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Year : 2014  |  Volume : 25  |  Issue : 4  |  Page : 535-538
Novel in vitro methodology for induction of Enterococcus faecalis biofilm on apical resorption areas

1 Department of Restorative Dentistry, Institute of Science and Technology of Sao Jose dos Campos Dental School, Sao Paulo State University, India
2 Department of Microbiology, Institute of Science and Technology of Sao Jose dos Campos Dental School, Sao Paulo State University, India

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Date of Submission07-Feb-2014
Date of Decision03-Mar-2014
Date of Acceptance17-Jun-2014
Date of Web Publication10-Oct-2014


Context: Teeth with periapical lesion usually present external root resorption around the apical foramen. These areas facilitate adhesion and co-aggregation of microorganisms developing biofilms. Up to the present moment, there is no methodology in the literature that enables the in vitro evaluation of endodontic irrigants and intracanal dressings on biofilms located in apical external root resorptions of human teeth.
Aims: This study aimed to describe a new in vitro methodology for Enterococcus faecalis biofilm development in external apical reportion areas of human extracted teeth in different periods of time.
Settings and Design: In vitro qualitative laboratory study.
Subjects and Methods: Thirty roots from human extracted teeth presenting external apical resorption had their root canal diameters standardized by means of instrumentation. Next, the roots were randomly divided into three groups (n = 30) according to E. faecalis strains (ATCC 29212) exposure time as follows: Group T5, with 5-day exposure; Group T10, with 10-day exposure, and Group T15, with 15-day exposure. The roots were attached to 24-well culture plates so that only their apices could be in contact with bacteria for induction of biofilm formation. At the end of these exposure times, the roots were qualitatively evaluated with scanning electron microscope to observe the presence of biofilm in external resorptions around the apical foramen.
Results: It was found that microorganisms were present in all exposure times, although structures suggesting the presence of biofilm with great conglomerate of bacteria showing structures similar to polysaccharide extensions were observed at the 10 th day of exposure.
Conclusions: By means of this new methodology, it was possible to observe biofilm formation in the areas of external apical resorption after 10 days of exposure.

Keywords: Biofilm, Enterococcus faecalis, external resorption

How to cite this article:
Albuquerque MT, Junqueira JC, Coelho MB, de Carvalho CT, Valera MC. Novel in vitro methodology for induction of Enterococcus faecalis biofilm on apical resorption areas. Indian J Dent Res 2014;25:535-8

How to cite this URL:
Albuquerque MT, Junqueira JC, Coelho MB, de Carvalho CT, Valera MC. Novel in vitro methodology for induction of Enterococcus faecalis biofilm on apical resorption areas. Indian J Dent Res [serial online] 2014 [cited 2023 Mar 30];25:535-8. Available from:
The microbial biofilm is one of the main causes of unsuccessful endodontic treatment, since it is located in regions of difficult access for procedures of root canal decontamination. [1] Among these regions, root resorption areas observed in the external surface of the apical portion of periapically compromised teeth, may be considered an important niche for accumulation of microorganisms. [2]

Microorganisms can survive chemo-mechanical preparation as well as intracanal medications. They become resistant and capable of surviving in adverse conditions [3],[4] due to efficient survival mechanisms. [4],[5],[6] Most of these microorganisms are located in the apical third of the root canal. [7] They can reach the resorption areas surrounding the apical foramen in the root external surface and organize themselves into microbial biofilm. This is one of the factors contributing to the failure in the endodontic treatment. [8]

Several in vitro studies have evaluated the efficacy of disinfection methods used in the endodontic treatment to eliminate bacterial biofilm. In this context, biofilms are developed in wells of culture plates, glass substrates, membrane filters, polystyrene plates, hydroxyapatite discs, and root dentin. [3],[5],[9],[10],[11],[12],[13],[14],[15],[16],[17] However, in general, these models do not reproduce anatomical irregularities such as isthmus and apical resorption. In these regions, biofilms can protect themselves from the effects of disinfecting agents. [18] Therefore, it is very important to develop methodologies using human extracted teeth in order to enable in vitro biofilm formation in areas of external apical resorption, thus simulating in vivo conditions in the cases of teeth with periapical lesion and allowing disinfection methods to be tested.

