This scientific study was performed in Dental Faculty of Azad University in Tehran, Iran. The study was performed from 2011-2013.

Root MTA (Baran Darou Parse, Tabriz, Iran) and Biodentine cements (Septodont, Lyon, France) were used in this study. Each cement powder was mixed according to the manufacturer’s instruction and packed into a cylindrical polyethylene tube with an internal diameter of 20 mm and a height of 5 mm using a non surgical manual MTA carrier (Dentsply, TulsaDental, USA) and hand pressure according to the previous studies [13, 14]. The cylindrical tubes were then stored in an incubator for 72 hour; two groups in DW as control groups and two groups in STF. The STF was prepared as follows: 1.7 g of KH₂PO₄, 11.8 g of Na₂HPO₄, 80.0 g of NaCl and 2.0 g of KCl in 10 L of H₂O (pH=7.2) according to a previous study [4]. The STF was renewed every day. There were four experimental groups:

Group A1: 10 tubes of MTA stored in DW.

Group A2: 10 tubes of MTA stored in STF.

Group B1:10 tubes of Biodentine stored in DW.

Group B2:10 tubes of Biodentine stored in STF.

Finally, all the specimens were stored in a desiccator for 24 hours prior to SEM analysis.

After drying, specimens were polished and sputter-coated with 10 nm of gold and evaluated under a scanning electron microscope (TESCAN, Tesla, Czech Republic). Secondary Electron (SE) and Back-Scattered Electron (BSE) detectors at ×500,×1000, ×2000 and ×5000 magnifications were selected. EDS color dot map analysis was performed for each sample at each magnification. Photomicrographs were taken and analyzed at desired magnifications. Digital images were recorded using a Microsoft picture manager (Redmond, WA, USA) to standardize each picture at 600×800 pixels. Surface porosity was calculated using the Image J program (Rasband WS, ImageJ; US National Institute of Health, Bethesda, MD, USA) according to the previous studies [12, 15]. Each figure was inverted [Table/Fig-1a-c] by this program and brightness was adjusted to select the orifices of each figure [Table/Fig-1c,d]. Binary images were made considering the orifices of the pores as outlined and the total number of outlines and the total pore surface in each micrograph was calculated in μm² [Table/Fig-1e-g].

[Table/Fig-1]:

Sequence of image analysis using Image J. a) Micrograph of White Mineral Trioxide Aggregate (WMTA) polished perpendicularly; b) Image is inverted for easy calculation; c) Enhanced contrast. d) Conversion to 8-bit type for better calculation. e) Threshold adjustment for better calculation; f) Calculation of the amount of circles or ellipses in the micrograph in regards to the scale; g) The mean ± standard deviation of the number of outlines of total pores in μm2 in each micrograph were calculated for each sample in experimental groups.

Data means and SD were elaborated by a spreadsheet (Excel; Microsoft Corp, Redmond, Wash). The statistical program of SPSS 9.0 (SPSS Inc., Chicago, IL, USA) was used. The data were analyzed by using one-way analysis of variance (ANOVA) test.

The means and standard deviations of the number of total porosities and the surface area covered by porosities in μm² of experimental groups are shown in [Table/Fig-2]. In ×500, ×1000 and ×2000 magnification micrographs, significant differences were noted for the number of total porosities and the surface area covered by porosities in both DW and STF storage medium inside each experimental group and between groups (p<0.001). However, in ×5000 magnification micrographs, the number of total porosities and the surface area covered by porosities of Biodentine and MTA samples stored in DW and storage medium did not show any significant differences (p<0.2). While at ×5000 magnification, the number of total porosities and the surface area covered by porosities of Biodentine and MTA samples stored in STF, showed significant differences (p<0.01).

The SEM images of the samples at ×1000 are presented in [Table/Fig-3]. As shown in this figure, samples stored in DW indicate more irregularities and porosities at the surface of cement [Table/Fig-3a, c]; please note arrows). However, the SEM images of surface of samples stored in STF medium showed more homogenous and regular contribution of cement material and lesser porosities [Table/Fig-3b,d]; please note arrows). The comparison of SEM images of experimental groups showed that MTA samples stored in DW and STF storage medium showed lesser irregularities and surface porosities compared to Biodentine samples [Table/Fig-3a,b]. The SEM images of Biodentine samples stored in DW showed large irregularities and porosities with large amount to unmixed cement powder, while the images of sample stored in STF showed better hydration and mixture of powder with lesser porosities [Table/Fig-3c,d].

[Table/Fig-3]:

The SEM images of MTA and Biodentine samples at ×1000 magnification (200 μm scale). a) SEM image of MTA sample stored in DW, Please note the pores formed at the surface of cement due to storage in DW (green arrows). b) SEM image of MTA sample stored in STF. Please note the uniform distribution of cement materials on the surface, which presents lesser irregularities and porosities (red arrows).c) SEM image of the Biodentine sample stored in DW. Please note the large amount of irregularities and unmixed powder particles at the surface due to storage in DW (orange arrows).d) SEM image of the Biodentine sample stored in STF. Please note the large size and also cleft like irregularities and porosities shown on the surface (blue arrows).

