|Year : 2020 | Volume
| Issue : 7 | Page : 957-964
Effect of surface characteristic of different restorative materials containing glass ionomer on Streptococcus mutans biofilm
OS Kelten1, OK Hepdeniz2, Y Tuncer3, DA Kankaya3, O Gurdal4
1 Department of Restorative Dentistry, Faculty of Dentistry, Biruni University, Istanbul, Turkey
2 Department of Restorative Dentistry, Faculty of Dentistry, Suleyman Demirel University, Isparta, Turkey
3 Department of Food Engineering, Faculty of Engineering, Suleyman Demirel University, Isparta, Turkey
4 Department of Biostatistics and Medical Informatics, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey
|Date of Submission||04-Oct-2019|
|Date of Acceptance||20-Feb-2020|
|Date of Web Publication||3-Jul-2020|
Dr. O K Hepdeniz
Department of Restorative Dentistry, Faculty of Dentistry, Suleyman Demirel University, Isparta
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aims of this study were to evaluate the surface morphology and surface roughness of restorative materials containing glass ionomer, analyze Streptococcus mutans biofilm formation on the surface of materials, and determine the correlation between surface roughness and biofilm. Materials and Methods: Four restorative materials: resin-modified glass ionomer; giomer; amalgomer; and glass carbomer were used and for each material, 6 mm in diameter and 2 mm in thickness disc-shaped specimens were prepared to evaluate the surface morphology (n = 3), surface roughness (n = 16), and biofilm (n = 20). Surface morphology was analyzed with a scanning electron microscope. Surface roughness was evaluated via an atomic force microscope. The biofilm was evaluated by counting the colony-forming units. Surface roughness measurements were evaluated using a one-way analysis of variance and Tukey HSD test. Biofilm parameters were analyzed using the Kruskal-Wallis H and Mann-Whitney U test. Pearson's correlation test was used to determine the correlation between surface roughness and biofilm. Results: While the highest roughness values were obtained for amalgomer and glass carbomer, the lowest roughness values belonged to giomer and resin-modified glass ionomer. Statistically significant differences in the number of adherent bacteria were observed between the materials only on day 1. No statistically significant correlation was determined between surface roughness and biofilm. Conclusions: The resin content and small filler particle size of material positively affect surface roughness. However, there is no direct relationship between surface roughness and biofilm.
Keywords: Atomic force microscopy, biofilm, glass ionomer, scanning electron microscopy, surface properties
|How to cite this article:|
Kelten O S, Hepdeniz O K, Tuncer Y, Kankaya D A, Gurdal O. Effect of surface characteristic of different restorative materials containing glass ionomer on Streptococcus mutans biofilm. Niger J Clin Pract 2020;23:957-64
|How to cite this URL:|
Kelten O S, Hepdeniz O K, Tuncer Y, Kankaya D A, Gurdal O. Effect of surface characteristic of different restorative materials containing glass ionomer on Streptococcus mutans biofilm. Niger J Clin Pract [serial online] 2020 [cited 2020 Aug 14];23:957-64. Available from: http://www.njcponline.com/text.asp?2020/23/7/957/288882
| Introduction|| |
Glass ionomer cement (GICs) have gained wide popularity in dental practice and continued to evolve with the development of new materials such as giomer and amalgomer. Giomer was launched as a restorative material containing pre-reacted glass ionomer (PRG). The use of PRG fillers ensures rapid fluoride release from giomers by ion exchange in the wet siliceous hydrogel that forms due to the pre-reaction of fluoroaluminosilicate glass fillers with polyacrylic acid., Although several studies on the clinical performance and physical and surface properties of giomers exist, there has been no sufficient information on the effect of surface topography on biofilm development., Amalgomer, which has the advantages of GICs in combination with amalgam strength, has been introduced to reinforce GICs via ceramic additive., The latest restorative material developed based on GIC technology is glass carbomer, which contains a specially designed carbomer filler and fluorapatite/hydroxyapatite nanoparticle. These nanoparticles were reported to enhance mechanical and remineralization properties.,
