Journal of Dental Research

http://jdr.sagepub.com/

In vitro Infection and of Dentinal Tubules

M. Haapasalo and D. Ørstavik
J DENT RES 1987 66: 1375
DOI: 10.1177/00220345870660081801

The online version of this article can be found at:

http://jdr.sagepub.com/content/66/8/1375

Published by:

http://www.sagepublications.com

On behalf of:
International and American Associations for Dental Research

Additional services and information for Journal of Dental Research can be found at:

Email Alerts: http://jdr.sagepub.com/cgi/alerts

Subscriptions: http://jdr.sagepub.com/subscriptions

Reprints: http://www.sagepub.com/journalsReprints.nav

Permissions: http://www.sagepub.com/journalsPermissions.nav

Citations: http://jdr.sagepub.com/content/66/8/1375.refs.html

>> Version of Record - Aug 1, 1987

What is This?

Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on March 25, 2014 For personal use only. No other uses without permission.
In vitro Infection and Disinfection of Dentinal Tubules
M. HAAPASALO1 and D. ORSTAVIK2

NIOM -Scandinavian Institute of Dental Materials, Forsktiingsveien 1, N-0371 Oslo 3, Norwos

An in vitro model for dentinal tuhide infection oJ root canals smas Materials and methods.
developed. Cylindrical dentin specimens, 4 inm high with a diameter
ofJ6 mm and a canal 2.3 min wide, sere preparedfJrom freshly ex- Freshly extracted, intact bovine incisors were used for the
tracted bovine incisors. The cenentum was removedfrom all detitit experiments. The teeth were kept in 0.5% NaOCI for surface
blocks. The tubules were opened bh four-minute treatments with / 7% disinfection overnight. The apical 5 mm and two-thirds of the
LDTA and 5.25c NaOCI before being infected swith Entercoccus crown were cut off with a rotating diamond saw at 700 rpm
faecalis ATCC 29212 in east extract-glutc se broth. Bacteria rapidly (Accutom, Struers, Copenhagen, Denmark) under water cool-
Invaded the tuhules. After three weeks of incbahaon, a heavy' infection ing. With a reamer bur, the canal was widened to 2 mm in
a s joufund 4(00 jim from thce cana I lu men, and the Jront oJ the infection
reached 1000 pLm in some block s. Canmphorated parainonochloro- diameter. The root cementum was removed with a cylindrical
phenol (CMCP) and a calcium hldroride compound, Cola septc, 1wvere diamond hole-bur at low speed (<100 rpm) in water bath at
testedl for their dis infe acting efjficacv toward E. faccalis- infeced dentin. + 25 C, leaving a center-holed root dentin piece of 6 mm outer
Liquid CMCP rapidly and completeR disinfected the dentinal tubules, diameter and approximately 15 mm long. The root was then
wvher eas CMCP in gaseous form disinfected tubules less rapidly. Ca- cut into slices 4 mm thick with a diamond saw (700 rpm, water
laseptc failed to eliminate, even superficially, E. taccalis in the Il- cooling). The canals of the 4-mm blocks were widened with
hiles. The method used in hacteriological sampling allowed far an ISO 023 round bur, finalizing the mechanical preparation
sequential removal of 100-Vjm-thick zones of dentin from the central of the test specimens (Fig. 1).
canal toward the periphery. Control specimens stere unifortnly in-
fected and1 yielded growth in bur samples up to some 500 jitn from The teeth and the dentin blocks were kept in tap water during
the surface. The model proved quite sensitive and seems suitable for all procedures to avoid dehydration. The smear layer xvas re-
in vitro testing of root canal medicaments. moved by treatment in an ultrasonic bath (Metason 120, Struers)
in 17% EDTA (pH 7.8) (four min) and 5.25% NaOCI (four
,J Dent Res 66(8):1375-1379, August, 1987 min). The effectiveness of this treatment was confirmed in the
scanning electron microscope (Philips SEM 515. Philips, Eind-
hoven, The Netherlands), and the ensuing dentinal tubule
openings are illustrated in Fig. 2.
Introduction.
The blocks were sterilized by being autoclaved in water for
i5 min at 1211C. The sterile test specimens were then trans-
Bacteria are the main causative factors in pulpal and periapical ferred to yeast extract-glucose broth [YG broth; Yeast Extract
inflammation (Kakehashi et al., 1965; Sundqvist, 1976). Me- (Oxoid, Hampshire, England), 10 g/L. glucose 10 g/L] and
chanical preparation and chemical disinfection of the root canal incubated for 24 hr at 370C as a test for sterility. Following
of the affected tooth remain the most important procedures in this incubation, the blocks in YG broth were subjected to ul-
endodontics (Grossman. 1978). Disinfecting agents are used
as irrigants and as intracanal dressings between the appoint-
ments, and a wide variety of medicaments is commonly used.
In vitro tests have shown that bacteria are usually killed rapidly
when they are in direct contact with various medicaments, even
in high dilutions (Spangberg, 1982). However, data on the
efficacy of endodontic medicaments against bacteria in viva
are limited. In routine cases, it seems that success can be
obtained with several methods and medicaments; however,
sometimes an infection is resistant to normal treatment, and
the therapy cannot be successfully completed. One possible
reason for the persistent infection may be that bacteria have
invaded the dentinal tubules. A few attempts have been made
to establish in vitro models of root canal infections (Akpata
and Blechman, 1982; Foley et al., 1983). However, there are
hardly any reports on the efficacy of root canal medicaments
against infected tubules under controlled conditions, and a sim-
ple and reliable method for studying the elimination of tubule
infection is lacking.
The aim of the present study was to develop an in vitro
model for infection of dentinal tubules which would allow for
testing of the efficacy of root canal medicaments.

