Physicochemical properties and effect of bioceramic root canal filling for primary teeth on osteoblast biology

Abstract Bio-C Pulpecto (Bio-CP) was recently developed as the first bioceramic root filling material for primary teeth. Objective To evaluate the physicochemical properties of radiopacity, setting time, pH, cytocompatibility and potential of Bio-CP to induce mineralisation, compared with (1) Calen thickened with zinc oxide (Calen-ZO), and (2) zinc oxide and eugenol (ZOE). Methodology Physicochemical properties were evaluated according to ISO 6876. Saos-2 (human osteoblast-like cell line) exposed to extracts of the materials were subjected to assays of methyl thiazolyl tetrazolium, neutral red, alkaline phosphatase (ALP) activity and mineralised nodule production. The results were analysed using one-way or two-way ANOVA and Tukey’s or Bonferroni’s post-tests (α=0.05). Results All the materials showed radiopacity higher than 3 mm Al. Bio-CP had lower pH than Calen-ZO, but higher pH than ZOE. Calen-ZO and Bio-CP did not set. The setting time for ZOE was 110 min. The cytocompatibility order was Calen-ZO > Bio-CP > ZOE (1:2, 1:4 dilutions) and Calen-ZO > Bio-CP = ZOE (1:12, 1:24 dilutions) and Calen-ZO = Bio-CP > ZOE (1:32 dilution). Bio-CP induced greater ALP activity at 7 days, and greater mineralised nodule production, compared to Calen-ZO (p<0.05). Conclusions Bio-CP showed adequate physicochemical properties, cytocompatibility and potential to induce mineralisation.


Introduction
An ideal root canal filling for primary teeth should not hinder the eruption of permanent successor teeth, but rather, should be resorbed as the deciduous tooth roots are physiologically resorbed, and should also resorb readily if pressed beyond the apex, be easily removed if necessary, be radiopaque and not discolour the tooth. 1 Zinc oxide and eugenol (ZOE) are a combination that has been used as a root canal filling material in primary dentition for a long time. 2,3 However, ZOE is genotoxic and cytotoxic, 4 and cannot be completely phagocytised, leaving particles in periapical tissues when extravasated beyond the apex, and after physiological root resorption of the deciduous teeth. 2 Calen (S.S. White, Rio de Janeiro, RJ, Brazil) is a commercial calcium hydroxide-based paste with a viscous vehicle (i.e., polyethylene glycol 400), and has suitable biological properties. 5 Calen thickened with zinc oxide (Calen-ZO) has been used as a root canal filling material in the primary dentition. [6][7][8] The addition of zinc oxide (ZO) reduces the phagocytosis of Calen, thus allowing Calen to accompany the physiological resorption of primary tooth roots, and improve its physicochemical properties. 7,9 Calen-ZO induces good tissue response, 7,10 has antimicrobial activity 6 , and is more biocompatible than ZOE. 7,8 New endodontic cements have been developed, especially highlighting the advance in improved bioceramics. The main characteristics of bioceramic materials for endodontic use are their alkaline pH, shrink-free property, chemical stability within the biological environment, 11,12   After handling of the specimens, 100 mg of each material ( Figure 1) was placed in a 1.5 mL microtube (Eppendorf, Hamburg, Germany), to which 1.2 mL of DMEM was added, followed by placement of the microtubes in an oven at 37°C for 24 hours. 18 Afterwards, the supernatant was transferred to new microtubes centrifuged for 10 min at 20,800 g (5430, Eppendorf AG, Hamburg, Germany) to decant any particles of material left in the supernatant. The supernatant was transferred to a new tube and was considered as the "stock solution/extract". It was then diluted and placed in contact with Saos-2 cells. 19 Cell viability analysis by methyl thiazolyl tetrazolium (MTT) and neutral red (NR) assays Saos-2 cells were cultured at a density of 1x10 5 cells/mL in a 96-well plate containing DMEM with 10% SFB for 24 hours to adhere to the plates. Thereafter, the cells were exposed to the cement extracts at the following dilutions in DMEM (v: v) 1:2; 1:4; 1:12; 1:24; 1:32. Cells exposed to DMEM were considered as the control group.
After 24 hours of cell contact with the material extracts and the control, 100 μL of 5 mg / mL MTT solution (Sigma-Aldrich) was added to each well, followed by incubation at 37°C, 95% humidity and 5% CO 2 for 3 hours. The colorimetric product was solubilised in 100 µl of 0.04 N acidified isopropanol (Sigma-Aldrich). A spectrophotometer (Elx800; Bio-Tek Instruments, Winooski, VT, USA) at 570 nm was used to measure the optical densities of the solutions.
Absorbance readings were normalised to readings of cells exposed to DMEM, and represented the succinate dehydrogenase activity (cellular metabolism).
The NR assay was performed by applying 100 μL DMEM containing NR at 50µg / mL (Sigma-Aldrich) after 24 hours of cell contact with the materials and control extracts. The cells were incubated at 37°C, 95% humidity and 5% CO 2 for 3 hours, the contents of the wells were removed, and the colorimetric product was solubilised with 100 μL of a solution containing 50% ethanol and 1% acetic acid (Sigma-Aldrich). A spectrophotometer (Elx800; Bio-Tek Instruments, Winooski, VT, USA) at 570 nm was used to measure the optical densities of the solutions. Absorbance readings were normalised to readings of cells exposed to DMEM, and represented the ability to incorporate the dye into viable cell lysosomes. Alkaline phosphatase (ALP) activity Saos-2 (5x10 4 cells / mL) were cultivated in 96-well plates and exposed to the control (DMEM) and the extract of the materials at 1:24 dilution. This dilution was selected after observing the results of MTT assays performed after exposure of cells to cement extracts for 1, 3 and 7 days (data not shown). The 1:24 dilution was the highest extract concentration without cytotoxic effects. This is an essential consideration, since dead cells do not show ALP activity. ALP activity was evaluated at periods of 1, 3 and 7 days, using the commercial kit (Labtest, Lagoa Santa, MG, Brazil).
Extracts of the materials were renewed every two days.

