UMEÅ UNIVERSITY ODONTOLOGICAL DISSERTATIONS Abstract No 27, ISSN 0345—7532

From the Departments of Endodontics and Oral Microbiology University of Umeå, Umeå, Sweden

EVALUATION OF ENDODONTIC TREATMENT OF TEETH

WITH APICAL PERIODONTITIS

ANDERS BYSTRÖM

Umeå 1986

EVALUATION OF ENDODONTIC TREATMENT

OF TEETH

WITH APICAL PERIODONTITIS

AKADEMISK AVHANDLING

som med vederbörligt tillstånd av Odontologiska Fakulteten vid Umeå Universitet för avläggande av odontologie doktorsexamen

kommer att offentligen försvaras i föreläsningssal B, Odontologiska kliniken, 9 tr, Umeå,

lördagen den 6 december 1986, kl 09.00

ANDERS BYSTRÖM

Avhandlingen baseras på följande delarbeten:

I BYSTRÖM A, SUNDQVIST G. Bactériologie evaluation of theefficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981; 89: 321-328.

II BYSTRÖM A, SUNDQVIST G. Bactériologie evaluation of theeffect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Path 1983; 55: 307- 312.

III BYSTRÖM A, SUNDQVIST G. The antibacterial action ofsodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J 1985; 18: 35-40.

IV BYSTRÖM A, CLAESSON R, SUNDQVIST G. The antibacterialeffect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985; 1: 170-175.

V BYSTRÖM A, HAPPONEN R-P, SJÖGREN U, SUNDQVIST G.Healing of periapical lesions of pulpless teeth after endodontic treatment with controlled asepsis. Endod Dent Traumatol 1987; In press.

Umeå 1986

ABSTRACTByström, Anders, 1986. Evaluation of endodontic treatment of teeth with apical periodontitis. Umeå University Odontological Dissertations. Abstract No. 27 ISSN-0345-7532.

Apical periodontitis, an acute or chronic inflamination around the apex of the tooth, is caused by bacteria in the root canal. In Sweden the dentists devote around 10X of their total time to treating this disease. The treatment usually requires 3 to 5 sessions. The treatment may fail in up to 25X of the cases. In the present study various treatment regimens were evaluated. One hundred and forty single­rooted teeth with apical periodontitis were treated. The importance of mechanical instrumentation, irrigating solutions and antibacterial dressings in eliminating bacteria from the infected root canals was studied using bacteriological techniques. The healing of the apical periodontitis after treatment was followed for 2 to 5 years on recall radiographs.

Bacteria were found in all 140 root canals at the beginning of the treatment. Most of these bacteria were anaerobes and they represented a restricted group of bacteria compared to the bacteria present at other sites in the oral cavity. Mechanical instrumentation with files and reamers in combination with saline irrigation reduced the number of bacterial cells in the root canal 100- to 1000-fold during one treatment session. Bacteria could be eliminated from about half the number of root canals if this treatment was performed at 4 sessions.

Mechanical instrumentation and irrigation with 0.5X or 5X sodium hypochlorite solutions or with the 5X solution in combination with 15X EDTA solution wa3 more efficient and the bacteria were eliminated from about half the treated canals after one treatment session. The bacteria which persisted in the root canal after this treatment usually increased in number during the interval up to the next session and reached levels which were often as high as in the initial sample at the previous session.

All bacteria persistent in the root canals after the previous treatment regimens were with 2 exceptions eliminated by dressing the root canals for 1 to 2 months with calcium hydroxide paste. Thirty-four out of 35 root canals treated at the first session with mechanical instrumentation, irrigation with sodium hypochlorite solution and dressed with calcium hydroxide paste were free of bacteria at the second session. Calcium hydroxide paste was superior to camphorated phenol and camphorated paramonochlorophenol as dressing.

Healing of 79 out of the 140 treated teeth was followed for 2 to 5 years. The majority of the lesions healed completely or decreased in size in such a way that they could be expected to heal. There was no or only an insignificant decrease in the size of the lesions in 5 cases. In 2 of these cases bacteria were demonstrated in the periapical tissues and in a third case dentin chips. Periapical lesions may thus fail to heal in a few cases due to an establishment of bacteria outside the root canal, and in that site the bacteria are inaccessible to conventional endodontic treatment.

The present study showed that treatment of the majority of infected non-vital teeth can be completed in only 2 sessions, if mechanical instrumentation, sodium hypochlorite irrigation and calcium hydroxide dressing are combined.

Key words: Endodontic treatment, bacteriological evaluation.

UMEÅ UNIVERSITY ODONTOLOGICAL DISSERTATIONS Abstract No 27, ISSN 0345—7532

From the Departments of Endodontics and Oral Microbiology University of Umeå, Umeå, Sweden

EVALUATION OF ENDODONTIC TREATMENT OF TEETH

WITH APICAL PERIODONTITIS

ANDERS BYSTRÖM

Umeå 1986

ABSTRACT

Byström, Anders, 1986. Evaluation of endodontic treatment of teeth with apical periodontitis. Umeå University Odontological Dissertations. Abstract No. 27 ISSN-0345-7532.

Apical periodontitis, an acute or chronic inflammation around the apex of the tooth, is caused by bacteria in the root canal. In Sweden the dentists devote around 10X of their total time to treating this disease. The treatment usually requires 3 to 5 sessions. The treatment may fail in up to 25X of the cases. In the present study various treatment regimens were evaluated. One hundred and forty single­rooted teeth with apical periodontitis were treated. The importance of mechanical instrumentation, irrigating solutions and antibacterial dressings in eliminating bacteria from the infected root canals was studied using bacteriological techniques. The healing of the apical periodontitis after treatment was followed for 2 to 5 years on recall radiographs.

Bacteria were found in all 140 root canals at the beginning of the treatment. Most of these bacteria were anaerobes and they represented a restricted group of bacteria compared to the bacteria present at other sites in the oral cavity. Mechanical instrumentation with files and reamers in combination with saline irrigation reduced the number of bacterial cells in the root canal 100- to 1000-fold during one treatment session. Bacteria could be eliminated from about half the number of root canals if this treatment was performed at 4 sessions.

Mechanical instrumentation and irrigation with 0.5X or 5X sodium hypochlorite solutions or with the 5X solution in combination with 15X EDTA solution was more efficient and the bacteria were eliminated from about half the treated canals after one treatment session. The bacteria which persisted in the root canal after this treatment usually increased in number during the interval up to the next session and reached levels which were often as high as in the initial sample at the previous session.

All bacteria persistent in the root canals after the previous treatment regimens were with 2 exceptions eliminated by dressing the root canals for 1 to 2 months with calcium hydroxide paste. Thirty-four out of 35 root canals treated at the first session with mechanical instrumentation, irrigation with sodium hypochlorite solution and dressed with calcium hydroxide paste were free of bacteria at the second session. Calcium hydroxide paste was superior to camphorated phenol and camphorated paramonochlorophenol as dressing.

Healing of 79 out of the 140 treated teeth was followed for 2 to 5 years. The majority of the lesions healed completely or decreased in size in such a way that they could be expected to heal. There was no or only an insignificant decrease in the size of the lesions in 5 cases. In 2 of these cases bacteria were demonstrated in the periapical tissues and in a third case dentin chips. Periapical lesions may thus fail to heal in a few cases due to an establishment of bacteria outside the root canal, and in that site the bacteria are inaccessible to conventional endodontic treatment.

The present study showed that treatment of the majority of infected non-vital teeth can be completed in only 2 sessions, if mechanical instrumentation, sodium hypochlorite irrigation and calcium hydroxide dressing are combined.

Key words: Endodontic treatment, bacteriological evaluation.

45899912172020202429293031343536

CONTENTS

PREFACE..................................INTRODUCTION.. ..........................AIMS.....................................MATERIALS AND METHODS..................Clinical material......................Bacteriological methods................Endodontic treatment...................Healing..................................RESULTS..................................Microorganisms in infected root canalsEndodontic treatment...................Healing..................................DISCUSSION .........................Microbial changes during treatment....Comparison of treatment regimens.....Healing..................................SUMMARY..................................ACKNOWLEDGEMENTS........................REFERENCES..............................PAPER I..................................PAPER II.................................PAPER III...............................PAPER IV.................................PAPER V ..................................

PREFACE

This thesis is based on the following Papers, which will bereferred to in the text by their Roman numerals.

I BYSTRÖM A, SUNDQVIST G. Bactériologie evaluation of theefficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981; 89: 321-328.

II BYSTRÖM A, SUNDQVIST G. Bactériologie evaluation of theeffect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Path 1983; 55: 307- 312.

III BYSTRÖM A, SUNDQVIST G. The antibacterial action ofsodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J 1985; 18: 35-40.

IV BYSTRÖM A, CLAESSON R, SUNDQVIST G. The antibacterialeffect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985; 1: 170-175.

V BYSTRÖM A, HAPPONEN R-P, SJÖGREN U, SUNDQVIST G.Healing of periapical lesions of pulpless teeth after endodontic treatment with controlled asepsis. Endod Dent Traumatol 1987; In press.

4

1 INTRODUCTION

Apical periodontitis is an acute or chronic inflammatory lesion around the apex of a tooth. Generally there are no clinical symptoms in the chronic state of this disease and it is therefore not identified until a radiograph is taken. The chronic state may, however, become acute with pain and swelling, sufficient to send the patient to a dentist.

