Vol. 99 No.

2 February 2005

ENDODONTOLOGY Editor: Larz S. W. Spångberg

Microbial status of apical root canal system of human mandibular first molars
with primary apical periodontitis after ‘‘one-visit’’ endodontic treatment
P. N. R. Nair, BVSc, DVM, PhD (Hon),a Stéphane Henry, DMD,b Victor Cano, DDS,b
and Jorge Vera, DMD,b Zurich, Switzerland, and Tlaxcala, Mexico
UNIVERSITY OF ZURICH AND UNIVERSITY OF TLAXCALA

Objective. To assess the in vivo intracanal microbial status of apical root canal system of mesial roots of human mandibular first
molars with primary apical periodontitis immediately after one-visit endodontic treatment. The residual intracanal infection was
confirmed by correlative light and transmission electron microscopy.
Study design. Sixteen diseased mesial roots of mandibular first molars were treated endodontically, each in one visit. Mesio-
buccal canals were instrumented using stainless steel hand files and mesio-lingual canals with a nickel-titanium rotary system.
The canals were irrigated with 5.25% sodium hypochlorite (NaOCl) during the instrumentation procedures, rinsed with 10 mL
of 17% ethylenediamine tetraacetic acid (EDTA), and obturated with gutta-percha and zinc oxide eugenol cement. Thereafter,
the apical portion of the root of each tooth was removed by flap-surgery. The specimens were fixed, decalcified, subdivided in
horizontal plane, embedded in plastic, processed, and evaluated by correlative light and transmission electron microscopy.
Results. Fourteen of the 16 endodontically treated teeth revealed residual intracanal infection after instrumentation,
antimicrobial irrigation, and obturation. The microbes were located in inaccessible recesses and diverticula of instrumented
main canals, the intercanal isthmus, and accessory canals, mostly as biofilms.
Conclusions. The results show (1) the anatomical complexity of the root canal system of mandibular first molar roots and (2) the
organization of the flora as biofilms in inaccessible areas of the canal system that cannot be removed by contemporary
instruments and irrigation alone in one-visit treatment. These findings demonstrate the importance of stringent application of all
nonantibiotic chemo-mechanical measures to treat teeth with infected and necrotic root canals so as to disrupt the biofilms and
reduce the intraradicular microbial load to the lowest possible level so as to expect a highly favorable long-term prognosis of the
root canal treatment.
(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:231-52)

Apical periodontitis is a disease of persistent microbial in an extracellular matrix material.4,5 Therefore, the
infection of the root canal system of the affected tooth. logical goal of treatment of the disease has been to
The microbial etiology of the disease has long been well eliminate or substantially reduce the microbial popula-
established.1,2 The infected and necrotic root canal tion within the root canal system and to prevent
system acts as a selective habitat for the causative reinfection by a tight seal of the root canal space. The
organisms3 that grow mostly in sessile biofilms, ag- disruption of the biofilms and reduction of the micro-
gregates, and coaggregates in which they are embedded bial load are achieved by a combination of mechanical
instrumentation, irrigation with various tissue lytic and
microbicidal solutions, and application of antimicrobial
The authors are indebted to Mrs Margrit Amstad-Jossi for skillful and
efficient technical assistance.
medicaments in the root canal.
a
Institute of Oral Biology, University of Zurich, Zurich, Switzerland. Traditionally, the mechanical cleaning, enlarging, and
b
Department of Endodontics, Dental School, University of Tlaxcala, shaping of the root canal have been done using a stainless
Tlaxcala, Mexico. steel hand instrument involving a filing and reaming
Received for publication Aug 23, 2004; returned for revision Sep 27, motion. Since the introduction of instruments made out
2004; accepted for publication Oct 7, 2004.
1079-2104/$ - see front matter
of highly flexible alloys to endodontics,6 nickel-titanium
Ó 2005 Elsevier Inc. All rights reserved. (NiTi) rotary systems7-10 have achieved widespread pop-
doi:10.1016/j.tripleo.2004.10.005 ularity among clinicians because of their convenience

231
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232 Nair et al February 2005

