Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules.
It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability.
The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation.
The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively.
An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules.
Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells.
The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.
| Identifer | oai:union.ndltd.org:ADTP/217792 |
| Date | January 2007 |
| Creators | Parmar, Dikesh, n/a |
| Publisher | University of Otago. School of Dentistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Dikesh Parmar |
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