Comparison of the Antibacterial Effect of Nano-calcium Hydroxide and Nano-chlorhexidine Against Enterococcus Faecalis

Authors

  • Mamak Adel Department of Endodontics, Dental Cariers Prevention Research Center, Qazvin University of Medical Siences, Qazvin, Iran
  • Zohreh Asgari Department of Endodontics, School of Dentristry, Qavin University of Medical Sciences, Qazvin, Iran
  • Neda Hajihassani Department of Endodontics, School of Dentristry, Qavin University of Medical Sciences, Qazvin, Iran
  • Zohreh Bahrami Department of Nanotechnology, Faculty of New Sciences and Technology, Semnan University, Semnan, Iran
  • Mehdi Ranjbaran Non-communicanle Diseases Research Center, Research Institute for Prevention of Non-communicable Diseases, Qavin University of Medical Sciences, Qazvin, Iran

Keywords:

Dentinal Tubule Disinfection, Enterococcus Faecalis, Intracanal Medicament, Nano-chlorhexidine Gel, Nano-calcium Hydroxide

Abstract

Background: Intracanal medicaments are used to eliminate residual microorganisms and provide a suitable environment for the healing of periapical tissues. This study aimed to compare the antibacterial effect of nano-calcium hydroxide and nano-chlorhexidine as intracanal medicaments against Enterococcus faecalis. Materials and Methods: In this study, 170 single-rooted mandibular premolars were included. After crown removal, canal preparation was performed. Five teeth were randomly selected as the negative control group. Nano-intracanal medicaments were synthesized using the encapsulation method. Drug release tests were carried out, and particle sizes were determined using electron microscopy and dynamic light scattering analysis. Samples were inoculated with E. faecalis for 21 days. Subsequently, five teeth were randomly selected as the positive control group. The remaining samples were randomly divided into four groups of 40 teeth each (20 for day 3 and 20 for day 7). The intracanal medicaments used in each group were: calcium hydroxide, nano-Ca(OH)2-PLGA, 2% chlorhexidine gel, and 2% nano-chlorhexidine gel (MCM-41), respectively. After 3 and 7 days, microbial biofilm samples were collected, and bacterial colony-forming units (CFU) were counted. Two-way ANOVA was used to compare the mean CFU counts among the groups at different time intervals. The significance level was set at 0.05. Results: Based on statistical analysis, on day 3, the 2% nano-chlorhexidine gel (MCM-41) and nano-Ca(OH)2-PLGA groups showed a significantly greater reduction in CFU/ml (P<0.05) compared to the other groups. On day 7, the 2% nano-chlorhexidine gel (MCM-41) and 2% chlorhexidine gel groups demonstrated significantly greater reductions in CFU/ml (P<0.05) than the remaining groups. Over time, the antibacterial efficacy of the nano-formulations declined, whereas the effectiveness of the 2% chlorhexidine gel and calcium hydroxide increased. Conclusion: According to the findings of the present study, nano-formulated medicaments were more effective in eliminating E. faecalis biofilm from the root canal only in short-short term.

References

RossiFedele G, Roberts AP. A preliminary study investigating the survival of tetracyclineresistant Enterococcus Faecalis after root canal irrigation with high concentrations of tetracycline. Int Endod J. 2007;40(10):772-7.

https://doi.org/10.1111/j.1365-2591.2007.01285.x

PMid:17697106

Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. Journal of endodontics. 2006 Feb 1;32(2):938.

https://doi.org/10.1016/j.joen.2005.10.049

PMid:16427453

BYSTRÖM A, SUNDQVIST G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. European Journal of Oral Sciences. 1981 Aug;89(4):3218.

https://doi.org/10.1111/j.1600-0722.1981.tb01689.x

PMid:6947391

Scand J Dent Res 1981;89:3218. Card SJ, Sigurdsson A, Orstavik D, Trope M. The effectiveness of increased apical enlargement in reducing intracanal bacteria. J Endod 2002; 28: 779-83.

https://doi.org/10.1097/00004770-200211000-00008

PMid:12470024

Siqueira Jr JF, Rôças IN, Santos SR, Lima KC, Magalhães FA, de Uzeda M. Efficacy of instrumentation techniques and irrigation regimens in reducing the bacterial population within root canals. Journal of endodontics. 2002 Mar 1;28(3):1814.

https://doi.org/10.1097/00004770-200203000-00009

PMid:12017176

Dutta B, Dhull KS, Das D, Samir PV, Verma RK, Singh N. Evaluation of antimicrobial efficacy of various intracanal medicaments in primary teeth: an in vivo study. International journal of clinical pediatric dentistry. 2017 Feb 27;10(3):267.

