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Keywords:

  • Antibacteriology;
  • biofilm;
  • chlorhexidine;
  • glass-ionomer cement;
  • resin-modified glass-ionomer cement

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background:  This pilot study investigated the antibiofilm effects of glass-ionomer cements (GICs) and resin-modified glass-ionomer cements (RMGICs) incorporated with chlorhexidine (CHX) in vivo.

Methods:  Experimental GICs and RMGICs containing 2% CHX were obtained by mixing CHX with the powder of GICs (CHXGIC) and RMGICs (CHXRMGIC). Four groups of specimens were prepared in a standardized size. After polishing and sterilization, they were bonded to the buccal surface of the molars in the first and second quadrant of volunteers and left untouched for 4 hours and 24 hours, respectively. The bacterial vitality of plaque was then analysed by confocal laser scanning microscopy (CLSM). The bacterial morphology and biofilm accumulation were determined by scanning electron microscopy (SEM). The pH value of biofilm was assessed by Plaque Indicator Kits.

Results:  CLSM analysis revealed that bacterial vitality of the biofilm on CHXGIC and CHXRMGIC was significantly lower than that on GIC and RMGIC. SEM analysis indicated that the morphology of bacteria on CHXGIC and CHXRMGIC was irregular. The pH value of biofilm on the experimental materials presented no statistically significant difference.

Conclusions:  Twenty-four hour bacterial vitality on GICs and RMGICs with CHX are lower in micro-organisms than on conventional GICs and RMGICs.


Abbreviations and acronyms:
CHX

chlorhexidine

CLSM

confocal laser scanning microscopy

FI

fluorescence intensity

GIC

glass-ionomer cement

HMDS

hexamethyldisilazane

PBS

phosphate-buffered saline

RMGIC

resin-modified glass-ionomer cement

SEM

scanning electron microscopy

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Glass-ionomer cements (GICs) are widely used in dentistry because of their unique advantages, such as fluoride release, anticariogenic properties, direct adhesion to tooth structure, minimal microleakage and low cytotoxicity.1 However, there are also obvious disadvantages including low mechanical strength and moisture sensitivity during setting.1,2 To improve the mechanical properties and handling facilities, resin-modified glass-ionomer cements (RMGICs) were introduced.3

An ideal restorative material should have a property of adhesion to tooth structure and an ability to withstand the trauma of occlusion. However, some therapeutic and caries-preventing efficacy is also desirable.4 With regard to the release of fluoride, GICs have been shown to produce some degree of antibacterial effect,5 but the evidence is weak. The incorporation of chlorhexidine (CHX) into GICs and RMGICs has resulted in an excellent antibacterial effect in vitro.6,7 However, it has also been reported that CHX may interact with the fluoride ions in GICs and thus interfere with the antibacterial effect.8 Hoszek and Ericson9 suggested that adding CHX and CHX-tartaric acid to GICs might increase the antibacterial properties of the material but the release of fluoride ions would decrease. Micro-organism counts in both affected and infected dentine under CHX-containing GIC restorations were significantly lower than those under conventional GIC restorations.10 As RMGIC is considered to be superior to conventional GIC in physical terms,11 there was a need to investigate whether RMGIC is also superior to GIC in its antibacterial character by comparing the level of biofilm development on the surfaces of both types of GICs.

The acid production of cariogenic bacteria serves as a virulence factor because the organic acids, as a by-product formed during the metabolism of dietary carbohydrates, are essential for the development of dental caries.12 This lowers the pH value and promotes a higher enamel and dentine mineral loss. An increase in the antibacterial activity of the material influences the pH value of the dental biofilm, decreases mineral loss and reduces the incidence of caries.