In restorative procedures, surface characteristics play a crucial role in determining the quality and clinical behavior of the material. Surface roughness is an important characteristic of the material as it affects the esthetics, wear, optical quality, and bacterial colonization and maturation of oral biofilm., While the decrease in surface roughness lowers bacterial adhesion by reducing the contact area between the surface and microbial cells, restorations with rough surfaces enhance plaque accumulation by promoting the retention, survival, and proliferation of many caries-inducing microorganisms such as Streptococcus mutans. Oral biofilms may harbor many bacteria, but S. mutans has been considered as the first to be involved in bacterial colonization and one of the main pathogens involved in the development of secondary caries.,S. mutans is also involved in the transition from nonpathogenic to cariogenic biofilms, and their presence on the pellicular surface allows other colonies to migrate to this area and remain there., These features have led to S. mutans being the most widely studied microorganism in oral biofilms.,,
There is a lack of information in the literature on the effects of surface characteristics of new materials such as amalgomer and glass carbomer on biofilm formation. Therefore, the present study aimed 1) to assess the surface morphology and surface roughness of four materials classified as resin-modified glass ionomer cement (RMGIC), giomer, amalgomer, and glass carbomer using a scanning electron microscope (SEM) and an atomic force microscope (AFM), 2) to analyze and compare the formation of S. mutans biofilm on the surface of the materials, and 3) to determine the correlation between surface roughness and S. mutans biofilm formation of the materials. In this study, we hypothesized that there would be a significant correlation between surface roughness and biofilm.
| Materials and Method|| |
Four restorative materials: RMGIC (Fuji II LC), giomer (Beautifil II), amalgomer (Amalgomer CR), and glass carbomer (GCP Glass Fill) were used in this study. Information such as material type, composition, and manufacturer based on manufacturers' data is given in [Table 1].
For each restorative material, 19 disc-shaped specimens (6 mm in diameter and 2 mm in thickness) were prepared using a cylindrical Teflon mold, and 76 specimens were obtained for surface morphology and surface roughness test (n = 19). Further, 60 specimens, 15 of each restorative material, were prepared for the biofilm assay (n = 15). The materials were manipulated according to the manufacturers' instructions and inserted into the molds. The material surface in the mold was covered on each side with a mylar strip (Kerr Hawe Stop strip, Kerr Hawe, Bioggio, Switzerland) and placed between two glass slides. RMGIC and giomer were polymerized through the glass slide using a LED-curing unit (Kerr Demi Ultra, Kerr Corporation, Orange, USA) for 20 s and 30 s, respectively. The amalgomer was allowed to set at room temperature for 10 min. For the glass carbomer cement, a capsule mixer (Linea Tac, Montegrosso d'Asti, Italy) was used for 10 s of mixing before the application of the material. The specimens were polymerized for 90 s with a LED-curing unit (1400 mW/cm2, Kerr Demi Ultra). After the curing procedures following the manufacturer's instructions, the specimens were stored in distilled water at 37°C for 24 h.
Surface morphology evaluation
Three specimens of each material were used to evaluate surface morphology. Qualitative evaluation of the materials was performed using SEM (QUANTA FEG 250, FEI, Brno, Czech Republic). The surfaces were examined with a low-vacuum, high-voltage technique at 20.00 kV and 10–11 mm working distance at 500×, 1000×, and 2000 × magnifications.
Surface roughness analysis
Sixteen specimens were used to evaluate the surface roughness. Surface roughness measurements were obtained using the AFM's profilometer mode (Nanomagnetics Inst, Oxford, UK). The measurement length was set to 2 cm. Three measurements were performed from the center of each specimen. The mean of the three measurements was recorded in terms of Ra value as the surface roughness value of that specimen. The device was calibrated after every three successive measurements.
Fifteen specimens of each restorative material were divided into three subgroups according to the evaluation period of 1, 7, and 21 days. Specimens were packed separately and sterilized using the ethylene oxide gas sterilization system (Steris Amsco Eagle, Mentor, USA). The specimens were exposed to ethylene oxide gas at 37–54°C for 4 h with cold steam and dry system ethylene oxide sterilization technique and then ventilated for 12 h.