https://doi.org/10.5005/jp-journals-10005-1448

PMid:29104387 PMCid:PMC5661041

Vasudeva A, Sinha DJ, Tyagi SP, Singh NN, Garg P, Upadhyay D. Disinfection of dentinal tubules with 2% Chlorhexidine gel, Calcium hydroxide and herbal intracanal medicaments against Enterococcus faecalis: An invitro study. Singapore dental journal. 2017 Dec 1;38:3944.

https://doi.org/10.1016/j.sdj.2017.06.001

PMid:29229073

Komabayashi T, D'souza RN, Dechow PC, Safavi KE, Spångberg LS. Particle size and shape of calcium hydroxide. Journal of endodontics. 2009 Feb 1;35(2):2847.

https://doi.org/10.1016/j.joen.2008.11.017

PMid:19166791 PMCid:PMC2656936

Haapasalo HK, Sirén EK, Waltimo TM, Orstavik D, Haapasalo MP. Inactivation of local root canal medicaments by dentine: an in vitro study. International endodontic journal. 2000 Mar;33(2):12631.

https://doi.org/10.1046/j.1365-2591.2000.00291.x

PMid:11307453

Siqueira JF, Rôças IN, Ricucci D. Biofilms in endodontic infection. Endodontic Topics. 2010 Mar;22(1):3349.

https://doi.org/10.1111/j.1601-1546.2012.00279.x

Kayaoglu G, Orstavik D. Virulence factors of Enterococcus Faecalis: relationship to endodontic disease. Crit Rev Oral Biol Med. 2004;15(5):30820.

https://doi.org/10.1177/154411130401500506

PMid:15470268

Gomes BP, Ferraz CC, ME V, Berber VB, Teixeira FB, Souza‐Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. International endodontic journal. 2001 Sep;34(6):4248.

https://doi.org/10.1046/j.1365-2591.2001.00410.x

PMid:11556507

Mohammadi Z, Abbott P. The properties and applications of chlorhexidine in endodontics. International Endodontic Journal. 2009;42(4):288302.

https://doi.org/10.1111/j.1365-2591.2008.01540.x

PMid:19220510

Wu D, Fan W, Kishen A, Gutmann JL, Fan B. Evaluation of the antibacterial efficacy of silver nanoparticles against Enterococcus Faecalis biofilm. Journal of Endodontics. 2014 Feb 1;40(2):28590.

https://doi.org/10.1016/j.joen.2013.08.022

PMid:24461420

Parolia A, Kumar H, Ramamurthy S, Davamani F, Pau A. Effectiveness of chitosanpropolis nanoparticle against Enterococcus faecalis biofilms in the root canal. BMC Oral Health. 2020 Nov 25;20(1):339.

https://doi.org/10.1186/s12903-020-01330-0

PMid:33238961 PMCid:PMC7690148

Balto H, Bukhary S, AlOmran O, BaHammam A, AlMutairi B. Combined Effect of a Mixture of Silver Nanoparticles and Calcium Hydroxide against Enterococcus Faecalis Biofilm. J Endod. 2020 Nov;46(11):16891694.

https://doi.org/10.1016/j.joen.2020.07.001

PMid:32679241

Afkhami F, Rostami G, Batebi S, Bahador A. Residual antibacterial effects of a mixture of silver nanoparticles/calcium hydroxide and other root canal medicaments against Enterococcus faecalis. Journal of Dental Sciences. 2022 Jul 1;17(3):12605.

https://doi.org/10.1016/j.jds.2021.11.013

PMid:35784127 PMCid:PMC9236898

SanchezLopez E, Ettcheto M, Egea MA, Espina M, Calpena AC, Folch J, Camins A, García ML. New potential strategies for Alzheimer's disease prevention: pegylated biodegradable dexibuprofen nanospheres administration to APPswe/PS1dE9. Nanomedicine: Nanotechnology, Biology and Medicine. 2017 Apr 1;13(3):117182.

https://doi.org/10.1016/j.nano.2016.12.003

PMid:27986603

SanchezLopez E, Ettcheto M, Egea MA, Espina M, Calpena AC, Folch J, Camins A, García ML. New potential strategies for Alzheimer's disease prevention: pegylated biodegradable dexibuprofen nanospheres administration to APPswe/PS1dE9. Nanomedicine: Nanotechnology, Biology and Medicine. 2017 Apr 1;13(3):117182.

