2.1

Physicochemical tests

2.1.1

Setting time

The setting time was evaluated based on ISO Standard 6876/2012 [ ]. The cements were mixed and placed into stainless steel rings measuring 10 mm in diameter and 1 mm high ( n = 6). A Gilmore needle having 100 ± 0.5 g and a tip diameter of 2 ± 0.1 mm was used, and the materials were kept in an oven (37 °C and 95% humidity) throughout the analysis. The setting time was the mean time elapsing from the time the materials were mixed up to when the needle did not leave any indentation on the specimen surface.

2.1.2

Radiopacity

For the radiopacity test, specimens of each material measuring 10 mm in diameter and 1 mm high ( n = 6) were kept in an oven at 37 °C and 95% humidity for 24 h. The specimens and an aluminum scale were placed on an occlusal film (Insight – Kodak Comp, Rochester, NY, USA) to take the radiograph (X-ray appliance -X GE 1000 – General Electric, Milwaukee, WI, USA). The parameters used were 60 kV, 7 mA, 0.32 pulses per second and focal distance of 33 cm. The exposed films were processed, digitized and evaluated by using Image J for Windows software, to determine the radiopacity equivalence of the cements in millimeters of aluminum (mm Al).

2.1.3

Solubility

Solubility test was evaluated based on a previous study [ ]. Specimens of each material measuring 1.5 mm high and 7.75 mm in internal diameter were manufactured ( n = 6) with a nylon thread included in the cement mass and kept in an oven at 37 °C for 24 h. The specimens were weighed on a precision balance (Adventurer AR2140, Ohaus Corporation, Parsippany, NJ, USA). Then, they were suspended and fixed by means of nylon threads inside plastic flasks containing 7.5 mL of distilled, and kept in an oven at 37 °C for 7 days. The specimens were removed from the distilled water, dried with absorbent paper, and placed in a dehumidifying chamber until the mass was stabilized. The loss of mass was expressed as a percentage of the original mass.

2.1.4

pH

For the pH test, polyethylene tubes 10 mm long and 1 mm in diameter were filled with each material ( n = 10) and immersed in 10 mL distilled water and kept in an oven during the time intervals of 3, 12 and 24 h, 7, 14 and 21 days. At each period, the tubes were removed from the flasks and put into a new flask with 10 mL distilled water. Distilled water was used as control. The pH of the solutions was measured with a previously calibrated digital pHmeter (Digimed Analítica Ltda., Grupo Digicrom Analítica, Sao Paulo, Brazil).

2.2

Biological tests

2.2.1

Preparation of material extracts

This method was carried out in accordance with the ISO Standard 10993-5/2005 [ ]. 0.5 g of each material was manipulated and placed on the bottoms of 12-well culture plates (Corning, New York, NY, USA). The culture plates were kept at 37 °C at 95% humidity, 5% CO ₂ , for 24 h to allow complete setting of the materials. After this period, the plates with the cements were exposed to U.V. light for 30 min. 5 mL DMEM culture medium (Sigma/Aldrich) serum-free (without the presence of fetal bovine serum – FBS) were placed in each well of the plates, in which the material was accommodated, and maintained for 24 h (37 °C, 95% humidity and 5% CO ₂ ) to create the extract of each material. The extracts were collected and diluted in DMEM without FBS, thereby obtaining the 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32 dilutions for performing the cytotoxicity tests. As negative control, DMEM culture medium serum-free; and as positive control 20% DMSO were used.

2.2.2

MTT

Human osteoblastic cells (Saos-2) were plated (1 × 10 ⁵ cells/mL) in 96-well plates (Corning) and kept at 37 °C at 95% humidity, 5% CO ₂ , for 24 h. After this period, the cells were exposed to the cement extracts in cell culture for 24 h. The cement extracts were replaced with 100 μL of a 5 mg/mL MTT solution (Sigma-Aldrich), and cells were incubated at 37 °C at 95% humidity, 5% CO ₂ , for 3 h. After this period, each well was washed with 100 μL phosphate buffer solution (PBS) and 100 μL isopropyl alcohol (acidified 0.04 N HCl). The optical density was measured at 570 nm in an automatic microplate reader (ELx800; Instruments Bio-Tek, Winooski, VT, USA). Three independent experiments were performed.

2.2.3

Neutral Red assay (NR)

The cells (1 × 10 ⁵ cells/mL) were plated in 96-well plates (Corning) in DMEM medium supplemented with 10% FBS and kept at 37 °C at 95% humidity, 5% CO ₂ , for 24 h. After the cells had remained in contact with the cement extracts in their different concentrations for 24 h, the extracts were replaced by 100 μL DMEM serum free, containing 50 μg NR/mL (Sigma-Aldrich), followed by incubation at 37 °C, 95% humidity and 5% CO ₂ for 3 h. The coloring agent was removed and the colorimetric product was solubilized in 100 μL of 50% ethanol and 1% acetic acid solution (Sigma-Aldrich). The optical density was measured in the plate reader at 570 nm (Asys-UVM 340, Biochrom – MikroWin 2000, USA). Three independent experiments were performed.

