Promotion of Endodontic Lesions in Rats by a Novel ExtraradicularBiofilm Model Using Obturation Materials

Katsutaka Kuremoto,a Yuichiro Noiri,a Takuya Ishimoto,b Naomichi Yoneda,a Reiko Yamamoto,a Hazuki Maezono,a

Takayoshi Nakano,b Mikako Hayashi,a Shigeyuki Ebisua

Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japana; Division of Materials and ManufacturingScience, Osaka University Graduate School of Engineering, Suita, Osaka, Japanb

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular bio-film development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an in vivo extraradicularbiofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extra-radicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular firstmolars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into themesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks ofpulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria wereidentified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites.Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, inthe extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulpexposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experi-mental sites than in the control sites (P < 0.05 by Welch’s t test). These results suggest that we developed an extraradicular bio-film model in rats and that extraradicular biofilms affect developing periapical lesions.

In clinical practice, we encounter patients in whom periapicalperiodontitis does not heal despite a root canal treatment fol-

lowing general dental procedures. The etiology is considered to bethe result of residual bacteria in the lateral branch of the rootcanal, endodontic reinfection, or both. Recent studies have shownthat biofilms that form outside the apical foramen, which aretermed “extraradicular biofilm,” are also involved in refractoryperiapical periodontitis (1–4). As the bacteria in biofilms are re-sistant to the host’s immunoreaction and to antibiotics (5), dentalbiofilm diseases such as caries or periodontitis have been treatedby mechanical removal. However, extraradicular biofilm is im-possible to remove mechanically with nonsurgical endodontictreatments. As extraradicular biofilms cannot be accessed fromthe root canal for mechanical removal, the only treatment meth-ods are endodontic surgery and tooth extraction.

Current research is focused on developing methods to preventor control biofilm formation (6, 7). We recently found three ana-logues of N-acyl homoserine lactone that participate in quorumsensing, a system of bacterial cell-to-cell communication, and in-hibit Porphyromonas gingivalis biofilm formation (8). Further-more, azithromycin, a 15-membered macrolide antibiotic, has anantibiofilm effect on P. gingivalis (9). For clinical applicability,these in vitro effects should be examined in animals as well.

Many researchers have induced periapical periodontitis in an-imals by exposing the dental pulp (10, 11). However, a model ofextraradicular biofilm after induction of periapical periodontitishas not yet been reported. Therefore, the mechanism of extrara-dicular biofilm development, the process by which extraradicularbiofilm becomes refractory to treatment, and the effects of extra-radicular biofilm on periapical lesions are not well understood.

We have found circumstantial evidence that biofilms form inthe extraradicular area and proposed that extraradicular biofilms,developed from the root canal by the apical foramen and com-

prised of multiple morphotypic bacteria, were attached to the ce-mentum around the root apex (2, 12). In addition, we suggestedthat Gram-positive facultative anaerobes such as Enterococcusfaecalis, Streptococcus sanguinis, and Streptococcus intermediuscould colonize and form extracellular matrices on the surface ofgutta-percha points, while serum plays a crucial role in biofilmformation in vitro (13). Therefore, we surmised that the surface ofgutta-percha points extruding from the root canal could also serveas a scaffold for biofilm formation, and thus, we attempted todevelop an extraradicular biofilm model in rats by inserting gutta-percha points out of the apical foramen.

Traditionally, periapical bone changes are assessed by usingradiographs or histological sections (14, 15). These two-dimen-sional projections of three-dimensional structures often do notadequately represent the region of interest and therefore may beinaccurate. In addition, radiographs are usually interpreted visu-ally, a process which is subjective in nature (16). In a few recentstudies, the periapical lesion volume of rodents was three-dimen-sionally analyzed by micro-computed tomography (micro-CT)(17, 18). Micro-CT can produce high-spatial-resolution images ofperiapical lesions.

In this study, on the basis of in vitro studies, we aimed to de-velop a novel rat model of extraradicular biofilm and to evaluate

Received 11 February 2014 Accepted 9 April 2014

Published ahead of print 18 April 2014

Editor: G. T. Macfarlane

Address correspondence to Yuichiro Noiri, noiri@dent.osaka-u.ac.jp.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00421-14

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the effect of extraradicular biofilms on in vivo periapical lesionsover time by using micro-CT.

MATERIALS AND METHODSEthics statement. The Animal Care and Use Committees of the OsakaUniversity Graduate Schools of Dentistry and Engineering (permit no.22-003-1 and 23-2) approved this study. All surgeries were performedwith sodium pentobarbital anesthesia, and all efforts were made to mini-mize the animals’ suffering.

