1Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey.
2Department of Biostatistics, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Copyright © 2021. The Korean Academy of Conservative Dentistry
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
1. In vitro studies performed on fully formed human permanent teeth
2. Teeth that had not received any endodontic treatment previously
3. Teeth contaminated with microorganisms
4. Studies comparing the efficacy of reciprocating and rotary instrumentation for the removal of microorganisms from root canals
5. Studies that quantified the antimicrobial effect and reported the outcome as reduction in microbial load
Studies | Tooth type | No. | Sterilization procedure | Preparation before contamination | Smear layer removal before contamination | Microorganism type | Incubation period following contamination | Confirmation of contamination |
---|---|---|---|---|---|---|---|---|
Alves et al. [3] | Mandibular incisors and maxillary second premolars with single root canals | 34 | Autoclave | 25 K-file | 17% EDTA and 2.5% NaOCl | E. faecalis strain (ATCC 29212) | 30 days | SEM |
Alves et al. [13] | Distobuccal canals of maxillary molars | 43 | Autoclave | Rotary instrument, size 10/0.04 | 17% EDTA | E. faecalis strain (ATCC 29212) | 30 days | Culture technique and SEM |
Basmaci et al. [14] | Mandibular premolars with single root canals | 81 | Autoclave | 20 K-file | NM | E. faecalis strain (ATCC 29212) | 24 hours | Culture technique |
Dagna et al. [15] | Single-rooted teeth | 60 | Autoclave | 20 K-file | 10% EDTA | E. faecalis strain (ATCC 19433) | 120 hours | Culture technique |
de Brito et al. [16] | Mandibular premolars | 100 | Autoclave | 20 K-file | NM | E. faecalis strain (ATCC 29212) | 28 days | Culture technique |
de Oliveira et al. [17] | Mandibular premolars | 60 | Autoclave | NM | NM | E. faecalis (ATCC 6057), P. aeruginosa (ATCC 27853), S. aureus (ATCC 29213) and C. albicans (ATCC 10231) | 48 hours | Culture technique |
Ferrer-Luque et al. [18] | Single-rooted mandibular premolars | 76 | Autoclave | 25 K-file | 17% EDTA and then irrigated with 1% NaOCl followed by DW | E. faecalis strain (ATCC 29212) | 4 weeks | Culture technique |
Guillen et al. [19] | Distobuccal canals of maxillary molars | 56 | Ethylene oxide | 15 K-file | 17% EDTA | E. faecalis strain (ATCC 29212) | 21 days | Culture technique |
Karatas et al. [20] | Mandibular incisor teeth | 70 | Autoclave | 20 K-file | NM | E. faecalis strain (ATCC 29212) | 48 hours | Culture technique |
Krokidis et al. [21] | Canines, lower incisors and premolars with single root canals | 50 | Autoclave | 25 K-file | 17% EDTA and 2.5% NaOCl | E. faecalis strain (ATCC 29212) | 30 days | Culture technique |
Machado et al. [4] | Distobuccal canals of maxillary molars | 65 | Ethylene oxide | 15 K-file | 17% EDTA and DW | E. faecalis strain (ATCC 29212) | 21 days | Culture technique |
Marinho et al. [9] | Mandibular premolars | 40 | Gamut radiation and autoclave | 15 K-file | 17% EDTA, 5.25% NaOCl and DW | E. coli strain (ATCC 25922) | 21 days | Culture technique and SEM |
Nabeshima et al. [22] | Distobuccal canals of maxillary molars | 51 | Ethylene oxide | 15 K-file | 17% EDTA and DW | E. faecalis strain (ATCC 29212) | 21 days | Culture technique |
Nakamura et al. [23] | Mandibular premolars | 50 | Autoclave | 30 K-file | 17% EDTA-T, 5.25% NaOCl and DW | E. faecalis strain (ATCC 29212) | 28 days | Culture technique and SEM |
Siqueira et al. [8] | Mesial canals of mandibular molars | 36 | Autoclave | 20 K-file | 17% EDTA and 2.5% NaOCl | E. faecalis strain ATCC 29212 | 30 days | Culture technique and SEM |
Üreyen Kaya et al. [24] | Mandibular premolars | 74 | Autoclave | NM | NM | E. faecalis | 4 weeks | Culture technique and SEM |
Vasconcelos et al. [25] | Mandibular incisors | 84 | Autoclave | 20 K-file | 1% NaOCl, 17% EDTA and saline | E. faecalis strain ATCC 29212 | 5 days | Culture technique and SEM |
