Abstract
Objectives
Remineralizing experimental composites based on amorphous calcium phosphate (ACP) were investigated. The impact of curing time (20 and 40 s), curing depth (1, 2, 3 and 4 mm) and addition of inert fillers (barium glass and silica) on the conversion and temperature rise during curing were examined.
Methods
Five ACP-composites and two control composites were prepared based on the light-curable EBPADMA-TEGDMA-HEMA resin. For temperature measurements, a commercial composite was used as an additional control. Conversion was assessed using FT-Raman spectroscopy by comparing the relative change of the band at 1640 cm −1 before and after polymerization. The temperature rise during curing was recorded in real-time using a T-type thermocouple.
Results
At 1 mm depth, the ACP-composites attained significantly higher conversion (77.8–87.3%) than the control composites based on the same resin (60.5–66.3%). The addition of inert fillers resulted in approximately 5% lower conversion at clinically relevant depths (up to 2 mm) for the curing time of 40 s. Conversion decline through depths depended on the added inert fillers. Conversion values higher than 80% of the maximum conversion were observed for all of the ACP-composites at depths up to 3 mm, when cured for 40 s. Significantly higher total temperature rise for the ACP-composites (11.5–13.1 °C) was measured compared to the control composites (8.6–10.8 °C) and the commercial control (8.7 °C).
Conclusions
The admixture of inert fillers represents a promising strategy for further development of ACP-composites, as it reduced the temperature rise while negligibly impairing the conversion.
Clinical significance
High conversions of ACP-composites are favorable in terms of mechanical properties and biocompatibility. However, high conversions were accompanied with high temperature rise, which might present a pulpal hazard.
1
Introduction
Composite materials based on amorphous calcium phosphate (ACP) represent an experimental class of remineralizing materials envisioned as a possible solution for combating secondary caries, one of the main causes of composite restoration failure . When exposed to an aqueous environment, composite materials incorporating ACP release calcium and phosphate ions in concentrations that are supersaturated with respect to hydroxyapatite, allowing the remineralization of dental hard tissues . Remineralizing effects of ACP-composites have been reported in vitro , in situ and in vivo , rendering them a promising future alternative to conventional composites. However, the clinical applicability of ACP-composites is limited by insufficient mechanical properties; their flexural strength ranges from 20 to 70 MPa , while at least 80 MPa is required for placement in load-bearing areas . The less than optimal mechanical properties are explained by the agglomeration of ACP particles, high water sorption of ACP-composites, intra-composite conversion of ACP to hydroxyapatite and lack of chemical bonding between the resinous matrix and ACP particles .
The issue of low mechanical properties has been addressed by introducing the ACP-composites with reinforcing silanized fillers similar to those used in commercial composites . This approach yields a “hybrid” material, whose filler consists of a bioactive part (ACP) and an inert part (barium glass or silica). Since the ACP filler compromises mechanical properties, its load should be reduced to a minimal level which is sufficient for the remineralizing effect. This has led to the optimization of the composition to 40 wt% of ACP . By adding up to 10 wt% of silanized barium glass or silica fillers, the flexural strength of ACP-composites was improved for up to 46% (from 22 to 32 MPa), while the ion release capability was unaffected . Despite the improvements, the flexural strength of the ACP-composites was still insufficient for use in load-bearing areas . Fine-tuning of the filler size, increasing the load of inert fillers and incorporating the nano-sized ACP particles may further improve mechanical properties , possibly leading to a remineralizing composite that would be able to withstand masticatory forces .
Adequate conversion is a basic prerequisite for a composite material, since it underlies virtually every property of the cured material, including its biocompatibility and mechanical features. ACP composites formulated with ethoxylated bisphenol A dimethacrylate (EBPADMA)-based resin attain high conversions (around 80%), as previously reported . Since high temperature rise might be a consequence of high conversion , this otherwise favorable property implies a potential hazard to pulpal health. However, the temperature rise does not depend simply on the conversion, but is affected by other factors, such as sample geometry, thermal capacity, translucency and polymerization kinetics . Thus it was of interest to examine the temperature rise that occurs during curing of ACP composites, as a possible side-effect of their high conversion.
