Introduction

Successful endodontic therapy hinges on the effective elimination of microbial pathogens from the complex root canal system, followed by the promotion of periradicular healing1. However, the intricate anatomy of the root canal—characterized by a dense network of dentinal tubules—poses a significant challenge to complete disinfection. Microorganisms persisting within these microscopic channels can compromise long-term treatment outcomes2. As such, the selection of an intracanal medicament with robust antimicrobial properties and effective penetration is critical for optimal disinfection.

CH remains one of the most extensively used intracanal medicaments due to its established antibacterial activity and clinical effectiveness1,3,4. Its therapeutic action is largely attributed to the sustained release of hydroxyl ions, which elevate pH and disrupt microbial enzyme systems5. However, the efficacy of CH is inherently dependent on its ability to diffuse through dentinal tubules and reach bacteria residing deep within the dentin. Factors such as tubule diameter, smear layer presence, and the duration of medicament placement significantly influence its diffusion5. These limitations raise concerns regarding the ability of CH to eradicate deeply embedded pathogens, particularly those such as Enterococcus faecalis, which are commonly associated with persistent infections.

Nanoparticles (NPs), defined as materials with dimensions ranging from 1 to 100 nm6, have gained considerable attention in dentistry due to their high surface-area-to-volume ratio and superior interaction with microbial surfaces7,8. Their small size enhances penetration into microanatomical spaces, including dentinal tubules, while their surface properties promote stronger antimicrobial effects through membrane disruption and oxidative stress9,10,11. Certain NPs also exhibit favourable biological responses, such as antioxidant activity and enhanced tissue regeneration12, and have been utilized in various biomedical applications, including UV protection and drug delivery13.

Among these, TNPs have emerged as promising candidates for endodontic disinfection. TNPs demonstrate potent antimicrobial properties through the generation of reactive oxygen species and possess favourable aesthetic and biocompatible profiles14,15,16. The incorporation of TNPs into CH may offer synergistic benefits—enhancing antimicrobial efficacy while simultaneously improving the material’s diffusion within dentinal tubules.

The present study aimed to evaluate the impact of TNP incorporation on the physicochemical and biological performance of CH. Specifically, this investigation assessed: (1) the pH, cytocompatibility, and antibacterial activity of the nano-modified CH formulation; and (2) its penetration depth into dentinal tubules, using SEM to compare its performance against conventional CH. The findings may contribute to the development of more effective intracanal medicaments with improved disinfection profiles.

Materials and methods

Ethical approval

This study was approved by the Institutional Review Board of AB Shetty Memorial Institute of Dental Sciences, Nitte Deemed to be University, India (Approval No: Ethics/ABSMIDS/405/2024). All procedures were conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained for the use of extracted human teeth.

Materials

Commercially available CH was obtained from ProDent (Ratnagiri, India), and TNPs (average particle size: 20 nm) were procured from SRL Chem Laboratory, India.

Preparation of Nano-Modified CH

An experimental nano-modified CH was prepared by incorporating 0.01 g of TNPs into 1 g of CH powder. The components were weighed using an analytical balance and manually homogenized in a porcelain mortar, followed by ball milling for 2 h to ensure uniform dispersion. Double-distilled water was added incrementally to achieve a paste-like consistency suitable for intracanal application.

Characterization by SEM

Surface morphology of the nano-modified CH was analysed using SEM (Zeiss Gemini). Samples were gold sputter-coated and examined under high-vacuum conditions to assess nanoparticle distribution and surface features.

Characterization by FTIR

FTIR analysis was conducted using the KBr pellet method (Bruker Alpha 400 spectrometer). Spectra were acquired in the transmission mode across a range of 500–4000 cm⁻¹ to identify characteristic functional groups and confirm the presence of Ti–O bonds.

Experimental Groups.

Two experimental groups were established:

  • Group 1: Nano-modified CH containing 1 wt% TNPs.

  • Group 2: Conventional CH.

Sample size calculation

Power analysis indicated that a sample size of 11 per group would detect a 0.5 unit difference in pH with 80% power at a 5% significance level, assuming a standard deviation of 0.4. To account for potential data loss, 12 samples per group were used.

pH Measurement17

Twelve polyethylene tubes (3 mm × 1 mm) were filled with freshly prepared samples of nano-modified CH and CH, respectively. Each tube was immersed in 10 mL of distilled water and stored at room temperature. After 24 h, the pH of each solution was measured using a calibrated digital pH meter (Merck Life Science Pvt Ltd.).