Studies | Instrumentation systems tested (final apical diameter/taper) | Irrigation techniques and irrigants | Sampling time | Evaluation method | Main findings |
---|---|---|---|---|---|
Alves et al. [3] | Reciproc (40/0.06), BioRace (40/0.04) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU, qPCR | No difference was found between the instrumentation systems. |
Alves et al. [13] | Reciproc (25/0.08), XP-endo Shaper (30/0.04) | Saline | S1 and S2 | qPCR | XP-endo Shaper resulted in higher bacteria reduction. |
Basmacı et al. [14] | SAF (1.5 mm), Reciproc (25/0.08), ProTaper Universal (30/0.09) | a) PBS | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
b) 5% NaOCl and 15% EDTA | |||||
c) 5% NaOCl and 7% maleic acid | |||||
Dagna et al. [15] | Mtwo (30/0.05), Revo-S (25/0.06), Reciproc (25/0.08), OneShape (25/0.06) | 5.25% NaOCl and 17% EDTA | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
de Brito et al. [16] | ProTaper Next (40/0.06), ProTaper Universal (40/0.06), WaveOne Large (40/0.08) | a) 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU | WaveOne resulted in a lower level of bacterial reduction when saline solution was used. No difference was found among the instrumentation systems when NaOCl and EDTA were used. |
b) Saline | |||||
de Oliveira et al. [17] | ProTaper Universal (30/0.09), Reciproc (40/0.06) | a) 1% NaOCl | S1 and S2 | Presence/Absence | ProTaper Universal showed the best results when NaOCl was used. |
b) Saline | |||||
Ferrer-Luque et al. [18] | Mtwo (40/0.04), Twisted File (40/0.04), WaveOne (40/0.08) | a) DW | S1 and S2 and S3 (after 60 days) | CFU | No difference was found among the instrumentation systems after S2. Mtwo showed the best results when NaOCl was used at 60 days (S3). |
b) 5.25% NaOCl | |||||
Guillen et al. [19] | WaveOne Gold (25/0.07), WaveOne (25/0.08), One Shape New Generation (25/0.06), One Shape (25/0.06) | DW | S1 and S2 and S3 (after 7 days) | CFU | WaveOne Gold and One Shape New Generation promoted higher bacterial reduction than WaveOne and One Shape systems. |
Karatas et al. [20] | ProTaper Next (25/0.06), Twisted File Adaptive (25/0.06), SAF (1.5 mm), WaveOne (25/0.08), Reciproc (25/0.08), OneShape (25/0.06) | DW | S1 and S2 | CFU | No difference was found between the rotary and reciprocating instrumentation. |
Krokidis et al. [21] | BT-Race (40/0.04), WaveOne (40/0.08) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU | BT-RaCe resulted in higher bacteria reduction. |
Machado et al. [4] | WaveOne (25/0.08), Reciproc (25/0.08), ProTaper Universal (25/.08), Mtwo (25/0.06), K-file (35/0.02) | DW | S1 and S2 and S3 (after 7 days) | CFU | No difference was found among the instrumentation systems. |
Marinho et al. [9] | Reciproc (25/0.08), Mtwo (25/0.06), ProTaper Universal (25/0.08), Race (25/0.04) | Endotoxin-free water (LAL water) | S1 and S2 | CFU, LAL assay (for endotoxin reduction) | No difference was found among the instrumentation systems. |
Nabeshima et al. [22] | WaveOne (25/0.08), One Shape (25/0.06), K-file (35/0.02) | DW | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
Nakamura et al. [23] | K-file (50/0.02), Mtwo (50/0.04), Reciproc (50/0.05) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
Siqueira et al. [8] | Reciproc (25/0.08), SAF (1.5 mm), Twisted File (25/0.06) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU, PCR | No difference was found among the instrumentation systems. |
Üreyen Kaya et al. [24] | WaveOne Gold (25/0.07), Hyflex EDM One File (25/variable), XP-endo Shaper (30/0.04) | Saline | S1 and S2 | CFU | Hyflex EDM and XP-endo Shaper resulted in significantly greater bacteria reduction than WaveOne Gold. |
Vasconcelos et al. [25] | ProTaper Universal (25/0.08), BioRaCe (25/0.06), Reciproc (25/0.08) | Saline | S1 and S2 | CFU | ProTaper Universal was the most effective system in bacteria reduction. |
