L. B. Peters & P. R.Wesselink
Department of Cardiology Endodontology Pedodontology, Amsterdam Academic Centre for Dentistry, Amsterdam, Netherlands.

Introduction.
Root-canal treatment aims to eliminate bacteria from the infected root-canal system to create an environment that is most favourable for healing. Several studies have shown that it is impossible to achieve a bacteria free root-canal space in all cases, even after thorough cleaning, shaping and irrigation with disinfectants or antiseptics (Bystrom & Sundqvist 1981, Bystrom et al. 1985, Orstavik et al.1991, Sjogren et al. 1997, Peters et al. 2002).Therefore, concern exists as to the fate and consequences of the remaining microorganisms in the canal. They may multiply rapidly, in some cases, to almost the initial numbers in 2-4 days, if the canal is left empty (Bystrom & Sundqvist1981).
It is generally believed that the remaining bacteria can be eliminated or be prevented from repopulating the root-canal space by introducing an interappointment dressing such as calcium hydroxide in the root canal  (Bystrom et al.1985, Chong & Pitt Ford1992). However, it has been shown that calcium hydroxide fails to consistently produce sterile root canals and even allows regrowth in some cases (Reit & Dahle. n 1988, Orstavik et al.1991, Peters et al.2002). It is unclear in which cases failure occurs, because the root-canal treatment with an interappointment calcium hydroxide dressing or a negative culture before obturation gives no guarantee of healing in all cases (Sjogren et al. 1990, Nair et al. 1990, Sjogren et al. 1997, Trope et al. 1999, Weiger et al. 2000).
Another approach is to eliminate the remaining microorganisms or to render them harmless by entombing them by complete obturation immediately after preparing and irrigating the canal space at the same visit. This way, the remaining microorganisms may be killed by the antimicrobial activity of the sealer or the Zn2ю ions of gutta-percha (Moorer & Genet1982, Kaplan et al. 1999, Fuss et al. 2000, Siqueira et al. 2000) or may be deprived of nutrition and space to multiply (Soltanoff 1978, Oliet1983,Weiger et al. 2000).
The healing potential for teeth that are treated in one or two visits with placement of an intracanal disinfectant appears similar (Trope et al. 1999, Weiger et al. 2000). Some studies report the presence of remaining microorganisms after cleaning and shaping and dressing with calcium hydroxide, but do not relate this to healing (Bystrom1986,Yared & Bou Dagher1994, Shuping et al. 2000). One study (Sjogren et al.1997) has compared the healing rate (after 5 years) between the canals that were obturated in the presence or absence of cultivable microorganisms after instrumentation and irrigation. A lower healing rate was found for teeth that harboured microorganism sat the time of root-canal obturation compared to those that had a negative culture at the time of obturation (68% vs.94%).
The purpose of this study is to evaluate the healing of periapical lesions of teeth with positive and negative canal cultures at the time of obturation and to evaluate periapical healing of teeth treated in one visit (without) or two visits with an interappointment dressing of calcium hydroxide.

Patient selection.
Thirty-nine, patients with a non-contributory medical history, referred to the endodontic clinic of the Academic Centre for Dentistry in Amsterdam for root-canal treatment, were selected according to the following criteria. All the selected roots (17 incisors, 6 canines, 5 premolars and 11 distal roots of mandibular molars) had one canal, were asymptomatic, did not respond to sensitivity testing, had not received any endodontic treatment previously, and showed radiographic evidence of periapical bone loss. Maxillary molars were not included, because of radiographic overlaps hindering reproducible observation of changes in the periapical lesion size.
The mean age of the participants (19 females and 20 males) was 40 years and ranged from 19 to 86 years. The teeth were randomly divided into two treatment groups, every second patient was assigned to group 2. Group1teeth (n ј 21) were treated in one visit, group 2 teeth (n ј18) were treated in two visits with intracanal disinfection by calcium hydroxide for 4 weeks. The size of the periapical lesions was determined from the preoperative radiograph on a light box by measuring the largest diameter in millimeters with a ruler to an accuracy of 0.5 mm.

