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Biofilms and Apical Periodontitis: Study of Prevalence and Association with Clinical and Histopathologic Findings
Clinical Research
Biofilms and Apical Periodontitis: Study of Prevalence and Association with Clinical and Histopathologic Findings Domenico Ricucci, MD, DDS,* and Jose´ 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 histobacteriologic 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 epithelialized lesions (cysts and epithelialized granulomas or abscesses) (p < 0.001). The overall prevalence of biofilms 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 biofilminduced 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 infection, endodontic treatment
I
n their natural habitats, microorganisms almost invariably live as members of metabolically 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 metabolic 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, biofilms 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 clinical 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 twofold: (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 periodontitis 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
From *Private Practice, Rome, Italy; and †Department of Endodontics, Faculty of Dentistry, Esta´cio de Sa´ University, Rio de Janeiro, Brazil. Address requests for reprints to Dr Domenico Ricucci, Piazza Calvario, 7, 87022 Cetraro (CS), Italy. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.04.007
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0/7 (0) 2/52 (4) 0/10 (0) 2/8 (25) 1/2 (50) 0/3 (0) 1/21 (5) 0/3 (0) 3/8 (37.5) 6/69 (9) 0/1 (0) 1/32 (3) — — — — 1/7 (14) 0/2 (0) 2/6 (33) 2/19 (10.5) 0/6 (0) 1/20 (5) 0/10 (0) 2/8 (25) 1/2 (50) 0/3 (0) 0/14 (0) 0/1 (0) 1/2 (50) 4/50 (8) 1/1 (100) 22/32 (69) — — — — 7/7 (100) 1/2 (50) 5/6 (83) 16/19 (84) 5/6 (83) 13/20 (65) 10/10 (100) 7/8 (87.5) 2/2 (100) 3/3 (100) 10/14 (71) 1/1 (100) 2/2 (100) 41/50 (82)
6/7 (86) 35/52 (67) 10/10 (100) 7/8 (87.5) 2/2 (100) 3/3 (100) 17/21 (81) 2/3 (67) 7/8 (87.5) 57/69 (83)
6/106 (6) 2/42 (5) 4/64 (6) 82/106 (77) 31/42 (74)
Total (untreated + treated) (%) Treated (%)
Extraradicular biofilm
Untreated (%) Total (untreated + treated) (%) Treated (%)
51/64 (80)
Overall prevalence Lesion type Granuloma, epithelialized Granuloma, nonepithelialized Cyst, true Cyst, pocket (bay) Cyst, unclassified Abscess, epithelialized Abscess, nonepithelialized Unclassified Sinus tract Symptoms
Evaluation Criteria Slides were examined by two evaluators. Evaluations were performed separately, and whenever disagreement occurred, it was resolved by joint discussion. The following aspects were specifically looked for in the examination: 1) the presence and location of bacteria in the apical segment of the root canal, including the main canal, lateral canals, apical ramifications, and isthmuses (intraradicular infection) or within the body of the apical periodontitis lesion or adhered to the external apical root surface (extraradicular infection). The parameter used for classification of bacterial community structures as biofilms followed the biofilm definition given by Hall-Stoodley et al (22): ‘‘Microbial biofilms are populations of microorganisms that are concentrated
Untreated (%)
Tissue Processing Immediately after removal (by periradicular surgery or extraction), 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 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 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 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 specimens, cross-cut sections were taken. Every fifth slide was stained with hematoxylin-eosin for screening purposes and for assessment of inflammation. A modified Brown and Brenn technique for staining 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).
Intraradicular biofilm
five specimens lesions had to be removed separately. All untreated teeth 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 presence/absence of a sinus tract was also recorded. For 33 teeth, conventional periapical radiographs were available before extraction, and the largest diameter of the periradicular radiolucency was measured. Teeth with periodontal pockets or longitudinal fractures 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 in this study for the presence of biofilms following the parameters established 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 previous endodontic treatment was judged as inadequate. Radiographs were not available for four of the new 18 cases. All patients had given consent for examination of their 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 Features
Clinical Research
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Clinical Research at an interface and typically surrounded by an extracellular polymeric substance matrix.’’ Also, according to these authors, aggregates or coaggregates 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 periradicular 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 epithelium, 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 periodontitis (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 consideration only untreated root canals, only treated canals, and all specimens 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).
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 proportions 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 virtually 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 inflammatory 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).
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 distribution of bacterial cells within the extracellular material (original magnification 1,000).