Alizarin Red Staining (ARS)
Saos-2 cells were cultivated (1x10 4 cells / mL) in 12-well culture plates with DMEM, supplemented with 50 µg / mL L-ascorbic acid (Sigma-Aldrich) and 10 mM β glycerophosphate (Sigma-Aldrich). The cells were exposed to material extracts for 21 days at the same dilution used for the ALP activity assay.
Material extracts were renewed every two days.
Then the cells were fixed with 4% paraformaldehyde (Sigma) and stained with 2% ARS (pH 4.1). The cells were incubated at room temperature for 20 minutes, and the dye was aspirated. The wells were washed 4 times with 1 mL of distilled water for 5 minutes.
The water was removed and mineralisation was quantified by dissolving the nodules with 1 mL of a 10% cetylpyridinium chloride solution (Sigma Aldrich) added to each well, after which the plate was incubated for 15 minutes under stirring at room temperature.
Three 100 μL aliquots of the suspension from each well were transferred to a 96-well plate and read with a 562 nm wavelength filter on a spectrometer (Elx800; Bio-Tek Instruments).

Statistical analyses
The results were shown as mean and standard

Physicochemical properties
According to Table 1, all the materials showed radiopacity higher than 3 mm Al, in agreement with ISO 6876:2012. 15 There was no difference between Bio-CP and Calen-ZO (p>0.05), and ZOE showed higher radiopacity than the other materials (p<0.05).

The ZOE setting time was 110 minutes. Calen-ZO and
Bio-CP were considered to "set" (maximum hardening) at 192 hours and 240 hours, respectively. Although Calen-ZO and Bio-CP pastes had alkaline pH, Calen-ZO had a higher pH than Bio-CP (p<0.05). ZOE had a neutral pH (Table 2).

Cell viability
As shown in Figure 2 Table 1-Different letters in the lines indicate statistically significant differences among the materials (P<0.05) *The material did not set after the elapsed "setting" time. ** Partial areas of the material set at the evaluated time Different letters in the lines indicate statistically significant differences among the materials (P<0.05). *The material did not set after the elapsed "setting" time. ** Partial areas of the material set at the evaluated time.
Physicochemical properties and effect of bioceramic root canal filling for primary teeth on osteoblast biology J Appl Oral Sci. 2021;29:e20200870 5/9 cell viability (p>0.05). As for the NR assay, there was no difference between the materials and the control (p>0.05), except Calen-ZO at 1:24 dilution, that had higher cell viability than the other groups (p<0.05).
At the highest dilution (1:32), there was no difference between Calen-ZO and Bio-CP (p>0.05) in the MTT assay, and both groups had higher cell viability than ZOE. In the NR assay, there was no difference among the materials evaluated at 1:32 dilution (p>0.05).   On the seventh day, there was no difference in the viability of the cells exposed to all the materials and the control (p>0.05). On the first day of cell culture, Calen-ZO induced higher ALP activity than Bio-CP and the control (p<0.05), whereas Bio-CP promoted ALP activity similar to that of the control (p>0.05).

Bio-CP
On the third day, there was no difference among the groups (p>0.05). On the seventh day, Bio-CP induced higher ALP activity than Calen-ZO (p<0.05), whereas Calen-ZO was no different from the control (p>0.05).
In 21 days of cell exposure to the materials, Bio-CP and Calen-ZO induced higher mineralised nodule production than the control (p<0.05), although that of Bio-CP was higher than that of Calen-ZO (p<0.05).