and superiority in negotiating curved root canals as The complexity of the root canal system formed by
compared to stainless steel instruments. The NiTi the main canals, an isthmus communicating between
instrumentation has been evaluated and found to be them, accessory canals, apical ramifications, and
effective in maintaining the original shape of the canal.11 anastomoses26,27 cannot be adequately visualized by
It is assumed that NiTi instruments shape the apical radiographs28,29 or resolved by microcomputed tomog-
portion of root canals so as to facilitate a better flow of raphy.30,31 Therefore, the problem of microorganisms
the irrigants into the area compared to instrumentation surviving in the inaccessible, remote areas of the canal
with traditional stainless steel files. It has also been system that are beyond the reaches of contemporary
suggested12,13 that the size of apical instrumentation sampling procedures remains a serious concern. Thus,
may be important for the effective removal of micro- the purpose of this study was to assess the in vivo
organisms from the canal. Molar canals have been found intracanal microbial status of the apical root canal
to be cleaner ‘‘. . .by means of counting the remaining system of mesial roots of human mandibular first molars
surface debris. . .’’ when instrumentation reached a size with primary apical periodontitis immediately after a
#45 apical file.14 On the other hand, when the mesial one-visit endodontic treatment by 2 different instru-
canals communicate with each other (about 40% of the mentation techniques, namely, a NiTi rotary instrumen-
cases), it was difficult to obtain culture-negative canals tation and a standard step-back procedure using stainless
even after having enlarged the apical portion of the canal steel hand K-files. The presence of residual intracanal
to a size #60 apical file.15 microorganisms was confirmed by modern correlative
Teeth without apical radiolucency at the time of root light and transmission electron microscopy (TEM).
canal treatment have a higher rate of success than those
with apical radiolucent lesions.16 Further, teeth that are MATERIAL AND METHODS
root-filled after obtaining a negative microbial culture Subjects and specimens
have a better long-term prognosis than those root-filled Patients considered for inclusion in this study had
with microbes known to be present in the root canals at teeth with pulpal necrosis and apical periodontitis. The
the time of obturation.17,18 In any case, for maximum criteria for tooth selection included presence of a distinct
long-term clinical success of root canal treatment, it is periapical radiolucent lesion, a negative response to
desirable to obturate the canal without any infectious thermal pulp sensitivity test, presence of enough coronal
agent present or with a microbial load reduced to a level tissues for adequate isolation of the tooth with rubber
that cannot be detected by contemporary growing dam, and no prior endodontic treatment on the involved
techniques. However, in the absence of routine micro- tooth. All the subjects were treated in accordance with
bial monitoring of root canals before obturation, the Helsinki Declaration (www.cirp.org/library/ethics/
clinicians can expect an optimal outcome of treatment helsinki). Informed consent of each patient was obtained
only when the treatment procedures follow a protocol after explaining the clinical procedures and risks
that had established efficient microbial reduction of the involved and clarifying all questions raised by the
infected canals. patients. Only mature mandibular first molars were used
The efficiency of reducing the quantity of intracanal in this study. A total of 16 mesial roots of mandibular first
microorganisms has been investigated during various molars were treated endodontically, each in one visit,
stages of root canal treatment by techniques using and apical surgery was performed immediately there-
traditional stainless steel hand files13,19-23 and modern after (Table I). Only 1 tooth per patient was sampled.
rotary NiTi instruments.15,24,25 The microbiological
method of evaluation in these studies consisted of Clinical procedures
taking intracanal samples with paper-points at various All clinical procedures were done by 2 experienced
stages of treatment, culturing the samples, and quanti- endodontists. After local anesthesia and isolation of
tatively estimating the viable microbes in the samples. the tooth by rubber dam, disinfection of the tooth and
These studies indicate that negative microbial cultures the rubber dam was done using 5.25% of NaOCl with
can be expected with high repeatability only after a circular movement starting on the tooth and going
thorough instrumentation and rinsing of the root canals outwards to the rubber dam. Thereafter, carious tissues
with sodium hypochlorite (NaOCl) and ethylenedi- were removed and endodontic access cavities were
amine tetraacetic acid (EDTA), and application of an prepared with sterile high-speed carbide burs. Gates
interappointment microbicidal intracanal dressing such Glidden drills were used for coronal flaring with copious
as calcium hydroxide (Ca(OH)2). The therapeutic action irrigation using 5.25% NaOCl. Thereafter, the working
of the microbicidal dressing requires sufficient time to lengths of the root canals were determined using 2
be effective and thus cannot be completed in 1 session or stainless steel hand-files (size 15) and the Root Zx
‘‘one-visit endodontic treatment.’’ Electronic Apex Locator (J. Morita & Co, Tustin, Calif).
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Volume 99, Number 2 Nair et al 233

Table I. Summary of the clinical data of subjects whose teeth were investigated
No. Specimens Patient Age Sex Tooth Group
01 MX-01 IM 40 M 19 (36) Trial
02 MX-02 MES 18 F 19 (36) Trial
03 MX-03 AL 21 F 30 (46) Trial
04 MX-04 MM 42 F 19 (36) Trial
05 MX-05 JCC 20 M 19 (36) Trial
06 MX-06 MIS 48 F 30 (46) Trial
07 MX-07 EG 42 M 30 (46) Trial
08 MX-08 YM 42 F 30 (46) Trial
09 Mx-09 JSL 25 F 19 (36) Trial
10 MX-10 MIH 27 F 30 (46) Trial
11 MX-11 VC 21 M 30 (46) Trial
12 MX-12 MR 33 F 19 (36) Trial
13 MX-13 CS 39 M 19 (36) Trial
14 MX-14 MT 30 F 30 (46) Trial
15 MX-15 DSP 13 F 19 (36) Trial
16 MX-16 HA 27 F 30 (46) Trial