In addition, due to the difficulties in creating an environment that allows biofilms to grow within root canals, there are few studies evaluating biofilm formation inside root canal and at the external root surface and the majority of the literature have used only one bacterial species (i.e. Enterococcus faecalis) for in vitro biofilm formation. [5],[19] Therefore, this study aimed to describe a new methodology that provides apical biofilm formation by E. faecalis in external apical resorption areas in human extracted teeth in different periods of time.

   Subjects and methods Top

Thirty roots from human extracted teeth presenting external apical resorption were used in the study. Teeth were stored in 10% formaldehyde solution for 48 h before being left in 1% solution of sodium hypochlorite for further 24 h in order to promote dissolution of the tissue rests adhered to dental surfaces. Next, the teeth were washed and brushed under tap water for complete removal of tissue remnants and sodium hypochlorite solution.

The teeth had their roots analyzed by using a stereomicroscope magnifying glass (Zeiss Stemi 2000-C, Gottingen, Germany) with Χ5 magnification and by scanning electron microscope (SEM) (JEOL-Modelo JSM T330A, Tokyo, Japan) [Figure 1] to observe the presence of erosions around the apical foramen, characterizing areas of external resorption. After this initial evaluation, the teeth had their clinical crowns sectioned at the cementum-enamel junction using a carborundum disc mounted on a hand piece, resulting in roots with standardized lengths of 13 ± 0.5 mm.
Figure 1: Images of external apical root surface: (a) Stereomicroscopic image of the external apical resorption at ×5 magnification; (b) scanning electron microscope showing resorption in apical area

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The selected samples were submitted to rotary instrumentation (MTwo, VDW, Munich, Germany) for the standardization of their root canal diameter so that the samples had a final apical diameter of 0.40 mm and taper of 0.07 mm/mm. At the end of the preparation, the root canals were irrigated with 17% ethylene diamine tetraacetic acid (EDTA) solution (Inodon Ind. Edit. Impe Exp. de Produtos Odontolσgicos, Porto Alegre, RS, Brazil) for 3 min, which was stirred with a Kerr no. 20 file (Dentsply/Maillefer, Petrσpolis, Rio de Janeiro, Brazil). The roots were then irrigated with 5 mL of saline solution in order to neutralize and remove the EDTA solution as well as the residues produced during instrumentation. Next, the root canals were dried with paper points (Dentsply/Maillefer, Petrσpolis, Brazil) and then attached to 24-well culture plates (Costar Corning, New York, USA) before being randomly distributed into experimental groups.

Attaching the roots to the 24-well culture plates

The roots were randomly distributed into 24-well culture plates (Costar Corning, New York, USA), with 10 specimens in each plate. Three 24-well cell plates (Costar, New York, USA) were used for mounting each set of plates. The cover of the other plates had their edges cut by using a carbide cylindrical bur (No. 557, Beavers Jet Burs, Canada) mounted on a counter-angle head, forming a platform for supporting the roots. Next, orifices were made in the circles corresponding to each well in the modified cover by using the same bur (No. 557, Beavers Jet Burs, Canada). Each orifice was then enlarged until the root was accordingly fitted. This procedure was followed for all roots. After fitting all the roots into the orifices, the modified cover was placed on the wells and then sealed with other 24-well culture plate's cover. On the whole, six sets of plates were sent for sterilization by gamma radiation with cobalt-60

(20 KGy for 6 h), since plastic materials are deformed by the heat of autoclave or greenhouse.

Experimental groups

After preparing the root canals, the samples were randomly divided into three experimental groups according to the exposure period of the areas of external apical resorption to microorganisms [Table 1].
Table 1: Experimental groups division

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Biofilm formation

The roots mounted on the 24-well culture plates (Costar Corning, New York, USA) were exposed to

