Clinical Research
Biofilms and Apical Periodontitis: Study of Prevalence
and Association with Clinical and Histopathologic Findings
Domenico Ricucci, MD, DDS,* and José F. Siqueira, Jr., DDS, MSc, PhD†
Abstract
Introduction: This study evaluated the prevalence of
bacterial biofilms in untreated and treated root canals
of teeth evincing apical periodontitis. The associations
of biofilms with clinical conditions, radiographic size,
and the histopathologic type of apical periodontitis
were also investigated. Methods: The material
comprised biopsy specimens from 106 (64 untreated
and 42 treated) roots of teeth with apical periodontitis.
Specimens were obtained by apical surgery or extraction
and were processed for histopathologic and histobacter-
iologic techniques. Results: Bacteria were found in all
but one specimen. Overall, intraradicular biofilm
arrangements were observed in the apical segment of
77% of the root canals (untreated canals: 80%; treated
canals: 74%). Biofilms were also seen covering the walls
of ramifications and isthmuses. Bacterial biofilms were
visualized in 62% and 82% of the root canals of teeth
with small and large radiographic lesions, respectively.
All canals with very large lesions harbored intraradicular
biofilms. Biofilms were significantly associated with epi-
thelialized lesions (cysts and epithelialized granulomas
or abscesses) (p < 0.001). The overall prevalence of bio-
films in cysts, abscesses, and granulomas was 95%,
83%, and 69.5%, respectively. No correlation was found
between biofilms and clinical symptoms or sinus tract
presence (p > 0.05). Extraradicular biofilms were
observed in only 6% of the cases. Conclusions: The
overall findings are consistent with acceptable criteria
to include apical periodontitis in the set of biofilm-
induced diseases. Biofilm morphologic structure varied
from case to case and no unique pattern for endodontic
infections was identified. Biofilms are more likely to be
present in association with longstanding pathologic
processes, including large lesions and cysts. (J Endod
2010;36:1277–1288)
Key Words
Apical periodontitis, bacterial biofilm, endodontic infec-
tion, endodontic treatment
From *Private Practice, Rome, Italy; and †Department of Endodontics, Faculty of Dentistry, Estácio de Sá University, Rio de Janeiro, Brazil.
Address requests for reprints to Dr Domenico Ricucci, Piazza Calvario, 7, 87022 Cetraro (CS), Italy. E-mail address: dricucci@libero.it.
0099-2399/$0 - see front matter
Copyright ª 2010 American Association of Endodontists.
doi:10.1016/j.joen.2010.04.007
JOE — Volume 36, Number 8, August 2010
I n their natural habitats, microorganisms almost invariably live as members of meta-
bolically integrated communities usually attached to surfaces to form biofilms (1).
The biofilm community lifestyle provides microorganisms with a series of advantages
and skills that are not observed for individual cells living in a free-floating (planktonic)
state including establishment of a broader habitat range for growth; increased meta-
bolic diversity and efficiency; protection against competing microorganisms, host
defenses, antimicrobial agents, and environmental stress; and enhanced pathogenicity
(2). The study of microbial biofilms assumes a great importance in different sectors of
industrial, environmental, and medical microbiology. In medical microbiology, bio-
films have been increasingly studied and estimates indicate that biofilm infections
comprise 65% to 80% of the human infections in the developed world (3). As for
the oral cavity, caries, gingivitis, and marginal periodontitis are examples of diseases
caused by bacterial biofilms in the form of supragingival or subgingival dental plaque.
Mounting evidence indicates that apical periodontitis is also a biofilm-induced
disease (4–6). In situ investigations using optical and/or electron microscopy have
allowed observations of bacteria colonizing the root canal system in primary or
persistent/secondary infections as sessile biofilms covering the dentinal walls (7–
12). Apical ramifications, lateral canals, and isthmuses connecting main root canals
have all been shown to harbor bacterial cells, which are also frequently organized in
biofilm-like structures (13–15). In addition, biofilms adhered to the apical root
surface (extraradicular biofilms) have been reported and regarded as a possible
cause of posttreatment apical periodontitis (16, 17).
Although the concept of apical periodontitis as a biofilm-induced disease has been
built upon these observations, the prevalence of biofilms and their association with clin-
ical and histopathologic findings have not yet been reported. Before this information
becomes available, it may seem somewhat imprecise to generalize and categorize apical
periodontitis as a biofilm-induced disease. The purpose of the present study was two-
fold: (1) evaluate the prevalence of intraradicular and extraradicular bacterial biofilms
in untreated and treated root canals of human teeth evincing apical periodontitis
through a histobacteriologic approach and (2) look for associations of biofilms with
some clinical conditions, radiographic size, and the histopathologic type of apical pe-
riodontitis lesions.
Materials and Methods
Clinical Specimens
The material for this study consisted of sequential biopsies of roots or root tips
together with surrounding apical periodontitis lesions. Specimens were part of the
histologic collection of one of the authors (DR). The material comprised 106 roots
from 100 human teeth. Of these, 58 were teeth with untreated root canals (6 incisors,
3 canines, 18 premolars, and 31 molars) from 52 patients (25 females, 27 males) aged
18 to 75 years (mean, 42 years). In total, 64 roots from untreated teeth were available,
of which 59 were extracted with apical periodontitis lesions attached while in the other
Biofilms and Apical Periodontitis 1277
Clinical Research
five specimens lesions had to be removed separately. All untreated teeth

TABLE 1. Prevalence of Intraradicular Biofilms at the Apical Segment and Extraradicular Biofilms in Untreated and Root Canal–treated Teeth According to the Histopathologic Type of Apical Periodontitis and Clinical

