Abstract
Objectives
The aim of the study was to evaluate antimicrobial inhibition zone, degree of conversion (DC) and Knoop hardness (KH) of experimental infiltrants. Experimental low viscosity monomer blends were prepared and chlorhexidine diacetate salt (CHX) (0.1% or 0.2%) was added comprising the groups: G1) TEGDMA; G2) TEGDMA/0.1CHX; G3) TEGDMA/0.2CHX; G4) TEGDMA/UDMA; G5) TEGDMA/UDMA/0.1CHX; G6) TEGDMA/UDMA/0.2CHX; G7) TEGDMA/BISEMA; G8) TEGDMA/BISEMA/0.1CHX; G9) TEGDMA/BISEMA/0.2CHX. Icon ® was used as control group.
Methods
Specimens of resin blends were made ( n = 9) to accomplished DC and KH. Pour plate was accomplished to evaluate antimicrobial groups’ activity against Streptococcus mutans (SM) and Lactobacillus acidophilus (LA). Data obtained were submitted to two-way ANOVA and Tukey tests for blends comparisons and Dunnett’s test for comparisons between experimental infiltrants and Icon ® ( p < 0.05).
Results
In relation to antimicrobial effect, uncured blends showed higher antibacterial activity than cured ones for the most of blends. After polymerization, G5 showed the highest inhibition zone against SM and, G3 and G6 against LA. Concerning KH, TEGDMA/UDMA-based blends showed the highest values of KH number and it was influenced by monomeric base, regardless CHX concentration. DC was not affected by monomer blend composition, neither for CHX concentration. The antimicrobial activity was affected by monomeric base, CHX concentration and polymerization. Experimental infiltrants presented similar or higher performance than Icon ® for the properties evaluated.
Significance
White spot lesion infiltration with low viscosity monomer blends (infiltrants) is an alternative to stop initial caries lesions progression. The incorporation of an antimicrobial agent as chlorhexidine diacetate salt in infiltrants composition could enhance the performance of these materials.
1
Introduction
Minimally invasive dentistry is a concept that involves dental tissue preservation, preferably by preventing disease from occurring and intercepting its progress, but also by removing and replacing dental tissues with as little tissue loss as possible . In the beginning of enamel caries lesions development, the mineral is removed from enamel structure leaving porosities (subsurface lesion), whilst the lesion surface visually remains relatively intact . These lesions are clinically recognized as opaque white spots areas on enamel surface.
The pores of enamel caries act as diffusion pathways for acids and dissolved minerals, and an occlusion of these pores by low viscosity monomer blends (“infiltrants”) could arrest the lesion progression and mechanically stabilize the fragile lesion structure . In order to obtain a deep penetration of the porous layer, the surface must be etched with hydrochloric acid gel and filled with infiltrants as the dimethacrylate triethylene glycol dimethacrylate (TEGDMA) .
TEGDMA is an extremely fluid monomer and its high flexibility structure chain results in resins with high conversion rate . It forms a polymer network with high cross-link density, although presenting a more heterogeneous structure, with higher water sorption, which causes greater polymer chains entanglement . In this way, TEGDMA-based materials solvent-free have appropriate characteristics for an infiltrant material, once the low viscosity and high degree of conversion also promote good results in mechanical properties as elastic modulus and KH . However, they also show high water sorption and polymerization shrinkage , as well as high hydrolysis potential in oral environment. Therefore, the addition of hydrophobic monomers as bisphenol A ethoxylate dimethacrylate (BISEMA) or urethane dimethacrylate (UDMA) is interesting, once it could improve the mechanical properties and the long term durability of resin infiltrants in oral environment . The monomers UDMA and BISEMA show a significantly lower viscosity when compared to BisGMA (bisphenol A diglycidyl methacrylate) .
Adding antibacterial agent in dental resin materials would improve the ability of arresting incipient caries lesions and inhibit plaque accumulation on surface of the material and on surrounding dental tissue . In this way, the addition of an antimicrobial agent such as CHX into resin materials with infiltrant characteristic would include antibacterial properties for these materials, which could reduce biofilm growth over the infiltrated enamel. This would be an important strategy; especially considering that resin infiltrants are indicated for high caries risk patients .
