Abstract
Objectives
Bioactive glass (BAG) is known to possess antimicrobial properties and release ions needed for remineralization of tooth tissue, and therefore may be a strategic additive for dental restorative materials. The objective of this study was to develop BAG containing dental restorative composites with adequate mechanical properties comparable to successful commercially available composites, and to confirm the stability of these materials when exposed to a biologically challenging environment.
Methods
Composites with 72 wt% total filler content were prepared while substituting 0–15% of the filler with ground BAG. Flexural strength, fracture toughness, and fatigue crack growth tests were performed after several different soaking treatments: 24 h in DI water (all experiments), two months in brain–heart infusion (BHI) media + Streptococcus mutans bacteria (all experiments) and two months in BHI media (only for flexural strength). Mechanical properties of new BAG composites were compared along with the commercial composite Heliomolar by two-way ANOVA and Tukey’s multiple comparison test ( p ≤ 0.05).
Results
Flexural strength, fracture toughness, and fatigue crack growth resistance for the BAG containing composites were unaffected by increasing BAG content up to 15% and were superior to Heliomolar after all post cure treatments. The flexural strength of the BAG composites was unaffected by two months exposure to aqueous media and a bacterial challenge, while some decreases in fracture toughness and fatigue resistance were observed. The favorable mechanical properties compared to Heliomolar were attributed to higher filler content and a microstructure morphology that better promoted the toughening mechanisms of crack deflection and bridging.
Significance
Overall, the BAG containing composites developed in this study demonstrated adequate and stable mechanical properties relative to three successful commercial composites.
1
Introduction
While the use of dental restorative composites has increased dramatically for posterior teeth, annual failure rates up to 15% have been reported depending on restoration class , and a review of the literature has suggested the average lifetime of posterior dental composites is only six years . Secondary caries at margins has been considered for over twenty years the most common reason for restoration replacement . The second most common reason is partial or complete fracture of the composite restoration, while other significant causes are erosion and discoloration . It has been reported that the replacement of posterior composites is primarily due to fracture of the restoration within the first five years, but as a response to secondary caries thereafter , although this has not been observed in all clinical studies . A review of the numerous causes identified for restoration replacements based on multiple surveys may be found in Deligeorgi et al. .
One of the most common reasons for secondary caries is biofilm (plaque) formation on the margin of the tooth and restoration. Bacteria in the plaque (e.g., Streptococcus mutans ) metabolize sugars to lactic acid which can demineralize tooth tissue . Resin based composites may ideally provide good sealing of the cavity with no marginal gaps; however, polymerization shrinkage during placement, combined with cyclic mechanical loading during function, may lead to local interface failure and gap development. These marginal gaps can serve as suitable anchorage sites for bacterial colonies . A minimum gap size exceeding 0.4 mm has been suggested for significant bacterial colonization of dental amalgam , but a similar relationship has not been discerned for composites. Moreover, increased roughness of the restoration increases the ability of bacteria to colonize a given area, by affecting pellicle formation and causing a favorable environment, often resulting in secondary caries formation . Microfloral analysis of marginal biofilms revealed that anaerobic bacteria are dominant with S. mutans , Actinomyces naeslundii and Lactobacillus casei being the most abundant bacterial species . Svanberg et al. found significantly larger S. mutans colony counts at the tooth interface with composite restorations compared to interfaces with amalgam .
One possible approach to increasing the resistance of restorations to secondary caries formation is to add agents that (1) negatively influence the micro-organisms and/or (2) promote remineralization of tooth structure after damage has occurred. In this regard, there is a substantial amount of published literature demonstrating the antibacterial qualities of various bioactive glass (BAG) compositions against many different bacterial species . However, to date there have been no published studies of dental restorative composites containing bioactive glasses. There are several concerns regarding the development of a successful bioactive glass dental restorative composite. First, there is a concern that BAG fillers not well adhered to the composite matrix will result in unsuitably low mechanical properties. Second, because the composite will leach ions there is a concern about the stability of the mechanical properties over time. Finally, it must be confirmed that sufficient antimicrobial and/or remineralization activity can be achieved in BAG containing composites to slow secondary caries at the marginal gaps of tooth restorations. The goal of this study is to address the former two issues, while the latter will be addressed in the future by ongoing studies. Accordingly, the objective of this paper was to test the hypotheses that new BAG-containing dental restorative composites can be developed with mechanical properties comparable to successful commercially available composites, and that the properties will remain adequately stable after aging in a bacterial environment.