1. Was the calculation of the required minimum sample size performed before experiments?
2. Were the samples randomly distributed to groups?
3. Was specimen sterilization confirmed after the sterilization procedures?
4. Was specimen contamination confirmed after the procedure of root canal contamination with microorganisms?
5. Were the root canal preparation procedures performed by a single operator?
6. Was the total irrigant volume standard in all groups?
7. Were the analyses performed by evaluators blinded to the groups?
8. Were one or more outcomes of interest reported incompletely?
Studies | Sample size calculation | Teeth randomization | Confirmation of sterilization | Confirmation of contamination | Single operator | Standardization of total irrigant volume | Blinding of the evaluator | Complete outcome reporting | Risk of bias |
---|---|---|---|---|---|---|---|---|---|
Alves et al. [3] | N | N | N | Y | N | Y | N | Y | Moderate |
Alves et al. [13] | Y | Y | Y | Y | N | Y | N | Y | Low |
Basmaci et al. [14] | N | Y | N | Y | N | N | N | Y | Moderate |
Dagna et al. [15] | N | Y | Y | Y | Y | Y | N | N | Moderate |
de Brito et al. [16] | N | Y | Y | Y | Y | Y | N | Y | Low |
de Oliveira et al. [17] | N | Y | Y | Y | Y | N | N | Y | Moderate |
Ferrer-Luque et al. [18] | N | N | Y | Y | N | Y | N | Y | Moderate |
Guillen et al. [19] | Y | Y | Y | Y | N | Y | N | Y | Low |
Karatas et al. [20] | N | Y | N | Y | N | Y | N | N | Moderate |
Krokidis et al. [21] | N | Y | N | Y | Y | Y | N | Y | Moderate |
Machado et al. [4] | N | N | Y | Y | N | Y | N | Y | Moderate |
Marinho et al. [9] | N | Y | Y | Y | N | Y | N | Y | Moderate |
Nabeshima et al. [22] | N | Y | Y | Y | N | Y | N | Y | Moderate |
Nakamura et al. [23] | N | Y | Y | Y | N | Y | N | Y | Moderate |
Siqueira et al. [8] | N | Y | N | Y | N | Y | N | Y | Moderate |
Üreyen Kaya et al. [24] | N | Y | Y | Y | N | Y | N | Y | Moderate |
Vasconcelos et al. [25] | N | Y | Y | Y | Y | N | N | N | Moderate |
Conflict of Interest: No potential conflict of interest relevant to this article was reported.
Author Contributions:
Conceptualization: Küçükkaya Eren S, Uzunoğlu-Özyürek E.
Data curation: Küçükkaya Eren S, Uzunoğlu-Özyürek E, Karahan S.
Formal analysis: Küçükkaya Eren S, Uzunoğlu-Özyürek E, Karahan S.
Funding acquisition: Küçükkaya Eren S, Uzunoğlu-Özyürek E, Karahan S.
Investigation: Küçükkaya Eren S, Uzunoğlu-Özyürek E, Karahan S.
Methodology: Küçükkaya Eren S, Uzunoğlu-Özyürek E, Karahan S.
Project administration: Küçükkaya Eren S.
Resources: Küçükkaya Eren S, Uzunoğlu-Özyürek E, Karahan S.
Software: Karahan S.
Supervision: Küçükkaya Eren S.
Validation: Karahan S.