Epidemiological studies (Bergenholtz et al. I, II 1973, Molven 1974, Erselius et al. 1975, Kerekes and Bervell 1976, Lavstedt 1978, Bergman et al. 1979, Hugosson and Koch 1979, Holm et al. 1982, Petersson et al. 1986) have shown that apical periodontitis is very common. Erselius et al. (1975) found from radiographs that in Sweden about 60% of evaluated adult patients had some form of apical periodontitis. Epidemiological studies have also shown that 25 to 30% of root-filled teeth have apical periodontitis (Bergenholtz et al. I, II 1973, Molven 1974, Kerekes 1978, Hugosson and Koch 1979). Such failures in endodontic treatment are, however, only recorded in 10 to 20% of teeth treated by students under supervision or by dentists who specialize in endodontics (Strindberg 1956, Grahnén and Hansson 1961, Engström et al. 1964,Seltzer et al. I, II 1967, Oliet and Sorin 1969, Kerekes 1978, Kerekes and Tronstad 1979).

It has been established beyond all doubt that bacteria cause apical periodontitis (Kakehashi et al. 1965, Sundqvist 1976, Dahlén and Bergenholtz 1980, Möller et al. 1981). This implies that the goal of endodontic treatment should be to eliminate all bacteria from the root canal (White 1976, Grossman 1981, Schilder 1984). This is achieved by mechanical instrumentation supported by various irrigating solutions and the placement of antibacterial dressings in the canals between appointments.

A higher failure rate for endodontic treatment has been found when the presence of bacteria has been demonstrated in the root canal just before root-filling (Zeldow and Ingle 1963, Engström et al. 1964, Oliet and Sorin 1969, Heling and Shapira 1978). In a follow-up study of teeth with such persistent infection (Engström et al. 1964) 24% of the root-fillings were judged as failures whereas the corresponding figure for teeth without persistent infection at the time of root-filling was 11%. The reasons for failure in endodontic treatment may be related to faults in the root-fillings themselves such as overfilling, or gaps

5

apical to the root-filling or between the root-filling and the canal walls (Strindberg 1956, Bergenholtz et al. II 1973, Molven 1974, Kerekes 1978, Bergman et al. 1979). It has also been found that a persistent infection in combination with root-filling excesses significantly increased the failure rate (Engström et al. 1964).

Mechanical instrumentation of the root canal has been considered the most important step in eliminating these bacteria (Grossman 1981, Schilder 1984, Ingle et al. 1985). There are, however, divergent results concerning the efficacy of mechanical instrumentation and irrigation in rendering the root canals free of bacteria (Ingle and Zeldow 1958, Grahnén and Krasse 1963, Stewart et al. 1969).

The instrumentation techniques have shortcomings in that some sites in the root canal may not be reached by the files, and that a smear layer is formed on the canal walls by the files (Baker et al.1975, McComb and Smith 1975, McComb et al. 1976, Bolanos and Jensen 1980, Goldman et al. 1981, 1982). This smear layer consists of organic and inorganic tissue components. It covers the dentinal tubules in which bacteria may be ensconced (Baker et al. 1975, McComb et al. 1976, Lester and Boyde 1977, Rubin et al. 1979).

Irrigating solutions are an important adjunct in the preparation of the root canal. The main objective of the irrigating solution is to wash out debris from the root canal during instrumentation. Other properties considered desirable are that the solution be antibacterial, will assist in the removal of the smear layer and be capable of digesting organic matter and debris. Sodium hypochlorite and ethylenediaminetetra-acetic acid (EDTA) are commonly recommended as irrigating solutions. Sodium hypochlorite has a strong antibacterial effect in vitro (Shih et al. 1970, Spångberg et al. 1973, Blomfield and Miles 1979, Flink et al. 1981, Foley et al. 1983), but its efficacy under clinical conditions has not yet been fully established. Sodium hypochlorite also has the capacity to degrade organic matter (Handt et al. 1978, Thé 1979, Koskinen et al. 1980, Moorer and Vesselink 1982), although some scanning electron microscopic studies have produced conflicting results. Baker et al. (1975) and Bolanos and Jensen (1980) have reported that sodium hypochlorite was not effective, while McComb and Smith (1975), McComb et al. (1976), Goldman et al. (1981, 1982) found it highly effective.

6

EDTÂ has been used to degrade the inorganic matter/debris remaining in the root canal (for review see Dow, 1984). It has been claimed that the combination of sodium hypochlorite and EDTA removes the smear layer from the root canal walls (Goldman et al, 1982). It is not known whether this regimen improves the efficacy of the endodontic treatment in eliminating bacteria from the root canal.

In addition to adequate mechanical instrumentation and irrigation of the root canal, endodontic treatment also involves an antibacterial dressing placed in the canal between the appointments for treatment. The chosen dressing should have a long-lasting antibacterial effect and should be able to reach all parts of the root canal system (Chirnside 1958, Shovelton 1964, Åkpata and fclechman 1982, Armitage et al, 1983). Calcium hydroxide, camphorated phenol and camphorated paramonochlorophenol are frequently used as dressings. The antibacterial effect of these agents has been reported to be strong in vitro (Hermann 1936, Proell 1949, Vander Wall et al. 1972, Spångberg et al. 1973), but clinically camphorated phenol and camphorated paramonochlorophenol have been shown to be inefficient (Ingle and Zeldow 1958, Sommer et al. 1962, Goldman and Pearsson 1969). Calcium hydroxide, however, has also been shown to be efficient under clinical conditions (Cvek et al. 1976).

The ultimate aim of endodontic treatment is to eliminate bacteria from the root canal before filling takes place. Surprisingly little, however, is known about the antibacterial efficacy of mechanical instrumentation, irrigation, and placement of an antibacterial dressing in the root canal. In recent years improved bacteriological techniques have been successfully used to reveal the presence of bacteria in the root canals (Kantz and Henry 1974, Wittgow and Sabiston 1975, Sundqvist 1976, Dahlén and Bergenholtz 1980). This means that it is now possible to evaluate by bacteriological methods the efficacy of various steps in endodontic treatment.

7

2 AIM OF THE INVESTIGATION

The aim of this investigation was, using bacteriological methods, to evaluate the antibacterial effect of the various regimens in the treatment of infected root canals with apical periodontitis. The following stages of root canal preparation were studied:

1. mechanical instrumentation2. irrigation3. dressing

The ultimate goal of successful treatment is the healing of the periapical lesion. Therefore the state of healing after the various treatment regimens was studied.

8

3 MATERIALS AND METHODS

3.1 CLINICAL MATERIALOne hundred and forty single-rooted non-vital teeth with intact pulp chamber walls were treated. All teeth showed radiographic evidence of apical periodontitis when examined with a modified long cone technique (Oralix 65 Philips) with Kodak ultra-speed film (24x36 mm) and a film holder (Eggen 1974).

3.2 BACTERIOLOGICAL METHODS3.2.1 Sampling from the tooth surface (I, II, III, IV)The procedure outlined by Möller (1966) was followed. An aseptic technique was used throughout the treatment. In all phases of the treatment sterile instruments, medicaments and filling materials wereused. The operator wore surgical gloves. The tooth was cleaned withpumice and isolated with rubber dam. The tooth, the clamp and the rubber dam were cleansed with 30Z hydrogen peroxide and then swabbed with 5Z tincture of iodine. After the tincture had dried the tooth surface was swabbed with sterile 5% sodium thiosulphate solution to inactivate the tincture of iodine before the sterility of the tooth surface was tested. A sterile paper point was then scrubbed against the lingual tooth surface and transferred to a fluid thioglycollate medium USE (11260,BBL; Carlsson and Sundqvist 1980). Entrance to the pulp chamber was effected from the lingual surface. Before the pulp chamber was opened a second sample for testing sterility was taken. At each appointment a similar sterility control sample of the lingual tooth surface was taken before the pulp chamber was entered.

3.2.2 Sampling from the root canal (I, II, III, IV)When access to the pulp chamber had been gained a small amount of sterile saline was introduced into the canal by syringe. A measurement file was placed in the root canal and a radiograph was taken to determine the working length. The canal was instrumented and enlarged with Hedström files (Sjödings, Sweden) until a No. 40 file could be introduced approximately 1 mm short of the tooth apex. The fluid in the canal was soaked up into charcoaled paper points and transferred to a tube containing 5 ml peptone yeast glucose (PYG) broth (Holdeman et al.

9

1977). Three subsequent points were used and each was le£t in the canal until it could absorb no more fluid. To avoid oxidation of the PYG-broth during sampling, the tube was flushed with oxygen-free gas (97Z 3XH^)* A ”mobile anaerobe laboratory” as described by Fulghum (1971) was used to facilitate this procedure. The canal was instrumented and irrigated (see below). When the instrumentation was completed a second bacterial sample was taken (I, II, III) in the manner described above.At subsequent appointments bacterial samples were taken at the beginning and at the end of each appointment in the manner described (I, II, III). The interval between the appointments was 2-4 days.

When sodium hypochlorite was used in the irrigating solution (II, III) the canal was irrigated with saline after the instrumentation was completed. Then the canal was dried with sterile paper points and refilled with 5Z sodium thiosulphate solution for 2 min to inactivate any remaining sodium hypochlorite (Möller 1966). The canal was then filled with saline and a bacterial sample was taken in the manner described.

When calcium hydroxide, camphorated phenol and camphorated paramonochlorophenol (see below) were used as intracanal dressings, the dressing was removed by irrigation with saline at the following appointment. Thereafter a bacterial sample was taken in the manner described above. Then the canal was sealed without dressing, to provide an opportunity for persistent bacteria in the root canal to multiply before the final sample was taken at the next appointment, 2 to 4 days later.