E. faecalis (ATCC 29212) for biofilm formation in the areas of external apical resorption. In a new 24-well culture plate (Costar Corning, New York, USA), 1800 μL of brain heart infusion (BHI) broth with 5% saccharose and 200 μL of a standardized E. faecalis suspension containing 10 6 cells/mL were placed into the wells. Approximately, 3 mm of the apical third of each root was in contact with the E. faecalis suspension in the wells to induce biofilm formation. The plates were sealed with their respective covers and were incubated at 37°C for the period of time corresponding to each experimental group (5, 10, and 15 days) under agitation at 75 rpm (Quimis, Diadema, SP, Sγo Paulo, Brazil) [Figure 2]. The growth medium was replaced with fresh BHI broth (BHI, Difco, Detroit, USA) every 2 days to avoid nutrient depletion and accumulation of toxic products.
Figure 2: Induction of biofilm formation: (a) Insertion of brain heart infusion broth and Enterococcus faecalis suspension; (b) modified cover containing the roots positioned in the wells; (c) approximately 3 mm of the apical third in contact with the suspension; (d) shaker incubator

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Analysis with scanning electron microscope

For SEM analysis, three of the ten roots of each experimental group were randomly chosen. The samples were fixed in 2.5% glutaraldehyde solution for 2 h before being dehydrated in solutions of alcohol at ascending concentrations (10%, 25%, 50%, 70%, 90%, and 100%) through successive 20 min immersions in each solution. Next, the roots were washed with distilled water and dried in the stove at 37°C ± 1°C for 24 h. With their apices positioned upward, their sectioned bases were attached to 30 mm diameter stubs with double-faced adhesive carbon tapes. Next, the roots were metalized in a vacuum chamber (Desk II-Denton Vacuum-Buffalo, New Jersey, USA) for gold sputtering during 120 s. A SEM (JEOL-Model JSM T330A, Tokyo, Japan) was used for analysis of the areas of external apical resorption in which the presence of biofilm was verified in all the experimental groups. Disposition, extension, and morphology were qualitatively evaluated at the apical region with the SEM images being transferred to the Windows Paint (image editor) for evaluation.

   Results Top

Images from SEM showed that the amount of microorganisms had increased proportionally to the exposure time of the external apical root surface. Therefore, in a group where areas of apical resorption were exposed to microorganisms for 5-day (Group T5), it was not possible to observe an organization of microbial biofilm because of the small amount of E. faecalis [Figure 3]. After 10 days of exposure (Group T10), it was possible to observe a great conglomerate of bacteria showing structures similar to polysaccharide extensions, thus suggesting the presence of biofilm (→). After 15 days of exposure (Group T15), it was observed a larger and more organized conglomerate of bacteria compared to Group T10, also showing structures suggestive of the presence of biofilm (→). The images were compared, and it was observed that 10-day microbial exposure was sufficient to present structures suggesting the presence of biofilm.
Figure 3: Scanning electron microscope images: (a) 5-day biofilm; (b) 10-day biofilm, with presence of extensions suggestive of polysaccharide matrix (arrow); (c) 15-day biofilm, with presence of extensions suggestive of polysaccharide matrix (arrow) and great agglomerate of bacteria

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   Discussion Top

Biofilm formation is regulated by three main steps: (1) Adsorption of macromolecules to the surface in the planktonic phase, leading to the formation of a conditioning pellicle consisting of proteins, glycoproteins, and some microbial byproducts, which is selective for certain types of microorganisms; (2) Adhesion and co-adhesion of microorganisms, which may be reinforced by the production of polymers, leading to formation of micro-colonies. Streptococcus are usually the first microorganisms to adhere to the newly-formed pellicle; (3) Multiplication and metabolism of the adhered microorganisms while they organize themselves, resulting in a mature biofilm. [20],[21] In general, it consists of a highly organized structure of micro-colonies embedded in a matrix with open canals to allow water entry and distribution of nutrients into the biofilm. [13],[22]

Once formed, the biofilm is often found in adverse environments and the bacteria composing it can survive because they resist to the stress of these environments. [5],[23],[24] Some places contribute to the agglomeration of microorganisms and consequently formation of microbial biofilm. Areas of resorption present in the external root surface, mainly in the region surrounding the apical foramen, are observed in all cases of teeth with periapical lesions. [2],[8] Furthermore, considering the difficult to eliminate the biofilm, in vitro studies simulating clinical conditions are needed to test the efficacy of instrumentation techniques and chemical substances that can act on these sites. It can be observed that the present methodology allowed microbial biofilm formation in the apical resorption areas, since there was a great concentration of bacteria embedded in a disorganized mass. This bacterial community showed extensions suggestive of polysaccharide matrix [Figure 3] after 10 days of exposure to E. faecalis strains. These features of the formed biofilm are similar to those found in previous studies. [16]