The current study investigated antibacterial activities on the early established biofilm of GICs and RMGICs incorporated with CHX diacetate in vivo. The null hypotheses tested were that: (1) the addition of CHX to GICs and RMGICs has no greater effect on the inhibition of early biofilm formation than that of GICs and RMGICs containing no CHX after 4 hours and 24 hours; and (2) the antibacterial effect of RMGICs with incorporated CHX is not superior to that of GICs with incorporated CHX.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study design

This pilot study was an experimental clinical in situ trial using a split-mouth design (Fig. 1). After flipping a coin, the first quadrant of the dentition was allocated to the experimental site and the second quadrant was allocated to the control site. Eight healthy volunteers (four males and four females) were selected from a group of 10 postgraduate students at the dental school of Wuhan University. For inclusion in the study, participants were required to be in good health; not to have used any kind of mouthrinses or undergone any antibiotic therapy during the six-month period preceding the start of the present experiment; not to have signs of destructive periodontitis or any other inflammatory conditions of the soft tissues; and to have plaque-free dentition. The exclusion criteria were: presence of carious lesions in upper first and second molars; signs of periodontitis or any other inflammatory conditions of the surrounding soft tissues; and poor oral hygiene.

image

Figure 1.  CONSORT flow chart of the study design.

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The study was approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University [2010 (044)]. All volunteers signed an informed consent form. The study design was registered at the Chinese Clinical Trial Registry (ChiCTR-TRC-10001161).

Sample preparation

The control specimens were a powder–liquid version of a conventional high-viscosity GIC (Fuji IX GC Corporation, Tokyo, Japan) and a RMGIC (Fuji II LC, GC Corporation, Tokyo, Japan) (Table 1). The experimental specimens comprised a high-viscosity GIC (CHXGIC) and a RMGIC (CHXRMGIC) to which 2% CHX diacetate (Sigma, St Louis, MO, USA) was added according to the ratio presented in Table 2. Control and experimental samples were both mixed in a coulter mixer and placed in silicon rubber moulds (4.0 mm × 4.0 mm × 1.2 mm). The RMGICs and CHXRMGICs were cured for 20 seconds, using a halogen light (Spectrum, Dentsply, York, USA). After 24 hours, the specimens were polished under wet conditions in 1.0 mm thick slabs, washed in sterile distilled water under ultrasonification and disinfected under ultraviolet light before being stored in sterilized glass bottles until ready for use.

Table 1.   Materials used in this study
MaterialsTypeMain compositionManufacturer
Fuji IXautocured GICAluminium-calcium-lanthanum fluorosilicate glass; Polycarboxylic acidGC Corporation, Tokyo, Japan
Fuji II LCresin-modified light-curing GICAlumina-silicate glass; Polyacrylic acid; HEMA; TEGDMAGC Corporation, Tokyo, Japan
Table 2.   Weight ratios and powder-to-liquid ratios used for the study groups
GroupGIC (%)RMGIC (%)CHX (%)P/L (g/g)
GIC100  3.6
CHXGIC98 23.5
RMGIC 100 3.2
CHXRMGIC 9823.1

Generation of oral biofilms

According to the study design, CHXGIC and CHXRMGIC specimens were respectively bonded to the buccal surfaces of the maxillary first and second molars in the first quadrant, whereas GIC and RMGIC were respectively bonded to the buccal surfaces of the maxillary first and second molars in the second quadrant in the same volunteer, using zinc polycarboxylate cement (GC Corporation, Tokyo, Japan). Volunteers were required to maintain their normal dietary habits during the entire experiment. Neither cleaning procedures nor agents for chemical plaque control were applied to the specimens for the complete duration of the test period. The specimens were removed after 4 hours and processed immediately. The process was repeated one week later but differed in that the specimens were kept in situ for 24 hours instead of 4 hours.

