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JOURNAL OF ENDODONTICS
Copyright © 1996 by The American Association of Endodontists
A Scanning Electron Microscopic Evaluation of In
Vitro Dentinal Tubules Penetration by Selected
Anaerobic Bacteria
Jose F. Siqueira, Jr., CD, MSc, Milton De Uzeda, CD, MSc, DSc, and
Maria Evangelina F. Fonseca, MSc, DSc
In vitro root canal dentinal tubule invasion by se-
lected anaerobic bacteria commonly isolated from
endodontic infections was evaluated. Dentinal cyl-
inders obtained from bovine incisors were inocu-
lated with bacteria, and microbial penetration into
tubules was demonstrated by scanning electron
microscopy.
The results indicated that all bacterial strains
tested were able to penetrate into dentinal tubules,
but to different extents.
Root canal infections are multibacterial, mixed infections, domi-
nated by anaerobic bacteria (1). Anaerobes seem to play an im-
portant role in development and perpetuation of endodontic patho-
sis. Tani-Ishii et al. (2) have observed that, during the period of
rapid periapical lesion expansion, the root canal microbiota be-
comes increasingly anaerobic. Fukushima et al. (3) have reported
that cases of unsuccessful outcome of endodontic therapy were
related to persistent anaerobic infections.
A pulpal infectious process of long duration allows bacteria to
propagate to the entire root canal system, including ramifications,
isthmuses, apical deltas, and dentinal tubules. According to several
authors (4-6), persistent infection may be caused by microorgan-
isms that have invaded dentinal tubules before or during endodon-
tic treatment. In these locations, they are often protected from the
action of endodontic instruments and irrigation solutions.
Studies conducted in vitro have attempted to show dentinal
tubule penetration by a diverse spectrum of microorganisms ( 6 -
10). Nevertheless, obligate anaerobic bacterial species that are
frequently associated with endodontic diseases were not used in
these studies. Studies that used anaerobes failed to show intratu-
bular penetration. Akpata and Blechman (11) investigated the
human dentinal tubules invasion by two obligate anaerobes and
two facultative anaerobic bacteria. Their results revealed that the
obligate anaerobes tested did not invade tubules. In a recent study,
Perez et al. (12) examined the migration of one obligate and two
facultative anaerobes commonly found in root canal infections.
They observed that Prevotella intermedia, the obligate anaerobe
tested, had not penetrated into dentinal tubules.
308
Printed in U.S.A.
VOL. 22, No. 6, JUNE 1996
The purpose of the present study was to investigate in vitro
dentinal tubules penetration by selected anaerobic bacteria com-
monly isolated from root canal infections and associated with
endodontic pathosis using scanning electron microscopy (SEM).
M A T E R I A L S AND M E T H O D S
Freshly extracted, mature bovine incisors were used in this
study. The teeth were kept in 0.5% sodium hypochlorite solution
overnight for surface disinfection and later stored in phosphate-
buffered saline solution (pH 7.4) until used. The apical end and the
crown were cut off perpendicular to the long axis of the tooth with
a rotating diamond saw under water cooling. Root canals were
widened to a standard diameter of - 2 . 0 mm with a round tungsten
carbide bur. The root cementum was removed by means of a
tapering diamond bur in a high-speed handpiece, with copious
water spray. The root canal portion retained was sectioned trans-
versely, so that two 4-mm dentin cylinders were obtained from
each tooth. Specimen preparation was done based on Haapasalo
and Orstavik (6).
The specimens were placed in a glass flask containing a 10%
solution of citric acid, where they remained for 3 min, under
agitation, for smear layer removal. Two specimens were examined
in the scanning electron microscope to confirm the dentinal tu-
bules' patency after citric acid treatment. Specimens were then
sterilized in an autoclave for 20 min at 121°C. To ensure specimen
sterilization, they were placed in tubes containing Brain Heart
Infusion broth (Difco, Detroit MI)-prereduced anaerobically ster-
ilized (BHI-PRAS), supplemented with hemin (5 mg/L) and men-
adione (0.5 mg/L), and incubated at 37°C for 1 wk. Each tube
contained one dentinal specimen. This allowed dentinal tubules
penetration by the broth.
After incubation, tubes containing BHI-PRAS and dentinal cyl-
inders were divided into six groups: six tubes were inoculated with
Porphyromonas endodontalis (BN 1 la-f, strain kindly supplied by
Dr. G. Sundqvist); another six tubes were inoculated with Fuso-
bacterium nucleatum (ATCC 10953); five tubes were inoculated
with Actinomyces israelii (ATCC 12103); five tubes were inocu-
lated with Porphyromonas gingivalis (ATCC 33277); and five
tubes were inoculated with Propionibacterium acnes (clinical iso-
late). Five tubes were inoculated with Enterococcus faecalis
Vol. 22, No. 6, June 1996
FIG 1. Scanning electron microscopic micrographs of bacterial pen-
etration into dentinal tubules. (A) Cells of P. acnes inside dentin
(original magnification x4900). (B) Tubule invasion by A. israelii
(original magnification ×4500).
(ATCC 29212) because this bacteria has been reported to penetrate
within dentinal tubules (6, 11). Therefore, it served as a control
group. All microorganisms used in this study are obligate anaer-
obes, except E. faecalis, a facultative anaerobe. All of them were
cultured under anaerobic conditions in BHI-PRAS.