Animals. Fifty-two 5-week-old male Wistar rats (Clea Japan, Inc.,Tokyo, Japan) were used. The animals were maintained in the animalfacility of the Osaka University Graduate School of Dentistry with a 12-h-light–12-h-dark cycle. Food and water were freely available.

Extraradicular biofilm formation and confirmation (experiment 1).Periapical lesions were induced by exposing the pulp of the rats’ mandib-ular first molars to the oral environment (Fig. 1) (19). Four weeks later,5-mm-long gutta-percha points (Pearl Dent Co., Ltd., Ho Chi Minh, Viet-nam), sterilized with ethylene oxide gas, were excessively inserted into themesial root canals of the right first molars on a clean bench (experimentalsites). Micro-CT images confirmed the extruding, 1-mm-long gutta-per-cha points. No gutta-percha point was inserted into the left first molars(control sites).

To confirm the presence of extraradicular biofilms, the rats were sac-rificed 6 and 8 weeks after pulp exposure (n � 4 per time point). Mandib-ular samples containing the first molars were dissected, fixed in 4% para-formaldehyde and 0.1% glutaraldehyde for 12 h at 4°C, and decalcified in10% EDTA containing 15% glycerol at 4°C. After preparation of 8-�m-thick serial frozen sections, some sections were stained by using a Brown-Brenn-modified Gram staining procedure (20) and observed under a lightmicroscope (Optiphot-2; Nikon Corporation, Tokyo, Japan).

For scanning electron microscopic observations, after a 6- or 8-weekpulp exposure, the mandibular first molars (n � 4 per time point) wereextracted and prepared according to a previously described method (21).In brief, the specimens were immersed in half-strength Karnovsky’s solu-tion (2% paraformaldehyde and 2.5% glutaraldehyde [pH 7.4]) for 30min. After fixation, the specimens were dehydrated with ascending gradesof ethanol and then freeze-dried. After sputter coating with platinum, thespecimens were observed by using a scanning electron microscope (JSM-6390LV; JEOL, Tokyo, Japan).

Cultivation, identification, and quantification of extraradicularbiofilm-forming bacteria (experiment 2). The rats were sacrificed 6 and 8weeks after pulp exposure (n � 5 per time point), and their mandibularfirst molars were extracted and immediately washed three times with ster-ile saline to remove planktonic bacteria. We then cut off the extrudinggutta-percha points and performed curettage of the root surfaces within 1mm of the periapical region by using a spoon excavator (catalog no. 10-506; YDM Corporation, Tokyo, Japan) under a stereomicroscope in orderto obtain an extraradicular sample. Intraradicular samples were obtainedby performing circumferential filing of the inside of the root canals with#30 H files (Mani, Inc., Tochigi, Japan). Both sample types were immersedin transport medium (ANA Port; Research Institute for Microbial Dis-ease, Osaka University, Japan), fully agitated, cultivated by inoculating 1ml of each sample onto thioglycolate medium, and incubated at 37°C for1 to 7 days. If bacterial growth was observed, the bacteria were separatedby using Columbia agar with 5% sheep blood (BD Japan, Tokyo, Japan) at37°C in an anaerobic atmosphere for 1 to 5 days or Trypticase soy agar IIwith 5% sheep blood, Columbia CNA agar with 5% sheep blood, andMacConkey agar (all from BD Japan) in an aerobic atmosphere at 35°C for24 to 48 h. Isolates were identified by using API strips (bioMérieux Clin-ical Diagnostics, Lyon, France) and RapID ANA kits (Amco, Inc., Tokyo,Japan).

The identified bacteria were quantified by using real-time PCR as-says. In brief, the rats were sacrificed 6, 8, 12, 16, and 20 weeks afterpulp exposure (n � 4 per time period), and their mandibular firstmolars were extracted and immediately washed three times with sterile

saline to remove planktonic bacteria. Extraradicular samples werethen obtained as described above for bacterial cultivation. GenomicDNA from the samples was isolated by using an InstaGene matrix(Bio-Rad Laboratories, Hercules, CA), according to the manufactur-er’s instructions. The assays were performed with a 20-�l solutioncontaining 1 �l of DNA extract (Applied Biosystems Power SYBRgreen PCR master mix; Life Technologies, Grand Island, NY) andbacterial universal primers 357F and 907R (22) (0.5 �l each), preparedin parallel reaction mixtures for each target sequence. The thermalcycling conditions for the Applied Biosystems 7500 Fast real-time PCRsystem (Life Technologies) were 95°C for 10 min, 40 cycles at 95°C for15 s, and 65°C for 1 min, with collection of the fluorescence signal atthe end of each cycle. Melting-curve analysis consisted of a denatur-ation step at 95°C for 15 s and a temperature reduction to 60°C for 1

FIG 1 Experimental protocol. Experiment 1, development and confirmationof the extraradicular biofilm model; experiment 2, cultivation, identification,and quantification of extraradicular biofilm-forming bacteria; experiment 3,measurement of periapical lesion volume. P, pulp exposure; GP, gutta-perchapoint insertion (experimental sites only); LM, light microscopy; SEM, scan-ning electron microscopy; micro-CT, micro-computed tomography.