https://doi.org/10.1016/j.nano.2016.12.003

PMid:27986603

Zhang Y, Huo M, Zhou J, Xie S. PKSolver: An addin program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Computer methods and programs in biomedicine. 2010 Sep 1;99(3):30614.

https://doi.org/10.1016/j.cmpb.2010.01.007

PMid:20176408

Montrucchio G, Corcione S, Sales G, Curtoni A, De Rosa F, Brazzi L. Carbapenemresistant Klebsiella pneumoniae in ICUadmitted COVID19 patients: keep an eye on the ball. J Global Antimicrob Resist. 2020;23:398-400.

https://doi.org/10.1016/j.jgar.2020.11.004

PMid:33242674 PMCid:PMC7682477

Ashima Nadar, Vidya Saraswathi Muliya, Swathi Pai, Kalyana Chakravarthy Pentapati. A comparative evaluation of calcium ion release and pH change using calcium hydroxide nanoparticles as intracanal medicament with diÓerent vehicles - An in vitro study. J Conserv Dent. 2022 Dec 8;26(1):47-51.

https://doi.org/10.4103/jcd.jcd_387_22

PMid:36908721 PMCid:PMC10003273

Vasudeva A, Sinha DJ, Tyagi SP, Singh NN, Garg P, Upadhyay D. Disinfection of dentinal tubules with 2% Chlorhexidine gel, Calcium hydroxide and herbal intracanal medicaments against Enterococcus faecalis: An invitro study. Singapore dental journal. 2017 Dec 1;38:3944.

https://doi.org/10.1016/j.sdj.2017.06.001

PMid:29229073

Chockattu SJ, Deepak B, Goud KM. Comparison of antibacterial efficiency of ibuprofen, diclofenac, and calcium hydroxide against Enterococcus Faecalis in an endodontic model: an in vitro study. Journal of conservative dentistry: JCD. 2018;21(1):80.

Batinić M, Ročan M, Budimir A, Anić I, Bago I. Comparison of final disinfection protocols using antimicrobial photodynamic therapy and different irrigants after singlefile reciprocating instrumentation against intracanal bacterial biofilm-An in vitro study. Photodiagnosis and photodynamic therapy. 2018 Dec 1;24:1537.

https://doi.org/10.1016/j.pdpdt.2018.10.006

PMid:30308309

Varshini R, Subha A, Prabhakar V, Mathini P, Narayanan S, Minu K. Antimicrobial efficacy of Aloe vera, lemon, Ricinus communis, and calcium hydroxide as intracanal medicament against Enterococcus faecalis: a confocal microscopic study. Journal of Pharmacy and Bioallied Sciences. 2019 May 1;11(Suppl 2):S2569.

https://doi.org/10.4103/JPBS.JPBS_5_19

PMid:31198348 PMCid:PMC6555306

Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, Souza‐Filho FJ. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. International endodontic journal. 2003 Apr 1;36(4):1.

https://doi.org/10.1046/j.1365-2591.2003.00634.x

PMid:12702121

Basrani B, Tjäderhane L, Santos JM, Pascon E, Grad H, Lawrence HP, Friedman S. Efficacy of chlorhexidineand calcium hydroxide-containing medicaments against Enterococcus faecalis in vitro. Oral surgery, oral medicine, oral Pathology, oral Radiology, and endodontology. 2003 Nov 1;96(5):61824.

https://doi.org/10.1016/S1079-2104(03)00166-5

PMid:14600699

Seneviratne CJ, Leung KC, Wong CH, Lee SF, Li X, Leung PC, Lau CB, Wat E, Jin L. Nanoparticleencapsulated chlorhexidine against oral bacterial biofilms. PloS one. 2014 Aug 29;9(8):e103234.

https://doi.org/10.1371/journal.pone.0103234

PMid:25170958 PMCid:PMC4149348

Soni KS, Desale SS, Bronich TK. Nanogels: An overview of properties, biomedical applications, and obstacles to clinical translation. J Control Release. 2016; 240:109126.

https://doi.org/10.1016/j.jconrel.2015.11.009

PMid:26571000 PMCid:PMC4862943

Qureshi MA, Khatoon F. Different types of smart nanogel for targeted delivery. Journal of Science: Advanced Materials and Devices. 2019 Jun 1;4(2):20112.

https://doi.org/10.1016/j.jsamd.2019.04.004

Kazemi F, Divsalar A, Saboury AA. Structural analysis of the interaction between free, glycated, and fructated hemoglobin with propolis nanoparticles: a spectroscopic study. Int J Biol Macromol. 2018;109:1329-37.