Cytocompatibility assessment (MTT Assay)

Human gingival fibroblasts (HGFs), cultured at the Nitte University Centre for Stem Cell Research and Regenerative Medicine, were used to assess cytocompatibility. Cells were maintained in DMEM supplemented with 5% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL), and L-glutamine (2 mmol/L), and incubated at 37 °C in 5% CO₂.

Cylindrical pellets of each material were fabricated and allowed to set in an incubator for 24 h. HGFs were seeded into 96-well plates (2.7 × 10⁴ cells/well) and exposed to the pellets. Cell viability was quantified using the MTT assay at 24, 48, and 72 h. Absorbance was measured at 630 nm using an ELISA microplate reader.

Antibacterial Assay18

Antibacterial activity against E. faecalis was evaluated via the agar diffusion method. Bacterial suspensions were standardized to 1 × 10⁷ CFU/mL in brain heart infusion broth. Sterilized samples of both materials were placed on agar plates pre-inoculated with the bacterial suspension and incubated at 37 °C for 24 h. The diameter of the inhibition zone was measured using a sliding calliper, and net inhibition was calculated by subtracting the disc diameter (10 mm) from the total halo diameter.

Intratubular penetration assessment

Twenty-four extracted, mature single-rooted premolars with intact roots were collected and stored in 0.5% chloramine-T solution. After decoronation, working length was established using a size 10 K-file. Root canals were instrumented with ProTaper rotary files and irrigated with 2.5% sodium hypochlorite and 17% EDTA.

Specimens were randomly allocated to two groups (n = 12). Each medicament was mixed with normal saline and delivered using a lentulo spiral. After placement, canals were sealed with utility wax and stored for two weeks at 37 °C and 100% humidity.

SEM evaluation of penetration depth

Roots were grooved longitudinally and split using end-cutting pliers, ensuring preservation of the canal filling. SEM imaging was performed at the coronal, middle, and apical thirds. The depth of medicament penetration was measured using ImageJ software by a blinded examiner.

Statistical analysis

Data were analysed using SPSS (v23.0; IBM, USA). Repeated measures ANOVA was used to evaluate differences in penetration depth. Independent t-tests were used to compare pH values and antibacterial activity between groups. Post hoc analysis for cytocompatibility testing was performed using Tukey’s test. Statistical significance was set at p < 0.05.

Results

Surface characterization by SEM

The SEM micrograph of the nano-modified CH revealed a heterogeneous surface morphology, characterized by uniform dispersion of TNPs within the CH matrix. Distinct agglomerates of TiO₂ were visible, embedded on the Ca(OH)₂ substrate, along with enhanced surface roughness indicative of nanoscale modification (Fig. 1).

Fig. 1
figure 1

Scanning Electron Microscope image of nano modified CH under 200 μm.

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Chemical characterization by FTIR

The FTIR spectrum of nano-modified CH (Fig. 2) demonstrated characteristic absorption bands at 3356.73 cm⁻¹ and 1628.86 cm⁻¹, corresponding to O–H stretching and bending vibrations, respectively, indicating retained moisture content. A distinct absorption peak at 662.73 cm⁻¹ confirmed Ti–O stretching, characteristic of TiO₂. Additional peaks at 1415.22 cm⁻¹ and 871.47 cm⁻¹ indicated carbonate presence, suggestive of atmospheric CO₂ interaction and calcium carbonate formation.

Fig. 2
figure 2

FTIR spectrum of nano-modified calcium hydroxide, showing characteristic peaks for O–H, Ti–O, and carbonate groups, confirming the presence of TiO₂ and atmospheric carbonate incorporation.

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pH measurement

Independent t-test analysis revealed that nano-modified CH exhibited a significantly higher mean pH (11.21 ± 0.62) compared to CH (10.67 ± 0.44), with a mean difference of 0.54 units (95% CI: 0.0824–1.0009, p = 0.0222) as shown in Table 1.

