|Year : 2017 | Volume
| Issue : 1 | Page : 28-33
Antibacterial effect and physical properties of chitosan and chlorhexidine-cetrimide-modified glass ionomer cements
Apurva Mishra1, Ramesh Kumar Pandey1, Natesan Manickam2
1 Department of Paediatric and Preventive Dentistry, Faculty of Dental Sciences, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Environmental Biotechnology Division, n Institute of Toxicology Research, Lucknow, Uttar Pradesh, India
|Date of Web Publication||31-Jan-2017|
Ramesh Kumar Pandey
Department of Paediatric and Preventive Dentistry, Faculty of Dental Sciences, King George's Medical University, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims: To compare antibacterial effect and physical properties of chitosan (CH) modified glass ionomer cement (GIC) (10% v/v), chlorhexidine-cetrimide (CHX-CT) modified GIC (2.5/2.5% w/w) and conventional GIC. Materials and Methods: A total of fifty healthy children of age 7–12 years were selected and randomly assigned to class A and B for in vivo analysis. Slabs of CH modified GIC (Group II) along with slabs of conventional GIC (Group I, control) were cemented on buccal surfaces of maxillary molars (split-mouth technique) for class A children. Similarly, slabs of CHX-CT modified GIC (Group III) were cemented against control (Group I, control) in class B children. Slabs were assessed after 48 h for microbial load of Streptococcus mutans and Lactobacillus (LB) on mitis salivarius-bacitracin and Man Rogosa Sharpe agar media, respectively. Agar diffusion test was done to access the antibacterial effect of each group against Streptococcus muatns and LB. Slabs and cylinders of GICs were made for in vitro evaluation of compressive and flexure strength in each group. Results: Comparison was done by nonparametric Kruskal–Wallis analysis followed by Dunn's multiple comparison test. Categorical groups were compared by Chi-square test. The increase in antibacterial activity (Group II > III > I) (P < 0.001) and marked increase in compressive and flexure strength (Group II > I > III) were observed. Conclusions: In the view of findings, it is concluded that CH modified GIC would be effective in inhibiting the bacteria associated with dental caries along with improved physical properties when compared with CHX-CT modified GIC and conventional GIC.
Keywords: Antibacterial agent, atraumatic restorative treatment, cetrimide, chitosan, chlorhexidine, glass ionomer cement
|How to cite this article:|
Mishra A, Pandey RK, Manickam N. Antibacterial effect and physical properties of chitosan and chlorhexidine-cetrimide-modified glass ionomer cements. J Indian Soc Pedod Prev Dent 2017;35:28-33
|How to cite this URL:|
Mishra A, Pandey RK, Manickam N. Antibacterial effect and physical properties of chitosan and chlorhexidine-cetrimide-modified glass ionomer cements. J Indian Soc Pedod Prev Dent [serial online] 2017 [cited 2017 Mar 23];35:28-33. Available from: http://www.jisppd.com/text.asp?2017/35/1/28/199224
| Introduction|| |
Atraumatic restorative treatment (ART) is an approach introduced to provide dental care to less-affluent populations, making caries management possible not only in clinics but also in remote areas. However, its success depends on judicious application of the technique, respecting the physicochemical properties of the glass ionomer cement (GIC) along with the adequate removal of the carious tissue. Studies ,,, have shown that cavities treated by ART might have residual infected dentine as hand excavation of dentine would be less effective when compared with rotary burs and if a GIC was unable to arrest the carious process, restoration could fail with the elapse in time.
Several studies have been undertaken, which constitute new ideas for enhancing the antibacterial effect of GICs without compromising their basic physicomechanical characteristics.,, Chitosan (CH) is a linear bio-polyaminosaccharide derived from alkaline deacetylation of chitin, having properties such as biocompatibility, biodegradability, and mucoadhesion. Studies have shown that the flexural strength of commercial GICs could be improved considerably by the addition of a 10% v/v of CH along with an increase in fluoride release and antibacterial properties., Chlorhexidine (CHX) is a widely used antibacterial agent and has been used to enhance the antibacterial properties of GICs.,, In addition to this, other cationic disinfectants such as cetrimide (CT) have been incorporated into the GICs. CT is a quaternary ammonium salt containing cetrimonium bromide. It is a cationic surfactant and is known to prevent colonization of bacteria in the biofilm. Moreover, studies ,, have demonstrated that CHX-GIC combinations have shown increased susceptibility in reducing mutans streptococci (MS), whereas CT-GIC combinations have exerted increased susceptibility in Lactobacillus (LB). The addition of CHX diacetate/CT at 2.5% w/w concentration each to the conventional GIC have shown an antibacterial effect against the MS and LB bacteria.
