Journal of Dental Sciences (2015) 10, 95e101

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ORIGINAL ARTICLE

Status of bacterial colonization in teethassociated with different types of pulpaland periradicular disease: A scanningelectron microscopy analysis

Yan-Hua Huang, Si-Jing Xie, Nan-Nan Wang, Jiu-Yu Ge*

Department of Cariology and Endodontics, Institute and Hospital of Stomatology,Nanjing University Medical School, 30 Zhongyang Road, Nanjing, Jiangsu 210008, China

Received 25 January 2014; Final revision received 19 April 2014Available online 24 July 2014

KEYWORDSbiofilms;dentin;infected root canal;SEM

* Corresponding author. DepartmenSchool, Nanjing Stomatological Hospit

E-mail address: JYGe@nju.edu.cn

http://dx.doi.org/10.1016/j.jds.2014.01991-7902/Copyrightª 2014, Associatio

Abstract Background/purpose: The purpose of this study was to use scanning electron mi-croscopy (SEM) to investigate the status of bacterial colonization in differently infected rootcanals and the damage to radicular dentin.Materials and methods: Twenty-five freshly extracted teeth were selected for this study(Group A: 8 teeth with pulpitis; Group B: 10 teeth with periapical lesions; and Group C: 7 teethwith failed root canal treatment). After fixation, the teeth were longitudinally split into twohalves. The halves were then dehydrated, sputter-coated with gold, and viewed using SEM,descriptively dividing their lengths into apical, middle, and coronal thirds.Results: In Group A, bacterial infection was mainly located in the coronal third of the root canalsand bacteria failed to penetrate into the dentinal tubules. In Group B, bacterial infection wasdistributed over the entire length of the root canal. The invasion depth of bacteria into thedentinal tubules was approximately 300 mm. In Group C, bacterial infection was mainly focusedontheapical thirdof theroot canals.Most of thedentinal tubules hadcollapsed,andthe rootcanalwallswere heavily colonizedwith densebacterial biofilm, primarily consisting of cocci. Comparedto Group B, the invasion depths were deeper in the apical thirds of root canals (P < 0.05).Conclusion: Bacterial infectionwas lighter in the root canalswith pulpitis than in thosewith apicalperiodontitis, which might require special considerations regarding different stages of pulp andperiapical pathology in root canal treatment.Copyrightª 2014, Association for Dental Sciences of the Republic of China. Published by ElsevierTaiwan LLC. All rights reserved.

t of Cariology and Endodontics, Institute and Hospital of Stomatology, Nanjing University Medicalal, 30 Zhongyang Road, Nanjing 210008, China.(J.-Y. Ge).

5.002n for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.

96 Y.-H. Huang et al

Introduction

Pulpal and periradicular disease is one of the most commoninfectious diseases threatening human health. Bacteria,usually from dental caries, are the major etiological agentsof pulpal and periradicular disease.1 Related research aspreviously described has indicated that infected root canalsact as habitats for bacteria, which exist in planktonicforms, aggregates, and coaggregates and in the biofilmstatus in complex communities that are composed of bac-terial cells and extracellular matrix.2 Root canal therapy(RCT) is the most commonly applied, effective method inclinical treatment for pulpal and periradicular disease. Thegoal of treatment of the disease has been total eradicationfrom the infected root canal systems and prevention ofreinfection.3 According to statistics, RCT has failure ratesbetween 4% and 15%,4,5 whereas the rate of successful rootcanal retreatment was approximately 74%.6,7 The mostimportant factor in endodontic failure is the incompleteeradication of bacteria.8 With improved bacterial cultureand analysis techniques, intracanal bacteria have beenextensively studied and exactly classified.9e11 However,these investigations have not provided sufficient informa-tion concerning the bacterial colonization status in thedentinal wall of different types of pulpal and periradiculardisease. Knowledge of the status of bacterial colonizationcould provide us with a better understanding of thedisease process. Scanning electron microscopy (SEM) hasexcellent resolution and can reveal details regardingthe bacterial colonization status in natural environ-ments.10,12e14 Therefore, this study sought to characterizethe process of disease and to provide a theoretical basis forthe establishment of effective treatment strategies usingSEM to examine the bacterial colonization of infected rootcanals associated with different types of pulpal and peri-radicular disease. Thus, it can provide us with clinicalguidance.