Microbial procedure.
All the canals were cultured at the start of treatment (S1, n ј 39); after instrumentation (S2, n ј 39); after removal of calcium hydroxide (S3, n ј18); and before obturation with gutta-percha (S4, n ј18) as described previously (Peters et al. 2002). After cleaning the tooth with pumice and isolation with rubber dam, the crown and the surrounding rubber dam were disinfected with 80% ethanol for 2 min. An access cavity was prepared with sterile high-speed diamond burs under irrigation with sterile saline. Before entering into the pulp chamber, the access cavity was disinfected again for 2 min with 80% ethanol. Sterility was checked by sampling with a cotton swab over the cavity surface and streaked on blood agar plates. All subsequent procedures were performed aseptically. The pulp chamber was accessed with burs and rinsed with Reduced Transport Fluid (RTF) (Syed & Loesche 1972) which was aspirated with suction tips. RTF was then introduced to the root canal with a syringe and 27-gauge needle. Care was taken not to over fill the canal. The canal was enlarged to a size 20 Hedstrom file to the estimated working length as calculated from the preoperative radiograph. Five sterile paper points were consecutively placed in the canal and left for 10 s (sample 1, S1). Then, these were placed in sterile tubes containing 1mL RTF and transferred to the laboratorywithin15 min for microbiological processing.
Ten-fold serial dilutions of the samples were prepared and100 mL of each dilution was inoculated on blood agar  plates supplemented with 5%horse blood,5 mg L  haemin and 1mg L  menadione. Plates were incubated anaerobically (80% N2, 10% H2, 10% CO2) at 37 8C for 7 days. After incubation, the total colony forming units (CFU) and the different colony types were counted using a stereomicroscope at 16x magnification (Zeiss, Oberkochen, Germany).
All the colony types were streaked to purity and incubated aerobically in air with 5% CO2 (BBL Gaspak CO2 systems, Becton Dickinson & Co., Cockeysville, MD, USA) as well as anaerobically to determine strict anaerobic and facultative anaerobic growth. Identification of the isolates was made on the basis of Gramstain, catalase activity and a commercially available identification kit - ATB rapid ID32A (Biomerieux SA, Lyon, France), for strict anaerobes and ATB rapid ID32Strep for facultative anaerobic cocci (Biomerieux SA).
In order to allow the slow-growing species to develop the blood agar plates with the total samples were kept under anaerobic conditions up to 14 days. The newly emerging colonies were also streaked to purity and were identified.

Root-canal procedure.
All procedures were performed by one endodontist. The working length (1mm from the radiographic apex) was checked with a radiograph after inserting a size15 K-file (Dentsply Maillefer, Ballaigues, Switzerland) in the canal to the estimated working length, or shorter if the attached electronic apex locator (Apit, Osada, Japan) indicated that the apical foramen had been reached. After the first microbiologic sample (S1), the canal was enlarged using Flexofiles (Dentsply Maillefer) with the modified double flare technique (Saunders & Saunders 1992), to a master apical file of at least size 35 (range 35-60). Each file was followed by irrigation of the canal with 2 mL sodium hypochlorite (2%) in a syringe with a 27-gauge needle. After preparation, the canal was irrigated with 5 mL sodium hypochlorite (2%). Then, inactivation of the sodium hypochlorite was accomplished with 5 mL sterile sodium thiosulphate, before a second microbiological sample (S2) was taken from the root canal in the same manner as the first sample.
After drying the canal with paper points, the teeth in group 1 (n ј 21) were obturated using the warm lateral- compaction technique with gutta-percha and AH- 26 sealer (Dentsply, Konstanz, Germany). At the end of the first visit all these teeth were restored. Teeth that did not receive a permanent restoration were restored with a temporary filling of two layers of Cavit (ESPE, Seefeld, Germany) and a glass ionomer restoration (Fuji-II, GC Corporation, Tokyo, Japan).
After drying the canal, the teeth in group 2 (n ј18) were dressed with a thick mix of calcium hydroxide (Merck, Darmstadt, Germany) in sterile saline. The calcium hydroxide slurry was inserted in the canal with a size 30 lentulo spiral (Dentsply Maillefer) and packed with the blunt end of a paper point. The access cavities in group 2 were filled with two layers of Cavit and a glass ionomer restoration. In the mandibular molars, the entrance of the distal canal was isolated with Cavit from the remaining pulp chamber in order to prevent contamination by microorganisms from the mesial canals (that had been instrumented but were not included in the study). A radiograph was taken to ensure proper placement of the calcium hydroxide in the canal.
The patients in group 2 returned after 4 weeks. The canal was aseptically accessed under rubber dam isolation and the calcium hydroxide removed with RTF and careful filing of the canal with the master apical file. Removal of calcium hydroxide from the canal was checked with an operating microscope at 16x (Zeiss, Oberkochen, Germany). A third bacteriological sample (S3) was taken as described previously. After sampling, the canal was rinsed with 5 mL of sodium hypochlorite (2%) andgently instrumented with the master apical file. After inactivation of the sodium hypochlorite with sodium thiosulphate, a fourth sample (S4) was taken from the root canal. The canal was dried and obturated withgutta-perchaandAH-26 sealer using the warm lateral compaction technique. After obturation of the canal, the tooth was restored in the same manner as the teeth in group1. A  final radiograph was taken using the paralleling technique with the aid of a beam guiding device (X-Act, Oral Diagnostic Systems, Amsterdam, the Netherlands), followed by control radiographs at 3, 6,12 and 24 months. If complete healing had not taken place at 24 months the patients returned annually up to 4.5 years. At the first follow-up appointment, all temporary restorations were replaced by a permanent restoration.

