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. 2024 Sep 11;75(2):1213–1222. doi: 10.1016/j.identj.2024.08.007

The Impact of Silver Nanoparticles’ Size on Biofilm Eradication

Ghazal Fakeeha a, Sarah AlHarbi b, Sayed Auda c, Hanan Balto a,⁎
PMCID: PMC11976549  PMID: 39266402

Abstract

Introduction

Efficient intracanal disinfection is required for a successful regenerative endodontic treatment. Thus, this study aimed to identify the silver nanoparticles’ (NPs) size (AgNPs) with the highest antibiofilm efficacy when mixed with calcium hydroxide [Ca(OH)2] to eradicate an in vitro endodontic biofilm.

Methods

The various sizes of AgNPs and mixtures were characterized by scanning electron microscopy, transmission electron microscopy, and ultraviolet-visible spectroscopy. A total of 168 dentin root segments were prepared, sterilized, and inoculated for 3 weeks with Actinomyces naeslundii and Fusobacterium nucleatum. Samples were randomly allocated to 4 experimental groups (n = 28/group): 2 nm AgNPs + 35% Ca(OH)2, 5 nm AgNPs + 35% Ca(OH)2, 10 nm AgNPs + 35% Ca(OH)2, and 35% Ca(OH)2 alone. Samples exposed to saline and triple antibiotic paste (TAP) acted as negative and positive control groups, respectively. After 1 and 2 weeks, samples were stained with LIVE/DEAD BacLight dye and examined under a confocal laser scanning microscope to determine the proportion of dead bacteria.

Results

The characterization procedure revealed a spherical NP's structure with minor aggregations. Except for Ca(OH)2 group, all groups had significantly higher antibiofilm efficacy at 2 weeks. Both the 10 nm mixture (99.5%) and TAP (99.2%) exhibited the highest antibiofilm efficacy at 2 weeks and were not significantly different from one another (P > .05). No significant difference was noted between the 2 and 5 nm mixtures at 1 week (81% and 84%) and 2 weeks (89% and 91%).

Conclusion

The 10 nm AgNPs (0.02%) + 35% Ca(OH)2 mixture exhibited the highest antibiofilm efficacy at 2 weeks compared to all other mixtures at both observation periods. Interestingly, the 10 nm mixture performed similarly to TAP at 2 weeks. Excluding Ca(OH)2 group, longer application significantly improved the antibiofilm efficacy of all tested medicaments.

Clinical Relevance

The 10 nm AgNPs + 35% Ca(OH)2 mixture revealed promising results as an intracanal medicament in the regenerative endodontic treatment protocol.

Key words: Calcium hydroxide, Intracanal medication, Multispecies biofilm, Silver nanoparticles, Triple antibiotic paste

Introduction

Traumatic injuries and infections of immature permanent teeth can lead to pulpal necrosis and root development arrest. Subsequently, an increased risk of tooth fracture is anticipated by the deteriorated root structure and open apex.1 Such cases were traditionally treated by apexification procedures using calcium hydroxide [Ca(OH)2] or mineral trioxide aggregate to induce an apical barrier prior to root canal filling. However, this does not restore the vitality of pulpal tissue, nor can it promote root maturation. Consequently, regenerative endodontic treatment (RET) of the dentin-pulp complex was proposed as an alternative. RET implements the triad concept of tissue engineering with stem cells, biomimetic scaffolds, and bioactive growth factors, all of which are required for successful regeneration.2 Later, this concept was upgraded to include disinfection as a fourth element to further improve the procedural outcome.3 Verma et al4 demonstrated that residual bacterial infection restricted the formation of mineralized tissue and led to radicular growth cessation, thus highlighting the significance of disinfection.