17 MX-17 RMA 33 M 30 (46) + Control

18 MX-18 AQR 32 M 19 (36) + Control
19 MX-19 NAC 53 F 30 (46) + Control
20 MX-20 JVP 67 M 19 (36) + Control

21 MX-21 JTG 14 F 21 (34) ÿControl

22 MX-22 MRA 15 F 28 (44) ÿControl
23 MX-23 MRA 15 F 21 (34) ÿControl

Mesio-angled and/or disto-angled radiographs were round sterile carbide bur, a shallow groove was prepared
taken to visualize and confirm the presence of 2 separate on the buccal surface of the exposed apical root portion.
canals. The mesial canals were divided into 2 groups. The groove was meant to identify the buccal aspect of
All mesio-buccal canals were instrumented using the resected root-segment so as to recognize and
a standardized technique with K hand files (Kerr orientate the mesio-buccal canal that had been hand-
Manufacturing Co, Romulus, Mich) until the apical file prepared. The apical third of the mesial root was
preparation reached a #25 apical size. Thereafter, a carefully removed using a Endo-Zekria (Maillefer,
step-back technique was used by withdrawing 1 mm of Ballaigues, Switzerland) high-speed sterile carbide bur
successive file tips from that of the previous until a operating with saline irrigation. The flap was reposi-
#40-sized hand file was reached (3 mm from the tioned and sutured. The patients were given oral and
working length). All mesio-lingual canals were instru- written postsurgical instructions. The entire clinical
mented with a standardized technique using Lightspeed procedures were done in one visit.
(Lightspeed Technology Inc, San Antonio, Tex) NiTi
instruments until a #40 apical size was reached. Copious Controls
irrigation with 5.25% NaOCl during the instrumentation The apical third of the distal root of 4 mandibular first
procedure and a final rinse with 10 mL of 17% EDTA molars with necrotic pulps and radiographic signs of
were done in both groups. All canals were subsequently apical periodontitis and scheduled for extraction were
obturated by lateral condensation of gutta-percha and used as positive controls (Table I). The apical third of 3
zinc-oxide eugenol cement (Roth 801). Thereafter, the clinically healthy mandibular first bicuspid roots served
access cavities were sealed with Cavit (3M ESPE, AG as the negative controls (Table I). The teeth had to be
Seefeld, Germany) and posttreatment control radio- removed for orthodontic reasons. The clinical status of
graphs were taken. the 3 teeth was determined by a combination of physical
On completion of the root canal treatment on each examination, radiography, and tooth sensitivity test.
patient, a full-thickness intrasulcular flap was raised Physical and radiographic examinations revealed that
under strict antiseptic conditions so as to expose the the teeth were caries-free and asymptomatic. Pulp
periapical lesion and the surrounding tissues. An sensitivity was determined by the application of a cotton
osteotomy was performed until the apical third of the pellet with EndoIce (Hygenic Corporation, Akron,
mesial root could be clearly identified. Using a # ¼ Ohio) to the cervical area of the involved tooth. All 3
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234 Nair et al February 2005

Table II. Summary of certain histo-antomical features with lead and uranium salts34,35 and examined in a
of the apical root canal system of the specimens Philips EM400T transmission electron microscope
presented in Table I. (Philips, Endoven, The Netherlands). The negative
Anatomical feature n % micrographs were scanned in a UMAX Powerlook
Total number of specimens 16 100 3000 (UMAX Systems, Willich, Germany) scanner
Number of specimens with mesio-lingual canal 16 100 and the digitized electron micrographs were further
Number of specimens with mesio-buccal canal 16 100 processed using an image-processing program (Photo-
Number of specimens with isthmus 11 69 shop 7.1; Adobe, San Jose, Calif).
Number of specimens with accessory canal(s) 8 50

RESULTS
teeth were positive for cold thermal sensitivity test. A Endodontically treated (trial) specimens
caries-free, asymptomatic, and cold-sensitive tooth was Histologically all the 16 endodontically treated teeth
assumed to be clinically healthy with a vital pulp. revealed the presence of mesio-lingual and mesio-
buccal canals (Table II). Four of them showed
Tissue processing confluence towards the apices. Eleven of the 16
Immediately after removal, the surgically removed specimens revealed communications (isthmus) between
apical third was fixed by immersion in a modified the 2 main canals. The root discs of 8 of the 16
Karnovsky’s fixative, consisting of 2% paraformalde- specimens showed cross-sectional profiles of varying
hyde and 2.5% glutaraldehyde buffered in 0.02 M sizes and numbers of accessory canals and/or anasta-
sodium cacodylate for several days.32 Thereafter, the mosing segments of the root canal system.
specimens were decalcified in solutions containing 0.25
mol/L EDTA and 4% glutaraldehyde. The demineral- Microbial status
ized specimens were orientated with the help of the In general, microorganisms were found in the
‘‘buccal groove’’ prepared during surgery and were complex, apical root canal system of 14 (88%) of the
subdivided into about 0.3-mm-thick to 0.5-mm-thick 16 root canal treated specimens (Tables III and IV).
horizontal discs. As the buccal groove was not extending Intracanal infection. Although 14 (88%) of the 16
to the full length of the root tip, an insignificant por- specimens showed the presence of microbes in the
tion of each root disc on the buccal aspect was cut off apical root canal system, the organisms were not
with a sharp razor blade for clear orientation during uniformly distributed and located in all specimens.
further processing of the specimen. The apical root discs Eight of the 16 specimens revealed microbes in both the
were post-fixed in 1.33% osmium tetroxide (OsO4), in mesio-lingual (hand filed) and mesio-buccal canals
s-collidine, dehydrated in ascending grades of ethanol (NiTi prepared). Seven of the 8 microbe-positive mesio-
and embedded in EponÒ (Fluka AG, Buchs, Switzerland). lingual and mesio-buccal canals belonged to identical
The correlative light and TEM investigation was root-specimens. Of the 11 root tips that revealed an
performed as described in detail before.33 Briefly, from isthmus between the 2 main canals, 10 showed the
each Epon block, 10 to 12 survey sections of about 1 mm presence of microorganisms within. Accessory root
to 2 mm thickness and from selected blocks serial canals were present in 8 of the 16 specimens, of which 5
sections were prepared using glass or histodiamond contained a mixed microbial flora.
knives (Diatome AG, Biel, Switzerland) and the As a representative case, one of the specimens in
Reichert Ultracut E microtome (Leica, Glattbrugg, which microorganisms were observed in the main and
Switzerland). The sections were stained in periodic accessory canals is illustrated radiographically (Fig 1),
acid-Schiff (PAS) and methylene blue-Azur II and photo- histologically (Fig 2), and transmission electron micro-
micrographed in a Dialux 20 photomicroscope (Leica) scopically (Fig 3). The specimen originated from the
equipped with the digital camera Progress C14 mesial root of the left mandibular first molar of a 40-
(Jenoptik, Eching, Germany) and a digital imaging year-old male patient (MX-1, Table I). The coronally
system (ImageAccess, Imagic, Glattbrugg, Switzerland). divergent mesio-lingual and the mesio-buccal canals
The sections were studied thoroughly in a light micro- converged toward the apical-most portion of the root
scope for the presence of microorganisms in the root (Fig 1, B; Fig 2, A and B). At the section plane of
canals and/or sites that are likely to contain microbes the illustration (Fig 2), signs of canal instrumentation
such as the presence of neutrophils. Thereafter, such and presence of gutta-percha and sealer components
areas in the Epon-blocks were determined for ultra- were clearly recognizable. The canals also revealed in-
sectioning. The selected tissue sites were target-trimmed accessible pulp recesses and irregular diverticula that
and thin-sectioned with the Reichert Ultracut E micro- contained a slurry of microbes, organic materials, and
tome (Leica). The thin sections were double contrasted dentinal chips. The striking feature of the specimen was
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Volume 99, Number 2 Nair et al 235