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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 magnification 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 intracanal 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 1280
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typical of actinomycotic colonies (Fig. 7c and d). Serial sections revealed 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 assessed as showing a biofilm, not because of the unusual large floc suspended 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
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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.
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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% Biofilms and Apical Periodontitis
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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 (Taylor’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 epithelialized 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 epithelialized or nonepithelialized granulomas exhibited intraradicular biofilms in 86% and 67%, respectively, this 20% difference did not reach signif1282
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icance for the sample size evaluated (Fisher test, p = 0.4). When lesions were grouped as granulomas (epithelialized or not), abscesses (epithelialized or not), and cysts (true, pocket, and unclassified), intraradicular 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 biofilms. 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
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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 symptoms (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 specimens (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-
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ciated with an intraradicular biofilm. Two of these extraradicular biofilms 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 forming 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
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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 neutrophils (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 structure 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 considering 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 periodontitis along with the observed morphological features of these biofilms 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 encased 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 inflam1284
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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 susceptible 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 explanations for antibiotic ineffectiveness. As for criterion 6 proposed in this study, effects of treatment on biofilms and how they influence the treatment outcome were not evaluated herein and await further investigations 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 criterion. 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 important 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 intraradicular biofilms may be related to the fact that bacteria in the apical canal compose the advanced front of infection and then directly face an inflamed 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 conditions for biofilm formation and help explain the many exuberant biofilms 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
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Figure 7. (a) Untreated maxillary first premolar extracted with the apical periodontitis lesion attached to the root tip. The longitudinal section passing approximately 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 magnification 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 extracellular 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 harbor intraradicular biofilms. Large lesions have been associated with complex intraradicular infections characterized by bacterial communities with increased species richness and high populational density (27, 28). Because it takes time for apical periodontitis to develop and become radiographically visible, it is conceivable to assume that large lesions represent a longstanding pathologic process caused by an even ‘‘older’’ intraradicular infection. In a longstanding infectious process, involved bacteria may have had enough time and conditions to adapt to the environment and set a mature and organized biofilm community. The fact that infected root canals of teeth with large lesions harbor a large number of cells and species almost always organized in biofilms may help explain the long-held concept that the treatment outcome may be influenced by the lesion size (29).
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The present study revealed that intraradicular biofilms were significantly more frequent in root canals of teeth with epithelialized lesions (cysts and epitheliazed granulomas or abscesses). Ninety-three percent of the lesions exhibiting some level of epithelial proliferation were in TABLE 2. Prevalence of Intraradicular Biofilms at the Apical Segment of Untreated and Treated Canals of Teeth with Apical Periodontitis According to the Lesion Size as Determined Radiographically Lesion size
Untreated (%)
Treated
Total (untreated + treated)
#5 mm >5 mm $10 mm*
10/17 (59) 14/16 (87.5) 2/2 (100)
13/20 (65) 14/18 (78) 3/3 (100)
23/37 (62) 28/34 (82) 5/5 (100)
*These specimens were also included in the group >5 mm.
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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 occurrence, 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 mineralized with relatively few bacteria (original magnification 1,000).
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Clinical Research 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 suggested to play a role in the pathogenesis of acute clinical forms of apical periodontitis (5). The ability of endodontic bacteria to organize themselves in biofilm 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 definitely more difficult to reach. Many bacteria under the biofilm were seen invading dentinal tubules (Fig. 6), which also pose a problem for disinfection. 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 HallStoodley 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 environment (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 environmental 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 biofilms. 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 nutrients, 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 reported in this study indicates that when properly and meticulously performed, conventional paraffin techniques and so-called ‘‘old’’ bacterial
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staining protocols are still valuable tools for studying bacterial colonization 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 determined. Bacterial biofilms are still more expected to be present in association 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 lifestyle. J Clin Periodontol 2005;32(suppl 6):7–15. 3. Costerton B. Microbial ecology comes of age and joins the general ecology community. 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, Roˆc¸as 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, Roˆc¸as 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 Traumatol 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 Endod 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 biofilms 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 treatment 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 infection 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 environment to infectious diseases. Nat Rev Microbiol 2004;2:95–108.
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32. Siqueira JF Jr, Arau´jo MC, Garcia PF, et al. Histological evaluation of the effectiveness of five instrumentation techniques for cleaning the apical third of root canals. J Endod 1997;23:499–502. 33. Wu M-K, van der Sluis LWM, Wesselink PR. The capability of two hand instrumentation techniques to remove the inner layer of dentine in oval canals. Int Endod J 2003;36:218–24. 34. Paque´ F, Balmer M, Attin T, et al. Preparation of oval-shaped root canals in mandibular molars using nickel-titanium rotary instruments: a micro-computed tomography 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. Cha´vez de Paz LE, Bergenholtz G, Svensa¨ter 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.
