Investigation of the radiopacity and cytotoxicity of ALBO-DENT – novel strontium carbonate incorporated calcium silicate based dental cement

Introduction Materials and methods Novel CS based dental cement with incorporation of SrCO 3 radiopacifier named ALBO-DENT was used as an experimental cement material while Portland cement (Aalborg, Denmark) and ProRoot MTA (Tulsa Dental, USA) were used as controls. The radiopacity evaluation was performed using digital Trophy Radiographic sys-tem with an intention to precisely determine the minimum of radiopaque agent needed to confer to ISO radiopacity requirement. Thereafter, biocompatibility of material was tested in in vitro conditions in mouse fibrosarcoma L929 cell culture treated with materials’ extracts. Cell morphology was observed using phase-contrast microscopy, while cell viability was measured using crystal violet (CV) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays. mm Al). Cytotoxicity analysis using CV and MTT assays revealed that pure extracts of ALBO-DENT presented superior biocompatibility when compared to PC and MTA controls while serial dilutions of experimental cements’ extracts as well as that of PC and MTA did not influence L929 cell viability. Conclusions Novel formulation of CS cement – ALBO-DENT presented satisfactory radiopacity and adequate biocompatibility.

Our research group has demonstrated the satisfactory properties of two novel CS formulations: one consisting of CS, nano-particulated hydroxyapatite (nano-hydroxyapatite, nHA) and BaSO 4 -ALBO MPCA 1 and another composed of CS, calcium carbonate (CaCO 3 ) and Bi 2 O 3 -ALBO MPCA 2 . Their mechanical properties and in vivo safety, after both acute and sub-chronic administration, are documented previously [13][14][15][16][17][18][19]. These materials have shown satisfactory setting time, increased pH value, adequate biocompatibility and enhanced neutralization of the bacterial biofilm [20,21]. It was confirmed that CS enriched with nHA was associated with YbF 3 as radiopacifiers leading to adequate physicochemical and biological characteristics [9,19].
This study generally served to further improve the quality of ALBO MPCA cements by incorporating the potentially bioactive radiopacifier -strontium carbonate (SrCO 3 ). The idea is rooted in proofs of numerous beneficial effects of strontium (Sr) on bone and dental tissue, among which are: osteoproliferative and odontoproliferative effects, stimulation of bone formation and angiogenesis, inhibition of cell differentiation and activity of osteoclasts and induction of human dental pulp stem cells by promoting their odontogenic differentiation, proliferation and mineralization [22,23,24]. The aim of this study was to determine the minimal ratio of SrCO 3 capable to satisfy ISO required radiopacity standard and investigate the biocompatibility of this material in L929 cell culture.

Synthesis of inorganic phases
The novel experimental cement -ALBO-DENT was composed of the following components: calcium silicate, zirconium oxide, strontium carbonate, magnesium silicate, mesosilica and hexaphosphate. Silicate active phase was synthesized from calcium chloride pentahydrate (CaCl 2 ·5H 2 O) (Merck, Germany) and silica sol obtained by hydrothermal treatment. Aluminium acetate (Al(CH 3 COO) 3 ) was added to the mixture to provide the production of a small amount (3.01 %) of active tricalcium aluminate (C 3 A) phase. Detailed procedure of used CS synthesis is given in investigations of Jokanović et al. [14,15]. SrCO 3 (Sigma-Aldrich, St. Louis, Missouri, USA) was added into the mixture at 10%, 20% and 30% wt. ratio. PC (Aalborg, Denmark) and MTA+ (thereafter referred to as MTA) (Cerkamed, StalowaWola, Poland) served as control.

Specimen preparation
All experimental cements and PC were hand-mixed at a powder/liquid ratio of 1 g cement/0.3 ml distilled water, while MTA preparation was performed in accordance with manufacturer's instructions, using glass mixing pad and stainless steel spatula for cement mixing. The specimens were made using polytetrafluoroethylene (PTFE) ring molds incorporating a cavity of various internal diameter and height depending on the used test. Molds were filled to a level surface with mixed cement.

Radiopacity assessments
Radiopacity was determined in accordance with ISO 6876 [25]. Specimens (n=5) measuring 8 mm in diameter and 1 mm thickness were placed alongside an aluminum step-wedge (99.6 % pure) varying in thickness from 1 to 10 mm in increments of 1 mm each and radiographed by CCD sensor and X-ray unit (Trophy Radiology, Cedex, France) operating at 65 kV, 7 mA, for 0.07 s and at the focus to target distance of 35 cm. Image J for Windows software (National Institutes of Health (NIH), Bethesda, MD, USA) was used to calculate the gray scale values of each specimen and of each aluminium step-wedge thickness. The mean grey scale values were plotted against the number of aluminum steps, the plots were linearly regressed and regressions were used to convert mean grey scale values into millimeters of aluminum.