Table III. Microbial status and location of microbial presence (+) or lack of observation (ÿ) assessed by light and
transmission electron microscopy in the apical root canal system of the trial specimens shown in table I.
No. Specimen MLC MBC IST AC Overall* Remarks
01 MX-01 + + N + + Canals confluent
02 MX-02 + + + + +
03 MX-03 + + + + +
04 MX-04 ÿ ÿ ÿ ÿ ÿ
05 MX-05 ÿ ÿ + N + Found in TEM only
06 MX-06 ÿ ÿ + N + Found in TEM only
07 MX-07 + ÿ N + +
08 MX-08 ÿ ÿ + N +
09 MX-09 + + + + +
10 MX-10 ÿ + + N +
11 MX-11 ÿ ÿ + + + Canals confluent
12 MX-12 + + + N +
13 MX-13 ÿ ÿ + N +
14 MX-14 + + N N + Canals confluent
15 MX-15 + + N ÿ + Canals confluent
16 MX-16 ÿ ÿ N N ÿ

MLC, mesio-lingual canal; MBC; mesio-buccal canal; AC, accessory canal; IST, isthmus; N, absence of the structural entity.
*Denotes the presence of microbes (+) or lack of observation of such (ÿ) in 1 or more of the 4 anatomical locations examined.

Table IV. Summary of the microbial status of the specimens detailed in Table III.
Microbial status n %
Total number of specimens 16 100
Overall specimens with microbes in the apical canal system 14 88
Number of specimens with microbes in mesio-lingual canal 8 50
Number of specimens with microbes in mesio-buccal canal 8 50
Number of specimens with microbes in isthmus 10 63
Number of specimens with microbes in accessory canals 5 31

the presence of cross-sectional profiles of numerous dentinal structures (Fig 5). Depending on the location
accessory canals and anastomosing segments of a com- and plane of sectioning, the isthmus appeared in
plex root canal system that were uninstrumented and segments and sizes that were difficult to be observed
clogged with microorganisms (Fig 2, E and F). In TEM in low magnification bright field microscopy (MX-8,
(Fig 3) the flora consisted of a mixed microbial Fig 6). However, in TEM, even a very narrow isthmus
population of cocci, rods, and predominantly filamen- revealed numerous bacteria and neutrophils, the latter in
tous organisms. Numerous dividing forms of cocci and varying stages of disintegration (Fig 7).
rods could be recognized. The flora appeared to be held Plant celleassociated infection. One of the speci-
together by an electron lucent extracellular matrixlike mens (MX-15, Table I), illustrated radigraphically (Fig
substance. The microbes and the extracellular material 8), histologically (Fig 9), and electron microscopically
clogged the entire lumen of the accessory canal. In (Fig 10), contained intraradicular foreign particles of
random ultrathin sections, the dentinal wall in isolated vegetable nature (Fig 9) that revealed large plant cells
segments revealed multilayered microbial condensation with distinct electron lucent cell walls and disintegrated
that resembled biofilms attached to the firm dentinal cellular contents (Fig 10). The intracanal and intra-
base. Spirochetes were present in the root canal system isthmus bacteria in this case were not only found in the
of this and another (MX-7, Table II) endodontically root canal space but were also present within the
treated specimen. disintegrated plant cells (Fig 10). Whereas some plant
Intra-isthmus infection. In 11 of the 16 specimens, cells were free of bacteria, others showed varying
the main canals were found to communicate through an numbers of coccoid bacterial cells of which many were
isthmus of varying width. The dentinal wall of the in dividing forms (Fig 10, B).
isthmus often showed arcading profiles of Howship’s
lacunae that were colonized and filled with micro- Positive controls
organisms (MX-11, Fig 4). The microbe-clogged, All the 4 nontreated, necrotic root canals that served
tortuous isthmus often contained ‘‘islands’’ of fibro- as positive controls revealed the presence of a mixed
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236 Nair et al February 2005