2
Materials and methods
2.1
Materials
The bioactive glass (BAG) used in this study had the composition 65% SiO 2 , 31% CaO and 4% P 2 O 5 (mol%) and was produced by a sol–gel process, as previously described . In brief, the BAG was produced from high-purity metal alkoxides including tetraethyl orthosilicate (TEOS, Si(OC 2 H 5 ) 4 ), calcium methoxyethoxide (CMOE, C 6 H 14 O 4 Ca), and triethyl phosphate (TEP, (C 2 H 5 ) 3 PO 4 ). All reagents were purchased (Sigma Aldrich), except the CMOE was synthesized from pure Ca metal and methoxyethanol, to produce a 20% solution in methoxyethanol, and this alcohol served as a mutual solvent for all of the alkoxides as well as being the water source used to initiate hydrolysis and glass formation. The solutions were prepared in a dry nitrogen-environment glovebox, aged in distilled water, air-dried and stabilized in a dedicated furnace at 600 °C to completely remove residual alcohols and alkoxide components, while retaining high surface area (between 200 and 300 m 2 /g). After rapidly cooling, the glass was ball milled in ethanol and sieved to a gross particle dimension of less than 38 μm. The particles were then further processed to a fine particle size (0.04–3.0 μm) using a Micronizer Jet Mill (Sturtevant Inc., Hanover, MA).
Three BAG-containing composites were produced by mixing the glass into a 50:50 mixture of bisphenol A glycidyl methacrylate (BisGMA):triethylene glycoldimethacrylate (TEGDMA) monomers with 0.4 wt% of camphorquinone (CQ), 0.8 wt% of 4-dimethylaminobenzoic acid ethyl ether (EDMAB), and 0.05 wt% of 3, 5-di-tertbutyl-4-hydroxytoluene (BHT). Samples denoted as 5BAG, 10BAG, and 15BAG were produced by combining the resin with 3.0 μm average size silanated strontium glass (Bisco Inc.) and 5, 10, or 15 wt% unsilanated bioactive glass, respectively, to a total filler of 72 wt% and mixed in a DAC-150 speed mixer (FlackTek Inc., Landrum, SC) at 3000 rpm for 2 min. Control samples (denoted 0BAG) had the same formulation as 5BAG with 5 wt% silane treated aerosol-silica filler (OX-50, Degussa) substituted for the BAG.
Mechanical property values were compared to published literature values and also the full set of mechanical property experiments were conducted on the commercial composite Heliomolar (Ivoclar Vivadent AG, batch # 4432). Heliomolar has a composition of 19 wt% Bis-GMA + urethane dimethacrylate, 3 wt% decandiol dimethacrylate, 66.7 wt% total filler content (highly dispersed silicon dioxide + ytterbium trifluoride) along with prepolymer and <1 wt% stabilizers, catalysts and pigments. Heliomolar is classified as an inhomogeneous microfilled composite (<1 μm filler size) and was chosen for this study because it is a clinically successful example of a composite for anterior and posterior restorations .
2.2
Specimen preparation for mechanical testing
For flexural strength testing, three-point bend beams ( N = 10 for each composition) were prepared by dispensing the composite paste into 25 mm long quartz tubes (square 2 mm × 2 mm cross-section) followed by curing for 40 s on two opposite sides in a visible light curing unit (Triad II, Dentsply International, York Division, PA, USA). Compact-tension, C(T), specimens were made for fracture toughness ( N = 5 for each composition) and fatigue crack growth ( N = 3 for each composition) experiments. The composites were dispensed into a stainless-steel rectangular split mold, pressed flat, and cured for 40 s on each side as described above. The cured rectangular blanks were then machined into C(T) specimens as shown in Fig. 1 a . To enable observation and measurement of cracks, samples were ground and polished using progressively finer SiC grinding papers and alumina oxide polishing compounds down to 0.05 μm and finally finished with MasterPolish (Buehler, Lake Bluff, IL, USA). A sharp pre-crack ( Fig. 1 b) was introduced by manually extending the starter notch using a razor blade until a pre-crack formed from the notch.