S. mutans ATCC 25175 (Liofilchem, Roseto delgi, Abruzzi, Italy) was used as the reference bacterium for biofilm formation. The S. mutans strain was developed in two successive passages at 37°C for 18 h in brain heart infusion (BHI) broth (Lab M, Bury Lancashire, UK). Bacterial suspensions to be inoculated into the discs were prepared by diluting the active culture (8.85 log colony-forming units [CFU]/mL) at 1:100 in the BHI broth.
Bacterial adhesion test was performed on sterile 24-well plates (Costar 3524, Corning Inc., New York, USA) in accordance with a previously described procedure. Five discs of each subgroup were separately placed in 20 wells containing 2 mL of sterile BHI broth under aseptic conditions. Then, 20 μL (5.29 log CFU/mL) of the cell suspension was inoculated into each well. Four different plates were incubated at 37°C for 1, 7, and 21 days to ensure the adhesion of S. mutans to the test material discs. 1 mL of used broth was removed from the wells in the plates incubated at 7 and 21 days, and 1 mL of sterile fresh BHI broth was added every alternate day. The specimens used to determine the number of nonadherent cells were obtained by washing each disc with 5 mL of sterile physiological saline (FTS, NaCl 0.85%, w/v) three times. The specimens used to determine the number of adherent cells were obtained by sonication of each disc within 1 mL FTS in an ultrasonic water bath (Euronda Energy, Vicenza, Italy) for 10 min. This ensured that bacteria in the biofilm transformed into the planktonic forms. The number of nonadherent and adherent cells to the material discs that developed in the culture broth was determined on BHI agar plates using the drop plate method. The number of CFUs was counted and recorded after incubation at 37°C for 48 h.
Surface roughness measurements were evaluated using a one-way analysis of variance and Tukey HSD test due to the satisfied assumptions of normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene's test). Biofilm parameters were not normally distributed; therefore, the Kruskal-Wallis H test and Mann-Whitney U test were used to analyze statistical differences. A comparison of the number of adherent bacteria on the materials between days was assessed using Wilcoxon Signed-Rank and Friedman tests. Dunn-Bonferroni post-hoc tests were carried out for each nonparametric pair of groups. Pearson's correlation test was used to determine the correlation between surface roughness and bacterial adhesion at 1, 7, and 21 days (P < 0.05).
| Results|| |
The SEM images showed that the surfaces of RMGIC and giomer were smoother and more homogenous than those of amalgomer and glass carbomer [Figure 1]. While a large number of shallow and short microcracks were observed on the surface of amalgomer, deeper and continuous microcracks were observed on the surface of glass carbomer. Small cavities randomly distributed on the surface were noted only on amalgomer surfaces [Figure 1]c.
|Figure 1: Scanning electron microscopy images (×1000 magnification), representing the surfaces of the tested materials. (a) Resin-modified glass ionomer, (b) Giomer, (c) Amalgomer, (d) Glass carbomer|
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According to the AFM images, RMGIC and giomer demonstrated a flat and smooth surface despite some small particles and grain clusters. Minimal roughness was observed on the surfaces of these materials, as expected [Figure 2]. While nonuniform surfaces with pores were observed on amalgomer surfaces, glass carbomer surfaces were the most irregular with peaks and valleys [Figure 2].
|Figure 2: Representative three-dimensional high-resolution atomic force microscopy images of the tested materials. (a) Resin-modified glass ionomer, (b) Giomer, (c) Amalgomer, (d) Glass carbomer|
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Descriptive statistics of surface roughness values of the materials are presented in [Table 2]. While the highest roughness values were obtained for amalgomer and glass carbomer materials, the lowest roughness values were obtained for giomer and RMGIC materials. The difference between the surface roughness values of the materials was statistically significant (P < 0.05). According to pairwise comparisons, no statistically significant difference was observed between amalgomer and glass carbomer (P > 0.05) and between RMGIC and giomer (P > 0.05) [Table 2]. Both RMGIC and giomer exhibited a statistically significant difference compared with amalgomer and glass carbomer (P < 0.05).