Visualization: Küçükkaya Eren S, Uzunoğlu-Özyürek E.
Writing - original draft: Küçükkaya Eren S.
Writing - review & editing: Küçükkaya Eren S, Uzunoğlu-Özyürek E, Karahan S.
No. | Search strategy | Results |
---|---|---|
1 | bacteria OR microbial OR microorganism OR microorganisms OR microbiota OR antibacterial OR antifungal OR antimicrobial OR CFU OR colony forming unit OR colony forming units OR PCR OR faecalis OR polymerase chain reaction OR toxin OR toxins OR infection | 6,358,577 |
2 | reciproc OR reciprocating OR reciprocal OR waveone | 59,954 |
3 | root canal OR root canals OR endodontic OR endodontics OR canal OR canals OR tooth OR teeth OR endodontology | 350,001 |
4 | #1 AND #2 AND #3 | 216 |
Studies | Tooth type | No. | Sterilization procedure | Preparation before contamination | Smear layer removal before contamination | Microorganism type | Incubation period following contamination | Confirmation of contamination |
---|---|---|---|---|---|---|---|---|
Alves et al. [ | Mandibular incisors and maxillary second premolars with single root canals | 34 | Autoclave | 25 K-file | 17% EDTA and 2.5% NaOCl | E. faecalis strain (ATCC 29212) | 30 days | SEM |
Alves et al. [ | Distobuccal canals of maxillary molars | 43 | Autoclave | Rotary instrument, size 10/0.04 | 17% EDTA | E. faecalis strain (ATCC 29212) | 30 days | Culture technique and SEM |
Basmaci et al. [ | Mandibular premolars with single root canals | 81 | Autoclave | 20 K-file | NM | E. faecalis strain (ATCC 29212) | 24 hours | Culture technique |
Dagna et al. [ | Single-rooted teeth | 60 | Autoclave | 20 K-file | 10% EDTA | E. faecalis strain (ATCC 19433) | 120 hours | Culture technique |
de Brito et al. [ | Mandibular premolars | 100 | Autoclave | 20 K-file | NM | E. faecalis strain (ATCC 29212) | 28 days | Culture technique |
de Oliveira et al. [ | Mandibular premolars | 60 | Autoclave | NM | NM | E. faecalis (ATCC 6057), P. aeruginosa (ATCC 27853), S. aureus (ATCC 29213) and C. albicans (ATCC 10231) | 48 hours | Culture technique |
Ferrer-Luque et al. [ | Single-rooted mandibular premolars | 76 | Autoclave | 25 K-file | 17% EDTA and then irrigated with 1% NaOCl followed by DW | E. faecalis strain (ATCC 29212) | 4 weeks | Culture technique |
Guillen et al. [ | Distobuccal canals of maxillary molars | 56 | Ethylene oxide | 15 K-file | 17% EDTA | E. faecalis strain (ATCC 29212) | 21 days | Culture technique |
Karatas et al. [ | Mandibular incisor teeth | 70 | Autoclave | 20 K-file | NM | E. faecalis strain (ATCC 29212) | 48 hours | Culture technique |
Krokidis et al. [ | Canines, lower incisors and premolars with single root canals | 50 | Autoclave | 25 K-file | 17% EDTA and 2.5% NaOCl | E. faecalis strain (ATCC 29212) | 30 days | Culture technique |
Machado et al. [ | Distobuccal canals of maxillary molars | 65 | Ethylene oxide | 15 K-file | 17% EDTA and DW | E. faecalis strain (ATCC 29212) | 21 days | Culture technique |
Marinho et al. [ | Mandibular premolars | 40 | Gamut radiation and autoclave | 15 K-file | 17% EDTA, 5.25% NaOCl and DW | E. coli strain (ATCC 25922) | 21 days | Culture technique and SEM |
Nabeshima et al. [ | Distobuccal canals of maxillary molars | 51 | Ethylene oxide | 15 K-file | 17% EDTA and DW | E. faecalis strain (ATCC 29212) | 21 days | Culture technique |
Nakamura et al. [ | Mandibular premolars | 50 | Autoclave | 30 K-file | 17% EDTA-T, 5.25% NaOCl and DW | E. faecalis strain (ATCC 29212) | 28 days | Culture technique and SEM |
Siqueira et al. [ | Mesial canals of mandibular molars | 36 | Autoclave | 20 K-file | 17% EDTA and 2.5% NaOCl | E. faecalis strain ATCC 29212 | 30 days | Culture technique and SEM |
Üreyen Kaya et al. [ | Mandibular premolars | 74 | Autoclave | NM | NM | E. faecalis | 4 weeks | Culture technique and SEM |
Vasconcelos et al. [ | Mandibular incisors | 84 | Autoclave | 20 K-file | 1% NaOCl, 17% EDTA and saline | E. faecalis strain ATCC 29212 | 5 days | Culture technique and SEM |
EDTA, ethylenediaminetetraacetic acid; SEM, scanning electron microscopy; DW, distilled water; NM: not mentioned.
