2020, Number 3
Evaluation of the antibacterial activity of glass ionomers modified by the incorporation of chlorhexidine and its impact on the compressive strength and bond strength
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Language: English/Spanish [Versión en español]
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ABSTRACT
Introduction: Even though glass ionomer cements have excellent properties for various clinical applications, they have been shown not to have sufficient antimicrobial activity. Objective: To determine if the incorporation of chlorhexidine into glass ionomer cements provides them with greater antimicrobial activity and if this incorporation affects their bond strength and compressive strength. Material and methods: Different proportions of 0.2% chlorhexidine were incorporated to ionomers type I and II. Compressive strength, bonding to dentin and agar diffusion tests were performed. Results: When incorporating chlorhexidine, a statistically significant increase in the antimicrobial activity of the cements was observed and no significant differences were found in compressive strength or bonding to dentin. Conclusion: The incorporation of chlorhexidine in the proportions studied seems to be an option to provide greater antimicrobial activity to glass ionomer cements without affecting some of their physical properties.INTRODUCTION
Glass ionomer cement (GICs) are widely used in preventive and restorative dental treatments, as their excellent properties make them a versatile material with very diverse clinical applications.1 The fluoride ions they release and their low pH during setting contribute to the remineralization of dentin and its antibacterial effect.2,3
However, it has been shown that this effect is not sufficient to prevent adherence or formation of biofilms, mainly due to Streptococcus mutants.4,5
For a long time, various works have been carried out in order to improve GICs by incorporating different additives such as chitosan,6 casein,7 hydroxyapatite,8 bioactive glass particles,9 zirconia,10 and chlorhexidine (CHX),11-15 the latter mainly seeking to increase their antibacterial properties.
CHX is an antiseptic substance widely used in dentistry. It has a broad antibacterial spectrum, both for Gram-positive and Gram-negative and even against some fungi. It is considered a safe compound with minimal adverse effects. It can be found in various presentations, the main ones being liquids, gels, and aerosols.16 However, these incorporations of other substances have often altered other properties of GICs, mainly physical and/or mechanical.12,15,17
Available studies reporting the incorporation of CHX to GICs use reagent grade CHX and/or in formulas that are not easily available to the dentist. For this paper, it was decided to use commercial dental CHX in order to determine if this mixture of common materials has greater antimicrobial activity and if it affects its bond strength and compressive strength.
MATERIAL AND METHODS
The materials used for this research were type I GIC (GC Gold Luting & Lining Cement®) and type II GIC (GC Gold Label Universal Restorative®) and CHX at 0.2% (Viarclean-up®). The preparations were made according to the manufacturer's instructions: for cement type I (1:2) and type II (1:1). To achieve this accurate powders and liquids were weighed.
All preparations were made in a multidisciplinary dental research laboratory at 25 ± 1 oC and relative humidity of 50 ± 10%. Different specimens were used for each test and with the necessary characteristics for each one, which are detailed further on.
In each test and for each type of GIC, 4 groups were analyzed. Group 1 in which CHX was not incorporated into the materials; group 2 in which 5% (w/w) of CHX was incorporated to the GIC liquid just before mixing; group 3 to which 10% (w/w) was incorporated and group 4, to which 15% (w/w) of CHX was incorporated.
ANTIBACTERIAL ACTIVITY
To evaluate the antibacterial activity, the agar diffusion test was used, widely used to determine the antibacterial potential of the materials.18
Twenty culture boxes (10 to evaluate type I and 10 for type II GIC) were used with brain-heart agar. Streptococcus mutans (ATCC 35665) were grown in a 10 mL infusion at 37 oC. 24 hours later, the suspension was adjusted to turbidity comparable to McFarland's 0.5 scale corresponding to approximately 1.5 × 108 CFU/mL, which was verified with a spectrophotometer.
300 uL of the suspension was placed in each of the boxes and distributed evenly on the surface, left for 30 minutes at room temperature while the components of all the GIC mixtures were weighed. 6 mm diameter filter paper discs were soaked on one side with each of the mixtures. A disc corresponding to each group was placed per culture box. Subsequently, in the center of each of the boxes, a filter paper with 6 µL of CHX as control, was placed.
All boxes were placed in a hermetically sealed container with partial anaerobiosis at 37 oC for 24 hours and then the inhibition halos of each of the mixtures were measured and the values were registered in a database.