3.2.3 Cultivation of the specimen

The bacterial samples were transported to the laboratory and were dealtwith within 30 min of sampling. The PYG-broth which contained thespecimen was introduced into an anaerobic box (Sundqvist 1976). Theatmosphere in the box was 10Z hydrogen and 5Z carbon dioxide in nitrogen. The tube, which contained glass beads, was agitated in a mechanical mixer until the paper points disintegrated and three 10-fold serial dilutions were made in dilution blanks (Holdeman et al. 1977). Aliquots of 0.5, 0.2, and 0.1 ml from the PYG-broth were inoculated onto duplicate blood agar plates (Holdeman et al. 1977), and aliquots of 0.1 ml onto one mitis salivarius agar (Difco 0298-01) and one Rogosa SL medium (Difco 0480-01). From each serial dilution tube aliquots of

10

0.1 ml were inoculated onto duplicate blood agar plates. One set of blood agar plates was incubated aerobically for 48 h at 37!C and the other set of blood agar plates, the mitis salivarius agar and the Rogosa SL medium, anaerobically in the box for 10 days at 37*C. The plates vere prepared outside the box. They vere placed in the box as soon as they had solidified and kept for at least 48 h before use. The plates incubated in the box vere observed daily for grovth. When no grovth occurred on the second day another blood agar plate vas inoculated from the PYG-broth. If no grovth occurred the process vas repeated three times at intervals of 1 veek before the sample vas judged free of living bacteria.

3.2.4 Isolation and identification of bacterial strains In Papers I and II bacterial samples taken from 15 root canals irrigated vith saline and 15 root canals irrigated vith 0.5% sodium hypochlorite solution vere analysed for the total number of bacterial strains and all strains vere identified.

In Paper III bacterial samples taken at the first and second appointment from 5 root canals irrigated vith 0.5% sodium hypochlorite solution, 20 root canals irrigated vith 5% sodium hypochlorite solution, 20 root canals irrigated vith 5% sodium hypochlorite solution and EDTA solution (see belov) vere analysed for the number of bacterial cells and strains. The bacterial strains vere preliminarily characterized, frozen and stored. Bacterial strains isolated at the third appointment vere identified.

In Paper IV bacterial samples taken at the first appointment from root canals subsequently dressed vith calcium hydroxide paste, camphorated phenol or camphorated paramonochlorophenol vere analysed for the number of bacterial cells. The isolated strains vere frozen and stored. Bacterial strains, isolated at the second and third appointments, vere identified. When bacteria persisted at the final appointment of the treatment regimen (III, IV) the bacterial strains, isolated at the first appointment, vere identified.

Each type of colony vas registered after 2 days on the aerobic and after 7-10 days on the anaerobic plates. The frequency of various colony types and the total number of colonies on the plates vere recorded. A representative number of strains of each colony type vas isolated. The isolated anaerobic bacteria vere characterized by the

11

methods described in the manual of the Virginia Polytechnic Institute and State University Anaerobe Laboratory, USA (Holdeman et al. 1977).Strains of uncertain taxonomic status were identified only as regardsgenus. Unless otherwise stated, anaerobic bacteria were characterized according to Holdeman et al. (1977), facultatively anaerobic streptococci according to Hardie and Bowden (1976) and.other facultative strains according to Cowan (1974). All isolated strains were preserved by freezing (-80*C).

3.3 ENDODONTIC TREATMENT3.3.1 Mechanical instrumentation

Entrance to the pulp chamber was effected from the lingual surface using a diamond bur in the low-speed handpiece with the water spray shut off. When access to the pulp chamber had been gained a small amount of saline was introduced into the canal by syringe. Then the canal was instrumented and enlarged with Hedström files (Sjödings, Sweden) until a No. 40 file could be inserted. The initial bacterial sample was taken as described above. The canal was thoroughly instrumented using the step back technique and Hedström files. Reamers (Maillefer Co, Switzerland) were used to enlarge the coronal part of the canal. The same set of files and reamers was used throughout the instrumentation at the appointment. The instrumentation was performed during 15 min (25 min when EDTA was used as the irrigating solution). After the instrumentation was completed, and another bacterial sample had been taken (I, II, III), the access cavity was sealed. At subsequent appointments the canal was instrumented and irrigated for 10 min.

3.3.2 Irrigation

During the mechanical instrumentation the root canal was frequently irrigated with 6 ml of irrigating solution. The solution was introduced into the canal by means of a syringe with a 23-gauge needle and soaked up by means of a vacuum ejector (Aga, Sweden). To ensure adequate exchange of the irrigating solution the needle of the syringe was introduced into the root canal approximately 3 mm short of the tooth apex. The root canal was repeatedly dried with sterile paper points.

12

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3.3.2.1 Saline (I)Fifteen teeth were instrumented and irrigated with saline at 4 appointments (Fig 1.1 refers to Paper I in Fig 1). No antibacterial dressing was used in the root canal in the period between the appointments. The interval between the appointments was 2 to 4 days. At the fifth appointment 7 root canals, from which no bacteria were recovered were filled (see below). One root canal without and 7 root canals with persistent bacteria were instrumented, irrigated with 0.5% sodium hypochlorite solution and dressed (see below) with calcium hydroxide paste (Calasept Scania Dental AB, Knivsta, Sweden) at the fifth appointment. The treatment regimen for the root canals irrigated with saline is presented in Fig 1:1.

3.3.2.2 Sodium hypochlorite solution (II, III)Fifteen teeth were instrumented and irrigated with 0.5% sodium hypochlorite solution at 4 appointments. In all other respects the treatment was in accordance with the treatment described for the 15 teeth irrigated with saline. At the fifth appointment 7 bacteria-free root canals were filled. Five root canals without and 3 root canals with persistent bacteria were instrumented, irrigated with 0.5% sodium hypochlorite solution and dressed with calcium hydroxide paste (Fig 1:11).

Five teeth were instrumented and irrigated with 0.5% sodium hypochlorite solution at 2 appointments (Fig 1:111). In other respects the treatment was in accordance with the treatment described for the 15 teeth instrumented and irrigated with 0.5% sodium hypochlorite solution at 4 appointments. At the third appointment 3 root canals which were free of bacteria were filled. Two root canals with persistent bacteria were instrumented and irrigated with 0.5% sodium hypochlorite solution and dressed with calcium hydroxide paste (Fig 1:111).

Twenty teeth were instrumented and irrigated with 5% sodium hypochlorite solution at 2 appointments (Fig 1:111). In other respects the treatment was in accordance with the 5 teeth instrumented and irrigated with 0.5% sodium hypochlorite solution at 2 appointments. At the third appointment the root canals were dressed with calcium hydroxide paste. When it was established that no bacteria could be recovered from 14 root canals at the third appointment, these canals were filled. Six root canals with persistent bacteria at the third

14

appointment were instrumented and irrigated with 0.5% sodium hypochlorite solution and dressed with calcium hydroxide paste (Fig I ïIII).

3.3.2.3 Etylenediaminetetra-acetic acid (III)Twenty teeth were instrumented and irrigated with 5% sodium hypochlorite solution and 15% EDTA solution for 25 min at 2 appointments (Fig 1:111). The treatment was initiated by instrumentation and irrigation with sodium hypochlorite solution for 10 min. Thereafter an EDTA solution was applied to the canal and repeatedly changed for 10 min. Finally the canal was instrumented and irrigated with the sodium hypochlorite solution for 5 min. In other respects the treatment was in accordance with the treatment described for the teeth irrigated with sodium hypochlorite solutions alone at 2 appointments. When it was established that no bacteria could be recovered from 17 root canals at the third appointment these canals were filled. Three root canals with persistent bacteria at thq third appointment were instrumented, irrigated with 0.5% sodium hypochlorite solution and dressed with calcium hydroxide paste (Fig 1:111).

3.3.3 Dressing

3.3.3.1 Bactericidal effect of calcium hydroxide, in vitro (IV)The sensitivity of 27 bacterial strains to a calcium hydroxidesuspension was tested. The bacteria were grown on the surface of bloodagar (Holdeman et al. 1977) and harvested after 1 to 4 days depending onthe growth rate of the actual strain. They were suspended in dilution

6 8solution (IV) to a density of 10 -10 bacterial cells per ml. The bacterial suspension (0.1 ml) was added to 4 ml of dilution solution (pH 7.2) and to 4 ml of dilution solution in which 150 mg calcium hydroxide-paste (Calasept ) had been suspended. The pH of the latter suspension was 12.5. From the suspension of bacteria and calcium hydroxide in dilution solution, aliquots of 0.1 ml were taken after various time intervals and the survival rate was determined by viable count on blood agar plates. The time required to decrease the number of living bacteria to less than 0.1% of the initial number was determined for each strain. The killing of Enterococcus faecalis (Schleifer and Kilpper-Bälz 1984) was also tested in the dilution solution adjusted to

15

various pH-levels by 1 N sodium hydroxide and in horse serum in which calcium hydroxide paste had been suspended.

3.3.3.2 Clinical effect of calcium hydroxide paste (IV)Fifteen teeth were instrumented and irrigated with 0.5% sodium hypochlorite solution at the first appointment (Fig 1:IV). After completion of the instrumentation and irrigation the root canals were dressed with calcium hydroxide paste (Calasept ) by means of a lentulo spiral, packed with the blunted end of a sterile paper point and sealed. At the second appointment 1 month later the dressing was removed by irrigation with saline. The canal was dried with sterile paper points and sealed without dressing. At the third appointment 2 to 4 days later another bacterial sample was taken. Thereafter the canal was dressed with calcium hydroxide paste in the manner described above.

Five teeth were treated in the above manner except that these root canals were irrigated with an EDTA solution for 10 min at the second appointment before sealing (Fig 1:IV).

Fifteen teeth were instrumented and irrigated with 5% sodium hypochlorite solution (Fig 1:IV). In other respects the treatment was in accordance with the treatment described for the 15 teeth irrigated with 0.5% sodium hypochlorite solution and dressed with calcium hydroxide paste for 1 month.

When it was established that no bacteria could be recovered from the samples taken at the third appointment the root canals were filled. One root canal with persistent bacteria at the third appointment was dressed with calcium hydroxide paste (Fig 1:1V).