Evaluation of biofilms by confocal laser scanning microscopy

The specimens were carefully rinsed with sterilized phosphate-buffered saline (PBS) to dislodge loosely adherent bacteria. The LIVE/DEAD BacLight Bacterial Viability Kit L-7012 (Molecular Probes, Eugene, OR, USA) was applied directly to each specimen surface according to the manufacturer’s instructions. The submerged specimens were incubated in the dark for 15 minutes at room temperature to allow for stain development during image scanning. After gentle rinsing with sterilized PBS, they were observed under confocal laser scanning microscopy (CLSM) (Leica Microsystems, Heidelberg, Germany). The kit stained viable bacteria green while those with damaged membranes stained red. The optical section of biofilms was obtained at three representative sites for each specimen. Leica confocal software LCSLite (Leica Microsystems, Heidelberg, Germany) was used to evaluate the fluorescence intensity (FI) within the biofilms. The average FI of each image at the green (FIg) or red channel (FIr) was expected to be directly proportionate to the number of bacteria with integral or compromised membranes, respectively. The percentage of vital bacteria was calculated by FIg/(FIg + FIr).

Evaluation of biofilms by scanning electron microscopy

Biofilms were generated as mentioned above. After placement for 4 hours or 24 hours, the specimens were removed and washed with sterilized PBS. The samples were fixed in 2.5% glutaraldehyde in a 0.1 mol/L cacodylate buffer at pH 7.2 for 4 hours at room temperature. Specimens were then dehydrated in ascending ethanol series and dried with hexamethyldisilazane (HMDS). Finally, they were mounted on the microscope stubs and coated with gold. Spatial distribution and architecture of biofilm were observed with scanning electron microscopy (SEM) (Sirion 200, FEI, Netherlands).

Evaluation of pH value of the biofilms by Plaque Indicator Kit

The pH values of the dental plaque were measured with a Plaque Indicator Kit (GC Corporation, Chicago, IL, USA), in accordance with the manufacturer’s instructions, at 4 hours and 24 hours, respectively. The disposable plaque collection instrument was used to harvest plaque from the experimental materials. The instrument with attached plaque was then dipped into the Plaque Indicator solution for 1 second. The instrument was then placed in the groove of the dispensing dish and the samples left to ferment for 5 minutes. The pH values were measured by comparing the colour of those on the chart with those on the dispensing dish.

Statistical analysis

Statistical analysis was performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Homogeneity of variance was assessed by using Levene’s test. Two-way ANOVA repeated measures were applied to evaluate the CLSM and pH value results, considering different materials as main effect and time as the repeated measure. Post hoc multiple comparisons were used to isolate and compare the significant results, using least significant difference or Tamhane’s test at a 5% significance level.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial vitality of the biofilms

Results of the LCSLite analysis are presented in Table 3. Green fluorescence intensity ratio FIg/(FIg + FIr) analysis showed that the intensity on CHX-incorporated materials was significantly lower than that of non-CHX-incorporated materials after 4 hours and 24 hours (p < 0.05). The live bacteria ratio of the biofilms on the CHXRMGIC material was significantly lower than that on the CHXGIC material (p < 0.05). CLSM images are shown in Fig. 2. The 4-hour images and 24-hour images both showed that live bacteria were reduced dramatically after the addition of CHX to both RMGIC and GIC.

Table 3.   The percentage of vital bacteria [FIg/(FIg + FIr) ratio] (%) of the biofilm on the materials studied for 4 and 24 hours (mean ± SD)
 GICCHXGICRMGICCHXRMGIC
  1. Values with same superscript letters are not significantly different at p < 0.05 level.

4 h42.91 ± 9.24a24.30 ± 3.80b42.83 ± 9.72a13.81 ± 4.31c
24 h45.27 ± 15.83a24.21 ± 9.12b53.06 ± 16.68a14.95 ± 2.32c
image

Figure 2.  Representative CLSM images of the biofilms formed on the different material surfaces. After 4 hours, bacteria began to adhere to the surface of the materials. The live bacteria on the non-CHX-incorporated materials (a and c) were more than those on the CHX-incorporated materials (b and d). After 24 hours, a mature biofilm was generated on the surface of materials; the green fluorescence of the biofilm on the non-CHX incorporated materials (e and g) was higher than that on the CHX-incorporated materials (f and h). Bar scale = 150 μm.