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min followed by a temperature increase to 95°C at a rate of 1%, withcontinuous fluorescence reading. Data were acquired and analyzed byusing Applied Biosystems 7500 system SDS v2.0.2 software (Life Tech-nologies).

Measurement of periapical lesion volume (experiment 3). After 1 to4, 6, 8, 12, 16, and 20 weeks of pulp exposure, the induced periapicallesions (n � 6; each rat had a control site [left] and an experimental site[right] per time point) were scanned with a micro-CT scanner (R_mCT2;Rigaku, Tokyo, Japan) at 90 kV and 160 �A. A total of 500 consecutivetomographic slices, each with a thickness of 20 �m, were acquired. Afterscanning, image data were reconstructed by using the Three-DimensionalReconstruction Imaging for Bone (TRI/3D-BON) system (Ratoc SystemEngineering, Tokyo, Japan). The axes were standardized; radiolucent ar-eas on the mesial side of the y axis and below the x axis were assumed to beperiapical lesions (Fig. 2).

Statistical analysis. Differences in bacterial counts and periapical le-sion volumes between the experimental and control sites at each timepoint were analyzed by using one-tailed Welch’s t test. Differences inbacterial counts among the time points were determined by one-way anal-ysis of variance (ANOVA). A P value of �0.05 was considered significant.

RESULTSMicroscopic observations. Both 6 and 8 weeks after pulp expo-sure, extraradicular biofilms with predominantly Gram-negativebacteria were observed on the periapical root surfaces at all exper-imental sites (Fig. 3A to D). No extraradicular biofilm was foundat any of the control sites by light microscopy (Fig. 3E to G).

Extraradicular biofilms were also observed in all experimen-tal sites (Fig. 3H to J), but not in any control site (Fig. 3K), byscanning electron microscopy at 6 and 8 weeks after pulp expo-sure. Figure 3H is a scanning electron microscope image of awhole tooth from an experimental site at 8 weeks. In this speci-

men, a small colony of short rods was observed at the crack in thebiofilm structure on the surface of the extruding gutta-perchapoint (Fig. 3I). Short rods on the superficial layer of the glycoca-lyx-like structure on the periapical root surface were also noted(Fig. 3J).

Intra- and extraradicular biofilm-forming bacteria. The bac-terial isolates recovered from intraradicular and extraradicularbiofilm samples of the experimental sites are presented in Table 1,and those recovered from the control sites are presented in Table2. Bacteria were detected in all 20 intraradicular biofilm samplesand in 19 of the 20 extraradicular biofilm samples. Strains of Pro-teus mirabilis (15 of 20 samples), Escherichia coli (14 of 20 sam-ples), Enterococcus avium (13 of 20 samples), and Enterococcusgallinarum (13 of 20 samples) were frequently detected in all in-traradicular biofilms. Furthermore, strains of E. avium (8 of 10samples), E. coli (7 of 10 samples), Klebsiella pneumoniae (6 of 10samples), Morganella morganii (6 of 10 samples), and P. mirabilis(6 of 10 samples) were frequently detected in the extraradicularbiofilms.

Extraradicular bacterial counts. The bacterial count was sig-nificantly higher in the experimental sites than in the controlsites after 12 to 20 weeks of pulp exposure (P � 0.05). In theexperimental and control sites, the detected bacterial countswere 57.9 � 103 cells/ml to 127.9 � 103 cells/ml and 3.05 � 103

cells/ml to 10.7 � 103 cells/ml, respectively. Although there wasno significant difference among the time points, the bacterialcount at the experimental sites demonstrated an upward ten-dency (Fig. 4).

Periapical lesion volume. In the experimental and controlsites, the periapical lesion volume reached a peak after 4 weeks ofpulp exposure (4.94 mm3 and 4.78 mm3, respectively); thereafter,the volume showed a slight narrowing trend. Furthermore, after 8to 20 weeks of pulp exposure, the volumes in the experimental andcontrol sites reached a plateau. The average volumes after 6 to 20weeks of pulp exposure in the experimental and control sites were4.04 mm3 and 3.15 mm3, respectively. After 8 weeks of pulp expo-sure, the induced periapical lesions in the experimental sites weresignificantly larger than those in the control sites. Pulp exposure of6 weeks or less did not result in lesions with significant volumetricdifferences (Fig. 5).

