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. 2026 Mar 30;29(4):440–445. doi: 10.4103/JCDE.JCDE_87_26

Antimicrobial efficacy of chitosan nanoparticles as intracanal medicament against Enterococcus faecalis and Staphylococcus aureus: An in vitro study

Lalitha Sri Roja Nallamilli 1, Kantheti Sirisha 1,✉, Hema Prakash Kumari Pilli 1, Ravi Kumar Konagala 1, Anupreeta Anwarullah 1, Vedamani Chandolu 1
PMCID: PMC13086389  PMID: 42004792

Abstract

Background:

Persistence of microbes is the main cause for root canal failure. Nanoparticles such as chitosan can prevent reinfection as chemo-mechanical preparation cannot abolish all the microbes.

Aim:

To investigate the antimicrobial efficacy of chitosan nanoparticles when they were combined with calcium hydroxide (Ca[OH]2) and 2% chlorhexidine digluconate against Enterococcus faecalis and Staphylococcus aureus.

Materials and Methods:

One hundred and forty single-rooted teeth were collected and root canal treatment was done. E. faecalis and S. aureus (n = 70 each) were inoculated and incubated for 21 days at 37°C. The teeth were then divided into seven experimental groups (n = 10 each) negative control, positive control, Ca (OH) 2, chlorhexidine digluconate (2%), chitosan nanoparticles, Ca (OH) 2 plus chitosan nanoparticles, and chlorhexidine digluconate (2%) plus chitosan nanoparticles. The above-mentioned intracanal medicaments were then placed and incubated at 37°C for 7 days. Colony-forming units (CFUs) were estimated using the standard loop method. Scanning electron microscope was used to confirm the microbial penetration and distribution. Analysis of data was dealt with the one-way ANOVA (P < 0.05) and pairwise comparison was done by Tukey’s post hoc procedure.

Results:

Chitosan nanoparticles samples exhibited fewer CFUs of all the experimental groups and Ca (OH)2 exhibited highest number.

Conclusion:

All the four medicaments showed significant reduction in the bacterial count. Chitosan nanoparticles exhibited greatest antimicrobial efficacy against E. faecalis and S. aureus than other experimental groups.

Keywords: Calcium hydroxide, chitosan nanoparticles, chlorhexidine, colony-forming units, Enterococcus faecalis, intracanal medicament, Staphylococcus aureus

INTRODUCTION

Persistence of microbes within the root canals is the most common cause for failure of root canal treatment.[1] Because of the complex nature of root canal anatomy, cleaning and shaping may not abolish all the microbes in all cases. Hence, interappointment medicaments play an important role in microbial eradication.[2] In nonsurgical endodontic retreatment, intracanal medicaments can enhance root canal disinfection and provide a conducive environment for periapical tissue repair.[3] Apical periodontitis is linked to viruses, fungi, and archaea, but bacteria are the primary microorganisms.[4] The microbiota of failed root canals include both obligate and facultative anaerobes. Enterococcus faecalis, a Gram-negative bacteria and Staphylococcus aureus a Gram-positive bacteria are the predominant organisms found in the root canals of refractory lesions.[5]

Interappointment bacterial growth can be decreased by using an appropriate dressing of intracanal medicaments which have good antimicrobial efficacy. Many intracanal medicaments such as calcium hydroxide (Ca[OH] 2), formocresol, and chlorhexidine have been used in the past for root canal disinfection.[6] Recently, nanoparticles of chitosan have gained considerable attention as antimicrobial and antifungal agents due to their biocompatibility. Thus, the objective of the present study is to determine the antimicrobial effectiveness of chitosan nanoparticles when used alone and when added to Ca (OH) 2/chlorhexidine.

MATERIALS AND METHODS

Periodontally compromised, recently extracted anatomically matched 140 noncarious, human single-rooted teeth with single canal were selected for this study. The teeth were examined for cracks and fracture lines with the help of optical loupes (2.5×). Radiographs were taken to confirm the presence of single canal. Teeth with root canal calcifications were excluded from the study. All the teeth were made to equal length by trimming at the occlusal surfaces. Based on the data from previous in vitro studies, the sample size calculation was done using the G*Power software version 20.0 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). The study design power was set at 80% with type I error of 5%. Minimal sample size for each group is eight specimens, with a statistically significance level of 0.05.[1]

Access cavity preparation

Access cavity preparation was done with #4 round bur (Mani Inc., Tochigi, Japan). The internal diameter of the canal was prepared up to the file size X4 using Pro-Taper Next (Dentsply; Mallifer; Switzerland) in all the teeth. During instrumentation, teeth were debrided with 5 mL of 3% sodium hypochlorite (NaOCl) for 2 min and then 9% Etidronic acid (MAARC, New Delhi) (Twin Kleen) to remove the smear layer. The tooth’s outer surface was painted with nail varnish. All the specimens were autoclaved (Confident, India) at a temperature of 121°C and 15 psi for 40 min, i.e. for two cycles for sterilization. Later, the teeth were mounted in Eppendorf tubes (Sigma Aldrich; United States) [Figure 1b] containing brain–heart infusion (BHI) broth (Sigma Aldrich; United States).