Total (untreated + treated) (%)

had necrotic pulps and gross carious lesions and were extracted
because they were judged unrestorable or the patient did not agree
to save the tooth. Records were made of any symptoms that the patient
experienced or was experiencing in relation to the affected tooth. The

3/8 (37.5)
2/8 (25)
1/2 (50)
presence/absence of a sinus tract was also recorded. For 33 teeth,

6/106 (6)

0/10 (0)

0/3 (0)
0/7 (0)
2/52 (4)

1/21 (5)
0/3 (0)

6/69 (9)
conventional periapical radiographs were available before extraction,
and the largest diameter of the periradicular radiolucency was
measured. Teeth with periodontal pockets or longitudinal fractures

Extraradicular biofilm
or cracks involving the root were excluded from analysis. Some of
the examined teeth were included in previous studies (18, 19).
The 42 biopsies of roots/root tips from root canal-treated teeth
were from 42 patients, 24 (12 symptomatic and 12 asymptomatic) of
which took part in a recent publication (10) and were re-evaluated

Treated (%)

2/19 (10.5)
1/7 (14)

2/6 (33)
in this study for the presence of biofilms following the parameters es-

2/42 (5)

0/1 (0)
1/32 (3)

0/2 (0)
—
—
—
—
tablished herein (see later). All 42 cases were categorized as treatment
failures on the basis of clinical and/or radiographic follow-ups, after
a minimum recall period of 4 years for the asymptomatic cases and 1
year for the symptomatic ones. For 10 of the new cases, the quality of

Untreated (%)
previous endodontic treatment was judged as inadequate. Radiographs

2/8 (25)
1/2 (50)

1/2 (50)
were not available for four of the new 18 cases. All patients had given

4/64 (6)

0/6 (0)
1/20 (5)
0/10 (0)

0/3 (0)
0/14 (0)
0/1 (0)

4/50 (8)
consent for examination of their teeth.

Tissue Processing
Immediately after removal (by periradicular surgery or extrac-

Total (untreated + treated) (%)

tion), the biopsy specimen was immersed in 10% neutral-buffered
formalin for at least 48 hours. Demineralization was performed in an
aqueous solution consisting of a mixture of 22.5% (vol/vol) formic
acid and 10% (wt/vol) sodium citrate for 3 to 4 weeks, with the

7/8 (87.5)

7/8 (87.5)
10/10 (100)

2/2 (100)
3/3 (100)
82/106 (77)

6/7 (86)
35/52 (67)

17/21 (81)
2/3 (67)

57/69 (83)
endpoint being determined radiographically. All specimens were
washed in running tap water for 24 to 48 hours, dehydrated in
ascending grades of ethanol, cleared in xylene, infiltrated, and
embedded in paraffin (melting point, 56 C) according to standard
Intraradicular biofilm

procedures. To produce sections parallel to the long axis of the root

canal, special precautions were taken. Roots in multirooted teeth
were dissected free and processed separately. If curved, roots were
separated in two pieces, one encompassing the coronal two thirds
and the other including the apical one third. These two pieces were
Treated (%)

1/1 (100)

7/7 (100)
31/42 (74)

22/32 (69)

1/2 (50)
5/6 (83)
16/19 (84)
embedded separately, but only the apical segment was evaluated in
this study.
—
—
—
—

With the microtome set at 4 to 5 mm, meticulous longitudinal serial

sections were taken until each specimen was exhausted. For some spec-
imens, cross-cut sections were taken. Every fifth slide was stained with
hematoxylin-eosin for screening purposes and for assessment of
Untreated (%)

7/8 (87.5)
10/10 (100)

2/2 (100)
3/3 (100)

1/1 (100)
2/2 (100)

inflammation. A modified Brown and Brenn technique for staining

51/64 (80)

5/6 (83)
13/20 (65)

10/14 (71)

41/50 (82)

bacteria (20) was used for selected slides. The accuracy of the bacterial
staining method was tested using the protocol described by Ricucci and
Bergenholtz (21).

Evaluation Criteria
Granuloma, nonepithelialized

Slides were examined by two evaluators. Evaluations were per-

Abscess, nonepithelialized
Granuloma, epithelialized

formed separately, and whenever disagreement occurred, it was

Abscess, epithelialized

resolved by joint discussion. The following aspects were specifically

looked for in the examination: 1) the presence and location of bacteria
Cyst, pocket (bay)
Cyst, unclassified
Overall prevalence

in the apical segment of the root canal, including the main canal, lateral
canals, apical ramifications, and isthmuses (intraradicular infection) or
Unclassified
Sinus tract
Symptoms

within the body of the apical periodontitis lesion or adhered to the

Cyst, true
Lesion type

external apical root surface (extraradicular infection). The parameter

used for classification of bacterial community structures as biofilms fol-
Features