CHX has been incorporated in dental materials, such as glass-ionomer cements, resin-modified glass-ionomer cements and methacrylates, improving and/or extending the antimicrobial properties of these materials against cariogenic bacteria . Therefore, adding soluble antimicrobials into resin matrix is a way to release the agent from the materials in a wet environment as oral one, and CHX has been the most frequently used .
Chlorhexidine has been described as the gold standard for antibacterial application because it is wide spectrum of action . It can suppress the growth of Streptococcus mutans and, consequently, prevent dental caries . Chlorhexidine is a symmetrical cationic molecule consisting of two 4-chlorophenyl rings and two biguanide groups connected by a central hexamethylene chain, which is considered a strong base and it is stable in the form of salts . At low chlorhexidine concentrations, small molecular weight substances, such as potassium and phosphorus, leach out, exerting a bacteriostatic effect . Nevertheless, in higher concentrations, chlorhexidine shows bactericidal action due to precipitation or coagulation of bacteria’s cytoplasm, probably caused by protein cross-linking .
Therefore, the incorporation of antimicrobial agents such as CHX in infiltrant composition would allow antimicrobial activity for these materials, especially in relation to cariogenic microorganisms present in incipient carious lesions, which also could decrease bacterial colonization on infiltrated area. Satisfactory results with experimental TEGDMA/UDMA/BISEMA blends with addition of CHX were obtained in a previous study that evaluated properties as: sorption/solubility, flexural strength, elastic modulus and softening. The addition of CHX in two concentrations (0.1% and 0.2%) did not interfere significantly in evaluated testes and experimental blends tested had better performance when they were compared with a commercial control group. Thus, the purpose of this study was to evaluate the antimicrobial activity and the polymerization characteristics of experimental resin blends with CHX addition. It is prudent to investigate whether the incorporation of CHX into experimental infiltrants can modify their polymerization, thereby affecting their DC and KH properties. The hypotheses tested in this study were: (1) CHX added to low viscosity monomer blends would enable its antimicrobial activity; (2) adding CHX would not interfere in DC and in KHN of experimental TEGDMA/UDMA/BISEMA-based blends; (3) the addition of hydrophobic monomers, such as BISEMA and UDMA in TEGDMA based-blends would increase mechanical properties of experimental infiltrants.
2
Materials and methods
2.1
Resin blends formulation
In this study, nine low viscosity monomer blends with infiltrant characteristic were prepared using TEGDMA (Sigma–Aldrich, St. Louis, MO, USA, Batch 36296HK), UDMA (Sigma–Aldrich, St. Louis, MO, USA, Batch MKBD1130), and BISEMA (Sigma–Aldrich, St. Louis, MO, USA, Batch 03514HF) as demonstrated in Table 1 . The photoinitiator system used in the blends was 1.0 wt.% DMAEMA (2-Dimethylaminoethyl Methacrylate, Sigma–Aldrich, St. Louis, MO, USA, Batch 1437599) and 0.5 wt.% CQ (Camphorquinone, Sigma–Aldrich, St. Louis, MO, USA, Batch 083K0014). The inhibitor BHT (butylated hydroxytoluene, Sigma–Aldrich, St. Louis, MO, USA, Batch 04416KD) was added in the blends in a concentration of 0.1 wt.% in order to prevent spontaneous initiation and propagation of the free-radical polymerization reaction. 9 Two different concentrations (0.1 wt.% and 0.2 wt.%) of CHX (Sigma–Aldrich, St. Louis, MO, USA, Batch 083K0014) were tested as demonstrated in Table 1 . In order to avoid premature polymerization, the resins were dark stored in opaque recipients at 4 °C until use. The infiltrant Icon ® (DMG – Hamburg, Germany, Batch 633102) was used as commercial control group.