Studies | Instrumentation systems tested (final apical diameter/taper) | Irrigation techniques and irrigants | Sampling time | Evaluation method | Main findings |
---|---|---|---|---|---|
Alves et al. [ | Reciproc (40/0.06), BioRace (40/0.04) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU, qPCR | No difference was found between the instrumentation systems. |
Alves et al. [ | Reciproc (25/0.08), XP-endo Shaper (30/0.04) | Saline | S1 and S2 | qPCR | XP-endo Shaper resulted in higher bacteria reduction. |
Basmacı et al. [ | SAF (1.5 mm), Reciproc (25/0.08), ProTaper Universal (30/0.09) | a) PBS | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
b) 5% NaOCl and 15% EDTA | |||||
c) 5% NaOCl and 7% maleic acid | |||||
Dagna et al. [ | Mtwo (30/0.05), Revo-S (25/0.06), Reciproc (25/0.08), OneShape (25/0.06) | 5.25% NaOCl and 17% EDTA | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
de Brito et al. [ | ProTaper Next (40/0.06), ProTaper Universal (40/0.06), WaveOne Large (40/0.08) | a) 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU | WaveOne resulted in a lower level of bacterial reduction when saline solution was used. No difference was found among the instrumentation systems when NaOCl and EDTA were used. |
b) Saline | |||||
de Oliveira et al. [ | ProTaper Universal (30/0.09), Reciproc (40/0.06) | a) 1% NaOCl | S1 and S2 | Presence/Absence | ProTaper Universal showed the best results when NaOCl was used. |
b) Saline | |||||
Ferrer-Luque et al. [ | Mtwo (40/0.04), Twisted File (40/0.04), WaveOne (40/0.08) | a) DW | S1 and S2 and S3 (after 60 days) | CFU | No difference was found among the instrumentation systems after S2. Mtwo showed the best results when NaOCl was used at 60 days (S3). |
b) 5.25% NaOCl | |||||
Guillen et al. [ | WaveOne Gold (25/0.07), WaveOne (25/0.08), One Shape New Generation (25/0.06), One Shape (25/0.06) | DW | S1 and S2 and S3 (after 7 days) | CFU | WaveOne Gold and One Shape New Generation promoted higher bacterial reduction than WaveOne and One Shape systems. |
Karatas et al. [ | ProTaper Next (25/0.06), Twisted File Adaptive (25/0.06), SAF (1.5 mm), WaveOne (25/0.08), Reciproc (25/0.08), OneShape (25/0.06) | DW | S1 and S2 | CFU | No difference was found between the rotary and reciprocating instrumentation. |
Krokidis et al. [ | BT-Race (40/0.04), WaveOne (40/0.08) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU | BT-RaCe resulted in higher bacteria reduction. |
Machado et al. [ | WaveOne (25/0.08), Reciproc (25/0.08), ProTaper Universal (25/.08), Mtwo (25/0.06), K-file (35/0.02) | DW | S1 and S2 and S3 (after 7 days) | CFU | No difference was found among the instrumentation systems. |
Marinho et al. [ | Reciproc (25/0.08), Mtwo (25/0.06), ProTaper Universal (25/0.08), Race (25/0.04) | Endotoxin-free water (LAL water) | S1 and S2 | CFU, LAL assay (for endotoxin reduction) | No difference was found among the instrumentation systems. |
Nabeshima et al. [ | WaveOne (25/0.08), One Shape (25/0.06), K-file (35/0.02) | DW | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