Materials and methods

The study protocol was reviewed and approved by theEthics Committee of Nanjing University StomatologicalHospital (Nanjing, China). Verbal and written consent wasobtained from all of the study participants prior toextraction of teeth.

Patients and specimen collection

The examined material consisted of 25 extracted teethrandomly collected from 25 patients at Nanjing UniversityStomatological Hospital. Healthy adult volunteers were age18e70 years. A detailed medical and dental history wasobtained from each patient. All patients were in goodhealth and were not taking any medication that would alterbacteria status during the past 3 months. Teeth withperiodontitis or fractures were excluded from the study.Teeth were extracted for reasons not related to this study.All samples for the pulpitis group were from the thirdmolars. For primary and secondary apical periodontitis, wecollected samples from these cases as the third molars,those that could not meet with the lowest conditions for

oral rehabilitation, and the extracted cases for personalreasons.

The extracted teeth were divided into three groups ac-cording to the clinical and radiographic examination: GroupA (8 teeth): pulpitis; Group B (10 teeth): primary apicalperiodontitis; and Group C (7 teeth): secondary apicalperiodontitis.

The inclusion criteria were as follows. Group A: Pulpitisis characterized by a history of provoked and spontaneousdull, heavy, and lingering thermal pain that can bereproduced clinically. Radiographs show the depth of thecaries or cavity preparation. The periodontal ligamentspace and lamina dura are normal.15 Group B: Presence ofclinical signs and symptoms of chronic apical periodontitis(pain, swelling, or sinus tract); no previous endodontictreatment; and diameter of the periapical radiolucentarea of at least 3 mm.16 Group C: Endodontic treatmentperformed for > 2 years and radiographically visible fillingof root canal: (1) the presence of obvious clinical signs andsymptoms (pain on palpation, discomfort to percussion,and pain of the sinus tract); and (2) the persistent oremergent periapical radiolucency. The appearance of oneof these two items is considered to be a treatmentfailure.10

Sampling procedures

After disinfection of the tooth crown and the adjacenttissues with 2% chlorhexidine digluconate solution, thetooth was carefully extracted. Subsequently, the peri-odontal ligament and other attachments were removedwith a scalpel, and the clinical crown was sectioned at thecementoenamel junction with carborundum disks. Then,the teeth were immediately immersed in 2.5% phosphate-buffered glutaraldehyde solution. The sample teeth werestored at 4�C to provide a total fixation period of 1 week.After fixation, longitudinal grooves (approximately 2 mm)were cut along the entire root length with tapered diamondburs under a water spray. The roots were then split with achisel into two halves. The root canal length was measuredand divided equally into coronal, middle, and apical thirds,which were marked with a scalpel blade.

SEM preparation and observation

Each root half was then gently washed in phosphate buff-ered saline (pH Z 7.2, 4�C), dehydrated in increasingconcentrations of ethanol (50%, 60%, 70%, 80%, 90%, and2 � 100% for 15 minutes each), critical-point dried usingliquid CO2 replacement, coated with gold, and imaged in ascanning electron microscope (S-3400N, Hitachi, Tokyo,Japan) at a voltage of 20 kV. Thereafter, the entire roothalf, the radicular dentinal wall, and the dentinal tubuleswere examined from low to high magnification using SEM.Observation was conducted, and photographic images wereobtained.

Statistical analysis

Statistical evaluation of the study results was performedusing SPSS 15.0 software (SPSS Inc., Chicago, IL, USA) and

Table 1 Comparison of positive rates of bacterial detec-tion in all root canal segments.a

Group Number Positive rates of bacterial detection (%)

Crown third Middle third Apical third

A 8 6 (75) 1 (13)** 0 (0)**B 10 8 (80) 9 (90)* 10 (100)*C 7 4 (57) 4 (57) 7 (100)*

Data are presented as n (%).*P < 0.05 compared with Group A.**P < 0.05 compared with the coronal third.a The bacteria or bacterial aggregates for areas of each root

canal part were observed by scanning electron microscopyunder �600 magnification. If we found more than five bacteriaor bacterial aggregates, we defined this root canal part ashaving positive results for bacterial colonization.