DISCUSSION

In this study, we aimed to develop an extraradicular biofilm modelin rats. A model of this endodontic infection could be valuable forimproving the understanding of disease etiology and progressionand effective treatments. There are many reports regarding theinduction of periapical lesions and their histological observationin animals including rats (23, 24), dogs (25), cats (26), and mon-keys (27). Rat models are the most frequently used models forsuch studies: the morphology of a rat molar is similar to that of ahuman molar, and the genetic background of these animals isclear, with practically no individual specificity (28). Moreover,many individuals can be easily obtained for experimentation overa short period. Therefore, we considered rats the ideal animals todevelop an extraradicular biofilm model in vivo.

Histological evaluations and scanning electron microscope ob-servations evidenced the formation of extraradicular biofilms bythe method used in this study. As extraradicular biofilms wereobserved after 6 and 8 weeks of pulp exposure in all experimentalspecimens in this study, the extraradicular biofilm model was con-

FIG 2 Standardization of each axis for micro-computed tomography anal-ysis. The x axis (green line) passes through the top edge of the periapicallesions, the y axis (pink line) passes through the center of the buccal andlingual root canals of the mandibular first molars, and the z axis (yellowline) passes through the center of the mesial and distal root canals of themandibular first molars.

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FIG 3 Light microscopic images at week 8 (A to G) and scanning electron microscopy (SEM) images at week 6 (H to K). (A and E) Periapical areas at theexperimental (A) and control (E) sites. (B) Enlarged image of the inset in panel A. (C and F) High-magnification view of the solid insets in panels B andE, respectively. (D and G) High-magnification view of the dotted insets in panels B and E, respectively. Panel C shows a biofilm (arrows) consisting ofGram-negative bacteria (stained red) on the root surface, and panel D shows an intraradicular biofilm (arrowheads) consisting of Gram-positive andGram-negative bacteria on the root canal wall at the root apex. Panel F shows host cells around the root apex but not in the biofilm, and panel G showsa periapical lesion with an inflammatory cell infiltration. AF, apical foramen; GP, gutta-percha point; RC, root canal. (H) Overall image of an experimentalsite with the gutta-percha point extruding from the mesial root apex of the right mandibular first molar. (I and J) High-magnification views of thearrowhead (I) and arrow (J) in panel H. Panel I shows a biofilm composed of short rods or cocci with an extracellular matrix-like structure (arrows). PanelJ shows a biofilm comprising mostly rods with an extracellular matrix-like structure (arrows). (K) High-magnification view of a control site. No biofilmcan be seen on the periapical root surface.

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sidered successfully developed by using the methods describedabove. The extraradicular biofilm comprised mainly Gram-nega-tive bacteria, similar to human extraradicular biofilms (12, 29). Inthe strict sense, an extraradicular biofilm model developed with-out extruding gutta-percha points is optimal to eliminate the pos-sibility of a foreign-body reaction to a gutta-percha point in theperiapical region (30, 31). However, in our pilot studies, no extra-radicular biofilm formed without projecting gutta-percha points,even after 20 weeks of pulp exposure (data not shown). Further-more, it was technically difficult to remove an extruding gutta-percha point after insertion. Therefore, such an experimentalmodel probably cannot be established without excessively insert-ing gutta-percha points. Moreover, gutta-percha points project-ing from the apical foramen may serve as a scaffold for extrara-dicular biofilm formation in clinical practice. As we did notperform a root canal filling, the available dead space may havefacilitated extraradicular biofilm formation. A loose root canalfilling and overfilling greatly increase the possibility of extrara-dicular biofilm formation in clinical practice. Consideration of theexistence of extraradicular biofilms is necessary in these cases,given the state shown in an X-ray photograph.

E. faecalis is frequently detected in the root canals of patientswith refractory periapical periodontitis (32–34). Similarly, wenoted a high frequency of Enterococcus spp. in rat root canals(Tables 1 and 2). The finding of similar species inside and outsidethe root canals in the experimental and control sites provides cir-cumstantial evidence that the intraradicular biofilm progressedand that an extraradicular biofilm had formed. Therefore, in clin-ical practice, the residual bacteria of intraradicular biofilms mayincrease the possibility of extraradicular biofilm formation; intra-radicular biofilms should be removed as thoroughly as possibleto inhibit the formation of extraradicular biofilms. We did notidentify Fusobacterium spp., Campylobacter spp., or Bacteroidesendodontalis, which are generally detected in human root canals;this result is in agreement with a previous report of rat root canalmicroflora (35). Although the proportion of aerobic and anaero-bic bacteria in rats’ root canals is similar to that in human rootcanals (35), rats are coprophagous animals. This likely contributesto the high representation of enteric species detected. When cou-pled with the unique aspects of the indigenous rat oral microflora,performing a microbial comparison to humans would have limi-tations, even if there is some similarity in offending species.