COMPRESSIVE STRENGTH
48 cylindrical specimens (6 specimens per group for each GIC) of 4 mm in diameter and 6 mm thick were manufactured using a precast aluminum mold in which the materials were compacted immediately after mixing and incorporation (if applicable) of the CHX. Once the specimens had been set, they were removed from the mold and checked under a stereomicroscope for irregularities or defects on their surface, discarding those defective specimens.
Each specimen was weighed on an analytical balance and those whose weight was ± 0.005 g of the average of each group were also discarded. After that, each specimen was transferred to an Eppendorf tube with 1 mL of sterile distilled water and kept at 37 oC for 24 hours.
After 24 hours, each sample was placed between the compression attachments of the universal testing machine (CMS Metrology) which was programmed at a cruising speed of 1 mm/min. The maximum compression force that was recorded in Newtons (N) was until the specimen fracture and this was considered as the "compressive strength" of each one.
BONDING TO DENTIN
For this test, 40 intact maxillary and mandibular anterior teeth were used, which were donated by patients from the dental clinics of the Facultad de Medicina de la Universidad Autónoma de Querétaro (during 2018-2019) that required extractions for periodontal and/or prosthetic reasons. All were stored in distilled water until completing the final number.
Teeth with fissures and/or fractures and those with caries or restorations on the vestibular surface were excluded.
Extracted teeth were placed in plastic molds and covered with auto-curing acrylic, leaving the coronal portion free to wear and expose the dentin.
A high-speed part with a 0.4 mm diameter frusto-conical carbide bur was used to standardize wear.
Subsequently, a 3.6 mm diameter carbide disc was used to uniform the dentin surface. The entire wear process was carried out with constant irrigation and inside an extraction hood.
Each tooth was evaluated under a stereomicroscope to verify that the surface was uniform and that there were no traces of enamel. They were sanded with a fine-grit "600" sandpaper vertically and uniformly for 30 seconds and with constant irrigation, they were washed and placed in an ultrasonic tub with sterile distilled water for 10 minutes.
1.5 squares per side were designed in 3 mm thick pink wax and a 3 mm diameter circular perforation was made in the center using a standardized tip. Using these molds on the dentin surface the different respective GICs mixtures were placed and compacted.
Finally, the teeth with GICs were immersed in sterile distilled water at 36 oC for 24 hours. And then the bonding strength test (shear) was carried out.
A shear load was applied at the junction between the GICs and the dentin at a cruising speed of 0.5 mm/min, recording the Newtons (N) of force required until total detachment.
The bond strength was calculated in MPa, according to the formula: bond strength = F/A, where F is the force recorded in N and A is the area in mm2 of the surface of the GIC placed on the dentin.
Once the tests had been carried out, the dentin surfaces where the GICs was incorporated were inspected using the stereomicroscope and classified according to the type of failure: 1. Adhesive failure, where 0 to 5% of the remaining GIC was observed on the dentin surface. 2. Cohesive failure, where a remnant of the GICs was observed over 90% of the dentin surface, and 3. Mixed failure where between 6 and 89% of the cement remainder on the dentin was observed.
STATISTICAL ANALYSIS
All the results were collected in a database. The distribution of quantitative values was determined through the Smirnov Kolmogorov test. The results are presented as means, standard deviation and range in tables and graphs.
To determine statistically significant differences between the groups, ANOVA and Tukey's post hoc test were applied. For the only qualitative variable (type of failure) frequency and percentage are presented. Statistical significance was considered when p ≤ 0.05.
RESULTS
The results of the agar diffusion test for antibacterial activity are shown in Figure 1, where it can be seen that both GIC without CHX have zero inhibition of the growth of Streptococcus mutans (the 6 mm disc diameter is only reported). This inhibition was detectable from 5% CHX incorporation. However, this was not statistically significant.
From 10% CHX incorporation, a statistically significant increase in antibacterial activity against Streptococcus mutants was observed, and it was the 15% CHX incorporation that presented the greatest inhibition. However, there was no statistically significant difference between the inhibition shown with 10 and 15%. The control with CHX showed the highest inhibition, being statistically different from all of the groups.
Table 1 shows the comparison between the different groups regarding the compressive strength (N) and the bond strength (MPa) of both types of cements with the incorporation of the different percentages of CHX. In no case was there a statistically significant difference.