lowed the biofilm definition given by Hall-Stoodley et al (22): ‘‘Micro-

bial biofilms are populations of microorganisms that are concentrated

1278 Ricucci and Siqueira Jr. JOE — Volume 36, Number 8, August 2010

at an interface and typically surrounded by an extracellular polymeric
substance matrix.’’ Also, according to these authors, aggregates or coag-
gregates of bacterial cells not apparently attached to the surface were
classified as ‘‘flocs’’; and 2) the presence and distribution of acute
and chronic inflammatory cells and epithelium in the inflamed perira-
dicular tissues; lesions were diagnosed histologically as follows: apical
abscess (epithelialized or nonepithelialized), granuloma (epithelialized
or nonepithelialized), or cyst (true, pocket, or unclassified; the latter
diagnosis was made when the lesion showed a cavity lined by epithe-
lium, but the soft tissue did not remain attached to the root tip and
had to be removed separately).
Statistical Analysis
The Fisher exact test or the chi-square test was used to check for
associations of intraradicular biofilms with the following parameters:
radiographic size of the apical periodontitis lesion (grouped as lesions
#5 mm and >5 mm), histopathologic general type of apical periodon-
titis (granuloma, cysts, and abscesses with no distinction of subtypes),
presence and absence of epithelial proliferation (irrespective of the
lesion type), sinus tract, and symptoms. Every analysis took into consid-
eration only untreated root canals, only treated canals, and all speci-
mens together. The prevalence of biofilms in untreated and treated
teeth was also compared by the chi-square test.
Results
Biofilm Overall Prevalence
Bacteria were found in all specimens, except for one asymptomatic
root canal–treated tooth in which disease emerged probably because of
a foreign body reaction. This case was reported in a previous study
(10). Overall, bacterial arrangements as intraradicular biofilms were
observed in the apical segment of 82 of 106 (77%) root canals. Of these,
51 of 64 (80%) were from untreated canals and 31 of 42 (74%) from
treated canals (Table 1). This difference was not statistically significant
(c2, p = 0.6).
Figure 1. Examples of intracanal biofilms with different bacterial cell morphologies. (a) The predominance of cocci. Note the high concentration of cells in contact
with the root canal wall (Taylor’s modified Brown and Brenn, original magnification 1,000). (b) Predominance of filamentous forms. Note the irregular distri-
bution of bacterial cells within the extracellular material (original magnification 1,000).
JOE — Volume 36, Number 8, August 2010
Clinical Research
Morphologic Description of Bacterial Colonization
Intraradicular bacterial biofilms were usually thick and composed
of several layers of bacterial cells. At high magnification, three basic
bacterial cell morphologies could be recognized in most cases: cocci,
rods, and filaments (Figs. 1a and b, 2c and d, 3c and d, 4d, and 5c and
d). These morphotypes were often present together in varied propor-
tions in the same biofilm. However, a single morphotype appeared to
dominate each biofilm (Figs. 1a and b, 2c and d, 3c, 4d, and 5c and d).
In the biofilm structure, the proportion between bacterial cells and
extracellular matrix was highly variable. In some instances, bacterial
cells appeared so clumped that the extracellular component was virtu-
ally not visible (Figs. 2d and 5c and d). In other cases, the extracellular
matrix was abundant, and less bacterial cells were seen distributed in an
uneven pattern (Figs. 1a and b, 2c, 3c, and 4c and d). In many biofilms,
cells were abundant in the deepest layers (Figs. 1a and 4c). In some
other cases, however, they were prevalent in the most superficial layers.
In many specimens, multilayered biofilms covered uniformly the
root canal walls to a long extent. In some cases, opposite root canal
walls covered by biofilms were faced with necrotic debris or inflamma-
tory cells in the canal lumen (Figs. 3b and 5b). In teeth with severe
caries destruction and longstanding pulp necrosis, the observation
that the entire apical canal was filled by a dense biofilm was not
uncommon. In some instances, however, although some areas of the
canal were completely covered by biofilms, others were apparently
free from bacterial colonization (Fig. 2g). This pattern was more
commonly observed in treated root canals but also seen in a few
untreated specimens.
In some instances, bacterial colonization was restricted to the root
canal wall surface, and no deep dentinal invasion was observed. This
was probably because of the reduced number and small diameter or
even the lack of dentinal tubules in certain regions of the apical root
segment (Figs. 1a and b, 2c and d, and 3c). However, when dentinal
tubules were present and abundant, they usually appeared colonized
by bacteria spreading from the biofilm and penetrating at varying depths
(Fig. 6a and b).
Biofilms and Apical Periodontitis 1279
Clinical Research

Figure 2. (a) Mandibular molar with the crown destroyed by a gross carious process and a radiolucency around the mesial root assessed as >5 mm. Several
exacerbations developed over the previous months, but the tooth was asymptomatic at the time of extraction. (b) Sections were taken on a mesiodistal plane. The
section passing through the apical third of the mesiobuccal canal. The lumen appears partly filled by large fuchsin-stained bodies, likely food remnants (large
vegetable cells). More apically the walls appeared covered by a dense bacterial biofilm (Taylor’s modified Brown and Brenn, original magnification 100).
(c) High magnification of the area of the root canal wall indicated by the left arrow in b. Rods are the prevailing bacterial morphotype at this level (original magni-
fication 1,000). (d) High magnification of the area of the root canal wall indicated by the right arrow in b. The high bacterial population density seems to obscure
the extracellular matrix. Note the polymorphonuclear neutrophils in contact with the biofilm surface (original magnification 1,000). (e) Cross-cut section taken
at the transition between the apical and the middle third of the mesial root. The low-magnification overview shows that the two mesial canals are connected by a wide
isthmus, clogged with bacteria (original magnification 8). (f) Detail of the isthmus. Its lumen is filled by a dense biofilm (original magnification 100). (g)
Magnification of the left canal in e. The majority of the root canal circumference is covered by a bacterial biofilm (original magnification 100).