https://doi.org/10.1016/j.ijbiomac.2017.11.143

PMid:29175521

Ahmed KS, Hussein SA, Ali AH, Korma SA, Lipeng Q, Jinghua C. Liposome: composition, characterisation, preparation, and recent innovation in clinical applications. Journal of drug targeting. 2019 Aug 9;27(7):74261.

https://doi.org/10.1080/1061186X.2018.1527337

PMid:30239255

Wilczewska AZ, Niemirowicz K, Markiewicz KH, Car H. Nanoparticles as drug delivery systems. Pharmacological reports. 2012 Sep 1;64(5):102037.

https://doi.org/10.1016/S1734-1140(12)70901-5

PMid:23238461

Aslani A, Malekpour N. Design, formulation, and physicochemical evaluation of periodontal propolis mucoadhesive gel. Dental Research Journal. 2016 Nov;13(6):484.

https://doi.org/10.4103/1735-3327.197037

PMid:28182054 PMCid:PMC5256011

Wang Y, Fang L, Wang P, Qin L, Jia Y, Cai Y, Liu F, Zhou H, Wang S. Antibacterial effects of silica nanoparticles loading nanosilver and chlorhexidine in root canals infected by enterococcus faecalis. Journal of Endodontics. 2025 Jan 1;51(1):5463.

https://doi.org/10.1016/j.joen.2024.11.004

PMid:39547456

Tülü G, Kaya BÜ, Çetin ES, Köle M. Antibacterial effect of silver nanoparticles mixed with calcium hydroxide or chlorhexidine on multispecies biofilms. Odontology. 2021 Oct;109(4):80211.

https://doi.org/10.1007/s10266-021-00601-8

PMid:34047872

Sireesha A, Jayasree R, Vidhya S, Mahalaxmi S, Sujatha V, Kumar TS. Comparative evaluation of micronand nanosized intracanal medicaments on penetration and fracture resistance of root dentin-An in vitro study. International journal of biological macromolecules. 2017 Nov 1;104:186673.

https://doi.org/10.1016/j.ijbiomac.2017.05.126

PMid:28545965

Elmsmari F, Delgado LM, DuranSindreu F, Pérez RA, García ML, Trull MT, Afrashtehfar KI, González JA, SánchezLópez E. Novel strategies enhancing endodontic disinfection: Antibacterial biodegradable calcium hydroxide nanoparticles in an ex vivo model. International journal of pharmaceutics. 2023 Dec 15;648:123627.

https://doi.org/10.1016/j.ijpharm.2023.123627

PMid:37984620

Elmsmari F, Delgado LM, DuranSindreu F, Pérez RA, García ML, Teulé Trull M, Afrashtehfar KI, González JA, SánchezLópez E. Novel strategies enhancing endodontic disinfection: antibacterial biodegradable calcium hydroxide nanoparticles in an ex vivo model. Int J Pharm. 2023;648:123627.

https://doi.org/10.1016/j.ijpharm.2023.123627

PMid:37984620

Shirur KS, Padya BS, Pandey A, Hegde MM, Narayan AI, Rao BS, Bhat VG, Mutalik S. Development of lipidic nanoplatform for intraoral delivery of chlorhexidine: characterization, biocompatibility, and assessment of depth of penetration in extracted human teeth. Nanomaterials. 2022 Sep 27;12(19):3372.

https://doi.org/10.3390/nano12193372

PMid:36234500 PMCid:PMC9565570

Wu HQ, Wang CC. Biodegradable smart nanogels: A new platform for targeting drug delivery and biomedical diagnostics. Langmuir. 2016; 32:62116225.

https://doi.org/10.1021/acs.langmuir.6b00842

PMid:27255455

Ruparel NB, Teixeira FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. Journal of endodontics. 2012 Oct 1;38(10):13725.

https://doi.org/10.1016/j.joen.2012.06.018

PMid:22980180

Downloads

Published

2026-05-17

How to Cite

Adel , M., Asgari , Z., Hajihassani, N., Bahrami, Z., & Ranjbaran, M. (2026). Comparison of the Antibacterial Effect of Nano-calcium Hydroxide and Nano-chlorhexidine Against Enterococcus Faecalis. Galen Medical Journal, 15, e4023. Retrieved from https://journals.salviapub.com/index.php/gmj/article/view/4023

Issue

Vol. 15 (2026): 2026: In Progress

Section

Original Article

License

Copyright (c) 2026 Galen Medical Journal

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

https://creativecommons.org/licenses/by/4.0/