| Abstract|| |
Background: Many attempts have been made to enhance the anti-cariogenic properties of the conventional glass ionomer through incorporation of variable materials. However, most importantly, the incorporation of such materials should not jeopardise the physical or mechanical properties of the final restoration. Aims: To investigate the effect of adding silver nanoparticles (Ag-NPs) and titanium dioxide nanoparticles (TiO2-NPs) to conventional glass ionomer cement (GIC) on its anti-bacterial effect against Streptococcus mutans (S. mutans), fluoride ion release, and compressive strength (CS). Settings and Design: This study was an in vitro investigation where 30-disc specimens were prepared in each of the three studied groups. Materials and Methods: The specimens were categorized into the control group (Group C), in which conventional GIC was used, and Group Ag and Group Ti, in which 5 wt% of Ag-NP and TiO2-NP were added, respectively, to GIC powder. In each group, the anti-bacterial effect against S. mutans, fluoride ion release at 24 hours and 14 and 28 days, and CS were assessed. Data were analysed using one-way analysis of variance, followed by Tukey honest significant difference post-hoc test. Results: Both Ag and Ti groups showed a significantly higher anti-bacterial effect than the control group. Ag-NP increased fluoride ion release, whereas TiO2-NP decreased fluoride release; however, cumulative ion release of both experimental groups did not differ significantly compared to the control group. Adding Ag-NP and TiO2-NP increased CS, yet only the Ti group showed the highest CS mean value that was statistically significant compared to other groups. Conclusions: Adding 5 wt% TiO2-NP or Ag-NP to conventional GIC significantly increased its anti-bacterial effect and its CS without affecting fluoride release.
Keywords: Glass ionomer cement, nanoparticles, silver, titanium dioxide
|How to cite this article:|
Wassel MO, Allam GG. Anti-Bacterial effect, fluoride release, and compressive strength of a glass ionomer containing silver and titanium nanoparticles. Indian J Dent Res 2022;33:75-9
|How to cite this URL:|
Wassel MO, Allam GG. Anti-Bacterial effect, fluoride release, and compressive strength of a glass ionomer containing silver and titanium nanoparticles. Indian J Dent Res [serial online] 2022 [cited 2022 Oct 4];33:75-9. Available from: https://www.ijdr.in/text.asp?2022/33/1/75/353528
| Introduction|| |
Glass ionomer cement (GIC) is a popular restorative material in pediatric dentistry because of its chemical adhesion to dental tissues, fluoride release, low thermal expansion coefficient, which is close to the tooth structure, and biocompatibility.
Despite fluoride release from GICs, secondary caries was cited as the main reason for GIC failure, indicating fluoride's low anti-bacterial potential. Considering this fact, restorative materials, especially those used in high-caries-risk children, should ideally have an anti-bacterial activity to reduce the adhesion and proliferation of cariogenic bacteria, thus decreasing the occurrence of primary and secondary caries. Anti-bacterial properties are also important when GIC is used in common pediatric dentistry techniques such as atraumatic restorative treatment or indirect pulp capping, where the residual carious dentin can be left in the prepared cavities.
Nanoparticles (NPs) are particles smaller than 100 nm. Their unique advantage of being smaller-sized particles results in a higher surface area to volume ratio and a stronger anti-bacterial activity than conventional fillers, which make their use in dentistry of particular interest. Studies mainly investigated their ability to enhance the mechanical properties and anti-bacterial effect of restorative materials.
Silver nanoparticles (Ag-NPs) have been investigated in dentistry because of their sustained ion release and long-term antibacterial property, which is 25-fold higher than that of chlorhexidine. In low concentrations, Ag-NPs demonstrated broad-spectrum anti-bacterial and anti-viral properties owing to multiple anti-bacterial mechanisms such as adherence and penetration into the bacterial cell, resulting in an increase of cell wall permeability, loss of cell wall integrity, inactivation of bacterial vital enzymes, and loss of DNA replication ability. TiO2 has anti-bacterial properties and is chemically stable, biocompatible, and non-toxic. Titanium dioxide nanoparticles (TiO2-NPs) have been added as reinforcing fillers to dental resin composites and epoxy.