Nakamura et al. [ | K-file (50/0.02), Mtwo (50/0.04), Reciproc (50/0.05) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU | No difference was found among the instrumentation systems. |
Siqueira et al. [ | Reciproc (25/0.08), SAF (1.5 mm), Twisted File (25/0.06) | 2.5% NaOCl and 17% EDTA | S1 and S2 | CFU, PCR | No difference was found among the instrumentation systems. |
Üreyen Kaya et al. [ | WaveOne Gold (25/0.07), Hyflex EDM One File (25/variable), XP-endo Shaper (30/0.04) | Saline | S1 and S2 | CFU | Hyflex EDM and XP-endo Shaper resulted in significantly greater bacteria reduction than WaveOne Gold. |
Vasconcelos et al. [ | ProTaper Universal (25/0.08), BioRaCe (25/0.06), Reciproc (25/0.08) | Saline | S1 and S2 | CFU | ProTaper Universal was the most effective system in bacteria reduction. |
CFU, colony forming unit; DW, distilled water; EDTA, ethylenediaminetetraacetic acid; NM, not mentioned; NaOCl, sodium hypochlorite; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; qPCR, quantitative polymerase chain reaction; S1: Sampling after cavity preparation immediately before root canal preparation; S2, Sampling immediately after root canal preparation; S3, Sampling after a period of time following root canal preparation (for regrowth evaluation).
Studies | Sample size calculation | Teeth randomization | Confirmation of sterilization | Confirmation of contamination | Single operator | Standardization of total irrigant volume | Blinding of the evaluator | Complete outcome reporting | Risk of bias |
---|---|---|---|---|---|---|---|---|---|
Alves et al. [ | N | N | N | Y | N | Y | N | Y | Moderate |
Alves et al. [ | Y | Y | Y | Y | N | Y | N | Y | Low |
Basmaci et al. [ | N | Y | N | Y | N | N | N | Y | Moderate |
Dagna et al. [ | N | Y | Y | Y | Y | Y | N | N | Moderate |
de Brito et al. [ | N | Y | Y | Y | Y | Y | N | Y | Low |
de Oliveira et al. [ | N | Y | Y | Y | Y | N | N | Y | Moderate |
Ferrer-Luque et al. [ | N | N | Y | Y | N | Y | N | Y | Moderate |
Guillen et al. [ | Y | Y | Y | Y | N | Y | N | Y | Low |
Karatas et al. [ | N | Y | N | Y | N | Y | N | N | Moderate |
Krokidis et al. [ | N | Y | N | Y | Y | Y | N | Y | Moderate |
Machado et al. [ | N | N | Y | Y | N | Y | N | Y | Moderate |
Marinho et al. [ | N | Y | Y | Y | N | Y | N | Y | Moderate |
Nabeshima et al. [ | N | Y | Y | Y | N | Y | N | Y | Moderate |
Nakamura et al. [ | N | Y | Y | Y | N | Y | N | Y | Moderate |
Siqueira et al. [ | N | Y | N | Y | N | Y | N | Y | Moderate |
Üreyen Kaya et al. [ | N | Y | Y | Y | N | Y | N | Y | Moderate |
Vasconcelos et al. [ | N | Y | Y | Y | Y | N | N | N | Moderate |
EDTA, ethylenediaminetetraacetic acid; SEM, scanning electron microscopy; DW, distilled water; NM: not mentioned.
CFU, colony forming unit; DW, distilled water; EDTA, ethylenediaminetetraacetic acid; NM, not mentioned; NaOCl, sodium hypochlorite; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; qPCR, quantitative polymerase chain reaction; S1: Sampling after cavity preparation immediately before root canal preparation; S2, Sampling immediately after root canal preparation; S3, Sampling after a period of time following root canal preparation (for regrowth evaluation).