Fig 1. Radiographs of a left mandibular left first molar (MX-01, Table I) with primary apical periodontitis (A). The mesio-angled
images recorded during instrumentation (B) and after obturation (C) reveal 2 major root canals of the mesial root. The apical third
of the root was removed by surgery immediately after the root canal treatment in ‘‘one visit’’ (D). The histological and transmission
electron microscopic findings of the apical root segment are illustrated in Fig 2 and Fig 3, respectively.

microbial flora both in the main and accessory canals. ovoid and elongated microbial cells that were often held
Histologically, the main canals of the 4 specimens together to form chains. These organisms were found to
contained disintegrated radicular pulp tissue and be about 10 to 12 mm in diameter and possessed distinct
a loosely distributed mixed microbial flora. The electron lucent cell walls and electron dense nuclear
accessory canals, on the other hand, were clogged area. These structures had the characteristic morphol-
with microorganisms. One of the specimens (MX-19, ogy of fungus.
Table I) is illustrated in detail (Figs 11 to 14). The loose
and clogged distribution of the intraradicular contents of Negative controls
the main and accessory canals respectively, could be No microorganisms were encountered in the canals of
observed even in low magnifications (Fig 11). In high the 3 root tips that originated from clinically healthy
magnifications the flora consisted of cocci, rods, and teeth. These showed typical healthy radicular pulp that
filamentous organisms (Fig 12). An exceptional feature consisted of peripheral cuboidal odontoblasts, soft
of the specimen MX-19 was the presence of ovoid and connective tissue, and neurovascular bundles. In mor-
filamentous microbial cells that were several times phological works, absence of something cannot be con-
larger in size than that of the bacterial cells. Even in vincingly illustrated with validity, so no illustration of
a light microscope (Fig 12), these cells appeared to exist negative controls of healthy radicular pulp is presented.
throughout the canal lumen, but they seemed to be
attached to the entire pulpal dentinal wall so as to form DISCUSSION
multileveled sessile biofilm (Fig 13). In the midcanal Because microorganisms are the essential etiological
region these cells appeared loosely mixed with the much agents of primary apical periodontitis1,2 and also the
smaller sized bacterial cells. In higher magnification major cause of posttreatment apical periodontitis,33,36-41
(Figs 13 and 14), they appeared as dividing forms of the cherished goal of treatment of the disease has been
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Volume 99, Number 2 Nair et al 237

Fig 2. Photomicrograph of a transverse section (A) through the apical portion of the mesial root of the tooth removed by surgery
from the radiolucent area in Fig 1, D. The rectangular demarcated are in A is magnified in B. The mesio-lingual (ML) and mesio-
buccal (MB) canals (magnified in C) communicate and are root-filled (GP). The rectangular demarcated area in B is magnified in E.
The main canals show recesses and diverticulations; those in the rectangular demarcated area in C are magnified in D. One
noninstrumented accessory canal (AC in E) is enlarged in F. Note the diverticulation of ML in D and the larger accessory canal (E,
F) clogged with bacteria (BA); the transmission electron microscopic view of the latter is shown in Fig 3. Black arrowheads in F
show cross-sectioned profiles of anastomoses of the root canal system. Original magnifications: A, 316; B, 340; C and E, 3100;
D, 3400; F, 3260.
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238 Nair et al February 2005

Fig 3. The composite transmission electron micrograph shows bacterial mass in the noninstrumented necrotic accessory canal of
Fig 2, E and F. Note the filamentous bacteria (BA) sectioned in various planes and embedded in an extracellular matrix material.
D = Dentine. Original magnification 33200.

a total elimination of the intraradicular infection and ule, is an ongoing topic of debate.42,43 This study
prevention of reinfection. Whether this ideal goal can be provides morphological evidence that 14 of the 16
achieved or not with contemporary instruments and (88%) mandibular molars that were root-treated in
procedures, particularly in a one-visit treatment sched- a single session harbored intracanal microorganisms
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Volume 99, Number 2 Nair et al 239

Fig 4. Light microscopic view of a transverse section through the apical portion of the mesial root of a right mandibular first molar
(MX-11, Table I). The surgical artifact (AT) into the root dentine did not reach or damage the instrumented mesio-lingual and
mesio-buccal canals, which were incompletely obturated with gutta-percha cones. The isthmus (IS) connecting the canals is
magnified in B; the area indicated with the black arrowhead is further magnified in stages in C and D, respectively. Note the
uninstrumented isthmus with arcading profiles of Howship’s lacune (HL) clogged with blue stained bacterial mass (BA). A
transmission electron microscopic view is shown in Fig 5. Original magnifications: A, 316; B, 344; C, 3240; D, 3400.

immediately after completion of the treatment. Further, compared the efficiency of the 2 techniques in reducing
it reconfirms the long-known anatomical complexity of intracanal microbes.
the apical root canal system26,27 that probably is the The fact that 14 of the 16 root-treated teeth contained
most important factor that makes the task of total residual intracanal infection after instrumentation,
elimination of intracanal microbes difficult and allows irrigation with NaOCl followed by EDTA, and obtura-
for the presence of residual infection posttreatment. tion demonstrates the difficulty of total eradication of
This study assessed the intracanal microbial status of microbes from infected canals. There were 2 specimens
endodontically treated teeth immediately after such in which intracanal microbes were not detected after the
treatment in one visit for which 2 commonly used treatment. It does not mean that the apical canals of the 2
instrumentation techniques were used on separate specimens were free of microbes. It is very much likely
canals of first human mandibular mesial roots. It should that the 2 cases also contained residual microbes that
be emphasized that the study did not attempt to make were not encountered by the methods used. In the 14
a comparison between the 2 instrumentation techniques. specimens that were positive for microbes, the orga-
Nevertheless, it is worth noting that the outcome of nisms were located not only in the inaccessible recesses
treatment, in terms of the microbial status, was almost and diverticula of instrumented main canals but also in
identical with both techniques. This finding is com- the isthmus and accessory canals that could not be
pletely in agreement with the results of a study24 that cleaned by the treatment procedures. The presence of
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240 Nair et al February 2005