Temperature rise due to the composite polymerization presents a continuing concern for pulpal health . There is currently no consensus regarding the maximum allowed intrapulpal temperature , nor the maximum temperature in the bulk of polymerizing composite material . In a classic and frequently cited study performed on monkey teeth, an intrapulpal temperature rise of 5 °C was proposed as critical for pulpal recovery . The findings of this study were critically evaluated , pointing out that the pulpal tolerance to temperature rise may have been underestimated. During that experiment, the heat source was kept in contact with the tooth for a predetermined time (5–20 s). Upon reaching a desired time point, the intrapulpal temperature was recorded. At this time, there was a temperature gradient from the point of application of heat source on the tooth surface towards the pulp, the temperature at the tooth surface being much higher than recorded in the pulp. The intrapulpal temperature continued to rise after its value was recorded in the experiment. It is thus likely that the intrapulpal temperature rise required for irreversible damage is higher than the reported 5.5 °C. Another study evaluated the pulpal response in premolar teeth scheduled for orthodontic extraction and found no histological changes indicative of pulpal damage upon the temperature increase up to 11 °C. However, the study was performed on young intact teeth and the findings may not apply to teeth whose pulp is already traumatized by caries and restorative procedures. Moreover, the temperature recorded in the bulk of the composite cannot be simply translated into the intrapulpal temperature. In the absence of reliable reference values for maximum allowed temperature rise, the researchers often resort to comparison with the temperature rise produced by existing commercial composite materials, for which there is clinical evidence of being tolerated by the pulp .
This study is a sequel to our investigations of ACP-composites modified with inert fillers . The impact of curing time, curing depth and addition of inert fillers on the conversion of ACP-composites is examined and the temperature rise during curing was recorded in real-time and compared to that of a commercial composite. The hypotheses tested were: (I) ACP-composites attain higher conversion than the control composites based on the same resin and photoinitiator; (II) conversion of ACP-composites is influenced by the following factors: curing time (20 and 40 s), curing depth (1, 2, 3 and 4 mm) and addition of inert fillers (5 formulations); (III) ACP-composites can be adequately cured up to 3 mm depth; (IV) high conversion of ACP-composites is accompanied with a high temperature rise; (V) ACP-composites produce higher temperature during curing than a commercial composite.
2
Materials and methods
2.1
Composite materials
The synthesis of zirconia-hybridized ACP (Zr-ACP) fillers followed the procedure by Skrtic et al. . Briefly, Zr-ACP precipitated from a water solution upon mixing of Ca(NO 3 ) 2 , Na 2 HPO 4 , Na 4 P 2 O 7 and ZrOCl 2 . The suspension was filtered, the solid phase washed with ice-cold ammoniated water and acetone and then lyophilized.
The experimental resin contained 67 wt% of EBPADMA (Esstech, Essington, PA, USA), 23 wt% of tri-ethylene glycol dimethacrylate (TEGDMA; Esstech) and 10 wt% of 2-hydroxyethyl methacrylate (HEMA; Esstech). The resin was rendered photosensitive by the addition of 0.2 wt% of the photo oxidant camphorquinone (CQ; Aldrich, Milwaukee, WI, USA) and 0.8 wt% of photo reductant ethyl-4- (dimethylamino) benzoate (4E; Aldrich). The monomers and photoactivators were mixed in the lightproof container using a magnetic stirrer for 48 h in order to assure a homogeneous mixture.