The increasing knowledge on the role of biofilms on human infections has led to a change in the direction of the studies. Currently, most of the studies have been focused in the evaluation of disinfection methods and antimicrobial agents against biofilms rather than studying planktonic microorganisms only. [24] It is known that the presence of biofilms in the apical region is one of the main causes of failure in the endodontic treatment. [1],[7] Most of the studies have evaluated the antimicrobial action of irrigating substances and intracanal medications on biofilms developed in hydroxyapatite discs, [24] samples of dentin, [5] membrane filters, [17] and root apex. [8] Considering this limitation, in an attempt to increase the reproducibility of in vivo conditions, this study sought to reproduce the biofilm in vitro by using the entire root of extracted human teeth with apical resorption.

Therefore, this work has managed to develop a method that allows biofilms formation in areas of apical resorption, thus enabling further studies to test chemical substances, as well as methods of irrigation and instrumentation of root canals in human teeth during the conventional endodontic treatment (i.e., through root canal). Moreover, in the future this model may be used for multi-species biofilm formation and for testing intracanal medications and irrigation agents, thus reproducing situations as close as possible to the in vivo condition.

Within the limits of this study, it can be concluded that the 10-day period of exposure to E. faecalis strains was enough to assess the presence of biofilm formed in areas of apical resorption, according to the proposed model.

   References Top

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2.Leonardo MR, Rossi MA, Silva LA, Ito IY, Bonifácio KC. EM evaluation of bacterial biofilm and microorganisms on the apical external root surface of human teeth. J Endod 2002;28:815-8.  Back to cited text no. 2
3.Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod 2002;28:689-93.  Back to cited text no. 3
4.Donlan RM, Costerton JW. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167-93.  Back to cited text no. 4
5.George S, Kishen A, Song KP. The role of environmental changes on monospecies biofilm formation on root canal wall by Enterococcus faecalis. J Endod 2005;31:867-72.  Back to cited text no. 5
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7.Nair PN, Sjögren U, Krey G, Kahnberg KE, Sundqvist G. Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: A long-term light and electron microscopic follow-up study. J Endod 1990;16:580-8.  Back to cited text no. 7
8.Tronstad L, Kreshtool D, Barnett F. Microbiological monitoring and results of treatment of extraradicular endodontic infection. Endod Dent Traumatol 1990;6:129-36.  Back to cited text no. 8
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10.Kishen A, Upadya M, Tegos GP, Hamblin MR. Efflux pump inhibitor potentiates antimicrobial photodynamic inactivation of Enterococcus faecalis biofilm. Photochem Photobiol 2010;86:1343-9.  Back to cited text no. 10
11.Williamson AE, Cardon JW, Drake DR. Antimicrobial susceptibility of monoculture biofilms of a clinical isolate of Enterococcus faecalis. J Endod 2009;35:95-7.  Back to cited text no. 11
12.Lima KC, Fava LR, Siqueira JF Jr. Susceptibilities of Enterococcus faecalis biofilms to some antimicrobial medications. J Endod 2001;27:616-9.  Back to cited text no. 12
13.Hope CK, Garton SG, Wang Q, Burnside G, Farrelly PJ. A direct comparison between extracted tooth and filter-membrane biofilm models of endodontic irrigation using Enterococcus faecalis. Arch Microbiol 2010;192:775-81.  Back to cited text no. 13
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20.Stanley NR, Lazazzera BA. Environmental signals and regulatory pathways that influence biofilm formation. Mol Microbiol 2004;52:917-24.  Back to cited text no. 20
21.Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science 1999;284:1318-22.  Back to cited text no. 21
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24.Shen Y, Stojicic S, Haapasalo M. Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development. J Endod 2011;37:657-61.  Back to cited text no. 24

Correspondence Address:
Maria Tereza Pedrosa Albuquerque
Department of Restorative Dentistry, Institute of Science and Technology of Sao Jose dos Campos Dental School, Sao Paulo State University
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.142569

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