|Table 2: Descriptive statistics of surface roughness values of the used restorative materials|
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The median, maximum, and minimum values determined for S. mutans (log CFU/mL) on the materials at 1, 7, and 21 days are shown in [Table 3]. Statistically significant differences in the number of adherent bacteria were observed between the materials at 1 day (Kruskal-Wallis H test, P = 0.013). According to the 1st-day analysis, a significant difference was determined only between RMGIC and giomer (Mann-Whitney U, P < 0.05), and RMGIC had a statistically significant lower bacterial count than giomer. The differences between the other materials were not statistically significant. Furthermore, there were no statistically significant differences between the materials in terms of the number of adherent bacteria at the end of day 7 (Kruskal-Wallis H test, P = 0.106) and at the end of day 21 (Kruskal-Wallis H test, P = 0.095).
|Table 3: The median, maximum, and minimum values determined for Streptococcus mutans (log CFU/mL) on the materials at the evaluation periods|
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As a result of the comparison of the number of adherent bacteria for all materials among the evaluation periods, it was determined that the highest number of adherent bacteria was reached at the end of day 7 (2.70 log CFU/mL) and the lowest on day 1 (1.05 log CFU/mL). There was a greater decrease in the number of bacteria on day 21 (2.25 log CFU/mL) than on day 7. The difference between these values was statistically significant (Wilcoxon Signed-Rank test, P < 0.05).
Considering all the materials used, Pearson's correlation test showed no statistically significant correlation between surface roughness and the number of adherent bacteria on the material surface for each evaluation period [Table 4].
|Table 4: Correlation values of materials between surface roughness (Ra) and biofilm (CFU) for each evaluation period|
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| Discussion|| |
Surface characteristics of a restorative material have a significant impact on the clinical life and esthetic features of a restoration due to its effect on bacterial accumulation and secondary caries formation on the surface. Therefore, in the literature, there are many studies on the surface properties of materials, and in these studies, SEM has been used to examine surface morphology and structure., Likewise, in the present study, SEM was used to analyze surface morphology.
The structure of the resin matrix and the characteristics of filler particles have a direct effect on surface smoothness. In this study, RMGIC and giomer surfaces were thought to exhibit a smoother and more homogenous structure than glass carbomer and amalgomer because of the resin content of RMGIC and giomer. The rough and nonhomogeneous structure of amalgomer could also be explained by the voids in the material induced by mixing the material as a powder-liquid form, which results in the formation of micropits in the specimens. In the current study, microcracks were also extensively observed on the surfaces of both amalgomer and glass carbomer. This result is in accordance with those of previous studies., According to the manufacturer's recommendation, the best results for glass carbomer can be achieved with heat application at a temperature of up to 60°C/140°F during the setting reaction. Hard and brittle materials have been previously reported on heat applications at a temperature of 60–70°C. In light of this information, deep cracks in glass carbomer surfaces may be attributed to high-temperature exposures.
In the literature, various techniques including qualitative methods, such as optical and SEM, and quantitative methods, such as surface profile analysis and atomic force microscopy, can be used for assessing the surface roughness of restorative materials. Although profilometry is a widely preferred method for the evaluation of surface roughness in in-vitro studies, more accurate information with three-dimensional detailed topographical images of surface roughness at a nanometer resolution can be obtained using an AFM. In the present study, surface roughness was determined using AFM's profilometer mode to achieve more accurate and reliable results.
Differences in surface roughness results were found among the tested materials, which may reflect variations in composition and size distribution of the fillers. In this study, resin-containing materials showed low surface roughness values. Hence, it can be concluded that the resin content of material positively influences surface roughness. The present study results on the surface roughness of RMGIC were similar to those reported by other studies., In a previous study, giomer showed the lowest surface roughness values, whereas conventional GIC without resin content showed high roughness values. The researchers concluded that surface roughness could vary according to the structure of the material. Another study demonstrated that the surface roughness values of resin-based restorative materials were significantly lower than those of non-resin-based materials.