One of the untreated teeth exhibited an unusual pattern of intraca-
nal bacterial colonization and deserves a more detailed description. It is
a maxillary first premolar from a 58-year-old man. The patient reported
several pain episodes, and the tooth, judged nonrestorable because of
gross coronal destruction, was extracted. Sections passing through the
buccal canal showed that the apical canal lumen was clogged with
necrotic debris and bacteria (Fig. 7a). At the center of the main root
canal, a large bacterial floc composed of ramifying filamentous bacterial
cells was present and surrounded by a distinct layer of an amorphous
material (Fig. 7b-d). Bacteria appeared particularly condensed in this
structure, and at the periphery they exhibited a starburst appearance
typical of actinomycotic colonies (Fig. 7c and d). Serial sections re-
vealed that this large colony was not contiguous with the root canal
walls. Actually, it was apparently free floating in the root canal lumen,
enmeshed in the remainder of the canal content. This case has been as-
sessed as showing a biofilm, not because of the unusual large floc sus-
pended in the canal, which according to the definition criteria used
herein cannot be strictly assessed as a biofilm, but rather because the
bacterial condensations present more apically were clearly adhered
to the root canal walls forming a biofilm-like structure (Fig. 7b).
Biofilms were also seen covering the walls of apical ramifications,
lateral canals, and isthmuses in both untreated and treated canals. In

1280 Ricucci and Siqueira Jr. JOE — Volume 36, Number 8, August 2010
 Clinical Research

Figure 3. Grossly carious single-rooted mandibular second molar extracted with the apical periodontitis lesion attached. The radiographic size of the lesion was
<5 mm (inset). The tooth was symptomatic. (a) The section passing approximately at the center of the foramen. The overview shows granulomatous tissue ingrowth
at the very apical canal (Taylor’s modified Brown and Brenn, original magnification 16). (b) Detail of the apical foramen region. A biofilm is present covering the
root canal walls, and a dense bacterial aggregate is evidenced more coronally. Empty spaces are shrinkage artefacts (original magnification 100). (c) Higher
magnification of the area demarcated by the rectangle in b. Bacterial filamentous forms prevail, and the extracellular component is abundant at this level (original
magnification 400). (d) Higher magnification of the bacterial aggregate indicated by the arrow in b. Different morphotypes are present. Note the concentration of
polymorphonuclear neutrophils in contact with the biofilm surface (original magnification 400).

some untreated canals, bacteria in the form of biofilms or flocs did not
reach the apical foramen because vital inflamed tissue was observed
occupying the very apical canal. In these cases, the front of infection
was located some millimeters short of the foramen.
Small bacterial flocs and planktonic cells were found in virtually all
specimens including those negative for the presence of biofilms. Flocs
and planktonic cells were usually observed in the lumen of the main
canal, ramifications, and isthmuses, either apparently floating or
more commonly enmeshed in the necrotic pulp tissue.
Association with Diverse Conditions
Radiographs available for 71 specimens were used to check for an
association between intraradicular biofilms and the radiographic size of
apical periodontitis. Bacterial biofilms were visualized in 23 of 37
(62%) root canals with small lesions (#5 mm), whereas in specimens
with large lesions (>5 mm), biofilm structures were present in 28 of 34
(82%) (Table 2). Statistical analysis disclosed a p value very close to the
level of significance used (c2, p = 0.059). Specifically for untreated
canals, the prevalence of intraradicular biofilms was 59% and 87.5%

JOE — Volume 36, Number 8, August 2010 Biofilms and Apical Periodontitis 1281
Clinical Research

Figure 4. (a) Maxillary second premolar with a periapical radiolucency whose diameter was measured >5 mm. The tooth was extracted in the presence of clinical
symptoms (pain and swelling). (b) The apical periodontitis lesion remained attached to the root tip. Section passing through the main wide apical foramen (Tay-
lor’s modified Brown and Brenn, original magnification 16). (c) Detail of the foramen area. A biofilm is present in the most apical canal, faced with inflammatory
tissue. Note the resorption of the canal walls (original magnification 100). The inset shows PMNs from the center of the lesion, one of which exhibits a cytoplasm
engulfed with several bacterial fragments (original magnification 1,000). (d) Higher magnification of the area from the cementum fragment indicated by the
arrow in c. Bacterial filamentous forms are dominant at this level. The biofilm is surrounded by PMNs (original magnification 400).

for specimens with small and large lesions, respectively. Biofilms in
treated teeth were disclosed in 65% and 78% of the canals with small
and large lesions, respectively. All five canals associated with lesions
larger than 10 mm harbored intraradicular biofilms (Table 2).
Regarding the histopathologic diagnosis, intraradicular biofilms
were significantly associated with epithelialized lesions (cysts and epi-
thelialized granulomas or abscesses) (Fisher test, p < 0.001). Of the 30
lesions exhibiting epithelial proliferation, 28 (93%) were associated
with intraradicular biofilms. Although canals associated with epithelial-
ized or nonepithelialized granulomas exhibited intraradicular biofilms
in 86% and 67%, respectively, this 20% difference did not reach signif-
icance for the sample size evaluated (Fisher test, p = 0.4). When lesions
were grouped as granulomas (epithelialized or not), abscesses (epithe-
lialized or not), and cysts (true, pocket, and unclassified), intraradic-
ular biofilms were found significantly more frequently in cysts than in
granulomas (Fisher test, p = 0.03). Actually, only one out of the 20
(5%) cystic lesions was negative for the presence of intraradicular bio-
films. No other significant differences were observed for lesion types (p
> 0.05). The overall prevalence of biofilms in granulomas was 41 of 59
(69.5%), and in abscesses it was 20 of 24 (83%). In general, findings
from separate analysis of untreated canals were similar to the overall
findings. Because cysts were not observed in association with root