3.3.3.3 Camphorated phenol (IV)Fifteen teeth were instrumented and irrigated with 0.5% sodium hypochlorite solution at the first appointment. After completion of the instrumentation and irrigation the root canals were dressed with camphorated phenol by means of a syringe and sealed. At the second appointment 2 weeks later the dressing was removed by irrigation with saline. The root canal was then dried with sterile paper points and sealed without dressing. At the third appointment 1 week later another bacterial sample was taken and the canal was dressed with camphorated phenol in the manner described above. When it was established that no bacteria could be recovered from the samples taken at the third

16

appointment 7 root canals were filled. Three root canals without and 5 root canals with persistent bacteria were instrumented and irrigated with 0.5X sodium hypochlorite solution and dressed with calcium hydroxide paste (Fig 1:IV).

3.3.3.4 Camphorated paramonochlorophenol (IV)Fifteen teeth were instrumented and irrigated with 0.5% sodium hypochlorite solution and dressed with camphorated paramonochlorophenol at the first, appointment (Fig 1:IV). In other respects the treatment was in accordance with the treatment described for the 15 teeth dressed with camphorated phenol for 2 weeks. When it was established that no bacteria could be recovered from the samples taken at the third appointment 7 root canals were filled. Three root canals without and 5 root canals with persistent bacteria were instrumented and irrigated with 0.5% sodium hypochlorite solution and dressed with calcium hydroxide paste (Fig 1 s IV).

3.3.4 Sealing of the access cavity

In the period between the appointments the access cavity was sealed with a more than 3 mm thick layer of zinc oxide-eugenol cement in all cases. Minifoams (Minnesota Mining and Manufacturing Co., St. Paul, Minnesota) in the pulp chamber prevented contact between the temporary filling and the bacteria in the root canal.

3.3.5 Filling of the root canal

All root canals were filled with gutta percha using the lateral condensation technique. The master cone was adapted to the canal by dipping it in rosin chloroform and then multiple accessory cones were laterally condensed using rosin chloroform as a sealing agent.

3.4 HEALINGThe healing of the apical periodontal lesion was recorded 2 to 5 years after the treatment for 79 of the 140 teeth treated initially. Thirty- nine teeth were excluded because the observation period was shorter than 2 years. Twenty-two teeth were lost to follow-up for other reasons.

17

3.4.1 Clinical and radiographic examination (V)At the clinical examination, pain, swelling, tenderness to apical and gingival palpation, as well as tenderness to percussion were recorded. Radiographs were taken before and during treatment, 6 and 12 months after the root canals were filled, and thereafter once a year. The radiographs were studied separately by 2 oral radiologists and 3 endodontists using a viewer with a magnifying glass (Mattson 1953). The radiographs were coded prior to evaluation by the examiners. In the radiographic evaluation the examiners determined the size of the lesion on each radiograph by measuring the greatest extent of the lesion in mm using a ruler. The interpretation of the treatment results was then based on the change in size of the lesions as determined for the entire series of recall radiographs. If there was disagreement between the evaluations of the 5 examiners the median value for each radiograph was used. The criterion for complete healing was that the radiographic width of the periodontal space was normal or slightly widened (<0.5 mm).

3.4.2 Histological examination (V)

Seven of the cases were operated on. The surgical specimens from 6 ofthese were studied histologically. Tissue specimens were fixed in 4%buffered formalin and embedded in paraffin. Multiple sections werestained with hematoxylin-eosin, Gram, Grocott's stain and PAS.Immunocytochemical demonstration of Actinomyces israelii, Actinomycesnaeslundii and Arachnia propionica was made according to Happonen et al.(1985). The avidin-biotin immunoperoxidase technique (Hsu and Raine

TM1981) was employed using Vectastain ABC Kit (Vector Laboratories Inc., Burlingame, Ca). The specific rabbit antisera were obtained from the Center for Disease Control (CDC), Atlanta, Ga. The substitution controls were made with the normal sera of two rabbits.

3.4.3 Statistical analysis (V)Student's t-test and the chi-square test were used for testing correlations between the outcome of treatment and the apical level of the root-filling.

18

Table 1. Bacteria initially recovered from non-vital teeth withapical periodontitis and bacteria persistent in the root canals at the fifth appointment when various irrigants were used.

Bacteria Irrigant recovered No of teeth

Appointment

Saline15

1

ì

5

0 . 5%

1

NaOCl15

5

Streptococcus milleri 1 1S. mitior 3 1S. mutans 1 1 1 1S. sanguis 2 2 1S. constellatus 1 1 2S. intermedius 1 1S. morbillorum 2 1 1

Peptostreptococcus sp 3 1 1P. anaerobius 5 3 4P. micros 6 3 3Peptococcus magnus 1P. prevotii 1P. niger 1

Eubacterium alactolyticum 5 2 5 1E. brachy 1 1 1E. lentum 2 2 5E . nodatum 1 1E. timidurn 1 1 1

Arachnia propionica 1 1Actinomyces spA. israelii 1 2

Lactobacillus sp 7 3 3 1

Fusobacterium sp 5 1 5 1F. nucleatum 6 2 10 1

Bacteroides sp 9 3 9 1B. corrodens 1B. gingivalis 1 1B. endodontalis 1 1B. oralis 5 2 2 1B. intermedius 5 4 1

Capnocytophaga ochracea 2 3Selenomonas sputigena 3Wolinella recta 2 5Enterobacter agglomerans 1 1Eikenella corrodens 1Veillonella parvula 1 1

19

4 RESULTS4.1 MICROORGANISMS IN INFECTED ROOT CANALSBacteria were found in all Initial samples taken from the 140 treatedroot canals. The median number of bacterial cells present in the initial

5 2samples from these canals before treatment was 3 x 10 (range < 1 0 - 2X 107). In total 165 different bacterial strains were isolated from 30 bacterial samples taken at the beginning of the first appointment (I, II). The isolated strains are presented in Table 1. The number of bacterial species in these 30 root canals ranged between 1 and 11. The most commonly isolated species were Fusobacterium nucleatum (16 strains), Eubacterium alactolyticum (10 strains), Peptostreptococcus anaerobius (9 strains), Peptostreptococcus micros (9 strains), Bacteroides intermedius (9 strains), Bacteroides oralis (7 strains), Eubacterium lentum (7 strains), and Wolinella recta (7 strains). Thirty- eight isolates could not be identified to the species level. They represented Fusobacterium (10 strains), Bacteroides (18 strains) and Lactobacillus (10 strains).

4.2 ENDODONTIC TREATMENTNo bacteria could be isolated in any samples taken from the operationfields or in samples collected during the preparation of the entrance into the pulp chambers.

4.2.1 Mechanical instrumentation (I)Fifteen teeth were treated. In these teeth mechanical instrumentationreduced the number of bacterial cells recovered from the root canals 100 - 1000-fold during the first appointment. Bacteria persisted, however, in all canals at the end of the first appointment (Fig 1:1). No bacteria could be recovered from 3 root canals at the second appointment, from5 canals at the third and fourth appointment, and from 8 canals at the fifth appointment. The bacteria persistent in the root canals at the end of an appointment usually increased in number by the next appointment. One such case is illustrated in Table 2. Most of the bacterial species found at the first appointment could also be recovered at the fifth appointment (Table 1).

Mechanical instrumentation and irrigation with saline did not reduce the number of bacteria in the root canal so effectively that the

20

bacteria could be eliminated in a predictable manner within five appointments.

Table 2. Bacteria recovered from the root canal during the treatment of one case.

Bacteria Appointment recovered

Sample

Relative proportion of the microbiota (%)

1 2 3 4 5

a* b+ a b a b a b a b

Anaerobic streptococcus 1 17 100 100 30 96 100 100 23 94S. mutans 1Peptostreptococcus sp 7 25 2 3P. anaerobius 27 4 3P. micros 13 17 54 2 40 6Lactobacillus sp 4 8 4 10Eubacterium lent urn 32 33 2 2 18Bacteroides sp 7 1B. oralis 4 2Fusobacterium sp 4 2 2

Total no. o£ 1.,8xl06 2.6xl04 1.2xl05 45x10 49x10bacterial cells

7. 5x10 1x10 2.1x10 1.75x10 3. 4x10

* a, Sample taken at the beginning of the appointment.+ b, Sample taken at the end of the appointment.

4.2.2 Irrigation (II, III)4.2.2.1 0.5Z sodium hypochlorite solution (II, III)Twenty teeth were treated. Of these teeth 15 were treated at 4 appointments (Fig 1:11) and 5 teeth at 2 appointments (Fig 1:111). No bacteria could be recovered from 12 out of 20 root canals after treatment at 2 consecutive appointments. After treatment at 4 consecutive appointments no bacteria could be recovered from 12 out of 15 root canals. Bacteria present in the root canals at the third appointment are presented in Table 3 and at the fifth appointment in Table 1.

4.2.2.2 5Z sodium hypochlorite solution (III)Twenty root canals were treated. No bacteria could be recovered from 14 out of 20 root canals after treatment at 2 consecutive appointments

21

(Fig 1:111). Bacteria persistent at the third appointment are presented in Table 3.

Table 3. Bacteria persistent in the root canals at the thirdappointment when various irrigants were used.

Bacteria Irrigant 0.5% NaOCl 5% NaOCl 5% NaOCl & EDTA

recovered No of teeth 8 6 3

Streptococcus milleri 1 1S. mutans 1S. sanguis 1S. intermedius 2 1S. morbillorum 1

Peptostreptococcus anaerobius 1P. micros 1

Arachnia propionica 1 1Eubacterium alactolyticum 1E. brachy 2E. lentum 1 1 1E. timidum 1

Lactobacillus sp 2 2

Fusobacterium sp 5 1F. nucleatum 1 2 1

Bacteroides sp 2 1B. gingivalis 1B . endodontalis 1B, intermedius 2 1B. oralis 1

Capnocytophaga ochracea 1Wolinella recta 1

4.2.2.3 Sodium hypochlorite - EDTA solution (III)Twenty teeth were treated. No bacteria could be recovered from 17 out of 20 root canals after 2 consecutive appointments (Fig 1:111). Bacteria present at the third appointment are presented in Table 3.