Preparation of the materials extracts
Cell viability was carried out in accordance with the ISO Standard 10993-5/2005 [26]. Cements were manipulated under sterile conditions. Immediately after mixing, materials were placed into pre-sterilized PTFE molds (12 mm in diameter and 2 mm thick) to set for 24 h in a humidified atmosphere. Thereafter, discs were sterilized by ultraviolet irradiation for 2 h, then immersed in 1 ml complete medium -Dulbecco's modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 5 % fetal bovine serum (FBS), 2 mM L-glutamine and penicillin/streptomycin (all from Capricorn Scientific, Ebsdorfergrund, Germany) and incubated for 24 h at 37 o C. To prepare eluents for treatment, extracts were diluted with complete culture medium that was used for cultivation of control/non-treated cells.

Cell culture and treatment
The mouse fibrosarcoma L929 cell line (European Collection of Animal Cell Cultures, Salisbury, UK) was cultivated in complete medium and maintained at 37°C, in a humidified atmosphere with 5% CO 2 . Cells were prepared for experiments using the conventional trypsinization procedure with trypsin/EDTA and seeded in 96-well flat-bottom plates (5×10 3 cells/well) for the cell viability assessment. Cells were treated 24 h post-seeding with pure extract (1) and serial dilutions (1:2, 1:4, 1:8, 1:16 and 1:32 (v:v)). Cell viability was assessed after 24, 48 and 72 h treatment.

Cell viability assessment
The number of adherent cells was determined using crystal violet (CV) while mitochondrial dehydrogenase activity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. The CV assay was based on the inability of dead cells to remain adherent. After treatment, the adherent, viable cells were fixed with methanol and stained with 10 % CV solution for 15 min at room temperature. CV dye was dissolved in 33 % acetic acid after rigorous washing with water. MTT test measures mitochondrial-dependent reduction of MTT to formazan by metabolically viable cells. MTT solution was added to the cell cultures in the final concentration of 0.5 mg/ml and cells were incubated for an additional hour. Subsequently, the solution was removed and cells were lysed by dimethyl sulfoxide. The absorbance of dissolved CV dye, corresponding to the number of adherent (viable) cells and the conversion of MTT to formazan, corresponding to the number of cells with an active mitochondria were measured in automated micro-plate reader at 570 nm (Sunrise; Tecan, Dorset, UK). The results were presented as percentage of viability relative to untreated, control cultures, considered as 100 % viable. The experiments were performed in triplicates.

Phase contrast microscopy analysis
Morphological changes in mouse fibrosarcoma L929 cell line were observed and cells photographed under Leica DCF320 phase contrast microscope (Leica Microsystems DMIL, Wetzlar, Germany) equipped with Leica Microsystems DFC320 camera and Leica Application Suite software (version 2.8.1), with 20× magnification.

Statistical analysis
The SPSS software program (ver. 20, IBM Corp., Armonk, NY, USA) was employed for statistical analysis. The Shapiro-Wilk test was used to check the normality of data distribution. Afterwards, one-way ANOVA with Bonferroni post-hoc tests was employed to compare obtained radiopacity and cytotoxicity outcomes (p<0.05).

RESULTS
The Shapiro-Wilk test for normality found that data were normally distributed and thus they were subjected to oneway ANOVA analysis followed by Bonferroni test.
The results of the radiopacity evaluations are presented in Figure 1. One-way ANOVA revealed that the addition of different percentage of radiopacifiers statistically influenced the obtained values of radiopacity. The lowest value of radiopacity was found in PC that was not statistically different when compared with CS+10%SrCO 3 addition, while it was statistically different that all other investigated cements. On the other hand, MTA presented the greatest radiopacity value, statistically higher than in all other cements. Results revealed that 30% wt addition of SrCO 3 conferred the ALBO-DENT radio-density of 3.45±0.09 mm Al that was in accordance with ISO 6876 requirement, while 10 % addition and 20 % addition of SrCO 3 did not conform with ISO standard for 3 mm Al (1.79±0.06 mmAl, 2.3±0.07 mmAl, for 10 % and 20 %, respectively).
Cytotoxicity data are given in Figure 2 and Figure 3, while representative phase-contrast images of the cells treated with extracts of investigated materials are presented in Figure 4. For CV assay (Figure 2), one-way ANOVA showed the statistical difference among tested cements after 24 h (pure extracts, 1:2 and 1:4), 48 h (pure extracts, 1:2 and 1:4) and 72 h (pure extracts and 1:2) (p<0.05). For MTT assay (Figure 3), one-way ANOVA showed the statistical difference for all time points for pure, 1:2 and 1:4 dilutions (p<0.05), while significance was not found for 1:8, 1:16 and 1:32 dilutions (p>0.05). The results obtained for CV and MTT assays are highly complementary. Pure extract of ALBO-DENT presented lower cytotoxicity than PC and MTA for all time points, showed by both CV and MTT assays. For 1:2 dilution, MTA presented significant proliferative potential after 24h. Similarly, treatment with 1:2 and 1:4 dilutions of PC extract exerted statistically higher proliferative potential after 48 h. The rest of dilutions (1:8, 1:16 and 1:32) had no effect on cell viability.
Consistent with results obtained using cell viability assays, treatment of L929 cells with ALBO-DENT pure extract for 24 h had no effect on cell morphology, but slightly decreased cell proliferation. Contrary, MTA and PC pure extracts triggered morphological changes typical for cell death, cell shrinkage and rounding and detachment of cells from bottom well (Figure 4).

