The Effect of 4% Lignocaine gel, 5% Amiloride HCl and 10% Chlorpromazine on E.faecalis.

INTRODUCTION: Thorough disinfection of the root canal system is essential for the success of root canal therapy. Enterococcus faecalis is the most frequently found species in persistent/secondary intracanal infection associated endodontic treatment failure. The aim of this study was to evaluate the disinfection of dentinal tubules using 10% Chlorpromazine, 4% Lignocaine gel, 5% Amiloride hydrochloride in comparison with 2% chlorhexidine gel.
MATERIALS AND METHODS: The antibacterial efficacy of the four medicaments against Enterococcus faecalis was assessed in vitro using extracted human first and second mandibular premolar teeth at the depths of 200 μm and 400 μm.
RESULTS: The overall percentage inhibition of bacterial growth was 100% with 2% chlorhexidine gel followed by 10% chlorpromazine (88.8%), 4% lignocaine gel (76.4%) and 5% amiloride hydrochloride (71.4%).
CONCLUSION: 2% chlorhexidine gel was most effective against E. faecalis followed by the newer non- antibiotic medicament 10% chlorpromazine when compared to the other medicaments tested.

INTRODUCTION

Microorganisms play a fundamental role in the pathogenesis and progression of pulp and periapical diseases. The primary aim of endodontic treatment is to remove as many bacteria as possible from the root canal system and then to create an environment in which any remaining organisms cannot survive.[1] Aerobic and facultative anaerobic microorganisms are usually minor constituents of primary infections and are found in higher frequency in endodontic flare-ups and in failed cases.[2] Enterococcus faecalis is a microorganism commonly detected in asymptomatic, persistent endodontic infections. Its prevalence in such infections ranges from 24% to 77%.[3] It has the capacity to endure prolonged periods of starvation, which increases the resistance of E.faecalis 1000-fold to 10,000 fold[4] and has collagen-binding protein (Ace), which help it bind to dentin.[5]

Mechanical instrumentation of the root canal reduce bacterial population but do not completely eliminate them. Microorganisms in the dentinal tubules may constitute a reservoir from which root canal and surrounding tissue infection and re-infection may occur.[1] Hence, the use of intra canal medicament helps in the elimination of bacteria that remain even after cleaning and shaping, thereby providing an environment conducive for periapical tissue repair.[6]

Calcium hydroxide is the most widely used intracanal medicament, requiring a disinfection period of seven days.[7] The high pH of calcium hydroxide formulations’, alters the biologic properties of bacterial lipopolysaccharides in the cell walls of gram-negative species and inactivates membrane transport mechanisms, resulting in bacterial cell toxicity.[8] However, certain strains of E. faecalis has been reported to be resistant to this effect as a result of its ability to penetrate the dentinal tubules and to maintain pH by proton pump activity.[3] The search for a better alternative has lead to the introduction of antimicrobial agents like chlorhexidine (CHX) and newer non-antibiotics like chlorpromazine, lignocaine and amiloride hydrochloride.

Non-antibiotics are a variety of compounds, which are employed in the management of pathological conditions of non-infectious etiology have also been shown to modify cell permeability and to exhibit broad spectrum antimicrobial activity in-vitro against bacteria and other microorganisms.[9]

Kristiansen et al, found that there is evidence that a few non-antibiotic compounds like lignocaine, amiloride, and chlorpromazine may play a useful role in the inhibition of E.faecalis.[9]

Hence, this study was undertaken to evaluate the disinfection of dentinal tubules contaminated with E. faecalis by using lignocaine gel (4%), amiloride HCl (5%) and chlorpromazine (10%) in comparison with 2% Chlorhexidine gel.

MATERIALS AND METHODS

The model proposed by Haapasalo and Orstavik[10] was modified for this study, 50 freshly extracted single rooted first and second mandibular premolar teeth were selected.

