New findings about releasing of chloride ions and quaternary ammonium compounds from conventional and experimental glass ionomers

Introduction

Glass-ionomer cements (GICs) are essential mate­rials in clinical practice, especially in pediatric dentistry, due to their versatility, self-adhesion to enamel and dentine, and good bio-compatibility. They are very useful in full restorations application, particularly in children, liners and bases, fissure sealants and also, to a lesser extent, adhesives for orthodontic brackets and as cementitious sealers in endodontics. For more than 60 years, GICs have been used in restorative dentistry, especially in pedodontics. By the late 1960s, they began to develop as a result of early research conducted by Alan Wilson and Brian Kent in the Government Chemist’s Laboratory in London [1, 2].
GICs have a list of advantages, including a similar thermal expansion coefficient of dental structures, adhering strongly to dental hard tissues, releasing fluoride for a considerable amount of time (at least five years), and being bio-compatible. When applied as bases in deep cavities, their ability to release fluoride makes them excellent materials to substitute dentine [3-9]. Despite these benefits, traditional GICs still have several drawbacks, such as brittleness, low fracture resistance, and low resistance to abrasion and wear. Additionally, they have a limited working time and a very long setting time as well as even worse aesthetic properties than composite materials [10-14]. However, GICs are one of the most analyzed materials in dentistry. The PubMed database, the most relevant search engine in the field of medical sciences, lists about 8,000 papers for the query term “Glass Ionomer Cements” [15]. The release and uptake process is one of the most important properties of GICs [16-19]. In that direction, the release of fluoride ions and the property of GICs to act as a kind of reservoir for those ions, emphasize them as the main restorative materials, which participate in the process of re-mineralization of de-mineralized tooth surfaces [20-23]. However, another crucial characteristic of GICs is that the released fluoride ions act on local bacteria by decreasing cariogenic micro-organisms [24-30].
GICs may serve as models for the release of other active anti-microbial substances in addition to fluoride ions. Different anti-microbial chemicals have been added to conventional and resin-modified GICs to enhance their anti-bacterial properties. In a number of studies, the most frequently analyzed anti-microbial agent is chlorhexidine (CHX), described as a golden standard for anti-bacterial application. The addition of CHX in different combinations and different concentrations in conventional GICs increases anti-bacterial activity against cariogenic bacteria [31-36]. Unfortunately, there is not much published data on the use of other additio­nal anti-microbial components in GICs.
The group of quaternary ammonium compounds consists of anti-microbial agents with proven anti-microbial activity. Only several papers described the incorporation of quaternary ammonium anti-microbial compounds, such as benzalkonium chloride (BKC) and cetylpyridi­nium chloride (CPC) [37-43]. As an active ingredient in oral antiseptics, CPC has a broad anti-bacterial spectrum, a potent bactericidal impact on Gram-positive bacteria and fungicidal effect on fungi [37-40]. BKC acts against bacteria, some viruses, fungi, and protozoa. Bacterial spores are considered to be resistant. Solutions are bacteriostatic and bactericidal activity, depending on their concentration. Gram-positive bacteria are more sensitive than Gram-negative ones [38, 39]. Both BKC and CPC contain chloride ions in their structure. Chloride exhibits anti-microbial properties through its inorganic and organic compounds, by rupturing chemical bonds in pathogens’ molecules, such as bacteria and viruses, and destroying them. It has been demonstrated that chlorine-based inorganic compounds, such as sodium chloride, potassium chloride, chlorine dioxide, calcium hypochlorite, sodium hypochlorite, and lithium hypochlorite, have anti-bacterial activity [44-46]. Inorganic and organic chlorine compounds are also widely used in dentistry. The elimination of biofilm by chlorine compounds is an essential step for successful therapy of implant-related infections [47]. Several clinical studies have confirmed the efficacy of sodium hypochlorite and chlorine dioxide oral rinse in combating dental plaque and gingival inflammation [48]. Root canal irri­gants using chlorine solutions play a significant role in eliminating micro-organisms, tissue dissolution, and removing debris and smear layer [49]. Treatment of candidiasis using chlorine dioxide, especially in geriatric patients, has also been proven [50].

Objectives

The idea for this study developed from the knowledge that glass ionomer cements are frequently used in pediatric dentistry, and may be employed as a medium for delayed and slow release of additional active anti-bacterial components apart from fluoride.
The objectives of the study were to determine the level of released chloride ions from conventional GICs in a medium consisting of deionized water over diffe­rent time periods. Also, to determine the level of released chloride ions from the experimental glass-ionomer cement with 2% anti-microbial agents in a medium consisting of deionized water over different time periods, and to measure the quantity of released anti-microbial components from the conventional and experimental GICs, in which 2% concentrations of anti-microbial agents were previously incorporated.
The study’s null hypothesis was that neither conventional nor experimental GICs release chloride ions or anti-microbial agents.