The medium was renewed every 3 days, and the purity of
cultures checked at every medium change. After incubation of 21
days, the infected specimens were prepared for SEM examination,
based on the method described by Dykstra (13). Dentinal cylinders
were fixed in 2.5% glutaraldehyde for 2 h. In sequence, they were
washed three times, 10 min each, in a buffer solution of 0.2 M
sodium cacodylate (pH 7.2) with 10 nM CaC12 and 0.2 M sucrose,
followed by a postfixation in 1% osmium in cacodylate buffer for
30 min. Specimens were washed three times in the buffer solution
and longitudinally split. They were dried with ascending ethanol
concentrations, in 10% steps, starting with a 50% concentration
and ending with a 100% concentration. Specimens remained 10
rain in each ethanol concentration. Dentinal cylinders were then
transferred to glass flasks containing acetone. Afterward, they were
dehydrated to their critical point with a drying apparatus (Bio-Rad,
E3000, Watford, UK), fixed on microscope slides, and gold-coated
using a sputter coater (Bio-Rad). Root dentinal tubules penetration
by the bacterial species tested was examined using a scanning
electron microscope (Cambridge, Stereoscan 200, Cambridge,
UK).
RESULTS
The SEM analysis revealed that all bacteria used in this exper-
iment were able to penetrate into root dentinal tubules to different
depths. It was not possible to measure the maximum bacterial
penetration, because tubule orientation and defects created in the
slices by the splitting procedure prevented the entire view of each
dentinal tubule.
Root canal walls and dentinal tubules were heavily infected by
E. faecalis, P. acnes, and A. israelii. Most tubules, but not all,
contained bacterial cells inside them to a great extent (Fig. 1 ). Very
Intratubular Penetration by Anaerobes 309
few tubules were invaded and colonized by P. gingivalis. How-
ever, whenever penetration was observed, it was to a great depth.
Although P. endodontalis also invaded dentinal tubules, penetra-
tion was restricted to a very small percentage of tubules just within
the circumpulpal dentin. Colonization of root canal walls by this
bacteria was very slight. F. nucleatum heavily colonized the root
canal walls, but just some tubules of the dentin surrounding the
canal were invaded by few cells. Further cellular migration was
apparently prevented because spindle-shaped cells of F. nucleatum
clustered into tubule entrances, forming bundles.
No bacterial invasion was observed in the peripheral exposed
dentin.
DISCUSSION
Several studies have used bovine teeth to examine dentinal
tubules penetration by bacteria (6, 7, 12). They are easy to obtain;
their size made handling easier; and their dentinal tubules are in
size, morphology, and density similar to those of human teeth (6,
14). Because of the qualitative nature of the present study, we did
not think it necessary to use a larger number of dentinal samples
for each bacterial strain tested, because all bacterial species used
herein were able to penetrate into tubules.
Although the comparison of types of broth was not done in this
study, our results are in disagreement with those of Haapasalo and
Orstavik (6), which reported weaker infections of dentin cylinders
by E. faecalis when BHI broth was used. In our study, BHI broth
allowed good results for all bacteria tested. In addition to growth
media used, differences between results of various studies may be
caused by bacterial species tested, incubation time, inoculum size,
inoculum age, and incubation conditions.
Apparently, bacteria penetrate into dentinal tubules by way of
cell division, using as nutrition the culture medium that has pen-
etrated into tubules. According to Perez et al. (7), another phe-
nomena may occur. Bacteria grow and multiply in the culture
medium and, as this medium penetrates dentinal tubules, bacterial
cells could also penetrate deeply by a passive process. Although
the tubule invasion theoretically could be facilitated by the size of
bacteria, which is lower than tubule diameter, different patterns of
invasiveness were observed. These differences may, in part, be
related to differences in growth rate of the bacterial species tested.
Differences may also be attributable to bacterial morphologic
factor, such as surface appendages (capsules, pili, and fimbriaes)
that may make difficult the tubule penetration to a great depth.
Cellular arrangement may also play an important role in permitting
bacterial migration into dentinal tubules. An interesting finding
was observed in dentinal cylinders infected with F. nucleatum. F.
nucleatum cells were long, slender filaments that colonized root
canal walls in an arrangement like a palisade. Some bacterial cells
were seen inside tubules, but further penetration seems to have
been impeded by other cells that clustered into tubule entrances,
fon-ning a bundle of cells. Tubular constrictions and odontoblastic
process inside tubules may act as obstacle to bacterial penetration,
although the latter was not observed by SEM in this study, sug-
gesting that they were eliminated from samples before bacterial
inoculation.
The bacterial species used in this study are commonly isolated
from infected root canals and may be implicated in endodontic
therapy failure. In truth, bacteria that penetrate dentin to a shallow
depth may be removed by mechanical preparation of the root canal
or be destroyed by the chemical action of irrigant solutions. Con-
310 Siqueira et al.
versely, those localized deep in dentin may remain even after
cleaning and shaping procedures. If root canal obturation fails to
seal the entire root canal system, the percolation of tissue fluids or
saliva may supply substrate to remaining bacteria that growing and
multiplying may cause periradicular tissue damage and, conse-
quently, treatment failure. Torabinejad et al. (15) reported that in
vitro bacteria infiltrated the entire root canal system along root
fillings, when coronal sealing was not placed and coronal portions
of root fillings were in contact with bacterial cultures. Fortunately,
in most clinical situations, sclerotic apical dentin of adult humans
may prevent the bacterial penetration of tubules and may help
explain the high success rate of endodontic therapy (16).
We gratefully acknowledge the valuable assistance of Mr. Geraldo Baeta
da Cruz, research assistant of Empresa Brasileira de Pesquisa Agropecuaria,
during the use of the scanning electron microscope.
Dr. Siqueira is professor, Department of Postgraduate Endodontics, Gama
Filho School of Dentistry; and Drs. Uzeda and Fonseca are professors of
Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Address requests for reprints to Dr. Jos6 F. Siqueira, Jr., Rua Herotides de
Oliveira 61/601, Icarai, Niteroi, Rio de Janeiro, Brazil.
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