Preparation of blocks

A rotary diamond disk was used to decoronate the teeth 5 mm below the cementoenamel junction. The remaining root was then sectioned such that 6 mm of the middle third of the root was obtained. Cementum was removed from the root surface to standardize the external diameter to 4 mm. The internal diameter was standardized using gates glidden drill no 3. Organic and inorganic debris was removed by treating the blocks in an ultrasonic bath of 17% ethylenediamine tetraacetic acid (EDTA) for 5 minutes followed by 3 % sodium hypochlorite (Merck Limited, Mumbai, Maharashtra, India) for 5 minutes. The blocks were immersed in an ultrasonic bath of distilled water for 5 minutes to remove all traces of the chemicals used and sterilized in an autoclave at 121 °C. The blocks were subjected to a second cycle of sterilization, with the blocks immersed in 1 ml of tryptone soy (TS) broth (HiMedia, Mumbai, India) in individual micro centrifuge tubes. This allows better penetration of the broth into the dentinal tubules.[11]

Contamination of the blocks

E. faecalis was used as the test organism in this study. This gram -positive facultative anaerobic bacterium is the most common isolate found in endodontically failed cases. Isolated 24-hour colonies of pure culture of E. faecalis (ATCC 29212) grown on tryptone soy agar were suspended in 5 mL of TS broth and incubated for 4 hours at 37°C . The culture suspen-sion was adjusted to match the turbidity equivalent to 0.5 McFarland standard. Fifty micro liters of the inoculum was transferred to presteril-ized individual microcentrifuge tubes containing 1 mL of the TS broth and dentin block. The dentin blocks were transferred to fresh broth containing E.faecalis every second day. All the procedures were carried out under laminar flow (Clean Air, Mumbai, India). The purity of the culture was checked by sub culturing 5 ml of broth from the incubated dentin block in TS broth on tryptone soy agar plates (HiMedia). The dentin blocks were contaminated during a period of 21 days. Five blocks were picked randomly and assessed for the depth of penetration of E. faecalis by using light microscopy.

Antimicrobial assessment

After the incubation period, the blocks were irrigated with 5mL of sterile saline to remove the incubation broth. The dentin blocks (n= 50) were as-signed to the following groups, with 10 blocks in each group: group 1, saline (negative control); group 2, 2% chlorhexidine gel (positive control)(Kem Colour International, India); group 3, 4% lignocaine gel (Warren Laboratories, Abbott); group 4, 5% amiloride hydrochloride (Glaxo Smithkline, India); and group 5, 10% chlorpromazine (Sun Pharmaceutical Industries Limited, India).According to Fava and Saunders[12] the antibacterial activity of intracanal medicaments is enhanced by the vehicle used. Hence, appro-priate vehicles were chosen for the individual-medicament as described below.

Group l received no medicament. Polyethylene glycol was used as a vehicle in group 2 and 3. Methyl cellulose was used as a thickening agent in both groups. In groups 4 and 5, the powder was mixed with dimethyl sulfoxide (DMS) solution in ratio 1.5:1 (wt/vol.) to obtain a paste like consis-tency. This paste was placed in the canal with a plastic instrument and condensed with a hand plugger. The orifice of all the blocks after medica-tion were sealed with paraffin wax and incubated in an anaerobic environment at 37° C.

Antibacterial assessment was performed at the end of 1,3,5 days with 10 blocks from each group. The blocks were washed with 5mL of sterile saline combined with ultrasonics to remove the medicament. Dentin debris was harvested at the depths of 200μm and 400μm by using Gates Glidden drills nos. 4 and 5 (Mani Inc) and collected in 1 mL of sterile TS broth and incubated in an anaerobic environment at 37°C for 24 hours. After the incubation period, the content of each micro centrifuge tube was serially diluted, 100 μL of broth in 100 μL of normal saline for 5 times. Five microliters of this diluted sample was plated on TS agar plates and incubated for 24 hours. Colonies were counted, and readings were tabulated.