Evaluation.
Clinically, all the patients were free of symptoms and periodontal disease. The coronal restorations were of good quality during the entire follow-up period.
Three experienced endodontists who had not been involved in the treatment or follow-up appointments were asked to analyse the radiographs. Thirty  radiographs (not included in the study) were used for calibration of the evaluators.
One set of radiographs consisted of four to six radiographs from one patient, taken at different follow-up appointments and projected in an at-random sequence. Each radiograph only showed the root and apical bone structure, the rest of the radiograph was masked. The 39sets (one set of radiographs per patient) wereprojected on a screen in a dark room. The evaluators were asked to indicate the largest periapical radiolucency and the smallest periapical radiolucency of the set that was projected. Both images were given a periapical score from 1 to 5 (Table 1) (Reit & Grondahl 1983). The evaluators also indicated a score for the treatment outcome according to the criteria presented in Table 1 (A, B or C). After each evaluator had given his individual periapical scores and treatment outcome, a joint evaluation was made to reach a consensus.

Table 1. Definitions of periapical scores and treatment outcome.

Statistics.
A Student’s t-test for independent samples or chi-square test (when appropriate) was performed for differences between patient groups related to gender, age and the clinical parameters, such as the tooth type, size of radiolucency, preparation length, master apical file size and apical extent of the root-canal filling.
Differences amongst evaluators and consensus for periapical scores and treatment outcome were tested using the Friedman’s test for ordinal data.
The indicated largest and smallest periapical consensus scores were compared using the Wilcoxon’s signed ranks test to indicate if there was a significant reduction of the periapical bone lesions over time.
The time needed by each tooth included in the study to be assigned to the‘success’group was of interest. Ananalysis of event times that also accounts for the observation period of teeth associated with ‘failure’ was applied as described by Weiger et al. (1998). This approach also considers the individual time span within which the tooth under observation that is scored as B may show complete healing (A), although this time is cut off before the event occurs. The distribution of the event times for both treatment groups were separately calculated on the basis of the Kaplan-Meier method (Kaplan&Meier 1958) and presented as step functions. The log-rank test was applied for comparison of the two treatment groups.
A chi-squared test with Yate’s correction was performed for healing results of canals that showed bacterial growth at the time of obturation and canals that showed a negative culture.
For all tests, the P-values<0.05 were considered statistically significant. When no differences were found power statistics (power set at 80%) were conducted to determine the numbers required to find significant differences (P ј 0.05) between healing of teeth obturated with a positive canal culture and teeth obturated with a negative canal culture as well as differences in healing results of teeth treated in one and two visits.

During the follow-up period of 4.5 years, none of the patients had any discomfort and all teeth were functional. All patients returned for follow-up. One series of radiographs was excluded because of imperfections of radiographic technique.
There were no significant differences between patient groups related to gender, age and (the clinical parameters) tooth type, size of radiolucency, preparation length, master apical file size and the apical extent of the root-canal filling (P > 0.05).
Kappa scores between observers were 0.7-0.9. The agreement between treatment outcome scores of the individual observers and consensus score was at least 94% with kappa scores of 0.8-0.9.
The consensus on periapical scores and treatment outcome are presented in Tables 2 and 3.Before the treatment, all teeth had a periapical score 4 or 5. At the end of the follow-up period18% stillhada radiolucency (periapical score 4 and 5), in 82% a clear radiolucency was not present (periapical score 1 and 2). The periapical scores after the follow-up period were significantly lower (P < 0.05) than the scores before the start of treatment, indicating that the lesions had reduced significantly over the time.

