Periapical bacterial plaque in teeth refractory toendodontic treatmentTronstad L, Barnett F, Cervone F. Periapical bacterial plaque inteeth refractory to endodontic treatment. Endod Dent Traumatol1990; 6: 73-77.

Abstract - It has recently been found that bacteria are able tosurvive and maintain an infectious disease process in periapicallesions of nonvital teeth. The purpose of this study was to examinethe surfaces of root tips removed during surgical-endodontictreatment for the presence of microorganisms. A full thicknessflap was reflected under strict surgical asepsis and the periapicallesions were enucleated and removed. About 2-3 mm of the rootwas cut off, rinsed in sterile saline and placed in 10% neutral-buffered formalin. Upon fixation, the root tips were dehydrated,air-dried and given an electrically conducting coat of gold in avacuum evaporator. The root tips were then studied in a Jeol,JSM-U3 scanning electron microscope, usually operated at 20 kV.The root surfaces were covered with soft tissue, except at theapex of the roots, where a continuous, smooth and structurelesscoating was seen, apparently adjacent to the apical foramen. Athigher magnification a variety of bacterial forms were recognizedin the smooth coating. A bacterial plaque was observed in irregu-larities of the surfaces between fiber bundles and cells and incrypts and holes. The bacteria were held together by an extracel-lular material and the plaque was dominated by cocci and rods.Fibrillar forms were recognized as well, often with cocci attachedto their surfaces.

Leif Trenstad, Frederic Barnett,Frank CerveneDepartment of Endodontics, University of Pennsyl-vania School of Dental Medicine, Philadelphia,Pennsylvania, USA

Key words: endodontic infection; extraradicular in-fection; bacterial plaque; periapical plaque.

Leif Tronstad, DM.D, Ph.D, Professor and Chair-man, University of Pennsylvania, School of DentalMedicine, Department of Endodontics, 4001 SpruceSt., Philadelphia, PA 19104, USA.

Accepted for publication July 7, 1989.

In recent studies it has been shown that bacteria areable to survive and maintain an infectious diseaseprocess in periapical tissues (1, 2). Periapical lesionsrefractory to conventional endodontic therapy ex-clusively yield anaerobic bacteria or are heavilydominated by anaerobes. Black-pigmented andnon-pigmenting Bacteroides species as well as anaer-obic gram-positive rods and cocci are isolated, asare facultative Staphylococcus and Streptococcus species.The flora of longstanding lesions with fistulae oftenis dominated by enteric bacteria or Pseudomonas aeru-ginosa. Yeast, mostly Candida albicans, is isolated aswell. The purpose of the present study was to exam-ine microscopically the surface of root tips removedduring surgical-endodontic treatment of teeth forthe presence of microorganisms.

Materiai and methods

The patients participating in this study had all beenreferred to the Postdoctoral Clinic of the Depart-

ment of Endodontics, University of PennsylvaniaSchool of Dental Medicine for diagnosis and treat-ment of endodontic diseases. The fmdings in 10patients are reported here.

Each patient had a tooth with a periapical lesionthat had not responded to previous endodontictreatment. Fistulae were present in conjunction with5 of the teeth. Periodontal probing was within nor-mal limits. Four patients were retreated conservati-vely without success. In 6 patients this was notfeasible because of large posts in the root canals andextensive prosthetic restorations.

All patients were therefore treated surgically. Fol-lowing a sulcular incision, a full thickness flap wasreflected and the periapical lesion was exposedunder aseptic conditions. An incision was made intothe lesion and bacterial sampling of the lesion wascarried out using sterile endodontic paper points,which were inserted into the lesion towards thelocation of the root tip. Upon removal, the paperpoints (at least 3) were placed in vials with 3 ml of

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Moller's VMGA III transport medium (3). Thesamples were processed using well-established aero-bic and anaerobic techniques (4).

The periapical lesion was then carefully enu-cleated and removed. An effort was made not totouch the root surfaces with curettes or other instru-ments. About 2-3 mm of the root was then cut offusing a sterile fissure bur in a high speed handpieceunder irrigation with sterile saline. The root tipswere removed by means of sterile pliers, rinsed offin sterile saline and placed in 10% neutral-bufferedformalin. Retrograde fillings were then placed inthe roots (5) and the flaps were repositioned andsutured in place. All patients received penicillin (orerythromycin in case of penicillin allergy) for 7days and the immediate postoperative healing wasuneventful in all instances.