Fig 5. Transmission electron micrograph of bacterial (BA) mass in Fig 4, D, in the tortuous space of the isthmus that reveals
numerous ‘‘islands’’ of fibro-dentinal (D) structures. Original magnification: 33200.
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Volume 99, Number 2 Nair et al 241

Fig 6. Light photomicrographs of apical root discs of the mesial root of a right mandibular first molar (MX-08, Table I). The
identification notch is on the buccal aspect in A. The mesio-lingual (ML) and mesio-buccal (MB) canals are wide apart in the more
cervical section (A) but only the mesio-buccal canal is still present in the more apical root segment (B). The rectangular demarcated
area in B is magnified in C. The tangentionally cut segment of a very narrow isthmus (IS) is further magnified in stages in D and E.
The contents of the isthmus cannot be resolved at the respective magnifications but are distinctly clear in the electron micrograph of
the area presented in Fig 7. Original magnifications: A and B, 315; C, 3100; D, 3180; E, 3300.
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242 Nair et al February 2005

mitotic forms of cocci and rods is a clear indication of

the viability of the organisms at the time of surgery and
fixation of the specimens. The observation of numerous
neutrophils, even in the very narrow isthmus of some
specimens, indicates migration of the cells from blood
vessels of the apical periodontitis into the necrotic and
infected root canal system by chemotaxis against
intracanal microbial antigens. It is also a sign of
presence of a fluid phase of the apical canal system.
These observations are fully in accordance with similar
findings reported before.4 For technical reasons, the
possibility that microbes remained in dentinal tubules
of the treated teeth was not investigated in this study.
Morphologically, the trial specimens contained a poly-
microbial flora consisting of cocci, rods, filaments, and
spirochetes. This is fully in accordance with the findings
of investigations using the precise correlative micro-
techniques on the apical root canal system of teeth with
asymptomatic posttreatment apical periodontitis.33,44
One of the 14 teeth with posttreatment residual
infection also showed the presence of foreign particles
of vegetable origin in the canal system. The particles
were most probably derived from vegetable foods of
nonleguminous nature that got trapped preoperatively in
the pulp chamber of the tooth that had an open carious
cavity. Cellulose containing food particles and clinical
materials (paper-points, cotton wool) is known to be
transported through the root canal system into the
periapical area to cause granulomatous lesions by
a foreign body reaction that is commonly referred to
as cellulose granuloma.45,46 Further, the foreign par-
ticles passing through an infected root canal can also
carry microorganisms with them, as was the case with
the specimen MX-15, into the periapical area and allow
a foreign bodyerelated biofilm47 to grow around them.
This has been clearly demonstrated to occur also in the
periapex of a human tooth.45
The intracanal microorganisms existed, both in the
positive controls and in the uninstrumented areas of the
main treated canals and untouched accessory canals of
the 14 trial specimens, as matrix-embedded multi-
leveled structures attached to the dentinal pulpal walls and
also as planktonic cells, aggregates, or coaggregates of
the species suspended in the fluid phase of the infected
and necrotic root canal. This is consistent with the
observations made in 1987 that ‘‘electron microscopi-
Fig 7. A composite transmission electron micrograph of the cally a condensed bacterial layer could be identified on
isthmus shown in Fig 6, E. The narrow uninstrumented the dentinal wall of the root canal which when light
isthmus shows plenty of bacteria and numerous neutrophils microscopically visible, gave the palisade structure of
(NG) in varying stages of disintegration. The small circular bacterial plaques adhering to tooth surfaces.’’4 In
demarcated area is magnified in the inset. Note the distinct
a contemporary microbiological sense, this means that
(arrowheads) filamentous bacterial profiles sectioned in
the undisturbed intracanal flora of an infected tooth with
various planes. Original magnification: 32800; inset 35600.
apical periodontitis is mostly organized as a matrix-
embedded collection of multispecies organisms in
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Volume 99, Number 2 Nair et al 243

Fig 8. Radiographs (A and B) of the mandibular left first molar (MX-15, Table I) with primary apical periodontitis during root
canal instrumentation (A) and postobturation (B). Note the slight extrusion of the root-filling material into the periapical area,
which was removed by apical surgery (C). Eight months after surgery (D), there is evidence of bone regeneration. The histological
and transmission electron microscopic findings of the apical root segment are illustrated in Fig 9 and Fig 10.