The resin was blended with fillers in lightproof containers using a dual asymmetric centrifugal mixing system (Speed Mixer TM DAC 150 FVZ, Hauschild & Co KG, Hamm, Germany) at 2700 rpm. The mixing was performed in five one-minute intervals separated by one-minute breaks. After mixing, the obtained composite pastes were deaerated in vacuum for 12 h. Seven experimental materials were prepared: five were ACP-based, while two materials were based on conventional inert fillers and served as a control ( Table 1 ).
| Material | Filler (wt%) | Resin (wt%) | Filler load (vol%) | |
|---|---|---|---|---|
| ACP-based materials | ACP40 | 40% Zr-ACP | 60 | 27.6 |
| ACP40-Ba10 | 40% Zr-ACP, 10% Ba-fillers | 50 | 35.1 | |
| ACP40-Si10 | 40% Zr-ACP, 10% Si-fillers | 50 | 36.6 | |
| ACP40-Ba5Si5 | 40% Zr-ACP, 5% Ba-fillers, 5% Si-fillers | 50 | 35.9 | |
| ACP40- Ba9Si1 | 40% Zr-ACP, 9% Ba-fillers, 1% Si-fillers | 50 | 35.3 | |
| Control materials | Ba40 | 40% Ba-fillers | 60 | 22.0 |
| Ba40Si10 | 40% Ba-fillers, 10% Si-fillers | 50 | 31.5 |
2.2
Degree of conversion
Five cylindrical samples (d = 3 mm, h = 5 mm) were made for each composite and each curing time using a custom-made stainless steel split-mold. Uncured composite was placed into the mold, both mold apertures were covered with a polyethylene terephthalate (PET) film and curing was performed for 20 s or 40 s through the upper aperture with a LED curing unit (Bluephase G2, Ivoclar-Vivadent, Schaan, Liechtenstein; wavelength range 380–515 nm, irradiance 1200 mW/cm 2 ). The curing unit tip was positioned directly on top of the PET film covering the sample. The samples were cured at 21 ± 1 °C and transferred to the incubator (Cultura, Ivoclar-Vivadent, Schaan, Liechtenstein) for dark storage at 37 ± 1 °C. After 24 h, Raman spectra were collected from four depths: 1, 2, 3 and 4 mm.
FT-Raman spectroscopy measurements were performed using a Spectrum GX spectrometer (PerkinElmer, Waltham, USA). The excitation was an NdYaG laser at 1064 nm wavelength, with laser power of 400 mW and resolution of 4 cm −1 . The samples were mounted on a universal holder that enabled translation along the axis of cylindrical sample thereby exposing different depths to the excitation laser light. The exposed sample surface was 0.5 mm in diameter. For each spectrum, 100 scans were recorded. Spectra of the uncured composites (n = 5) were recorded in the same manner. The spectra were processed with the Kinetics add-on for Matlab (Mathworks, Natick, Massachusetts, USA).
Degree of conversion (DC) calculation was performed by comparing the relative change of the band at 1640 cm −1 , representing the aliphatic C C stretching mode to the aromatic C C band at 1610 cm −1 , before and after polymerization. Peak heights of aliphatic C C and aromatic C C bands were used for DC calculation by the following equation: DC = 1–R polymerized /R unpolymerized , where R = (aliphatic C C peak height)/(aromatic C C peak height) .
2.3
Temperature rise
Uncured composite materials were cast into cylindrical Teflon molds (d = 6 mm, h = 2 mm), covered from both sides with PET film (0.05 mm thick) and sandwiched between two glass plates (h = 1 mm). The curing unit tip was positioned immediately below the glass plate, i.e. at 1.05 mm distance from the sample surface. At the opposite side of the sample, T-type thermocouple was positioned centrally between the PET film and glass plate. This assembly was held together by a custom-made brass holder. The sample was enclosed by the Teflon mold and PET films, so that it was not in direct contact with the brass holder. Five repeats were made for each material (n = 5).
Curing was performed using the Bluephase G2 curing device (1200 mW/cm 2 ) for 30 s and a custom-made computer program was used to record the temperature change at the rate of 20 points per second. Two minutes after the end of curing, temperature returned to the baseline and the curing device was activated again for 30 s. Two illuminations were performed to separate the heating effect of the curing unit from the reaction exotherm. Preliminary experiments showed that the second illumination did not significantly increase the conversion, thus it was assumed that the polymerization was completed during the first illumination.