Figure 1.

Figure 1

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(a) Comparision of % reduction of colony-forming unit (CFU) counts in Enterococcus faecalis and Staphylococcus aureus, (b) Teeth submerged in Eppendrof tubes for bacterial cultivation, (c and d) Box and Whisker’s plot – mean value of CFU count in E. faecalis and S. aureus, respectively, (e) Mechanical magnetic stirrer

Microbial inoculation into root canals

Standard suspensions of E. faecalis (ATCC 29212) and S. aureus (ATCC 25923) were taken from the American Type Culture Collection. The bacteria were grown and maintained for a period of 24-h at 37°C in BHI Broth (Sigma Aldrich; United states). The sterilized tooth specimens were inoculated with bacterial culture grown in BHI broth. A standardized suspension of 10 µL (0.5 McFarland) was introduced into the canals using sterile micropipette. The access cavities were sealed with parafilm over a sterile cotton pellet and they were cultured at 37°C for 21 days. Fresh inoculum was replenished every 48–72 h to ensure sustained bacterial colonization.[6]

Grouping of the specimens

Following the inoculation period of 21 days, of 140 tooth specimens, 70 samples were inoculated with E. faecalis (n = 70) and 70 samples with S. aureus (n = 70). Subsequently, the specimens in each microbial group were subdivided randomly into seven experimental groups based on intracanal medicaments.

  • Group I (n = 10): negative control (uninfected and unmedicated root canals)

  • Group II (n = 10): positive control (infected with microbes and unmedicated root canals)

  • Group III (n = 10): Ca (OH) 2

  • Group IV (n = 10): Chlorhexidine digluconate (2%)

  • Group V (n = 10): Chitosan nanoparticle gel (Nano research lab, Jharkhand)

  • Group VI (n = 10): Ca (OH) 2 plus chitosan nanoparticle gel

  • Group VII (n = 10): Chlorhexidine digluconate (2%) plus chitosan nanoparticle gel.

Chitosan nanoparticles intracanal medicament preparation

In Group 5, 2% chitosan nanoparticle gel was made by dissolving 2 g of chitosan nanoparticles powder (Nano research lab, Jharkhand) in 100 mL of a solution-containing 2% (v/v) glacial acetic acid and 0.3 g of methylcellulose under mechanical agitation using magnetic stirrer (Coral Labtech, Vapi; Gujarat) [Figure 1e] for 2 h at 430 rpm. The pH of the 2% chitosan gel was found to be 4.05 in pH/meter. The pH was adjusted to 6.0–7.0 by adding 0.1N NaOH. In Group 6, Ca (OH) 2 paste (1 g) (RCT pex, Waldent; New Delhi) was mixed with an equal amount of (1 g) 2% chitosan gel. In Group 7, 1 mL of 2% chlorhexidine digluconate (Glucochex, Cerkamed; Poland) was mechanically mixed with 1 mL of 2% chitosan gel in a 1:1 ratio to obtain a homogenous mixture. This entire procedure was carried out according to the methodology by Nunes et al.[7]

Assessment of microbial colony-forming units

After incubation of bacteria for 21 days, the specimens were washed with saline and transferred to another Eppendorf tubes. The above-mentioned intracanal medicaments (n = 10 for each group) were inserted into root canals until they are completely filled. Teeth were kept in an incubator at 37°C for 7 days. After incubation for 7 days, the dentin debris was removed with Hedstrom files and placed in 0.5 ml of BHI broth. A serial five-fold dilutions of microbial suspensions were prepared using sterile saline. Tubes were vortexed for 15 s to ensure the homogenous mixture of microorganisms. 0.1 ml was taken from the dilution tubes and then it was plated onto blood agar for 24 h incubation and subsequent colony counting. Colony-forming unit (CFU) counting was performed using the standard loop method. The microbial concentration was calculated by multiplying the number of colonies on a plate by the inverse of the dilution factor. For example, if 10 colonies were counted on a 10 − 2 dilution plate, the total count is 10 × 10 − 2 CFU/mL. For example, if 10 colonies were counted on a 10 − 2 dilution plate, the total count is 10 × 10 − 2 CFU/mL [Figure 2].

Figure 2.