Enhancing the mechanical, physical, and anti-bacterial properties of restorative materials helps improve their clinical serviceability. Accordingly, the goal of the current study was to investigate the effect of Ag-NP and TiO2-NP addition to the conventional GIC restorative material on its in vitro anti-bacterial effect against S. mutans, fluoride ion release, and compressive strength (CS).
| Methods|| |
The present study was an in vitro study. A power analysis was designed for sample size calculations to have adequate power to apply a two-sided statistical test of the research hypothesis that there was no difference between the three groups. According to the results of Elsaka SE et al. 2011 and El-Negoly et al. 2014, using an alpha (α) level of 0.05 (5%) and a beta (β) level of 0.20 (20%), that is, power = 80%, the predicted sample size (n) was a total of nine samples per tested parameter. Ten samples per parameter were used to gain extra power. Sample size calculations were performed using G*Power version 188.8.131.52. The approval from the ethics committee was obtained from Faculty of Dentistry Ain Shams University and the date of the approval was 18-12-2019.
The study consisted of three groups: Group C (n = 30) or the control group, in which conventional GIC was used, Group Ag (n = 30), in which 5 wt% Ag-NPs were added to GIC powder, and Group Ti (n = 30), in which 5 wt% TiO2-NPs were added to GIC powder. The anti-bacterial effect, fluoride ion release, and CS were assessed in each group (n = 10 specimens/parameter).
Preparation of group C specimens
The conventional self-cure GIC (Riva, SDI, Australia) powder and liquid were mixed at a powder/liquid ratio of 2.17:1 according to the manufacturer's instructions. The powder and liquid weights were measured using a digital balance (Precisa 205A series, Superbal, Germany) accurate to 0.0001 gm. Mixing of the powder and liquid was performed using a metal spatula and a glass slab at room temperature. The mix was then placed in a prefabricated Teflon trough having five holes (10 mm in diameter and 2 mm in height) and then covered with a celluloid strip and a microscopic glass slab under hand pressure. The glass ionomer was allowed to set at room temperature for 15 minutes; then, the bottom of the trough was pushed upwards using finger pressure to remove the disc specimens. The excess material was removed, thereafter, gently with a scalpel.
Preparation of group Ag and Ti specimens
Ag-NP powder with a <100 nm particle size (Sigma-Aldrich Co, St. Louis, MO, USA) and TiO2-NP with a <20 nm particle size (Nanostreams–Egypt; Batch No.: NS0021) were used. Experimental GIC was prepared by evenly mixing 5 wt% of Ag-NP or TiO2-NP powder with GIC powder. GIC powder containing 5 wt% NPs was mixed according to the manufacturer's instructions as previously mentioned to prepare the tests' specimens.
Agar diffusion test was used for anti-bacterial evaluation where ten-disc specimens (10 mm in diameter and 2 mm in height) were prepared using a Teflon trough in each of the three groups. Six mitis salivarius Agar (Ralin BV, Netherlands) plates were prepared, with two plates for each group. S. mutans ATCC 25127 (Microbiologics, Inc, USA) was plated onto the plates using calibrated loops; the medium formulation was prepared by adding 1 ml of 20% Chapman Tellurite solution. Twenty percent sucrose and 5 μg of bacitracin were also added to the medium according to Gold et al., 1973. The plates were incubated at 37°C using an anaerobic gas pack system for 72 hours to obtain a culture of S. mutans that was identified based on the S. mutans characteristic colony morphology. Five-disc specimens were placed on each plate. GIC specimens were prepared in a laminar cabinet to ensure sterile conditions. The plates were then incubated at 37°C for 48 hours and then visually inspected for the presence of inhibition zones around each specimen. The diameters of bacterial inhibition zone halos were measured using a ruler and expressed in millimetres. The average of three measurements was calculated for each specimen.
