Bacterial Invasion of Pulpal Dentin Wall in vitro
E.S. AKPATA and H. BLECHMAN
Department of Restorative Dentistry, College of Medicine, University ofLagos, P.M.B. 12003, Lagos, Nigeria and Department of Endodontics,
New York University College of Dentistry, 421 First Avenue, New York, New York 1 0010
Instrumented root canals of extracted human teeth were inoculated
with known pulpal bacterial isolates. The inoculated teeth were
immersed in the appropriate culture media and incubated at 3 7C
for varying periods. Streptococci multiplied in the root canals and
invaded the radicular dentinal tubules. The extent of bacterial in-
vasion was time-dependent. This experimental model may be useful
in investigating the effect of intra-canal medicaments on micro-
organisms lodged in the pulpal dentin wall.
J Dent Res 61(2):435-438, February 1982
Introduction.
The population of microorganisms in an infected root canal
may be reduced significantly by reaming, filing, and irriga-
ting. However, some microorganisms lodged in the pulpal
dentin wall may not be removed by instrumentation, and
little is known of their continued viability and resistance to
intra-canal medicaments in situ.
Chirnsidel aseptically exposed the pulp of a freshly-
extracted sound canine tooth, and inoculated it with a cul-
ture of Serratia marcescens. After being incubated in a
humid atmosphere at 25°C for 28 d, the organism was
observed in the radicular dentinal tubules. Chirnside also
examined the radicular dentin of 50 extracted teeth with
clinically infected pulps and disclosed the invasion of the
pulpal dentin wall by microorganisms in 62% of the teeth.
Bacilli penetrated the dentin more frequently than did
cocci, and, in a few cases, lysis of dentin by gram-negative
bacilli was observed.
Shovelton2 demonstrated the presence of bacteria in the
dentin wall in 46 out of 61 teeth with clinically infected
root canals. In some of these teeth, the microorganisms
penetrated halfway through the thickness of dentin. While
the number of invaded dentinal tubules varied from one
tooth to another, there was no significant difference in the
degree of bacterial invasion at different levels of the same
tooth.
The studies of Chirnsidel and Shovelton 2,3 were uncon-
trolled clinical investigations. From these studies, therefore,
it is difficult to interpret the rate and extent of microbial
invasion of radicular dentinal tubules.
The purpose of this study was to investigate the invasion
of the pulpal dentin wall by known pulpal microbial iso-
lates, using an experimental model.
Materials and methods.
Teeth. Fourteen recently-extracted single-rooted
human teeth with intact dental pulp, extracted for perio-
dontal or prosthodontic reasons, were selected for study.
Nine of the teeth were from patients aged 30-40 yr, with
the remaining five teeth from those aged 60-70 yr. Some of
the teeth had small carious lesions which were excised by a
Received for publication February 9, 1981
Accepted for publication September 30, 1981
This work was done during E.S.A.'s Fulbright Fellowship at the
New York University.
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#4 round bur at low speed. Conventional access cavities
were prepared, and the dental pulps were extirpated. The
root canals were filed to at least three sizes above the instru-
ment which began to bind to the unprepared canal wall. The
instrumented canals were irrigated with physiological saline
in a standardized manner. A small retentive cavity prepared
at the apical foramen was plugged with a cotton pledget.
The prepared teeth were sterilized in ethylene oxide gas.
Inoculation of the teeth. - Two obligate anaerobic
bacteria4 - Bacteroides melaninogenicus ss. melaninogeni-
cus (ATCC 25847) and Peptococcus asaccharolyticus
(ATCC 14963) - and two facultative anaerobes - Strepto-
coccus faecalis (ATCC 19433) and Streptococcus sanguis
(ATCC 10556) - were used. The obligate anaerobes were
maintained in cooked meat medium,* and the facultative
anaerobes in yeast extract glucose broth.*
The prepared pulp canals were inoculated with approxi-
mately 0.1 ml aliquots of fresh turbid culture of the test
organism, and the access cavity was sealed with a sterile
cotton pledget (Fig. 1). The inoculated tooth was then
immersed in the appropriate culture medium in a petri dish
and incubated for one, two, or three wk. The obligate anae-
robes were incubated in an anaerobic jar. The facultative
anaerobes were incubated aerobically. To check on the
growth of microorganisms inoculated into the experimental
teeth, the test bacteria (obtained from the fresh turbid cul-
ture) were inoculated into blood agar plates and were
incubated along with the experimental teeth. After incuba-
tion, the teeth were prepared for histological sections.
As a control, two teeth contralateral to two of those
used in the experiment were treated as described above,
except that the pulp canals were inoculated with sterile
yeast extract glucose broth and cooked meat medium,
respectively. To evaluate the sterilization of the experimen-
tal teeth by ethylene oxide gas, the two prepared teeth
were cultured in yeast extract glucose broth (aerobically)
and cooked meat medium (in an anaerobic jar). After
incubation, the broths were subcultured in blood agar.
Incubation was at 37° C for 72 h throughout.
Preparation of histological sections. - The experimental
teeth were fixed in 10% neutral formalin, demineralized in
formic acid, dehydrated, and infiltrated with paraffin. The
clinical crown and the apical 2-3 mm of each tooth were
removed, and the remaining root was cut into thirds -
cervical, middle, and apical - which were then embedded in
paraffin. Sections 7 Jim thick were then prepared. Alto-
gether, 12 sections each of the cervical, middle, and apical
thirds of each root were prepared and stained by the Brown
and Brenn technique.5
Results.
Timedependence of bacterial invasion (Table and Figs.
2-3). The extent of bacterial invasion of the root dentin
-
was related to the incubation period. Few dentinal tubules
in the cervical third of the root were invaded by S. sanguis
*Difco
435
 .:'*r~
436 AKPATA & BLECHMAN J Den t Res February 1 982