Discussion
In the present study, a comparison was made of the biological and physicochemical properties of Bio-CP, Calen-ZO and ZOE. Bio-CP exhibited adequate physicochemical properties, had cytocompatibility and revealed potential to induce mineralisation, when compared to the other evaluated root canal filling materials for primary teeth.
Radiopacity is an essential property for endodontic materials, because it allows endodontic filling material to be viewed by radiographic examination to evaluate the obturation quality, 12 and make a clear distinction among the materials and the surrounding anatomical structures. 20 ISO 6876:2012 15 establishes that the root canal sealers must have at least 3 mm Al. In the present study, all the materials evaluated showed higher radiopacity than 3 mm Al. However, the radiopacity value of Bio-CP (3.50 mm Al) was less than that stated by the manufacturer (9 mm AL). On the other hand, it agrees with a previous study that showed that an experimental MTA-based material, with Figure 3-Effect of root canal filling materials (1:24 dilution) of primary teeth on the biology of Saos-2. MTT assay (a) and alkaline phosphatase (ALP) activity (b) were performed 1, 3 and 7 days after exposure of Soas-2 to extracts of materials and serum-free culture medium (control), whereas alizarin red stain (ARS ) assay (c) was evaluated after 21 days of cell exposure to material extracts and osteogenic culture medium (control). Bio-CP=Bio C Pulpecto; Calen-ZO=Calen mixed with zinc oxide and eugenol; ZOE=Zinc oxide and eugenol. Different letters indicate statistically significant differences among the materials (p<0.05) J Appl Oral Sci. 2021;29:e20200870 7/9 similar composition of Bio-CP, had radiopacity of 3.28. 8 The greater radiopacity of ZOE (9.25 mm Al) than that of the other materials (3.5 mm Al) is attributed to its zinc oxide used as a radiopacifier. 21 An alkaline medium not only neutralises lactic acid from osteoclasts, but also prevents the mineral components of dentine from dissolving. In addition, it promotes antimicrobial activity, and may activate alkaline phosphatases, which play an important role in hard-tissue formation. 22 Both Bio-CP and Calen-ZO had alkaline pH; however, Calen-ZO had a higher alkaline pH compared with Bio-CP over the evaluated periods. Calcium hydroxide dissociates into calcium and hydroxyl ions, which raises the pH of the medium. that Bio-CP does not set is not a disadvantage, since this phenomenon is observed in materials considered suitable for filling root canals of primary teeth. 8,9,23 Because Bio-CP was developed as a root filling material for primary teeth, is desirable that the material can be Bio-CP induced lower ALP activity in 1 day, but greater ALP activity in 7 days, than Calen-ZO. In addition, Bio-CP induced greater mineralised nodule production than Calen-ZO. Calen-ZO induced higher calcium deposits than the control group. A recent study showed that Bio-CP was biocompatible and induced biomineralization similar to MTA in the subcutaneous tissue of rats. 16 Regarding Calen-ZO, the results of the present study are in line with previous studies performed on dogs' teeth and the subcutaneous tissue of mice, which showed that Calen-Zo was more biocompatible than ZOE. 7,10 ZOE was not evaluated in either mineralisation assay conducted in the present study, because it has no bioactivity. 29 The high mineralisation potential of calcium-silicate-based bioceramic materials is attributed to their hydration, that is, a chemical reaction with tissue fluids yields hydrated silicate gel (C-S-H) and calcium hydroxide. 30 2021;29:e20200870 8/9 These compounds (C-S-H and calcium hydroxide) each have a mineralisation pathway. Calcium hydroxide induces the formation of calcite crystals that induce the formation of calcified areas. Calcite crystals are formed from the dissociation of calcium ions from calcium hydroxide, and carbon dioxide ions from tissues. 31 On the other hand, C-S-H induces hydroxyapatite deposition; this reaction is promoted by the hydration of tissue fluids. In bioceramic materials, calcium hydroxide reacts with the phosphate ions in tissue fluids to deposit calcium phosphate. 4,30 The results obtained for cytocompatibility and mineralization induction should be confirmed by further in vivo studies. In addition, to complement the research on Bio-CP, studies should be carried out to evaluate its antibiofilm activity and the capacity to be reabsorbed to accompany the physiological resorption of deciduous teeth roots.
It was concluded that Bio-CP did not set, presented adequate radiopacity and alkaline pH, was cytocompatible and had the potential to induce mineralisation. Considering its good physicochemical and biological properties, Bio-CP has the potential to become an adequate material for the root canal filling of primary teeth.