Table 2. Consensus periapical score.


Score1-5, see Table 1. The values in parentheses are in per cent.

Table 3. Consensus treatment outcome.


Score A, B, C see Table 1. The values in parentheses are in per cent.

Consensus treatment outcome A or B was assigned in 97% of the cases. One case was judged as a failure (Table 3). Upon extraction a vertical root fracture was diagnosed in this case.
Of the 21 teeth treated in one visit (group1), 17 showed complete radiographic healing (score A, 81%), and 4 teeth a reduction in lesion size (score B, 19%). In group 2, 12 teeth showed complete radiographic healing (score A, 71%), and 4 had a reduction in the lesion size (score B, 23%) whilst one tooth had failed (score C, 6%) (Table 4).
The time necessary to complete the periapical healing used as basis for calculation ranged from 7 to 55 months. The individual observation times varied between 12 and 35 months for those teeth associated with incomplete healing or failure. The probability that complete healing occurred within a certain time span increased continuously with the length of the observation period (Table 5, Fig.1) The log-rank test did not reveal any significant difference between the one and two visit treatment groups (P > 0.05). Power statistics showed that the presented differences (91% vs. 94%) between one and two-visit root-canal treatment could be significant if one experimental group would comprise of1275 participants (power set at 80%).
Prior to the final obturation, there were eight teeth (seven in group 1, one in group 2) with a positive rootcanal culture (Table 6). Six positive root canals contained <102 CFU mL, one canal contained 2 x103 and one canal harboured 8 _104 CFU mL. The latter case scored a treatment outcome B. Of the 30 cases that were filled with a negative culture prior to obturation, 22 (74%) healed (11 from group 1 and 11 from group 2) whereas this was the case for seven out of eight cases (six from group 1 and one from group 2) with a positive root-canal culture prior to obturation (87.5%). The chi squared analysis showed no significant difference in healing whether cultivable bacteria were present or not prior to filling. Power statistics showed that the presented differences (74% vs. 87%) between negative and positive cultures at the time of obturation can be significant if one experimental group would comprise 160 patients (power set at 80%, P < 0.05).

Table 4. Treatment outcome scores related to one-and two-visit endodontic treatment.


Score A, B, C see Table 1. The values in parentheses are in per cent.

Table 5. Probability of success (95% confidence interval).



Figure 1. Estimation of the fraction of teeth [p(t)] that are successful following one-o r two-visit root-canal treatment with in 4 years.



Table 6. Positive and negative root-canal cultures related to treatment outcome.