TABLE 1 Prevalence of bacterial species identified from teeth atexperimental sites after 6- and 8-week pulp exposures

Bacterial species

No. of samples with bacterial speciesisolated

Extraradicular Intraradicular

6 wka 8 wk 6 wk 8 wk

Actinomyces sp. 1Bacteroides distasonis 2 1Bacteroides fragilisBacteroides thetaiotaomicron 1 1 1Bacteroides uniformisEnterococcus avium 4 4 5 2Enterococcus casseliflavusEnterococcus faecalis 1 2 1 2Enterococcus gallinarum 2 3 4 2Enterococcus sp. 3Escherichia coli 4 3 4 4Klebsiella pneumoniae 4 2 4 1Leuconostoc spp.Micrococcus sp.Morganella morganii 4 2 4 2Peptostreptococcus microsPeptostreptococcus prevotii 1 1Proteus mirabilis 4 2 4 4Streptococcus agalactiae 3 2 2Streptococcus bovis 2 3Streptococcus parasanguinisAlpha Streptococcus 1Coagulase-negative

staphylococci1 1

Veillonella parvula 1Anaerobic Gram-negative rod 1Anaerobic Gram-positive

coccusAnaerobic Gram-positive rodAerobic Gram-negative rod 2 2Nonfermenting Gram-negative

rod, uncultured2 1

a Five samples at each time point with pulp exposure.

TABLE 2 Prevalence of bacterial species identified from teeth at controlsites after 6- and 8-week pulp exposures

Bacterial species

No. of samples with bacterial speciesisolated

Extraradicular Intraradicular

6 wka 8 wk 6 wk 8 wk

Actinomyces sp. 1Bacteroides distasonis 2 4Bacteroides fragilisBacteroides thetaiotaomicron 1 1Bacteroides uniformis 1Enterococcus avium 1 2 4 2Enterococcus casseliflavusEnterococcus faecalis 1 2 2Enterococcus gallinarum 3 2 3 4Enterococcus sp. 1 1 2Escherichia coli 4 4 2Klebsiella pneumoniae 4 1 4 2Leuconostoc spp. 2Micrococcus sp.Morganella morganii 3 4 4Peptostreptococcus microsPeptostreptococcus prevotiiProteus mirabilis 3 5 2Streptococcus agalactiae 3 1Streptococcus bovis 1 3Streptococcus parasanguinisAlpha Streptococcus 1Coagulase-negative

staphylococci2

Veillonella parvulaAnaerobic Gram-negative rodAnaerobic Gram-positive

coccus1

Anaerobic Gram-positive rod 1Aerobic Gram-negative rod 2 3Nonfermenting Gram-negative

rod, uncultured1 2

a Five samples at each time point with pulp exposure.

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In this study, although the bacterial counts at the experimentalsites were not significantly different among the assessment peri-ods, an upward tendency was noted, suggesting that extraradicu-lar biofilm grows in a time-dependent manner. The bacteria iso-lated from the extraradicular samples and the few bacterial genesthat were identified in the control sites may be attributed to con-tamination during extraradicular sampling.

The slightly narrowing trend of the periapical lesions after 4weeks of pulp exposure is in agreement with previous reports oftwo-dimensionally measured periapical lesions in Wistar rats (36,37). Four weeks of pulp exposure is regarded as the expansionterm of rat periapical lesions, which stabilize thereafter (38, 39).Considering that bacteria exist around the root apex and inducean inflammatory reaction in periapical tissue (40, 41) and becauseof the existence of extraradicular biofilms, a shift to a stabilizedperiod from a lesion expansion period may be affected. This as-sumption may also explain the significant difference in periapicallesion volumes between the experimental and control sites after 8weeks of pulp exposure.

Both in vitro and in vivo research regarding the local immunereaction to biofilm has been conducted in the dental field (42–44).However, as an in vivo extraradicular biofilm model was unavail-able, studies regarding the host immune reaction to extraradicularbiofilm have not yet been performed. The model developed in thisresearch should enable investigations pertaining to the influenceof extraradicular biofilm on the host immune reaction and theformative mechanism of periapical lesions.

It has been reported that three types of compounds similar toN-acyl homoserine lactone participate in quorum sensing and in-hibit biofilm formation in vitro (8) and that a 15-membered mac-rolide antibiotic, azithromycin, has an antibiofilm effect (9). Ourmodel may serve as a bridge for the clinical application of thesestudies. We believe that the results from this research are useful to

create an evaluation system as well as to devise a new method tohelp control extraradicular biofilm development.

ACKNOWLEDGMENTS

We thank Tomotaka Nagayama and Wakako Kiba for their technical ad-vice.

This work was supported by grants-in-aid for scientific research (grantno. 2490424) from the Japan Society for the Promotion of Science (JSPS).