According to Nagata et al,5 Actinomyces naeslundii (A. naeslundii) was the most prevalent bacterial species (66.67%) isolated from immature necrotic teeth, followed by Parvimonas micra, Fusobacterium nucleatum (F. nucleatum), and Porphyromonas endodontalis (33.34%). A. naeslundii is a gram-positive facultative that exhibits resistance to sodium hypochlorite (NaOCl) and Ca(OH)2,6 and is considered among the initial commensal colonizers of human dental biofilm.7 The gram-negative obligatory anaerobe, F. nucleatum, acts as a bridging species, linking initial commensal colonizers to secondary colonizing pathogens.8 RadD, an outer membrane protein expressed by F. nucleatum, was found to mediate arginine-inhibitable adhesion to A. naeslundii and therefore contributes to multispecies biofilm formation.9 The notable ability of such biofilm-producing bacteria to escape host immune mechanisms, along with their concomitant tendency for horizontal gene transfer between different strains, both demonstrate a challenge for successful infection eradication.10

Several intracanal medicaments were recommended during RET, including triple antibiotic paste (TAP), double antibiotic paste (DAP), and Ca(OH)2. TAP demonstrated strong antimicrobial activity against bacterial species in the infected root canals.11 Yet, a concentration of no greater than 1 mg/mL was recommended to avoid damage to the residing stem cell population of the apical papilla.12 Dentin discoloration was also listed among the disadvantages of its use. On the other hand, the resistance of Enterococcus faecalis to the hydroxyl ion in Ca(OH)2, the buffering effect of dentin against Ca(OH)2, and its low solubility have all been reported as valid concerns related to the application of Ca(OH)2.13

Therefore, the search for an innovative, less harmful, and more potent intracanal medicament is still ongoing. In this regard, nanotechnology is considered a breakthrough in the field of dentistry by producing novel biomaterials with unique properties.14 Nanoparticles (NPs) possess improved physicochemical characteristics, including their ultrasmall size, large surface area/mass ratio, enhanced chemical activity with minimal doses, and distinct reactivity against antibiotic-resistant bacteria.15,16 Among the various NPs available, silver NPs (AgNPs) are known to impair cell membrane integrity, increase cellular permeability, and trigger internal oxidative stress mechanisms to generate reactive oxygen species; which can interfere with various cellular metabolic pathways, destroy constituent proteins, and eventually damage DNA by the electrostatic interaction with organelles.17 Those NPs have been implemented in endodontics in irrigation strategies, as intracanal medicaments, as obturation materials, and as part of the RET protocol.18 Reports have confirmed their broad-spectrum activity when applied as an antibacterial agent, along with their ability to damage lipoteichoic acids and peptidoglycans.19,20 It's interesting to note that this activity was size- and dose-dependent, with smaller particles displaying a much stronger bactericidal potential.21 AgNPs with an average size of 5 to 10 nm exhibited a distinct antibacterial activity along with a higher reported cytocompatibility when compared to sodium hypochlorite (NaOCl) and chlorhexidine (CHX).22 Furthermore, it was shown that only 1 to 10 nm AgNPs were effective in eradicating gram-negative strains across a diameter range of 1 to 100 nm.23 Interestingly, AgNPs mixed with Ca(OH)2 yielded a synergistic effect with improved action. This mixture was found to surpass the bactericidal activity of Ca(OH)2 alone.24, 25, [26] Tülü et al27 successfully implemented 2.5 nm AgNPs with Ca(OH)2 or CHX and showed a significant antibiofilm activity of the mixture against a multispecies biofilm of E. faecalis, Streptococcus mutans, Lactobacillus acidophilus, and A. naeslundii after only 7 days of application. Thus, the optimal size of silver NPs to be used with Ca(OH)2 is yet to be determined. The aim of this in vitro study was to identify the size of AgNPs that should be mixed with Ca(OH)2 in order to achieve the highest antibiofilm efficacy against multispecies biofilm.