Received for publication July 23, 1986

Accepted for publication January 4, 1987
'Permanent address: Department of Cariology, Institute of Den-
tistry, University of Helsinki, Finland Fig. I -Appearance and dimensions ot a prepared dentin test speci-
2To whom correspondence and reprint requests should be addressed men.
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on March 25, 2014 For personal use only. No other uses without permission.
1375
1376 HAAPASALO & 0RSTAVIK
Fig. 2 - Appearance ot dentinal
tubules (A) and their pLilpal openings
(B) after complctcd EDTA-NaOCl
treatment. Scanning electron 1 icros-
copy.
trasonic treatment for 10 min to facilitate the permeation of
the dentinal tubules with the broth.
Enute.otocucs ftwcif/is ATCC 29212 (Difco Laboratories.
Detroit) was used as a test organism aind maintained by regular
transfer in YG broth. For infection, two blocks were kept in
each test tube with 1.5-2 mL YG broth. which was changed
daily. Strict asepsis was necessary to avoid contamination. The
purity of the cultures was checked at intervals. The develop-
ment of tubule infection up to 28 days was evaluated in the
scanning electron microscope (Fig. 3) and in Brown and Brenn-
stained (B&B) sections of demineralized fixed, and paraffin-
embedded specimens (Fig 4).
Specimens infected for three weeks were used for the testing
of the medicaments. Prior to the actual test procedure, the outer
surfaces of the specimens were covered with nail varnish, and
the specimens were mounted with sticky-wax (Amalgamated
Dental. London) at the bottom of cell culture wells (Falcon
FTi. 3 Cells tf E. foieclir.s in dentinal
tubLules (A) and on the pulp canal wall (B)
matter a three-week infection period. Scanning
electron microscopy.
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on March 25, 2014 For personal use only. No other uses without permission.
J Dzewl Res Augus.t 1987
I
I
I
3047. Becton-Dickinson. Oxnard. CA). The medicamnCIt to be
tested was then applied to the canal lumen and filled to cover
the fr-ee. top suif-ace of the dentin block well. as
The medicatcd blocks incubated under humid condi-
were
tions at 37 C in air. The two medicaments tested were a cal-
ciurm hydroxide CompoLund. Calasept'1 (Swcdia. Knivsta.
Sweden). and camphorated pariamonochlorophenol (CMCP: 60%
camphor. 30% p-monochlorophenol. 10%c/ ethanol), obtained
on prescription from aL pharmacist. As controls, infected spec-
mens with no medicament but With varnish on the outer surface
only or on all surfaces used. Incubation times foi the
were
controls were seven and 10 days (outer surface varnished) and
20 and 60 min (all surfaces varnished). Calasept paste and
CNICP in liquid form were placed centrally in the test speci-
men. For testing of CMCP vapor, we placed the infected blocks
in seal-tight containers with liquid CMCP present but placed
outside the specimen.
Vol. 66N No1. oI)INTINAL TUBUlE INFECTION I1377

A
04
.

.
O
Al
.:, B:

PL
F
w .
N5% *,.
.a..

EF

FLi. 4- Infection of dentinal tubules hy E.

cci co/s. B&B stain, light microscopy. A. pulpal

v
..K end: B. 400 WiLn into dentin. EF. cells of f.
fi cil, UET. unninfectcd tubuile; PL. pul pal u-
WO~s
I''
GZ~A~x '0
11Ccen.