No specific bacteria were found which survived treatment with all the various irrigating solutions. Irrigation with sodium hypochlorite solutions (II, III) was more efficient than saline (I) in

22

eliminating bacteria from the root canals. There was, however, no difference between 0.5% and 5% sodium hypochlorite solutions. The combined use of EDTA and sodium hypochlorite solutions was somewhat more effective than the other irrigation regimens.

4.2.3 Dressing (IV)4.2.3.1 Calcium hydroxide paste (IV)Calcium hydroxide has a strong antibacterial effect in vitro. The killing of various bacterial strains by a calcium hydroxide suspension is presented in Table 4. Most strains were killed within less than 1 min. The most resistant strain belonged to the species Enterococcus faecal is. The rate at which E. faecalis was killed was dependent on the pH and the bacterial cells survived at pH 11.5 but not at pH 12.5. Horseserum caused an insignificant delay of the killing of E. faecalis.

Table 4. The time required to kill 99.9% or in a calcium hydroxide suspension.

more of bacterial cells

<1 min 1-3 min

Streptococcus sanguis S. salivarius S. milleri S. mitis S. intermed ius Campylobacter fetus Capnocytophaga ochracea Bifidobacterium dentium Fusobacterium nucleatum Lactobacillus acidophilus Selenomonas sputigena'Wolinella recta Bacteroides melaninogenicua Peptostreptococcus anaerobius Actinobacillus actinomycetemcomitans

Streptococcus mutans S. morbillorum Lactobacillus casei Actinomyces israelii A. naeslundii A. odontolyticus A. viscosus Veillonella parvula

3-6 min >6 min

Arachnia propionica Eubacterium alactolyticum Propionibacterium acnes

Enterococcus faecalis

23

Thirty-five teeth vere treated with calcium hydroxide paste.No bacteria could be recovered from any samples taken from root canals after they had been dressed with calcium hydroxide paste for 1 month. In samples taken 2 to 4 days after the dressing had been removed, bacteria were recovered from 1 of the 35 samples. That sample contained Wolinella recta and Fusobacterium nucleatum. That case (OD) did not heal (Fig 3b). Bacteria persisted in 21 root canals after the treatment regimens of studies I, II and III and in 10 root canals after dressing with camphorated phenol or camphorated paramonochlorophenol in study IV.These bacteria were eliminated by dressing the root canals with calcium hydroxide paste in all but 2 canals.

4.2.3:2 Camphorated phenol/paramonochlorophenol (IV)Thirty teeth were treated with camphorated phenol/paramonochlorophenol for 2 weeks (Fig 1:IV). No bacteria could be recovered from 20 out of 30 root canals after 2 weeks. Bacteria present in the 10 root canals are presented in Table 5.

4.3 HEALING (V)In all 140 teeth were treated using the various regimens. Of these teeth 79 could be followed-up for at least 2 years. Radiographs were regularly taken during this period and there was complete healing in 67 teeth within 5 years. The criterion for complete healing was a normal or slightly widened apical periodontal space (<0.5 mm). The size of the lesions decreased progressively over the review period. In Fig 2 this healing pattern is illustrated for lesions of various initial sizes.

Seven lesions also decreased in size, but after 2 years the size of the apical periodontal space was more than 0.5 mm (Fig 3a). The treatment regimen for these teeth before root-filling is presented in Table 6. In 3 of the 7 cases (A, B, C, Fig 3a) the healing followed the same pattern as for those which healed completely. In one case (LL12) there was a slower decrease in the size of the lesion. The remaining 3 cases (LL31, LL41, LL42, Fig 3a) were treated surgically. They were all involved in a large confluent lesion in the mandibular anterior region and histological examination of the tissue removed at surgery showed scar tissue which was almost free of inflammatory cells.

In 5 lesions, there was no or only an insignificant decrease in the size of the lesions (Fig 3b). The treatment regimen for these

24

teeth before root filling is presented in Table 6. Four cases (IL, JV, ABg, LB) were treated surgically and tissue samples from IL, JV and ABg were subjected to histological examination. The histological examination of the tissue sample from IL shoved a radicular cyst vith the presence of Actinomyces israelii and Arachnia propionica. The histological examination of the tissue sample from JV shoved a periapical abscess vith Actinomyces israelii. In case ABg there vas a radicular cyst vith chips of dentin in the tissue.

Table 5. Bacteria persistent in 10 root canals after treatment vhen camphorated phenol (CP) or camphorated paramonochlorophenol (CPCP) vere used as dressing betveen appointments.

Case Dressing No of bacteria Appointment 2 3

Persistent bacteria

ACS CP 1.5xl05 45x10 Bacteroides intermedius Fusobacterium sp Propionibacterium acnes Peptostreptococcus micros

EU CP - <io2 Streptococcus milleri

LB CP 1.8xl03 4.2xl02 Eubacterium timidurn Fusobacterium nucleatum Lactobacillus catenaforme Peptostreptococcus anaerobius Streptococcus constellatus Wolinella recta

OL CP <102 2.5xl03 Actinomyces israelii

TN CP 2.3xl03 5.6xl03 Eubacterium alactolyticum Fusobacterium sp

SU CPCP 2.2xl02 l.lxlO2 Bacteroides sp Eubacterium alactolyticum Peptococcus prevotii

KK CPCP <102 <102 Lactobacillus salivarius

KGS CPCP <102 2.lxlO2 Wolinella recta

RA CPCP <102 1.2xl02 Bifidobacterium eriksonii Lactobacillus salivarius

BR CPCP 2xl02 9xl02 Enterococcus faecal is

25

Table 6. Treatment regimens and the outcome o£ the endodontic treatment for the 79 followed-up teeth.

Teeth Treatment regimens Bacteria Root Outcome of treatment+no Irrigant Dressing after

treatment+*

filleddressing*

+

Heale'd "Healing" Not healed

11 NaCl4

74

746 A

13 NaOCl(0.51)11

27 4

2111 ABg

14 NaOCl(5Z)9

595

7 LL12 LB 5

15 NaOCl/EDTA12

3 CO)—*3 8 B,LL41,LL42 JW 2 IL

13 NaOCl(0.5%) Ca(0H)212

1121

11 COD

13 NaOCl(0.5Z) Ca(0H)213

013 12

LL31

79 61 18 11 68 67 7 5

* All teeth in this column subsequently dressed with calcium hydroxide paste.• Dressing with calcium hydroxide paste after ordinary treatment regimen.+ Healing and not healed cases are presented in Fig 3a and 3b.

Of the 79 cases followed-up for at least 2 years 12 cases had acute apical abscesses at the beginning of the treatment and 9 cases developed acute exacerbations during the treatment. Nineteen of these 21 cases healed completely. The remaining 2 cases were JW and ABg (Fig 3b).

In 38 out of 79 teeth the apical level of the root-fillingswas within 0.5 to 2 mm from the radiographic apex of the teeth(Table 7). Eleven teeth were filled to the radiographic apex and 30teeth were overfilled. Three of the 5 cases, with no or only aninsignificant decrease in the size of the lesions, were overfilled. However, the root-filling excesses did not significantly influence the healing of the apical lesions.

26

Table 7. The apical level of the root filling and the outcome of the endodontic treatment.

Number Completely healed or healing

Not healed

Root filled to apex 11 11

Root filled short of apex 38 36 2

Root filled with excess 30 27 3

4

3

21

6543

2

1

22 1 21 1

O B SE R V A T IO N P E R IO D (Y E A R S)

Fig 2 The change in size of the apical lesions followingtreatment. Healed lesions grouped according to initial sizes. Number of cases in each group are given in the figure. Sizes given as mean + 2 standard deviations.

27

SIZE

OF

TH

E LE

SION

S (m

m)

12

1110

JW

9876 ABg

54

OD

3 LB

21

1 32

12

11

10987 L L 42

65432

L L 12

1 B,C

3 421

OBSERVATION PERIOD (YEARS) OBSERVATION PERIOD (YEARS)

Fig 3 Change in size of the apical lesions following treatment.a. The decrease in the size of "healing" lesions, b. Not healed lesions, e.g. lesions with no or insignificant decrease in the size.

28

5 DISCUSSION

5.1 MICROBIAL CHANGES DURING TREATMENTThe bacteria initially isolated from root canals were predominantly anaerobic and the variety of bacterial species found was similar to that reported in previous studies (Kantz and Henry 1974, Wittgow and Sabiston 1975, Sundqvist 1976, Dahlén and Bergenholtz 1980, Zavistoski et al. 1980). These bacteria represented a restricted group compared with the total potential pool of bacteria present in the oral cavity. This indicates that an ecological balance is established in closed root canals. Möller and associates (1981) have also demonstrated this by experimental infection of root canals in monkeys. They found an increase in the number and proportions of anaerobic bacteria in samples taken at the end of experimental periods compared to the initial infecting microbiota.

The median number of bacterial cells recovered from the rootcanals at the beginning of the first appointment was 3 x 105 (range <

2 710 - 2 x 10 ). Mechanical instrumentation and irrigation reduced thenumber of bacterial cells recovered from the root canals 100 - 1000-fold irrespective of the treatment regimen. Bacteria which survived the treatment usually multiplied in the undressed root canals. Similar results have been reported by Stewart and collaborators (1969) and Bence and collaborators (1973). When few bacterial cells were recovered at the end of one appointment these bacteria in some cases did not survive until the next appointment.

During a treatment period the composition of the microbiota recovered from the root canal varied. There was, however, no indication that specific bacteria were implicated in persistent infections at the end of the treatment period. In previous studies (Bender and Seltzer 1952, Grahnén and Krasse 1963, Engström 1964, Goldman and Pearson 1969) enterococci have been implicated in persistent root canal infections.We, however, could identify enterococci in only one out of 140 root canals. These enterococci were not found in the initial sample but only after the root canal had been dressed with camphorated paramonochlorophenol for 2 weeks.

