Abstract
Context:
Persistent endodontic infections caused by Enterococcus faecalis and Candida albicans often resist standard intracanal medicaments. Chitosan nanoparticles (CNPs), with their antimicrobial and drug-delivery potential, may improve disinfection efficacy.
Aims:
To evaluate and compare the antimicrobial efficacy of calcium hydroxide and chlorhexidine intracanal medicaments, with and without chitosan nanoparticle incorporation, against E. faecalis and C. albicans at different time intervals.
Setting and Design:
In vitro microbiological study.
Materials and Methods:
An eighty in vitro agar well-diffusion model was used to assess the antimicrobial efficacy of four formulations – calcium hydroxide (Ca[OH]2), Ca (OH)2 + CNPs, 2% chlorhexidine (CHX), and CHX + CNPs against E. faecalis and C. albicans at 3- and 7-day intervals.
Statistical Analysis Used:
Data were statistically analyzed using the one-way analysis of variance and Tukey’s post hoc test (P < 0.05).
Results:
CNP incorporation significantly enhanced and prolonged antimicrobial activity. CHX + CNPs showed the largest inhibition zones (28.0 ± 1.9 mm vs. C. albicans, P = 0.042), while Ca (OH)2 + CNPs exhibited a time-dependent increase (0 → 23.0 ± 2.1 mm vs. E. faecalis, P = 0.003).
Conclusion:
CNPs synergistically potentiate conventional medicaments via deeper diffusion, sustained release, and intrinsic antimicrobial action.
Keywords: Calcium hydroxide, Candida albicans, chitosan nanoparticles, chlorhexidine, endodontics, Enterococcus faecalis, intracanal medicament
INTRODUCTION
The keystone of endodontic therapy is to expunge microbial colonization within the root canal system and prevent its recurrence. Although contemporary dentistry has witnessed substantial progress in instrument design, irrigant chemistry, and obturation science, complete disinfection of the canal system remains an enduring challenge.[1,2]
Among the various pathogens associated with persistent or secondary endodontic infections, Enterococcus faecalis and Candida albicans are the most clinically consequential species linked to treatment failures.[3]
E. faecalis, a Gram-positive, facultative anaerobe, is renowned for its exceptional environmental adaptability with the prevalence rate of 4%–77%. Similarly, C. albicans, a dimorphic opportunistic fungus, is increasingly recognized in refractory and posttreatment disease with the prevalence rate of 18%.[2,3] Their ability to tolerate high alkalinity, penetrate dentinal tubules, form resilient biofilms, and survive long-term nutrient scarcity allows them to persist even after meticulous chemomechanical preparation.[1,4]
Intracanal medicaments serve as chemical adjuncts to mechanical instrumentation, aiming to eliminate remaining microbes, neutralize endotoxins, and create favorable conditions for periapical repair. Conventionally, calcium hydroxide has been regarded as the “gold standard” owing to its high alkalinity (pH ≈ 12.5) and ability to produce hydroxyl ions that disrupt microbial membranes, denature proteins, and inhibit essential enzymatic pathways.[5]
However, numerous studies demonstrate that E. faecalis and C. albicans can endure highly alkaline environments, thereby diminishing the efficacy of Ca (OH)2.[6] This limitation has encouraged the use of chlorhexidine (CHX), a cationic bisbiguanide with broad-spectrum antibacterial and antifungal properties, notable substantivity, and prolonged dentin binding.[7] Yet, CHX lacks tissue-dissolving capacity and exhibits reduced residual activity once it diffuses out of the canal.[8] Furthermore, both Ca (OH)2 and CHX, when used alone, show limited effects on mature biofilms, which are inherently more resistant than planktonic microbial forms.[9]
Nanotechnology has recently opened new avenues in endodontics by enabling manipulation of materials at the nanoscale for enhanced biological interaction.[10] Chitosan, a naturally derived cationic polysaccharide produced by chitin deacetylation, has garnered considerable interest due to its inherent antimicrobial activity, excellent biocompatibility, biodegradability, muco-adhesion, and drug-delivery potential.[11]
When converted into nanoparticles, chitosan exhibits dramatically increased surface area, improved capacity to penetrate dentinal tubules and biofilms, and the ability to facilitate controlled and sustained release of therapeutic agents.[12]
Despite their promising potential, literature remains limited regarding direct comparisons of Ca (OH)2 and CHX used alone or in combination with chitosan nanoparticles against resistant endodontic species.