Materials and methods

Characterization of AgNPs: Suspensions of 2 nm (US Research Nanomaterial, Inc.), 5 nm, and 10 nm (Nano Composix, Inc., Suite K) AgNPs were purchased and characterized using scanning electron microscopy (SEM) (JEOL, JSM-7610F Schottky Field Emission), transmission electron microscopy (TEM) (JEOL, JEM, 1400 Plus), and ultraviolet-visible spectroscopy (UV-VIS) (Perkin Elmer) at a range of 200 to 900 nm wavelength before and after medicament preparation to examine the size, shape, and possible aggregation of particles in the suspension. The pH of the colloidal suspension was measured before and after medicament preparation using a laboratory pH meter (Thermo Scientific Orion 2 Star pH Meter).

Dentin root segment preparation: At a level of significance α = 0.05, an estimated standard deviation of 0.4, and a power of 90%, the total sample size was determined to be 168 dentin segments. Single-rooted human teeth extracted for reasons not related to this study were collected. Roots with anatomical variations, curvatures, or previous endodontic treatment were excluded. Dentin segments’ preparation was adopted from a previous study.24 Briefly, roots were sectioned using a diamond disc to utilize the middle third. The canals were prepared with a size of 35/0.06 ProFile (Dentsply-Maillefer) and a final dimension of 4 × 4 × 1 mm (width × length × thickness) of dentin segment was obtained. Dentin segments were treated with 17% EDTA, rinsed with sterile saline, and sterilized with gamma irradiation.28 Two dentin segments were randomly selected from each group and incubated in fastidious anaerobic broth (FAB) (NEOGEN Culture Media) for 24 hours to exclude possible contamination.

Biofilm generation:A. naeslundii (ATCC 12104) and F. nucleatum (ATCC 25586) were streaked from previously prepared 15% frozen glycerol stocks on sheep blood agar plates (Cat No. 333, SAMCO Media) and were incubated for 24 and 48 hours, respectively, under anaerobic conditions. A single colony from each strain was extracted, gram-stained, and examined under light microscopy to confirm the strain morphology. After confirmation, a single colony of each strain was isolated and separately inoculated into 20 mL (FAB) (FAB, SAMCO Media). Strains were incubated overnight in a shaker under anaerobic conditions. Later, overnight cultures were adjusted to 0.7 (1 × 106 CFU/mL) and 0.4 (1 × 109 CFU/mL) optical densities for A. naeslundii and F. nucleatum, respectively, which corresponds to the midexponential phase of each strain. An amount of 5 mL of each mono-culture suspension was transferred into a sterile 50 mL centrifuge tube to create the multiculture suspension. Dentin segments were placed in a sterilized 12-well culture plate (Nunc; Thermo Scientific) with the pulpal sides oriented outward. Each well was inoculated with 0.5 mL of the multiculture suspension and 1.5 mL of fresh (FAB) and incubated under anaerobic conditions at 37°C for 21 days. Fresh (FAB) was replenished every 3 days. The biofilm mass was assessed for uniformity and thickness by means of (SEM), a confocal laser scanning microscope (CLSM), and gram staining using three additional dentin root segments.

Dentin root segment treatment: Infected dentin segments (n = 168) were placed in a sterilized 12-well culture plate and were randomly allocated to 4 experimental groups and 2 control groups (n = 28 segment/group). The experimental groups (2 nm group, 5 nm group, 10 nm group, and Ca(OH)2 group) were prepared by mixing 2 nm, 5 nm, and 10 nm AgNPs colloidal suspensions (provided at 0.02 mg/mL in a 2 mM sodium citrate solution, 0.02%) with 35% Ca(OH)2 paste, in a proportion of 1:1 (w/w), while the Ca(OH)2 group was prepared by mixing 35 gm of Ca(OH)2 powder (Somatco) with 65 g of a prepared viscous aqueous vehicle (propylene glycol: glycerin, 1:1 proportion, w/w) to produce a consistency similar to that of toothpaste. TAP acted as a positive control and was prepared by mixing metronidazole, ciprofloxacin, and minocycline powders (Xi'an Sgonek Biological Technology Co., Ltd) at a ratio of 1:1:1 (w/w/w) with distill water using a magnetic stirrer, reaching a concentration of 1 mg/mL, while saline acted as a negative control. All medicaments were prepared by a single investigator, and 0.3 mL of the assigned medicament was injected on top of each dentin segment. Dentin segments in each group were equally subdivided into 2 subgroups and were incubated anaerobically for 1 week (1W) and 2 weeks (2W) at 37°C.

