Comparative Evaluation of Antibacterial Property of Bioactive Glass Alone and its Combination with Chlorhexidine against Enterococcus faecalis - An In vitro Study.

CONTEXT: One of the most common organisms responsible for root canal failure even after the usage of intracanal medicament is Enterococcus faecalis. Newer medicaments with higher antibacterial efficacy against E. faecalis need to be analyzed. AIM: The aim of this study was to compare the antibacterial property of bioactive glass (BAG) alone and its combination with chlorhexidine (CHX) against E. faecalis.
MATERIALS AND METHODS: Eighty permanent maxillary single-rooted teeth were included in the study. These teeth were randomly divided into three groups of ten teeth each. Dentin blocks were prepared. After debris and smear layer removal, dentin blocks were inoculated with E. faecalis and incubated for 7 days for biofilm formation. Medications including BAG alone and combination of CHX with BAG were placed into the dentin blocks and incubated at 37°C for 72 h. Dentinal shavings were collected, and the mean bacterial count was recorded. STATISTICAL ANALYSIS: The comparison of colony-forming unit (CFU) counts was done using one-way analysis of variance, Tukey's post hoc test, and unpaired t-test. The analysis was done using the Statistical Package for the Social Sciences version 20.0 for Windows.
RESULTS: Based on the CFU count obtained after 72 h, compared to the control group, both Group 1 and Group 2 reduced the bacterial load effectively. In comparison with Group 1, Group 2 (combination) was found to be more effective against E. faecalis.
CONCLUSION: It can be concluded that although BAG was effective against E. faecalis, its efficacy increased significantly in combination with CHX. Copyright:

INTRODUCTION

Root canal treatment can be done in single or multiple visits. Placement of intracanal medicaments is an essential step in clinical conditions requiring multiple visits including weeping canals.

Although 4%–40% of primary endodontic infections exhibit the presence of Enterococcus faecalis, root canal treated teeth with secondary infection are nine times more likely to contain E. faecalis than primary endodontic infections. The microorganism commonly isolated from asymptomatic, persistent endodontic infections is E. faecalis, with a prevalence of 24%–77%.[1] Till date, none of the intracanal medicaments completely eradicated the E. faecalis biofilm.

Bioactive glass (BAG) is a four-component oxide system containing SiO2, Na2O, CaO, and P2O5. BAG induces dentin mineralization and may not negatively affect the mechanical properties of teeth when used as an intracanal dressing compared to calcium hydroxide.[2]

Chlorhexidine (CHX) gluconate has proven efficacy against E. faecalis. It has excellent biocompatibility and property of substantivity which may protect the canal against microbial colonization beyond the actual medication period. The combination of drugs was shown to exhibit synergistic action resulting in increased efficacy.

Thus, the aim of the study was to compare the antimicrobial property of BAG alone and its combination with CHX against E. faecalis.

MATERIALS AND METHODS

The present study was conducted in the Conservative Dentistry and Endodontics Department, Noorul Islam College of Dental Sciences, Trivandrum.

Materials

The medicaments used in the study were BAG (PerioGlas, NovaBone products LLC, Alachua, USA) and 2% CHX gel (PREVEST Dental Products).

Methods

Thirty permanent maxillary anterior, noncarious, completely developed, and single-, straight-rooted teeth extracted for therapeutic reasons were selected.

Preparation of dentin blocks

All the thirty specimens collected were disinfected. A rotary diamond disk was used to decoronate the teeth 5 mm below the cementoenamel junction, and the apical portion of the root was also removed to keep the length of dentin block as 10 mm. The cementum was removed from the root surface using an ISO 016 bur, and the root canals were enlarged to a standard diameter of 1.6 mm using an ISO 016 bur.

Organic and inorganic debris including smear layer were removed in an ultrasonic bath of 17% ethylenediaminetetraacetic acid for 10 min followed by sterilization in an autoclave.

Grouping

Based on the medicament to be used, dentin blocks were grouped into three groups of ten each:

Group 1: BAG

Group 2: BAG with CHX

Group 3: No medication.

Infecting the specimens

E. faecalis (ATCC29212) was initially procured from ATCC and maintained in brain–heart infusion media (HiMedia). The sterilized media containing sterilized teeth samples were inoculated with primary inoculum and incubated for 7 days for formation of matured biofilm.

Medicating the specimens

After the incubation of 7 days, the culture media were removed and medicaments were applied and the lumen was sealed on both sides with paraffin wax and kept it for incubation at 37°C for 3 days in a moistened environment. The untreated teeth remained as the control teeth.

Group 1 – Specimens were filled with freshly prepared mixture of BAG with sterile saline

Group 2 – Specimens were filled with mixture of BAG with 2% CHX gel. BAG with saline was mixed with equal amount of CHX in a ratio of 1:1 to form the mixture

Group 3 – Specimens were not filled with any medication.