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Biofilms and Apical Periodontitis: Study of Prevalence and Association with Clinical and Histopathologic Findings Domenico Ricucci, MD, DDS,* and Jose´ 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 histobacteriologic 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 epithelialized lesions (cysts and epithelialized granulomas or abscesses) (p < 0.001). The overall prevalence of biofilms 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 biofilminduced 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 infection, endodontic treatment
I
n their natural habitats, microorganisms almost invariably live as members of metabolically 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 metabolic 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, biofilms 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 clinical 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 twofold: (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 periodontitis 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
From *Private Practice, Rome, Italy; and †Department of Endodontics, Faculty of Dentistry, Esta´cio de Sa´ University, Rio de Janeiro, Brazil. Address requests for reprints to Dr Domenico Ricucci, Piazza Calvario, 7, 87022 Cetraro (CS), Italy. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.04.007
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0/7 (0) 2/52 (4) 0/10 (0) 2/8 (25) 1/2 (50) 0/3 (0) 1/21 (5) 0/3 (0) 3/8 (37.5) 6/69 (9) 0/1 (0) 1/32 (3) — — — — 1/7 (14) 0/2 (0) 2/6 (33) 2/19 (10.5) 0/6 (0) 1/20 (5) 0/10 (0) 2/8 (25) 1/2 (50) 0/3 (0) 0/14 (0) 0/1 (0) 1/2 (50) 4/50 (8) 1/1 (100) 22/32 (69) — — — — 7/7 (100) 1/2 (50) 5/6 (83) 16/19 (84) 5/6 (83) 13/20 (65) 10/10 (100) 7/8 (87.5) 2/2 (100) 3/3 (100) 10/14 (71) 1/1 (100) 2/2 (100) 41/50 (82)
6/7 (86) 35/52 (67) 10/10 (100) 7/8 (87.5) 2/2 (100) 3/3 (100) 17/21 (81) 2/3 (67) 7/8 (87.5) 57/69 (83)
6/106 (6) 2/42 (5) 4/64 (6) 82/106 (77) 31/42 (74)
Total (untreated + treated) (%) Treated (%)
Extraradicular biofilm
Untreated (%) Total (untreated + treated) (%) Treated (%)
51/64 (80)
Overall prevalence Lesion type Granuloma, epithelialized Granuloma, nonepithelialized Cyst, true Cyst, pocket (bay) Cyst, unclassified Abscess, epithelialized Abscess, nonepithelialized Unclassified Sinus tract Symptoms
Evaluation Criteria Slides were examined by two evaluators. Evaluations were performed separately, and whenever disagreement occurred, it was resolved by joint discussion. The following aspects were specifically looked for in the examination: 1) the presence and location of bacteria in the apical segment of the root canal, including the main canal, lateral canals, apical ramifications, and isthmuses (intraradicular infection) or within the body of the apical periodontitis lesion or adhered to the external apical root surface (extraradicular infection). The parameter used for classification of bacterial community structures as biofilms followed the biofilm definition given by Hall-Stoodley et al (22): ‘‘Microbial biofilms are populations of microorganisms that are concentrated
Untreated (%)
Tissue Processing Immediately after removal (by periradicular surgery or extraction), 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 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 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 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 specimens, cross-cut sections were taken. Every fifth slide was stained with hematoxylin-eosin for screening purposes and for assessment of inflammation. A modified Brown and Brenn technique for staining 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).
Intraradicular biofilm
five specimens lesions had to be removed separately. All untreated teeth 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 presence/absence of a sinus tract was also recorded. For 33 teeth, conventional periapical radiographs were available before extraction, and the largest diameter of the periradicular radiolucency was measured. Teeth with periodontal pockets or longitudinal fractures 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 in this study for the presence of biofilms following the parameters established 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 previous endodontic treatment was judged as inadequate. Radiographs were not available for four of the new 18 cases. All patients had given consent for examination of their 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 Features
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Clinical Research at an interface and typically surrounded by an extracellular polymeric substance matrix.’’ Also, according to these authors, aggregates or coaggregates 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 periradicular 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 epithelium, 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 periodontitis (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 consideration only untreated root canals, only treated canals, and all specimens 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).
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 proportions 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 virtually 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 inflammatory 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).
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 distribution of bacterial cells within the extracellular material (original magnification 1,000).