| Experimental blends | Composition |
|---|---|
| G1 | TEGDMA (100 wt.%) |
| G2 | TEGDMA (99.9 wt.%), CHX 0.1 wt.% |
| G3 | TEGDMA (99.8 wt.%), CHX 0.2 wt.% |
| G4 | TEGDMA (75 wt.%), UDMA (25 wt.%) |
| G5 | TEGDMA (74.9 wt.%), UDMA (25 wt.%), CHX 0.1 wt.% |
| G6 | TEGDMA (74.8 wt.%), UDMA (25 wt.%), CHX 0.2 wt.% |
| G7 | TEGDMA (75 wt.%), BISEMA (25 wt.%) |
| G8 | TEGDMA (74.9 wt.%), BISEMA (25 wt.%), CHX 0.1 wt.% |
| G9 | TEGDMA (74.8 wt.%), BISEMA (25 wt.%), CHX 0.2 wt.% |
2.2
Microbiological assays – pour plate technique
The strains used were S. mutans UA159 and Lactobacillus acidophilus LYO50DCU-S. Pour plate technique was used to observe the inhibition zone formed from experimental low viscosity monomer blends and commercial control group in two different situations: uncured and cured. For this assay, 0.12% chlorhexidine digluconate (Proderma, Piracicaba, SP, Brazil) was used as negative control for inhibition of strain growth. Each inoculum was adjusted at absorbance of 0.6–0.7 A and 550 nm in spectrophotometer (Genesys 10uv, Thermo Electron Corporation, Madison, WI, USA), according to the Mc Farland 0.5 scale , and a serial dilution was made reaching a final concentration of 1.0 × 10 6 cells/mL in BHI broth culture (Difco Laboratories, Detroit, MI, USA). To pour plate assay, 1 mL of each adjusted inoculum was transferred to a vial containing 50 mL of fused BHI agar (Difco Laboratories, Detroit, MI, USA) at 45 °C and dispensed into a Petri dish (140 mm × 15 mm). After agar solidification, wells (5 mm diameter × 1.5 mm height) were made in equidistant points using sterile metal molds (5 mm diameter). The wells were then completely filled with the tested materials. Since a large number of groups and with intention to avoid the intersection of possible inhibition halos, the groups were divided in two Petri dishes: one with groups G1–G9 and 0.12% chlorhexidine digluconate and other with Icon ® , experimental blends without CHX (G1, G4 and G7) and 0.12%chlorhexidine digluconate. To the assay with cured materials the same process described above was conducted, however, soon after the blends were put in the wells, they were cured for 60 s with light curing Elipar Free Light 2 (3M ESPE, St. Paul, MN, USA) with power density of 1000 mW/cm 2 . Petri dishes were kept at room temperature for 2 h in order to allow pre-diffusion of substances. Subsequently, they were stored at 37 °C in an anaerobic chamber for 24 h. The inhibition zones diameter of microbial growth formed around the wells was measured in millimeters with a digital caliper (Mitutoyo, Tokyo, Japan) under reflected light.
2.3
Degree of conversion and hardnesss analyses
2.3.1
Specimen preparation
Cylindrical specimens (7 mm diameter and 1 mm thick) were prepared from experimental infiltrants and Icon ® ( n = 9) into polyvinyl siloxane mold (Express, 3M ESPE, St. Paul, MN, USA). The mold was completely filled with the blend; then, a polyester strip was placed over and covered with a glass slide until light curing in order to obtain a smooth and standard flat surface. Each specimen was cured for 60 s with Elipar Free Light 2 LED device (3M ESPE, St. Paul, MN, USA) with power density of 1000 mW/cm 2 . After light curing, the specimens were stored in 100% humidity at room temperature for 24 h before evaluations of DC and KH.
2.3.2
Degree of conversion
The DC of infiltrants was measured using Fourier Transformed Infrared Spectroscopy (FTIR) with an attenuated total reflectance (ATR) device (Spectrum 100, PerkinElmer, Shelton, CT, USA). The specimens were placed on zinc selenite pellet surface and the absorption spectra of non-polymerized and polymerized specimens were obtained from region between 650 and 4000 cm −1 wavelength with 32 scans at 4 cm −1 resolution. For resin blends and Icon ® the intensity of the carbonyl peak 1716 cm −1 was used as the reference and the peak 1638 cm −1 was used as reference of aliphatic carbon double bonds of the methacrylate functional group. The degree of conversion was calculated according to the following formula: DC (%) = 100 × [1 − (R polymerized/R non-polymerized)], where R represents the ratio between the absorbance peak at 1638 cm −1 and 1716 cm −1 .