We declare that we have no conflicts of interest with respect to theresearch described in this article.

REFERENCES1. Sunde PT, Olsen I, Debelian GJ, Tronstad L. 2002. Microbiota of

periapical lesions refractory to endodontic therapy. J. Endod. 28:304 –310.http://dx.doi.org/10.1097/00004770-200204000-00011.

2. Noiri Y, Ehara A, Kawahara T, Takemura N, Ebisu S. 2002. Participation ofbacterial biofilms in refractory and chronic periapical periodontitis. J. Endod.28:679–683. http://dx.doi.org/10.1097/00004770-200210000-00001.

3. Leonardo MR, Rossi MA, Silva LA, Ito IY, Bonifácio KC. 2002. EMevaluation of bacterial biofilm and microorganisms on the apical externalroot surface of human teeth. J. Endod. 28:815– 818. http://dx.doi.org/10.1097/00004770-200212000-00006.

4. Nair PN. 2004. Pathogenesis of apical periodontitis and the causes ofendodontic failures. Crit. Rev. Oral Biol. Med. 15:348 –381. http://dx.doi.org/10.1177/154411130401500604.

5. Stewart PS, Costerton JW. 2001. Antibiotic resistance of bacteria inbiofilms. Lancet 358:135–138. http://dx.doi.org/10.1016/S0140-6736(01)05321-1.

6. Kagan S, Jabbour A, Sionov E, Alquntar AA, Steinberg D, Srebnik M,Nir-Paz R, Weiss A, Polacheck I. 26 September 2013, posting date.Anti-Candida albicans biofilm effect of novel heterocyclic compounds. J.Antimicrob. Chemother. http://dx.doi.org/10.1093/jac/dkt365.

7. Ma H, Darmawan ET, Zhang M, Zhang L, Bryers JD. 2013. Develop-ment of a poly(ether urethane) system for the controlled release of twonovel anti-biofilm agents based on gallium or zinc and its efficacy to pre-vent bacterial biofilm formation. J. Control. Release 172:1035–1044. http://dx.doi.org/10.1016/j.jconrel.2013.10.005.

8. Asahi Y, Noiri Y, Igarashi J, Asai H, Suga H, Ebisu S. 2010. Effects ofN-acyl homoserine lactone analogues on Porphyromonas gingivalis bio-film formation. J. Periodont. Res. 45:255–261. http://dx.doi.org/10.1111/j.1600-0765.2009.01228.x.

9. Maezono H, Noiri Y, Asahi Y, Yamaguchi M, Yamamoto R, Izutani N,Azakami H, Ebisu S. 2011. Antibiofilm effects of azithromycin and eryth-

FIG 4 Comparison of bacterial counts in extraradicular samples. Extraradicu-lar bacteria at the experimental and control sites were quantified at 6, 8, 12, 16,and 20 weeks by real-time PCR assays. Data represent the means of four samplemeasurements; error bars denote standard deviations. After 12 weeks of pulpexposure, the bacterial count was significantly higher in the experimental sitesthan in the control sites (P � 0.05 by Welch’s t test). There were no significantdifferences among the time points (P � 0.05 by one-way ANOVA). ES, exper-imental site; CS, control site.

FIG 5 Comparison of periapical lesion volumes. Periapical lesion volumes atthe experimental and control sites were measured at 1 to 4, 6, 8, 12, 16, and 20weeks by micro-computed tomography analysis. Data represent the means ofsix sample measurements; error bars denote standard deviations. As of 8 weeksafter pulp exposure, the periapical lesion volume was significantly larger in theexperimental sites than in the control sites (P � 0.05 by Welch’s t test). ES,experimental site; CS, control site.

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romycin on Porphyromonas gingivalis. Antimicrob. Agents Chemother.55:5887–5892. http://dx.doi.org/10.1128/AAC.05169-11.

10. Kakehashi S, Stanley HR, Fitzgerald RJ. 1965. The effects of surgicalexposures of dental pulps in germ-free and conventional laboratory rats.Oral Surg. Oral Med. Oral Pathol. 20:340 –349. http://dx.doi.org/10.1016/0030-4220(65)90166-0.

11. Tagger M, Massler M. 1975. Periapical tissue reactions after pulp expo-sure in rat molars. Oral Surg. Oral Med. Oral Pathol. 39:304 –317. http://dx.doi.org/10.1016/0030-4220(75)90234-0.

12. Noguchi N, Noiri Y, Narimatsu M, Ebisu S. 2005. Identification andlocalization of extraradicular biofilm-forming bacteria associated with re-fractory endodontic pathogens. Appl. Environ. Microbiol. 71:8738 – 8743.http://dx.doi.org/10.1128/AEM.71.12.8738-8743.2005.