Key: B: Bacteria C=Cotton pledget M= Culture medium

Fig. 1 - Inoculated tooth immersed in the appropriate culture medium in a petri dish.

TABLE (e
i.- .
-7d...E5.M .X ;.: .:'. ::
. :.
BACTERIAL INVASION OF TUBULES IN DENTIN i, B
t
..

SURROUNDING THE DENTAL PULP

Bacterial Invasion ..
.:....v.: :
Incubation Cervical Middle Apical :'::i;: ? : ':::
:":'''
.:i::...:...:
Period Microorganism 1/3 1/3 1/3 ~~.;igig:e
?:.iiE':p.;:
EE:%p^ ^::?:*s ?:p ^'^...
--.... ....i

1 WK B. melaninogenicus
P. asaccharolyticus :'..:..:a:.
:..." :."
..::.':
.: s

S. sanguis +
S. faecalis
2 WK B. melaninogenicus ..;'
: R" i ' ......:"
'~
P. asaccharolyticus ......

S. sanguis + +
i;: ....
.... ..

S. faecalis ++ ++ +
. , . ...l:..e:
.: .........

3 WK B. melaninogenicus
P. asaccharolyticus
S. sanguis + + +
S. faecalis +++ f++ +

No invasion.
+ Bacteria invading approximately one third of the thickness of
the pulpal dentin wall and less than a quarter of its perimeter. o:i9~:: ..'
:..:
.':'I
.:
.: ....:: ....

++ Bacteria invading about half-way through the thickness of the '

N *

pulpal dentin wall and about half its perimeter. - *;

+++ Bacteria invading over half-way through the thickness of the
pulpal dentin wall and nearly its entire perimeter.

after one wk of incubation. In the tooth incubated for two

wk, the dentinal tubules in the cervical and middle thirds of
the root were invaded by S. sanguis, while the entire length
of the pulpal dentin wall was invaded by the organism in
NIl
the tooth incubated for three wk.
S. faecalis did not invade the pulpal dentin wall until
after two wk of incubation, when the entire root length
showed isolated areas of bacterial invasion. Compared with
S. sanguis, however, S. faecalis invaded more dentinal Fig. 2 Invasion of dentinal tubules by S. sanguis after one wk
tubules after three wk of incubation. Accordingly, numer- of incubation. X640.

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Vol. 61 No. 2

Olt~

m4.f
..^
...

Fig. 3
.,
WA, a:sa

Invasion of dentinal tubules

t),^*
BACTERIAL INVASION OFPULPAL DENTIN WALL

by S. faecalis after three wk of incubation. X640.