2.3.3
Hardness test
The KH test was performed using the hardness Future Tech FM-100 indenter (FUTURE-TECH CORP., Kawasaki-City, Japan) at automatic procedure with a load of 10gF applied for 6 s, using 10× magnification lens. Three readings were performed for each specimen. The values obtained in micrometers were converted to Knoop Hardness Number (KHN), by indenter software. The mean of three indentations was considered for statistical analysis.
2.4
Statistical analysis
In order to evaluate and compare the different experimental resin blends, data from physical, mechanical and antimicrobial properties were submitted to two-way ANOVA (factor 1: monomeric base; factor 2: CHX) and Tukey’s test was applied ( p < 0.05). In order to compare the properties between the experimental resin blends and the commercial control group (Icon ® ), the Dunnett’s test was performed ( p < 0.05) using the Assistat statistical software (Statistical Assistance Software, version 7.6 beta, 2011, Campina Grande, Brazil).
2
Materials and methods
2.1
Resin blends formulation
In this study, nine low viscosity monomer blends with infiltrant characteristic were prepared using TEGDMA (Sigma–Aldrich, St. Louis, MO, USA, Batch 36296HK), UDMA (Sigma–Aldrich, St. Louis, MO, USA, Batch MKBD1130), and BISEMA (Sigma–Aldrich, St. Louis, MO, USA, Batch 03514HF) as demonstrated in Table 1 . The photoinitiator system used in the blends was 1.0 wt.% DMAEMA (2-Dimethylaminoethyl Methacrylate, Sigma–Aldrich, St. Louis, MO, USA, Batch 1437599) and 0.5 wt.% CQ (Camphorquinone, Sigma–Aldrich, St. Louis, MO, USA, Batch 083K0014). The inhibitor BHT (butylated hydroxytoluene, Sigma–Aldrich, St. Louis, MO, USA, Batch 04416KD) was added in the blends in a concentration of 0.1 wt.% in order to prevent spontaneous initiation and propagation of the free-radical polymerization reaction. 9 Two different concentrations (0.1 wt.% and 0.2 wt.%) of CHX (Sigma–Aldrich, St. Louis, MO, USA, Batch 083K0014) were tested as demonstrated in Table 1 . In order to avoid premature polymerization, the resins were dark stored in opaque recipients at 4 °C until use. The infiltrant Icon ® (DMG – Hamburg, Germany, Batch 633102) was used as commercial control group.
| Experimental blends | Composition |
|---|---|
| G1 | TEGDMA (100 wt.%) |
| G2 | TEGDMA (99.9 wt.%), CHX 0.1 wt.% |
| G3 | TEGDMA (99.8 wt.%), CHX 0.2 wt.% |
| G4 | TEGDMA (75 wt.%), UDMA (25 wt.%) |
| G5 | TEGDMA (74.9 wt.%), UDMA (25 wt.%), CHX 0.1 wt.% |
| G6 | TEGDMA (74.8 wt.%), UDMA (25 wt.%), CHX 0.2 wt.% |
| G7 | TEGDMA (75 wt.%), BISEMA (25 wt.%) |
| G8 | TEGDMA (74.9 wt.%), BISEMA (25 wt.%), CHX 0.1 wt.% |
| G9 | TEGDMA (74.8 wt.%), BISEMA (25 wt.%), CHX 0.2 wt.% |
2.2
Microbiological assays – pour plate technique
The strains used were S. mutans UA159 and Lactobacillus acidophilus LYO50DCU-S. Pour plate technique was used to observe the inhibition zone formed from experimental low viscosity monomer blends and commercial control group in two different situations: uncured and cured. For this assay, 0.12% chlorhexidine digluconate (Proderma, Piracicaba, SP, Brazil) was used as negative control for inhibition of strain growth. Each inoculum was adjusted at absorbance of 0.6–0.7 A and 550 nm in spectrophotometer (Genesys 10uv, Thermo Electron Corporation, Madison, WI, USA), according to the Mc Farland 0.5 scale , and a serial dilution was made reaching a final concentration of 1.0 × 10 6 cells/mL in BHI broth culture (Difco Laboratories, Detroit, MI, USA). To pour plate assay, 1 mL of each adjusted inoculum was transferred to a vial containing 50 mL of fused BHI agar (Difco Laboratories, Detroit, MI, USA) at 45 °C and dispensed into a Petri dish (140 mm × 15 mm). After agar solidification, wells (5 mm diameter × 1.5 mm height) were made in equidistant points using sterile metal molds (5 mm diameter). The wells were then completely filled with the tested materials. Since a large number of groups and with intention to avoid the intersection of possible inhibition halos, the groups were divided in two Petri dishes: one with groups G1–G9 and 0.12% chlorhexidine digluconate and other with Icon ® , experimental blends without CHX (G1, G4 and G7) and 0.12%chlorhexidine digluconate. To the assay with cured materials the same process described above was conducted, however, soon after the blends were put in the wells, they were cured for 60 s with light curing Elipar Free Light 2 (3M ESPE, St. Paul, MN, USA) with power density of 1000 mW/cm 2 . Petri dishes were kept at room temperature for 2 h in order to allow pre-diffusion of substances. Subsequently, they were stored at 37 °C in an anaerobic chamber for 24 h. The inhibition zones diameter of microbial growth formed around the wells was measured in millimeters with a digital caliper (Mitutoyo, Tokyo, Japan) under reflected light.
2.3
Degree of conversion and hardnesss analyses
2.3.1
Specimen preparation
Cylindrical specimens (7 mm diameter and 1 mm thick) were prepared from experimental infiltrants and Icon ® ( n = 9) into polyvinyl siloxane mold (Express, 3M ESPE, St. Paul, MN, USA). The mold was completely filled with the blend; then, a polyester strip was placed over and covered with a glass slide until light curing in order to obtain a smooth and standard flat surface. Each specimen was cured for 60 s with Elipar Free Light 2 LED device (3M ESPE, St. Paul, MN, USA) with power density of 1000 mW/cm 2 . After light curing, the specimens were stored in 100% humidity at room temperature for 24 h before evaluations of DC and KH.
2.3.2
Degree of conversion
The DC of infiltrants was measured using Fourier Transformed Infrared Spectroscopy (FTIR) with an attenuated total reflectance (ATR) device (Spectrum 100, PerkinElmer, Shelton, CT, USA). The specimens were placed on zinc selenite pellet surface and the absorption spectra of non-polymerized and polymerized specimens were obtained from region between 650 and 4000 cm −1 wavelength with 32 scans at 4 cm −1 resolution. For resin blends and Icon ® the intensity of the carbonyl peak 1716 cm −1 was used as the reference and the peak 1638 cm −1 was used as reference of aliphatic carbon double bonds of the methacrylate functional group. The degree of conversion was calculated according to the following formula: DC (%) = 100 × [1 − (R polymerized/R non-polymerized)], where R represents the ratio between the absorbance peak at 1638 cm −1 and 1716 cm −1 .
2.3.3
Hardness test
The KH test was performed using the hardness Future Tech FM-100 indenter (FUTURE-TECH CORP., Kawasaki-City, Japan) at automatic procedure with a load of 10gF applied for 6 s, using 10× magnification lens. Three readings were performed for each specimen. The values obtained in micrometers were converted to Knoop Hardness Number (KHN), by indenter software. The mean of three indentations was considered for statistical analysis.
2.4
Statistical analysis
In order to evaluate and compare the different experimental resin blends, data from physical, mechanical and antimicrobial properties were submitted to two-way ANOVA (factor 1: monomeric base; factor 2: CHX) and Tukey’s test was applied ( p < 0.05). In order to compare the properties between the experimental resin blends and the commercial control group (Icon ® ), the Dunnett’s test was performed ( p < 0.05) using the Assistat statistical software (Statistical Assistance Software, version 7.6 beta, 2011, Campina Grande, Brazil).
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