Effects of dodecacalcium hepta-aluminate content on the setting time, compressive strength, alkalinity, and cytocompatibility of tricalcium silicate cement

Abstract Objective This study aimed to investigate the effects of dodecacalcium hepta-aluminate (C12A7) content on some physicochemical properties and cytocompatibility of tricalcium silicate (C3S) cement using human dental pulp cells (hDPCs). Material and Methods High purity C3S cement was manufactured by a solid phase method. C12A7 was mixed with the cement in proportions of 0, 5, 8, and 10 wt% (C12A7-0, −5, −8, and −10, respectively). Physicochemical properties including initial setting time, compressive strength, and alkalinity were evaluated. Cytocompatibility was assessed with cell viability tests and cell number counts. Statistical analysis was performed by using one-way analysis of variance (ANOVA) and Tukey's test (p<0.05). Results The initial setting time of C3S-based cement was shorter in the presence of C12A7 (p<0.05). After 1 day, C12A7-5 showed significantly higher compressive strength than the other groups (p<0.05). After 7 days, the compressive strength of C12A7-5 was similar to that of C12A7-0, whereas other groups showed strength lower than C12A7-0. The pH values of all tested groups showed no significant differences after 1 day (p>0.05). The C12A7-5 group showed similar cell viability to the C12A7-0 group (p>0.05), while the other experimental groups showed lower values compared to C12A7-0 group (p<0.05). The number of cells grown on the C12A7-5 specimen was higher than that on C12A7-8 and −10 (p<0.05). Conclusions The addition of C12A7 to C3S cement at a proportion of 5% resulted in rapid initial setting time and higher compressive strength with no adverse effects on cytocompatibility.


Introduction
Since its introduction in the 1990s, mineral trioxide aggregate (MTA) has been widely used in the endodontic field for purposes including retrograde filling, perforation repair, apexification, and vital pulp therapy. 1 Considerable evidence has demonstrated the excellent biocompatibility and sealing ability of MTA and promising outcomes for endodontic procedures. 2,3 Nevertheless, because of drawbacks including long setting time and initial wash-out possibility, poor handling characteristics, discoloration of teeth, and heavy metal content, the development of novel MTAlike calcium silicate (CS) cement to overcome these drawbacks and improve biocompatibility and clinical convenience of MTA is an important goal. 4 The composition of MTA is similar to that of Portland cement except for the presence of radiopacifierlike bismuth oxide, which is generally composed of tricalcium silicate (C3S), dicalcium silicate (C2S), tricalcium aluminates (C3A), tetracalcium aluminoferrite, and other ingredients. 5 Among these, C3S is one of the major constituents of MTA. 6 C3S forms into calcium silicate hydrates (C-S-H) through hydration reactions with water, which contribute to the spontaneous development of strength. 7 Despite its excellent in vitro bioactivity and biocompatibility, 8 C3S alone has limitations in clinical contexts due to its long setting time and low mechanical strength during the early stages of hydration. 9 Tricalcium aluminate (C3A) is the most reactive part of Portland cement. 10 When C3A is mixed with calcium silicate, it contributes to the initial hydration process of the cement. 10 In studies, C3A mixed with calcium silicate cement showed a faster hydration rate and higher initial mechanical strength than non-mixed cement. 7,9,10 Dodecacalcium hepta-aluminate (C12A7), one of the stable phases of calcium aluminates (CA), is also expected to react rapidly with water and may provide beneficial properties during the early hydration stages of calcium silicate. However, there have been no attempts to mix C12A7 in C3S cement and evaluate the effects of C12A7 on cement in dental contexts. This study aimed to evaluate the effects of C12A7 on C3S cement regarding some physicochemical properties and cytocompatibility with hDPCs. We manufactured high-purity cement with uniformly fine particles to determine the optimal component ratio of C3S/ C12A7 for use as an endodontic biomaterial. The null hypothesis was that cements with different C12A7 contents would not significantly differ in practical properties.