Therefore, the present in vitro investigation aims to evaluate and compare the antimicrobial efficacy of calcium hydroxide and chlorhexidine intracanal medicaments, with and without chitosan nanoparticle incorporation, against E. faecalis and C. albicans at different time intervals.
MATERIALS AND METHODS
Source of materials
The experimental medicament formulations calcium hydroxide and chlorhexidine incorporated with chitosan nanoparticles (CNPs) were sourced from Swakit Biotech Pvt. Ltd., Bangalore, India. Standard strains of E. faecalis (MTCC 29212) and C. albicans (MTCC 24433) were procured from the Microbial Type Culture Collection (MTCC), Chandigarh. These cultures were subsequently kept under the controlled laboratory conditions at Dextrose Technologies Pvt. Ltd., Bangalore, for use in the study. The study got an ethical clearance vide Reference number 248-IRB-2024 from the Institutional review board Dayananda Sagar College of Dental science on 24-04-2024.
Preparation of test samples
Formulation of 0.2% chitosan nanoparticle (CSN) solution
A 0.3 N acetic acid solution was freshly prepared by diluting 1.8 mL of glacial acetic acid in 100 mL of distilled water. Precisely, 20 mg of chitosan nanoparticle powder was weighed and dispersed into this medium. Continuous magnetic stirring yielded a uniform, translucent 0.2% CNP solution, which was stored in a sterile, airtight container at ambient temperature until further use [Figures 1-4].[13]
Figure 1.
Prime dental RC Calcium Hydroxide (control group 1)
Figure 4.
Chlorexidine + CNPs (Test material 2)
Formulation of CNP enhanced calcium hydroxide
A commercially available calcium hydroxide paste (Prime Dental RC Cal) was combined with the freshly prepared 0.2% CSN solution to achieve a final CNP concentration of 100 mg/mL within the paste. This formulation was designated as Ca (OH)2 + CSN (0.2%) [Figures 1 and 2].[13]
Figure 2.
Calcium Hydroxide + CNPs (Test material 1)
Formulation of CNP enhanced chlorhexidine
Similarly, a 2% chlorhexidine gel (Waldent Chlorhe × 2%) was blended with the 0.2% CSN solution to reach a final nanoparticle concentration of 100 mg/mL. This formulation was labelled CHX + CSN (0.2%) [Figures 3 and 4].[13]
Figure 3.
Waldent chlorhe × 2% chlorhexidine gel (control group 2)
The study incorporated two experimental and two control groups, as delineated below [Chart 1]:
Table 1.
Comparing the zones of inhibition for both the micro-organism in both the time interval for all four medicament (analysis of variance with post hoc Tukey test)
| Medicament (groups) | E. faecalis | C. albicans | ||||
|---|---|---|---|---|---|---|
|
|
|
|||||
| 3rd day (mean±SD) | 7th day (mean±SD) | P (<0.05) | 3rd day (mean±SD) | 7th day (mean±SD) | P (<0.05) | |
| Ca(OH)2 | 0.0±0.0 | 12.0±1.8 | 0.038 | 0.0±0.0 | 0.0±0.0 | 1.000 |
| Ca(OH)2 + CNPs | 0.0±0.0 | 23.0±2.1 | 0.003* | 15.0±1.2 | 17.0±1.5 | 0.047* |
| CHX | 14.0±1.5 | 15.0±1.3 | 0.705 | 25.0±2.0 | 25.0±1.8 | 0.500 |
| CHX + CNPs | 15.0±1.4 | 17.0±1.7 | 0.046* | 26.0±2.1 | 28.0±1.9 | 0.042* |
*Represents the values are statistically significant (P<0.05) and all the values are measured (mm). CHX: Chlorhexidine, Ca(OH)2: Calcium hydroxide, E. faecalis: Enterococcus faecalis, C. albicans: Candida albicans, SD: Standard deviation, CNPs: Chitosan nanoparticles
Preparation of culture media
E. faecalis was cultured in brain–heart infusion broth, whereas C. albicans was grown on Sabouraud’s Dextrose Agar. Both organisms were incubated aerobically at 37°C to promote optimal proliferation. Microbial suspensions were standardized to match 0.5 McFarland turbidity, ensuring consistent inoculum density throughout the experiment.