CLSM examination: At the end of each observation period, dentin segments were irrigated with 5 mL sterile PBS to remove the medication, stained with the fluorescent LIVE/DEAD BacLight dye (Molecular Probes) according to the manufacturer instructions, and analyzed by a blinded operator under CLSM. The four corners of each dentin segment were scanned with a 2-μm step size at a resolution of 1024 × 1024 pixels. Simultaneous dual-channel imaging was used to display the green (live cells) and red (dead cells) fluorescence using the software NIS-Elements AR v4.0 (Nikon confocal C2+, Nikon Instruments Inc). Biofilm viability was assessed by calculating the proportion of dead bacteria and quantified using a validated software.29

Statistical analysis

Data were analyzed using SPSS version 26.0 (IBM Corp.). Kruskal–Wallis followed by post-hoc tests and a Wilcoxon sign rank test, were used to compare the percentage of dead bacteria between the groups and within the same groups at both observation periods (P < .05).

Results

Characterization of AgNPs: SEM images of the colloidal suspensions demonstrated a relatively spherical NPs’ structure with minor clusters/aggregation (Figure 1A-C). TEM images further confirmed the spherical morphology and the uniform distribution of NPs within the suspension (Figure 1D-F). A prominent absorption band was observed by UV-VIS spectroscopy at 408, 422, and 430 nm in the 2, 5, and 10 nm AgNPs samples, respectively (Figure 1G-I). After medicament preparation (the mixture of AgNPs and Ca(OH)2), AgNPs maintained their original spherical shape with no obvious aggregation when examined under TEM (Figure 2A-C). Calcium hydroxide particles appeared to be coagulating, forming a backbone structure, where AgNPs were found to be precipitated. Moreover, AgNPs were visualized on the surface of Ca(OH)2 particles as dots. UV-VIS analysis of the mixture revealed a shift in the primary absorption peak to a shorter wavelength with a higher energy in all three tested samples (Figure 2D-F). The reported pH of the AgNPs, the mixture of Ca(OH)2 and AgNPs, and Ca(OH)2 was 7.7, 12.9, and 12.5, respectively.

Fig. 1.

Fig 1

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(A-C) SEM images of AgNPs alone illustrating the spherical shape and size range of each colloidal suspension (× 200,000); (D-F) TEM images of AgNPs alone showing no particles’ aggregations (× 300,000); (G-I) absorption bands generated by UV-VIS spectroscopy, displaying the position of Lambda max of the 2, 5, and 10 nm AgNPs colloidal suspensions.

Fig. 2.

Fig 2

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(A-C) TEM images of AgNPs + Ca(OH)2 mixtures of (A) 2 nm, (B) 5 nm and (C) 10 nm AgNPs samples (× 100,000-150,000); (D-F) UV-VIS spectroscopy analysis of AgNPs + Ca(OH)2 mixtures of (D) 2 nm, (E) 5 nm and (F) 10 nm AgNPs samples. The abscenc of a second absorption peak potentially excludes particles’ aggregation in all tested samples.

Assessment of the biofilm mass: At the end of 21 days, SEM (Figure 3A-L), CLSM (Figure 3M-P), and gram-stained images (Figure 3Q-T) clearly demonstrated a homogenous, dense, and intact viable multispecies biofilm of the two strains.

Fig. 3.