Figure 2

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Blood agar plates showing colony-forming units of Enterococcus faecalis and Staphylococcus aureus with different intracanal medicaments

graphic file with name JCDE-29-440-g002.jpg

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Scanning electron microscopic analysis

To study the microbial penetration and distribution into the root canals, evaluation by using scanning electron microscopic (SEM) was done. Teeth were dehydrated by soaking them in ethanol. After 20 min of dehydration, the specimen was immediately placed in the pressure chamber of a critical point drying machine. The specimens were then put on aluminum stubs using conductive tape. Sputtering was performed for 2 min with a 30 nm thick gold coating. Later, the specimens are examined at × 2000 magnification with a scanning electron microscope [Figure 3].

Figure 3.

Figure 3

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Scanning electron microscopic images of intracanal medicaments infused with Enterococcus faecalis at ×2000 magnification (a) negative control, (b) calcium hydroxide, (c) 2% chlorhexidine gel, (d) chitosan nanoparticle gel, (e) chitosan gel plus calcium hydroxide, (f) chitosan gel plus 2% chlorhexidine gel

Statistical analysis was applied to the obtained data deploying software (SPSS Statistics for Windows, version 22.0, IBM, Armonk, NY, USA). Intergroup comparison of number of CFUs/mL was calculated using an Independent t-test and intragroup comparisons were formulated using the one-way analysis of variance. Pairwise comparisons were done using Tukey’s multiple post hoc procedures. Entire data were analyzed with 95% confidence interval where P < 0.05 was contemplated significant.

RESULTS

A significant difference was seen among groups with log CFU counts (P < 0.001) [Tables 1 and 2]. Chitosan nanoparticles gel has shown lowest CFU count among all the tested groups of Ca (OH) 2, chlorhexidine, Ca (OH) 2 + chitosan and chlorhexidine + chitosan in both E. faecalis and S. aureus. Ca (OH) 2 was found to be ineffective against E. faecalis and (0.9186) S. aureus (P = 0.9998). Ca (OH) 2 was found to be ineffective against E. faecalis and (0.9186) S. aureus (P = 0.9998) [Figure 1a, c and d]. SEM images verified the presence of thick biofilm of residual E. faecalis and S. aureus bacteria on the canal dentinal wall. In both the micro-organisms categories, all the tested groups exhibited decrease in bacterial counts. The highest coverage of the canal walls with bacteria was seen in Ca (OH) 2 group followed by the positive control. Least coverage with microorganisms was exhibited by negative control and chitosan groups [Figure 3]. No significant difference was observed between positive control and Ca (OH) 2 group which suggests the low efficacy of Ca (OH) 2.

Table 1.

Comparison of colony-forming unit analysis of Enterococcus faecalis and Staphylococcus aureus by independent t-test

Intracanal medicament protocol Group I - Enterococcus faecalis
 Group II - Staphylococcus aureus
Mean SD Mean SD
Negative control 1.01 0.97 0.57 0.85
Positive control 4.62 0.10 4.73 0.04
Calcium hydroxide 4.23 0.19 4.59 0.07
Chlorhexidine 3.12 0.62 2.67 1.03
Chitosan nanoparticle gel 1.86 1.15 1.23 1.46
Calcium hydroxide + Chitosan 3.43 0.86 3.77 0.58
Chlorhexidine + Chitosan 3.26 0.69 3.11 0.16
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SD: Standard deviation

Table 2.

Comparision of colony-forming unit analysis of Enterococcus faecalis and Staphylococcus aureus by the one-way analysis of variance

Sources of variation Sum of squares df Mean square F P
Group I - Enterococcus faecalis
  Intragroup 86.5260 6 14.4210 25.8636 0.0038*
  Intergroup 31.2244 56 0.5576
Group II - staphylococcus aureus
  Intragroup 137.3553 6 22.8925 37.3798 0.0046*
  Intergroup 34.2961 56 0.6124
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*P<0.05 indicates significant difference. df: Degrees of freedom

DISCUSSION

Endodontic therapy may fail to entirely remove the bacteria, resulting in chronic apical periodontitis. Several factors contributed to recurrent apical periodontitis such as inadequate root canal debridement, incorrect access cavity design, the existence of missing root canals, insufficient root canal disinfection, and restorative leakage.[8] Studies have revealed diverse microorganisms with unique properties that allow them to evade chemo-mechanical debridement during endodontic therapy.[9] If microbial removal cannot be achieved physically, it should be accomplished chemically with the help of antimicrobial agents such as intracanal medicaments.[10] The microbes which are resistant to antimicrobial therapy are Campylobacter rectus, Prevotella spp. E. faecalis, Streptococci, Staphylococci, Lactobacilli, and Actinomyces spp. etc., E. faecalis is the most common species in chronic apical periodontitis. E. faecalis can also suppress lymphocytes, thereby causing endodontic failure. It can bind to dentin as it possesses gelatinase, serine protease, and collagen-binding protein (Ace). Its small size can invade and live in dentinal tubules enduring prolonged starvation periods. The starved microbial cells can recover after getting adequate nutrients.[11] Staphylococci species such as S. aureus and S. epidermidis are frequently identified as etiological agents in medical bacteriology. Hence, in this study, E. faecalis and S. aureus were chosen because of their role as possible causative microbial factors in persistent apical periodontitis. Ca (OH) 2 is a gold-standard intracanal medicament. The downside of Ca (OH) 2 is that it is ineffective against E. faecalis, which possesses buffering capacity and can maintain pH homeostasis.[12] Chlorhexidine is both an irrigant and an intracanal medicament. It is efficient against E. faecalis, fungi, and yeasts.[13]