Agar well-diffusion method
This experimental analysis was done is Dextrose Technologies, Kengeri, Bangalore, India where a total of 80 sterile agar plates were prepared in borosilicate Petri dishes and confirmed for sterility by overnight incubation at 37°C and standardized cultures of E. faecalis and C. albicans were evenly spread across the agar surfaces using a sterile L-shaped spreader to create consistent lawn cultures.
Four wells were then created in each plate (6 mm diameter × 4 mm depth). Precisely, 50 μL of each test medicament was dispensed into the respective wells with a calibrated micropipette.
The plates were incubated for two predetermined intervals 72 h (Day 3) and 168 h (Day 7). After incubation, the zones of inhibition (ZOI) around each well were measured in millimetres using a digital Vernier caliper to ensure high measurement accuracy.[11]
Statistical analysis
All the data were statistically analyzed using IBM Statistical package for social sciences (SPSS) version 23. Descriptive statistics were expressed as mean ± standard deviation. To assess intergroup and intragroup variations, one-way analysis of variance was performed, followed by Tukey’s post hoc test to determine the level of significance. P < 0.05 was considered statistically significant, corresponding to a 95% confidence level.
Antimicrobial efficacy
The mean ZOI obtained for each medicament against E. faecalis and C. albicans at both observation periods (3rd and 7th days) are presented in Table 1.The findings revealed that the incorporation of chitosan nanoparticles significantly enhanced the antimicrobial performance of both Ca (OH)2 and CHX formulations.
Chart 1.
Study of two experimental and two control groups
| Test material | Micro-organisms | Time interval | |
|---|---|---|---|
| Group 1 | Ca (OH)2 (control) (20) | E. faecalis (10) | 3rd day (5)–7th day (5) |
| C. albicans (10) | 3rd day (5)–7th day (5) | ||
| Group 2 | Ca (OH)2 incorporated with | E. faecalis (10) | 3rd day (5)–7th day (5) |
| chitosan nanoparticle (20) | C. albicans (10) | 3rd day (5)–7th day (5) | |
| Group 3 | CHX (control) (20) | E. faecalis (10) | 3rd day (5)–7th day (5) |
| C. albicans (10) | 3rd day (5)–7th day (5) | ||
| Group 4 | CHX incorporated with chitosan | E. faecalis (10) | 3rd day (5)–7th day (5) |
| nanoparticle (20) | C. albicans (10) | 3rd day (5)–7th day (5) |
CHX: Chlorhexidine, Ca(OH)2: Calcium hydroxide, E. faecalis: Enterococcus faecalis, C. albicans: Candida albicans
Antimicrobial efficacy against Enterococcus faecalis
On Day 3, neither Ca (OH)2 nor chitosan-enhanced counterpart demonstrated any inhibitory effect against E. faecalis, both producing ZOI measuring 0.0 ± 0.0 mm. In contrast, the (CHX) and CHX + CNP formulations exhibited clear antimicrobial activity, yielding inhibition zones of 14.0 ± 1.5 mm and 15.0 ± 1.4 mm, respectively [Figure 5].
Figure 5.
(a-d) Antimicrobial susceptibility test of the given samples against Enterococcus faecalis at 3rd day
By Day 7, all medicament groups showed a notable enhancement in antimicrobial performance. Ca (OH)2 alone produced a moderate inhibition zone of 12.0 ± 1.8 mm, whereas the Ca (OH)2 + CNP group demonstrated a markedly superior response, generating a mean inhibition zone of 23.0 ± 2.1 mm a statistically significant improvement (P = 0.003). Similarly, the CHX + CNP combination yielded an increased inhibition zone of 17.0 ± 1.7 mm, significantly outperforming CHX alone (P = 0.046) [Figure 6].
Figure 6.
(a-d) Antimicrobial susceptibility test of the given samples against Enterococcus faecalis at 7th day
Antimicrobial efficacy against Candida albicans
For C. albicans, Ca (OH)2 demonstrated no detectable antifungal activity at either time interval, producing inhibition zones of 0.0 ± 0.0 mm on both day 3 and day 7. In contrast, the Ca (OH)2 + CNP formulation exhibited clear antifungal action, yielding inhibition zones of 15.0 ± 1.2 mm on day 3 and 17.0 ± 1.5 mm on day 7 an increase that reached statistical significance (P = 0.047) [Figure 7].