.S
i. ':
:*. in

from the cervical third of the root

quently invaded.
..
:
.. --.
a r

that portion of the

root near the access cavity through which the organisms
were inoculated into the root canal. Transverse sections
taken from the apical third of the root were the least fre-
Control experiments. - Microorganisms were not ob-
served in the dentinal tubules of the control teeth inocu-
lated with sterile culture media. Furthermore, the root
canals of these teeth were found to be free of organisms.
The control teeth sterilized in ethylene oxide gas showed
;%
HE
c.*

.sll
:
:
'.
z: :l.:z.;.
:
:
il ::';'! z'
:: :.
En
...i ..;

a
437

E...

..
i.:
i...

a......
.i:->
. . :;St*
::g.x ::: nfiit

no growth in the test media under the given experimental

*:::: conditions. However, colonies of the test aerobic and anae-
f 9f /y
i. **-- x
--, robic bacteria grew on the control blood agar plates which
..
!.:!:'..
.e ,;
:: wr;~
t-
...
were incubated along with the experimental teeth.

Discussion.
Fig. 4 Microorganisms in the root canal inoculated with P. Strains of bacteria selected for use in this experiment
asaccharolyticus after three wk of incubation. The dentinal tubules have been isolated from infected pulp canals and periapical
are not invaded. X1,600.
infections. Winkler and van Amerongen6 isolated S. faecalis
from 15% of 4000 root canal cultures. Furthermore,
Feldman and Larje7 reported the proportion of S. faecalis
in root canals to be the same as in periapical infections.
ous dentinal tubules were densely infiltrated by S. faecalis Mejare8 estimated S. sanguis to be present in 11.6% of 587
after three wk of incubation. positive root canal cultures. Sundqvist,9 who isolated B.
Invasiveness of different bacteria. - Microorganisms melaninogenicus and peptococci from infected root canals,
were present largely in small numbers in the root canals emphasized the importance of obligate anaerobes in the
inoculated with the anaerobic bacteria, and the dentinal pathogenesis of acute periapical infections.
tubules were not invaded (Fig. 4). In contrast, S. sanguis The obligate anaerobes used in this study are known to
and S. faecalis multiplied to form thick masses of bacteria grow slowly, as compared with streptococci, which have a
abutting the pulpal dentin wall invaded by the bacteria relatively rapid growth rate. Thus, thick masses of S. sanguis
(Fig. 3). and S. faecalis were generally seen to be abutting the
The B. melaninogenicus was not, however, distinctly prepared root canal wall and invading the dentinal tubules.
stained gram-negatively by the Brown and Brenn tech- On the contrary, relatively few bacteroides and peptococci
nique.5 were seen in the root canals of the experimental teeth. The
Bacterial invasion at different levels of tooth root (Ta- observed difference in the invasiveness of the bacteria may,
ble).- In
general, bacterial invasion of the dentinal tubules in part, be attributable to the difference in growth0l rate,
was observed most frequently in the transverse sections taken and this deserves further investigation.
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438 AKPA TA & BLECHMAN
To simplify instrumentation and make the experimental
conditions relatively uniform, incisors and canines were
selected for this study. The model system described above
may be useful in investigating the effects of root canal
irrigants and intra-canal medicaments on the microorga-
nisms remaining in the dentinal tubules of the pulpal dentin
wall. This may be accomplished by first allowing bacterial
invasion of the pulpal dentin wall. The pulp chamber could
then be cleaned and medicated, and further progress and
viability of the invading microorganisms could be studied.
Since the microbial invasion of the pulp canal system
may be time-related, early endodontic treatment of a tooth
with an infected root canal should minimize the number
of residual microorganisms lodged in the dentinal tubules.
This study suggests that the extended exposure of the
pulp canal to the oral microbial milieu may result in signif-
icant increased microbial invasion of the root canal system.
Conclusion.
The invasion of the pulpal dentin wall by bacteria
inoculated into the prepared root canal in vitro is time-
dependent and related to the growth rate of the micro-
organisms. Where the bacteria multiply and abut the pulpal
dentin wall in great number, the radicular dentinal tubules
may be invaded by the microorganisms.
Acknowledgment.
Support from the Council for International Exchange of
Scholars, U.S.A., is acknowledged.
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J Dent Res February 1 982
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