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Bacterial morphology and accumulation of the biofilms

Representative SEM images of the biofilms are shown in Fig. 3. After 4 hours, a few bacteria were generated on the surface of non-CHX-incorporated materials (Figs. 3a and 3c), while bacteria were scattered sporadically and the profile was not regular on the CHX-incorporated materials (Figs. 3b and 3d). After 24 hours, a mature biofilm was generated on the surface of non-CHX-incorporated materials with a great quantity of extracellular matrix (Figs. 3e and 3g). However, bacterial morphology was irregular and extracellular matrix was absent on the CHX-incorporated materials (Figs. 3f and 3h).

image

Figure 3.  Representative SEM images of the biofilm formed on different material surfaces. After 4 hours, a few bacteria were generated on the surface of conventional materials (a and c); while bacteria were scattered sporadically and their profile were not regular on the CHX-incorporated materials (b and d). After 24 hours, a mature biofilm was generated on the surface of non-CHX incorporated materials, with a great quantity of extracellular matrix (e and g). However, most of the bacteria were deformed on the CHX-incorporated materials (f and h). Original magnification ×5000, bar scale = 5 μm.

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pH value

No statistically significant difference was observed between the pH values of the biofilms on the non-CHX-incorporated and CHX-incorporated materials after 4 hours and 24 hours (p > 0.05) (Table 4).

Table 4.   pH values of the biofilm on the materials studied for 4 and 24 hours (mean ± SD)
 GICCHXGICRMGICCHXRMGIC
  1. No statistically significant difference was observed.

4 h6.75 ± 0.266.75 ± 0.266.75 ± 0.266.75 ± 0.26
24 h6.25 ± 0.266.25 ± 0.266.25 ± 0.266.25 ± 0.26

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There are several ways to evaluate the antibacterial effects of plaque flora, such as the culturing of plaque and measuring the vitality of plaque smears with fluorescence techniques.13,14 The method used in this experiment is one of the models widely accepted for biofilm study in vivo. CLSM, used in the present study, is a powerful tool for investigating oral biofilm in situ.15 It was the first time that the non-destructive visualization of dental biofilm was combined with an assessment of the antibacterial effect of GICs and RMGICs incorporated with CHX in vivo by CLSM.

Dental biofilm, being an important factor in the occurrence of dental caries and periodontal diseases, comprises complex three-dimensional structures consisting of diverse communities of microbial multispecies formed on oral tissue.16 Bacterial adhesion to, and subsequent colonization of, the surfaces of teeth and restorative materials are the first steps toward the formation of dental biofilm.17 In this study, the antibacterial effect was assessed through the bacterial vitality on the dental biofilm. The 4-hour and 24-hour time measurements were chosen because the acquired pellicle is formed after about 4 hours on a thoroughly cleansed tooth surface and oral bacteria begin to adhere to the acquired pellicle. Then co-adhesion of later colonizers to already attached early colonizers takes place. The attached micro-organisms multiply and the oral biofilm is matured about 24 hours later.16

The CLSM result showed that the live bacteria ratio of the biofilms on the experimental materials was lower than that on the control materials. This provides evidence that the antibacterial activities of the CHX-incorporated materials were higher than those of the non-CHX-incorporated materials. Therefore, the first null hypothesis was not confirmed. The pH values exhibited no significant differences among the biofilms on tested materials. This indicates that the acid-producing ability of the biofilms did not change.

