Calcium silicates have attracted great attention in orthopaedics and dentistry because of their excellent biocompatibility and osteogenesis. Prior to the clinical applications, some issues need to be clarified. In this project, first we investigated the effect of two solutions differing by pH (7.4 and 4.0) on the physicochemical properties of the radiopaque dicalcium silicate cement. Second, the effects of bismuth oxide (Bi2O3) radiopacifier on the physicochemical properties and in vitro osteogenic activities of the cements were examined.
The results indicated that, after soaking in a pH 7.4 solution for 1 day, the particle size of precipitated apatite spherulites on the cement surfaces was greater than that obtained in a pH 4.0 solution. Solution pH did not result in significant differences (P > 0.05) in diametral tensile strength of cement specimens at the same soaking time-point. On day 30, the sample was associated with a weight loss of 0.8% in a pH 4.0 solution, whereas in a pH 7.4 solution, a weight increase of 0.2% occurred. A greater porosity of the cement soaked in a pH 4.0 was found compared with that in the solution with pH 7.4. Regarding Bi2O3 effect, the setting time increased significantly (P < 0.05) with increasing Bi2O3 content. The pH value and diametral tensile strength of the cements were slightly affected by the introduction of Bi2O3. After the addition of 5, 10, and 20 wt% Bi2O3 the radiopacity of the cements became significantly (P <
0.05) higher, with values equivalent to 3.3, 5.8, and 8.4 mm of Al, respectively. The solubility of the three radiopaque cements ranged between 0.8% and 1.1%, which is significantly (P <
0.05) lower than that of white-colored mineral trioxide aggregate (WMTA) (1.4%). 20 wt%
Bi2O3 led to lower cell proliferation, differentiation, and calcium deposits of MG63 cells on the cement surfaces at all culture times compared those obtained with the other cements. The addition of 10 wt% Bi2O3 to dicalcium silicate cement improves the setting time, radiopacity, and osteogenic activity, making the cement a potential alternative to WMTA as a root-end filling material.
Keywords: Calcium silicate cement, preclinical study, root-end filling material, acid environment, bismuth oxide
3. Introduction and purpose
Calcium silicates have attracted great attention in orthopaedics and dentistry because of their excellent biocompatibility and osteogenesis [1-3]. A variety of Portland cement-based materials consisting mainly of calcium silicate have been developed for endodontic use as an alternative to calcium silicate-based mineral trioxide aggregate (MTA), on the basis of reducing setting time [4-8]. In recent studies [9,10], aluminium-free hydraulic and radiopaque dicalcium silicate cement displayed a shorter setting time and better biocompatibility than white-coloured MTA and thus may have the potential to be a root-end filling material.
During clinical practice, the periradicular environment may have varying pH from a neutral pH of 7.4 to an acidic pH as low as 5.0, because of bacterial-induced local metabolic acidosis or tissue inflammation [11]. The root-end filling materials may be exposed to an inflammatory environment with relatively low pH values [12,13]. A low pH could potentially inhibit setting reactions, affect adhesion, or increase the solubility of the materials such as MTA [11-14]. For example, Shie et al. reported that pH 4.0 had a deleterious effect on the morphology of white-colored MTA mixed with water [14]. Hence, it is worthwhile to evaluate changes in the characteristics of the root-end filling material after implantation or after soaking in a physiological solution with different pH values.
On the other hand, MTA powder is basically a mixture of calcium-silicate-based Portland cement and bismuth oxide (Bi2O3). Bi2O3 is introduced to improve radiopacity, which is important for endodontic treatment [15,16]. Endodontic materials should have sufficient radiopacity to be distinguished from the peripheral anatomical structures. ProRoot MTA has 20 wt% Bi2O3 as reported by the manufacturer [17]. However, the addition of radiopacifiers might be detrimental to some of the physical, mechanical, and biological properties of endodontic materials [18]. The addition of Bi2O3 has been questioned as it could influence the properties of the cement. The inclusion of Bi2O3 in Portland cement leads to an increase in the amount of unreacted water in the cement, which in turn increases the porosity of the cement [15]. More importantly, Bi2O3 extends the setting time and reduces the compressive strength of the cement [15,16].