Chitosan is deacetylated form of chitin. It is the second abundant natural biopolymer available which can be chemically modified.[14] It has excellent antimicrobial, antiviral, and antifungal characteristics. This efficacy of chitosan nanoparticles is because of the electrostatic interaction leading to the cell membrane disruption of the microbes. Chitosan nanoparticles were developed especially as an antibacterial agent. They have certain properties such as very small size (1–100 nm), greater surface area to mass ratio and greater reactivity with the chemicals.[15,16] A homogenous chitosan nanoparticles gel was obtained with the help of magnetic stirring. Nadar A et al. reported that gel form of chitosan has greater antibacterial efficacy compared to solution form.[15] According to Parolia et al. and Nunes et al., chitosan was prepared by using ionotropic gelation method. The gel form of chitosan can exhibit less toxicity and solubility to periapical tissues. Its viscosity will help the medicament in maintaining continuous contact with the canal walls as well as within the dentinal tubules.[1,7] In earlier studies done by Mannan et al.,[1,17] the specimens were decoronated to obtain a standardized length to obtain the greatest proportion of root canal wall instrumentation.

NaOCl cannot remove the smear layer alone. Earlier, many researchers used ethylenediaminetetraacetic acid (EDTA) as chelating agent, but EDTA has the disadvantage of reducing the dentin microhardness. Hence, this study used a weaker chelating agent, 9% etidronic acid (HEBP). This irrigating agent can remove smear layer like EDTA and can be used with NaOCl without losing its antibacterial properties.[18] ProTaper Next was chosen for mechanical debridement. Polineni S et al. found that ProTaper Next outperformed K3XF and Hyflex CM, in terms of microbial load reduction.[19] Ca (OH) 2 and chlorhexidine were mixed with chitosan in 1:1 ratio for a synergistic effect. This was according to Savitha et al.[20] and Kapadia et al.,[21] the 1:1 ratio of chlorhexidine gluconate gel and chitosan has greater decrease in the bacterial load. H files were employed to collect dentinal debris. Awawdeh et al. discovered that H files were effective to reach bacteria that have infiltrated the dentinal tubules.[22]

This study revealed that chitosan nanoparticle gel exhibited better antibacterial efficacy compared with other tested medicaments against both the organisms; E. faecalis and S. aureus. These findings were similar to the results reported by earlier researchers Savitha et al.[7,20] The superior antimicrobial properties of chitosan nanoparticles were due to electrostatic inhibition of microbial cell wall, disruption of the microbial DNA, inhibition of microbial enzymes, and by blocking the nutrient supply for the bacteria. By using chitosan, smear layer can be removed very effectively from the root canals. It can chelate the calcium ions from the smear layer, thereby reducing the inorganic content of the smear layer.[23] Ca (OH) 2 + chitosan nanoparticle gel exhibited better antibacterial activity compared with Ca (OH) 2 alone. The better activity of Ca (OH) 2 and chitosan was due to the aqueous nature of chitosan. The aqueous suspension permitted more efficient release of hydroxyl ions.[24]

In the present study, although chlorhexidine exhibited better efficacy in contrast to its combination with chitosan nanoparticles, it was not statistically significant. This was similar to an earlier study by Kapadia et al.[21] The results of the current study are in contrary to the results reported by Ballal et al. where the chlorhexidine and chitosan combination exhibited synergistic effect.[25] The difference in the results could be due to methodological differences. Although chitosan nanoparticles exhibited superior antibacterial effect, further animal and clinical studies should be done before using it as potential intracanal medicament. Further studies on multispecies biofilm and clinical trials should be performed to develop chitosan as potential intracanal medicament for future clinical applications.

CONCLUSION

Within the limitations of the current study, the five intracanal medicaments chitosan nanoparticles, Ca (OH) 2, chlorhexidine, chitosan + chlorhexidine, and chitosan + Ca (OH) 2 showed greater reduction in the bacterial count in the experimentally infected root canals. Of them, nanoparticles of chitosan had demonstrated the greatest antimicrobial efficacy against microbes E. faecalis and S. aureus, compared to all other groups. Chlorhexidine showed promising antibacterial activity next to chitosan against E. faecalis and S. aureus. The combination of chitosan nanoparticles + Ca (OH) 2 had the advantage of synergism than Ca (OH) 2 alone.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.

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