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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 2  »  Vitality status of microorganisms in infected human root dentine
Vitality status of microorganisms in infected human root dentine
Introduction - Materials and methods.



Introduction.
Conventional therapy of endodontically induced periapical lesions aims at eliminating the infection as completely as possible. However, there are indications that despite adequate instrumentation of the root canal, bacteria can be left in the root canal system in some cases (Chong & Pitt Ford 1992, Oguntebi 1994, Peters et al . 1995). These microorganisms and/or their by-products may interfere with the periapical healing process and can be a major cause for failures.
For this reason, experimental studies have been initiated to explore the potential of different bacteria to migrate into root canal dentine and to test the antibacterial efficacy of various intracanal medicaments (Akpata & Blechman 1982, Haapasalo & Ørstavik 1987, Ørstavik & Haapasalo 1990, Safavi et al . 1990, Buck et al . 1999, Komorowski et al . 2000, Siqueira et al . 2000). Haapasalo & Ørstavik (1987) were the first to introduce a model for infection and disinfection of root dentine using artificially infected bovine root segments. Circumpulpal root dentine was sampled and conventional microbiological culture techniques were utilized to identify bacteria. The plate count method applied in most studies gives information on the number of microorganisms able to divide at a sufficient rate to form colonies under selected laboratory conditions. However, several reports described bacteria that decreased their culturability under starvation conditions and maintained metabolic activity (Mason et al . 1986, Kaprelyants et al . 1993, Oliver 1995). Also, bacterial cell wall remnants from dead bacteria still present in dentinal tubules may interfere with periapical healing from an immunological point of view. In comparison to the classical cultivation of microorganisms, rapid direct methods detecting different physiological states of bacteria become increasingly important.
Recently, a sensitive assay for monitoring bacterial viability was introduced. It contains the nucleic acid dye Syto 9, capable of penetrating bacterial cells with intact cell walls and the counterstain propidium iodide that only labels dead bacterial cells. This two-colour fluorescent assay has already been applied in microbiological experiments with promising results (Bogosian et al . 1998, Decker & Weiger 1998, Weiger et al . 1999).
The aim of this in vitro study was to characterize the vitality status of bacteria that penetrated dentinal tubules of human root dentine by fluorescence labelling in order to differentiate between viable and dead microorganisms.

Materials and methods.

Root specimens.
Twenty-four straight roots from teeth extracted for orthodontic or periodontal reasons were selected. The teeth were either not carious or had only small carious lesions and were stored in hydrogen peroxide (3%) for a maximum of 2 months. After cleaning the root surface with curettes, the roots were removed 2–3 mm below the cemento-enamel junction. A root segment with a length of about 7 mm was prepared by sectioning the root tip. Each root canal was enlarged to size 60 with Hedstroem files under irrigation with physiological saline. The smear layer was removed with 37% phosphoric acid by leaving it in the root canal for 30 s. The specimens were sterilized in the autoclave for 20 min at 121 C. Under sterile conditions the outer root surface was coated with nail varnish and the specimens placed in a sterile vial for 1 day. Sterility was then checked by incubating each specimen in 5 mL sterile Schaedler broth at 37 C for 24 h.
As target microorganisms, Streptococcus sanguinis (Nr. 20068, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) and Enterococcus faecalis (ATCC 29212) were selected. The root specimens were transferred into 5 mL Schaedler broth (BBL, Becton Dickinson Systems, USA) inoculated with 200 L of a 24-hour-old bacterial suspension containing either S. sanguinis (12 root specimens) or E. faecalis (12 root specimens). Under strict asepsis, the bacterial suspension was changed every second day for a period of 8 weeks. The specimens were incubated at 37 C. The purity of the infection was checked on days 28 and 56.

Study design.
The study design is outlined in Figure 1. The root dentine specimens were randomly split into the control group and the test group. Bacterial samples from root dentine (rd) were taken on days 28 (week 4), 56 (week 8, control group) and 84 (week 12, test group). On days 28 and 56, 1 mL from the environmental bacterial suspension (sus) harbouring the root specimens was taken for microbiological analysis.
At week 4, all 24 root canals were irrigated with 1 mL physiological saline and cleaned using sterile paper points to remove microorganisms attached to the root canal walls. Subsequently, the root canals were prepared from size 60 to 90. For this purpose, Hedstroem files size 25 were used to obtain fine dentine chips, which were collected in a tube filled with 1 mL Schaedler broth. The Hedstroem files size 25 were agitated thoroughly in this solution several times. Finally, two paper points were used to take up remaining dentine shavings on the root canal walls and then added to the Schaedler broth. The smear layer was again removed with 37% phosphoric acid to allow bacterial penetration into dentinal tubules.