Statistical analysis

The data were statistically analyzed with Mann-Whitney test followed by Tukey multiple comparison means to check the difference in bacterial inhibition between groups (P<0.01).The paired t test was used to check for differences in growth at different time intervals within the groups and for differences at two depths (P<0.01)

RESULTS

The current study showed that all four medicaments studied exerted antibacterial activity. The light microscopy evaluation of five dentin blocks showed invasion of the bacteria within the dentinal tubules. Infection of dentin blocks was confirmed when debris samples harvested from the saline group (negative control) yielded positive growth. Table 1 showed the antibacterial activity, which was measured at the depths of 200 μm and 400μm. The inhibition of growth in all the groups was statistically significant in comparison with control group (saline). Group 2 (2% chlorhexidine gel) was most effective against E.faecalis. Intergroup comparison showed that inhibition in group 5 (10% chlorpromazine) was statistically significant compared with group 3 (4% lignocaine gel) and group 4 (5% amiloride hydrochloride). No statistical difference was seen between group 3 (4% lignocaine gel) and group 4(5% amiloride hydrochloride). There was no statistical difference in the data between 200μm and 400μm.

Table 1

Mean colony counts for Different Intracanal Medicaments at 1,3,5 days time interval

To summarize the results, maximum inhibition was produced with 2% chlorhexidine gel (100%), followed by 88.8% inhibition with 10% chlorpromazine, 76.4% and 71.4% inhibition with respect to lignocaine gel and amiloride hydrochloride. [Figure 1].

Figure 1

Percentage reduction in bacterial growth at 200 μm and 400 μm for different medicaments

DISCUSSION

This article reports on the disinfection potential of four intracanal medicaments against E.faecalis. The model proposed by Happasalo and Orstavik has been modified for this study.[10] Human permanent teeth were used instead of bovine teeth as suggested by Basrani et al.[13]

The canal lumens of bovine blocks were three times larger than those of human blocks, thus influencing the antimicrobial activity of certain medicaments.[13] In addition, studies with human dentin blocks would definitely be more suitable to simulate the clinical scenario.[2]

Chlorhexidine (CHX) has a wide antibacterial spectrum and is effective against gram positive and gram negative bacteria as well as yeasts and candida species.[14] In the present study 2% CHX gel provided 100% inhibition of E.faecalis at depths of 200μm and 400μm from day 1 to day 5.

Its efficacy is based on the interaction between the positive charge of the molecule and negatively charged phosphate groups on the bacterial cell wall. This increases the permeability of the cell wall which allows the chlorhexidine molecule to penetrate into the bacteria with intracellular toxic effects. In addition CHX has a unique feature in that dentine medicated with it acquires antimicrobial substantivity.[15] In an in vitro study using human teeth, Ercan et al, showed 2% CHX was the most effective agent against E. faecalis inside dentinal tubules.[16]

Chlorpromazine is an antipsychotic/antiemetic drug. In this study, Chlorpromazine produced 89.8% and 87.7% inhibition of E.faecalis at depths of 200 μm and 400μm. The possible reason might be due to fraying of the cell wall, and by specific inhibition of membrane bound enzymes in this synthesis and construction.[1718]

Lignocaine which is a local anesthetic showed 78.6% and 74.2% inhibition of E.faecalis at depths of 200 μm and 400μm. The effect of lignocaine might be attributed to the presence of antibacterial agent sodium metabisulfite.[19] The gel form was chosen because the viscosity of the gel keeps the active agent in contact with the root canal walls and dentinal tubules.

Amiloride hydrochloride is a potassium-sparing diuretic. Amiloride produced 72.7% and 70% inhibition of E.faecalis at depths of 200 μm and 400μm.The probable antimicrobial effect of amiloride hydrocholoride could be by the blockade of sodium permeability of the cell which plays a role in cell multiplication and survival.[20]

In addition, these compounds have been found to enhance the in-vitro activity of certain antibiotics against specific bacteria to make in-vitro antibiotic resistant bacteria susceptible to previously ineffective drugs. These non-antibiotics show no drug resistance and are not toxic unlike the conventional antibiotics.[18]

The results obtained is based on an invitro study and caution must be exercised when drawing conclusions to in vivo situations. There is a plausibility of negative interactions between endodontic disinfecting agents and the various compounds present in the root canal environment. This might have a vital role in deciding the clinical effectiveness of antibacterial agents.[21]

CONCLUSIONS

Within the limitations of the present study, 2% chlorhexidine was most effective against E. faecalis followed by the newer non- antibiotic medicament 10% chlorpromazine.