Table 1 Comparison of pH values between groups (Independent t-test).
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Cytocompatibility assessment

The MTT assay revealed high cell viability for both experimental groups. At 72 h, Group 1 (nano-modified CH) and Group 2 (CH) demonstrated mean cell viability of 82.5% and 83.9%, respectively, with no statistically significant difference (p > 0.05). Both groups showed slightly reduced viability over time, though within acceptable biocompatibility limits. Post hoc analysis confirmed a significant difference only between both experimental groups and the control, but not between each other. (Tables 2 and 3). Figure 3 represents the microscopic images of viable human gingival fibroblasts following exposure to experimental materials during the MTT assay.

Table 2 Descriptive statistics of cell viability (%).
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Table 3 Post hoc comparison of cell viability (Tukey test).
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Fig. 3
figure 3

Representative microscopic images of viable human gingival fibroblasts following exposure to experimental materials during the MTT assay.

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Antibacterial activity

Nano-modified CH demonstrated a significantly larger zone of inhibition against E. faecalis (12.67 ± 0.61 mm) compared to CH (9.08 ± 0.55 mm). The mean difference of 3.58 mm was statistically significant (95% CI: 3.0862–4.0804, p < 0.001) (Table 4).

Table 4 Antibacterial activity against E. faecalis (Independent t-test).
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Intratubular penetration depth

Repeated measures ANOVA revealed that nano-modified CH penetrated significantly deeper into the dentinal tubules at all canal levels compared to CH. Penetration depths in the coronal, middle, and apical thirds were 374 ± 3.82 μm, 196 ± 2.66 μm, and 81.0 ± 3.02 μm, respectively, for nano-modified CH, and 318 ± 2.49 μm, 153 ± 2.39 μm, and 55.8 ± 2.41 μm for CH. Differences across all regions were statistically significant (p < 0.001) (Table 5).

As depicted in the graph (Fig. 4), nano-modified CH demonstrates significantly deeper and more uniform penetration across all three regions of the root canal—namely the coronal, middle, and apical thirds.

Table 5 Comparison of dentinal tubule penetration (Repeated measures ANOVA).
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Fig. 4
figure 4

Plot of Mean Penetration Depth.

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This graph illustrates significantly enhanced penetration of nano-modified CH into the coronal, middle, and apical thirds of dentinal tubules relative to conventional CH.

Fig. 5
figure 5

SEM image showing the penetration depth of nano-modified calcium hydroxide into dentinal tubules at coronal, middle, and apical levels.

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Fig. 6
figure 6

SEM image illustrating the comparatively limited penetration of conventional calcium hydroxide within dentinal tubules across all root levels.

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Discussion

The application of nanotechnology in endodontic materials has introduced promising strategies for enhancing disinfection efficacy and biological performance19,20,21. Among various nanomaterials, titanium dioxide nanoparticles (TNPs) have gained attention due to their favorable physicochemical properties, including high surface area, photoreactivity, and demonstrated antimicrobial effects22. In this study, incorporation of 1 wt% TNPs into calcium hydroxide (CH) resulted in a nano-modified formulation that exhibited enhanced pH, deeper dentinal tubule penetration, and superior antibacterial activity compared to conventional CH.

Biocompatibility remains a crucial criterion for materials that contact periapical tissues. As shown in Tables 2 and 3, the MTT assay results indicated no significant cytotoxic differences between nano-modified CH and CH at any of the evaluated time points. These findings align with earlier studies reporting acceptable cytocompatibility of CH-based formulations containing low-concentration TNPs12,20.

The antimicrobial effect of CH is primarily linked to its ability to release hydroxyl ions, creating an alkaline environment detrimental to microbial survival23,24. The current findings showed that nano-modified CH produced a significantly higher pH than CH (p = 0.0222), suggesting enhanced ion diffusion capacity. A high pH is essential for denaturing microbial proteins and disrupting enzymatic systems, thereby improving bactericidal efficiency.

Despite these properties, CH has shown limited efficacy against Enterococcus faecalis, a facultative anaerobe commonly implicated in persistent endodontic infections25,26,27,28,29. This resistance is attributed to its ability to penetrate deep into dentinal tubules (up to 300 μm), and to maintain internal homeostasis through a robust proton pump system30. Moreover, dentin’s intrinsic buffering capacity can reduce the external pH below the threshold required to inactivate E. faecalis27,28.