Figure 7.
(a-d) Antimicrobial susceptibility test of the given samples against Candida albicans at 3rd day
CHX alone showed robust antifungal efficacy from the beginning, with inhibition zones of 25.0 ± 2.0 mm on day 3 and 25.0 ± 1.8 mm on day 7, reflecting no significant temporal variation (P = 0.500). However, incorporation of chitosan nanoparticles further potentiated the effect of CHX. The CHX + CNP combination produced inhibition zones of 26.0 ± 2.1 mm on day 3 and 28.0 ± 1.9 mm on day 7, a statistically significant enhancement (P = 0.042) [Figure 8].
Figure 8.
(a-d) Antimicrobial susceptibility test of the given samples against Candida albicans at 7th day
Collectively, these findings reveal that chitosan nanoparticles significantly elevate the antifungal performance of both Ca (OH)2 and CHX, with the CHX + CNP formulation exhibiting the greatest inhibitory effect against C. albicans.
Upon comparing all medicament groups, it was observed
CHX + CNPs > CHX > Ca (OH)2 + CNPs > Ca (OH)2
The comparative bar graph [Figure 9] vividly highlights the superior antimicrobial performance of the nanoparticle-enhanced formulations. Among all the tested medicaments, the CHX + CNP combination demonstrated the greatest overall efficacy against both E. faecalis and C. albicans, followed sequentially by Ca (OH)2 + CNPs, CHX alone, and finally Ca (OH)2.
Figure 9.
Bar Graph for the overall antimicrobial efficacy of the intracanal medicament for both the micro-organisms and time interval
This progressive pattern clearly suggests that the incorporation of CNPs not only amplifies the intrinsic antimicrobial strength of the medicaments but also prolongs their activity over time. This effect is particularly evident in the delayed yet robust antifungal and antibacterial response exhibited by Ca (OH)2 + CNPs on day 7. The statistically significant improvements seen in all nanoparticle-containing groups further emphasize the value of CNPs as an efficient bioactive carrier system, enhancing both release dynamics and surface interaction of conventional intracanal medicaments.
DISCUSSION
The findings of this study clearly indicate that integrating CNPs with traditional intracanal medicaments namely calcium hydroxide and 2% chlorhexidine significantly strengthens both the intensity and longevity of antimicrobial activity against E. faecalis and C. albicans. Whereas Ca (OH)2 alone demonstrated virtually no antifungal effect and only a delayed antibacterial response, its CNP-enhanced counterpart exhibited a pronounced and time-dependent increase in inhibition by day 7. Similarly, although chlorhexidine produced strong immediate antimicrobial action, its efficacy was further amplified when combined with CNPs. These observations align with earlier reports by Thienngern et al. and Wassel et al.[14,15]
Mechanistic interpretation
The superior performance of CNP-modified medicaments observed in this investigation can be attributed to several interdependent mechanisms:
Electrostatic interactions and membrane destabilization
Chitosan, being a cationic biopolymer, carries positively charged amino groups. These groups interact strongly with the negatively charged surfaces of bacterial and fungal cells, increasing membrane permeability and triggering leakage of cellular contents. As reported by Zong et al. and Capuano et al., this interaction compromises microbial viability. When paired with CHX or Ca (OH)2, CNPs likely enhance localized membrane disruption, accelerating microbial death and strengthening antimicrobial potency.[16,17]
Enhanced penetration into dentinal tubules and biofilm matrices
The nano-size of CNPs provide a high surface area that facilitates deeper penetration into dentinal tubules and the dense architecture of biofilms areas traditionally shielded from conventional medicaments. Capuano et al. and Thienngern et al. demonstrated that nanoparticles can infiltrate regions inaccessible to larger molecules, enabling effective delivery of hydroxyl ions or chlorhexidine directly to embedded microorganisms which explains the delayed but powerful Day-7 activity of Ca (OH)2 + CNPs and the sustained antimicrobial effect seen in CHX + CNP formulations.[14,16]
Controlled and prolonged release of active agents
Chitosan-based matrices function as slow-release reservoirs, modulating drug delivery over extended periods. Findings from Wassel et al. and Ratih et al. highlight that incorporating medicaments into nanoparticle systems produce a controlled release profile rather than a rapid depletion. This sustained delivery accounts for the progressive increase in inhibition zones particularly with Ca (OH)2 + CNPs and the prolonged antifungal action of CHX + CNPs corresponding to the present study.[15,18]
