Mineral trioxide aggregate (MTA) has been widely used for root-end filling, perforation repair, pulp capping, pulpotomy, and apexification.1,2,3,4,5 It is a type of hydraulic cement that sets in an aqueous environment.6 According to the manufacturer's instructions, MTA must be allowed to set in the presence of moisture by placing a wet cotton pellet against the intracanal surface of the material for a minimum of 4 hours. Torabinejad and Chivian also recommended covering MTA with a wet cotton pellet when it is used for pulp capping and perforation repair, or as an apical plug.1

However, there are conflicting results regarding the need for placing a wet cotton pellet over the MTA. In an investigation comparing the effect of the setting condition on the flexural strength of MTA, it has been shown that the flexural strength of MTA was significantly higher in specimens that received moisture from two sides than that in the case of one-sided specimen moisture exposure after a setting time of 24 hours.7 In the case of apexification, although a single-visit procedure with MTA has been suggested, the findings of another study support the two-step technique involving the placement of a moist cotton pellet on the MTA.8,9 Some authors have shown the positive effect of moisture on the push-out strength of MTA.10 On the other hand, it has been reported that dry ProRoot MTA powder packed into the root canals could achieve a full set at 72 hours solely because of the moisture absorbed through the root without any extra moisture received from the coronal aspect of the MTA.11 The findings of recent investigations have also indicated that placing a moist cotton pellet on the MTA may be unnecessary to improve the setting of MTA.12,13,14

Besides the controversy over the need to place a wet cotton pellet over the MTA, the definition of wet conditions and the type of moisture are not consistent in the literature. According to the manufacturer's instructions, a wet cotton pellet should be placed on the MTA. However, the type of moisture has not been mentioned. Interaction of MTA with solutions used over the coronal aspect of the material might affect its physicochemical properties depending on the constituents of the moisture. Several studies have shown that the interaction of MTA with a phosphate-containing solution such as phosphate-buffered saline (PBS) results in the formation of apatite crystals.15,16,17 Calcium ions released by MTA react with phosphate in PBS, resulting in the formation of hydroxyapatite or carbonated apatite.15,16

In various clinical applications, different thicknesses of MTA are used. Although it has been recommended that 4 to 5 mm-thick MTA be used as an apical barrier in the apexification procedure, there is no recommended thickness for vital pulp therapy, perforation repair, or root canal obturation.9,18 Some properties of MTA, such as sealing ability, displacement resistance, and hardness, can be impacted by the material thickness.9,19 Microhardness tests can be used for the assessment of the progress and the quality of the hydration process during the setting reaction, as well as the evaluation of the microstructural gradient of MTA materials.20,21

The present study was aimed at comparing the surface microhardness of different thicknesses of ProRoot MTA exposed to human blood from one side and with or without a distilled water- or PBS-moistened cotton pellet on the other side of the material.

Polymethyl methacrylate (Plexiglass, Cho Chen Industry Co. Ltd., Tainan City, Taiwan) cylindrical molds with an internal diameter of 4 mm and three heights of 2, 4, and 6 mm (30 molds of each height) were fabricated by computerized numerical control (CNC) laser cutting (LaserProI, GCC, New Taipei City, Taiwan). Floral foams wetted with whole fresh human blood were placed on glass slabs and then transferred into plastic containers. The human blood collection procedure was approved by the Ethics Committee of the Tehran University of Medical Sciences (No. 19905). Then, the molds were placed on blood-wetted foams. Tooth-colored ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK, USA) was mixed with sterile distilled water according to the manufacturer's instructions and delivered into the molds. The details of the experimental groups are as follows:

Group 1: dry condition (n = 30)

This group included three subgroups with the heights of 2, 4, and 6 mm (10 molds of each). After the molds were filled with MTA, the upper surface of the material was not exposed to any additional moisture and was completely covered with a layer of parafilm (Parafilm 'M' Laboratory Film, American Can Company, Greenwich, CT, USA).22

Group 2: exposed to distilled water (n = 30)

Molds with the heights of 2, 4, and 6 mm (10 molds of each) were filled with MTA. A distilled water-moistened cotton pellet was placed on the upper surface of the MTA.

Group 3: exposed to PBS (n = 30)

The specimens were prepared in the manner described for the previous groups, but a PBS-moistened cotton pellet was placed on the upper surface of the MTA.

The specimens of each group were stored in separate plastic containers. The closed containers were kept at 37℃ and a relative humidity of 100% for 4 days to simulate physiological conditions.

The results of the microhardness tests are shown in Table 1. A significant difference was found between the surface microhardness of the three thicknesses of MTA with no additional moisture on the material (p < 0.001). In the dry condition (group 1), the 2 mm-thick MTA samples showed a significantly higher surface microhardness than the 4 and 6 mm-thick samples (p = 0.003 and p = 0.001, respectively). On the other hand, the surface microhardness of MTA samples having different thicknesses was not significantly different in the presence of distilled water and PBS (p = 0.210 and p = 0.112, respectively). There was also no significant difference between the surface microhardness of the samples in groups 2 and 3 (p > 0.05). Furthermore, the VHN of 2 mm-thick samples set in the dry condition was not significantly different from that obtained for each tested thickness of MTA that received additional moisture from a distilled water- or PBS-wetted cotton pellet (p = 0.201).

