The microstructure and surface morphology of radiopaque tricalcium silicate cement exposed to different curing conditions

Abstract

Objective

Tricalcium silicate is the major constituent phase in mineral trioxide aggregate (MTA). It is thus postulated that pure tricalcium silicate can replace the Portland cement component of MTA. The aim of this research was to evaluate the microstructure and surface characteristics of radiopaque tricalcium silicate cement exposed to different curing conditions namely at 100% humidity or immersed in either water or a simulated body fluid at 37 °C.

Methods

The materials under study included tricalcium silicate and Portland cements with and without the addition of bismuth oxide radiopacifier. Material characterization was performed on hydrated cements using a combination of scanning electron microscopy (SEM) with X-ray energy dispersive (EDX) analyses and X-ray diffraction (XRD) analyses. Surface morphology was further investigated using optical profilometry. Testing was performed on cements cured at 100% humidity or immersed in either water or Hank’s balanced salt solution (HBSS) for 1 and 28 days at 37 °C. In addition leachate analysis was performed by X-ray fluorescence of the storage solution. The pH of the storage solution was assessed.

Results

All the cements produced calcium silicate hydrate and calcium hydroxide on hydration. Tricalcium silicate showed a higher reaction rate than Portland cement and addition of bismuth oxide seemed to also increase the rate of reaction with more calcium silicate hydrate and calcium hydroxide being produced as demonstrated by SEM and XRD analysis and also by surface deposits viewed by the optical profilometer. Cement immersion in HBSS resulted in the deposition of calcium phosphate during the early stages following immersion and extensive calcification after 28 days. The pH of all storage solutions was alkaline. The immersion in distilled water resulted in a higher pH of the solution than when the cements were immersed in HBSS. Leachate analysis demonstrated high calcium levels in all cements tested with higher levels in tricalcium silicate and bismuth replaced cements.

Significance

Tricalcium silicate cement is more bioactive than Portland cement as demonstrated by various characterization techniques. The bioactivity was monitored by measuring the production of calcium hydroxide and the formation of calcium phosphate when in contact with simulated body fluids.

Introduction

Tricalcium silicate (TCS) is one of the main constituents of mineral trioxide aggregate (MTA). It constitutes approximately 52% of the un-hydrated material . Portland cement which is the main constituent of MTA is in turn made up of 67–75% of tricalcium silicate . This percentage composition differs depending on the type of cement and the manufacturer . It is thus hypothesized that pure tricalcium silicate will be a candidate to replace the Portland cement component of MTA. Both MTA and tricalcium silicate cement belong to a new class of dental materials namely the Hydraulic Silicate Cements .

Hydration of tricalcium silicate in Portland cement results in the formation of calcium silicate hydrate gel, calcium hydroxide and un-reacted tricalcium silicate . This analysis was confirmed by research performed on Portland cement intended for use as a dental material whereby after 28 days of hydration a small fraction of the tricalcium silicate in Portland cement remained unreacted on hydration, and the rest formed calcium silicate hydrate and calcium hydroxide . In Portland cement the calcium silicate hydrate and calcium hydroxide are also produced by the reaction of dicalcium silicate with water . Pure tricalcium silicate also reacts with water producing calcium silicate hydrate and calcium hydroxide leaving a small portion of un-reacted material . The calcium hydroxide produced from the tricalcium silicate hydration possesses antibacterial and anti-inflammatory properties mainly due to the high (alkaline) pH of the surrounding environment after it dissolves . Calcium hydroxide can further induce mineralization which results in repair of damaged dentin matrix .

Pure tricalcium silicate is prepared by the sol-gel method . Tetraethyl Orthosilicate (TEOS, Si(OC 2 H 5 ) 4 ) and nitric acid as catalyst are combined in water under continuous stirring. Calcium Nitrate (Ca(NO 3 ) 2 ·4H 2 O) is then added and the solution is maintained at 60 °C until gelation occurs. The gel is then dried at 120 °C and calcined for 8 h at 1450 °C. The resultant powder is finally ground and sieved. The advantage of this method is that the raw materials are pure, unlike those used in Portland cement manufacture. In addition the raw materials are free of aluminum which has been linked to Parkinson’s and Alzheimer’s disease . Tricalcium silicate cement is nowadays used as the main constituent of a number of proprietary brands namely Biodentine (Active Biosilicate Technology™, Septodont, Saint-Maur-des-Fossés Cedex, France) and Bioaggregate (Verio Dental, Vancouver, Canada).