DISCUSSION
This study showed that SrCO 3 might be a radiopacifying agent in CS-based dental cement. It has been shown that 30 % wt addition of SrCO 3 has met ISO requirement for radiopacity and at the same time cement mixture enriched with 30 % wt SrCO 3 showed satisfactory biocompatibility properties.
The idea and reason behind adding SrCO 3 into CSbased cement formulation originate from two reasons.   Firstly, Sr is nowadays accepted as a bioactive constituent of many dental materials and biomaterials used in orthopaedic surgery. In addition, modern strategies for bio-activation of the surfaces of titanium implants include their coating with Sr incorporated layers. Secondly, Sr is intentionally used in the form of carbonates since the addition of calcium carbonate (CaCO 3 ) into CS cements decreases setting time, as it was achieved in Biodentine (Septodont, France) [27].
The results of radiopacity have shown that 30 % wt addition of SrCO 3 was necessary to satisfy the radiopacity of ALBO-DENT. The radiopacity of ALBO-DENT was lower than previously found for CS+30%Bi 2 O 3 (~11 mm Al) and CS+25%Bi 2 O 3 (6.9 mm Al) [28]. The results demonstrated for radiopacity of MTA (6.9 mm Al) corroborate findings of previous studies: 4.86, 6.74, 7.0, 7.5 and 8.0mm Al [28][29][30][31]. The PC did not meet the ISO radiopacity requirement that is in line with previous studies (~0.9 mm Al) [28,29]. The influence of SrCO 3 on the radiopacity of endodontic ceramics has not been previously mentioned in the literature. The variations in radiopacity come as a consequence of the difference in the atomic number between the constituents [32]. Namely, atomic number of the compounds is directly proportional to the absorption of x-rays. The atomic number of Sr (Z=40) is lower than that in Bi (Z=83) and therefore higher percentage of SrCO 3 is needed to meet ISO radiopacity standard. This is not playing a negative role in the case of SrCO 3 such as with other radiopacifiers addition (i.e. Bi) because Sr may be considered not only as biologically safe, but also biologically active constituent.
Biological safety of dental materials is of paramount importance. Therefore, in vitro and in vivo tests are routinely performed to evaluate material's biocompatibility before it can be used in clinical practice. The cytotoxicity assessment was performed for the mixture with adequate radiopacity value (30% wt addition of SrCO 3 ). ISO 10993-5 stipulates that material can be considered as not cytotoxic if it causes less than 30% cells to die in in vitro assays. In our study, two cytotoxicity tests were used: MTT that measures mitochondrial activity of the metabolically active cells and CV that determines the number of the adherent, viable cells. It was demonstrated that novel experimental cement ALBO-DENT performed satisfactory behavior in cell culture, comparable to that of PC and MTA. Presented results showed significantly lower percentage of viable cells in MTA/PC treatments than those found in some studies (80-150%) [7,33,34], but they are in agreement with other studies [35,36]. For PC/ MTA 1:2 and 1:4 eluents, the CV and MTT tests showed similar outcomes and are in rough agreement with data documented in the literature [7,37]. The differences of cell viability results in different studies could arise from variations in specimens' size (5×3 mm [37], 5×2 mm [12] and 5×1 mm [29]). The toxic potential of PC and MTA pure elute may be a matter of debate, but it is presumably the consequence of its high alkalinity in the closed in vi tro cell viability assessment system. It may be speculated that in vivo, where the constant fluid uptake is ensured, these materials may not present negative effect on the surrounding tissue. In any case, novel cement mixture ALBO-DENT presented superior characteristics than widely commercially used ProRoot MTA.
From the clinical point of view, new CS-based experimental material has certain advantages since Sr incorporation may enhance the bone healing during root end canal surgery by activating osteoblasts for improved bone synthesis [22,23,24]. In addition, if used for pulp capping procedures, it may stimulate odontoblasts for faster formation of tertiary dentine. These assumptions should be confirmed in the future state of the art researches.

CONCLUSION
Newly synthesized CS-based dental cement with 30% wt addition of SrCO 3 as radiopacifying agent meets ISO standard for radiopacity. Biocompatibility of newly synthesized cement, assessed by analysis of cell viability via measurement of the number of adherent, viable cells and viable cells with active mitochondria, is satisfactory and indicates its biological safety.