ABSTRACT
Objective:
To Evaluate The Antimicrobial Properties of Double Antibiotic Paste and Morinda citrifolia And Propolis Paste Used In Regenerative Endodontics.
Materials and Method:
Using intact human teeth, radicular dentin samples were prepared (dimensions 4×4×2 mm) by following a standard method. The samples were cut using a diamond saw at minimal speed using water as coolant and smoothed with abrasive papers. To expose the dentin tubules and eliminate the smear layer, they were treated with NaOCl (1.5%), distilled water, and EDTA (17%). A total of 30 dentin samples went through a three-week anaerobic infection with bacterial biofilms sourced from the root canal of an underdeveloped tooth showing pulp necrosis. Group 1 acted as the control, while Group 2 received treatment with DAP, and Group 3 was treated with a paste of Morinda citrifolia and Propolis. After a week, pastes were cleaned, and specimens were dipped in phosphate-buffered saline for a day. The biofilm was released from each specimen by sonicating and vortexing it for 30 seconds. It was then diluted, spiral-plated on blood agar, and cultured anaerobically for a day. An automated colony counter was utilized to measure colony-forming units (CFU/mL), and statistical analysis was conducted using the Wilcoxon rank-sum test.
Result:
The wash from dentin samples treated with DAP and MCP showed minimal CFU counts, indicating both pastes’ high efficiency against biofilm-forming microbes from the canals of an immature tooth exhibiting pulp necrosis.
Conclusion:
Dentin samples infused with DAP and MCP showed minimal CFU counts, indicating their high effectiveness against biofilm-forming microbes from the root canal of an underdeveloped tooth with pulp necrosis. Additional research is required to validate these findings.
KEYWORDS: Biofilms, double antibiotic paste, immature tooth, Morinda citrifolia
INTRODUCTION
Regenerative endodontics constitutes a biologically-centered methodology aimed at the restoration of compromised dental structures, including dentin, root, and pulp–dentin complex cells. When treating underdeveloped permanent teeth with necrotic pulp, this method is gradually applied.[1]
Metronidazole and ciprofloxacin amalgamation formed DAP, offers antibacterial efficacy comparable to triple antibiotic paste (TAP) while reducing discoloration in endodontic regeneration.[2]
Propolis, a resinous mixture comprising resin, wax, essential oils, pollen, and impurities, is rich in flavonoids, which confer its antimicrobial properties. Its use in dentistry stems from its antimicrobial, antioxidant, anti-inflammatory, and anticancer activities.[3]
Morinda citrifolia (noni), a significant folk medicine, contains antibacterial compounds like asperuloside and alizarin. Its juice exhibits broad therapeutic effects, including its use as an intracanal medicament, proving effective against various gram-positive and gram-negative bacteria causing oral infections.[4]
METHODOLOGY
To conduct this experimental study, approval was obtained from the Institutional Ethical Committee (Reg. No.- RKDF/DC/PG/2024/844). The investigation utilized extracted human maxillary premolars for all procedural steps. The samples (n = 30) were divided into the following groups: GROUP I (n = 10), serving as the Control Group; GROUP II (n = 10), designated as DAP; and GROUP III (n = 10), designated as MCP.
Preparation of dentin samples
Thirty extracted human maxillary premolars were sectioned to prepare radicular dentin samples of dimensions 4 × 4 × 2 mm3 using a low-speed diamond saw. These specimens were then polished using abrasive papers and consecutively irrigation was done with NaOCl (1.5%), distilled water, and EDTA (17%) to expose the dentinal tubules and eliminate the smear layer. Sterilization was achieved by autoclaving at 121°C for 40 minutes.
Preparation of DAP and MCP
To formulate a DAP solution (25 mg/mL), equal amounts of metronidazole (125 mg) and ciprofloxacin (125 mg) were mixed in sterile water (10 mL). To create a paste, 0.7 g of methylcellulose was added at ambient temperature, followed by centrifugation at 7000 rpm for 15 minutes to ensure a bubble-free paste. Similarly, to prepare the MCP paste, 200 mg of propolis powder and 10 mL of M. citrifolia juice were combined. After adding 0.7 g of room-temperature methylcellulose, the mixture was centrifuged for 15 minutes at 7000 rpm to produce a uniform, bubble-free paste.