Although a variety of calcium-silicate-based materials have been developed for endodontic treatment [16,7,19,20], calcium silicate cement lacks sufficient radiopacity, limiting its application in endodontic use; therefore, Bi2O3 radiopacifier is added to it to reach an acceptable level of radiopacity.The effects of Bi2O3 on the physical properties, sealing ability, and biocompatibility of Portland cement are well documented [15,16]; however, there have been few systematic studies on its effects on physicochemical properties and in vitro osteogenic activities.
Following previous studies [9,19], the purpose of this study was to examine the physicochemical behaviours of a radiopaque dicalcium silicate cement soaked in physiological solutions with different pH values (4.0 and 7.4). The pH 4.0 condition was selected to simulate clinical conditions that would be considered extreme. The parameters of diametral tensile strength, morphology, porosity and weight change were determined, in addition to the pH of the solution as a function of soaking time. Additionally, the effects of Bi2O3 on the physicochemical properties and osteogenic activities of dicalcium silicate cement were examined systematically.
4. Experimental
4.1. Specimen preparation
Reagent-grade tetraethyl orthosilicate (Si(OC2H5)4; Sigma-Aldrich, St. Louis, MO) and calcium nitrate (Ca(NO3)2·4H2O; Showa, Tokyo, Japan) were used as precursors for SiO2 and CaO, respectively. The catalyst was 2 mol L-1 nitric acid, and absolute ethanol was used as the
solvent. The molar ratio of Ca(NO3)2·4H2O to Si(OC2H5)4 was 3 : 2. General sol-gel procedures, such as hydrolysis and aging, were adopted. A detailed description of the powder’s fabrication has been reported [19]. Briefly, Si(OC2H5)4 was hydrolyzed with the sequential addition of nitric acid and absolute ethanol with 1 h of stirring separately. The required amount of Ca(NO3)2·4H2O was added to the above solution, and the mixed solutions were stirred for an additional hour. The sol solution was sealed and aged at 60 ºC for 1 day.
After vaporization of the solvent in an oven at 120 ºC, the dried gel was heated in air to 800 ºC at a heating rate of 10 ºC min-1 for 2 h using a high-temperature furnace and then cooled to room temperature in the furnace to produce a powder. The sintered granules were ball-milled for 12 h in ethyl alcohol using a centrifugal ball mill (Retsch S 100, Hann, Germany) and then dried in an oven at 60 ºC. Bi2O3 (Sigma-Aldrich) was added to the ground powder at 20 wt%, as described by Torabinejad & White [17], using a conditioning mixer (ARE-250, Thinky, Tokyo, Japan). The cement specimens were prepared by hand mixing the powder with distilled water in a liquid-to-powder ratio of 0.4 mL g-1. The cements were placed in a cylindrical Teflon mould to form the cylindrical specimen with dimension of 6 mm (diameter) × 3 mm (height); the specimens were stored in an incubator at 100% relative humidity and 37 ºC for 1 day to set. Regarding Bi2O3 effect, the powder (Sigma-Aldrich) was added to the ground powder at 5%, 10%, or 20% by weight using a conditioning mixer. The cement specimens were prepared by hand mixing the powder with distilled water at liquid-to-powder ratios of 0.5 to 0.4 mL g-1 (Table 1). The specimen codes “CS control”, “CSB5”, “CSB10”, and “CSB20”
represent cements containing 0, 5, 10, and 20 wt% Bi2O3, respectively.
4.2. Soaking in physiological solution
The cement specimens were soaked in 10 mL physiological solution at 37 ºC. Ionic composition of the solution is similar to that of human blood plasma (Chen et al. 2009b), consisted of 7.9949 g NaCl, 0.3528 g NaHCO3, 0.2235 g KCl, 0.147 g K2HPO4, 0.305 g MgCl2·6H2O, 0.2775 g CaCl2, 0.071 g Na2SO4 in 1000 mL distilled H2O and was buffered to either pH 7.4 or 4.0 with hydrochloric acid (HCl; Merck, Darmstadt, Germany) and trishydroxymethyl aminomethane (CH2OH)3CNH2; Sigma-Aldrich). All chemicals used were of reagent grade and used as obtained. The solution in a shaker water bath was not changed daily. After soaking time periods of 1, 3, 7, 15 and 30 days, the specimens were removed from the vials to evaluate the in vitro physicochemical properties.