Both MTA and Portland cement have demonstrated bioactivity, i.e. both form a bone-like hydroxyapatite layer on the surface when immersed in physiological solution; this is a common characteristic observed in calcium-silicate based materials . This layer provides the benefits of increasing the sealing ability of MTA and promoting remineralization and regeneration of hard tissues . Tricalcium silicate also exhibits the ability to form hydroxyapatite in contact with simulated body fluid. This layer is also instrumental in maintaining the bone–biomaterial interface when implanted in the body . Furthermore, calcium silicate cements including tricalcium silicate were found to have advantageously shortened setting time compared with MTA, and postulated to be suitable for replacement of the cement component of MTA due to their similar composition and bioactivity . Tricalcium silicate cement possesses good injectability, good bioactivity and moderate in vitro degradability, meaning that ultimately the body may be able to replace the implanted cement by natural tissue . The addition of up to 30% calcium carbonate (CaCO 3 ), calcium sulphate (CaSO 4 ·0.5H 2 O) and calcium chloride resulted in improvement in physical properties of tricalcium silicate cement as well as enhancing the bioactivity and degradability of the resultant composite material . Tricalcium silicate exhibited a similar cell mortality and mutagenicity to that of MTA . It was thus concluded that tricalcium silicate was safe to use as a bulk restorative material, without requiring any surface treatments, as an alternative to traditional materials .

The aim of this research was to evaluate the microstructure and surface characteristics of radiopaque tricalcium silicate cement exposed to different curing conditions namely at 100% humidity or immersed in either water or a simulated body fluid at 37 °C.

Materials and methods

Materials used in this study were Portland cement (PC; CEM 1, 52.5 N; LaFarge Cement, Birmingham, UK), tricalcium silicate (TCS manufactured using the sol-gel method; Mineral Research Processing, Meyzieu, France), and bismuth (III) oxide, Bi 2 O 3 (10 μm 223891-100G – Sigma Aldrich, St. Louis, MO, USA). The prototype materials and their corresponding water/powder ratios for each mixture are shown in Table 1 . The bismuth oxide replaced the 20% of the cement by weight.

Table 1
Test materials corresponding water/powder ratios.
Material Acronym Cement proportion (%) Radiopacifier proportion (%) Water/powder ratio
Portland cement PC 100 0 0.30
Portland cement and bismuth oxide PCB 80 20 0.30
Tricalcium silicate TCS 100 0 0.37
Tricalcium silicate and bismuth oxide TCSB 80 20 0.37

Microscopy and elemental analysis of powders and hydrated cements

Cylindrical specimens with 10 ± 0.1 mm diameter and a height of 2 ± 0.1 mm were prepared. The resultant discs were incubated in an atmosphere with 100% relative humidity for 24 h at 37 ± 1 °C to allow the cement to cure. They were then removed from the molds and either allowed to cure at 37 ± 1 °C in an atmosphere of 100% relative humidity, or immersed in either distilled water or Hank’s balanced salt solution (HBSS; H6648, Sigma Aldrich, St. Louis, MO, USA). The composition of the HBSS was (g/l) 0.4 KCl, 0.06 KH 2 PO 4 anhydrous, 0.35 NaHCO 3 , 8.0 NaCl, 0.05 Na 2 HPO 4 anhydrous and 1.0 d -glucose.

After both 1 day and 28 days of curing, the specimens were taken out of solution, dried using a filter paper and placed in an oven at around 60 °C for 24 h to dry. They were then placed overnight in a vacuum chamber and subsequently mounted on aluminum stubs. The cements were then examined under the scanning electron microscope (SEM; Zeiss MERLIN Field Emission SEM, Carl Zeiss NTS GmbH, Oberkochen, Germany) and micro-structural analysis was performed.