Hence, the objective of the present study was to determine and compare the antibacterial effect (in vivo), compressive and flexure strength (in vitro) of CH modified GIC (10% v/v), CHX-CT modified GIC (2.5/2.5% w/w) and conventional GIC. The null hypothesis, regarding the modified GICs, stated:
- The addition of 10% v/v of CH into GIC does not improve antibacterial effect and physical properties of GIC when compared with CHX-CT modified GIC (2.5/2.5% w/w)
- CHX-CT mixture incorporation into GIC would have no significant effect on its antimicrobial and physical properties in comparison with CH modified and conventional GIC.
| Materials and Methods|| |
GIC, Ketac – Molar purchased from 3M ESPE, ST Paul, MN, USA was used in the present study [Table 1]. The low molecular weight CH purchased from Sigma-Aldrich, USA was dissolved in acetic acid, to prepare a solution of 0.1 mol/L. Aliquots of CH solution were added to the commercial liquid of GIC to prepare a liquid with CH concentration of 0.2 g/l (10% v/v). CHX diacetate powder and CT powder were purchased from Hi-Media Laboratories Pvt. Ltd., India. 2.5% w/w each of CHX and CT powders was added into GIC powder.
The present clinical trial was approved by the Institutional Ethical Committee (reference no. ECM II-B/P19). The sample size was calculated 16 for each group for biofilm collection assuming 80% power, 5% significance level with 95% confidence interval and 1 standard deviation as in a study conducted by Tüzüner et al.
Inclusion criteria for the present study were: Good health, plaque free dentition, no antibiotic history for the last 6 months preceding the commencement of the present study, and absence of any intraoral soft tissue or periodontal inflammation. Exclusion criteria were: Subjects with a history of any chronic systemic diseases, poor oral hygiene, multiple carious teeth, and child/parents unwilling to participate in this study.
The fifty healthy controls ranging from 7 to 12 years of age group reported to the Department of Paediatric and Preventive Dentistry were enrolled in the present study to evaluate the antibacterial effect of conventional and modified GICs. They were divided into class A and B (25 subjects for each category) using computer generated block randomization; allocation concealment was done in sealed opaque envelopes with allocation ratio 1:1. Envelopes were sequentially opened after writing the details of the subject. As per study design, double blinding was done for intervener and subject. Informed consents were obtained from parents/guardians accompanying the child patients.
Collection of biofilms
The study samples of GICs used were classified as:
- Group I – Conventional GIC (control group)
- Group II – CH modified GIC (experimental group)
- Group III – CHX-CT modified GIC (experimental group).
The control and experimental GIC slabs of 10 mm × 5 mm × 2 mm dimension were made using steel mold. After 24 h, the slabs were polished, sterilized under ultraviolet (UV) light and stored in sterilized glass bottles until ready for use.
Both control and experimental GIC slabs were cemented on buccal surfaces of permanent maxillary first molars using flip coin simple randomization (split-mouth design). Zinc polycarboxylate cement (Poly-F, Dentsply, Germany) was used for cementation of the slabs. Thus, 25 subjects have slabs of Group I as control against Group II (class A). Similarly, another 25 subjects have slabs of Group I as control against Group III (class B). Group I comprises of control for all the fifty patients. During the present study, subjects were instructed to maintain their normal dietary habits. Children were instructed to restrain from cleaning procedures (tooth brushing) over the cemented slabs or any chemical plaque control agents (mouthwashes/mouth rinses) during the test period of 48 h. The slabs were then removed and were evaluated for microbial load.
Evaluation of biofilms
The slabs were carefully rinsed and stored in sterilized phosphate buffer saline. Serial dilution of 103, 104, 105 of the suspensions was prepared and agar plating was done for both the experimental and control groups for the evaluation of Streptococcus mutans (SM) and LB on mitis salivarius-bacitracin (MSB) agar and Man Rogosa Sharpe agar, respectively. The agar plates were incubated for 48 h at 37°C in 5% CO2 atmosphere. The presence of SM on MSB agar plate was further confirmed by biochemical test such as sorbitol fermentation and catalase test. Similarly, the presence of LB was confirmed by catalase test and Gram-staining. The colonies were counted using an Andaman colony counter and were expressed as colony forming units per milliliter of the original suspension.