Status of bacterial colonization in teeth 97

using the Chi-square test. A P value of <0.05 was consid-ered to be statistically significant.

Results

The longitudinal distribution of bacterial cells in teethassociated with different types of pulpal and periradiculardisease is outlined in Table 1.

In the coronal third of the root canals, the positivenumber of bacterial colonization was in six of eight speci-mens in Group A, eight of 10 specimens in Group B, and fourof seven specimens in Group C. No significant differenceswere observed between the three groups. In the middlethird of the root canals, the statistical analysis revealedthat the bacterial detection rate was much lower in GroupA (1/8, 13%) than in Group B (9/10, 90%) or Group C (4/7,57%). However, no significant differences were observedbetween Group B and Group C. In the apical third of theroot canals, bacteria were present in all of the examinedspecimens in Group B and Group C, whereas the situation inGroup A (no bacteria were observed in Group A) was theopposite.

The results also revealed that bacterial infection withpulpitis was mainly confined to the crown (P < 0.05),whereas bacterial infection with primary apical periodon-titis was distributed over the entire root half (P > 0.05). Forsecondary apical periodontitis, 100% of the apical thirdsexhibited bacterial infection, but only approximately 50%

Table 2 Comparison of the invasion depths of bacteria into thcanals.

Group Extent of the diffe

Number Middle third

<100 mm 100e300 mm >300 mm

B 9 2 (22) 4 (44) 3 (34)C 4 2 (50) 2 (50) 0 (0)P >0.05

Data are presented as n (%).a Invasion depth: the perpendicular distance from the deepest bact

of the specimens were infected in the coronal and middlethirds (P > 0.05).

A comparison of the invasion depth of bacteria intodentinal tubules in the middle and apical thirds of primaryand secondary apical periodontitis is presented in Table 2.

In the middle third of the root canals, the invasion depthof bacteria into the dentinal wall was >300 mm in 34% of thespecimens in Group B, whereas the invasion depth was<300 mm in all of the specimens in Group C. However, nosignificant differences were observed in the situation(P > 0.05). In the apical third of the root canals, the in-vasion depth of bacteria into the dentinal wall was>300 mm in 86% of the specimens in Group C, whereas theinvasion depth was <300 mm in 80% of the specimens inGroup B, which had significant differences between GroupB and Group C (P < 0.05).

The patterns of bacterial colonization and dentinalstructural changes of different types of pulpal and peri-radicular disease are described in Figs. 1e3.

In the infected root canals with pulpitis, a small amountof bacterial aggregates was present in the root canal wallsand lumen, and the invasion depth was <100 mm in thecoronal area (Fig. 1A and B). In the middle and apicalthirds, no bacterial colonization or invasion of the dentinaltubules was observed, but erythrocytes were visible in theapical area (Fig. 1CeF).

In the coronal thirds of infected root canals with primaryapical periodontitis, a large number of amorphous materialsand bacterial aggregates were observed in the root canallumen (Fig. 2A). In addition, bacterial aggregates were pre-sent on thedentinalwalls and in thedentinal tubules (Fig. 2B).In the middle third, amorphous materials and bacterial ag-gregates were present in larger amounts, and the bacterialinvasion depth was >300 mm (Fig. 2C and D). In the apicalthird, a large number of bacterial aggregates had penetratedinto the dentinal tubules to approximately 300 mm, and bac-teria were colonized more heavily in the shallow dentinaltubules than in the deep dentinal tubules (Fig. 2E and F).

In the coronal and middle third areas of the infectedroot canals with secondary apical periodontitis, amorphousmaterials and bacterial aggregates consisting of cocci androds adhered to the root canal walls, colonized the dentinalsurface, and progressed toward the dentinal tubules(Fig. 3AeD). In the apical area, most of the dentinal tubuleshad collapsed, and the root canal walls were heavily colo-nized with a dense bacterial biofilm consisting of cocci,rods, or filaments (Fig. 3E and F).

e dentinal walls in the middle and apical thirds of the root

rent invasion depths (%)a

Number Apical third

<100 mm 100e300 mm >300 mm

10 3 (30) 5 (50) 2 (20%)7 0 (0) 1 (14) 6 (86%)

<0.05

erial aggregate to the root canal dentinal wall.