~ ~

The dentin specimens with applied medicament were incu- Dismf(etction. The results of the experiments with CMCP
bated for time intervals ranging from five min to 10 days. On and Calasept acting on tubules infected with f/weclis are
termination of each experiment, the blocks were blotted free presented in Figs. 5. 6. and 7. In this system. liquid CMCP
of excess medicament on sterile filter paper. Bacterial samples caused a rapid reduction ot bacteria which could be cultivated
were taken with sterile round burs mounted a handpiece andin from the pulpal side, and after one hour no live oreanismns
run at low speed. The specimen kept in place with sterile
was could be sampled. After one and seven days of incubation, the
forceps during the sampling. The following bur sizes were entire specimen appeared disinfected (Fig. 5). Gaseous CMCP
used: ISO 023. 025. 027, 029. 031. 033. 035, 037. 040. 042. was hardly effective for the first four hr, but complete disin-
045. and 050. The bur invaded the canal from the medicated tection was obtained after one day. In contrast, treatment with
surface and never penetrated to the non-medicated surface. The Ca(OH)1 failed to eliminate E. fcali~s even the inner-
dentin chips obtained with each bur were immediately col-
lected in separate test tubes containing brain-heart infusion
broth (BHI: Cibco Europe. Paisley. Scotland): the tubes were
incubated at 37 C in air, and inspected daily for days. seven
No growthI
When growth occurred. it was checked in every instance for
purity (o E. fiwecoi.s. * m m
z >700
F-
z 700 -
LL cn

Results. 0
0
a)

ai)
In/tuetion. -Bacteria invaded the dentinal tubules both from E
0 500 *
the pulpal side and to a lesser degree from the outcr side. 2, 0

Specimens infected for only one day revealed penetration of 0.

.Eui
bacteria to a 300 -400 p)m depth in a few canals when studied L-
in the light microscope. The main cffcct prolonged infection
of LL 300 - 0

was that more tubules became infected, whereas the average LU

c)
C)

<1:
depth of penetration of the infected tubules by bacteria in-
creased only slowly with tirme. After three weeks of incubation
z

uD 100 -
Uf)
with k:. joeccfi i.s, a dense infection from the canal side reached

up to 300-400 pim: a moderate infection was usually seen up

to 400-500 pim and the front of the infection could reach 800- 5 10 20 30-60 1-7
1000 ptm. From the outer surface, where the tubules were min. d
narrower, the infection usually reached 150-200 p9m. occa-
sionally extending to 280 pm from the outer surface. When
part of the cementum had unintentionally been left unremoved, TIME OF MEDICAMENT ACTION
no bacteria had penetrated throuTh it. and the infection in the ti fccccil/is from infected dcnitin after inLihbation
Groth of
corresponding tubules from the pulpal side was also consid- with liqLidl camphorated paranmonochlorophenol. Each dot represents one
erably shorter than that in those tubules which were patent test specimen and denotes the shortest distance frona the pulpodentin sur-
through the whole block. face where growth detected.
was

Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on March 25, 2014 For personal use only. No other uses without permission.
1378

z
p
z
w (j,
HAAPASALO & 0RSTAVIK

No growth [

>700

700
l 031, frequently
entire dentin block.

Discussion.
to bur size

Several simplifications and extrapolations were necessary

for the present model to be functional and reproducible. The
J Dent Res August 1987

040, and sometimes through the

a- a) use of bovine teeth was based on their availability and basic

E
0~
-JAL 0 500 - morphology (similar to that of human teeth). The dentinal tu-
0
bules were of size and density similar to those of human teeth,
-i
0r
us and giant tubules (Hals and Olsen, 1984) were not frequently
Cc Ll observed. To standardize the test specimens, we decided to
LL 300 -
remove the cementum with a special bur and remnants of pul-
w LL
z
cc pal tissue by filing and by treatment with sodium hypochlorite.
U) Treatment with EDTA removed hard tissue debris and facili-
Co
100 tated the cleaning action of NaOCl.
En 0 @00 E. faecalis was chosen as a test organism primarily because
it is among the few facultative organisms associated with per-
20m 1h 4h id sistent apical periodontitis (Engstrbm, 1964; Haapasalo et al.,
1983), and because previous experimental studies have used
TIME OF MEDICAMENT ACTION this organism (Akpata and Blechman, 1982; Stevens and
Fig. 6 - Growth of E. faecalis from infected dentin after incubation Grossman, 1983; Bystrdm et al., 1985).
with camphorated paramonochlorophenol in gaseous form. Each dot rep- During this study and in pilot experiments, it was noticed
resents one test specimen and denotes the shortest distance from the pul- that the presence of cementum affected the ability of E. fae-
podentin surface where growth was detected. calis cells to infect the dentinal tubules. When cementum was
removed from the blocks before infection, this resulted in blocks
which were better standardized and more easily infected. This
observation reflects on the findings by Akpata and Blechman
No growth L I (1982), who studied the invasion of E. faecalis and Strepto-
coccus sanguis into pulpal dentin in extracted human teeth with
cementum. They found that E. faecalis did not invade the
.