Material preparation
For the manufacture of C3S cement powder, calcium carbonate (Sigma-Aldrich, St. Louis, MO, USA) and silicon dioxide (Junsei Chemical, Kyoto, Japan) were uniformly mixed in a 3:1 molar ratio and stirred by ball milling for 1 h in ethanol. The powder (10 g) was put into a mold 15-mm in diameter and pressed uniaxially at pressures of 2 to 3 t. After the cylindertype pelletized samples were desiccated at 40°C for 12 h, the sample was heated at a rate of 10°C/min to the sintering temperature of 1400-1500°C, held for 1 to 20 h, and then cooled. Sintering process was repeated four times to achieve higher proportions of C3S in the cement. The sintered material was ground to powders less than 8 μm in diameter by using air jet mill (CGS16, NETZSCH GmbH, Selb, Germany) with 6000 rpm. The characteristics of prepared C3S cement powder were evaluated with X-ray diffraction

Alkalinity
Alkalinity was evaluated by measuring pH according to a previously published study. 11 In brief, we prepared specimen (1-mm thickness and 5-mm diameter) and allowed to set completely. After setting, we inserted one tablet into 10 mL of deionized water. Then, the pH value was measured using a pH meter (Orion 3 Star; Thermo Fisher Scientific, Singapore).

Primary culture of human dental pulp cells (hDPCs)
The experimental procedures with hDPCs of this study were approved by the institutional review

Cell viability test
After the powder was mixed with DW, the cement was allowed to set in disc shaped-paraffin wax molds

Statistical analysis
The results were statistically analyzed using oneway analysis of variance (ANOVA) and Tukey's tests

Characterization of C3S and C12A7
The characteristics of prepared C3S and C12A7 were identified by SEM and XRD (Figure 1). The SEM images of fabricated C3S powders showed homogeneous composition of particles ( Figure 1A and 1B). Phase analysis results by XRD indicated that the prepared powders were C3S and C12A7, respectively ( Figure 1C and 1F). Quantitatively, the proportion of C3S was 100%, and particle size was less than 8 μm (Table 1). Regarding the initial setting time, there were significant differences between control (C12A7-0) and other samples (C12A7-5, -8, and -10) (p<0.05) ( Figure 2A). As shown in Figure 2B, the compressive strength of C12A7-5 was significantly higher than that of other groups 1 day after the setting (p<0.05).
However, there was no statistical difference between the other groups (p>0.05). After 7 days, the strength of C12A7-5 was similar to C12A7-0, whereas other groups showed lower strength compared to C12A7-0 ( Figure 2B) (p>0.05). Regarding alkalinity, samples of all groups showed high pH values around 10 -12 with an increasing pattern for 7 days. Although pH values of C12A7-0 were higher than other groups within 6 h (p<0.05), there were no significant differences after 1 day ( Figure 2C). Furthermore, the C12A7-5 group showed higher pH value than C12A7-8 and -10 within 6 h (p<0.05).

Measurement of viability and the number of hDPCs
Viability and number of hDPCs were measured to investigate the cytocompatibiltiy of the cement.
Although the cell viability of C12A7-containing cement decreased as the proportion of C12A7 increased, the cell viability of C12A7-5 was similar to that of both negative and positive control (C12A7-0) (p>0.05).
Significantly lower cell viability than the control group in C12A7-10 was observed after 1 day and in C12A7-8 and -10 after 2 days ( Figure 3A). Differences in cell number were also evaluated for C3S cement

XRD-rietveld refinement method
Particle size (μm) d<10 0.84 Laser diffraction particle size analyzer  Table 1-Characteristics of prepared cement powder. "d<n" is defined as the diameter at which n% of the sample's cumulative mass is comprised of particles with a diameter less than this value in the particle size distribution, and "dmax" is defined as the maximum diameter of particle size