After 1 month, 1 patient came back with a fistulafrom the treated tooth. Based upon the results ofthe microbiological culturing, metronidazole (750mg X 4 for 10 days) was prescribed and the fistulaclosed. Complete bony healing was evident radio-graphically in all patients after 6 months.

Upon fixation, the root tips were dehydrated, air-dried and given an electrically conducting coat ofgold in a vacuum evaporator. The surfaces of theroot tips were then studied in a Jeol, JSM-U3, scan-ning electron microscope, usually operated at 20 kV.

Results

Microorganisms were recovered from all periapicalsamples. The flora was heavily dominated by anaer-obic bacteria as described in 2 previous studies (1,2).

To the naked eye, the surgically removed root

Fig. 1. Scanning electron micrograph of root tip of human toothremoved during endodontic surgery. The root surfaces are cover-ed with soft tissue except at the apex (A) where the root has asmooth structureless appearance. Note that the apical foramenis not visible, x 35.

Fig. 2. Higher magnification of apical area (A) in Fig. 1. Thesmooth appearance of the root in this area is due to the presenceof an extraneous, structureless coating layer visualized wherecracks and tears have led to a loosening of the layer from theunderlying tooth structure (arrow), x 110.

tips looked denuded. When examined in the micro-scope the root cementum was mostly not visible,but was covered with soft tissue or an extraneoussmooth and structureless coating (Fig. 1). Thesmooth coating was seen as a continuous layer atthe apex of the root, seemingly adjacent to theapical foramen, which was not visible in any of thespecimens. In particular, the presence of a coatinglayer was obvious when occasional cracks or tearshad led to a loosening of the layer from the underly-ing tooth structure (Fig. 2). In 9 of 10 specimensthe distribution of the structureless coating was asseen in Fig. 1. In the 10th specimen only smalldiscontinuous patches of the structureless materialwere seen in the apex area of the tooth. At higher

Fig. 3. Higher magnification of apical smooth coating demon-strated in Figs. 1 and 2. Colonies of bacteria are seen in thestructureless material, x 550.

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Fig. 4. Scanning electron micrograph from lateral aspect of roottip. Soft tissue with crypts and holes possibly representing vascu-lar canals is covering the root surface.

Fig. 6. Scanning electron micrograph from lateral aspect of roottip. A bacterial plaque comprising mainly cocci and rods is seenbetween periodontal cells and fibers, x 6500.

magnification a variety of bacterial forms were rec-ognized in the smooth coating (Fig. 3). The bacteriahad formed colonies and were completely coveredby the structureless material. In other areas of thesmooth coating there was no evidence of bacteria.

The soft tissue, which was seen on the root surfaceoutside the apical area with the smooth coating,appeared to be in various stages of degradation (Fig.4). Fibrillar structures were recognized and in someareas large fiber bundles were seen lying on theroot surfaces (Figs. 5, 6). Individual cells could berecognized as well (Fig. 6) and small areas with anapparent intact cementoblast layer were seen (Fig.7). In some specimens erythrocytes covered parts ofthe root in spite of a thorough rinsing prior tofixation. Only occasionally was the root surface vis-ible between the soft tissue components (Fig. 8).

Fig. 5. Scanning electron micrograph from lateral aspect of roottip. Fibrous structures of various sizes are seen. A bacterialplaque is present on and between the fiber bundles. Fibrillarforms with cocci attached to their surfaces are recognized.X 6500.

A bacterial plaque was observed in scatteredareas of the root surface. The plaque was located inirregularities in the surface between fiber bundlesand cells (Figs. 5, 6) and in crypts and holes (Figs.4, 9). The bacteria were embedded in or held to-gether by various amounts of extracellular material.This made it difficult to distinguish individual cells,although the plaque clearly was dominated by cocciand rods (Figs. 5, 6, 9).

However, in areas with minimal amounts of ex-tracellular material a large variation of bacterialforms were recognized (Fig. 8). Due to the fibrillarnature of much of the soft tissue covering the root,filamentous organisms may have been overlooked.Still filamentous or fibrillar forms were recognized,often with cocci attached to their surfaces (Figs. 5,6 ,8 ,9) .