so
z >700
tubules at all during the first week of the test. Valderhaug
z 700
(1974) induced root canal infections in monkey teeth in vivo
w and found that if the inflammation around the apex affected
0 a)
the cementum layer, bacteria invaded through the dentin. By
a.
-i E contrast, in areas where the tubules ended in histologically
a-
0
LU
500

sound cementum, bacteria, if present, only reached the inner

0 third of the tubule. Thus, it appears that for a controlled in-
0 fection in vitro, it is important that the cementum be removed
cc
LL 0
300
to facilitate the ingress of the infective organism.
w UL Four different broths were studied in preliminary tests to
z
cc determine whether the type of broth had any effect on the
co
100 S rapidity of development of the infection in the tubules. YG
Co) broth, tryptone soya broth (Oxoid), BHI broth, and a modifi-
0; 0 som es me cation of Carlsson's (1970) synthetic medium for streptococci
5 h 1 d 7 d 10 d
(M3) were tested. The use of BHI broth seemed to result in a
weaker infection compared with the other three, which appar-
ently performed equally well. YG broth was chosen because
TIME OF MEDICAMENT ACTION it was easy to make (compared with Carlsson's medium) and
Fig. 7Growth of E. faecalis from infected dentin after incubation had been used earlier by Akpata and Blechman (1982). Three
with calcium hydroxide. Each dot represents one test specimen and denotes weeks were used as the time for the infection on the basis of
the shortest distance from the pulpodentin surface where growth was de- studies with SEM and B&B-stained sections. However, the
tected. difference in the infection was not great among 1-, 2-, and 3-
week-old blocks. Thus, a shorter time for infection can also
be used.
most zones of dentin after prolonged incubation (Fig. 6). Con- Nail varnish was used to close the tubule openings on the
trol blocks with varnish and no medicament invariably showed outer surface of the block before application of the medica-
growth starting from the canal lumen. ment. This was done partly because many root canal medica-
Performance of the model. Scanning electron and light ments are volatile, like CMCP, and would act on the infection
microscopy documented the presence of bacteria in dentin tu- from both sides, which might complicate the evaluation of the
bules of all blocks inspected as controls (n - 30). However, results, but also because the diffusion of medicaments through
rather a small percentage of tubules was seen to be infected tubules opened at both ends is probably different from diffu-
(Fig. 4). Similarly, control specimens and specimens subjected sion when one end is covered with varnish or with cementum,
to medicaments for short time periods (Figs. 5 and 6) con- as in the clinical situation. While the nail varnish contains
stantly showed growth of bur samples from the pulpodentin solvents which may have some antibacterial effect, control
surface (bur no. 023) and the first scrapings (burs nos. 025- experiments showed that this effect was negligible.
031). Growth from these teeth continued at least to bur size The sampling procedures employed seemed quite sensitive
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on March 25, 2014 For personal use only. No other uses without permission.
Vol. 66 No. 8
and made it possible to follow the increase of the length of the
disinfected zone from the canal lumen. The technique was easy
to perform, and most of the dentin powder could be collected
into BHI tubes. In pilot experiments, two blocks were totally
embedded in Calasept® for two days. The blocks were then
rinsed in sterile water to clean them of Calasept® and dropped
into BHI-broth, where they were ultrasonically treated (Me-
tason 120, Struers) for 15 sec. Only one block yielded growth.
However, after being sampled with a bur, both blocks revealed
E. faecalis infection in the first 100-jim zone around the canal
lumen. This experiment clearly demonstrated the possibility of
false-negative results when a routine sampling from the main
canal is performed, and confirms that a negative test does not
necessarily mean a sterile canal (Akpata, 1976). This experi-