4.3. Surface morphology
The surface of the cement before and after soaking in the physiological solution was coated with gold using a JFC-1600 (JEOL, Tokyo, Japan) coater and observed under a JEOL JSM-7401F field emission scanning electron microscope (SEM) operating in the lower secondary electron image mode (LEI) at 3 kV accelerating voltage. The energy dispersive spectroscopy (EDS) was used to perform chemical analyses. Three samples were observed for each test condition.
4.4. Diametral tensile strength
Diametral tensile strength (DTS) testing was performed using an EZ-Test machine (Shimadzu, Kyoto, Japan) at a loading rate of 0.5 mm min1 after weight change and porosity measurements. The strength value of each cement specimen was calculated using the relationship defined in the equation DTS = 2P/πbw, where P is the peak load (N), b is the diameter (mm) and w is the thickness (mm) of the specimen. The peak load at failure was recorded from the load-deflection curves. Twenty specimens were tested for each time point.
Totally 200 specimens were examined.
4.5. Weight change
The degradation behavior of the cement specimens in the physiological solutions with different pH values was also determined through monitoring sample weight change. The samples were dried in an oven at 120 ºC for 3 h before (day 0) and after soaking and then weighed to constant weight using a four-digital balance (AE 240S, Mettler-Toledo AG, Greifensee, Switzerland). Twenty samples were tested for each condition.
4.6. Porosity
The measurement of the porosity was conducted by using a liquid displacement technique, according to the literature [20]. In this method, ethanol was used as the displacement liquid because water was the setting liquid. In order to enhance the precision, twenty samples for each condition were randomly divided into five subgroups. Four specimens were regarded as a subgroup and one measured value was obtained. The average value of five measurements was taken as the porosity of the cement specimens. Before the measurement, the set specimens were dried in an oven at 120 ºC for 3 h. The specimen was immersed in a graduated cylinder that contained a known volume (V1) of ethanol. Afterwards, it was ultrasonically stirred for at least 3 min to force the ethanol into the pores of the specimens until no air bubbles were observed emerging from the specimens. The total volume of ethanol and the ethanol-impregnated specimen was then recorded as V2. The ethanol-ethanol-impregnated specimen was removed from the cylinder, and the residual ethanol volume was recorded as V3. The volume differences, (V1 – V3) and (V2 − V3), were the pore and total volumes of the cement specimen, respectively. Thus, the porosity of the specimen was obtained by the following equation: Porosity = (V1 − V3)/(V2 − V3).
4.7. pH variation of the solution
The pH values of the solution were measured using a pH meter (SP-701, Suntex, Taipei, Taiwan). The electrode was inserted into the soaking solution at room temperature. Twenty measurements were performed.
4.8. Setting time
The setting time of each cement was tested using a 400-g Gillmore needle with a flat end, 1 mm in diameter, according to ISO 9917-1:2003. Each material was mixed and placed in a cylindrical Teflon mold (diameter = 6 mm and height = 6 mm). The tests were performed in an incubator maintained at 37 ºC with a relative humidity of at least 90%. One minute after mixing, the indenter needle was lowered vertically onto the surface of the test cement for 5 s.
The setting time was recorded as the time that elapsed between the end of mixing and the time when the needle failed to create an indentation of 1 mm in depth in three separate areas of the cement. The setting times of six specimens for each cement group were measured.
4.9. pH variation in the cement
The pH values of the cement specimens during setting were measured with an IQ120 miniLab pH meter (IQ Scientific Instruments, San Diego, CA). Triplicate measurements were used.
4.10. Phase composition
To investigate the phase composition, the cement specimens were characterized using X-ray diffraction (XRD, Shimadzu XD-D1, Kyoto, Japan). Three samples were used for each group.
