Biocompatibility and biomineralization of the experimental nanoparticulate mineral trioxide aggregate (MTA)

To investigate the tissue response and the biomineralization ability of the experimental nanoparticulate mineral trioxide aggregate compared to grey MTA and Fillapex MTA. Polyethylene tubes containing materials or empty tubes for control were inserted into the subcutaneous tissues of 30 rats. After 7, 15, 30, 60, and 90 days, the rats were killed and the tubes were removed for analysis using hematoxylin-eosin staining, von Kossa staining, and under polarized light. Inflammation was graded through a score system; the biomineralization ability was recorded as present or absent. The results were statistically analyzed using the Kruskal-Wallis test (p<0.05). On days 7 and 15 there was a significant difference between the Nano MTA (median score of 3) and MTA Fillapex groups (median score of 4), being MTA Fillapex the material with the highest number of inflammatory cells. At 30, 60, and 90 days there was no difference between the Nano MTA, Grey MTA, and MTA Fillapex groups. All materials induced the formation of mineralized tissue in all experimental periods. Nano MTA showed biocompatibility and biomineralization similar to grey MTA Angelus.

Materials based on mineral trioxide aggregate (MTA) are the gold standard for the aforementioned cases . These materials with a composition largely based on Portland cement components exhibit a hydrophilic nature, enabling its application even in the presence of moisture, once the main elements are tricalcium and dicalcium silicate, tricalcium aluminate, tricalcium oxide, and radiopacifying agents, such as bismuth oxide (J. Camilleri, 2008).
Although it is a material of excellent biological properties, some physicochemical and working properties are lacking.
As a result, infinite modifications and preparations have been developed and tested to circumvent these drawbacks. However, none of these new modifications presents powders in a nano-sized, that is particles with 1/1.000 of a micron. Nano-sized particles are of interest because of the high surface-to-volume ratio versus micron-sized particles. The surface-to-volume ratio changes from less than 10 % for micron-sized particles to more than 50 % for nanoparticles, which is essential because a higher surface-to-volume ratio can dramatically increase reactivity, such as hydration and MTA microhardness (Josette Camilleri, 2014).
Hence, this study evaluated a nanoparticulate mineral trioxide aggregate (Nano MTA), analyzing its in vivo reaction in the subcutaneous tissue of rats, and its power to induce mineralization assessed by von Kossa staining and structures birefringent to polarized light. Grey MTA and MTA Fillapex were used for comparison.

Animals
Thirty male Wistar rats aged 3 months were used in this study. Sample size estimates were based on data from previous studies using six animals per group (de Azevedo Queiroz et al., 2018;Viola et al., 2012). They were arranged into five groups, each group corresponding to an experimental time: 7, 15, 30, 60 and 90 days. The rats were kept in temperaturecontrolled housings receiving water and diet ad libitum. Animal care was provided according to the Ethics Committee of Araçatuba School of Dentistry-UNESP for animal use, which was approved before the beginning of the experiments (FOA no. 374-2017) We performed the study following the Animal Research Reporting In Vivo Experiment (ARRIVE) guidelines.

Statistical analysis
Using the GraphPad Prism (version 8.0) software (GraphPad Software Inc, La Jolla, USA), the Kruskal-Wallis test was performed, followed by Dunn's test. Values of p < 0.05 were considered significant.

Histological Analysis
Representative images of the groups can be observed in Figure     Mineral trioxide aggregate (MTA) is the primary hydraulic calcium silicate cement patented for endodontic applications and the most well known and most thoroughly investigated of all the hydraulic calcium cements available to date.
It is a clinker-derived Portland cement composed of different phases, including tricalcium silicate, dicalcium silicate, tricalcium aluminate, tetracalcium aluminoferrite and calcium sulphate, as well as bismuth oxide as a radiopacifier (Josette Camilleri, 2014).
The placement of the materials into the subcutaneous tissue of rats is considered a standardized and valid test for biocompatibility (Olsson, Sliwkowski, & Langeland, 1981), analyzing the inflammatory tissue response through hematoxylineosin staining (Bueno et al., 2018), within time periods of the endorsed standard practices for biological evaluation of dental the calcium carbonate formation from the calcium of the material and carbon dioxide from the surrounding tissue (Holland et al., 1999).
One possible reason for the adequate biological properties of the Nano MTA material containing is their high similarity to the conventional MTA composition, differing only in particle size. This change in particle size can play an important role in physical and chemical properties, leading to an increased surface area of powder that can reduce the setting time and increase the microhardness even at lower pH values after hydration. This fast setting prevents washout or dislodgement of MTA cement in clinical use. The bioactivity of these materials is due to the hydration of the calcium silicate leading to by-products formation. When in contact with the tissue, materials based on calcium silicate form calcium hydroxide and release calcium and hydroxyl ions promoting the increase of pH. The alkaline pH and the release of calcium ions initially promote a tissue inflammatory response, and later, these ions react with the carbon dioxide present in the tissue, giving rise to calcite crystals, which in turn are related to the decrease of inflammation and deposition of mineralized structures promoting repair (Cintra et al., 2017;Gomes-Filho et al., 2009;Holland et al., 1999).
Every new material needs to be tested priory being launched to the market. The sample of the Nano MTA used in this study was donated by a researcher from the São Paulo State University (UNESP), Ilha Solteira, Brazil. As in any preclinical experimental study, the results of the present investigation should be read with caution since the methodology used has limitations to its application in humans. The use of animals allows investigating different materials under controlled laboratory conditions prior to their use in humans (Browne, 1994). Although these results do not reflect a complete analysis of the reactions that occur in the human conditions, they are significant for the preliminary assessment of the biocompatibility and biomineralization of the tested material.
Considering that Nano MTA presented a biocompatibility and biomineralization capability similar to grey MTA, it seems to be a promising alternative for root canal treatment due to alleged high chemical physical properties as a nanoparticulate material. However, more studies are required to reinforce its physical and biological characteristics.

Conclusion
Nano MTA exhibited biocompatibility and biomineralization ability comparable to grey MTA Angelus.