From some root canals no bacteria could be recovered at the end of one appointment, but at the next appointment bacteria could again be found in these canals. The bacteriological technique might not reveal

29

all the bacteria present in the root canal or there might have been a reinfection of the root canal during the interval between the appointments. It is, however, unlikely that reinfection caused the reappearance of bacteria in the canals. All bacteria recovered from these root canals at subsequent appointments were, with one exception, recovered from the root canals at the beginning of the treatment. It is thus obvious that in spite of the very careful bacteriological technique used in the present study (Möller 1966), the result obtained by means of this technique cannot always be used as an ultimate proof of the bacteriological status of the root canal.

One possible improvement in the method for recovering bacteria from the root canal would be to increase the interval between appointments to more than 2 to 4 days. Presumably bacteria which remain in the root canal following treatment could multiply to a detectable level. This may, however, increase the risk of reinfection in the extended inter-appointment time. It has recently been shown that in cases of periodontal disease with loss of attachment, bacteria could invade the dentinal tubules and infect the root canal (Adriaens et al. 1986).

It has been proposed that the oxygen tension in the root canal is changed by the endodontic treatment so that the more oxygen tolerant bacteria are favoured and thus will outnumber the anaerobes (Naidorf 1985, Matusow 1986). The present results showed, however, that the proportion of anaerobes of the microbiota in the root canals did not change during the treatment. It is thus likely that anaerobiosis was reestablished in the root canals when they were sealed (Table 2). This is consistent with the findings of Möller et al. (1981), and Fabricius et al. (1982).

5.2 COMPARISON OF TREATMENT REGIMENSBacteria could not be eliminated in a predictable manner by combining mechanical instrumentation and irrigation with antibacterial solutions. Furthermore, there was no difference in efficacy between 0.5% and 5% sodium hypochlorite solutions. Similar results have been obtained by Cvek and collaborators (1976). This implies that other measures than using irrigants with increased antibacterial potential are required for the elimination of bacteria from the root canal.

30

An antiseptic only works if it comes into contact with the target - the bacteria. In root canals the antiseptic may not reach the bacteria if they are concealed within or behind a smear layer (Baker et al. 1975, McComb et al. 1976, Lester and Boyde 1977, Rubin et al. 1979, Bolanos and Jenen 1980, Goldman et al. 1982, Mader et al. 1984, Berg et al. 1986) or in dentinal tubules (Chirnside 1958, Shovelton 1964, Akpata and Blechman 1982, Armitage et al. 1983). By including EDTA in an irrigating solution this smear layer may be degraded (McComb et al.1976, Goldberg and Abramowich 1977, Bolanos and Jensen 1980, Goldman et al. 1981, 1982, Goldberg and Spielberg 1982, Berg et al. 1986), which might improve the access of the antiseptic to the bacteria. The alternate use of 5% sodium hypochlorite and EDTA solutions as irrigants did not, however, eliminate all bacteria from the infected root canals. Not even the combination of ultrasonic instrumentation and irrigation with 0.5% sodium hypochlorite solution has proved to be effective (Sjögren and Sundqvist 1986). Thus irrespective of instrumentation and irrigation regimens, bacteria could persist in the root canals. Although the need for antibacterial dressings in the period between the appointments has been questioned (Strindberg 1965, Weine 1982, Schilder 1984), our results showed that an antibacterial dressing is an essential part of the endodontic treatment.

Calcium hydroxide proved to be an excellent antibacterial dressing. This is consistent with the results reported by Cvek and collaborators (1976) and Strömberg and Allard (1976). The present study clearly showed that calcium hydroxide paste was superior to camphorated phenol and camphorated paramonochlorophenol. This confirms previous observations that calcium hydroxide paste maintains its antibacterial effect for weeks or months (Tronstad et al. 1981), while camphorated phenol and paramonochlorophenol quickly lose their antibacterial effect when they are sealed in the root canal (Messer and Chen 1984, Tronstad et al. 1985, Fager and Messer 1986). The use of these phenols as antibacterial dressings in endodontic treatment should therefore be discontinued.

5.3 HEALINGIn the present study no bacteria could be recovered from any root canal at the appointment when they were filled. The majority of the 79 evaluated lesions healed completely or decreased in size in such a way

31

that they could be expected to heal. Only 5 cases did not heal. In those cases there vas no or only an insignificant decrease in the size of the lesions 2 years after treatment. Persistent bacteria were demonstrated in the apical tissues in 2 of the 5 cases. Our results suggest that failure of apical lesions to heal may be due to an establishment of bacteria outside the root canal.

Survival of bacteria outside the root canal in the periodontal tissue has, however, been debated. It is known that in acute apical periodontitis bacteria are present in the periapical tissue (van Vinkelhoff et al. 1985, Lewis et al. 1986), but in the chronic state of the disease bacteria are rarely found and then only in phagocytizing cells or in association with necrotic tissue and particles of root filling materials (Block et al. 1976, Langeland et al. 1977, Pitt Ford 1982). Analysis of biopsy specimens from clinical cases (Block et al. 1976, Langeland et al. 1977) and from animal experiments (Malooley et al. 1979, Pitt Ford 1982) shows that bacteria may survive on the root surface especially in exposed dentinal tubules, in lacunae of the cellular cementum, and in apical foramina. A close association between the survival of bacteria at these sites and failure of the lesion to resolve was apparent in the animal experiments (Malooley et al. 1979, Pitt Ford 1982). Healing of a lesion may also be prevented by bacteria of the genera Actinomyces and Arachnia because they survive in the tissue without the support of a root surface, necrotic tissue or particles of root-filling materials (Sundqvist and Reuterwing 1980, Weir and Buch 1982, Happonen et al. 1985). In a recent report, Tronstad and associates (1986) claim that other anaerobic bacteria may also establish themselves in the apical tissue, inaccessible to conventional endodontic treatment.

In clinical work it may sometimes be a problem to judge when the endodontic treatment should be considered a failure. The present study provides some clues as to how this problem could be tackled. The treatment was evaluated by means of regular radiographic recordings. It was thus possible to demonstrate a variety of healing patterns among the lesions. The analysis of the healing patterns showed that the initial size of the lesion influenced the healing pattern and that complete healing of a lesion could take up to 5 years. Our results showed, however, that as long as there is a continuous decrease in the size of a lesion following treatment, there is no reason to designate a case a

32

failure. Judging success or failure after the endodontic treatment of teeth with an unknown background on one occasion (Bergenholtz et al. I 1973, Erselius et al. 1975, Bergman et al. 1979, Hugosson and Kock 1979, Holm et al. 1982, Petersson et al. 1986) may result in an overestimation of the failure rate of such treatment.

In routine clinical work this calls for radiographic examination before the endodontic treatment and at regular follow-ups until complete healing is achieved. In order to obtain comparable radiographs of high quality at different examinations the following recommendations are made: 1. that the long-cone technique with a filmholder is employed 2. that the same exposure time is used for the same tooth on each occasion a radiograph is taken 3. that standardized processing procedures in accordance with the manufacturers' guidelines should be followed. The judgment of the treatment result should be based on the change in the size of the lesion carefully registered on recall radiographs.

Every single step in the endodontic treatment has been evaluated from many angles over the years and this has resulted in an antibacterial treatment regimen consisting of mechanical instrumentation, irrigation and placement of antibacterial dressing. In the present study the efficacy of these measures has been evaluated using bacteriological methods and by recording the healing pattern of the lesions. From these results it can be concluded that it is possible to follow a treatment regimen at the first appointment which eliminates bacteria so effectively that the treatment can be completed in 2 appointments.

In Sweden dentists devote 12 to 13% of their total time to endodontic treatment (Andersson et al. 1986). More than 75% of this time is spent in treating non-vital teeth. The average number of appointments requested in the treatment is 3 to 5 for each tooth (Andersson et al. 1986). The treatment regimen finally applied in the present study might both significantly increase the rate of success and decrease the time needed for endodontic treatment.

33

SUMMARY

The present study is a clinical and bacteriological investigation of non-vital teeth with apical periodontitis. Bacteria play a decisive role in the development of this disease. The aim of the treatment is therefore to eliminate the bacteria. Earlier studies, however, provide limited information about the relative efficacy of the various measures used in this treatment. In the present study it was shown that mechanical cleansing with files in combination with saline as the irrigating solution, reduced the number of bacteria in the root canal during the treatment sessions but few root canals were completely freed from bacteria. The same technique but using sodium hypochlorite as the irrigating solution, resulted in more root canals free from bacteria. There was, however, no difference in the antibacterial effect between 0.5% and 5.0% sodium hypochlorite solutions, while the combination of 15% EDTA and 5% sodium hypochlorite solutions was somewhat more efficient than 5% sodium hypochlorite solution alone.

The bacteria which survived the mechanical cleansing and the irrigation usually increased in number in the period between treatment sessions. By dressing the root canal with calcium hydroxide paste the bacteria that persisted after the mechanical cleansing and the irrigation were efficiently eliminated. The healing of 79 lesions was followed-up. The majority of these lesions healed within 2 years, but in some cases the healing was not completed until 5 years after the treatment. Five lesions did not heal. Histological examination of these lesions showed that in 2 cases bacteria had become established outside the root canal.

The present study showed, that the treatment of infected non- vital teeth can be completed in only 2 sessions. At the first session the root canal is mechanically cleansed, irrigated with sodium hypochlorite solution and dressed with calcium hydroxide paste. At the second session, one month later, the calcium hydroxide paste is removed and the root canal is filled. This treatment may however fail in some cases because of bacteria may establish outside the root canal where they only can be eliminated by surgery.