2.2.4

Alkaline phosphatase

Alkaline phosphatase activity was determined by using the commercial Labtest kit (Labtest, Lagoa Santa, MG, Brazil). After plating for 24 h, (7 × 10 ⁴ cells/mL in 96-well plates), the Saos-2 cells were exposed to the cement extracts for 7 and 14 days. The extracts were renewed every two days. After each experimental period, the cells were washed with 200 μL of PBS 1X, followed by the addition of 200 μL of a sodium lauryl sulphate solution (1% in distilled water, Sigma-Aldrich). The samples remained at rest at room temperature for 30 min, and then 12.5 μL of each sample was transferred to Eppendorf tubes with reagents of the Labtest kit, according to the manufacturer’s instructions. The optical density was evaluated in an automatic miniplate reader (ELx800, Bio-Tek Instruments, Winooski, VT, USA) at 590 nm. The data were expressed as ALP activity normalized by the number of viable cells determined in the MTT assay, in the respective culture period. Three independent experiments were performed.

2.2.5

Quantitative Real Time PCR (qPCR)

For gene expression study, RNA extraction was performed from cells using Trizol (Life Technologies – Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s Kit instructions (PureLink™, RNA Mini Kit, Carlsbad, CA, USA). The transcripts were synthesized from total Saos-2 RNA (0,5 μg/μL) by reverse transcriptase reaction using the ImProm-II Reverse Transcription System (PROMEGA, Mandison, WI, USA) kit according to the manufacturer instructions. The gene expression was analyzed by qPCR (StepOne, Applied Biosystems, Life Technologies, Grand Island, NY, USA) using TaqMan chemistry and pre-designed primers and probe sets alkaline phosphatase (ALP) (Hs01029144_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (Hs02758991_g1) (Applied Biosystems, Life Technologies). Triplicates were prepared for each reaction, and the experiment was repeated three times independently. The levels of target gene expression for each sample group were calculated with the ΔΔCt method (fold expression = 2 ^{− (ΔΔCt± stdev)} ) compared to control (serum-free DMEM medium).

2.2.6

Alizarin Red

Saos-2 cells were plated (1 × 10 ⁴ cells/mL) in 24-well culture plates (Corning, New York, NY, USA) in DMEM medium supplemented with 10% FBS. For 21 days, the cement extracts prepared with osteogenic DMEM culture medium (DMEM 10% SFB; 100 IU/mL penicillin; 100 mg/mL streptomycin; 0.023 g/mL β-Glycerophosphate; 0.055 mg/mL ascorbic acid – Sigma Chemicals St Louis MO, USA) were renewed every 2 days. The medium was then aspirated, the wells washed with PBS 1X, and the cells fixed with 70% ethanol (Sigma/Aldrich) at 4 °C, for 1 h. The cells were washed twice with distilled water before the addition of 300 μL Alizarin Red S (at 2% and pH 4.1) and were incubated at room temperature for 2 min. After this they were washed 4 times with 1 mL distilled water/well for 5 min. For quantitative analysis of mineralization nodule formation, the nodules were solubilized in 0.5 mL Cetylpyridinium chloride (Sigma-Aldrich) under agitation for 15 min. After homogenization, three aliquots of 100 μL from each well were transferred to a 96-well plate. Mineral nodule formation was analyzed in accordance with the absorbance determined at 562 nm in a plate reader (ELx800, Bio-Tek Instruments). Three independent experiments were performed.

2.3

Antibacterial test

2.3.1

Test of direct contact on planktonic E. faecalis cells

The experimental materials were manipulated and inserted on the lateral wall of the well of 96-well plates. The materials were submitted to decontamination under ultraviolet light for 30 min. After this, 10 μL of inoculum of the E. faecalis (ATCC 29212) planktonic cells was deposited on the materials. In the positive control group, only inoculum was added. The plates were kept in an oven at 37 °C for 1 h and 30 min. Afterwards, 250 μL of brain heart infusion (BHI) were added to each contaminated well, decimal serial dilution was performed, and the plates containing tryptic soy agar (TSa) were seeded. After 48 h of incubation at 37 °C, the CFUs mL ⁻¹ were counted and logarithmic transformation (log10) was performed.

2.4

Statistical analysis

For the physicochemical tests and Alizarin red staining (ARS) the data obtained were submitted to a normality test, and afterwards to the ANOVA and Tukey tests, with 5% level of significance. For the cell viability and bioactivity, the data were analyzed by using Two-way ANOVA and Bonferroni tests ( α = 0.05). For the antibacterial test, the Kruskall–Wallis and Dunn multiple comparison tests were performed, with 5% significance, after logarithmic transformation (log 10).

3.1

Physicochemical properties

The radiopacity, setting time and solubility results are described in Table 2 . All the cements had values higher than 3 mm aluminum, except for pure TCS (p < 0.05). The highest value of radiopacity was observed in TCS + CaWO _4, followed by TCS + ZrO ₂ (p < 0.05), and the lowest value was observed in TCS (p < 0.05). TCS + CaWO ₄ presented the highest setting time and MTA HP had the shortest (p < 0.05). Low solubility values were observed for the materials. TCS, TCS + ZrO ₂ , and TCS + CaWO ₄ presented a mass loss, which TCS presenting higher values than TCS + CaWO ₄ (p < 0.05). TCS + Nb ₂ O ₅ and MTA HP showed a mass gain (p > 0.05). All the materials had alkalization ability, showing high pH values in the different time intervals evaluated. After 3 h the materials that had the highest values were TCS, TCS + ZrO ₂ and TCS + CaWO ₄ (p > 0.05). After 12 h, MTA HP, TCS and TCS + ZrO ₂ presented more alkalization capacity. After 7 days, MTA HP, TCS and TCS CaWO ₄ had the highest values (p > 0.05). At 14 days MTA HP, TCS, TCS + CaWO ₄ and TCS + Nb ₂ O ₅ presented more alkaline pH (p > 0.05). After 21 days, all materials had an alkaline pH and there was no significant difference among the groups ( Table 3 ).

Table 2

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