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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 magnification 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 intracanal 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 1280
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typical of actinomycotic colonies (Fig. 7c and d). Serial sections revealed 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 assessed as showing a biofilm, not because of the unusual large floc suspended 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
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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.
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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% Biofilms and Apical Periodontitis
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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 (Taylor’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 epithelialized 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 epithelialized or nonepithelialized granulomas exhibited intraradicular biofilms in 86% and 67%, respectively, this 20% difference did not reach signif1282
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icance for the sample size evaluated (Fisher test, p = 0.4). When lesions were grouped as granulomas (epithelialized or not), abscesses (epithelialized or not), and cysts (true, pocket, and unclassified), intraradicular 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 biofilms. 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
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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 symptoms (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 specimens (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-
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ciated with an intraradicular biofilm. Two of these extraradicular biofilms 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 forming 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
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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 neutrophils (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 structure 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 considering 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 periodontitis along with the observed morphological features of these biofilms 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 encased 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 inflam1284
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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 susceptible 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 explanations for antibiotic ineffectiveness. As for criterion 6 proposed in this study, effects of treatment on biofilms and how they influence the treatment outcome were not evaluated herein and await further investigations 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 criterion. 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 important 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 intraradicular biofilms may be related to the fact that bacteria in the apical canal compose the advanced front of infection and then directly face an inflamed 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 conditions for biofilm formation and help explain the many exuberant biofilms 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
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Figure 7. (a) Untreated maxillary first premolar extracted with the apical periodontitis lesion attached to the root tip. The longitudinal section passing approximately 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 magnification 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 extracellular 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 harbor intraradicular biofilms. Large lesions have been associated with complex intraradicular infections characterized by bacterial communities with increased species richness and high populational density (27, 28). Because it takes time for apical periodontitis to develop and become radiographically visible, it is conceivable to assume that large lesions represent a longstanding pathologic process caused by an even ‘‘older’’ intraradicular infection. In a longstanding infectious process, involved bacteria may have had enough time and conditions to adapt to the environment and set a mature and organized biofilm community. The fact that infected root canals of teeth with large lesions harbor a large number of cells and species almost always organized in biofilms may help explain the long-held concept that the treatment outcome may be influenced by the lesion size (29).
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The present study revealed that intraradicular biofilms were significantly more frequent in root canals of teeth with epithelialized lesions (cysts and epitheliazed granulomas or abscesses). Ninety-three percent of the lesions exhibiting some level of epithelial proliferation were in TABLE 2. Prevalence of Intraradicular Biofilms at the Apical Segment of Untreated and Treated Canals of Teeth with Apical Periodontitis According to the Lesion Size as Determined Radiographically Lesion size
Untreated (%)
Treated
Total (untreated + treated)
#5 mm >5 mm $10 mm*
10/17 (59) 14/16 (87.5) 2/2 (100)
13/20 (65) 14/18 (78) 3/3 (100)
23/37 (62) 28/34 (82) 5/5 (100)
*These specimens were also included in the group >5 mm.
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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 occurrence, 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 mineralized with relatively few bacteria (original magnification 1,000).
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Clinical Research 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 suggested to play a role in the pathogenesis of acute clinical forms of apical periodontitis (5). The ability of endodontic bacteria to organize themselves in biofilm 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 definitely more difficult to reach. Many bacteria under the biofilm were seen invading dentinal tubules (Fig. 6), which also pose a problem for disinfection. 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 HallStoodley 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 environment (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 environmental 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 biofilms. 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 nutrients, 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 reported in this study indicates that when properly and meticulously performed, conventional paraffin techniques and so-called ‘‘old’’ bacterial
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staining protocols are still valuable tools for studying bacterial colonization 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 determined. Bacterial biofilms are still more expected to be present in association with longstanding pathologic processes, including large lesions and cysts.
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32. Siqueira JF Jr, Arau´jo MC, Garcia PF, et al. Histological evaluation of the effectiveness of five instrumentation techniques for cleaning the apical third of root canals. J Endod 1997;23:499–502. 33. Wu M-K, van der Sluis LWM, Wesselink PR. The capability of two hand instrumentation techniques to remove the inner layer of dentine in oval canals. Int Endod J 2003;36:218–24. 34. Paque´ F, Balmer M, Attin T, et al. Preparation of oval-shaped root canals in mandibular molars using nickel-titanium rotary instruments: a micro-computed tomography 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. Cha´vez de Paz LE, Bergenholtz G, Svensa¨ter 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.
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