1282 Ricucci and Siqueira Jr. JOE — Volume 36, Number 8, August 2010
 Clinical Research

Figure 5. (a) Mandibular second premolar extracted after several pain episodes. The apical periodontitis lesion remained attached to the root tip at extraction.
The section passing approximately at the center of the root canal. The overview discloses a severely resorbed root apex. The canal appears filled with tissue, and
there are two bacterial masses on the opposite root canal walls (Taylor’s modified Brown and Brenn, original magnification 16). (b) Higher magnification of the
apical canal. The bacterial masses were biofilm structures. The canal lumen is filled with inflammatory cells (original magnification 100). (c and d) Higher
magnification of the left and right biofilm structures respectively. Both were apparently exclusively composed of the bacterial filamentous morphotype. A severe
inflammatory reaction surrounded these bacterial biofilms (original magnification 400).

canal–treated teeth, statistical analysis was not performed separately for
data from treated canals.
Intraradicular biofilms were found in seven of the eight (87.5%)
specimens associated with a sinus tract and in 57 of 69 (83%) of the
symptomatic cases (Table 1). However, despite the high prevalence,
these values were not statistically significant when compared with cases
with no sinus tract (75/98, 76.5%) (Fisher test, p = 0.7) and no symp-
toms (25/37, 68%) (c2, 0.08). No significance was found for data from
untreated canals and treated canals when examined separately (p >
0.05).
Extraradicular bacterial biofilms were observed in only six speci-
mens (6%), four from untreated canals and two from treated canals. All
these specimens were associated with clinical symptoms. Of the eight
cases with sinus tracts, extraradicular biofilms were detected in three
(37.5%). In all but one of the cases, the extraradicular biofilm was asso-
ciated with an intraradicular biofilm. Two of these extraradicular bio-
films showed areas of mineralization with a calculus-like appearance
(Fig. 8).
Discussion
Determination that a given human infectious disease is caused by
biofilms is not an easy task. Difficulties may be related to several
reasons, including the coexistence of biofilm and planktonic bacteria
in many infections, the absence of a definitive marker for bacteria form-
ing biofilms, and the loss of the biofilm phenotype when subject to
sampling and culturing procedures (23). By taking such difficulties
into account, Parsek and Singh (23) proposed the following criteria
to define infections caused by biofilms: (1) the infecting bacteria are
adhered to or associated with a surface (‘‘associated with’’ implies

JOE — Volume 36, Number 8, August 2010 Biofilms and Apical Periodontitis 1283
Clinical Research
Figure 6. Maxillary lateral incisor with a large periapical radiolucency (>5 mm) (inset). (a and b) The section taken at the transition between the apical and the
middle third showing a bacterial biofilm covering the dentinal walls. The dentinal tubules subjacent to the biofilm are heavily invaded and colonized to varying
depths (Taylor’s modified Brown and Brenn, original magnification 100 and 400).
that aggregates/coaggregates do not need to be firmly attached); (2)
direct examination of infected tissue shows bacteria forming clusters
or microcolonies encased in an extracellular matrix, which may be of
bacterial or host origin; (3) the infection is generally confined to
a particular site, and although dissemination may occur, it is a secondary
event; and (4) the infection is difficult or impossible to eradicate with
antibiotics despite the fact that the responsible microorganisms are
susceptible to killing in the planktonic state. The following criterion
was further added by Hall-Stoodley and Stoodley (24): (5) ineffective
host clearance evidenced by the location of bacterial colonies in
discrete areas in the host tissue associated with host inflammatory cells.
According to this last criterion, evidence of polymorphonuclear neutro-
phils (PMNs) and macrophages surrounding bacterial aggregates/
coaggregates in situ considerably increases the suspicion of biofilm
involvement with disease causation. We propose the following sixth
criterion: the elimination or significant disruption of the biofilm struc-
ture and ecology leads to remission of the disease process.
Although there are recognized limitations to these criteria, it is
assumed that they provide general characteristics that allow for consid-
ering the role of biofilms in the pathogenesis of a certain human disease
(24). The present findings showing biofilm structures in the great
majority of cases of primary (80%) and posttreatment (74%) apical pe-
riodontitis along with the observed morphological features of these bio-
films seem to fulfill four of the six criteria. Although adhesion and
strength of adhesion cannot be measured by the methods used in the
present study, the bacterial agreggates/coaggregates were observed to
at least be associated with the root canal dentin surface (criterion 1).
Bacterial colonies were seen in the huge majority of the specimens en-
cased in an amorphous extracellular matrix whose origin was, however,
not possible to determine (criterion 2). Endodontic biofilms were often
confined to the root canal, in a few cases extending to the external root
surface, but dissemination through the lesion never occurred (criterion
3). In the great majority of cases, biofilms were directly faced by inflam-
1284 Ricucci and Siqueira Jr.
matory cells in the very apical canal, in ramifications, and in isthmuses
(criterion 5). Criterion 4 was not assessed in this study, but it is widely
known that intraradicular endodontic infections are not treatable by
systemic antibiotics, even though most endodontic bacteria are suscep-
tible to currently used antibiotics (25). The problem with using systemic
antibiotics is related to the fact that endodontic infection occurs in an
avascular space with restricted access to antibiotics, but the recognition
of endodontic infections as biofilm infections still strengthens the expla-
nations for antibiotic ineffectiveness. As for criterion 6 proposed in this
study, effects of treatment on biofilms and how they influence the treat-
ment outcome were not evaluated herein and await further investiga-
tions in a longitudinal experimental design. Nonetheless, the frequent
observation of biofilms in treated canals with posttreatment disease
may at least suggest that there is a potential for fulfillment of this crite-
rion.
The apical root canal can be regarded as a critical territory for
pathogenetic and therapeutic reasons (26). This is because bacteria
located at the apical canal of teeth with apical periodontitis are in
such a strategic position that they may be regarded as the most impor-
tant infective agents related to the disease pathogenesis. With this in
mind, the present study restricted the investigation of biofilm prevalence
to the apical root canal system. The very high prevalence of intraradic-
ular biofilms may be related to the fact that bacteria in the apical canal
compose the advanced front of infection and then directly face an in-
flamed tissue area. Inflammatory exudate seeps into the apical canal
to create a fluid phase and provide bacteria with nutrients in the
form of glycoproteins and proteins. This may represent optimal condi-
tions for biofilm formation and help explain the many exuberant bio-
films observed in the apical canal, especially in primary endodontic
infections in which the untreated root canal may afford more space
for exudate seepage and stagnation into the canal.
Overall, intraradicular biofilms were 20% more frequent in teeth
with large radiographic lesions than in those with small lesions. All root
JOE — Volume 36, Number 8, August 2010
 Clinical Research