Additionally, Tetric EvoCeram (Ivoclar Vivadent, Schaan, Liechtenstein) of shade A2, LOT: S26173, EXP: 06/2017 was used as a commercial control for temperature measurements.
2.4
Scanning electron microscopy (SEM)
The light-cured (30 s), disc-shaped samples of 1 mm thickness, unpolished and uncoated were examined with FE-SEM JSM 7000 (JEOL, Peabody, MA, USA).
2.5
Statistical analysis
Results of DC were compared by means of mixed model ANOVA and a multivariate analysis was used to test the influence of parameters “material”, “curing depth” and “curing time” on the DC values within the group of ACP-composites. The DC values among curing depths within a material and curing time were compared using a repeated measures ANOVA with Bonferroni correction. For a given curing depth, DC values among the materials and curing times were compared using ANOVA with Tukey correction.
Results of temperature rise were compared using ANOVA with Tukey correction, while the temperature rises during first and second illumination for a given material were compared by a repeated samples t-test. Statistical analysis was performed in SPSS 20 (IBM, Armonk, NY, USA) with α=0.05.
2
Materials and methods
2.1
Composite materials
The synthesis of zirconia-hybridized ACP (Zr-ACP) fillers followed the procedure by Skrtic et al. . Briefly, Zr-ACP precipitated from a water solution upon mixing of Ca(NO 3 ) 2 , Na 2 HPO 4 , Na 4 P 2 O 7 and ZrOCl 2 . The suspension was filtered, the solid phase washed with ice-cold ammoniated water and acetone and then lyophilized.
The experimental resin contained 67 wt% of EBPADMA (Esstech, Essington, PA, USA), 23 wt% of tri-ethylene glycol dimethacrylate (TEGDMA; Esstech) and 10 wt% of 2-hydroxyethyl methacrylate (HEMA; Esstech). The resin was rendered photosensitive by the addition of 0.2 wt% of the photo oxidant camphorquinone (CQ; Aldrich, Milwaukee, WI, USA) and 0.8 wt% of photo reductant ethyl-4- (dimethylamino) benzoate (4E; Aldrich). The monomers and photoactivators were mixed in the lightproof container using a magnetic stirrer for 48 h in order to assure a homogeneous mixture.
The resin was blended with fillers in lightproof containers using a dual asymmetric centrifugal mixing system (Speed Mixer TM DAC 150 FVZ, Hauschild & Co KG, Hamm, Germany) at 2700 rpm. The mixing was performed in five one-minute intervals separated by one-minute breaks. After mixing, the obtained composite pastes were deaerated in vacuum for 12 h. Seven experimental materials were prepared: five were ACP-based, while two materials were based on conventional inert fillers and served as a control ( Table 1 ).
| Material | Filler (wt%) | Resin (wt%) | Filler load (vol%) | |
|---|---|---|---|---|
| ACP-based materials | ACP40 | 40% Zr-ACP | 60 | 27.6 |
| ACP40-Ba10 | 40% Zr-ACP, 10% Ba-fillers | 50 | 35.1 | |
| ACP40-Si10 | 40% Zr-ACP, 10% Si-fillers | 50 | 36.6 | |
| ACP40-Ba5Si5 | 40% Zr-ACP, 5% Ba-fillers, 5% Si-fillers | 50 | 35.9 | |
| ACP40- Ba9Si1 | 40% Zr-ACP, 9% Ba-fillers, 1% Si-fillers | 50 | 35.3 | |
| Control materials | Ba40 | 40% Ba-fillers | 60 | 22.0 |
| Ba40Si10 | 40% Ba-fillers, 10% Si-fillers | 50 | 31.5 |
2.2
Degree of conversion
Five cylindrical samples (d = 3 mm, h = 5 mm) were made for each composite and each curing time using a custom-made stainless steel split-mold. Uncured composite was placed into the mold, both mold apertures were covered with a polyethylene terephthalate (PET) film and curing was performed for 20 s or 40 s through the upper aperture with a LED curing unit (Bluephase G2, Ivoclar-Vivadent, Schaan, Liechtenstein; wavelength range 380–515 nm, irradiance 1200 mW/cm 2 ). The curing unit tip was positioned directly on top of the PET film covering the sample. The samples were cured at 21 ± 1 °C and transferred to the incubator (Cultura, Ivoclar-Vivadent, Schaan, Liechtenstein) for dark storage at 37 ± 1 °C. After 24 h, Raman spectra were collected from four depths: 1, 2, 3 and 4 mm.