Study design, root dentine, bacterial suspension
Figures 1. Study design (rd, root dentine; sus, bacterial suspension).

At week 8, the collection of ‘rd’ samples was limited to the 12 root specimens of the control group (Fig. 1). For this purpose, the root canals were enlarged to size 120. The test group consisted of the other 12 root specimens. Their root canals were irrigated with 1 mL of physiological saline and cleaned using sterile paper points. Afterwards, pure calcium hydroxide powder was mixed with distilled water in a powder-liquid ratio of 1 g mL –1 (pH = 13) and then condensed into the root canal with paper points. The cervical and apical openings of the filled root canals were closed with Cavit (ESPE, Seefeld/Oberbayern, Germany) and the root specimens stored in a wet gauze at 37 C for an additional 28 days.
At week 12, the calcium hydroxide was removed by rinsing the root canals twice with 5 mL of distilled water. After the first rinse the root canal walls were cleaned with sterile paper points. Finally, the root canals were enlarged to size 120 to provide ‘rd’ samples in the same way as described above.

Microbiological analysis.
The ‘rd’ and ‘sus’ samples were processed in the laboratory immediately. The portions of viable bacteria (PVB rd , PVB sus ) and the number of colony-forming units (CFU rd , CFU sus ) were determined as follows:

PVB rd and PVB sus

The samples were centrifuged for 5 min at 8240 g. After washing with sterile saline, a second centrifugation step followed. The pellet was then resuspended in 300 L staining solution containing two fluorescent stains, Syto 9 and propidium iodide (Live/Dead BacLight Bacterial Viability Kit, MoBiTec, Göttingen, Germany). These two substances allowed the differentiation between viable and dead microorganisms. After 15 min, the samples were again centrifuged and the stained pellet was analysed by a fluorescence photomicroscope (Zeiss, Jena, Germany) with a suitable filter combination (FITC: 450– 490 nm; Rhodamin: 540 nm) allowing to count the number of green (= viable) and red (= dead) bacterial cells in 10 visual fields at a magnification of 640 . PVB represents the number of viable microorganisms related to the total number of stained microorganisms (= sum of viable and dead bacteria). PVB was given as a percentage.

CFU rd and CFU sus

The original samples were diluted up to a concentration of 10 –5 for S. sanguinis and 10 –6 for E. faecalis . Portions of 20 L were inoculated onto Schaedler agar supplemented with sheep blood and Vitamin K 1 . Following anaerobic incubation (Anaerocult A, Merck, Darmstadt, Germany) for 48 h at 37 C, visible colonies were counted as colony-forming units (CFU) and related to 1 mL of the original sample.

Data analysis.
The CFU values were log transformed. The mean values of PVB rd and CFU rd with the corresponding 95% confidence intervals were calculated. As the ‘rd’ samples were of particular interest, the following differences PVB rd and CFU rd were given for each sample:

  • Control group: PVB rd (week 8) – PVB rd (week 4) log CFU rd (week 8)
    – log CFU rd (week 4), if growth was present.
  • Test group: PVB rd (week 12) – PVB rd (week 4). log CFU rd (week 12)
    – log CFU rd (week 4), if growth was present.

Positive values indicate that PVB rd or CFU rd increased within the given period of time, whilst for negative values PVB rd or CFU rd decreased.

Mean values with the corresponding 95% confidence intervals of the portion of viable bacteria
Table 1. Mean values with the corresponding 95% confidence intervals of the portion of viable bacteria (PVBrd) and the number of colony-forming units (CFUrd) in root dentine (rd) collected after 4, 8 and 12 weeks.

Comparison between baseline PVBrd values and PVBrd values recorded at week 8
Table 2. Comparison between baseline PVBrd values (week 4) and PVBrd values recorded at week 8 (control group) and at week 12 (test group). The individual differences  PVBrd = PVBrd (ti) - PVBrd (tbaseline) are depicted (d: mean value). Positive values indicate that PVBrd increased within the given period of time, whilst for negative values PVBrd decreased.

Positive values indicate that CFUrd increased within the given period of time, whilst for negative values CFUrd decreased
Table 3. Comparison between baseline CFUrd values (week 4) and CFUrd values recorded at week 8 (control group) and at week 12 (test group). The individual differences  log CFUrd = log CFUrd (ti) - log CFUrd (tbaseline) are depicted (d: mean value). Positive values indicate that CFUrd increased within the given period of time, whilst for negative values CFUrd decreased.