As mentioned above, the main factors affecting surface roughness are resin content and the size and type of the filler. Studies have also reported that surface roughness of materials with large particle size was high., In the current study, the materials containing small-particle fillers resulted in significantly smoother surfaces compared with materials including large particle fillers. The highest surface roughness was observed in amalgomer, which has a mean particle size of 5–10 μm, and glass carbomer, which has a mean particle size of 0.5–200 μm. In a recent study, the surface roughness of amalgomer was reported to be higher than that of other materials. This result was attributed to the large particles and ceramic particles in the material.
Interestingly, although RMGIC has particles of larger size than giomer, no statistically significant difference was determined between the surface roughness of RMGIC and giomer. This result may be related to the high-quality bond between the resin matrix and glass particles in RMGIC, which may positively affect surface properties. Indeed, it has been reported that the bond between the resin matrix and the filler particles also affect surface roughness, besides the amount of filler and filler particle size.
Biofilm formation on dental surfaces is a complex phenomenon that involves different key factors. The quantity of biofilm varies according to the surface characteristics of materials, and surface roughness is regarded as a parameter influencing biofilm formation on both oral tissues and dental materials. However, in the present study, the materials with lower surface roughness did not result in less S. mutans biofilm than the materials with higher surface roughness. Furthermore, RMGIC showed significantly less biofilm than giomer despite similar surface roughness values. Based on these results, it was concluded that surface roughness is not the only parameter influencing biofilm and that other surface features could also be efficacious. As previously stated, surface energy, surface hydrophilicity, surface chemistry, and fluoride release are the other factors affecting bacterial adhesion as well as surface roughness. This may also explain why no significant correlations were found between surface roughness and biofilm for the materials tested in this study, which is in agreement with the results of some previous studies.,,, Contrary to these studies, some researchers have indicated a correlation between surface roughness and biofilm formation., According to the results of the present study, the null hypothesis that there would be a correlation of biofilm formation with surface roughness was rejected.
Surprisingly, similar biofilm formation on amalgomer and glass carbomer with that on RMGIC was observed, even though the surface roughness of amalgomer and glass carbomer was higher than that of RMGIC. It may be speculated that fluoride content in the material's composition (amalgomer and glass carbomer) or its release was sufficient to result in a significant reduction in biofilm formation. However, glass carbomer's organic matrix is composed of glass nanoparticles enriched with fluorapatite/hydroxyapatite. The nano-fluorapatite/hydroxyapatite particles increase the surface area of the material for the reaction of the cement, leading to a better reactive process, whereas the inclusion of fluorapatite converts the glass ionomer into a fluorapatite-like material. In a study comparing fluoride emission of some materials, it was reported that the highest fluoride release was by amalgomer and was due to the presence of ceramic particles. In another study, glass carbomer was reported to exhibit similar fluoride release as conventional glass ionomer and RMGI.
At the end of days 7 and 21, no significant difference was determined between the materials. It is well-known that the high initial release of fluoride from GIC and RMGIC decreases after a few days. It has been previously stated that after the initial burst effect, constant fluoride release or reduction in fluoride release occurs because fluoride ions do not react chemically during the setting reaction. These results are in line with those of a previous study that the materials that released higher amounts of fluoride showed similar bacterial adhesion as the materials with lower fluoride release after 4 h. The other theoretical possibility was that surface roughness is more efficient for initial microbial adhesion than prolonged biofilm formation. Also, the results of this study should be evaluated considering the limitations of an in-vitro study. Indeed, the interaction between surface parameters on biofilm may be affected by the experimental set up of a laboratory study.
| Conclusion|| |
Resin-containing materials exhibit more homogeneous and smoother surfaces than resin-free materials. The resin content and small filler particle size of material positively affect surface roughness. However, there is no direct relationship between surface roughness and biofilm formation. The longer incubation period does not cause a statistically significant change in the number of adherent bacteria. Other factors, as well as surface roughness, should be considered in biofilm formation on restorative materials.
Financial support and sponsorship
This study was supported by the Suleyman Demirel University Scientific Research Projects Foundation (4992-DU1-17).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]