34

ACKNOWLEDGEMENTS

I want to express my sincere gratitude to my supervisor and co-author Professor Göran Sundqvist who introduced me to this field of research and guided me through it with inspiration, constructive criticism and support. I am also most grateful to Professor Jan Carlsson for his generous support, constructive critisism and advices which have been essential for this work. I also want to express my sincere thanks to Docent Kenneth Wing, Drs Jan Ahlqvist, Eva Borssén, Per Nelvig, Ulf Sjögren and Per Strandberg for analysing the roentgenograms, Mrs Eva Johansson and Mr Rolf Classon for excellent technical assistance, Fil. kand. Tomas Laitila for statistical analyses, Mrs Sonja Andersson for excellent assistance, and Mrs Katarina Wrethén for excellent secretarial assistance. I also want to thank Dr David Figdor and Mrs Patricia Shrimpton for linguistic revision of the manuscript.

This investigation was supported by grants from the Faculty of Odontology, University of Umeå and the Swedish Dental Society.

35

REFERENCES

ADRIAENS PA, LOESCHE WJ, DE BOEVER JA. Bacteriological study of the microbial flora invading the radicular dentin of periodontally diseased caries-free human teeth, in: Cimasoni G, Lehner T, eds. Borderland between caries and periodontal disaese III. 3rd European Symposium. Med Hyg, Chene-Bourg, 1986; In press.

AKPATA ES, BLECHMAN H. Bacterial invasion of pulpal dentin wall in vitro. J Dent Res 1982; 61: 435-438.

ANDERSSON K, JONSSON M, SJÖGREN U. Metoder och material vid endodontibehandling. Tandläkartidningen 1986; 78: 940-944.

ARMITAGE GC, RYDER MI, WILCOX SE. Cementai changes in teeth with heavily infected root canals. J Endod 1983; 9: 127-130.

BAKER NA, ELEAZER PD, AVERBACH RE, SELTZER S. Scanning electron microscopic study of the efficacy of various irrigating solutions.J Endod 1975; 4: 127-135.

BENCE R, MADONIA JV, WEINE FS, SMULSON MH. A microbiologie evaluation of endodontic instrumentation in pulpless teeth. Oral Surg Oral Med Oral Path 1973; 35: 676-683.

BENDER IB, SELTZER S. Combination of antibiotics and fungicides used in treatment of the infected pulpless tooth. J Am Dent Assoc 1952; 45: 293- 300.

BERG MS, JACOBSEN EL, BeGOLE EA, REMEIKIS NA. A comparison of five irrigating solutions: a scanning electron microscopic study. J Endod 1986; 12: 192-197.

BERGENHOLTZ G, MALMCRONA E, MILTHON R. Endodontisk behandling och periapikalstatus. I. Röntgenologisk undersökning av frekvensen endodontiskt behandlade tänder och frekvensen periapikala destruktioner. Tandläkartidningen 1973; 65: 64-73.

BERGENHOLTZ G, MALMCRONA E, MILTHON R. Endodontisk behandling och periapikalstatus. II. Röntgenologisk bedömning av rotfyllningens kvalitet ställd i relation till förekomst av periapikala destruktioner. Tandläkartidningen 1973; 65: 269-279.

BERGMAN J, DAHLHEIM M, LORIN C, NENNING J. Rotfyllningar och periapikalstatus. Tandläkartidningen 1979; 71: 848-855.

BLOCK RM, BUSHELL A, RODRIDGUES H, LANGELAND K. A histopathologic, histobacteriologic, and radiographic study of periapical endodontic surgical specimens. Oral Surg Oral Med Oral Path 1976; 42: 656-678.

BLOOMFIELD SF, MILES GA. The antibacterial properties of sodium dichloroisocyanurate and sodium hypochlorite formulations. J Appi Bacteriol 1979; 46: 65-73.

BOLANOS OR, JENSEN JR. Scanning electron microscope comparisons of the efficacy of various methods of root canal preparation. J Endod 1980; 6: 815-822.

36

CARLSSON J, SUNDQVIST G. Evaluation of methods of transport and cultivation of bacterial specimens from infected dental root canals.Oral Surg Oral Med Oral Path 1980; 49: 451-454.

CHIRNSIDE IM. The bacteriological status of dentine around infected pulp canals. New Zealand Dent J 1958; 54: 173-183.

COWAN ST. Cowan and Steel 's manual for the identification of medical bacteria. 2nd ed. Cambridge University Press, London, 1974.

CVEK M, NORD C-E, HOLLENDER L. Antimicrobial effect of root canal débridement in teeth with immature root. A clinical and microbiologiestudy. Odontol Revy 1976; 27: 1-10.

DAHLÉN G, BERGENHOLTZ G. Endotoxic activity in teeth with necrotic pulps. J Dent Res 1980; 59: 1033-1040.

DOW PR. EDTA - time for re-evaluation? Int Endod J 1984; 17: 2-5.

EGGEN S. Röntgenografiske tannmålinger i daglig praksis ved hjelp av standardisert parallell-teknikk og en kalibrert målelinjal. Tandläkartidningen 1974; 66: 10-12.

ENGSTRÖM B. The significance of enterococci in root canal treatment. Odontol Revy 1964; 15: 87-106.

ENGSTRÖM B, HÅRD af SEGERSTAD L, RAMSTRÖM G, FROSTELL G. Correlation of positive cultures with the prognosis for root canal treatment. Odontol Revy 1964; 15: 257-270.

ERSELIUS L, KOSCHKE T, LJUNGMAN A-M, PHILIP E, HISING P. JULIN P, PIOCH W. Röntgenfynd på 15000 helstatus. Periapikala förändringar, resorptioner och retinerade tänder i ett material på Tandläkarhögskolan, Stockholm. Tandläkartidningen 1975; 67: 342-350.

FABRICIUS L, DAHLÉN 6, ÖHMAN AE, MÖLLER ÅJR. Predominant indigenous oral bacteria isolated from infected root canals after varied times of closure. Scand J Dent Res 1982; 90: 134-144.

FAGER FK, MESSER HH. Systemic distribution of camphorated monochlorophenol from cotton pellets sealed in pulp chambers. J Endod 1986; 12: 225-230.

FLINK 0, OHLSSON I, REDMALM G. Apoteksberedda rotbehandlingspreparat - sortimentsutveckling. Tandläkartidningen 1981; 73: 200-203.

FOLEY DB, WEINE FS, HAGEN JC, deOBARRIO JJ. Effectiveness of selected irrigants in the elimination of Bacteroides melaninogenicus from the root canal system: An in vitro study. J Endod 1983; 9: 236-241.

FULGHUM RS. Mobile anaerobe laboratory. Appi Microbiol 1971; 21: 769- 770.

GOLDBERG F, ABRAMOVICH A. Analysis of the effect of EDTAC on the dentinal walls of the root canal. J Endod 1977; 3: 101-105.

GOLDBERG F, SPIELBERG C. The effect of EDTAC and the variation of its working time analyzed with scanning electron microscopy. Oral Surg Oral Med Oral Path 1982; 53: 74-77.

37

GOLDMAN LB, GOLDMAN M, KRONMAN JH, LIN PS. The efficacy of several irrigating solutions for endodontics : A scanning electron microscopic study. Oral Surg Oral Med Oral Path 1981; 52: 197-204.

GOLDMAN M, GOLDMAN LB, CAVALERI R, BOGIS J, LIN PS. The efficacy of several endodontic irrigating solutions: a scanning electron microscopic study: part 2. J Endod 1982; 8: 487-492.

GOLDMAN M, PEARSON AH. Postdébridement bacterial flora and antibiotic sensitivity. Oral Surg Oral Med Oral Path 1969; 28: 897-905.

GRAHNÉN H, HANSSON L. The prognosis of pulp and root canal therapy. A clinical and radiographic follow-up examination. Odontol Revy 1961; 12: 146-165.

GRAHNÉN H, KRASSE B. The effect of instrumentation and flushing of non- vital teeth in endodontic therapy. I. A clinical and bacteriological study. Odontol Revy 1963; 14: 167-177.

GROSSMAN LI. Endodontic practice. 10th ed, Lea & Febiger, Philadelphia, 1981; 200.

HAND RE, SMITH ML, HARRISON JW. Analysis of the effect of dilution on the necrotic tissue dissolution property of sodium hypochlorite. J Endod 1978; 4: 60-64.

HAPPONEN R-P, SÖDERLING E, VIANDER M, LINKO-KETTUNEN L, PELLINIEMI LJ. Immunocytochemical demonstration of Actinomyces species and Arachnia propionica in periapical infections. J Oral Pathol 1985; 14: 405-413.

HARDIE JM, BOWDEN GH. Physiological classification of oral viridans streptococci. J Dent Res 1976; 55: A166-A176.

HELING B, SHAPIRA J. Roentgenologic and clinical evaluation of endodontically treated teeth, with or without negative culture. Quintessence Int 1978; 11: 79-84.

HERMANN BW. Dentinobiiteration der Wurzelkanäle nach Behandlung mit Calcium. Zahnärztl Rundsch 1930; 21: 888-900.

HOLDEMAN LV, CATO EP, MOORE WE. Anaerobe laboratory manual. 4th ed. VPI Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg VA, 1977.

HOLM G, LAURELL L, HEDIN M. Tandhälsotillståndet hos den vuxna befolkningen i Gävleborgs län -X-länsundersökningen. Gävleborgs läns landsting, 1982.

HSU S-M, RAINE L. Protein A, avidin and biotin in immunohistochemistry.J Histochem Cytochem 1981; 29: 1349-1353.

HUGOSON A, KOCH G. Oral health in 1000 individuals aged 3-70 years in the community of Jönköping, Sweden. Swed Dent J 1979; 3: 69-87.

INGLE JI, MULLANEY TA, GRANDICH RA, TAINTOR JF, FAHID A. Endodontic cavity preparation. In: Ingle JI, Taintor JF, eds. Endodontics. 3rd ed. Lea and Febiger, Philadelphia, 1985; 102-222.