2.3
Post-cure treatments
Specimens for all experiments were treated in two different ways: 24 h aging in deionized (DI) water and 58 ± 3 days at 37 °C in brain–heart infusion (BHI) media with S. mutans (strain ATCC25175) cultures growing in logarithmic phase with the media changed every other day. The longer of the two aging times was chosen since previous studies have estimated that is the amount of time needed for the specimens to become fully (>98%) saturated with water . Lyophilized bacterial cultures were obtained from the American Type Culture Collection (ATTC, Manassas, VA, USA). All DI water aged samples were tested immediately after removal from the water. After the aging period in BHI media with S. mutans , composite samples were immersed in 50% bleach for 5 min and then rinsed with BHI three times. The specimens were then soaked in BHI without sucrose and stored in a refrigerator until mechanical testing. With the exception of the fatigue tests, all mechanical testing was performed within a day of removal from the test aging solution. Due to the time required for fatigue testing, some samples needed to be stored for days or weeks while waiting for testing, so these samples were continually stored in sterile BHI media at ∼4 °C. Finally, bending beams made from the experimental composites were also soaked in sterile BHI media without bacteria for ∼60 days and used for strength testing.
2.4
Mechanical testing
Flexure strength was tested in 3-point bending (20 mm span) on a universal testing machine at a cross-head speed of 0.254 mm/min, in general accord with ISO 4049 . The steel supports had rollers of 2 mm diameter and the loading piston was a steel ball of 2 mm diameter. The flexure strength was determined using the maximum load.
Fracture toughness tests were conducted on wet samples immediately after removal from the storage solution using a computer controlled hydraulic testing machine (Instron 8872, Canton, MA, USA). Tests were conducted in load control with a 1.1 N/s loading rate until fracture occurred. K IC was calculated from the peak load at fracture according to the standard stress intensity factor equation for the C(T) sample geometry .
Fatigue crack growth testing was done in general accordance with ASTM standard E647 , using computer controlled hydraulic testing machine (Instron 8872, Canton, MA, USA) and a sine waveform with frequency ν = 1.5 Hz, which corresponds to a typical human chewing frequency . A constant load ratio R = P min / P max = 0.1 was used, where P max and P min are the maximum and minimum loads experienced during the loading cycle, respectively. Fatigue crack growth rates, da / dN , were characterized as a function of the stress intensity range, Δ K = K max − K min , where K max and K min are the stress intensity values calculated from P max and P min , respectively. After initial establishment of a high crack growth rate of 10 −7 –10 −6 m/cycle, the test was conducted in decreasing Δ K control using a normalized K -gradient (1/Δ K [ d Δ K / da ]) of −0.08 mm −1 . Crack length was determined by measuring the load point compliance using a capacitance displacement gage (HPT150, Capacitec, Inc., Ayer, MA) attached to the clevises of the testing machine. The sample compliance was converted to crack length using published calibrations . Data points were collected roughly every 10 −5 m of crack extension. Samples were kept wet during the entire test using a sponge to surround the sample and a custom drip system to keep it wet. After the test, the final crack length was measured optically. When the final compliance and optically measured crack lengths differed, the crack length data was corrected by assuming the error accumulated linearly with crack extension. From the crack length data, the crack growth rates ( da / dN ) were determined as a function of Δ K by fitting over ranges of ∼100 μm of crack length change.
For statistical comparisons of data, ANOVA followed by Tukey’s multiple comparison test was used with p < 0.05 considered statistically significant.
After fatigue crack growth and fracture toughness experiments, both crack profiles and fracture surfaces, respectively, were examined using a scanning electron microscope (Quanta 600 FEG SEM, FEI Company, Hillsboro, OR) in order to discern crack-microstructure interactions and toughening mechanisms.
2
Materials and methods
2.1
Materials
The bioactive glass (BAG) used in this study had the composition 65% SiO 2 , 31% CaO and 4% P 2 O 5 (mol%) and was produced by a sol–gel process, as previously described . In brief, the BAG was produced from high-purity metal alkoxides including tetraethyl orthosilicate (TEOS, Si(OC 2 H 5 ) 4 ), calcium methoxyethoxide (CMOE, C 6 H 14 O 4 Ca), and triethyl phosphate (TEP, (C 2 H 5 ) 3 PO 4 ). All reagents were purchased (Sigma Aldrich), except the CMOE was synthesized from pure Ca metal and methoxyethanol, to produce a 20% solution in methoxyethanol, and this alcohol served as a mutual solvent for all of the alkoxides as well as being the water source used to initiate hydrolysis and glass formation. The solutions were prepared in a dry nitrogen-environment glovebox, aged in distilled water, air-dried and stabilized in a dedicated furnace at 600 °C to completely remove residual alcohols and alkoxide components, while retaining high surface area (between 200 and 300 m 2 /g). After rapidly cooling, the glass was ball milled in ethanol and sieved to a gross particle dimension of less than 38 μm. The particles were then further processed to a fine particle size (0.04–3.0 μm) using a Micronizer Jet Mill (Sturtevant Inc., Hanover, MA).