Intrinsic antifungal and antibiofilm properties of chitosan
Beyond acting as a carrier, chitosan itself has well-recognized antifungal and antibiofilm properties. Reports by Thienngern et al. and Balsaraf et al. (2023) confirm that chitosan can directly impair both E. faecalis and C. albicans, contributing an additional antimicrobial layer. This intrinsic effect becomes especially valuable when paired with medicaments of inherently lower efficacy, such as Ca (OH)2 against C. albicans.[14,19]
Correlation with existing literature
The results of the present study reinforce a growing body of evidence supporting the benefits of nanoparticle-enhanced intracanal medicaments. Numerous in vitro investigations have shown that chitosan nanoparticles interact favorably with microbial cell membranes, causing increased permeability and eventual cell lysis. Biofilm studies further reveal that CNPs efficiently infiltrate dentinal tubules and disrupt the robust extracellular matrix surrounding E. faecalis and C. albicans a barrier that often limits the efficiency of conventional agents like Ca (OH)2.[10,12]
Studies by Praveen et al., Alsubait et al., and Aswathy et al. have consistently demonstrated that chitosan-based nanoformulations deliver superior reductions in microbial viability, enhanced biofilm removal, and extended antimicrobial substantivity highlighting improved surface interaction, deeper penetration in complex canal anatomies, and sustained release of antimicrobial components findings that closely mirror the outcomes of the current investigation.[20,21,22]
Correspondingly, Harshitha et al. 2022 demonstrated that, chitosan acted as an effective carrier matrix for nisin, Ca (OH)2, and triple antibiotic paste, leading to greater zone-of-inhibition diameters and more pronounced bacterial reduction than when the medicaments were used alone, which strongly reinforces, chitosan nanoparticles do not merely serve as inert carriers but function as synergistic enhancers.[23]
Pandey et al. 2024 demonstrated that chitosan nanoparticles exhibit markedly superior efficacy over calcium hydroxide in eradicating E. faecalis biofilms due to their unique ability to infiltrate dentinal tubules, disrupt mature biofilm architecture, and provide a sustained antimicrobial release profile which corresponds to significantly enhanced performance of the Ca (OH)2 + CNP formulation observed in this study.[24]
The conclusions of the current study echo the insights of the systematic review and meta-analysis by Patil et al. 2025 reviewing that nano-encapsulation significantly amplifies the antimicrobial performance of calcium hydroxide by improving its stability, penetration, and controlled ion release.[25]
Ugalmugale et al. 2024 evaluating nitrofurantoin, chitosan, and calcium hydroxide with propylene glycol complement the present findings by reinforcing the synergistic value of combining Ca (OH)2 with an efficient carrier system yielding stronger antibacterial effects than Ca (OH)2 alone, emphasizing the importance of improved diffusion and sustained activity.[26]
Clinical implications
Augmented Ca (OH)2 formulations may help overcome its longstanding limitations against fungal pathogens and biofilm-associated microorganisms. CNP-modified CHX exhibits strong immediate and prolonged effects, a desirable characteristic during multi-visit treatments where microbial regrowth between appointments poses a significant risk.
Limitations
Despite promising results, interpretation should be cautious due to in vitro model limitations that fail to mimic the root canal milieu, reliance on limited outcome measures, and variability in chitosan nanoparticle characteristics. Lack of standardized nanoparticle parameters further restricts reproducibility and meaningful cross-study comparisons.
CONCLUSION
Essentially, this study demonstrates that the integration of chitosan nanoparticles strikingly enhances and prolongs the antimicrobial effects of both calcium hydroxide and chlorhexidine against E. faecalis and C. albicans. Which, likely attributable to a multifaceted synergy combining chitosan’s inherent antimicrobial properties with improved penetration and a controlled, sustained release of active agents.
These findings position CNP-fortified medicaments as promising candidates for next-generation endodontic disinfection strategies. Nonetheless, comprehensive mechanistic investigations, safety evaluations, and translational studies are essential before these formulations can be confidently incorporated into routine clinical practice.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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