ment also documented the efficacy of the bur sampling in de-
tecting growth.
CMCP in liquid form proved highly effective in eliminating
E. faecalis infection inside the tubules. As might be expected,
gaseous CMCP was slower in onset of antibacterial activity,
but gave complete disinfection after one day of incubation.
This may indicate that the effect of CMCP is rather dose-
dependent, and may aid in the interpretation of the variable
results obtained in clinical tests of CMCP (Bystrbm et al.,
1985).
By comparison, the calcium hydroxide paste, Calasept®,
was quite ineffective against tubule infection with E. faecalis
during the test period. Even after ten days, live E. Jaecalis
was found within the first 100-jim zone in the canal walls of
most blocks. Bystrdm et al. (1985) reported that a pH drop
from 12.5 to 11.5 of calcium hydroxide resulted in a high
recovery of E. faecalis in test tube conditions even after 24
hr, whereas saturated calcium hydroxide killed this organism
in a few minutes. Tronstad et al. (1981) showed, in monkey
teeth, a sharp increase of pH in root dentin after the canal had
been filled with calcium hydroxide. In conjunction with the
present work, a liquid pH indicator (thymol blue) was applied
on the fractured faces of some dentin blocks. Increased pH
values were recorded far beyond the inner infection after only
one day of calcium hydroxide in the canal (unpublished data).
It seems evident that in the case of E. faecalis this increase in
pH is not sufficient to disinfect the circumpulpal dentin. The
results of this study are consistent with those of the study of
Stevens and Grossman (1983), in which CMCP was found
Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on March 25, 2014 For personal use only. No other uses without permission.
DENTINAL TUBULE INFECTION 1379
more effective than calcium hydroxide in eliminating E. fae-
calis from artificially infected root canals of cat canines.
REFERENCES
AKPATA, E.S. (1976): Effect of Endodontic Procedures on the Pop-
ulation of Viable Microorganisms in the Infected Root Canal, J
Endo 2:369-373.
AKPATA, E.S. and BLECHMAN, H. (1982): Bacterial Invasion of
Pulpal Dentin Wall in vitro, J Dent Res 61:435-438.
BYSTROM, A.; CLAESSON, R.; and SUNDQVIST, G. (1985): The
Antibacterial Effect of Camphorated Paramonochlorophenol,
Camphorated Phenol and Calcium Hydroxide in the Treatment of
Infected Root Canals, Endod Dent Traumatol 1:170-175.
CARLSSON, J. (1970): Chemically Defined Medium for Growth of
Streptococcus sanguis, Caries Res 4:297-304.
ENGSTROM, B. (1964): The Significance of Enterococci in Root
Canal Treatment, Odont Revy 15:87-106.
FOLEY, D.B.; WEIDE, F.S.; HAGEN, J.C.; and deOBARRIO, J.J.
(1983): Effectiveness of Selected Irrigants in the Elimination of
Bacteroides melaninogenicus from the Root Canal System: an in
vitro Study, J Endo 9:236-241.
GROSSMAN, L.I. (1978): Endodontic Practice, 9th ed. Philadel-
phia: Lea & Febiger, pp. 197-255.
HAAPASALO, M.; RANTA, H.; and RANTA, K.T. (1983): Facul-
tative Gram-negative Enteric Rods in Persistent Periapical Infec-
tions, Acta Odontol Scand 41:19-22.
HALS, E. and OLSEN, H.C. (1984): Scanning Electron and Incident
Light Microscopy of Giant Tubules in Red Deer Dentin, Scand J
Dent Res 92:269-274.
KAKEHASHI, S.; STANLEY, H.R.; and FITZGERALD, R.J. (1965):
The Effects of Surgical Exposures of Dental Pulps in Germ-free
and Conventional Rats, Oral Surg 20:340-349.
SPANGBERG, L.S.W. (1982): Endodontic Medicaments. In: Bio-
compatibility of Dental Materials, D.C. Smith and D.F. Wil-
liams, Eds., Boca Raton: CRC Press, pp. 223-257.
STEVENS, R.H. and GROSSMAN, L.I. (1983): Evaluation of the
Antimicrobial Potential of Calcium Hydroxide as an Intracanal
Medicament, J Endo 9:372-374.
SUNDQVIST, G. (1976): Bacteriological Studies of Necrotic Den-
tal Pulps, Umea University, Odontol. Diss. No. 7.
TRONSTAD, L.; ANDREASEN, J.O.; HASSELGREN, G.; KRIS-
TERSON, L.; and RIIS, I. (1981): pH Changes in Dental Tissues
after Root Canal Filling with Calcium Hydroxide, J Endo 7:17-
21.
VALDERHAUG, J. (1974): A Histologic Study of Experimentally
Induced Periapical Inflammation in Primary Teeth in Monkeys,
Int J Oral Surg 3:111-123.