The microhardness of MTA is influenced by many factors such as the thickness of the material, condensation pressure, pH and acid etching of the material.9,21,23,24 In this study, the effect of different setting conditions on the surface microhardness of MTA samples having different thicknesses was assessed using a Vickers hardness test. Since some fundamental properties of the material, such as yield strength, tensile strength, modulus of elasticity, and stability of the crystal structure, influence material microhardness, the effect of the setting conditions on the overall strength of the material can be evaluated using the microhardness test as an indicator of the setting reaction.20,23,25

In the present study, the inferior surface of MTA was in contact with the human blood-wetted foam to partially simulate some clinical situations, such as root perforations, vital pulp therapy, and apexification. However, the inferior surface of the material was not investigated because the effect of blood contamination on the surface microhardness and the compressive strength of MTA had been studied previously.26,27 In the dry condition, the upper surface of the MTA was covered with a layer of parafilm to protect it from further moisture exposure. In this study, the specimens were incubated at 37℃ and 100% humidity to provide a similar environment to the clinical situation. The assessment of microhardness was performed after incubation for 4 days. It has been suggested that MTA be untouched for at least 72 - 96 hours to decrease the chance of displacement.21,24,28

In the present study, the surface microhardness of the three thicknesses of MTA (2, 4, and 6 mm) was not similar when no moist cotton pellet was placed on the material. In this condition, the 4 and 6 mm-thick samples showed significantly lower surface microhardness than the 2 mm-thick specimens. In samples with a higher thickness, it can be expected that the exposure of the material to moisture only from one side might not be sufficient for the setting of parts that are away from the moisture source. MTA consists of fine hydrophilic particles that set to a hard composition when brought into contact with water through the creation of a colloidal gel.6,20,29 In the dry condition, the higher surface microhardness of the thinner samples of MTA might be attributed to the moisture provided from the blood-wetted foam at the bottom of the materials that might act as a moisture source for the setting of the upper surface of the MTA. On the other hand, Eid et al. showed no significant difference in hardness between the 3.5 mm-thick samples of ProRoot MTA placed in plastic cylinders with or without the application of a wet cotton pellet on the material.13 The difference between the results for the 4 mm-thick MTA samples used in the current study and the 3.5 mm-thick MTA samples used in the study conducted by Eid et al. could be explained by some differences in the methodologies used.13 In the study conducted by Eid et al., the MTA base was in contact with sterile gauze wetted with normal saline and the upper surface of MTA was covered with glass ionomer cement in the dry condition group.13

In the current study, there were no significant differences between the surface microhardness of 2, 4, and 6 mm-thick MTA samples exposed to the cotton pellets moistened with either distilled water or PBS at the upper surface of the material. However, the microhardness of 4 and 6 mm-thick MTA samples that received no additional moisture was significantly lower than that of samples with similar thickness but exposed to cotton pellets moistened with distilled water or PBS. In contrast to our results, DeAngelis et al. showed no difference between the surface microhardness of 4 mm-thick MTA samples with or without a moist cotton pellet on the material in unvarnished roots.12 Budig and Eleazer also showed that ProRoot MTA powder packed within the roots could achieve a complete set because of the moisture absorbed through the roots submerged in saline for 72 hours.11 These controversial results might be attributed to the absorption of moisture needed for the hydration of MTA from the intrinsic moisture retained within the dentinal tubules without the need of a wet cotton pellet.12,30 Although many studies have confirmed that the interaction between MTA and phosphate-containing solutions results in the formation of apatite crystalline structures over MTA, this study showed no difference between the microhardness of MTA samples having different thicknesses and exposed to PBS, which was comparable to that of the samples exposed to distilled water.16,17,31 The precipitation of apatite has been shown to begin within the first few hours of exposure to phosphate-containing fluids, however, the aggregation of greater amounts of apatite crystals occurs with an increase in the exposure time.16,32 In this study, the exposure of MTA to PBS for 4 days might be insufficient for altering the properties of the material.

The findings of this study also showed no significant difference between the 2 mm-thick samples of MTA in dry and wet conditions. These results are not in agreement with Walker et al., who showed that 2 mm-thick MTA specimens that received moisture from two sides had a significantly higher flexural strength than those that received moisture only from one side after a setting time of 24 hours.7 However, the results for surface microhardness could not be extrapolated to those for flexural strength.

Under the conditions of this study, it could be concluded that placing a moist cotton pellet on a 4 to 6 mm-thick MTA sample was necessary for better hydration of the material. However, this might not be essential when a 2 mm-thick MTA sample is used.