For the fluoride ion release test, ten-disc specimens of the same previously mentioned dimensions were prepared for each group. Specimens were stored at 37°C and 100% relative humidity (R.H.) for 24 hours. Thereafter, each specimen was immersed in 10 ml of deionized water in a closed plastic container at 37°C. At the time of the fluoride ion measurement, each specimen was removed from its container and the storage solution was collected for analysis. The discs were plotted dry and then placed in a new container with fresh 20 ml of deionized water. The measurement of fluoride ion concentration was made using an ion-selective electrode (ISE) [Orion Research, Inc., Denmark] at 24 hours, 14 days, and 28 days from the start of the experiment. The results were calculated as the amount of fluoride per unit surface area of the specimen (ug/mm2). Fluoride levels in parts per million (ppm) were obtained using the ISE connected to a digital meter. Total fluoride in μg was calculated by multiplying 1 ppm = 1 μg/ml by the tested solution volume (20 ml). The total fluoride was then divided by the area of the sample disc to obtain the fluoride release in μg/mm2.
As for CS test, ten cylindrical GIC specimens in each of the three groups (6 mm in height and 4 mm in diameter) were made using a prefabricated Teflon trough and tested according to 1SO9917. The specimens were stored at 100% R.H. at 37°C for 1 hour and then immersed in small containers containing water at 37°C in an incubator (IFE Precision Incubator) for further 7 days. CS was assessed at 7 days after mixing. Wet specimens were placed in a vertical position with the force incident on their long axis and loaded in compression at a crosshead speed of 1.0 mm/min in a universal testing machine (Model WDW-20, Beijing Sinofound; Beijing, China) until fracture occurred. The CS was calculated by the formula P/πr2, where P is the load at fracture, r is the radius of the specimen, and π = 3.14. The CS values [kgf/mm2] were converted into megapascal (MPa) as follows:12 CS [MPa] = CS [Kgf/mm2] × 0.09807.
One-way analysis of variance (ANOVA) was used for the analysis. Tukey honest significant difference post-hoc test was used for pair-wise comparison when ANOVA was significant. The significance level was set at P ≤ 0.05. R Foundation for Statistical Computing, Vienna, Austria version 3.6.0, was used for the statistical analysis. R packages used in the analysis were “HH” version 3.1-35.
| Results|| |
Group Ag showed the highest anti-bacterial effect against S. mutans, followed by group Ti. Both the Ag and Ti groups had a significantly higher anti-bacterial effect compared to the control group, whereas no significant difference was evident between groups Ag and Ti [Table 1].
|Table 1: Mean and standard deviation (SD) values of bacterial inhibition zones (mm) for different tested groups|
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Fluoride ion release
The frequency of fluoride ion release at 1, 14, and 28 days and the cumulative release after 28 days were the highest in group Ag, followed by the control group, and the lowest in group Ti [Table 2] and [Table 3].
|Table 2: Mean and standard deviation (SD) values for fluoride ion release of different groups at different time points|
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|Table 3: Mean and standard deviation (SD) values of cumulative fluoride release (μg/mm2) for different tested sub-groups at 28 days|
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Post hoc analysis after 24 hours revealed that all three groups were significantly different. Post hoc analysis for fluoride release after 14 days revealed that a statistically significant difference only existed between Ag and Ti. After 28 days, Ti was significantly different from both C and Ag [Table 2].
Cumulatively, Ag showed an insignificant increase in released fluoride ions and Ti showed an insignificant reduction in fluoride ion release compared to the control. However, the Ag group significantly released more fluoride ions than the Ti group [Table 3].
In a descending order, CS was the highest in the Ti group, followed by Ag and then the control group. TiO2-NP containing GIC had a significantly higher CS mean value compared to the other two groups. However, the Ag group showed a comparable CS to the control [Table 4].
|Table 4: Mean and standard deviation (SD) values of CS (MPa) for different tested groups|
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| Discussion|| |
In the present study, addition of 5 wt% Ag-NP and TiO2-NP to self-cure GIC promoted GIC's anti-bacterial action against S. mutans and CS without impairing its overall fluoride ion release.