Agar diffusion test
The antibacterial effects of the set specimens against SM and Lactobacillus casei were assessed in vitro by agar diffusion test. Five specimens were prepared for each of the three groups. P/L ratio 3.6:1 of each GIC was dispensed on the mixing pad and mixed for 30 s with sterile plastic spatula and placed into steel mold of dimension 10 mm diameter and 2 mm thickness. The material was allowed to set for 30 min at room temperature and then removed. All specimens were then sterilized with UV before the subsequent procedure. Each strain from the stock culture stored in 50% glycerol at −20° was cultivated in brain heart infusion (BHI) broth (Hi-Media Laboratories Pvt. Ltd.) at 37°C, and a loopful of inoculum was transferred to 10 ml of BHI broth after incubation for 48 h. Bacterial suspension of 300 µl was then spread on BHI plate and left for 30 min at room temperature. The set disc-shaped specimens were placed onto BHI plate, inoculated with bacterial strain and left at 37°C for 48 h. The zones of inhibition were measured in millimeters using a digital caliper at three different points. The sizes of the inhibition zones were calculated by subtracting the diameter of the specimen, 10 mm, from the average of the three measurements of the halo.
Evaluation of compressive strength
The compressive strength was measured using the universal testing machine (Instron series 3382, USA). Ten cylindrical specimens per group were prepared using a plastic mold with an inner diameter of 6 mm and height of 9 mm. The inner surface of each mold was coated with a thin layer of petroleum jelly. The GIC was mixed and loaded into the molds with the help of a sterile dental instrument. The molds were stored at room temperature for 24 h before testing. The diameter of each specimen was determined using digital Vernier calipers. The specimens were placed in between the plates of universal testing machine. A compressive load along the long axis was applied using crosshead speed of 1 mm/min. The maximum force required to fracture the specimen was recorded.
Evaluation of flexural strength
The specimens were made by inserting the GIC into plastic mold of 10 mm × 5 mm × 3 mm dimension to determine flexural strength. After 30 min, the specimens were removed, coated with silicone wax and stored in distilled water at 37°C for 20 h. Ten samples of each group were prepared and weighed. Three-point bending tests were performed using Instron equipment at (25 ± 1°C) at a crosshead speed of 0.5 mm/min. The maximum force required to fracture the specimen was recorded.
The three independent groups were compared by nonparametric Kruskal–Wallis analysis of variance (ANOVA) followed by Dunn's multiple comparison test. Intraobserver reliability coefficient (r) was found to be very high, i.e., 0.89 and 0.91 for SM and LB respectively for in vivo study. Groups were also compared by repeated measures ANOVA followed by Tukey honestly significant difference test. Categorical groups were compared by Chi-square test. A two-sided (α = 2) P < 0.05 was considered statistically significant. All analyses were performed on STATISTICA software (Windows version 6.0, DELL software, Texas, USA).
| Results|| |
The mean colony count of both SM and LB were significantly lower in Group II followed by Groups III and I. Further, Mann–Whitney U-test revealed significantly higher (55.2%) SM colony count of Group III as compared to that of Group II (P < 0.001) and significantly lower (79.3%) LB colony count of Group II as compared to that of Group III (P < 0.001) [Table 2] and [Table 3]. The zone of inhibition against SM and LB of three groups are summarized in [Table 4]. The experimental groups showed significantly improved antibacterial action than individual control groups. Dunn's multiple comparison test revealed difference in compressive strength of both Group II (P > 0.05) and Group III (P > 0.05) as compared to Group I; though it was 4.3% higher in Group II and 27.2% lower in Group III as compared to Group I. However, the mean compressive strength of Group III was found significantly lower (30.4%) as compared to Group II (P < 0.05) [Table 5] and [Table 6]. Comparing the mean flexure strength among the groups, Dunn's multiple comparison test revealed similar flexure strength of both Group II (P > 0.05) and Group III (P > 0.05) as compared to Group I; resulting 3.8% higher in Group II and 12.8% lower in Group III as compared to Group I. Furthermore, the mean flexure strength of Group III was found significantly less (16.1%) as compared to that of Group II (P = 0.027) [Table 5] and [Table 6].