X-ray diffraction (XRD) analysis

Phase analysis was carried out on the cements using X-ray diffraction (Bruker D8 Advance, Bruker Corp., Billerica, MA, USA) in locked mode. The diffractometer was set with a Mo Kα radiation tube. The operating current and voltage was set at 35 mA and 45 kV respectively. Specimens were presented both as solid discs of diameter 10 ± 0.1 mm and height 2 ± 0.1 mm for surface analysis, and in powdered form. The intensity of X-rays from the sample were measured using a detector which was rotated between 5° and 25°. A step of 0.01° 2 and a step time of 0.4 s was used. For the powdered specimens the sample holder was spun at 15 rpm. Analysis was performed on cement samples following 1 day and 28 days of curing at 37 ± 1 °C in an atmosphere of 100% relative humidity, or immersed in either distilled water or HBSS. Phase identification was accomplished using a search-match software utilizing ICDD database (International Center for Diffraction Data, Newtown Square, PA, USA).

Evaluation of pH of leachate

Discs of diameter 15 ± 1 mm and a thickness of 1 ± 0.1 mm were prepared of each cement type. After storage in an incubator at 37 ± 1°C for 24 h and removal from the molds, the materials were immersed upright in either 10 ml water or Hanks balanced salt solution. The pH readings of the storage solution were taken using a pH meter (Hanna HI 9811, Hanna Instruments, Woonsocket, RI, USA) prior to immersion and after 1, 7, 14, 21 and 28 days post-immersion. Three replicate samples of each of the four materials were made for each of the two solutions (24 disks in total).

Chemical analysis of leachate

The chemical analysis of the cement products released in water and HBSS was performed using Energy Dispersive X-ray fluorescence (EDXRF). One disk of each of the four materials was immersed in 5 ± 0.001 ml of either distilled water or HBSS in an airtight polycarbonate container, for a period of either 1 day or 28 days from the immersion, at a constant 37 ± 1 °C. At each time point the disks were removed from solution and the solution was analyzed using EDXRF (Bruker S2 Ranger, Bruker Corporation, Madison, USA) using water as matrix and 4 μm liquid prolene film. Containers filled with water and HBSS were used as controls.

Profilometry

Surface analysis of pre-prepared samples was carried out using 3D optical profilometry. The profiler (Xyris 2000WL, TaiCaan Technologies, Southampton, UK) used a precision motion system and a confocal polychromatic (white) light optical probe to measure the displacement at the cement surface over a specified area. The region measured was a 1.000 mm by 1.000 mm area at the center of each cement sample with a measurement taken every 2.5 μm in the X and Y axes. This provided a 3D map of the cement surface consisting of 160,801 evenly spaced samples. Each measurement took approximately 12 min. Measurements of the cement samples were taken before (Day 0) and after (Day 28) either immersion in distilled water, immersion in HBSS or exposure to ambient atmosphere. The measurements were then analyzed for the changes in areal roughness, and 3D surface area.

Statistical analysis

The data were evaluated using SPSS (Statistical Package for the Social Sciences) software (SPSS Inc., Chicago, IL, USA). Parametric tests were performed as K–S tests on the results indicated that the data were normally distributed. Analysis of variance (ANOVA) with P = 0.05 and Tukey post hoc test were used to perform multiple comparison tests.

Materials and methods

Materials used in this study were Portland cement (PC; CEM 1, 52.5 N; LaFarge Cement, Birmingham, UK), tricalcium silicate (TCS manufactured using the sol-gel method; Mineral Research Processing, Meyzieu, France), and bismuth (III) oxide, Bi 2 O 3 (10 μm 223891-100G – Sigma Aldrich, St. Louis, MO, USA). The prototype materials and their corresponding water/powder ratios for each mixture are shown in Table 1 . The bismuth oxide replaced the 20% of the cement by weight.

Table 1
Test materials corresponding water/powder ratios.
Material Acronym Cement proportion (%) Radiopacifier proportion (%) Water/powder ratio
Portland cement PC 100 0 0.30
Portland cement and bismuth oxide PCB 80 20 0.30
Tricalcium silicate TCS 100 0 0.37
Tricalcium silicate and bismuth oxide TCSB 80 20 0.37

Microscopy and elemental analysis of powders and hydrated cements

Cylindrical specimens with 10 ± 0.1 mm diameter and a height of 2 ± 0.1 mm were prepared. The resultant discs were incubated in an atmosphere with 100% relative humidity for 24 h at 37 ± 1 °C to allow the cement to cure. They were then removed from the molds and either allowed to cure at 37 ± 1 °C in an atmosphere of 100% relative humidity, or immersed in either distilled water or Hank’s balanced salt solution (HBSS; H6648, Sigma Aldrich, St. Louis, MO, USA). The composition of the HBSS was (g/l) 0.4 KCl, 0.06 KH 2 PO 4 anhydrous, 0.35 NaHCO 3 , 8.0 NaCl, 0.05 Na 2 HPO 4 anhydrous and 1.0 d -glucose.