Figure 1 (AeF) Scanning electron microscopy of bacterial colonization with pulpitis. Coronal third portion: (A) a small amount ofbacteria in the root canal lumen (� 1000, red arrows). (B) The invasion depths were <100 mm (� 500); inset is � 5000 magnification.Middle and apical third portions: (C and E) no bacterial colonization, but erythrocytes were visible in the root canal lumen(� 3000, � 2000, red arrows); (D and F) no bacterial invasion into the dentinal tubules (� 500,� 500); inset is � 5000 magnification.

Figure 2 (AeF) Scanning electron microscopy of bacterial colonization with primary apical periodontitis. Coronal third portion:(A) a large number of amorphous materials and bacterial aggregates in the root canal lumen (� 1000, red arrows); (B) bacterialaggregates on the dentinal walls and in the dentinal tubules (� 600); inset is � 30,000 magnification. Middle and apical thirdportions: (C and E) amorphous materials and bacterial aggregates were present in larger amounts (� 1000, � 1000, red arrows);(D and F) bacterial invasion depth was equal to or greater than 300 mm (� 300, � 1000); inset is � 5000 magnification.

98 Y.-H. Huang et al

Figure 3 (AeF) Scanning electron microscopy of bacterial colonization with secondary apical periodontitis. Coronal and middlethird portions: (A and C) amorphous materials and bacterial aggregates consisting of cocci and rods in the root canal lumen(� 500, � 300, red arrows); (B and D) bacterial aggregates adhered to the root canal walls, colonized the dentinal surface, andprogressed toward the dentinal tubules (� 1000, � 1000); inset is � 5000 magnification. Apical third portion: (E) cocci and rodswere present in the root canal lumen (� 10,000, red arrows); (F) the root canal walls were heavily colonized with a dense bacterialbiofilm consisting of cocci, rods, or filaments (� 500); inset is � 5000 magnification.

Status of bacterial colonization in teeth 99

Discussion

To date, some studies of the location and distribution ofbacterial colonization in infected root canals have beenpublished, but the infected root canals have rarely beenclassified according to different stages of pulp and peri-apical pathology in these studies.3,17e20 Therefore, in ourcurrent research, we classified the infected root canals intothe following three groups: pulpitis, primary apical peri-odontitis, and secondary apical periodontitis. Richardsonet al,21 using different microscopy techniques [light mi-croscopy (LM), transmission electron microscopy (TEM), andSEM] and protocols (Eastman Dental Institute, London,Britain and Zurich, Switzerland), found that microorganismsorganized into biofilm mainly on the root canal wall in theapical third. Noiri et al22 found that bacteria were non-uniformly distributed in the same infected root canal, andthese bacteria were almost in the planktonic status in thecoronal and middle thirds but formed typical biofilm in theapical third. The observations in this study demonstratedthat there was no bacterial biofilm detected in the teethwith primary apical periodontitis and the bacteria in theteeth with failed RCT (secondary apical periodontitis) weremainly organized as biofilms in the apical third, whichdiffered from the results of Richardson et al21 and Noiriet al,22 who did not classify apical periodontitis into pri-mary and secondary types. As a result, they did not defin-itively indicate whether the visualized biofilm adhering to

the root canal wall was in teeth with periapical lesions orfailed RCT. Biofilms are >1000 times more resistant toantimicrobial medications than the same bacteria in theplanktonic status because biofilms can tolerate the harshintracanal environment and can multiply rapidly wheneverfeasible, causing intracanal reinfection, damage to theperiapical tissues, and eventually endodontic failure,23

which is not difficult to explain earlier findings that long-term residual bacteria in root canals after obturationwere a major cause of treatment failure.9,11e13