Material and methods

The study was performed using conventional glass ionomer cements, i.e., ChemFlex (ex. Dentsply, Germany) and Fuji IX (ex. GC, Japan), and experimental glass ionomer cement MP4 (Pilkington’s Ltd., UK).
Anti-microbial compounds used were cetylpyridi­nium chloride (CPC; Sigma-Aldrich, Dorset, UK) and benzalkonium chloride (BKC; ex Fluka, Germany). The composition of each cement is presented in Table 1.

Preparation of samples and incorporation of anti-microbial components

Specimens were prepared in compliance with the 1989 British Standards Institution Dental Glass Ionomer Cements Specifications. Anti-microbial compounds, BKC and CPC, were first incorporated into a glass ionomer cement’s polyacrylic acid by mixing, and then the powder was added gradually to previously prepared acid and anti-microbial compound mixture, while care was taken to mix them together until complete saturation. Then, this freshly prepared paste was poured into metal molds (4 mm in diameter and 6 mm in height). The molds were put in specialized G-clamps after being sealed on both sides by metal plates. Anti-microbial agents were used in precise amounts of 2% of the cements’ weight. By measuring specified proportion of the anti-microbial agent with an analytical scale (Mettler AE 200), the concentration (weight) of BKC and CPC was calculated. Previous investigations have established that the amount of anti-microbial agents at 2% was equal to 0.0044 grams of GIC ChemFlex, 0.0064 grams of GIC Fuji IX, and 0.0054 grams of experimental GIC MP4. After that, the samples were kept for an hour in an incubator at 37oC (maturation time). The specimens were removed from the clamps and molds after being removed from the incubator, and were kept individually in distinctly labeled plastic tubes with 5 ml of deionized water at a temperature of 22-24°C and air humidity of 40-50%.
The amounts of released Cl- ions were determined using ORION 4-star pH meter and an ion-selective electrode (ISE, Benchtop Thermo Electron Corporation, USA) optimized for chloride ion detection (Figure 1). Prior to use, an electrode was calibrated using standards with molarities that were within the range of ions’ concentrations to be measured (0.1, 1.0, 10.0, 100.0, and 1,000.0 ppm). The release of chloride ions was done in a water medium of deionized water. Then, the ion-selective electrode was subsequently sunk in marked plastic laboratory tubes for chloride ions determination. Prior to each subsequent measurement, the plastic tubes were shaken for a few seconds to obtain a uniform distribution of released ions. Levels of released chloride ions were determined at different time intervals, as follows: immediately, after 15 min, 30 min, 45 min, 1 hour, 2 hours, 3 hours, 4 hours, 24 hours, and after 7 days (Figures 1 A, B).

Determination of amounts of anti-microbial agents using spectrophotometer

Concentrations of the anti-microbial agents (quaternary compounds) were determined using Varian Cary 50 tablet UV/VIS spectrophotometer (Agilent Technologies Inc., USA). CPC and BKC solutions with pre-set concentrations were used to calibrate the spectrophoto­meter. The UV spectrophotometer was calibrated to detect BKC at a maximum absorption wavelength of 214 nm, and CPC at a maximum absorption wavelength of 259 nm. Before each sample was measured, the plastic tube was shaken for 10 sec in order to disperse the settled anti-microbial organic compound uniformly throughout the medium (settling is caused by the high molecular weight of quaternary ammonium compounds). A portion of deionized water was poured from the plastic tube into a cuvette, and the cuvette was placed into the spectrophotometer. The instrument was connected to a computer that used specialized software, and displayed the results of measurements in absorbance units (AU). Once the measurement of each sample was done, the water was poured back into the tube. Re-zeroing was performed when changing the anti-microbial agent. Measurements were carried out over a period of 9 sequential time intervals, including 15 min, 30 min, 45 min, 1 h, 2 hrs., 3 hrs., 4 hrs., 24 hrs., and 7 days. The deionized water, in which the samples were kept was unchanged during whole time. Six samples of each GIC and both anti-microbial compounds were utilized to create a total of 36 specimens. Additionally, six samples of identical cements without any anti-microbial agents were created to serve as a control group (Figure 2).
One-way ANOVA test was applied for statistical ana­lysis, and Mann-Whitney U test and post-hoc Tukey’s test were performed for honest significant difference (HSD).