microecosystems that are immobilized on dentinal Several investigators have studied the effect of the
surface48 as a biofilm,5,47 which cannot be eradicated crucial mechanical measures of instrumentation and
by host defenses or chemotherapy alone. Therefore, an irrigation with sterile distilled water or physiological
effective reduction of the mostly sessile intracanal flora saline and achieved substantial quantitative reduction of
can be achieved only by mechanical dislocation of the the intracanal flora.49-51 Current data on the efficiency of
biofilm, cleaning and planing of the infected inner reducing the microbial load from infected canals using
dentine using hand and/or rotary instruments, washing stainless steel hand files and associated antimicrobial
away of the organisms and debris by irrigating with procedures come from a series of carefully designed and
antimicrobial solutions, and deposition of microbicidal executed studies19-22,52 that evaluated the effect of
dressings in the canals.19 individual steps in the treatment procedures on the
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244 Nair et al February 2005

Fig 9. Photomicrographs of 2 transverse sections through the apical portion of the mesial root of the mandibular left first molar
(MX-15, Table I) removed by surgery from the radiolucent area in Fig 8, C. The identification notch is on the buccal side. Note the
circular profiles of gutta-percha cones incompletely obturating the mesio-lingual and mesio-buccal canals that are connected by
a wide isthmus. The latter contains blue-stained materials from the arrowed area in B that are magnified in stages in C and D,
respectively. Note the remnants of plant cells (PLC) with distinct cell walls and disintegrating cytoplasmic area that in many
contain dot-like structures resembling cocci. Original magnifications: A and B, 316; C, 3520; D, 3800.
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Volume 99, Number 2 Nair et al 245

Fig 10. Transmission electron micrographs of the plant cells (PLC) depicted in Fig 9, D. Note the distinct electron lucent cell walls
(CW). The intracellular area of the PLC reveal varying numbers of coccoid bacteria (BA), many among them show mitotic forms in
the higher magnification in B. Original magnifications: A, 32000; B, 33400.
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246 Nair et al February 2005

Fig 11. Photomicrographs (A-D) of sequential transverse sections at varying distances in corono-apical direction from the apical
portion of a necrotic, noninstrumented and nonobturated distal root of a mandibular first molar (MX-19, Table I) with primary
apical periodontitis (positive control). The main canal (MC) contains microorganisms. Note the accessory canals (AC) in B and C.
The AC on the right-hand side in B is magnified in Fig 12. The demarcated AC in C is magnified in stages in the right and left insets,
respectively, in C. Note the AC is clogged with bacteria (BA). Original magnifications: A-D, 315; right inset in C, 3110; left inset
in C, 3180.
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Fig 12. Stagewise magnifications of the accessory canal (AC) on the right side of Fig 11, B. The canal is filled with
microorganisms (MO) of varying morphological forms. Note the small dot-like bacteria (BA in C) and the large pleomorphic
organisms showing filamentous and dividing forms (arrowhead), which seem to be yeast stage of fungi. Original magnifications:
A, 3100; B, 3280; C, 3800.
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248 Nair et al February 2005

Fig 13. Composite transmission electron micrographs show the presence of bacteria (BA) and fungi (FU) in the accessory canal
illustrated in Fig 12. Note the distinct electron lucent cell wall and the larger size of fungal organisms in comparison to that of the
bacteria. The fungus shows several dividing forms, some of which form chains. The organisms marked FU are further magnified in
Fig 14. Original magnifications: A, 3750; B, 3600.
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Volume 99, Number 2 Nair et al 249

Fig 14. Higher magnification electron micrograph of the chain of large microorganisms (FU) in Fig 13, B. Note the much smaller
size of the bacteria (BA) that consist of cocci, rods, and filaments. CW = cell wall. Original magnification 31200.
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250 Nair et al February 2005