38

INGLE JI, ZELDOW BJ. An evaluation of mechanical instrumentation and the negative culture in endodontic therapy. J Am Dent Assoc 1958; 57: 471- 476.

KAKEHASI S, STANLEY HR, FITZGERALD RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Path 1965; 20: 340-349.

KANTZ WE, HENRY CA. Isolation and classification of anaerobic bacteria from intact pulp chambers of non-vital teeth in man. Arch Oral Biol 1974; 19: 91-96.

KEREKES K. Radiographic assessment of an endodontic treatment method. J Endod 1978; 4: 210-213.

KEREKES K, BERVELL SFA. En rtfntgenologisk vordering av endodontisk behandlingsbehov. Norske Tannlaegeforen Tid 1976; 86: 248-254.

KEREKES K, TRONSTAD L. Long-term results of endodontic treatment performed with a standardized technique. J Endod 1979; 5: 83-90.

KOSKINEN KP, STENVALL H, UITTO V-J. Dissolution of bovine pulp tissue by endodontic solutions. Scand J Dent Res 1980; 88: 406-411.

LANGELAND K, BLOCK RM, GROSSMAN LI. A histopathologic and histobacteriologic study of 35 periapical endodontic surgical specimens. J Endod 1977; 3: 8-23.

LAVSTEDT S. Behovet av tandhälsovård och tandsjukvård hos en normalpopulation. Rebusundersökning II. Tandläkartidningen 1978; 70: 971-991.

LESTER KS, BOYDE A. Scanning electron microscopy of instrumented, irrigated and filled root canals. Br Dent J 1977; 143: 359-367.

LEWIS MAO, MacFARLANE TW, McGOWAN DA. Quantitative bacteriology of acute dento-alveolar abscesses. J Med Microbiol 1986; 21: 101-104.

MADER CL, BAUMGARTNER JC, PETERS DD. Scanning electron microscopic investigation of the smeared layer on root canals walls. J Endod 1984; 10: 477-483.

MALOOLEY J, PATTERSON SS, KAFRAWY A. Response of periapical pathosis to endodontic treatment in monkeys. Oral Surg Oral Med Oral Path 1979; 47: 545-554.

MATTSON 0. A magnifying viewer of photofluorographic film. Acta Radiol 1953; 39: 412-414.

MATUSOW RJ. Acute pulpal-alveolar cellulitis syndrome. IV. Exacerbations during endodontic treatment: A clinical study of specific microbial isolates and their etiologic role. Part 1. Oral Surg Oral Med Oral Path 1986; 61: 90-95.

McCOMB D, SMITH DC, BEAGRIE GS. The results of in vivo endodontic chemomechanical instrumentation - A scanning electron microscopic study. J Br Endod Soc 1976; 9: 11-18.

McCOMB D, SMITH DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1975; 1: 238-242.

39

MESSER HH, CHEN R-S. The duration of effectiveness of root canal medicaments. J Endod 1984; 10: 240-245.

MOLVEN 0. The frequency, technical standard and results of endodontic therapy. Thesis, University of Bergen, Bergen, Norway 1974.

MOORER WR, WESSELINK PR. Factors promoting the tissue dissolving capability of sodium hypochlorite. Int Endod J 1982; 15: 187-196.

MÖLLER ÅJR. Microbiological examination of root canals and periapical tissues of human teeth. Thesis, Odontol Tidskr Spec Issue 1966; 74: 1- 380.

MÖLLER ÅJR, FABRICIUS L, DAHLÉN G, ÖHMAN AE, HEYDEN G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dent Res 1981; 89: 475-484.

NAIDORF IJ. Endodontic flare-ups: Bacteriological and immunological mechanisms. J Endod 1985; 11: 462-464.

OLIET S, SORIN SM. Evaluation of clinical results based upon culturing root canals. J Br Endod Soc 1969; 3: 3-6.

PETERSSON K, PETERSSON A, OLSSON B, HÅKANSSON J, WENNBERG A. Technical quality of root fillings in an adult Swedish population. Endod Dent Traumatol 1986; 2: 99-102.

PITT FORD TR. The effects on the periapical tissues of bacterial contamination of the filled root canal. Int Endod J 1982; 15: 16-22.

PROELL F. Über die Eigenschaften des Calxyls und seine Vorzüge vor anderen in der zahnärztlichen Praxis angewandten Medikamenten.Zahnärtztl Rundsch 1949; 58: 255-259.

RUBIN LM, SKOBE Z, KRAKOW AA, GRON P. The effect of instrumentation and flushing of freshly extracted teeth in endodontic therapy: a scanning electron microscope study. J Endod 1979; 5: 328-334.

SCHILDER H. Canal debridement and disinfection. In: Cohen S, Burns RC, eds. Pathways of the pulp. 3rd ed. The CV Mosby Company, St. Louis,1984; 175-202.

SCHLEIFER KH, KILPPER-BÄLZ R. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. asEnterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. IntJ Syst Bacteriol 1984; 34: 31-34.

SELTZER S, BENDER IB, SMITH J, FREEDMAN I, NAZIMOV H. Endodontic failures - An analysis based on clinical, roentgenographic, and histologic findings. Part I. Oral Surg Oral Med Oral Path 1967; 23: 500-516.

SELTZER S, BENDER IB, SMITH J, FREEDMAN I, NAZIMOV H. Endodontic failures - An analysis based on clinical, roentgenographic, and histologic findings. Part II. Oral Surg Oral Med Oral Path 1967; 23: 517-530.

SHIH M, MARSHALL J, ROSEN S. The bactericidal efficiency of sodium hypochlorite as an endodontic irrigant. Oral Surg Oral Med Oral Path 1970; 29: 613-619.

40

SHOVELTON DS. The presence and distibution of micro-organisms within non-vital teeth. Br Dent J 1964; 117: 101-107.

SJÖGREN U, SUNDQVIST G. Bactériologie evaluation of ultrasonic root canal instrumentation. Oral Surg Oral Med Oral Path 1986; In Press.

SOMMER RF, OSTRANDER FD, CROWLEY MC, eds. Clinical endodontics. 2nd ed.W B Saunders Company, Philadelphia, 1962; 364-365.

SPÅNGBERG L, ENGSTRÖM B, LANGELAND K. Biologic efects of dental materials. 3. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg Oral Med Oral Path 1973; 36: 856-871.

STEWART GG, KAPSIMALAS P, RAPPAPORT H. EDTA and urea peroxide for root canal preparation. J Am Dent Assoc 1969; 78: 335-338.

STRINDBERG LZ. The dependence of the results of pulp therapy of certain factors. An analytic study based on radiographic and clinical follow-up examinations. Thesis, Acta Odontol Scand 1956; 14: Suppl 21.

STRINDBERG LZ. Det antibakteriella inläggets effect vid konserverande rotbehandling. En jämförande bakteriologisk studie. Sven Tandlak Tidskr 1965; 58: 219-235.

STRÖMBERG T, ALLARD U. Ca(OH) efter primär utrensning av nonvitala tänder. Sven Tandlak Tidskr 1976; 69: 135-136.

SUNDQVIST G. Bacteriological studies of necrotic dental pulps. Umeå University Odontological Dissertation no. 7, University of Umeå, Umeå, Sweden, 1976.

SUNDQVIST G, REUTERVING C-0. Isolation of Actinomyces israelii from periapical lesion. J Endod 1980; 6: 602-606.

TANNER ACR, BADGER S, LAI C-H, LISTGARTEN MA, VISCONTI RA, SOCRANSKY SS. Wolinella gen. nov., Wolinella succinogenes (Vibrio succinogenes Wolin et al.) comb, nov., and description of Bacteroides gracilis sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov., and Eikenella corrodens from humans with periodontal disease. Int J Syst Bacteriol 1981; 31: 432-445.

THÉ SD. The solvent action of sodium hypochlorite on fixed and unfixed necrotic tissue. Oral Surg Oral Med Oral Path 1979; 47: 558-561.

TRONSTAD L, ANDREASEN JO, HASSELGREN G, KRISTERSON L, RIIS I. pH changes in dental tissues after root canal filling with calcium hydroxide. J Endod 1981; 7: 17-21.

TRONSTAD L, YANG Z-P, TROPE M, BARNETT F, HAMMOND BF. Controlled release of medicaments in endodontic therapy. Endod Dent Traumatol 1985; 1: 130- 134.

TRONSTAD L, BARNETT F, FLAX M, SLOTS J. Anaerobic bacteria in periapical lesions of human teeth. J Dent Res 1986; 65: 231.

VANDER WALL GL, DOWSON J, SHIPMAN JR C. Antibacterial efficacy and cytotoxicity of three endodontic drugs. Oral Surg Oral Med Oral Path 1972; 33: 230-241.

41

WEIR JC, BUCK WH. Periapical actinomycosis. Report of a case and review of the literature. Oral Surg Oral Med Oral Path 1982; 54: 336-340.

WHITE E. Microbiologie considerations in endodontics. In: Ingle I, Beveridge EE. (eds): Endodontics. 2nd ed. Lea and Febiger, Philadelphia, 1976; 564-579.

WEINE FS. Endodontic therapy. 3rd ed. The C.V. Mosby Company, St. Louis, 1982; 325

van WINKELHOFF A, CARLÉE AW, de GRAAFF J. Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses. Infect Immun 1985; 49: 494-497.

WITTGOW Jr WC, SABISTON Jr CB. Microorganisms from pulpal chambers of intact teeth with necrotic pulps. J Endod 1975; 1: 168-171.

ZAVISTOSKI J, DZINK J, ONDERDONK A, BARTLETT J. Quantitative bacteriology of endodontic infections. Oral Surg Oral Med Oral Path 1980; 49: 171-174.

ZELDOW BJ, INGLE JI. Correlation of the positive culture to the prognosis of endodontically treated teeth: a clinical study. J Am Dent Assoc 1963; 66: 9-13.

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