The self-cure powder and liquid forms of GIC were chosen to easily blend NPs powder with that of GIC. The 5% concentration of NPs was selected as higher concentrations were associated with reduced GIC flexural strength. It seems that in percentages higher than 5%, agglomerations of NPs in the matrix act as weak points that reduce the mechanical properties. Moreover, NPs in high weight percentages act as non-reactive fillers and may interfere with acid–base reactions.
Both the Ag and Ti groups had significantly higher values of inhibition zones than the control group. These results are in accordance with El-Wassefy et al., 2017, who reported that GIC containing 3% Ag-NPs showed higher inhibition of bacterial bio-films when compared to conventional GIC. Also, Garcia Contreras et al., 2015, showed that incorporation of 3% or 5% (w/w) of TiO2-NP to GIC had better anti-bacterial activity against S. mutans than conventional GIC.
The anti-bacterial effect of TiO2 might be related to the photocatalytic properties of TiO2, which involves the production of reactive oxygen species such as hydroxyl radicals and hydrogen peroxide that cause degradation of the cell wall and cytoplasmic membrane of the bacteria. At first, this leads to the leakage of cellular contents, followed by cell death, and may be proceeded by complete damage of the organism. It has been reported that TiO2 particles cause quick intra-cellular damage. Additionally, nano-sized particles of TiO2 lead to enhancement of its bactericidal effect.
On the other hand, Ag-NP containing GIC showed the highest anti-bacterial effect against S. mutans compared to other groups. This anti-bacterial effect of Ag-NPs can be explained by the presence of silver. Silver is a benign bactericidal metal as it is non-toxic to animal cells, although it is very toxic to bacteria.
The present study showed that none of the added NPs caused significant changes in fluoride ion release compared to the control. Ag-NPs increased fluoride release compared to the control, whereas TiO2-NPs reduced fluoride release throughout the experimental time periods. However, after 28 days, the cumulative fluoride release did not differ significantly among the three groups. The anti-bacterial property of TiO2 may compensate for the reduced fluoride ion release and maintain its overall anti-caries properties.
The results showed that the mean values for CS in the TiO2 and Ag groups were higher than that of the control group, with the TiO2 group having the significantly highest mean value. This finding agreed with El Saka et al., 2011, who reported that incorporation of 3% and 5% (w/w) TiO2-NP enhanced the CS of GIC. The authors attributed this finding to the small size of TiO2-NP and to the large range of particle size distribution of the experimental GIC. These small NPs can accordingly occupy the empty spaces between the larger GIC glass particles and act as additional bonding sites for the polyacrylic polymer, therefore strengthening the GIC material.
In contrast, El-Negoly et al., 2014, concluded that addition of 7% TiO2-NP to conventional GIC caused a significant reduction in CS when compared with conventional GIC. The authors attributed this reduction to the increased concentration of TiO2-NP, leading to an insufficient amount of glass ionomer particles that are needed to effectively hold the relatively large amount of TiO2-NP powder.
In the present study, Ag-NP containing GIC also exhibited an insignificantly higher CS compared to the control group. The present finding agreed with Zahra Jowkar et al., 2019, and may be explained by the small sizes of the Ag-NPs incorporated into GIC that improved packing of particles within the matrix of the set cement. As mentioned for TiO2-NP, the incorporation of Ag-NP into GIC may also result in a wider range of particle size distribution. Therefore, these small silver NPs can occupy the empty spaces between the larger glass particles and may provide an additional bonding site for the polyacrylic polymer, thereby reinforcing GIC. Moreover, the increased CS may be attributed to the high density of interfaces of the nanomaterial and the tendency of NPs to resist the compression forces. Therefore, based on the current results, the authors recommend carrying out future studies to assess other laboratory tests such as solubility, micro-leakage, and bond strength to the enamel and dentin as well as clinical performance and cost-effectiveness.
| Conclusions|| |
Adding 5 wt% TiO2-NP or Ag-NP to conventional GIC significantly increased its anti-bacterial effect against S. mutans and its CS without affecting its fluoride release, which makes them potential additives to GIC as an effort to promote its clinical performance.
My acknowledgement goes to technicians who helped us in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Gehan G Allam
Organization of African Unity St. Abbasia - Cairo, Post No. 11566
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]