After both 1 day and 28 days of curing, the specimens were taken out of solution, dried using a filter paper and placed in an oven at around 60 °C for 24 h to dry. They were then placed overnight in a vacuum chamber and subsequently mounted on aluminum stubs. The cements were then examined under the scanning electron microscope (SEM; Zeiss MERLIN Field Emission SEM, Carl Zeiss NTS GmbH, Oberkochen, Germany) and micro-structural analysis was performed.

X-ray diffraction (XRD) analysis

Phase analysis was carried out on the cements using X-ray diffraction (Bruker D8 Advance, Bruker Corp., Billerica, MA, USA) in locked mode. The diffractometer was set with a Mo Kα radiation tube. The operating current and voltage was set at 35 mA and 45 kV respectively. Specimens were presented both as solid discs of diameter 10 ± 0.1 mm and height 2 ± 0.1 mm for surface analysis, and in powdered form. The intensity of X-rays from the sample were measured using a detector which was rotated between 5° and 25°. A step of 0.01° 2 and a step time of 0.4 s was used. For the powdered specimens the sample holder was spun at 15 rpm. Analysis was performed on cement samples following 1 day and 28 days of curing at 37 ± 1 °C in an atmosphere of 100% relative humidity, or immersed in either distilled water or HBSS. Phase identification was accomplished using a search-match software utilizing ICDD database (International Center for Diffraction Data, Newtown Square, PA, USA).

Evaluation of pH of leachate

Discs of diameter 15 ± 1 mm and a thickness of 1 ± 0.1 mm were prepared of each cement type. After storage in an incubator at 37 ± 1°C for 24 h and removal from the molds, the materials were immersed upright in either 10 ml water or Hanks balanced salt solution. The pH readings of the storage solution were taken using a pH meter (Hanna HI 9811, Hanna Instruments, Woonsocket, RI, USA) prior to immersion and after 1, 7, 14, 21 and 28 days post-immersion. Three replicate samples of each of the four materials were made for each of the two solutions (24 disks in total).

Chemical analysis of leachate

The chemical analysis of the cement products released in water and HBSS was performed using Energy Dispersive X-ray fluorescence (EDXRF). One disk of each of the four materials was immersed in 5 ± 0.001 ml of either distilled water or HBSS in an airtight polycarbonate container, for a period of either 1 day or 28 days from the immersion, at a constant 37 ± 1 °C. At each time point the disks were removed from solution and the solution was analyzed using EDXRF (Bruker S2 Ranger, Bruker Corporation, Madison, USA) using water as matrix and 4 μm liquid prolene film. Containers filled with water and HBSS were used as controls.

Profilometry

Surface analysis of pre-prepared samples was carried out using 3D optical profilometry. The profiler (Xyris 2000WL, TaiCaan Technologies, Southampton, UK) used a precision motion system and a confocal polychromatic (white) light optical probe to measure the displacement at the cement surface over a specified area. The region measured was a 1.000 mm by 1.000 mm area at the center of each cement sample with a measurement taken every 2.5 μm in the X and Y axes. This provided a 3D map of the cement surface consisting of 160,801 evenly spaced samples. Each measurement took approximately 12 min. Measurements of the cement samples were taken before (Day 0) and after (Day 28) either immersion in distilled water, immersion in HBSS or exposure to ambient atmosphere. The measurements were then analyzed for the changes in areal roughness, and 3D surface area.

Statistical analysis

The data were evaluated using SPSS (Statistical Package for the Social Sciences) software (SPSS Inc., Chicago, IL, USA). Parametric tests were performed as K–S tests on the results indicated that the data were normally distributed. Analysis of variance (ANOVA) with P = 0.05 and Tukey post hoc test were used to perform multiple comparison tests.

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Nov 28, 2017 | Posted by in Dental Materials | Comments Off on The microstructure and surface morphology of radiopaque tricalcium silicate cement exposed to different curing conditions
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