Fig. 7. Scanning electron micrograph from lateral aspect of roottip. An apparent intact layer of cementoblasts is covering theroot surface, x 650.

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Fig. 8. Scanning electron micrograph from lateral aspect of roottip. A variety of bacterial forms are recognized on the rootsurface, x 6000.

Discussion

Previous findings (1,2) that microorganisms maybe recovered from periapical lesions refractory toendodontic treatment were confirmed in the presentstudy. Microscopically, this study had clear limi-tations in that the periapical lesions were removedfrom the root tips during the surgical operation sothat only remnants of granulomatous tissue left onthe root surfaces could be examined. However, inspite of this, interesting findings were made that

Fig. 9. Scanning electron micrograph from lateral aspect of roottip. A bacterial plaque dominated by cocci is present in crypt insoft tissue-covering of the root, x 5000.

further elucidated the phenomenon of extraradicul-ar endodontic infections.

Microorganisms were observed on the surfaces ofall specimens studied. Scattered bacteria of differentmorphological types were seen, but mostly the or-ganisms were present in aggregates or colonies increvices and crypts of the tissue left on the rootsurfaces. An extracellular material was present be-tween the individual bacterial cells, often obscuringtheir morphology. Still most colonies were domi-nated by cocci and short rods. Filamentous organ-isms appeared to be relatively scarce. However, thefibrillar character of much of the soft tissue coveringthe root may have made it difficult to distinguishboth spirochete clusters and filamentous forms. Stillfilaments with cocci attached to their surfaces wereseen (6) although fully developed "corn cobs" werenot recognized (7-9). On the whole, the periapicalbacterial plaque as seen in the scanning electronmicroscope appeared similar to periodontal plaque(7, 9, 10).

An interesting finding in this study was the con-spicuous presence of a smooth and structureless ma-terial outside the main apical foramen. The pres-ence of bacteria embedded in this material mightindicate that it represented an extracellular materialproduced by the bacteria, in all likelihood extracel-lular polysaccharide (11-13). Extracellular polysac-charide provides a reserve of substrate (14) andmay therefore be important for the survival andcontinuous growth of the bacteria outside the canal.It is also widely held that extracellular polysacchar-ide acts as an effective diffusion barrier (15, 16). Itis conceivable, therefore, that its presence could beone reason that it has proven difficult to eliminateextraradicular bacteria with systemic antibiotictreatment, even after accurate microbiologic diag-nosis (17).

However, the structureless material may also bethe result of a host response to the infecting organ-isms. In this regard it should be alluded to theapparent similarities between the material observedin this study and the structureless material observedon tooth surfaces adjacent to periodontal pockets(8, 9). Originally, this material was considered tobe a secretory product of the adjacent epithelialcells (18, 19), whereas the present evidence suggeststhat it may form by adsorption of components ofgingival exudate seeping out between the junctionalepithelium and the tooth surface incident to inflam-mation in the marginal periodontium (20). Sincethe structureless material in this study always wasobserved outside the apical foramen, it is temptingto suggest an analogous reaction in the marginaland the apical periodontium.

However, the similarities in the microbial flora ofthe periodontal pocket and the root canal should

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be considered in this regard (21). Clearly, the struc-tureless material both apically and marginally inthe periodontium may be a combination of bacteria-produced extracellular products as well as com-ponents reflecting the local inflammatory reaction.

The question then remains where the extraradic-ular bacteria came from. It seems logical that theorganisms observed embedded in the structurelessmaterial outside the apical foramen had come fromthe root canal. Even on the lateral aspects of theroot tip, the bacterial colonies were mostly found inor near crypts or holes in the soft tissue coating. Itis not clear what was the origin of these holes, butthey often had a regular form and originally mighthave been nerve-vascular channels leading to acces-sory foramina and root canals. Thus, the micro-organisms in these areas as well may have comefrom the root canal. However, in our microbiologi-cal studies of refractory periapical lesions, bacteriawhich are not considered common oral bacteria arefrequently found (1, 2). An example is the recoveryo{ Bacteroides fragilis from periapical lesions (2). Itappears as a distinct possibility, therefore, that he-matogenous spreading of bacteria to longstandingperiapical lesions may occur.

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