Three BAG-containing composites were produced by mixing the glass into a 50:50 mixture of bisphenol A glycidyl methacrylate (BisGMA):triethylene glycoldimethacrylate (TEGDMA) monomers with 0.4 wt% of camphorquinone (CQ), 0.8 wt% of 4-dimethylaminobenzoic acid ethyl ether (EDMAB), and 0.05 wt% of 3, 5-di-tertbutyl-4-hydroxytoluene (BHT). Samples denoted as 5BAG, 10BAG, and 15BAG were produced by combining the resin with 3.0 μm average size silanated strontium glass (Bisco Inc.) and 5, 10, or 15 wt% unsilanated bioactive glass, respectively, to a total filler of 72 wt% and mixed in a DAC-150 speed mixer (FlackTek Inc., Landrum, SC) at 3000 rpm for 2 min. Control samples (denoted 0BAG) had the same formulation as 5BAG with 5 wt% silane treated aerosol-silica filler (OX-50, Degussa) substituted for the BAG.
Mechanical property values were compared to published literature values and also the full set of mechanical property experiments were conducted on the commercial composite Heliomolar (Ivoclar Vivadent AG, batch # 4432). Heliomolar has a composition of 19 wt% Bis-GMA + urethane dimethacrylate, 3 wt% decandiol dimethacrylate, 66.7 wt% total filler content (highly dispersed silicon dioxide + ytterbium trifluoride) along with prepolymer and <1 wt% stabilizers, catalysts and pigments. Heliomolar is classified as an inhomogeneous microfilled composite (<1 μm filler size) and was chosen for this study because it is a clinically successful example of a composite for anterior and posterior restorations .
2.2
Specimen preparation for mechanical testing
For flexural strength testing, three-point bend beams ( N = 10 for each composition) were prepared by dispensing the composite paste into 25 mm long quartz tubes (square 2 mm × 2 mm cross-section) followed by curing for 40 s on two opposite sides in a visible light curing unit (Triad II, Dentsply International, York Division, PA, USA). Compact-tension, C(T), specimens were made for fracture toughness ( N = 5 for each composition) and fatigue crack growth ( N = 3 for each composition) experiments. The composites were dispensed into a stainless-steel rectangular split mold, pressed flat, and cured for 40 s on each side as described above. The cured rectangular blanks were then machined into C(T) specimens as shown in Fig. 1 a . To enable observation and measurement of cracks, samples were ground and polished using progressively finer SiC grinding papers and alumina oxide polishing compounds down to 0.05 μm and finally finished with MasterPolish (Buehler, Lake Bluff, IL, USA). A sharp pre-crack ( Fig. 1 b) was introduced by manually extending the starter notch using a razor blade until a pre-crack formed from the notch.
2.3
Post-cure treatments
Specimens for all experiments were treated in two different ways: 24 h aging in deionized (DI) water and 58 ± 3 days at 37 °C in brain–heart infusion (BHI) media with S. mutans (strain ATCC25175) cultures growing in logarithmic phase with the media changed every other day. The longer of the two aging times was chosen since previous studies have estimated that is the amount of time needed for the specimens to become fully (>98%) saturated with water . Lyophilized bacterial cultures were obtained from the American Type Culture Collection (ATTC, Manassas, VA, USA). All DI water aged samples were tested immediately after removal from the water. After the aging period in BHI media with S. mutans , composite samples were immersed in 50% bleach for 5 min and then rinsed with BHI three times. The specimens were then soaked in BHI without sucrose and stored in a refrigerator until mechanical testing. With the exception of the fatigue tests, all mechanical testing was performed within a day of removal from the test aging solution. Due to the time required for fatigue testing, some samples needed to be stored for days or weeks while waiting for testing, so these samples were continually stored in sterile BHI media at ∼4 °C. Finally, bending beams made from the experimental composites were also soaked in sterile BHI media without bacteria for ∼60 days and used for strength testing.