4.11. Radiopacity
The radiopacity and solubility of various cement specimens were determined according to the method in ISO 6876:2001. The radiopacity of each specimen was measured by irradiating specimens alongside an aluminium step wedge (10 steps, 1 mm per step). An Ashia G610S X-ray unit (Kyoto, Japan) with Kodak dental intraoral E-speed X-X-ray film (Carestream Health, Rochester, NY) was used, operating at 60 kV, 10 mA, 5 pulses/s, and a focus-surface distance of 200 mm. The developed film was transformed into digital images using a Canon EOS 350D digital camera (Tokyo, Japan). A standard curve of gray-level values versus thickness of aluminum was established to determine the radiopacity value of each specimen using ImageJ software (National Institutes of Health, Bethesda, Maryland). The corresponding gray-level value for each specimen was superimposed on the standard curve and the equivalent thickness of aluminum was recorded. Twelve parallel measurements were performed with the data of every group.
4.12. Solubility
The solubility of the cement was examined according to ISO 6876:2001. After mixing, the cements were placed into a plastic mold (diameter = 20 mm and thickness = 1.5 mm) and covered with a glass plate. The molds were stored in an incubator at 100% relative humidity and 37 ºC for 24 h after which the specimens were removed from the molds. One specimen was placed in a shallow dish and 25 mL of water was added. The dish was then covered and placed in an incubator at 37 ºC for 24 h. The specimens were then removed and rinsed with 3 mL of water; after which, the specimens were discarded. The container and the water were then weighed, followed by evaporation of the water until a constant mass was achieved. The containers were cooled in a desiccator and the weight of the containers before and after the placing of specimens was determined to an accuracy of 0.001 g. The solubility of the materials under test was calculated by recording the difference in mass as a percentage of the original mass of the shallow dish. Six samples were tested for each cement group.
4.13. Cell culture
MG63 human osteoblast-like cells (BCRC 60279; Hsinchu, Taiwan) were used to evaluate cell behavior. They were suspended in Dulbecco’s modified Eagle medium (DMEM; Gibco, Langley, OK) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 g/mL) solution (Gibco) in 5% CO2 at 37 ºC. Prior to cell incubation, the hardened cement specimens were sterilized by soaking in a 75% ethanol solution and exposure to ultraviolet (UV) light for 2 h. MG63 cell suspensions at a density of 5×103 cells/mL were seeded over each of the cement specimens. Cells cultured on the cement without Bi2O3 were used as a negative control and the CSB20 group was used as a positive control.
4.14. Cell proliferation
The reagent Alamar Blue (Invitrogen, Grand Island, NY) was used for real-time and repeated monitoring of cell proliferation, which is based on the detection of mitochondrial activity. The number of living cells can be estimated via redox reactions between the indicator dye and metabolically active cells. To assess proliferation, cells were cultured for 1, 3, and 7 days. The culture media were changed every 3 days. Briefly, at the end of the culture period, the medium was discarded and the cells were washed with phosphate-buffered saline (PBS) twice. Each well was filled with 350 L at a ratio of 1:99 Alamar Blue:fresh medium and incubated at 37 ºC for 2 h. 100 µL of the solution in each well was transferred to a 96-well
tissue culture plate. Plates were read in a Sunrise Microtiter Reader (Tecan Austria Gesellschaft, Salzburg, Austria) at 570 nm with a reference wavelength of 600 nm. The results were obtained from three separate experiments for each test and represented in terms of optical density (OD).
4.15. Cell differentiation
To evaluate the effect of Bi2O3 content on early cell differentiation, the alkaline phosphatase (ALP) activity assay was carried out using a TRACP & ALP assay kit (Takara, Shiga, Japan) according to the manufacture’s instructions. ALP catalyzes the hydrolysis of the colorless organic phosphate ester substrate, p-nitrophenyl phosphate (pNPP), to p-nitrophenol, a yellow product, and phosphate. To perform the assay, after 7 and 14 days of incubation, the cells were washed with physiological saline (150 mM NaCl) and lysed in 50 μL of lysis buffer (1% NP40 in 150 mM NaCl). For measurement purposes, 50 μL of the substrate solution (20 mM Tris-HCl, 1 mM MgCl2, 12.5 mM pNPP, pH = 9.5) was added to each well and allowed to react at 37 ºC for 30 min in the dark. The reaction was stopped by the addition of 50 μL of 0.9 M NaOH and read at 405 nm using a Sunrise Microplate Reader. Three dependent measurements were made.