FT-Raman spectroscopy measurements were performed using a Spectrum GX spectrometer (PerkinElmer, Waltham, USA). The excitation was an NdYaG laser at 1064 nm wavelength, with laser power of 400 mW and resolution of 4 cm −1 . The samples were mounted on a universal holder that enabled translation along the axis of cylindrical sample thereby exposing different depths to the excitation laser light. The exposed sample surface was 0.5 mm in diameter. For each spectrum, 100 scans were recorded. Spectra of the uncured composites (n = 5) were recorded in the same manner. The spectra were processed with the Kinetics add-on for Matlab (Mathworks, Natick, Massachusetts, USA).
Degree of conversion (DC) calculation was performed by comparing the relative change of the band at 1640 cm −1 , representing the aliphatic C C stretching mode to the aromatic C C band at 1610 cm −1 , before and after polymerization. Peak heights of aliphatic C C and aromatic C C bands were used for DC calculation by the following equation: DC = 1–R polymerized /R unpolymerized , where R = (aliphatic C C peak height)/(aromatic C C peak height) .
2.3
Temperature rise
Uncured composite materials were cast into cylindrical Teflon molds (d = 6 mm, h = 2 mm), covered from both sides with PET film (0.05 mm thick) and sandwiched between two glass plates (h = 1 mm). The curing unit tip was positioned immediately below the glass plate, i.e. at 1.05 mm distance from the sample surface. At the opposite side of the sample, T-type thermocouple was positioned centrally between the PET film and glass plate. This assembly was held together by a custom-made brass holder. The sample was enclosed by the Teflon mold and PET films, so that it was not in direct contact with the brass holder. Five repeats were made for each material (n = 5).
Curing was performed using the Bluephase G2 curing device (1200 mW/cm 2 ) for 30 s and a custom-made computer program was used to record the temperature change at the rate of 20 points per second. Two minutes after the end of curing, temperature returned to the baseline and the curing device was activated again for 30 s. Two illuminations were performed to separate the heating effect of the curing unit from the reaction exotherm. Preliminary experiments showed that the second illumination did not significantly increase the conversion, thus it was assumed that the polymerization was completed during the first illumination.
Additionally, Tetric EvoCeram (Ivoclar Vivadent, Schaan, Liechtenstein) of shade A2, LOT: S26173, EXP: 06/2017 was used as a commercial control for temperature measurements.
2.4
Scanning electron microscopy (SEM)
The light-cured (30 s), disc-shaped samples of 1 mm thickness, unpolished and uncoated were examined with FE-SEM JSM 7000 (JEOL, Peabody, MA, USA).
2.5
Statistical analysis
Results of DC were compared by means of mixed model ANOVA and a multivariate analysis was used to test the influence of parameters “material”, “curing depth” and “curing time” on the DC values within the group of ACP-composites. The DC values among curing depths within a material and curing time were compared using a repeated measures ANOVA with Bonferroni correction. For a given curing depth, DC values among the materials and curing times were compared using ANOVA with Tukey correction.
Results of temperature rise were compared using ANOVA with Tukey correction, while the temperature rises during first and second illumination for a given material were compared by a repeated samples t-test. Statistical analysis was performed in SPSS 20 (IBM, Armonk, NY, USA) with α=0.05.
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