Reformulated mineral trioxide aggregate components and the assessments for use as future dental regenerative cements

Abstract

Mineral trioxide aggregate, which comprises three major inorganic components, namely, tricalcium silicate (C3S), dicalcium silicate (C2S), and tricalcium aluminate (C3A), is promising regenerative cement for dentistry. While mineral trioxide aggregate has been successfully applied in retrograde filling, the exact role of each component in the mineral trioxide aggregate system is largely unexplored. In this study, we individually synthesized the three components, namely, C3S, C2A, and C3A, and then mixed them to achieve various compositions (a total of 14 compositions including those similar to mineral trioxide aggregate). All powders were fabricated to obtain high purity. The setting reaction of all cement compositions was within 40 min, which is shorter than for commercial mineral trioxide aggregate (~150 min). Over time, the pH of the composed cements initially showed an abrupt increase and then plateaued (pH 10-12), which is a typical behavior of mineral trioxide aggregate. The compression and tensile strength of the composed cements increased (2-4 times the initial values) with time for up to 21 days in an aqueous medium, the degree to which largely depended on the composition. The cell viability test with rat mesenchymal stem cells revealed no toxicity for any composition except C3A, which contained aluminum. To confirm the in vivo biological response, cement was retro-filled into an extracted rat tooth and the complex was re-implanted. Four weeks post-operation, histological assessments revealed that C3A caused significant tissue toxicity, while good tissue compatibility was observed with the other compositions. Taken together, these results reveal that of the three major constituents of mineral trioxide aggregate, C3A generated significant toxicity in vitro and in vivo, although it accelerated setting time. This study highlights the need for careful consideration with regard to the composition of mineral trioxide aggregate, and if possible (when other properties are satisfactory), the C3A component should be avoided, which can be achieved by the mixture of individual components.

Keywords: Mineral trioxide aggregate; dicalcium silicate; intentional replantation; tricalcium aluminate; tricalcium silicate.

Figure 1.

Characterization of the powder. After individual synthesis of the three major components of MTA (C3S, C2S, and C3A), (a) XRD, (b) SEM, and (c) laser diffraction analysis were performed to investigate the crystal structure, morphology, and size distribution of MTA, C3S, C2S, and C3A. Successful fabrication of the three powders (C3S, C2S, and C3A) with comparable size and shape was performed. All scale bars from SEM are 5 µm.

Figure 2.

Physicomechanical properties of the cement. (a) Compositions of 14 types of cement were analyzed for (b) setting time (n = 5), (c) pH change (n = 3) in DW, (d) PBS, (e) compressive strength (n = 5), and (f) diametrical tensile strength (n = 5) in SBF are shown. C3A accelerated setting time while it had little effect on pH and changes in strength within 20% share in cement formula. Error bars representing standard deviation are plotted in each graph. Sharp (#) in setting time results indicate significant difference compared to group 13 (C3A) at <0.05. For the observation of trend of the effect of C3A, statistical analysis results omitted in other graphs.

Figure 3.

Cytocompatibility of the elute from the cement against rMSCs. The 100% extraction from C3A showed severe cytotoxicity compared to the control, while the others significantly increased cell viability (asterisk, n = 4, p < 0.05). Error bars representing standard deviation are plotted in each graph.

Figure 4.

(a) Composition of the cements for the in vivo experiment. (b) Schematic image of the intentional implantation of the rat incisor after root-end filling with cement for investigation of the biocompatibility of the cement. (c) Images from the µCT after 4-week implantation. The red arrow in C3A indicates destruction of the thin radiopaque line at the material–tissue interface and the dashed rectangle shows alveolar bone surrounding the tooth, which was destroyed.

Figure 5.

H&E staining images after 4 weeks of implantation are shown. The dashed rectangle indicates the site of magnification (C: cement, AB: alveolar bone, T: developing teeth). The dotted black line shows the destruction of alveolar bone surrounding tooth end. Severe inflammatory responses noted with a number of macrophages.