bacteriological status of the treated canals. These It has also been recommended by others54-56 to
studies showed that mechanical instrumentation and complete endodontic treatment of nonvital teeth with
irrigation with physiological saline resulted in 100-fold infected root canals in 1 session, without any intracanal
to 1000-fold reduction of microbes from the root microbicidal dressing. Intraradicular microbes surviv-
canal.19 In the study,19 however, none of the 15 root ing root canal treatment are argued to be entombed by
canaletreated and sampled teeth gave negative micro- obturation of the root canal and die off as a result of
bial cultures after the first appointment of treatment, inadequate nutrients.55 ‘‘These microbes may no longer
which is fully in confirmation with the results of the interfere with the periapical healing process.’’56 The
present study. Even after 5 appointments and only long-standing popular notion of entombment and
mechanical instrumentation and saline irrigation, bac- perishing of intraradicular microbes posttreatment still
teria could be cultivated in 50% of the canals.19 Other awaits scientific validity, but in the light of contempo-
researchers13 reported that 10 of the 23 (43%) root rary understanding of apical periodontitis as an intra-
canaletreated teeth produced no microbial growth radicular biofilm-induced chronic disease would be
immediately after extensive instrumentation and irriga- a challenging task to realize. It has been shown that
tion with saline, a finding in discord with that reported periapical healing of some teeth occurs even when
here. Further reduction of intracanal viable microbes microbes are present in the canals at the time of
can be achieved by the application of tissue lytic and obturation.18 Although this may imply that the organ-
antimicrobial agents such as NaOCl alone20 or NaOCl isms may not survive posttreatment, it is more likely that
and EDTA in sequence.21 This series of studies also the microbes may be present in quantities and virulence
showed the importance of application of bactericidal that may be subcritical to sustain the inflammation of the
intracanal medicaments during the treatment.22,52 periapex, or that they remain in a location where they
The efficiency of reducing intracanal microbes by cannot communicate with the periapical tissues.
instrumentation with NiTi devices in comparison to that Taken together, there is substantial evidence19-25 that
of stainless steel hand files has been investigated24,25 by negative microbial cultures are consistently obtained
adopting methods ‘‘similar to the series performed by only after adequate instrumentation, rinsing of the canals
Byström et al.’’19-22 In one study,24 it was found that only with NaOCl and EDTA, and application of an intracanal
13 of the 46 (28%) teeth sampled were culture-negative microbicidal dressing. Short intra-appointment applica-
after instrumentation and saline irrigation. There was no tion of a bactericidal dressing has not so far resulted in
statistical difference between the outcomes of instru- a satisfactory reduction of root canal microbes.52,53
mentation with stainless steel hand files and NiTi rotary Further, certain sessile bacteria protected in biofilms
instruments. In the other investigation,25 it was shown have been found to be more than 1000 times resistant to
that addition of rinsing with NaOCl solution resulted in antimicrobial agents as the same organisms in plank-
better antibacterial effect when the instrumentation tonic form.57,58 Therefore, additional caution is neces-
exceeded ISO size #30-35. It was concluded that sary about the positive antimicrobial effects of potential
a predictable elimination of microorganisms from the root canal disinfectants reported based on short-term in
canal system cannot be achieved by NiTi instruments vitro experiments.59 Purely based on biological consid-
irrespective of the size of the final instrument used. erations, a treatment plan involving meticulous in-
Application of iodine-potassium-iodide (IKI) as a short- strumentation, irrigation with NaOCl, rinsing with
time dressing during one-visit treatment has shown some EDTA, and application of a microbicidal dressing for
promising results in terms of reducing intracanal a sufficient duration of time to be effective, cannot be
microbes. However, 15 of the 52 (29%) teeth that were completed in one treatment session with contemporary
treated in one visit, with IKI short-time dressing, still technology. Therefore, the results of this study do not
contained cultivable microbes posttreatment.53 In this provide the biological basis for treating teeth with
context, it has to be pointed out that root canals showing infected necrotic pulps in one visit. On the contrary, they
negative microbial cultures do not imply that the entire provide strong histomorphological evidence in support
canal system of the teeth involved are ‘‘rendered bacteria of following a treatment protocol based on carefully
free’’ as sometimes described.15,25 Rather, there were no controlled investigations that consistently give negative
microorganisms recoverable when an advanced bacte- microbial cultures of the treated canals.19-25
riological method was used. This is because the
microbial samples in these investigations were obtained CONCLUSIONS
from the instrumented main canal(s) only. It is obvious Fourteen of the 16 instrumented and root canale
from the study reported here that there are root canal treated mandibular molars showed residual infection
regions harboring microorganisms that cannot be of mesial roots after instrumentation, irrigation with
reached by such sampling procedures. NaOCl, and obturation were completed in a one-visit
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Volume 99, Number 2 Nair et al 251

treatment. The infectious agents were mostly located 14. Wu M, Wesselink PR. Efficacy of three techniques in cleaning
the apical portion of curved root canals. Oral Surg Oral Med Oral
in the uninstrumented recesses of the main canals, the Pathol 1995;79:492-6.
isthmus communicating between them, and in accessory 15. Card SJ, Sigurdsson A, Orstavik D, Trope M. The effectiveness
canals. The microbes in these untouched locations of increased apical enlargement in reducing intracanal bacteria.
existed primarily as biofilms that were not removed J Endod 2002;28:779-83.
16. Sjögren U, Hägglund B, Sundqvist G, Wing K. Factors affecting
by instrumentation and irrigation with NaOCl in 1 the long-term results of endodontic treatment. J Endod 1990;16:
treatment session. In view of the great anatomical 498-504.
complexity of the root canal system, particularly of 17. Engström B, Hård af Segerstad L, Ramström G, Frostell G.
Correlation of positive cultures with the prognosis for root canal
molars,26,27 and the ecological organization of the flora treatment. Odontol Revy 1964;15:257-70.
into protected sessile biofilms5,47 composed of microbial 18. Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection
cells embedded in a hydrated exopolysaccharide- at the time of root filling on the outcome of endodontic treatment of
teeth with apical periodontitis (Published erratum appears in Int
complex in microcolonies,4 it is very unlikely that an Endod J 1998;31:148). Int Endod J 1997;30:297-306.
absolutely microorganism-free canal system can be 19. Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy
achieved by any of the contemporary root canal prepara- of mechanical root canal instrumentation in endodontic therapy.
Scand J Dent Res 1981;89:321-8.
tion, cleaning, and root-filling procedures, particularly 20. Byström A, Sundqvist G. Bacteriologic evaluation of the effect
in 1 treatment session. These findings highlight the of 0.5 percent sodium hapochlorite in endodontic therapy. Oral
importance and necessity of stringently applying non- Surg Oral Med Oral Pathol 1983;55:307-12.
21. Byström A, Sundqvist G. The antibacterial action of sodium
antibiotic chemomechanical measures in order to dis- hypochlorite and EDTA in 60 cases of endodontic therapy. Int
rupt the biofilms and reduce the intraradicular microbial Endod J 1985;18:35-40.
load to the lowest possible level to ensure the most 22. Byström A, Claeson R, Sundqvist G. The antibacterial effect of
camphorated paramonochlrophenol, camphorated phenol and
favorable long-term prognosis for treatment of infected calcium hydroxide in the treatment of infected root canals
root canals. phenol. Endod Dent Traumatol 1985;1:170-5.
23. Byström A, Happonen RP, Sjögren U, Sundqvist G. Healing of
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