Fig 3

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(A-C) Actinomyces naeslundii observed under SEM showing dividing bacterial cells; (D-F) Fusobacterium nucleatum observed under SEM showing bacterial autoaggregation; (G-L) SEM images illustrating the biofilm structure forming on the surface of dentin segment with dentinal tubules observed at the periphery (red arrow), F. nucleatum forming water channels to permit water diffusion through the thick biofilm structure (red circles); (M-P) CLSM image confirming the generation of a viable biofilm on the surface of dentin segment after 3 weeks of incubation; (Q-T) gram-stained images of the multispecies biofilm displaying both strains with heavy F. nucleatum autoaggregation (black arrow).

CLSM Analysis: Among all groups, TAP at 1W and 2W (Figure 4 A1-A4) and the 10 nm group at 2W (Figure 4 B1-B4) destroyed all A. naeslundii and F. nucleatum biofilm, and no significant differences in the proportion of dead bacteria were observed between them (P > .05). On the contrary, most of the biofilm was intact in the saline group at 1W and 2W (Figure 4C1-C4). At 1W, there was no statistically significant difference in antibiofilm efficacy between the 2 nm, 5 nm, and Ca(OH)2 groups (P > .05). However, at 2W, a significantly greater proportion of dead cells was observed in dentin segments treated with the 2 nm (Figure 4D1-D4) and 5 nm (Figure 4E1-E4) groups than those treated with Ca(OH)2 alone (Figure 4F1-F4) (P < .05). With the exception of the Ca(OH)2 group, which performed similarly at 1W and 2W, all groups generally showed significantly better biofilm destruction at the 2W observation period (Table).

Fig. 4.

Fig 4

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CLSM images of the different medications at both observation periods. Green fluorescence indicates viable/live bacteria, while red fluorescence indicates damaged/dead bacteria. Following the application of TAP for 1W (A1-A2) and 2W (A3-A4), the biofilm was completely destroyed; (B1-B4) the biofilm was mostly destroyed after being exposed to a mixture of 10 nm; intact and viable biofilm after exposure to saline for 1W (C1-C2) and 2W (C3-C4); biofilm is partially destroyed after being exposed to a 2 nm (D1-D4) and 5 nm (E1-E4) mixture and Ca(OH)2 alone (F1-F4).

Table.

Percentage of dead bacteria in each group at the two observation periods.

Groups No. of segments Wk 1 (1W)
 Wk 2 (2W)
Mean ± SD
(%)		 Mean ranks P value Mean ± SD
(%)		 Mean ranks P value
2 nm AgNP mixture 14 seg/per wk 81.07 ± 11.41 77.16 89.33 ± 19.01 118.06
5 nm AgNP mixture 14 seg/per wk 84.86 ± 8.51 85.06 91.51 ± 8.23 115.00
10 nm AgNP mixture 14 seg/per wk 85.14 ± 11.39 90.34 99.50 ± 0.64 168.84§
Ca(OH)2 alone 14 seg/per wk 71.54 ± 26.26 77.13 <.0001 82.57 ± 14.10 87.34 <.0001
TAP 14 seg/per wk 94.04 ± 8.78 133.34† 99.20 ± 1.96 172.00§
Saline 14 seg/per wk 8.67 ± 6.41 11.44* 16.72 ± 7.91 22.28‡
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⁎

Significantly lower than other groups (P = .039).

†

Significantly higher than other groups (P = .018).

‡

Significantly lower than other groups (P = .026).

§

Significantly higher than other groups (P = .012).

Discussion

Biofilm eradication from the pulpal space is a crucial step for the success of endodontic treatment, as the structure of the biofilm renders the residing bacteria resistant to different antibacterial agents.30 AgNPs have demonstrated significant antibiofilm activity against biofilm-forming species, which justifies their potential application in RET.31

