O₃), providing a material with proper radiopacity. This percentage is in agreement with Collares et al. ⁹9 Collares FM, Ogliari FA, Lima GS, Fontanella VR, Piva E, Samuel SM. Ytterbium trifluoride as a radiopaque agent for dental cements. Int Endod J 2010;43:792-797. that associated 30% of the YbF3 in experimental dental cements.

The association of CSC/Yb₂O₃ did not change the mechanical strength of CSC, probably not changing the mechanism of hydration of CSC ¹⁸18 Bosso-Martelo R, Guerreiro-Tanomaru JM, Viapiana R, Berbert FL, Duarte MA, Tanomaru-Filho M. Physicochemical properties of calcium silicate cements associated with microparticulate and nanoparticulate radiopacifiers. Clin Oral Investig 2016;20:83-90. and the compressive strength of these materials was higher than MTA. On the other hand, Prentice et al. ⁸8 Prentice LH, Tyas MJ, Burrow MF. The effect of ytterbium fluoride and barium sulphate nanoparticles on the reactivity and strength of a glass-ionomer cement. Dent Mater 2006;22:746-751. evaluated the addition of ytterbium fluoride (YbF3) to commercial glass-ionomer cement, and observed that the compressive strength decreased with the addition of YbF3. All the materials showed an increase in the compressive strength values after 21 days. According to Islam et al. ³3 Islam I, Chng HK, Yap AU. Comparison of the physical and mechanical properties of MTA and portland cement. J Endod 2006;32:193-197. this occurred due to the continuous setting of the materials, which resulted in greater stability over the course of time.

The bioactive potential refers to the capacity of a determined material to be biocompatible and to stimulate the formation of mineralized tissue. These characteristics are observed in MTA and materials based on calcium silicate ²²22 Gomes-Cornelio AL, Rodrigues EM, Salles LP, Mestieri LB, Faria G, Guerreiro-Tanomaru JM, et al.. Bioactivity of MTA Plus, Biodentine and experimental calcium silicate-based cements in human osteoblast-like cells. Int Endod J 2017;50:39-47.. The bioactive potential of MTA is attributed to the capacity for mineralization of PC components ¹²12 Tay FR, Pashley DH, Rueggeberg FA, Loushine RJ, Weller RN. Calcium phosphate phase transformation produced by the interaction of the portland cement component of white mineral trioxide aggregate with a phosphate-containing fluid. J Endod 2007;33:1347-1351.. Hydration of PC results in the deposition of calcium hydroxide on the surface of the material, which produces hydroxyapatite in the presence of phosphate-containing fluids ¹³13 Bozeman TB, Lemon RR, Eleazer PD. Elemental analysis of crystal precipitate from gray and white MTA. J Endod 2006;32:425-428.. In the present study, structures were observed in the SEM analysis suggesting the presence of calcium phosphate deposits on the surface of all the materials. These results suggested the biological potential of CSC and its association CSC/Yb₂O₃.

Another essential property of a biomaterial is the biocompatibility allowing the tissue repair. Calcium silicate-based cements have demonstrated potential for use as reparative materials and the interaction of the reparative materials with osteogenic cells is important to repair processes ²²22 Gomes-Cornelio AL, Rodrigues EM, Salles LP, Mestieri LB, Faria G, Guerreiro-Tanomaru JM, et al.. Bioactivity of MTA Plus, Biodentine and experimental calcium silicate-based cements in human osteoblast-like cells. Int Endod J 2017;50:39-47.. In the 1:8 dilution, in comparison with the control group, higher cell viability was observed for CSC/Yb₂O₃. MTT assay showed that the MTA, CSC and CSC/Yb₂O₃ cements have no cytotoxic effect on human osteoblast SAOS-2 cell lineage. Antonijevic et al. ¹⁰10 Antonijevic D, Jeschke A, Colovic B, Milovanovic P, Jevremovic D, Kisic D, et al.. Addition of a Fluoride-containing Radiopacifier Improves Micromechanical and Biological Characteristics of Modified Calcium Silicate Cements. J Endod2015;41:2050-2057. evaluated the biocompatibility of bioactive endodontic cements on mesenchymal cell differentiation, and showed that materials with YbF3 did not change the integrity of cells from bones of rats.

ALP is one of the main enzymes expressed during the early maturation of osteoblasts, and plays an important role in mineral deposition. Although MTA and CSC had showed grater ALP activity after 3 days in comparison to CSC/Yb₂O₃), the addition of ytterbium oxide as radiopacifier did not affected ALP activity as it was similarly expressed by Saos-2 cells in comparison to the control in all evaluated periods. It has been reported in other studies that the addition of radiopacifying agents did not affect the ALP activity and also allow the anatomical structures differentiation ⁽²²22 Gomes-Cornelio AL, Rodrigues EM, Salles LP, Mestieri LB, Faria G, Guerreiro-Tanomaru JM, et al.. Bioactivity of MTA Plus, Biodentine and experimental calcium silicate-based cements in human osteoblast-like cells. Int Endod J 2017;50:39-47.^,2323 Tanomaru-Filho M, Andrade AS, Rodrigues EM, Viola KS, Faria G, Camilleri J. Biocompatibility and mineralized nodule formation of Neo MTA Plus and an experimental tricalcium silicate cement containing tantalum oxide. Int Endod J 2017;50Suppl 2:e31-e39.. The evaluation of this enzyme allows the determination of the material bioactivity and its potential to promote repair with formation of mineralized tissue ²⁰20 Lucas CP, Viapiana R, Bosso-Martelo R, Guerreiro-Tanomaru JM, Camilleri J, Tanomaru-Filho M. Physicochemical Properties and Dentin Bond Strength of a Tricalcium Silicate-Based Retrograde Material. Braz Dent J 2017;28:51-56.. The capacity of MTA to induce calcium deposition, as well as its bioactivity has been demonstrated ²³23 Tanomaru-Filho M, Andrade AS, Rodrigues EM, Viola KS, Faria G, Camilleri J. Biocompatibility and mineralized nodule formation of Neo MTA Plus and an experimental tricalcium silicate cement containing tantalum oxide. Int Endod J 2017;50Suppl 2:e31-e39.. MTA Angelus cement stimulated the formation of mineralized tissue by the formation of calcium carbonate ²⁴24 Gomes-Filho JE, Rodrigues G, Watanabe S, Bernabé PFE, Simonett C, Gomes AC, et al.. Evaluation of the Tissue Reaction to Fast Endodontic Cement (CER) and Angelus MTA. J Endod 2009;35:1377-1380..

The alizarin red is a stain that identifies calcium deposits in cell cultures ²²22 Gomes-Cornelio AL, Rodrigues EM, Salles LP, Mestieri LB, Faria G, Guerreiro-Tanomaru JM, et al.. Bioactivity of MTA Plus, Biodentine and experimental calcium silicate-based cements in human osteoblast-like cells. Int Endod J 2017;50:39-47.. The formation of calcified nodules has been related to the differentiation of stem cells into cells capable of forming mineralized tissues ²⁵25 Rosa V, Zhang Z, Grande RH, Nör JE. Dental pulp tissue engineering in full-length human root canals. J Dent Res 2013;92:970-975.. In the present study, all the materials produced mineralized nodules after 21 days of cell cultures in an osteogenic medium (Fig. 3D).

Based on the result, Yb₂O₃ may be considered a potential radiopacifying agent for use in association with CSC.

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