Isolation and cultivation of bacterial strain
An underdeveloped tooth’s infected root canal yielded a clinical bacterial isolate, which was kept in regular saline. After being cultivated in brain heart infusion broth with 5 g/L of yeast extract added, the sample was incubated for 48 hours at 37°C and then stored at −80°C until it was needed.
Analysis of antibacterial properties of DAP and MCP
Ten microliters of an overnight biofilm culture from an immature tooth with pulpal necrosis was added to sterile dentin specimens that were arranged in a 96-well microtiter plate. 190 μL of freshly prepared BHI-YE was incorporated after inoculation, and the preparations endured incubation for 3 weeks. The infectious specimens were then segregated into ten research cohorts and treated with either DAP or MCP for a week at 37°C.
Post-treatment procedures and biofilm analysis
Post-treatment, the infected dentin specimens were incubated for an additional week at 37°C in an environment with 100% humidity. Sterile water at the quantity of 3 mL was utilized further to rinse the treated samples for one minute to remove residual agents. Biofilm turbidity assessment was done for each sample by placing the specimen in 2 mL of sterile water in sterile test tubes and vortexing for 30 seconds to disperse the biofilm. The resulting suspensions were subsequently diluted and inoculated onto blood agar plates, and the bacterial count (CFU/mL) was determined using a digital colony counter following 24 hours of anaerobic incubation.
RESULT
It was observed that the bacterial matter was formed on the blood culture plate due to inoculum wash from control sets. This is considered as too numerous to count. In the control group, the highest bacterial count was observed (mean 96.1±97.1 CFU/ml). Both DAP and MCP treatments demonstrated a comparable antibacterial effect, with no statistically significant differences between them (mean 4.40±5.20 CFU/ml; P > .05). The wash obtained from the dentin samples treated with DAP and MCP presented only few CFU Count the indication that both the pastes were highly efficient against the biofilm-forming microbes acquired from the root canal. The bar graph compares the effectiveness of the Control, DAP (Double Antibiotic Paste), and MCP (M. citrifolia and Propolis Paste) groups. The Control group shows a high baseline mean value of 96.10, indicating significant bacterial presence or infection. In contrast, both DAP (5.20) and MCP (4.40) groups exhibit substantial reductions, with MCP showing slightly greater effectiveness. Overall, both treatments demonstrate strong antimicrobial effects compared to the untreated Control group [Graph 1].
Graph 1.
Intergroup comparison of control, DAP, MCP
DISCUSSION
Commercial intracanal antibiotic formulations, such as Ledermix and Odontopaste, are accessible; nevertheless, the application of Ledermix can lead to crown discoloration, particularly in younger teeth, while Odontopaste fails to eradicate all root canal bacteria, thereby restricting their utilization. Throughout regenerative endodontics, various disinfection methods, including TAP and DAP at different concentrations and calcium hydroxide, have shown success in promoting root development.[5]
Given the importance of disinfection in regenerative endodontic outcomes, the objective of current research is to assess the outcome and predictability of using DAP and MCP as intracanal medicaments in regenerative endodontic treatment.
This study used DAP, an intracanal medicament consisting of metronidazole and ciprofloxacin. When formulated as nanoparticles, the drugs’ effectiveness is enhanced, allowing deeper diffusion into radicular dentin tubules for a stronger antimicrobial effect.[6]
A paste composed of M. citrifolia and propolis is employed as an intracanal medicament. Propolis, a resinous material gathered by bees from plants to safeguard their hive, when combined with M. citrifolia juice, which is efficacious against both gram-positive and negative bacteria - augments antimicrobial efficacy. Dentin samples treated with DAP and MCP exhibited minimal colony-forming units, suggesting these pastes exhibit substantial effectiveness against biofilm-forming microorganisms within the root canal of an underdeveloped tooth experiencing pulpal necrosis.[7]
CONCLUSION
The dentin samples treated with DAP and MCP exhibited a minimal colony-forming unit count, indicating that both pastes were highly effective against biofilm-forming microorganisms isolated from the root canals of immature teeth with pulp necrosis.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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