Figure 7. (a) Untreated maxillary first premolar extracted with the apical periodontitis lesion attached to the root tip. The longitudinal section passing approx-
imately through the center of the buccal canal. Ingrowth of granulomatous tissue is evident at the apical foramen (Taylor’s modified Brown and Brenn, original
magnification 16). (b) Detail of the apical root canal showing the bacterial content. A large bacterial floc exhibiting a high bacterial density and surrounded by
amorphous material can be distinguished at the center of the canal lumen. Empty spaces are artifacts (original magnification 100). (c and d) Higher magni-
fication of the upper and the lower halves of the floc in b. A great amount of intertwining filaments radiating at the periphery and projecting into a distinct extra-
cellular surround is discernible. Note the totally different arrangement of the bacterial populations outside the floc (original magnification 400).

canals associated with very large lesions (>10 mm) were found to The present study revealed that intraradicular biofilms were signif-
harbor intraradicular biofilms. Large lesions have been associated icantly more frequent in root canals of teeth with epithelialized lesions
with complex intraradicular infections characterized by bacterial (cysts and epitheliazed granulomas or abscesses). Ninety-three percent
communities with increased species richness and high populational of the lesions exhibiting some level of epithelial proliferation were in
density (27, 28). Because it takes time for apical periodontitis to
develop and become radiographically visible, it is conceivable to
TABLE 2. Prevalence of Intraradicular Biofilms at the Apical Segment of
assume that large lesions represent a longstanding pathologic Untreated and Treated Canals of Teeth with Apical Periodontitis According to
process caused by an even ‘‘older’’ intraradicular infection. In the Lesion Size as Determined Radiographically
a longstanding infectious process, involved bacteria may have had
enough time and conditions to adapt to the environment and set Untreated Total (untreated
a mature and organized biofilm community. The fact that infected Lesion size (%) Treated + treated)
root canals of teeth with large lesions harbor a large number of cells #5 mm 10/17 (59) 13/20 (65) 23/37 (62)
and species almost always organized in biofilms may help explain the >5 mm 14/16 (87.5) 14/18 (78) 28/34 (82)
$10 mm* 2/2 (100) 3/3 (100) 5/5 (100)
long-held concept that the treatment outcome may be influenced by
the lesion size (29). *These specimens were also included in the group >5 mm.

JOE — Volume 36, Number 8, August 2010 Biofilms and Apical Periodontitis 1285
Clinical Research
association with root canals colonized by bacterial biofilms. As for the
lesion histopathologic type, intraradicular biofilms were significantly
more detected in cases diagnosed as cysts (95% as compared with
69.5% in granulomas and 83% in abscesses). Because apical cysts
develop as a result of epithelial proliferation in some granulomas
(30), it is reasonable to assume that the older the apical periodontitis
lesion, the greater the probability of it becoming a cyst. Similar to teeth
with large lesions, the age of the pathologic process may also help
explain the higher prevalence of biofilms in association with cysts.
Extraradicular bacterial biofilms were found in only six speci-
mens, and except for one case they were always associated with intra-
radicular biofilms. This low prevalence of extraradicular biofilms is in
accordance with previous studies (10, 31). These findings also suggest
that extraradicular biofilms are usually maintained by intraradicular
infection. All cases showing an extraradicular biofilm exhibited
clinical symptoms, and three of them were associated with sinus
tracts. In abscesses, individual bacterial cells were seen within the
inflamed periradicular tissue and commonly being phagocytosed by
PMNs (Fig. 4a). These findings indicate that extraradicular infections
in the form of biofilms or planktonic bacteria are not a common occur-
rence, are usually dependent on the intraradicular infection, and are
more frequent in symptomatic teeth.
Similar to other studies, bacteria were also seen in the lumen of the
main canal, ramifications, and isthmuses as flocs and planktonic cells,
either intermixed with necrotic pulp tissue or possibly suspended in
a fluid phase. Bacterial flocs in clinical specimens may have originated

Figure 8. Maxillary premolar with clinically necrotic pulp and a sinus tract buccally. No periodontal pockets were disclosed at probing. The largest diameter of the
lesion on the radiograph was >5 mm (inset). (a and b) After extraction, calculus is observed covering exclusively the root apex. The apical periodontitis lesion did
not remain attached to the root tip and was removed separately. (c) The section taken on a mesiodistal plane not passing through the main foramen. The apical
external surface is covered by a bacterial biofilm (Taylor’s modified Brown and Brenn, original magnification 16). (d) Higher magnification of the area indicated
by the upper left arrow in c. Biofilm with high bacterial density (original magnification 400). (e) Higher magnification of the area indicated by the lower left arrow
in c. A dense biofilm with a prevalence of filamentous morphotypes. Note the area apparently free of bacterial cells, which may be likely a focus of calcification
(original magnification 1,000). (f) Higher magnification of the area from the external radicular profile indicated by the right arrow in c. The biofilm is miner-
alized with relatively few bacteria (original magnification 1,000).