2.4
Mechanical testing
Flexure strength was tested in 3-point bending (20 mm span) on a universal testing machine at a cross-head speed of 0.254 mm/min, in general accord with ISO 4049 . The steel supports had rollers of 2 mm diameter and the loading piston was a steel ball of 2 mm diameter. The flexure strength was determined using the maximum load.
Fracture toughness tests were conducted on wet samples immediately after removal from the storage solution using a computer controlled hydraulic testing machine (Instron 8872, Canton, MA, USA). Tests were conducted in load control with a 1.1 N/s loading rate until fracture occurred. K IC was calculated from the peak load at fracture according to the standard stress intensity factor equation for the C(T) sample geometry .
Fatigue crack growth testing was done in general accordance with ASTM standard E647 , using computer controlled hydraulic testing machine (Instron 8872, Canton, MA, USA) and a sine waveform with frequency ν = 1.5 Hz, which corresponds to a typical human chewing frequency . A constant load ratio R = P min / P max = 0.1 was used, where P max and P min are the maximum and minimum loads experienced during the loading cycle, respectively. Fatigue crack growth rates, da / dN , were characterized as a function of the stress intensity range, Δ K = K max − K min , where K max and K min are the stress intensity values calculated from P max and P min , respectively. After initial establishment of a high crack growth rate of 10 −7 –10 −6 m/cycle, the test was conducted in decreasing Δ K control using a normalized K -gradient (1/Δ K [ d Δ K / da ]) of −0.08 mm −1 . Crack length was determined by measuring the load point compliance using a capacitance displacement gage (HPT150, Capacitec, Inc., Ayer, MA) attached to the clevises of the testing machine. The sample compliance was converted to crack length using published calibrations . Data points were collected roughly every 10 −5 m of crack extension. Samples were kept wet during the entire test using a sponge to surround the sample and a custom drip system to keep it wet. After the test, the final crack length was measured optically. When the final compliance and optically measured crack lengths differed, the crack length data was corrected by assuming the error accumulated linearly with crack extension. From the crack length data, the crack growth rates ( da / dN ) were determined as a function of Δ K by fitting over ranges of ∼100 μm of crack length change.
For statistical comparisons of data, ANOVA followed by Tukey’s multiple comparison test was used with p < 0.05 considered statistically significant.
After fatigue crack growth and fracture toughness experiments, both crack profiles and fracture surfaces, respectively, were examined using a scanning electron microscope (Quanta 600 FEG SEM, FEI Company, Hillsboro, OR) in order to discern crack-microstructure interactions and toughening mechanisms.
3
Results
3.1
Flexural strength results
Flexural strength and statistical test results are shown in Table 1 . BAG composites did not show a significant difference in flexural strength as a function of the various soaking treatments. However, Heliomolar composites did show a significant reduction in flexural strength between 24 h water and 2 months in bacteria. The experimental BAG composites all had superior flexural strength when compared to Heliomolar.
| Material | 24 h DI water | 2 months bacteria | 2 months media |
|---|---|---|---|
| Flexural strength (MPa) | Flexural strength (MPa) | Flexural strength (MPa) | |
| Heliomolar | 73.2 (4.4) a | 61.7 (7.9) a | – |
| 0BAG | 123.5 (16.2) | 114.9 (12.3) | 107.4 (12.8) |
| 5BAG | 112.8 (12.9) | 108.4 (12.2) | 107.4 (11.2) |
| 10BAG | 116.4 (14.2) | 112.6 (13.0) | 95.7 (16.2) |
| 15BAG | 116.9 (10.7) | 105.6 (17.7) | 101.2 (10.8) |
a Denotes value has statistically significant difference from rest of column.
3.2
Fracture toughness results
Fracture toughness data and statistical test results for both Heliomolar and BAG composites are shown in Table 2 .
| Material | 24 h DI water | 2 months bacteria | % Change |
|---|---|---|---|
| K IC (MPa √m) | K IC (MPa √m) | ||
| Heliomolar | 0.98 (0.17) a | 0.77 (0.02) a | −21% b |
| 0BAG | 1.45 (0.13) | 1.25 (0.15) | −13% |
| 5 BAG | 1.52 (0.17) | 1.12 (0.13) | −26% b |
| 10BAG | 1.54 (0.17) | 1.40 (0.01) a | −9% |
| 15BAG | 1.31 (0.14) | 1.10 (0.05) | −16% b |
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