Results

Chemical analyses with conventional glass ionomer cements

The average values of released chloride ions from all the groups showed a gradual increase during the investigated periods, and this was especially emphasized in the groups of GICs with anti-microbial agents, particularly in the combination of anti-microbial agents with GIC ChemFlex. The variance analysis (ANOVA) revealed that all three groups showed statistically significant differences between average values across the study period.
According to Tukey’s HSD test, GIC ChemFlex without anti-microbial agents demonstrated statistical insignificance only in one-time interval. The average value differences between all-time intervals in the group of ChemFlex with 2% BKC were statistically significant, except between the noted time intervals. The group of ChemFlex and 2% CPC demonstrated statistically significant differences in the average values between almost all-time intervals. In contrast to GIC ChemFlex, Fuji IX cement with no additional anti-microbial component showed a statistical significance in the majority of time periods. The combination of Fuji IX and 2% BKC was statistically insignificant in more time intervals than the combination of the same cement incorporated with 2% CPC, which had a high degree of statistical significance (Table 2).

UV spectrophotometric analyses with conventional glass ionomer cements

According to the variance analysis, all the groups showed statistically significant differences in average values over analyzed time (p < 0.05). Except for the combination of ChemFlex with an additional 2% BKC, there was substantial significant reliance according to Tukey’s HSD test (Table 3).

Chemical analyses with experimental glass ionomer cement MP4

The statistical analyses carried out with ANOVA test showed that there was a statistically highly significant difference in the average values for p = 0.00000. To provide a clearer picture of the findings of statistical analysis using Tukey’s HSD test, statistically insignificant differences were only reported for both combinations and all time intervals. According to Mann-Whitney U test, the difference in the average values between MP4 + CPC 2% and MP4 + BKC 2% was statistically significant (p < 0.05) between the average values for all time intervals, except at zero time and at fourth hour (Table 4).

UV spectrophotometric analyses with experimental glass ionomer cement MP4

The ANOVA test showed that the differences in average values were highly statistically significant (p = 0.00000). Individual comparisons of the average value differences (Tukey’s HSD test) in combination of MP4 + CPC indicated statistical significance for p < 0.05 only in the time intervals mentioned, but the combination of MP4 + BKC showed a statistically negligible difference across all time intervals. The difference in the average values between MP4 and both anti-microbials was statistically significant over all time periods for p < 0.05, according to Mann-Whitney U test (Table 5).

Comparative analyses between analyzed glass ionomer cements

The average values of released chloride ions and anti-microbial agents in samples prepared from all glass ionomer cements separately integrated with 2% anti-bacterial agents, are shown in Tables 6 and 7. The applied statistical analysis of variance revealed the existence of statistically significant differences between the GICs in all time periods (p < 0.05). Tukey’s HSD test showed that the combinations of the three GICs with both BKC and CPC resulted in a statistically significant difference in the average values (p < 0.05).