87% and 95.8% of the tests carried out with type I GICs and type 2 GICs, respectively, presented adhesive failures, while the rest were mixed, and in no case did the cohesive failure occurs.
DISCUSSION
Incorporating antibacterial compounds in GICs is promising and has great potential, as it would bring several benefits to patients. Recurrent caries would be prevented, especially on margins of restoration; the formation of plaque on their surfaces and dental surfaces close to the restoration would be inhibited, and it would even contribute to reducing the number of bacteria in saliva and the oral cavity in general.15
Several studies have been carried out that report that incorporating CHX into GICs confers them antibacterial properties. However, other investigations have concluded that it damages some of their physical properties.12
Two of the most important physical properties of GICs are compressive strength and bond strength. The first one provides information about the resistance that the material will have against the forces of mastication. The second one allows to have an idea of adequate retention to the dental structure and is also directly related to marginal sealing and therefore avoiding marginal microfiltration.
It is known that the physical properties of GICs can be affected by the way in which the cements are prepared, including the powder-liquid ratio, the particle size in the powder, and even the aging of the specimens. Besides, each GIC commercial brand presents variations in its components and could even vary in different production batches of the same brand. For all this, care must be taken when generalizing about properties of the GICs, especially when products are incorporated or their mixing proportions are modified.
In this research, the GICs type I GC Gold Luting & Lining Cement® and type II GC Gold Label Universal Restorative® were used in strict preparation, as indicated by the manufacturer and with the incorporation of CHX at 0.2% (Viarclean-up®) in different proportions, an antibacterial effect was obtained that increased as the proportion of CHX increased. This is consistent with previous reports by Botelho11 and Ribeiro14 but not with Jedrychowski15 or Takahashi.19
It was observed that although the effect increased, this was not statistically significant for all the cases, which would indicate that the ideal proportion to incorporate would be 10% for both GICs.
On the other hand, the same incorporation of CHX did not show statistically significant changes in compressive strength or bond strength. Our results are different from those reported by Palmer et al.12 who concluded that as the amount of CHX incorporated into GICs increases, a decrease in compressive strength is observed.
As for the in vitro studies, these do not contemplate all the variables present in the mouth, so their results cannot be extrapolated to a clinical situation, so more in vitro studies should be carried out that consider other variables and that evaluate other physical properties that could be altered. Clinical trials are also necessary to consider carrying out the practice of incorporating CHX during the preparation of these GICs.
At the moment, the GIC type I GC Gold Luting & Lining Cement® and GIC type II GC Gold Label Universal Restorative® modified by the incorporation of CHX at 0.2% (Viarclean-up®) seem to be a promising option that provides them with important antibacterial properties without altering their compressive strength and dentin bond strength significantly.
CONCLUSION
Incorporating CHX at 0.2% (Viarclean-up®) at 5%, 10%, or 15% increases the antibacterial activity of type I glass cements GC Gold Luting & Lining Cement® and type II GC Gold Label Universal Restorative® without significantly compromising the compressive strength and dentin bond strength.
ACKNOWLEDGEMENTS
Thanks to the support of the programs "Verano de la Ciencia UAQ 2019", "21o Verano de la Ciencia Región Centro 2019" and "XXIX Verano de la Investigación Científica de la Academia Mexicana de las Ciencias", three undergraduate students (authors of this article) were able to meet and work on this project of research in the Laboratorio de Investigación Odontológica Multidisciplinaria de la Universidad Autónoma de Querétaro.
REFERENCES
AFFILIATIONS
1 Laboratorio Multidisciplinario de Investigación Odontológica, Facultad de Medicina de la Universidad Autónoma de Querétaro. México.
2 Estudiante de Licenciatura en Odontología, Escuela Nacional de Estudios Superiores Unidad León, UNAM. México.
3 Estudiante de Licenciatura en Cirujano Dentista de la Universidad Autónoma de Ciudad Juárez. México.
4 Departamento de Especialización en Prostodoncia, Facultad de Medicina de la Universidad Autónoma de Querétaro. México.
CORRESPONDENCE
Rubén Abraham Domínguez Pérez, PhD, MSc, DDS. E-mail: dominguez.ra@uaq.mxRecibido: Septiembre 2019. Aceptado: Enero 2020.