1286 Ricucci and Siqueira Jr. JOE — Volume 36, Number 8, August 2010

from the growth of cell aggregates in a fluid or they may have detached
from biofilms (24). Flocs may exhibit many of the same characteristics
as biofilms (22) and along with planktonic bacteria have been sug-
gested to play a role in the pathogenesis of acute clinical forms of apical
periodontitis (5).
The ability of endodontic bacteria to organize themselves in bio-
film communities is of great therapeutic interest in endodontics.
Although bacteria present as flocs and planktonic cells in the main
root canal may be easily accessed and eliminated by instruments and
substances used during treatment, those organized in biofilms attached
to the canal walls or located into isthmuses and ramifications are defi-
nitely more difficult to reach. Many bacteria under the biofilm were seen
invading dentinal tubules (Fig. 6), which also pose a problem for disin-
fection. Some biofilm-covered walls of the main canal may remain
untouched by instruments, which is especially true when the root canal
is irregular, flattened, or oval in cross-section (32–34) (Fig. 2e and f).
Biofilm remnants were observed on the root canal walls of treated teeth
in the present study. This study confirmed that isthmuses, lateral canals,
and apical ramifications can be clogged with bacteria, including in
treated teeth (13–15). These areas are not expected to be reached
by instruments and antimicrobial irrigants. Even in the event that
antimicrobial agents reach the biofilm, this is no guarantee of
successful antimicrobial activity because bacteria arranged in
biofilms exhibit increased resistance to antimicrobials (35, 36).
Biofilms were classified morphologically as described by Hall-
Stoodley et al (22) in a comprehensive review on the subject. A very
similar definition is provided by Costerton in his ‘‘biofilm primer’’(1).
‘‘A biofilm is a multicellular community composed of prokaryotic and/
or eukaryotic cells embedded in a matrix composed, at least partially, of
material synthesized by the sessile cells in the community.’’ There are
obviously some features associated with biofilms such as differential
genetic expression and the presence of water and nutrient channels
in the matrix that could not be evaluated by the method used in this
study. However, biofilms significantly differ in structure according to
the overall physical, chemical, and biological features of the environ-
ment (1, 37–39). For instance, in an environment where there is low
shear force related to the passage of fluid or air, a strong adhesion to
the surface is not made necessary. Promoting such adhesion
would represent energy waste for the community. Therefore, our
morphologic findings support the inclusion of apical periodontitis in
the set of biofilm-induced diseases. Further studies are required to
compare the main structural and physiologic features of endodontic
biofilms to other biofilms in nature.
It is reasonable to surmise that the unique root canal environ-
mental conditions are expected to influence the biofilm structure and
function to the point of giving rise to endodontic biofilms with typical
features. However, although limited in resolution power, our study
failed to show any specific morphological pattern for endodontic bio-
films. Actually, endodontic biofilm morphology differed consistently
from individual to individual, and the reasons for that deserve further
investigations but may be conceivably related to different species
composition and resulting interactions, type and availability of nutri-
ents, and time of infection.
Although a very high prevalence of biofilms was observed in teeth
with apical periodontitis, the possibility exists that the figures reported
in this study still represent an underestimation. Some root canal
contents may have been washed away during histological processing
because of the numerous chemical solutions, and gram-negative
bacteria may sometimes be overlooked by the method used. Even
considering these limitations, the very high prevalence of biofilms as re-
ported in this study indicates that when properly and meticulously per-
formed, conventional paraffin techniques and so-called ‘‘old’’ bacterial
JOE — Volume 36, Number 8, August 2010
Clinical Research
staining protocols are still valuable tools for studying bacterial coloni-
zation of the root canal system.
In conclusion, the present study revealed a very high prevalence of
bacterial biofilms in the apical root canals of both untreated and treated
teeth with apical periodontitis. The pattern of bacterial community
arrangement in the canal, which adhered to or at least was associated
with the dentinal walls with cells encased in an extracellular amorphous
matrix and often surrounded by inflammatory cells, is consistent with
acceptable criteria to include apical periodontitis in the set of
biofilm-induced disease. Biofilm morphologic structure varied from
case to case, and no unique pattern for endodontic infections was deter-
mined. Bacterial biofilms are still more expected to be present in asso-
ciation with longstanding pathologic processes, including large lesions
and cysts.
References
1. Costerton JW. The biofilm primer. Berlin, Heidelberg: Springer-Verlag; 2007.
2. Marsh PD. Dental plaque: biological significance of a biofilm and community life-
style. J Clin Periodontol 2005;32(suppl 6):7–15.
3. Costerton B. Microbial ecology comes of age and joins the general ecology commu-
nity. Proc Natl Acad Sci U S A 2004;101:16983–4.
4. Chavez de Paz LE. Redefining the persistent infection in root canals: possible role of
biofilm communities. J Endod 2007;33:652–62.
5. Siqueira JF Jr, Rôças IN. Community as the unit of pathogenicity: an emerging