Discussion

The determination of chloride ions was carried out with an ion-selective electrode in a medium of deionized water, in which the GICs samples, without and with the addition of 2% concentration of anti-microbial compounds, were stored during ten time periods encompassing a week.
The purpose of this investigation was to determine if the GICs have an anti-bacterial impact because chlorine, the primary component of anti-microbial compounds BKC and CPC, has been proven to have such an effect. The results on the release of chloride ions from conventional GICs containing 2% anti-microbial agents indicated a continuous release of these ions over the course of a week, which is likely caused by chlorides in the anti-microbial compounds. However, chloride ion release occurs from the GICs with no anti-microbial compounds incorporated. Additionally, a continuous release of chloride ions was noted during all investigated periods. There have been several research on the release of elements from conventional cements. However, not a single study on the release of chloride ions from GICs, either in the references or in the Medline database, has been found. Additionally, manufacturers do not mention chloride ions in compositions of the examined cements in their specifications. This raises a question of where the chloride ions in the measured medium came from. Even when the cumulative release impact is examined, the amount of released chloride ions keeps increasing, providing proof of their ongoing release. Given that the application of a particular ISE does not demonstrate the existence of chloride ions in a medium devoid of cement, it is obvious that these concentrations are not caused by the medium itself (deionized water). The potential of an external (aero) introduction or secondary contamination of the medium, is also ruled out for two reasons: the concentrations of chlorine in the lab’s air were brought to zero, and the test tubes holding the samples and medium were shut after each measurement. The integration of ions from the air into a water medium in a very short amount of time would be doubtful, even if there was a precise calculation of the chlorine content in the air.
The cumulative effects of released anti-microbial compounds incorporated into conventional GICs were also performed in a medium of deionized water. The cumulative impact of released anti-microbial compounds is crucial, especially in situations where oral hygiene is lacking and dental plaque is persistently abundant. On the other hand, the cumulative effect of the modified “anti-microbial cements” applied in the cavities, which release anti-microbial compounds toward the interior of the cavity, i.e., at the interfaces between the restorations and the dentine, is of no lesser importance. The improvement of anti-microbial properties of GICs, without a loss in their chemical and physical characteristics, is of permanent interest in dental medical science. The incorporation of compounds with far stronger anti-microbial characteristics is still questionable with respect to the effect on the basic characteristics of the used GIC. A number of studies described the possibility of incorporating chlorhexidine [31-36]. It was the first anti-microbial compound incorporated into GICs. Data from the literature on the possibility of incorporating other compounds, especially from the group of quaternary ammonium compounds, is insignificant. The incorporation and release of anti-bacterial substances cetylpyridinium chloride and benzalkonium chloride are discussed in this work. Due to their chemistry, i.e., large and heavy organic chains, their determination with ion-selective electrodes is impossible, and therefore the analysis was conducted with the use of ultraviolet-visible spectrophotometry. This procedure is appro­priate for the detection of organic compounds contained in a medium, which were the target of the analysis.
There are few literature data describing the incorporation and release of other anti-microbial agents from other GICs prepared in a different way, in samples with different sizes, and using a different methodology and equipment [37-43]. Attempts by incorporating CPC in other restorative materials, such as resin matrix, have been made. Although the two restorative materials are incomparable, some similarities can be seen in the obtained results. The release of CPC was conducted in a water medium, the amount of the released CPC was determined with the use of a UV/VIS spectrophotometer, and the analyzed intervals were 1, 3, 5, and 7 days [42].
This study also included chemical analyses with the experimental glass ionomer cement MP4, developed by Pilkington’s Ltd., UK. Being a cement with no fluoride, MP4 is mainly used in laboratories for experiments on the incorporation and release of fluoride ions, but it is also used as a medical cement [5]. The intention was to see the possibility of the release of chloride ions and anti-microbial compounds of BKC and CPC from the MP4 experimental GIC as well as to compare the values obtained with the conventional GICs.
The outcomes of the release of chloride ions demonstrated their continual release over the investigated periods, until the seventh day for the CPC compound, i.e., until the 24^th hour for the BKC compound, when a drastic drop in the average values occurred. The results of the possibility for the release of anti-microbial compounds from MP4 into a water medium point to a constant slight increase in the average values of the released compounds. The differences in the average values at all time intervals were higher for the combination of MP4 + BKC, which was also statistically confirmed (Mann-Whitney test). For the combination of MP4 + CPC, the analysis of the variance revealed statistically significant variations between the average values in the studied period; however, this was not the case for the second tested compound. To achieve a comprehensive understanding of the experimental GIC MP4’s properties regarding the release of chloride ions, i.e., anti-microbial chemicals, the findings were compared to the results for conventional cements. According to the findings on the release of chloride ions between the conventional cements and the experimental cement, the release of ions was significantly greater for the MP4 + BKC combination until the fourth hour, and for the MP4 + CPC combination until the 24^th hour. All of the examined intervals’ resultant average value differences were statistically very significant (ANOVA). Differences in the values obtained from the combination of the MP4 cement with the two studied compounds with regard to the conventional cements were statistically significant, although at different levels of significance, according to the results acquired using Tukey’s test.
The comparative analysis of the data obtained on the release of anti-microbial compounds (UV-analyses) shows a drastically higher release of the BKC compound from the experimental cement at all time intervals with respect to the conventional cements, and a significantly higher release of the CPC compound. As no experiments with the incorporation of anti-microbial compounds in experimental GIC MP4 have been performed to date, and regarding the results obtained from our comparative analysis with conventional cements, numerous questions arise. Does the proportional composition of compounds constituting the cement, or the absence of fluoride ions in the composition, or perhaps the polyacrylic and tartaric acids, which are in this case mixed within the powder of the cement, contribute to the significant differences in conventional cements? Considering the fact that no analyses have been done so far of the release of chloride ions, i.e., of anti-microbial compounds, further research is needed in order to get answers to these issues.

Conclusions

Based on the study and obtained data, we may conclude that: conventional GICs without incorporated anti-microbial compounds release chloride ions at all time periods, and the concentration increases with time. Experimental GIC MP4 integrated with 2% anti-microbial agents releases a high level of chloride ions. Both conventional and experimental GICs containing 2% anti-microbials release anti-microbial agents that are proportionate to a time period. Fuji IX GIC releases more chloride ions on average than ChemFlex. In comparison with conventional GICs, MP4 GIC produces a significantly higher release both of chloride ions and anti-microbial compounds.

Disclosures

1. Institutional review board statement: Not applicable.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: None.

References