Present study intended to evaluate the effect of two storage medium including STF and DW on surface porosity of MTA and Biodentine cements. The result of this study showed that the number of porosities and the surface covered by porosities in Biodentine samples were higher than MTA in STF and DW. Infact, the presence of more porosity in Biodentine might be related to the different components [5,6] procedure of mixing and powder to liquid ratio compared to MTA [16]. The main component of the Biodentine powder is tricalcium silicate and dicalcium silicate, with the addition of CaCO₃ as filler and ZrO₂ as a radiopacifier. The liquid is a solution of CaCl₂ (accelerator of setting) with a water reducing agent [5,6]. The addition of zirconium oxide doesn’t react with base of calcium silicate cements so does not affect the hydration of the cements. In other words, the zirconium oxide is an inert filler in the matrix of Biodentine causes porosity [17]. CaCl₂ accelerates the setting reaction [5,6]. If a setting reaction occurs very fast, it causes more porosity. Also, one of the other strategies for reducing the setting time is decreasing of the liquid content in the system [5]. This results in incomplete hydration of powder that produces more porosity [18]. This issue was also detected in SEM images of Biodentine sample stored in DW [Table/Fig-3c] In relation to the powder to liquid ratio, as we know, MTA is hand-mixed using a 3:1 powder to liquid ratio.

For Biodentine, liquid from the single-dose container is added into the powder-containing capsule and triturated with an amalgamator [16]. These amounts show more liquid in Biodentine can also affect more porosity for this cement [18]. In comparison of hand-mixing preparation of MTA, Biodentine is mixed by trituration [16]. Mechanical mixing like trituration could increase compressive strength [19], but this factor reduces the setting time [16], which can affect the porosity indirectly. In some previous investigations, normal saline or tap water was used as storage medium for MTA cement [20-22]. However, in other studies, authors have used phosphate containing solution for this purpose to mimic the clinical situation [11,23-25]. It was indicated that STF as storage medium can reduce the microleakage value in MTA cement [11]. Other investigators showed that phosphate buffer solutions can decrease the bacterial penetration or microleakage of calcium silicate cements due to formation of crystals over these cements [25]. Similar results were reported by Taddei P et al., where these authors reported that superficial biocoating layer can be formed on the surface of these cements when they were soaked in phosphate-rich solution during a time period of one-six months. It was explained that when calcium silicate cements are soaked in lower concentration phosphate solution, the amount of apatite precipitation is decreased and the time for apatite formation is prolonged [26,27].

The high amount of phosphate represents the continuous replenishment of phosphate ions from tissue fluids [28,29] and the absence of calcium causes the release of calcium ions from the cements, the limiting parameter in the precipitation reaction [21]. The studies have reported that Portland cements release calcium and when soak in phosphate solutions, induce apatite formation and produces high alkalinity in surrounding environment [4]. The hydration reaction of calcium silicate cement produces calcium silicate (C–S–H) gel and a large amount of calcium hydroxide. Calcium-silicate cements like MTA aged in physiological-like phosphate solutions such as STF produce a superficial biocoating formed by carbonated apatite and calcite (calcium carbonate) deposits can affect surface porosity [30, 31]. Infact, the reaction between these cements and STF is as following: Calcium ions supplied by the rapid dissolution of calcium hydroxide can react with environmental carbonate ions to form a calcium carbonate (calcite) superficial layer. Calcium carbonate may precipitate at the surface and in the cement paste porosity forming a protective layer of calcium carbonate on the outer zone of cement discs [32]. This calcium rich layer may absorb phosphate from the STF allowing the crystallization of apatite crystals [30]. These reactions are as follow:

2(3CaO.SiO₂) + 6 H₂O→3CaO.2SiO₂ .3H₂O + 3Ca (OH) ₂

Ca (OH) ₂→Ca²⁺ + H₂O

Ca²⁺ + CO_3-→CaCO₃ (Calcite)

CaCO₃ + PO₄^3-→3 CaCO₃. PO₄ (Apatite)

The apatite content in the coating layer increases over soaking time [33]. This layer decreases microleakage by filling gaps in dentine or at the dentine-material interface [25,34]. In other words, presence of solution containing ion like phosphate can affect calcium silicate based cements and produce a superficial layer on the substance and therefore formation of this layer can affect chemical and physical properties of cement. Similar outcomes were noticed in present study as the SEM analysis of MTA samples showed better distribution of powder particles and uniform cement surface with lesser porosities specially in samples stored in STF medium [Table/Fig-3 a,b].

Goldberg M et al., showed the apatite formation after soaking in phosphate solution for Biodentine [35]. Han L and Okiji T demonstrated that both Biodentine and MTA caused the uptake of Ca and Si in the adjacent root canal dentine in the presence of phosphate buffered saline. The dentine ion uptake was more prominent for Biodentine than MTA at 30 and 90 days [36]. This result showed that more Ca can be released from Biodentine, which causes more porosity in this cement. These outcomes are consistent with present study results. Although, biological compounds, particularly the STF effect on bone metabolism has been proved [30], In our study, STF provided a significant positive effect in reducing the porosity, which can be regarded as a capacity to improve properties such as solubility and setting time in general. We recommend the immersion of calcium silicate cement in biological environment like STF in clinical scenario in order to reduce the surface porosity and leakage of set cement.

As an eye to the future, it will be beneficial to design more investigations, especially in vivo studies, in order to testify the outcomes of present study in clinical situation.

According to the present study results, it can be concluded that storage medium is an effective factor that can influence the porosity of tested calcium silicate-based cements. However, MTA showed lower surface porosity compared to Biodentine cement, which can result in lower microleakage in applied areas. Future studies in this area are needed to assess the storage medium may affect the bulk porosity of calcium silicate-based cements.