4.16. Calcium quantification
The mineralized matrix synthesis was analyzed using an Alizarin Red S staining method, which identifies calcium deposits. After culture for 7 and 14 days, the cells were washed with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at 4 ºC. This was followed by staining for 10 min in 0.5% Alizarin Red S (Sigma-Aldrich) in PBS at room temperature. Cells were completely washed with PBS and then observed using an optical microscope (BH2-UMA; Olympus, Tokyo, Japan). To quantify matrix mineralization, the calcium mineral precipitate was destained by 10% cetylpyridinium chloride (Sigma-Aldrich) in PBS for 30 min at room temperature. The absorbance of Alizarin Red S extracts was measured at 562 nm using a Sunrise Microplate Reader. The data were expressed as OD. Mean absorbance values were obtained from three independent experiments. To clarify the material effect, the cement specimens without cell culture (day 0) were also tested as the blank test.
Three measurements were made.
4.17. Statistical analysis
A two-way ANOVA statistical analysis was used to evaluate the significance of the differences between mean values. Scheffé multiple comparison testing was used to determine the significance of the deviations in the data. In all cases, the results were considered statistically significant at a P value <0.05.
5. Results
5.1. Surface morphology
Broad face SEM micrographs of the cements after soaking in a solution with different pH values for 1 and 30 days are shown in Fig. 1, in addition to Ca/P ratio.The Ca/P ratios of the cement specimen after 1 day of soaking in pH 7.4 and 4.0 solutions were 1.44 and 2.56 (Fig.
1a), respectively; on day 7, the Ca/P ratios became 1.68 and 1.84. Before soaking, the cement specimen essentially appeared rather smooth looking with particle entanglement and several micropores (Fig. 1b). After soaking in a physiological solution, i
t was
clear that precipitation took place on the cement surfaces, which were covered with clusters of precipitated apatite spherulites (Fig. 1cf). However, it is worth noting that after soaking in a pH 7.4 solution for 1 day (Fig. 1c), the size of apatite spherulites was greater than that formed in the pH 4.0 solution(Fig. 1d). With increasing soaking time, spherulites coalesced to form a surface apatite layer.
Greater spherule aggregates appeared on the cement surface under a pH 7.4 condition (Fig. 1e) compared to that under a pH 4.0 condition (Fig. 1f).
5.2. Diametral tensile strength
The changes in diametral tensile strength value of the cements before and after soaking in a pH 7.4 or 4.0 solution as a function of time are shown in Fig. 2. The cement specimen gradually increased in strength with an increase in soaking time, achieving a maximum on day 7, and thereafter decreased. The values at 0- and 30-day soaking in a pH 7.4 solution were 4.7
± 0.4 and 4.8 ± 0.8 MPa, respectively, indicating no significant difference (P > 0.05). In the case of pH 4.0, day 30 samples could achieve a value of 3.8 ± 0.6 MPa, which was comparable to that obtained in the environment of pH 7.4. Scheffé post hoc tests revealed that the difference between the strength values of cement specimens exposed to pH 7.4 and pH 4.0 was not statistically significant (P > 0.05) at the same soaking time point, although the cement soaked in a pH 7.4 solution had a higher strength than the pH 4.0 solution. Soaking time significantly (P < 0.05) affected the strength of the cement soaked in either pH 7.4 or 4.0 solution.
5.3. Weight change
There were statistically significant differences (P < 0.05) in the weight change among the groups because of solution pH and soaking time. Figure 3 shows the weight changes for the cements after exposure to the physiological solution. In terms of solution pH, the cement gained weight of 2.0% and 1.2% after 3 days of soaking in a pH 7.4 and 4.0 solution, respectively; afterwards, the sample weight reduced to -0.2% and 0.8% on day 30. Not only the initial solution pH values significantly (P < 0.05) affected the weight change of the cement
There were statistically significant differences (P < 0.05) in the weight change among the groups because of solution pH and soaking time. Figure 3 shows the weight changes for the cements after exposure to the physiological solution. In terms of solution pH, the cement gained weight of 2.0% and 1.2% after 3 days of soaking in a pH 7.4 and 4.0 solution, respectively; afterwards, the sample weight reduced to -0.2% and 0.8% on day 30. Not only the initial solution pH values significantly (P < 0.05) affected the weight change of the cement