Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubule

By: Orstavik D 1990 Endod Dent Traumatol

In vitro

Aim of the study:

The effect of endodontic irrigants and dressings on bacteria in bovine dentin specimens
experimentally infected with Enterococcusfaecalis, Streptococcus sanguis, Escherichia coli, or
Pseudomonas aerugi

Material and methods

Dentin test specimens

The roots of freshly extracted bovine incisors were cut and machined to cylindrical test pieces

The root cementum was intentionally removed during this procedure. Organic and inorganic
debris in- cluding the smear layer were removed by ultrasonic treatment for 4 min each in 17%
EDTA and 5.25% NaOCl, and the test pieces were steril- ized by autoclaving (121 °C, 15 min)
prior to infection with the test organism.

Bacteria

and
Enterococcus faecalis, Streptococcus sanguis, and clinical isolates f Escherichia coli
Pseudomonas aeruginosa were used

Infection of dentin specimens

Sterilized dentin pieces were incubated in 2 ml yeast extract glucose broth inoculated with the
test organism

Incubation periods ranged from 3 d to 6 weeks with fresh medium supplied every second day

The purity of the infecting organism was checked regularly.

On termination of the incubation for infection, the specimens were blotted dry and coated on
their outer, periodontal side with nail varnish.

They used ESM for selected samples and for the other specimens they used light microscope
following Brown and Brenn staining
Bacteriological testing of tubule infection

The dentin was ground off from the pulpal side of infected specimens with round burs

the dentin dust was collected from each bur and used to inoculate 2 ml of brain heart infusion
(BHI) broth.

The dentin shell with intact varnish coating, which remained after the burring sequence, was
separately inoculated into 2 ml BHI broth

** both tubes were incubated at 37°C for 7 d and inspected daily for growth

When growth occurred, the bacteria were sub cultured on blood agar and mitis-salivarius-agar
(S. sanguis) plates and checked for purity and identity with the infecting organism

Test for survival of infecting organism

infected specimens were washed and incubated in distilled, autoclaved water for periods up to
7 days. Bacteriological testing of tubule infection was per- formed on duplicate specimens ater
5 min, 20 min, 1 h, 4 h, 1 d and 7 d of incubation in distilled water

Speed of bacterial penetration

the tube of BHI broth with dentin dust from the largest bur dimension showing growth, was
recorded.

The depth of infection was also controlled by SEM analysis

Medicaments tested

 Calasept  a saturated solution/suspension of calcium hydroxide in a balanced salt

solution.
 Camphorated paramonochlorophenol (CMCP)  60% camphor, 30% p-
monochlorophenol, 10% ethanol.
 Hibitane  0.2% chlorhexidine gluconate in 7% ethanol in water
 Iodine potassium iodide  a 2% solution of iodine in 4% potassium iodide
 Sodium hypochlorite  5.25% solution in water.
 Ethylenediaminetetraacetic acid  17% solution of its tetrasodium salt

Procedure for testing with medicaments

Strict asepsis was maintained during handling of the infected dentin specimens.

They were blotted dry, coated with varnish, and then mounted with sticky wax to the bottom of
tissue culture dishes

The medicament to be tested was applied with a microsyringe to fill the pulpal lumen and cover
the top surface of the specimen

Since CMCP act as a vapor, it was tested by applying some 100 micro liter onto the culture dish
floor outside the specimen and sealing the lid of the well with wax.

All specimens were incubated at 37°C for periods from 5 min up to 7 days.

Tubes of BHI not showing growth after the 7-d incubation period were reinoculated with 10
micro liter of a 24-h broth culture of the originally infecting organism, to make sure that the
media in the tubes had not been rendered inhibitory via the possible transfer of medicaments
by the dentin dust

The effect of medicaments was monitored both by:

1. observing the gradual decrease in bacterial viability in the pulpal part of the tubules,
2. by registering the time necessary to obtain disinfection of the whole length of the
tubules

Smear layer production

The effect of the presence of a smear layer on medicament efficacy was tested in some
specimens

These were ground on their pulpal side after infection but prior to incubation with medicament

The effect of the smear layer was tested with S. sanguis- and E. faecalis infected tubules in
conjunction with CMCP in liquid and gaseous form and on S. sanguis- infected tubules and
Calasept.

Results

Speed and depth of infection

E. faecalis permeated the entire width of circum-pulpal dentin after only 2 d of incubation.
S. sanguis required up to 14 d to cover the whole length of the tubules

E. coli penetrated no deeper than 600 jim even after 14 d of incubation for infection.

infection could also be seen from the periodontal side, this occurred far less than from the
pulpal side

Survival of infecting organisms

E. faecalis persisted and remained viable throughout the length of the tubules for more than
7d.

S. sanguis survived post-infection incubation periods for more than 4 h.

P.aeruginosa regularly survived for at least 4 h, but viable cells were present in one block only
and over a very small tubular distance at 24 h

E. coli apparently succumbed quickly, with bacteria persisting over short distances only at 4 h,
and none at all after 1 d.

Effects of medicaments

Calasept.-->

E. faecalis  More that 10 d of incubation was necessary to achieve disinfection of dentinal

tubules infected with E. faecalis

S. sanguis  more rapid but somewhat variable 2-24 hrs

P.aeruginos  was eliminated after 20 min incubation

E. coli  were no longer recoverable from the tubules after 1 h of incubation

CMCP :

Liquid form: it killed S. sanguis in the tubules within 5 min.

60 min necessary to eliminate E. faecali

vapour form

it took longer to eliminate E. faecalis and S. sanguis

E. coli was killed within 20 min, whereas P. aeruginosa withstood disinfection up to 4 h

Irrigant

IKI effectively killed S. sanguis in the tubules after only 5 min incubation

NaOCl and Hibitane effectively reduced S. sanguis for 100 to 300 micron into the tubules.

No disinfecting action was observed with EDTA

Effect of a smear layer on medicament efficacy

Liquid CMCP was clearly impeded in its effect by the presence of a smear layer,

whereas gaseous CMCP and Calasept (with S. sanguis) performed with similar efficacy on
patent and occluded dentinal tubules

Discussion

The presence of a smear layer delayed, but did not eliminate, the effect of the medicaments.

disinfection logically follows, rather than precedes, chemo-mechanical cleansing and shaping of
the pulp canal.

Disinfection of the prepared root canal surface and- of the adjoining tubules thus becomes one
of the primary functions of antiseptics in endodontics, whether they are used as dressings or as
irrigating solutions.

Why these speciemens:

E. faecalis was chosen for several reasons: it occurs in some endodontic infections; it has been
extensively used in experimental work on endodontics including antiseptic susceptibility, and it
is a non fastidious and easy-to-grow bacterium

S. sanguis is less active than E. faecalis in infecting the tubules in their model.

E. faecalis to be inefficient in penetrating etched dentin slices compared with P. aeruginosa and
E. coli.

E.faecalis might survive in the dentinal tubules in the ap- parent absence of nutrients for
periods exceeding 7 d
other micro-organisms were quickly killed when withdrawn from the incubating medium.

The clinical relevance of this finding may be question- able, as in vivo conditions may permit
nutrient supply to bacteria in dentinal tubules.