Discussion.
The presence of bacteria in the root canal results in the development of apical periodontitis. In the present study, no correlation was seen between the healing of endodontic lesions and the presence orabsence of a positive canal culture after proper cleaning and shaping, and no correlation was seen between healing of endodontic lesions and treatment in one or two visits with intracanal dressing of calcium hydroxide. It is widely accepted that inclusion of calcium hydroxide renders the canal sterile and ready for obturation (Tronstad 1991, Friedman 1998).However, studies have shown that calciumhydroxide is not always effective and that its action is unreliable (Reit & Dahle. n 1988, Orstavik et al. 1991,Yared & Bou Dagher 1994, Peters et al. 2002). Comparisons of the success rate between the two-visit endodonticswith calcium hydroxide as an intracanal interappointment dressing and the one-visit endodontic treatment of teeth with necrotic pulps in this and other prospective studies do not show significant differences (Friedman et al. 1995,Trope et al.1999,Weiger et al. 2000). Other studies evaluating the healing of teeth with necrotic pulps after either one or two-visits report success rates between 75 and 90% for both treatment options (Bystrom et al. 1987, Murphy et al. 1991, Jurcak et al. 1993, Caliskan & Sen1996).
It seems more reliable to evaluate infection at the time of root filling. Studies by Zeldow & Ingle (1963) and Engstrom et al. (1964) showed inferior results for teeth with a positive culture at the time of root filling. On the other hand, Seltzer et al. (1963) and Matsumoto et al. (1987) could not ￠find any significant differences in healing results. All these studies are limited in bacteriological technique, because no strict anaerobic techniques were used and contamination cannot be ruled out in some reports owing to a lack of information about the treatment procedures. Sjogren et al. (1997) using strict anaerobic techniques, related infection at the time of obturation and success. They showed inferior healing results for root canals with a positive culture at the time of obturation. Our results do not indicate such differences. It is stressed that, in both studies, the number of CFU left in the canal at the time of obturation was very low. In our study, six out of the eight positive root canals contained <102 CFU mL_1, one canal contained 2 x103 and one canal harboured 8 _104 CFU mL_1. The latter case scored a treatment outcome B. In the study by Sjogren et al. (1997), 22 (40%) root canals contained bacteria at the time of obturation. In eight cases, bacteria could only be detected after the enrichment growth in fluid media, nine samples contained 102_103 CFU, three samples contained >104 CFU, and in two samples, no data were available. Of these 22 positive samples 7 failed. The authors did not relate the failed cases to the number of CFU found. If the failures are related to higher number of CFU (>103) in the canal, it could provide an explanation for the reason why cases with a positive culture appeared successful in both studies, 15/22 (Sjogren et al. 1997) and 7/8 in our study.
Comments have been made previously about the uncertainty of the bacteriological sampling procedure immediately after the removal of a calcium hydroxide dressing (Reit&Dahle. n1988). It has been suggested (Reit et al. 1999) that the microbiological samples should be taken after filling the canal with a sampling fluid (after removal of the calcium hydroxide) for 7 days. However, when the authors applied this procedure, culture reversals were seen in both directions. Thus, Reit et al. (1999) reported seven canals that turned from a negative to a positive culture after 1week, but also the seven canals that changed from a positive culture to a negative culture over the same period. It cannot be ruled out completely that some negative canals in the present study after calcium hydroxide removal (S3), may have become positive if evaluated 1 week later. The studies of Reit & Dahlen (1988) and Reit et al. (1999) demonstrated the limitations of microbiological root-canal sampling, and this should be taken into account when evaluating all root-canal procedures. Because it was found that removal of calcium hydroxide from the root canal with the aid of the operating microscope was enhanced, and because it has been shown previously that a second culture taken 7 days later did not result in more reliable data (Reit & Dahlen 1988, Molander et al. 1990, Reit et al. 1999), cultures were taken immediately after removal of the calcium hydroxide. This process was also less demanding for the patients, as it reduced the number of appointments.
Differences in healing can also be created because the size of the preoperative radiolucency, between canals with detectable and non-detectable microorganisms, is different in both groups. In the present study, these differences were not found.
Sjogren et al. (1997) found 60% negative root-canal samples after similar preparation of the root canals. The difference with our findings (79% negative) could be a result of the concentrations of sodium hypochlorite (0.5% vs. 2%) and different delivery systems used for irrigation. In addition, the sampling techniques and transport media used may have also created differences.
A question that remains to be answered is the reason for failure in cases of root canals from which no bacteria could be cultured. In Sjogren’s study (1997), two of those cases failed. In one of these, bacteria were found at the time of surgery in a lateral canal. This points to the in ability of current cleaning and shaping techniques to reach microorganisms present in lateral canals and dentinal tubules (Peters et al. 2001). These bacteria cannot be identified using current endodontic sampling procedures and seem inaccessible to an intracanal disinfectant like calcium hydroxide (Siqueira & Lopes1999).
The number of patients available in our study as well as most other prospective studies is limited. Given the high-success rates of both treatment options, the sample size per group required to detect a difference is very high. Weiger et al. (2000) showed that the probability for complete periapical repair over 5 years was 93%for two-visit root-canal treatment and 92% for one-visit root-canal treatment. When we calculate power statistics (power set at 80%) with these proportions, the numbers per group would have to exceed 10 000. Trope et al. (1999) calculated the need for a group size of 354 (power set at 80%) to show significance for the differences he found (74 vs. 64%) in comparing one- versus two-visit rootcanal treatment. However, the short observation period of 52 weeks in that study may lead to an underestimation of periapical healing over longer observation periods, as was shown by Weiger et al. (1998). In the present study, the probability for healing increased gradually to 91 and 94% over a period of 4 years, indicating the need for a group size of 1275 teeth to show significance at P ј 0.05. However, the cases that were selected in these studies, all present a relatively simple anatomy resulting in high success rates. The numbers that are needed in a prospective study with more complex cases maybe much lower because of lower success rates.

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