In in vitro studies, the invasion depth of bacteria into thetest tube could reach 900 mm,24 mainly because there was nosmear layer in the test tube. Such invasion depth was notever reported in tooth specimens. Peters et al25 found thatmicrobes could invade dentine tubules up to 500 mm or evendeeper. This study concluded that bacteria primarily foundin the apical third segment could invade the dentine tubulesto varying depths between 100 mm and 300 mm. In fact, thepermeability of root canal irrigation drugs is limited. Forexample, the permeability of sodium hypochlorite was onlyapproximately 130 mm into the dentine tubules.26 During theprocess of root canal cleaning and shaping, bacteria in theroot canal lumen and shallow dentinal walls were obviouslyeliminated as a result of the function of irrigation drugs andthe removal of diseased dentin. However, deep-seatedbacteria in the dentinal tubules are difficult to removeabsolutely. For this reason, ultrasound flushing is recom-mended. The application of ultrasound to a flooded canal

100 Y.-H. Huang et al

goes further by warming the solution and creating violentturbulence to scrub canal walls and dentinal tubules.27 Ul-trasonically activated sodium hypochlorite is probably themost effective cleaning regime.

In addition, we mainly analyzed the invasion depth ofbacteria in the middle and apical thirds of Group B andGroup C because the invasion depth of the bacteria in thecoronal area of each group was almost always �100 mm,and there were no bacteria in the middle and apical thirdsof Group A. The invasion depths were deeper in the apicalthirds of root canals with secondary apical periodontitiscompared to primary apical periodontitis. However, nosignificant difference was observed in the middle thirds.The results further suggested that the complete eradicationof bacteria is much more difficult to achieve in teeth withfailed RCT than in teeth with primary apical periodontitis.Because chemomechanical procedures are unlikely tocompletely eradicate the root canal infection, the appli-cation of new and effective antimicrobial intracanalmedication might be a valuable adjunct in the treatment ofteeth with failed RCT.

In this study, we added a pulpitis experimental group,which has been mentioned only rarely in previousstudies.22,28,29 The difference in bacterial distribution be-tween teeth with pulpitis and chronic apical periodontitiswas significant: teeth with pulpitis had bacteria mainlyconfined to the coronal third, failing to penetrate into thedentine tubules, whereas teeth with chronic apical peri-odontitis had bacteria distributed over the entire length ofthe root canal and penetrated into the dentine tubules. Thedifference clearly indicated that the removal of bacteria inteeth with apical periodontitis was much more difficultthan in teeth with pulpitis. These results guided us towardtargeted treatment for different types of pulpal and peri-radicular disease. For pulpitis, we could obtain favorableoutcomes after thorough crown-down preparation, which isless likely to be carried apically during subsequent prepa-ration, reducing the incidence of blockage and post-operative flare-up. If time allows, RCT for pulpitis may beperformed in one visit. There is a consensus that one-visittreatment is best to preserve an infection-free environ-ment and periapical health. However, for maximum elimi-nation of intracanal bacteria in the treatment of the teethwith apical periodontitis, we should combine more effec-tive chemomechanical instrumentation with the applica-tion of disinfectants such as an interappointment dressing.

In summary, this descriptive study presented the complexbacterial colonization status of different types of infectedroot canals, which could explainwhy complete eradication isoften unsuccessful. However, the quantity of the specimensused herein was somewhat limited. The specimens wererequired to conform to both the exact filter standards andextraction indications, which might have resulted in diffi-culty in taking samples and could have affected the experi-mental results. For that reason,wewill attempt to overcomethe limitation of sample acquisition in our future studies.

Conflicts of interest

The authors have no conflicts of interest relevant to thisarticle.

Acknowledgments

This work was supported by Construction Project of theNational Key Clinical Specialty, awarded to the Departmentof Cariology and Endodontics of the Hospital of Stomatologyof Nanjing University Medical School and by a grantfor basic and clinical research on root canal reinfection inthe Yangtze River Delta region (11495811000). The authorsare indebted to Professor Liang Jingping of the Departmentof Endodontics and Operative Dentistry of the Ninth Peo-ple’s Hospital, associated with the School of Medicine ofShanghai Jiao Tong University, for skillful and efficienttechnical assistance.

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