Discussion
Portland cement acquired from natural materials can vary in composition and include impurities such as heavy metals of leachable lead and arsenic. 1 According to several studies, commercially available MTAs may also contain heavy metals, although the amount released by MTAs is less than that of Portland cement. 12 This contamination is of major concern in the development of cements that have high-purity CS, which leads to compositional stability and reliability, and the exclusion of heavy metal elements. 13   shown that the addition of CA induces faster hydration reactions and improvements of the early mechanical strength of CS-based cement. 7,10 C3A has been used as a representative additive in CS-based cement.
However, the synthesis of non-blended C3A clinker is an energy-consuming process, because sintering must be repeated several times to avoid eutectic and peritectic reactions on the CaO-Al 2 O 3 phase diagram. In contrast, C12A7 is more efficient to fabricate than C3A, due to its favorable sinterability. In addition, C12A7, known as mayenite, is one of the intermediary phases of the CaO-Al 2 O 3 binary system and is also known to contribute to the first stage of strength development in aluminous cements like C3A. 16 Therefore, C12A7 was selected for use in this study and was fabricated by sintering processes (Figure 1D-F [17][18][19] In C12A7-containing groups (C12A7-5, 8 and 10) of this study, the setting time was significantly shortened compared to pure the C3S group (C12A7-0), resulting in a value less than 15 min similar to that of commercial products (Figure 2A). [17][18][19] In this respect, the addition of C12A7 in C3S cement showed significant benefit regarding the reduction of initial setting time. Furthermore, according to the results of the compressive strength test after 1 day, C12A7-5 showed higher early strength compared to C12A7-0. The compressive strength values obtained in this study were higher than those of MTA in another previous study. 20 The results indicated that the addition of C12A7 might be beneficial for the early strength of the cement. Although the values of C12A7-8 and -10 decreased compared to control after 7 days, C12A7-5 showed no significant difference compared to the control group ( Figure 2B). We also evaluated the pH of hydrated samples, since alkalinity is one of the most important characteristics of CS-based cement, as it is related to antimicrobial effect and dentin bridge formation near the pulp cells. 1,20,21 The pH values increased during the period until 7 days, retaining high alkalinity at around 10 -12, showing similar pattern and values with other previous studies using white MTA. 18,21 Although the alkalinity of C12A7-0 was significantly higher in the early period within 6 h, there were no differences among all groups after 24 h ( Figure 2C). Based on these findings from the initial setting time, compressive strength, and alkalinity test, we suggest that the addition of 5% C12A7 to C3S cement provide suitable benefit in terms of the clinical perspectives.
To evaluate the effect of C12A7 on the cytocompatibility of C3S-based cement in our study, we performed cell viability tests and measurements of cell number with hDPCs. Previously, several cell lines, 3,8,10 were used for cell viability tests of CS-based cement.
However, susceptibility to toxicity may vary between human-and animal-derived cells. Moreover, in clinics, as CS-based cement usually contacts exposed dental pulp when used as direct pulp capping materials, using the hDPCs can provide more exact informations. 22 In our study, although cell viability decreased with increasing C12A7 concentration, the cell viability of C12A7-5 was similar to that of the control group after 1 and 2 days (p>0.05) ( Figure 3A). Furthermore, the C12A7-5 group showed higher cell count than other groups ( Figure 3B). In general, CS-based cement has proven to be biocompatible and to stimulate cell proliferation. 23 It has been also reported that the dissolution of calcium and silicate ions from CS-based cements stimulates cell proliferation. 8,24 However, the addition of CA can also exhibit negative effects on cytocompatibility in measures such as cell viability, attachment, and growth. 7 It was also reported that, within the range of 10% of C3A in C3S, there was noncytotoxic in the L929 cell. 10 According to our results, we argue that adding 5% C12A7 to C3S cement does not negatively affect the cytocompatibility of hDPCs.

Conclusions
In our study, we fabricated purified C3S cement and evaluated the effects of C12A7 when added to Within the limitations of this study, we found that C3S-based cement with 5% C12A7 exhibited optimal characteristics, including faster initial setting time, improved compressive strength, and optimal alkalinity without adverse effects on cytocompatibility.
Therefore, C3S-based cement containing 5% C12A7 could be used as a base material for the further study and development of new biomaterials for endodontic procedures.