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 »  Home  »  Endodontic Articles 1  »  Effects of instrumentation, irrigation and dressing with calcium hydroxide on infection in pulpless teeth with periapical bone lesions
Effects of instrumentation, irrigation and dressing with calcium hydroxide on infection in pulpless teeth with periapical bone lesions
Introduction - Materials and methods.

Bacteria in the root canal system initiate and maintain periapical inflammatory lesions (Kakehashi et al . 1965). The bacterial microflora in root canal infections of untreated teeth is dominated by anaerobic bacteria, and several different species are commonly found (Bergenholtz 1974, Byström & Sundqvist 1981, Sundqvist et al . 1989, Brauner & Conrads 1995, Le Goff et al . 1997). Root canal treatment aims to eliminate bacteria from the infected root canal and prevent reinfection. Cleaning, shaping and irrigating the canal greatly reduces the number of bacteria (Byström & Sundqvist 1981).
However, it has been shown that it is impossible to obtain complete disinfection in all cases, even after thorough cleaning, shaping and irrigation with disinfectants or antiseptics (Byström & Sundqvist 1981, Byström et al . 1985, Ørstavik et al . 1991). Therefore, concern exists as to the fate and subsequent activity of the remaining microorganisms in the canal. It has been shown that, if the canal is not filled or dressed with a disinfectant between two visits, they may multiply rapidly within days, to near the original numbers (Byström & Sundqvist 1981).
It is generally believed that the number of remaining bacteria can be controlled by enclosing an interappointment dressing such as calcium hydroxide within the prepared canal (Byström et al . 1985, Chong & Pitt Ford 1992). Some authors therefore consider a two-visit root canal treatment with an interappointment disinfectant dressing mandatory in infected cases (Tronstad 1991). Another approach has been to entomb the remaining microorganisms, depriving them of nutrition and leaving no space to multiply, by the direct and complete filling of the prepared and disinfected canal space in one visit (Soltanoff 1978, Oliet 1983).
Residual bacteria in the apical part of the root canal have been held responsible for failures, even when no bacteria could be detected after the use of an interappointment dressing prior to filling (Sjögren et al . 1990, Nair et al . 1990). In the present study the number and identities of bacteria in root canals during root canal treatment and the influence of calcium hydroxide on regrowth of bacteria in the period between the first and second visits was investigated.

Materials and methods.

Patient selection.
Forty-three systemically healthy patients, 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 selected teeth (15 incisors, six canines, eight single-root canal premolars and 13 single-root canal distal roots of mandibular molars) were asymptomatic, did not respond to sensitivity testing, had not received previous endodontic treatment and showed radiographic evidence of periapical bone loss.
The mean age of the participants was 40 years (range 16–86 years). There were 19 females and 24 males. The teeth were randomly divided into two treatment groups. The size of the periapical lesions was determined from the preoperative radiograph by measuring the largest diameter in millimetres.

Microbiological sampling.
After cleaning the tooth with pumice and isolation with rubber dam, the crown and 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 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 in the root canal by a syringe with a 27-gauge needle. Care was taken not to overfill the canal. The canal was enlarged to a number 20 Hedström 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) and then placed in sterile tubes containing 1 mL RTF and transferred to the laboratory within 15 min for microbiological processing.

Endodontic procedure (Table 1).
The working length (1 mm from the radiographic apex) was checked with a radiograph after inserting a size 15 K-file 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, Ballaigues, Switzerland) with the modified double flare technique (Saunders & Saunders 1992), to a master apical file of at least size 35. Each file was followed by irrigation of the canal with 2 mL sodium hypochlorite (2%) in a syringe with a 27-gauge needle. Concentrations of hypochlorite were measured by the titration technique using 0.1 mol L –1 Na 2 S 2 O 3 and soluble starch as indicator (Moorer & Wesselink 1982). After preparation, the canal was irrigated with a rinse of 5 mL sodium hypochlorite (2%). Then, inactivation of the sodium hypochlorite was accomplished with a rinse of 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.

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Table 1. Endodontic procedure and time of sampling.

After drying the canal with paper points, the teeth in group 1 ( n 22) were obturated using the warm lateral compaction technique with gutta-percha and AH-26 sealer (Dentsply, Konstanz, Germany). After the first visit all these teeth were restored.
After drying the canal, the teeth in group 2 ( n 21) were dressed with a thick mix of calcium hydroxide (Merck, Darmstadt, Germany) in sterile saline. The calcium hydroxide slurry was plugged in the canal with the blunt end of a sterile paper point. If the canal could not be dried, the tooth was excluded from the study. The access cavities in group 2 were filled with two layers of Cavit (ESPE, Seefeld, Germany) and a glass ionomer restoration (Fuji- II, GC Corporation, Tokyo, Japan). 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. A radiograph was taken to ensure proper placement of the calcium hydroxide in the canal.
After 4 weeks the patients in group 2 returned. The canal was aseptically accessed under rubber dam isolation and the calcium hydroxide was 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 16 (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%) and gently 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 with gutta-percha and AH-26 sealer using the warm-lateral compaction technique. A final radiograph was taken using the paralleling technique with the aid of a beam guiding device (RINN, Rinn Corporation, Elgin, IL, USA). After obturation of the canal, the tooth was restored.

Microbiological procedures.
Tenfold serial dilutions of the samples were prepared and 100 L of each dilution was inoculated on blood agar plates supplemented with 5% horse blood, 5 mg L –1 haemin and 1 mg L –1 menadione. Plates were incubated anaerobically (80% N 2 , 10% H 2 , 10% CO 2 ) at 37 C for 7 days. After incubation, the total colony forming units (CFU) and the different colony types were counted with the use of a stereomicroscope at 16 magnification (Zeiss, Oberkochen, Germany).
All colony types were streaked to purity and incubated aerobically in air with 5% CO 2 (BBL Gaspak CO 2 systems, Becton Dickinson & Co., Cockeysville, MD, USA) as well as anaerobically to determine strict anaerobic and facultative anaerobic growth. Identification was made on the basis of Gram stain, catalase activity and a commercially available identification kit, ATB rapid ID32A (Biomerieux SA, Lyon, France), for strict anaerobes and ATB rapid ID32Strep for aerobic cocci (Biomerieux SA).
In order to allow slow-growing species to develop, the blood agar plates with the total samples were kept under anaerobic conditions from day 7 to day 14. Newly emerging colonies were also streaked to purity and identified.

Statistical comparisons were made between groups 1 and 2 for age distribution and size of the periapical lesion using a t -test for independent samples. CFU counts, number of strains, number of anaerobes, number of facultatives, percentage of gram-positive rods and cocci and the percentage of gram-negative rods and cocci between group 1 and 2 at the start of the experiment were compared using the Mann–Whitney test for nonparametric data.
Differences between samples 1–4 were compared using the Kruskal–Wallis test for non-parametric data (CFU counts, percentages of gram-positive and gram-negative rods and cocci) or with the anova -test for parametric variables (number of strains, anaerobic and facultative microorganisms). When significant differences were found in the Kruskal–Wallis test, Mann–Whitney tests were performed to demonstrate where the differences were located. When the anova -test showed differences a Scheffé posthoc test was used for the same purpose.
With a positive or negative bacterial sample 2 a t -test or Mann–Whitney test, respectively ( * ) was performed for differences between patients related to gender, age, microbiological differences in CFU count * , number of species, number of anaerobes, number of facultatives, percentages of cocci * , of rods * , and the clinical parameters tooth type, size of radiolucency, preparation length and master apical file size.
P -values 0.05 were considered statistically significant.