13. Takemura N, Noiri Y, Ehara A, Kawahara T, Noguchi N, Ebisu S. 2004.Single species biofilm-forming ability of root canal isolates on gutta-percha points. Eur. J. Oral Sci. 112:523–529. http://dx.doi.org/10.1111/j.1600-0722.2004.00165.x.

14. Yu SM, Stashenko P. 1987. Identification of inflammatory cells in devel-oping rat periapical lesions. J. Endod. 13:535–540. http://dx.doi.org/10.1016/S0099-2399(87)80033-X.

15. Liu L, Peng B. 2013. The expression of macrophage migration inhibitoryfactor is correlated with receptor activator of nuclear factor kappa B ligandin induced rat periapical lesions. J. Endod. 39:984 –989. http://dx.doi.org/10.1016/j.joen.2013.03.001.

16. Gelfand M, Sunderman EJ, Goldman M. 1983. Reliability of radio-graphical interpretations. J. Endod. 9:71–75. http://dx.doi.org/10.1016/S0099-2399(83)80079-X.

17. von Stechow D, Balto K, Stashenko P, Müller R. 2003. Three-dimensional quantitation of periradicular bone destruction by micro-computed tomography. J. Endod. 29:252–256. http://dx.doi.org/10.1097/00004770-200304000-00005.

18. Oseko F, Yamamoto T, Akamatsu Y, Kanamura N, Iwakura Y, ImanishiJ, Kita M. 2009. IL-17 is involved in bone resorption in mouse periapicallesions. Microbiol. Immunol. 53:287–294. http://dx.doi.org/10.1111/j.1348-0421.2009.00123.x.

19. Kawahara T, Murakami S, Noiri Y, Ehara A, Takemura N, Furukawa S,Ebisu S. 2004. Effects of cyclosporin-A-induced immunosuppression onperiapical lesions in rats. J. Dent. Res. 83:683– 687. http://dx.doi.org/10.1177/154405910408300905.

20. Taylor RD. 1966. Modification of the Brown and Brenn Gram stain forthe differential staining of gram-positive and gram-negative bacteria intissue sections. Am. J. Clin. Pathol. 46:472– 474.

21. Asahi Y, Noiri Y, Igarashi J, Suga H, Azakami H, Ebisu S. 2012.Synergistic effects of antibiotics and an N-acyl homoserine lactone analogon Porphyromonas gingivalis biofilms. J. Appl. Microbiol. 112:404 – 411.http://dx.doi.org/10.1111/j.1365-2672.2011.05194.x.

22. Favia G, Ricci I, Damiani C, Raddadi N, Crotti E, Marzorati M, RizziA, Urso R, Brusetti L, Borin S, Mora D, Scuppa P, Pasqualini L,Clementi E, Genchi M, Corona S, Negri I, Grandi G, Alma A, KramerL, Esposito F, Bandi C, Sacchi L, Daffonchio D. 2007. Bacteria of thegenus Asaia stably associate with Anopheles stephensi, an Asian malarialmosquito vector. Proc. Natl. Acad. Sci. U. S. A. 104:9047–9051. http://dx.doi.org/10.1073/pnas.0610451104.

23. Iwama A, Nishigaki N, Nakamura K, Imaizumi I, Shibata N, Yamasaki M,Nakamura H, Kameyama Y, Kapila Y. 2003. The effect of high sugar intakeon the development of periradicular lesions in rats with type 2 diabetes. J.Dent. Res. 82:322–325. http://dx.doi.org/10.1177/154405910308200416.

24. Astolphi RD, Curbete MM, Colombo NH, Shirakashi DJ, Chiba FY,Prieto AK, Cintra LT, Bomfim SR, Ervolino E, Sumida DH. 2013.Periapical lesions decrease insulin signal and cause insulin resistance. J.Endod. 39:648 – 652. http://dx.doi.org/10.1016/j.joen.2012.12.031.

25. de Paula-Silva FW, Santamaria M, Jr, Leonardo MR, Consolaro A, daSilva LA. 2009. Cone-beam computerized tomographic, radiographic,and histologic evaluation of periapical repair in dogs’ post-endodontictreatment. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 108:796 – 805. http://dx.doi.org/10.1016/j.tripleo.2009.06.016.

26. Torabinejad M, Kiger RD. 1980. Experimentally induced alterations in

periapical tissues of the cat. J. Dent. Res. 59:87–96. http://dx.doi.org/10.1177/00220345800590011601.

27. Walton RE, Ardjmand K. 1992. Histological evaluation of the presence ofbacteria in induced periapical lesions in monkeys. J. Endod. 18:216 –221.http://dx.doi.org/10.1016/S0099-2399(06)81263-X.

28. Page RC, Schroeder HE. 1982. Periodontitis in man and other animals: acomparative review, p 71–116. S Karger AG, Basel, Switzerland.