Paraffin wax was removed from each side of specimens, and the dentinal shavings were collected by doing a circumferential filing along entire length of canal using a number 40 K-file. The samples were serially diluted three times and were swabbed on to nutrient agar plate. The plates were incubated at 37°C for 24 h in a microbiological incubator (KEMI). After incubation, the plates were observed for colony-forming units (CFUs) [Figures 1–3]. The CFUs were counted using a digital colony counter and were expressed in CFUs/ml.

Figure 1

Colony-forming units in Group 1 specimen

Figure 3

Colony-forming units in control group

Colony-forming units in Group 2 specimen

Statistical analysis

The CFU counts in each group were summarized and tabulated as mean ± standard deviation. The comparison of CFU counts between the various groups was analyzed using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test and unpaired t-test for pairwise comparison. P <0.05 was considered statistically significant. The analysis was done using IBM (International Business Machines Corporation) Statistical Package for Social Sciences version 20.0 for Windows.

RESULTS

The CFU count of all the three groups, BAG alone, BAG with CHX, and control, is shown in [Table 1]. Based on the ANOVA test, a statistically significant difference exists between bacterial counts in all the three groups. The results suggest that both Group 1 and Group 2 are effective against E. faecalis. Compared to Group 1, Group 2 showed more efficacy in reducing bacterial count and the difference is statistically highly significant.(P < 0.05) [Table 2].

Table 1

Overall comparison

Group	CFU count	t	P
Bioactive glass (4A)	4.32±0.26	16.008	0.001*
Bioactive glass + CHX (4B)	2.39±0.28

* Significant(P<.05 is significant) CFU: Colony-forming unit, CHX: Chlorhexidine

Table 2

Group comparison

Group	CFU count (mean±SD)	ANOVA
Bioactive glass (Group 1)	4.32±0.26	F=40.225 P<0.001
Bioactive glass + CHX (Group 2)	2.39±0.28
Control (Group 3)	10.67±3.65

CFU: Colony-forming unit, CHX: Chlorhexidine, SD: Standard deviation, ANOVA: Analysis of variance

DISCUSSION

E. faecalis is seen in 4%–40% of primary endodontic infections. However, the failed endodontic treatment cases are nine times more likely to contain E. faecalis than primary endodontic infections. In failed root canal treatment cases, culturing methods reveal an E. faecalis prevalence of 24%–70% while it is 67%–77% when a polymerase chain reaction detection method is used.[1]

Distinct features which enable it to become an exceptional survivor in the root canal include its ability to adapt to poor nutrient conditions, survive the action of commonly used medications, form biofilms even in medicated canals, invade the dentinal tubules, convert into a viable but noncultivable state, antibiotic resistance, survive in low pH, high salinity, and high temperatures, survive prolonged periods of starvation, and utilize tissue fluid from the periodontal ligament.[3]

BAG was introduced by Hench in 1969.[4] The antibacterial property is due to its potential to raise the pH in aqueous solution due to the release of cation which affects the survival of most microbes.[5] According to Goel et al.,[6] the high pH levels of BAG were not tolerated by the bacterial cells and results in its inhibition. Silica will act as a surfactant at solid–liquid interfaces and thus directly inhibits the bacterial viability.

CHX gluconate is an antimicrobial agent widely used both as an endodontic irrigant and medicament. At 2% concentration, CHX is bactericidal causing precipitation of cytoplasmic contents of bacterial cell. CHX has a wide range of action against both Gram-positive and Gram-negative bacteria. Other characteristics include antifungal action, substantivity of up to 12 weeks, less tissue solubility, delayed coronal leakage and biocompatibility. It does not affect the apical seal of root canal when placed as medication.[7] 2% CHX is considered to be effective against 1-day and 3-day biofilms.[8]

According to various literatures, synergistic action of medicaments has improved efficacy against microbes in many instances.

In the present study, BAG was combined with CHX and the antimicrobial activity when used alone and in combination was evaluated.

Based on the results, BAG was found to be effective against E. faecalis. Waltimo et al.[9] also reported a strong antimicrobial activity for Bioglass 45S5 against enterococci isolated from persistent canal infections. Zehnder et al.[10] in their study concluded that BAG S53P4 powder suspension was more effective than calcium hydroxide against E. faecalis.

On combining CHX with BAG, its antibacterial property increased significantly. Various studies by Kapadia et al.,[11] Delgado et al.,[12] Ercan et al.,[13] and Evans et al.[14] have supported the fact that when individual medicaments were combined with CHX, their antibacterial efficacy increased significantly when used against E. faecalis.

According to Prabhakar and Kumar,[2] BAG and its combinations were highly effective against E. faecalis in an in vitro model.

One of the reasons for increased action of BAG in combination with CHX might be due to its particle size. The antibacterial effect of BAGs is related to their particle size. The lesser effect of the BAG suspension can be explained by the lesser alkaline capacity of the glass suspension. Although correct mechanism for increased efficacy of BAG in combination with CHX is unknown, more dissolution of glass particles in combination with 2% CHX gel facilitate more antimicrobial action.[26]

CONCLUSION

Within the limitations of the study, it can be concluded that,

In an in vitro model, BAG was relatively effective against E. faecalis

Addition of CHX to the BAG increased the antibacterial property of BAG significantly against E. faecalis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.