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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
Results - Discussion - References.



Results.
Fluorescent-labeled bacteria were found in all ‘rd’ samples During the period of infection, PVB sus ranged from 55 to 75% ( S. sanguinis ) and from 35 to 60% ( E. faecalis ), respectively. Likewise, viable as well as dead microorganisms were identified in root dentine at any time. In the majority of the cases, PVB rd values were lower than PVB sus values, regardless of the bacterial strain and the point of time.
In the control group, mean values for PVB rd did not markedly differ at week 4 and at week 8 for S. sanguinis and E. faecalis (Table 1). The individual PVB rd values varied between 13 and 61%. The differences PVB rd and CFU rd are specified in Figures 2 and 3. Mean values for CFU rd ranged between log 6.5 and 8.6 (Table 1).
In the test group, PVB rd decreased from 36% to 27% (mean values) after placement of calcium hydroxide in specimens infected with S. sanguinis (Table 1). The individual PVB rd values ranged from 13 to 52%. The differences PVB rd substantiated the decrease of PVB rd in most samples, indicating that viable S. sanguinis cells were still present in root dentine after 12 weeks, although in culture no growth was detectable in any sample (Figs 2, 3). By contrast, the majority of the samples infected with E. faecalis demonstrated somewhat higher PVB rd values after calcium hydroxide treatment than initially after 4 weeks (Fig. 2). CFU rd varied to a certain extent when comparing week 12 with week 4 (Fig. 3).

Discussion.
In the present study, extracted human teeth were selected as the test specimens, although bovine teeth were used in the majority of in vitro investigations related to infected root dentine (Haapasalo & Ørstavik 1987, Ørstavik & Haapasalo 1990, Vahdati et al . 1993, Perez et al . 1996, Siqueira & De Uzeda 1996, Heling & Chandler 1998, Komorowski et al . 2000). Bovine root dentine, however, differs from human dentine; dentinal tubules of bovine teeth have wider diameters (Perez et al . 1993) and in some cases giant tubules are present. This may affect the microenvironment of bacteria (e.g. diffusion of nutrients or medicaments into dentinal tubules) and their vitality status. Similarly, the presence of intact root cementum can limit bacterial ingrowth from the pulpal side in deeper layers of root dentine, due to limited availability of the nutrient source (Haapasalo & Ørstavik 1987, Adriaens et al . 1988). This indicates that microorganisms in dentinal tubules may be different in a nutrient-rich or nutrient-deficient environment. Therefore, infected human root specimens seem to be more suitable for investigating the microbial vitality status than infected bovine root dentine.
Under stressful conditions (e.g. starvation), bacterial populations may retain certain physiological functions although they do not grow under conventional culture conditions. These viable but non-culturable cells (VBNC) may survive and recover or go in a prelytic or lytic state. Undoubtedly, the existence of VBNC can only be suggested. An approach to substantiate the VBNC theory is to combine different viability tests examining, for example, respiration, enzymatic activitiy, membrane potential or membrane permeability. In the present study bacterial viability was characterized by conventional culturing and by applying the two stains, Syto 9 and propidium iodide. The latter provides information on the cytoplasmic membrane permeability that is a key element of cell viability. The two stains differ in their spectral characteristics and their ability to penetrate bacterial cell membranes. Viable bacteria with intact bacterial cell membranes stain fluorescent green. However, information is scarce as to whether or not these labelled organisms are able to divide under the environmental conditions prevailing in the root canal system. In growth cultures the majority of S. sanguinis cells that stained fluorescent green are viable, i.e. they form colonies (Decker & Weiger 1998). In contrast, microorganisms with damaged cell membranes accumulate propidium iodide in the cell body and stain fluorescent red; they are scored dead. Experimental results with S. sanguinis as the test organism showed that these cells were not capable of dividing after conventional culturing (Bogosian et al . 1998, Decker & Weiger 1998). A recent study demonstrated bacterial cells identified by propidium iodide to be permeabilized and irreversibly damaged (Nebe-von Caron et al . 1998).
Streptococcus sanguinis and Enterococcus faecalis were chosen as target microorganisms. Both are frequently encountered in infected root canal systems, and particularly Enterococcus faecalis is a species commonly recovered in teeth with failed root canal treatment (Siren et al . 1997, Sundqvist et al . 1998, Molander et al . 1998). In the present study smear layer was removed after root canal preparation of the specimens to facilitate bacterial migration into root dentine from the pulpal side as previously shown for S. sanguinis (Perez et al . 1996). Our experiments demonstrated that an infection time of 4 weeks allowed bacteria to penetrate human root dentine up to a depth of at least 150 m. The removal of infected circumpulpal root dentine with small files (size 25) resulted in very fine dentine shavings. These facilitated the microscopic analysis of the stained samples, namely the detection of green fluorescent bacteria, due to the reduced autofluorescence of the dentine dust, allowing the accurate determination of PVB rd . It appeared unlikely that the ‘rd’ samples still contained bacteria attached to the root canal walls, because the rinsing procedure prior to ‘rd’ sampling occurred under favourable conditions (straight and short root canal prepared to size 60 [at week 4] or 90 [at week 8] with a wide apical opening). Similarly, calcium hydroxide was thoroughly removed from the root canals at week 12. Preliminary experiments demonstrated that calcium hydroxide could neither be detected macroscopically nor microscopically on root canal walls. Furthermore, it might be speculated whether or not bacteria were pushed deep into dentine during preparation of the root canal walls with Hedström files. It is possible that some bacteria were pushed into the superficial dentine layers and were not included in the analysis.
Viable bacterial cells multiplying under the selected laboratory conditions, as well as dead microorganisms, were identified in all root dentine samples regardless of the bacterial strain used. However, whether all viable and green fluorescent microorganisms were capable of proliferating remains unanswered. Nevertheless, the portion of viable bacteria found in root dentine was reduced compared to the one present in the bacterial suspension in the root canal lumen. Most probably, some of the bacteria attached to the inner surfaces of dentinal tubules could not resist the specific conditions prevailing in root dentine, whilst they would have survived as planktonic cells in the surrounding bacterial suspension.
As expected, the degree of tubule infection varied from sample to sample. For that reason, the baseline dentine samples collected after 4 weeks served as individual controls for the samples taken after 8 weeks (control group) and 12 weeks (test group), respectively. When analysing each root specimen of the control group, the parameters PVB rd and CFU rd substantiated that the degree of infection established within the first 4 weeks largely corresponded to that re-established in the following 4 weeks after ‘rd’ sampling (weeks 5–8). Therefore, it was justified to take the ‘rd’ samples at week 4 as basis for those collected at week 12 in the test group. In these cases, no infected circumpulpal dentine was removed at week 8, as calcium hydroxide should demonstrate its efficacy toward infected root canal dentine.
With S. sanguinis , no growth on blood agar plates was observed after exposure of root dentine to calcium hydroxide for 4 weeks. However, in all these ‘negative’ ‘rd’ samples green fluorescent and viable bacterial cells were detected. Obviously, these were not culturable under the selected laboratory conditions and have not been detected in other comparable studies so far. These S. sanguinis cells may play a role in treatment failure when nutrients are available. With E. faecalis , PVB rd tended to increase, indicating the presence of a large number of viable E. faecalis cells in the root dentine despite calcium hydroxide treatment. These results confirmed various investigations showing that E. faecalis resists calcium hydroxide treatment (Haapasalo & Ørstavik 1987, Ørstavik & Haapasalo 1990, Siqueira & De Uzeda 1996).

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