29. Wang J, Chen W, Jiang Y, Liang J. 2013. Imaging of extraradicularbiofilm using combined scanning electron microscopy and stereomicros-copy. Microsc. Res. Tech. 76:979 –983. http://dx.doi.org/10.1002/jemt.22257.

30. Hatsuya H. 1978. Mouse peritoneal macrophage reaction to gutta perchain vivo and in vitro. Jpn. J. Exp. Med. 48:167–176.

31. Ricucci D, Langeland K. 1998. Apical limit of root canal instrumentationand obturation, part 2. A histological study. Int. Endod. J. 31:394 – 409.

32. Sundqvist G, Figdor D, Persson S, Sjögren U. 1998. Microbiologicanalysis of teeth with failed endodontic treatment and the outcome ofconservative re-treatment. Oral Surg. Oral Med. Oral Pathol. Oral Radiol.Endod. 85:86 –93. http://dx.doi.org/10.1016/S1079-2104(98)90404-8.

33. Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ.2003. Microorganisms from canals of root-filled teeth with periapical lesions.Int. Endod. J. 36:1–11. http://dx.doi.org/10.1046/j.1365-2591.2003.00603.x.

34. Gomes BP, Pinheiro ET, Gadê-Neto CR, Sousa EL, Ferraz CC, Zaia AA,Teixeira FB, Souza-Filho FJ. 2004. Microbiological examination of in-fected dental root canals. Oral Microbiol. Immunol. 19:71–76. http://dx.doi.org/10.1046/j.0902-0055.2003.00116.x.

35. Tani-Ishii N, Wang C-Y, Tanner A, Stashenko P. 1994. Changes in rootcanal microbiota during the development of rat periapical lesions. OralMicrobiol. Immunol. 9:129 –135. http://dx.doi.org/10.1111/j.1399-302X.1994.tb00048.x.

36. Yamasaki M, Kumazawa M, Kohsaka T, Nakamura H, Kameyama Y.1994. Pulpal and periapical tissue reactions after experimental pulpal ex-posure in rats. J. Endod. 20:13–17. http://dx.doi.org/10.1016/S0099-2399(06)80020-8.

37. Kohsaka T, Kumazawa M, Yamasaki M, Nakamura H. 1996. Periapicallesions in rats with streptozotocin-induced diabetes. J. Endod. 22:418 –421. http://dx.doi.org/10.1016/S0099-2399(96)80243-3.

38. Stashenko P, Yu SM. 1989. T helper and T suppressor cell reversal duringthe development of induced rat periapical lesions. J. Dent. Res. 68:830 –834. http://dx.doi.org/10.1177/00220345890680051601.

39. Okiji T, Kawashima N, Kosaka T, Kobayashi C, Suda H. 1994. Distri-bution of Ia antigen-expressing nonlymphoid cells in various stages ofinduced periapical lesions in rat molars. J. Endod. 20:27–31. http://dx.doi.org/10.1016/S0099-2399(06)80023-3.

40. Ricucci D, Pascon EA, Ford TR, Langeland K. 2006. Epithelium and bac-teria in periapical lesions. Oral Surg. Oral Med. Oral Pathol. Oral Radiol.Endod. 101:239–249. http://dx.doi.org/10.1016/j.tripleo.2005.03.038.

41. Ricucci D, Siqueira JF, Jr. 2010. Biofilms and apical periodontitis: studyof prevalence and association with clinical and histopathologic findings. J.Endod. 36:1277–1288. http://dx.doi.org/10.1016/j.joen.2010.04.007.

42. Bainbridge B, Verma RK, Eastman C, Yehia B, Rivera M, Moffatt C,Bhattacharyya I, Lamont RJ, Kesavalu L. 2010. Role of Porphyromonasgingivalis phosphoserine phosphatase enzyme SerB in inflammation, im-mune response, and induction of alveolar bone resorption in rats. Infect.Immun. 78:4560 – 4569. http://dx.doi.org/10.1128/IAI.00703-10.

43. Liang S, Krauss JL, Domon H, McIntosh ML, Hosur KB, Qu H, Li F,Tzekou A, Lambris JD, Hajishengallis G. 2011. The C5a receptor impairsIL-12-dependent clearance of Porphyromonas gingivalis and is required forinduction of periodontal bone loss. J. Immunol. 186:869 – 877. http://dx.doi.org/10.4049/jimmunol.1003252.

44. Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA,McIntosh ML, Alsam A, Kirkwood KL, Lambris JD, Darveau RP, CurtisMA. 2011. Low-abundance biofilm species orchestrates inflammatoryperiodontal disease through the commensal microbiota and complement.Cell Host Microbe 10:497–506. http://dx.doi.org/10.1016/j.chom.2011.10.006.

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