Different characterization techniques have been reported to evaluate the NPs’ properties. Their shape, size, and degree of aggregation are among the main parameters assessed.32 In this study, the number and position of absorption bands generated by UV-VIS spectroscopy were found to be determined by the NPs’ size and shape.33 The detected peaks at 408, 422, and 430 nm in the 2, 5, and 10 nm AgNPs colloidal suspensions, respectively, were found to fall within the reported range of spherical NPs’ structure. Moreover, the increase in band intensity and the redshift to longer wavelengths as AgNP size increases are two optical phenomena reported and explained in the literature.34 TEM images of the mixture of AgNPs and Ca(OH)2 showed no evidence of significant structural aggregation between the two chemical components, which may have compromised or attenuated the mixture's antibiofilm activity. Moreover, UV-VIS spectroscopy analysis of the mixtures demonstrated a change in the plasmon resonance properties of AgNPs. This change is mainly related to the addition of hydroxyl functional groups and calcium atoms to the colloidal suspensions.35, 36, 37

A biofilm is a bacterial population growing on a surface where 85% to 90% of its constituents are mainly extracellular polymeric substances.30 A. naeslundii and F. nucleatum were used as a multispecies biofilm model in this study to mimic the microbiota existing in immature necrotic teeth.5 The filamentous granules producing A. naeslundii are known to cohesively coaggregate, escaping phagocytosis by host defense mechanisms and impeding antibiotic accessibility.38 F. nucleatum, on the other hand, brings together the early and late colonizers that otherwise would not attach to each other. Its distinctive shape and length make it an ideal bridging partner to further stabilize and strengthen the biofilm structure.8 The structure of biofilm observed in this study confirms the high autoaggregation tendency of F. nucleatum and its need to coaggregate with the primary colonizer, A. naeslundii, for biofilm growth, stabilization, and maturation.39

The antibacterial and biological properties of AgNPs are strongly dependent on their size, shape, and surface character,15 and their bactericidal action is exerted mainly by the provoked oxidative stress and subsequent (reactive oxygen species) production.40 This activity was found to be more pronounced against gram-negative bacteria, as the multilayer mucopeptide structure of the cell wall of gram-positive bacteria was found to hinder the NPs’ penetration.17 Interestingly, the ability of AgNPs to disturb the biofilm structure was found to be related to their ability to retard bacterial quorum sensing and inhibit extracellular polymeric substances production.41 It was also proven that they could negatively alter the transcription of biofilm-related genes and prevent bacterial adhesion to surfaces by disrupting their intermolecular forces.31,42 It is worth noting that AgNPs’ penetration through the biofilm structure was found to be obstructed/retarded for larger NPs compared to their smaller counterparts.43,44 Although smaller NPs showed a higher level of bactericidal activity,23 others pointed to no correlation between the two.45 In this study, the 10 nm group exhibited the highest antibiofilm efficacy when compared to the 2 and 5 nm groups. This comes in agreement with several reports,23,46,47 which set 10 nm AgNPs as a threshold for the highest bactericidal activity when compared to larger sizes. However, sizes smaller than 10 nm were reported to be either less toxic or to have no size-dependent effect. This could be attributed to the higher aggregation tendency of smaller NPs, which can attenuate or, in extreme cases, completely abolish AgNPs’ bioactivity.45 Furthermore, it has been observed that bacterial resistance to AgNPs is associated with the production of adhesive flagellum protein, which triggers NPs’ aggregation and subsequently lowers the total quantity of Ag ions released.48, 49, 50 Due to their larger surface area, smaller NPs were found to release a higher amount of Ag ions, which are considered the bioactive molecule by which AgNPs mainly express their activity.43

All AgNPs + Ca(OH)2 mixtures (regardless of NP size) showed superior biofilm destruction when compared to Ca(OH)2 alone. This is in agreement with others and is mainly attributed to the bactericidal synergism exerted by the two medicaments.24, 25, [26] According to Siqueira et al,51 Ca(OH)2 primarily expresses its bactericidal activity through the slow and controlled release of both calcium and hydroxyl ions, whereas AgNPs act primarily through the release of (Ag) ions and their subsequent intracellular accumulation.52,53 The results of this study demonstrated that the addition of AgNPs did not lower the high pH of Ca(OH)2, which may indicate that the mixture's antibiofilm activity can also be attributed to its alkaline chemical nature in addition to the previously mentioned synergism.