CHX is well recognized for its antimicrobial activity against some gram-positive bacterial species found in the oral cavity.18 Application of the CHX/Thymol varnish clearly delays bacterial colonization and biofilm formation.19 In the present study, the CLSM and SEM images showed that the bacterial vitality on the surface of CHX-incorporated materials was lower than those on the non-CHX-incorporated materials at 4 hours, and the morphology of most bacteria on the surface of material containing CHX had changed accordingly. It is speculated that the CHX released from the material can persist in the mouth by binding to the pellicle and saliva, creating a bacteriostatic milieu which delays bacterial colonization and biofilm formation.20 After 24 hours, the live bacteria ratio of the biofilm on the CHX-incorporated materials was much lower than that on the conventional materials. Takahashi et al.7 suggested that GICs containing CHX were effective in inhibiting bacteria associated with caries in vitro. Our findings coincide with the findings of these previous investigations.6,7 Evidence showed that an increase in the plaque vitality over time is due to dead, rather than vital cells, attaching preferentially to solid surfaces, and that saliva has a pronounced antimicrobial effect on early plaque.21 However, in our study, only an increased tendency was noticed and the difference between the plaque vitality of 4 hours and 24 hours was not significant. We assume that the antibacterial effect of CHX had influenced the colonization of bacterially formed biofilm.

CHX may interact with fluoride and interfere with its antibacterial effect. A higher concentration of CHX may increase the release of fluoride and a lower concentration of CHX may decrease the release of fluoride. Studies have shown that CHX could facilitate the release of fluoride when its content is 11.5 wt% or greater in the resin. The increase in release may be attributed to the physical presence of CHX, which might soften the resin matrix pathway for the efficient release.22 Hoszek and Ericson9 suggested that the addition of 10 wt% CHX to GICs decreases the release of fluoride. This might be due to the interaction between fluoride and the cationic CHX molecule, resulting in the precipitation of salts with lower solubility and consequently, low fluoride concentration.

Our study showed no significant difference between the bacterial activity of the biofilm on the GICs and the RMGICs. However, when they were incorporated with CHX, the live bacteria ratio of the biofilms on the RMGICs was significantly lower than that on GICs incorporated with CHX. Therefore, the second null hypothesis was rejected. This is not consistent with results from previous studies which suggested that resin more easily accumulated bacteria.23 It is speculated that CHX reacted with some component in the RMGICs which influences the antibacterial activity of the material. More research is needed for further evaluation.

Higher cariogenic bacteria such as Streptococcus mutans in biofilms would induce a lower pH value, which consequently would promote a higher enamel mineral loss.24 The pH value of the biofilm on the materials used in the experiment should increase if the antimicrobial activities improve. However, in the present study the pH values showed no significant differences. This might have been due to the following reasons. Firstly, the sample size was too small. The pH value change might have been demonstrated if the sample size had been large enough. Secondly, several healthy non-carious volunteers were selected as the subjects; their plaque pH values were not low per se. Thirdly, after 4 hours and 24 hours the amount of dental plaque on the materials was too little to cause significant pH value changes. Further studies of longer duration are required to determine whether the acid production of biofilm is influenced after the application of materials incorporated with CHX.

The ability of the restorative dental material to withstand functional forces is an important requirement for their long-term clinical performance. To be clinically acceptable, modified materials must provide superior antimicrobial activity without compromising the mechanical properties.6,7 However, previous findings revealed that incorporating antimicrobials to GICs reduces the physical-mechanical performance of GICs.7 This could be because of the high elution rates of the CHX from the GICs or the synergistic interactions between the CHX and the GICs. Some of the studies showed that the addition of CHX did not seriously deteriorate the surface hardness of RMGICs.25 These different findings may be due to the different experimental designs. A related study conducted by our research group showed that after 2% CHX incorporation, the microhardness of the GICs and RMGICs presented no significant differences (data not shown) and this is why we chose this CHX concentration.

Within the limitations of this study, it can be concluded that the incorporation of CHX into GICs and RMGICs has the ability to lower the bacterial load on dental biofilm in vivo after 4 hours and 24 hours. This warrants further studies to examine the long-term antibacterial effect. Furthermore, the mechanical properties of GICs and RMGICs incorporated with CHX should be studied thoroughly.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank Dr Pan Boqun from the Medical Experimental Centre, Wuhan University for assisting with the CLSM examination. This work was supported by the National Nature Science Foundation of China (81070852) and the Open Research Fund Program of Hubei-MOST KLOS and KLOBME (200903).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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