|Table 2: Bacterial colony counts (Mean ± SD) of Group II as compared to respective Group I|
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|Table 3: Bacterial colony counts (Mean ± SD) of Group III as compared to respective Group I|
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|Table 4: Agar diffusion test (Zone of inhibition) (Mean ± SD) of three groups|
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|Table 6: Comparison (P value) of mean physical properties between the three groups by Dunn's multiple comparison test|
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| Discussion|| |
GIC is the mainstream bioactive restorative material and is the first choice for the ART technique, as it adheres chemically to the tooth structure and releases fluoride, hence it not only contributes to the reduction in the amount of residual bacteria underneath the restoration, but also favors re-mineralization of the affected dentin. Studies have shown that GICs release approximately 10 ppm of fluoride during the first 48 h following insertion into the cavity, but this is still regarded low for achieving the desired antibacterial effects. Furthermore, LB underneath the restoration has been found to be the most resistant oral microorganism to the inhibitory effects of GIC. To overcome this, many studies aiming to incorporate antibacterial agents to the GICs have been reported.,,,,, The incorporation of cationic disinfectants such as CHX or CT in various concentrations (1%–5%) has exhibited favorable antibacterial effects on certain microorganisms under in vitro conditions.,,
In the present study, antibacterial efficacy of modified GIC was evaluated against conventional GIC acted as control. The antibacterial efficacy in vivo was determined by estimating bacterial load on 2 days old dental biofilm. The rationale behind 48 h was that an undisturbed plaque up to 2 days would be more acidic as compared to immature plaque. The results revealed significantly marked antibacterial effect by CH-GIC followed by CHX-CT-GIC and conventional GIC against both SM and LB. Hence, null hypothesis was rejected. The observations depicted that degree of bacterial attachment or plaque accumulation was less on CH-GIC than on CHX-CT-GIC. Further, the agar diffusion test showed marked antibacterial effect by CH-GIC against both SM and LB when compared to CHX-CT-GIC and conventional GIC. The mechanism of anti-adherence activity of CH involves bacterial surface modifications, alterations in expression of bacterial surface ligands, and CH adsorption to host surfaces to change its hydroxyapatite ionic properties. Therefore, the significant antibacterial effect depicted by CH-GIC in the present study is in concurrence with previous studies., The addition of acidic solutions of CH in the polyacrylic acid liquid of GIC at v/v ratios of 5%–10% improved the antibacterial properties of conventional GIC against SM., In addition to this, incorporation of 10 v/v % CH in the glass ionomer restorations imparts a catalytic effect on the fluoride release. Fluoride ion, after their release from restoration, forms a weak acid HF, which is attributed to increase the antimicrobial properties of CH modified GIC than conventional GIC, since fluoride enters the cell as HF, it is then dissociated inside the cell. In addition, enhanced fluoride release is the reason for the increase in antibacterial effect as fluoride plays an important role in enolase inhibition in glycolysis. Petri et al. and Botelho  indicated that no combination of antibacterial agents appeared to be superior to any other, for cationic disinfectants against the MS and LB. They also advocated that it might be beneficial to use a combination of antibacterial agents that have a broader range of activity against an ecosystem of bacteria. These findings of previous studies ,,, were in agreement with the present studies as a CHX-CT combination in GIC showed a marked antibacterial effect against both SM and LB.
GIC, as a restorative material, should withstand functional forces for a long lasting clinical performance. Thus, any modification done in its composition to enhance its antibacterial effect should not compromise its physical properties. However, when an antibacterial agent is incorporated in the GICs, alterations have been observed in their physical properties, and it is accepted that the physical properties of GICs would be compromised with the addition of antibacterial agent.,, In the present study the mean compressive strength and mean flexure strength of CH-GIC was observed highest followed by GIC and CHX-CT-GIC. The difference could be explained by the fact that the network formed by CH and polyacrylic acid around the inorganic particles/fillers reduce the interfacial tension among glass ionomer particles, thereby improving its mechanical properties. However, statistically significant decrease in physical properties was found in CHX-CT-GIC as compared to CH-GIC. The decrease in the strength of CHX-CT-GIC might be attributed to the release of CHX from the restoration, compromising its physical properties. Another reason for the decrease in mechanical properties may be associated to CHX salts, which could hamper the reaction of polyacrylic acid as the amines present in the CHX molecule neutralize the polyacid during salt formation and consequently might interfere with the setting reaction of the GIC.,
Within the limitations of the present in vivo and in vitro study design, real-time longevity of GICs for restorative procedures could not be certainly determined because of certain factors such as saliva, pH changes, food, liquids, and masticatory functions in the oral environment. Since the oral cavity being a dynamic field where initiation and progression of dental caries becomes a long and continuous process with variations in microbial load in response to changes in oral environment. Thus, more clinical and longitudinal studies employing the use of CH incorporated in GIC should be undertaken to establish its use for ART procedures and also in dental practice.
| Conclusions|| |
The present study led to the conclusion that, the addition of 10% v/v of CH into GIC resulted in significant increase in antibacterial effect and physical properties when compared to CHX-CT GIC (2.5/2.5% w/w) combination and conventional GIC. Therefore, CH being biocompatible, a low-cost additive could easily be incorporated into traditional GICs, and the modified cement is being recommended as a restorative material of choice in high caries susceptibility patients and atraumatic restorative treatment procedures.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]