concept as to the microbial pathogenesis of apical periodontitis. Oral Surg Oral
Med Oral Pathol Oral Radiol Endod 2009;107:870–8.
6. Svensater G, Bergenholtz G. Biofilms in endodontic infections. Endod Topics 2004;
9:27–36.
7. Nair PNR. Light and electron microscopic studies of root canal flora and periapical
lesions. J Endod 1987;13:29–39.
8. Siqueira JF Jr, Rôças IN, Lopes HP. Patterns of microbial colonization in primary
root canal infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;
93:174–8.
9. Molven O, Olsen I, Kerekes K. Scanning electron microscopy of bacteria in the apical
part of root canals in permanent teeth with periapical lesions. Endod Dent Trauma-
tol 1991;7:226–9.
10. Ricucci D, Siqueira JF Jr, Bate AL, et al. Histologic investigation of root canal-treated
teeth with apical periodontitis: a retrospective study from twenty-four patients. J En-
dod 2009;35:493–502.
11. Carr GB, Schwartz RS, Schaudinn C, et al. Ultrastructural examination of failed molar
retreatment with secondary apical periodontitis: an examination of endodontic bio-
films in an endodontic retreatment failure. J Endod 2009;35:1303–9.
12. Schaudinn C, Carr G, Gorur A, et al. Imaging of endodontic biofilms by combined
microscopy (FISH/cLSM - SEM). J Microsc 2009;235:124–7.
13. Nair PN, Henry S, Cano V, et al. Microbial status of apical root canal system of human
mandibular first molars with primary apical periodontitis after ‘‘one-visit’’
endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;
99:231–52.
14. Ricucci D, Siqueira JF Jr. Apical actinomycosis as a continuum of intraradicular and
extraradicular infection: case report and critical review on its involvement with treat-
ment failure. J Endod 2008;34:1124–9.
15. Ricucci D, Siqueira JF Jr. Fate of the tissue in lateral canals and apical ramifications
in response to pathologic conditions and treatment procedures. J Endod 2010;36:
1–15.
16. Tronstad L, Barnett F, Cervone F. Periapical bacterial plaque in teeth refractory to
endodontic treatment. Endod Dent Traumatol 1990;6:73–7.
17. Ferreira FB, Ferreira AL, Gomes BP, et al. Resolution of persistent periapical infec-
tion by endodontic surgery. Int Endod J 2004;37:61–9.
18. Ricucci D, Pascon EA, Pitt Ford TR, et al. Epithelium and bacteria in periapical
lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:239–49.
19. Ricucci D, Mannocci F, Pitt Ford TR. A study of periapical lesions correlating the
presence of a radiopaque lamina with histological findings. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2006;101:389–94.
20. Taylor RD. Modification of the Brown and Brenn Gram stain for the differential
staining of gram-positive and gram-negative bacteria in tissue sections. Am J Clin
Pathol 1966;46:472–6.
21. Ricucci D, Bergenholtz G. Bacterial status in root-filled teeth exposed to the oral
environment by loss of restoration and fracture or caries—a histobacteriological
study of treated cases. Int Endod J 2003;36:787–802.
22. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural envi-
ronment to infectious diseases. Nat Rev Microbiol 2004;2:95–108.
Biofilms and Apical Periodontitis 1287
Clinical Research
23. Parsek MR, Singh PK. Bacterial biofilms: an emerging link to disease pathogenesis.
Annu Rev Microbiol 2003;57:677–701.
24. Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol
2009;11:1034–43.
25. Baumgartner JC, Xia T. Antibiotic susceptibility of bacteria associated with
endodontic abscesses. J Endod 2003;29:44–7.
26. Siqueira JF Jr. Reaction of periradicular tissues to root canal treatment: benefits and
drawbacks. Endod Topics 2005;10:123–47.
27. Sundqvist G. Bacteriological studies of necrotic dental pulps [Odontological Disser-
tation no.7]. Umeå, Sweden: University of Umeå; 1976.
28. Rôças IN, Siqueira JF Jr. Root canal microbiota of teeth with chronic apical perio-
dontitis. J Clin Microbiol 2008;46:3599–606.
29. Strindberg LZ. The dependence of the results of pulp therapy on certain factors. Acta
Odontol Scand 1956;14(suppl 21):1–175.
30. Lin LM, Ricucci D, Lin J, et al. Nonsurgical root canal therapy of large cyst-like
inflammatory periapical lesions and inflammatory apical cysts. J Endod 2009;35:
607–15.
31. Siqueira JF Jr, Lopes HP. Bacteria on the apical root surfaces of untreated teeth with
periradicular lesions: a scanning electron microscopy study. Int Endod J 2001;34:
216–20.
1288 Ricucci and Siqueira Jr.
32. Siqueira JF Jr, Araújo MC, Garcia PF, et al. Histological evaluation of the effectiveness
of five instrumentation techniques for cleaning the apical third of root canals. J En-
dod 1997;23:499–502.
33. Wu M-K, van der Sluis LWM, Wesselink PR. The capability of two hand instrumen-
tation techniques to remove the inner layer of dentine in oval canals. Int Endod J
2003;36:218–24.
34. Paqué F, Balmer M, Attin T, et al. Preparation of oval-shaped root canals in mandib-
ular molars using nickel-titanium rotary instruments: a micro-computed tomog-
raphy study. J Endod 2010;36:703–7.
35. Mah TF, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents.
Trends Microbiol 2001;9:34–9.
36. Chávez de Paz LE, Bergenholtz G, Svensäter G. The effects of antimicrobials on
endodontic biofilm bacteria. J Endod 2010;36:70–7.
37. Stoodley P, Dodds I, Boyle JD, et al. Influence of hydrodynamics and nutrients on
biofilm structure. J Appl Microbiol 1999;85:S19–28.
38. Purevdorj B, Costerton JW, Stoodley P. Influence of hydrodynamics and cell
signaling on the structure and behavior of Pseudomonas aeruginosa biofilms.
Appl Environ Microbiol 2002;68:4457–64.
39. Paramonova E, Kalmykowa OJ, van der Mei HC, et al. Impact of hydrodynamics on
oral biofilm strength. J Dent Res 2009;88:922–6.
JOE — Volume 36, Number 8, August 2010