The present study demonstrated a significantly greater inhibition zone (mean difference: 3.58 mm; p < 0.001) (Table 4) in the nano-modified CH group, indicating superior antibacterial efficacy. Previous studies have shown that nano CH demonstrates enhanced antimicrobial activity against E. faecalis, particularly at depths of 200 and 400 μm, compared to conventional CH31,32. This improvement is primarily attributed to its reduced particle size and enhanced physicochemical properties. Komabayashi et al. and Reyhani et al reported that smaller, spherical particles penetrate dentinal tubules more effectively than larger, irregular ones, supporting the findings of this study33,34

In the present formulation, incorporation of TNPs (~ 0.1 μm) likely contributed to deeper penetration (Table 5), particularly within the coronal, middle, and apical thirds of the canal system (Fig. 5) when compared to conventional calcium hydroxide (Fig. 6). This observation aligns with the findings of Mohamed et al., who demonstrated that TNPs can serve as efficient carriers, enhancing the diffusion of therapeutic agents into dentinal tubules35.

In addition to enhanced penetration, the antibacterial efficacy of nano-modified CH was significantly greater than that of CH. This may be attributed to the oxidative potential of TNPs, which generate reactive oxygen species capable of damaging microbial cell walls, proteins, and DNA36. Such mechanisms of action provide synergistic enhancement to the conventional high-pH-mediated bactericidal effect of CH. Furthermore, studies have confirmed that TNPs are effective even against resistant microorganisms and biofilms37,38.

The elevated pH observed in nano-modified CH (Table 1) may also reflect its improved chemical stability. CH tends to absorb atmospheric CO₂, forming calcium carbonate, which modulates its alkalinity39. However, TiO₂ is known to form a stable oxide layer that helps preserve the high pH environment, thereby sustaining antimicrobial activity40. This may explain the concurrent increase in both penetration depth and antibacterial efficacy observed in our study.

While our findings are promising, it is essential to recognize that differences in nanoparticle type, preparation method, and concentration across studies may yield varying outcomes. The current study employed a 1 wt% concentration of TNPs, which has been previously shown to offer a balance between efficacy and biocompatibility41. Further investigations are warranted to explore other nanoparticle systems and optimization strategies for clinical application.

Limitations and future directions

While this study provides compelling evidence for the enhanced antibacterial activity and dentinal tubule penetration of nano-modified CH, several limitations should be acknowledged. The antibacterial assessment was conducted using the agar diffusion method, which, while standardized and easy to implement—primarily captures material diffusion and may not accurately reflect bactericidal action under clinical conditions. Future investigations should incorporate more representative models, such as the direct contact test (DCT) or dentin block systems, to better simulate the complex root canal environment.

The evaluation of pH was limited to a single time point, aimed at capturing the initial alkalizing effect of the materials. However, monitoring temporal pH changes over extended periods would provide more insight into sustained release and long-term antimicrobial potential. Similarly, while the in vitro model allowed for a controlled analysis, it cannot fully replicate the biological complexity of the root canal system. In vivo or ex vivo investigations using simulated canals or animal models are necessary to confirm clinical relevance.

In this study, a fixed concentration of 1 wt% titanium dioxide nanoparticles was used to modify CH, based on previous reports and pilot validations. Exploring a broader range of nanoparticle concentrations could help fine-tune the balance between efficacy, biocompatibility, and physical handling characteristics.

Despite these limitations, the current findings establish a strong foundation for continued research on optimizing nano-modified CH formulations and evaluating their translational potential.

Conclusion

Within the limitations of the present study, nano-modified CH—prepared by incorporating 1 wt% titanium dioxide nanoparticles into conventional calcium hydroxide—demonstrated superior antibacterial efficacy and significantly deeper dentinal tubule penetration compared to unmodified CH. These enhancements may be attributed to the reduced particle size, improved dispersion, and sustained alkalinity provided by the nanoparticles, collectively enhancing both diffusion and antimicrobial performance.

Importantly, the formulation maintained comparable cytocompatibility to conventional CH, as confirmed through MTT assays using human gingival fibroblasts. These findings suggest that nano-modified CH holds promise as a next-generation intracanal medicament, particularly in the management of refractory endodontic infections.

To establish its full clinical utility, further investigations involving long-term in vivo testing, comparative evaluation with commercial products, and assessment against multispecies biofilms are recommended.