In this study, the antibiofilm efficacy of the 10 nm group at 2W was found to be comparable to TAP. This is in agreement with the findings of Balto et al,24 who showed that the antibacterial activity of Ca(OH)2 + 0.02% AgNPs was not significantly different from 1 mg/mL TAP after 2 weeks of application.

Despite the promising antibiofilm effect of AgNPs, their potential side effects, such as tooth discoloration and cytotoxicity/genotoxicity, make them controversial for in vivo application. Although medications’ impact on dentin discoloration was outside the scope of this study, TAP-treated dentin segments displayed heavy discoloration when compared to dentin segments treated with AgNPs + Ca(OH)2 mixtures and Ca(OH)2 alone at both observation periods (Figure 5). Moreover, several researchers have reported conflicting results regarding the cytotoxic/genotoxic impact of AgNP size on different cell lines.54, 55, 56, 57 A study by Souza et al58 indicated that due to the higher release of (Ag) ions from the surface of smaller AgNPs, 10 nm structures exhibited higher toxicity than 100 nm structures using the Comet assay. Interestingly, the same report displayed higher toxicity levels expressed by the 100 nm size using the Micronucleus assay. This was justified by the innate cellular tendency to preferentially endocytose larger particles ranging between 20 and 100 nm.58,59 Thus, the size-dependent cytotoxic/genotoxic influence of AgNPs exclusively exerted on the dental stem cell population remains elusive; further research is needed to optimize the size with the highest biocompatibility that permits the survival, proliferation, and differentiation of residing stem cells for a successful RET.

Fig. 5.

Fig 5

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Photographs of representative dentin segments treated with TAP (red), 2 nm AgNPs + 35% Ca(OH)2 (yellow), 5 nm AgNPs + 35% Ca(OH)2 (green), 10 nm AgNPs + 35% Ca(OH)2 (blue), 35% Ca(OH)2 alone (purple), and Saline (orange) for 1 week (1W) and 2 weeks (2W).

Conclusion

The mixture of 10 nm AgNPs (0.02%) + Ca(OH)2 exhibited the highest antibiofilm efficacy at 2 weeks compared to all other mixtures at both observation periods. Interestingly, the 10 nm mixture demonstrated a comparable antibiofilm activity to TAP at 2 weeks. Excluding Ca(OH)2 group, longer application significantly improved the antibiofilm efficacy of all tested medicaments. The results of this study suggest the possible application of the 10 nm AgNPs mixture in the regenerative endodontic treatment protocol after the assessment of its biocompatibility with the dental pulp stem cells population.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Acknowledgments

Ethical approval statement

This study was approved by the Institutional Review Board (IRB) at King Saud University (E-22-7126).

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions

Hanan Balto and Ghazal Fakeeha: Conceptualization, methodology. Ghazal Fakeeha: Software, data curation, formal analysis, investigation, resources, writing—original draft preparation, visualization, final approval of the version submitted. Hanan Balto: Supervision, project administration, writing—reviewing and editing, resources, visualization, final approval of the version submitted. Sarah Alharbi: Methodology, validation, reviewing and editing, final approval of the version submitted. Sayed Auda: Methodology, validation, reviewing and editing, final approval of the version submitted.

Acknowledgments

The authors acknowledge the Molecular and Cell Biology Laboratory-KSU-College of Dentistry, and Mr. Terrence Sumague for his technical support. This study is supported by Researchers Supporting Project Number (RSP2024R179), King Saud University, Riyadh, Saudi Arabia.

Data availability statement

The data that support the findings of this study are available from the corresponding author, (H. Balto), upon reasonable request.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.identj.2024.08.007.

Appendix. Supplementary materials

mmc1.docx (232.4KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (232.4KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, (H. Balto), upon reasonable request.

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