Radiographic evaluation of the effect of endotoxin (LPS) plus calcium hydroxide on apical and periapical tissues of dogs.

Abstract

The aim of this study was the radiographic evaluation of the apical and periapical region of dog teeth submitted to intracanal bacterial endotoxin (lipopolysaccharide, LPS), associated or not with calcium hydroxide. After removal of the pulp, 60 premolars were divided into four groups and were filled with bacterial endotoxin (group 1), bacterial endotoxin plus calcium hydroxide (group 2), saline solution (group 3), or periapical lesions were induced with no treatment (group 4), for a period of 30 days. Similar periapical lesions were observed in groups 1 and 4. The lamina dura was intact in groups 2 and 3. Bacterial endotoxin (LPS) caused radiographically visible periapical lesions, but when associated with calcium hydroxide, this endotoxin was detoxified.

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Radiographic Evaluation of the Effect of Endotoxin
(LPS) Plus Calcium Hydroxide on Apical and
Periapical Tissues of Dogs
Paulo Nelson-Filho, DDS, PhD, Mario Roberto Leonardo, DDS, PhD, Le ´a Assed Bezerra Silva, DDS, PhD,
and Sada Assed, DDS, PhD
The aim of this study was the radiographic evalu-
ation of the apical and periapical region of dog
teeth submitted to intracanal bacterial endotoxin
(lipopolysaccharide, LPS), associated or not with
calcium hydroxide. After removal of the pulp, 60
premolars were divided into four groups and were
filled with bacterial endotoxin (group 1), bacterial
endotoxin plus calcium hydroxide (group 2), saline
solution (group 3), or periapical lesions were in-
duced with no treatment (group 4), for a period of
30 days. Similar periapical lesions were observed
in groups 1 and 4. The lamina dura was intact in
groups 2 and 3. Bacterial endotoxin (LPS) caused
radiographically visible periapical lesions, but
when associated with calcium hydroxide, this en-
dotoxin was detoxified.
A fundamental role in the cause and maintenance of periapical
lesions has been attributed to the bacterial endotoxin, lipopolysac-
charide (LPS) (1–3). When released during bacterial multiplication
or death, this endotoxin, composed of lipopolysaccharides, causes
a series of important biological effects, which lead to an inflam-
matory reaction (4) and periapical bone resorption (2).
During the past 20 yr, a high prevalence of Gram-negative
anaerobic microorganisms (5, 6) and the chemical structure of
bacterial endotoxin commonly found in root canals with radio-
graphically visible periapical lesions has been reported (7). The
types of microorganisms and the level of intracanal endotoxin have
also been correlated with specific clinical findings (1, 8). However,
little research has evaluated the effect on periapical tissues of the
inoculation of endotoxin into root canals (9–12).
The medical and dental literature have emphasized research
attempting to obtain a medication that could inactivate bacterial
endotoxin: examples are caustic soda (9), formocresol (13), 1.2%
chlorhexidine (14), and sodium hypochlorite (15). Use of most of
these products is limited due to their lack of efficiency or high
toxicity, which causes undesirable effects when in contact with
tissue.
During in vitro research, Safavi and Nichols (16, 17), Barthel et
al. (3), and Olsen et al. (18) modified the concept of intracanal
dressings, showing that calcium hydroxide hydrolyzes lipid A,
which is the toxic part of endotoxin. Recently, we reported the
histopathological finding of inactivation of the toxic effects of
bacterial endotoxin by calcium hydroxide, in vivo (19). However,
there are no in vivo radiographic reports of this inactivation. Thus,
the purpose of this study was to evaluate radiographically the effect
of endotoxin and endotoxin plus calcium hydroxide on the apical
and periapical region in dogs.
MATERIAL AND METHODS
The methods used for this radiographic study were similar to
those used for the histopathologic evaluation of the effect of
endotoxin plus calcium hydroxide (19). In a laminar air flow, 100
mg of Escherichia coli endotoxin (lipopolysaccharide B, E. coli
055:B5-Lipid A, 9.2%; Difco, Bacto, Detroit, MI) was suspended
in 10 ml of phosphate buffered saline. Half of the 10-mg/ml
suspension was kept in sterile carpules and the other half was
mixed with 2.75 g of calcium hydroxide p.a. (550 mg/ml, Merck,
Whitehouse Station, NJ) and also kept in sterile carpules.
The second, third, and fourth mandibular premolars and the
second and third maxillary premolars of three dogs (age: 12–18
months; weight: 8–15 kg) were selected for treatment (total: 60
root canals). Twenty roots were used for each of the two experi-
mental groups (groups 1 and 2) and 10 for each of the control
groups (groups 3 and 4).
The animals were anesthetized intravenously with sodium thio-
pental (30 mg/kg body weight, Thionembutal, Abbott Laborato-
ries, Sa ˜o Paulo, SP, Brazil), and standardized radiographs were
taken by using a Heliodent RX machine (Siemens, Erlanger, Ger-
many) with 60 kVp, 10 mA, and 0.4 s exposure. Ultraspeed
periapical film (Eastman Kodak, Corp., Rochester, NY) was used,
and the radiographs were processed by the time/temperature
method.
After isolation with a rubber dam and disinfection of the oper-
ative field with 0.3% iodine/70% alcohol, access was made. The
working length was determined to 2-mm short of the radiographic
apex by using #30 K-files. The root pulp was removed and the root
canal was irrigated with saline solution (Laborme ´dica Industria
JOURNAL OF ENDODONTICS
Copyright © 2002 by The American Association of Endodontists
Printed in U.S.A.
VOL. 28, NO. 10, OCTOBER 2002
694

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Farmace ˆutica Ltda., Sa ˜o Jose ´ dos Campos, SP, Brazil) with a
minimum volume of 3.6 ml at each instrument change. The apical
foramen was enlarged by sequential use of #15 to #30 K-files
(Maillefer, Ballaigues, Switzerland) to the radiographic apex (al-
ways with saline irrigation). Instrumentation was then performed
to the working length up to a #50 K-file. A #30 K-file was used at
the total length of the root to ensure that no dentin chips or other
residue remained in the apical foramen. After irrigation, the root
canals were dried by aspiration and sterile paper points, filled with
14.3% buffered EDTA (pH 7.4; Odahcan-Herpo Produtos Den-
ta ´rios Ltda., Rio de Janeiro, RJ, Brazil) for 3 min, and then
irrigated with saline and dried.
Because all variables should be tested in the same animal and in
the different quadrants, each hemiarch was submitted, in an alter-
nate manner, to the experimental protocols. Group 1: 20 root canals
were filled with 0.1 ml of the endotoxin by using a threaded
syringe (S.S. White Artigos Denta ´rios Ltda., Rio de Janeiro, RJ,
Brazil) with a 27-gauge needle (Terumo, Tokyo, Japan). Group 2:
20 root canals were filled with 0.1 ml of the endotoxin and calcium
hydroxide suspension by using a threaded syringe with a Calasept
Kit needle (Scania Dental AB, Knivsta, Sweden). Group 3: 10 root
canals were filled with saline by using a carpule syringe with a
27-gauge needle. After these procedures, the pulp chambers of
groups 1, 2, and 3 were sealed with a sterile cotton pellet, and the
teeth were sealed with zinc oxide-eugenol cement (IRM, Dentsply,
Rio de Janeiro, RJ, Brazil) for a period of 30 days. Group 4: 10 root
canals were exposed to the oral environment for 5 days to allow
microbial contamination, after which, under general anesthesia, the
pulp chamber was cleared of all debris and sealed with a cotton
pledge and zinc oxide-eugenol cement to induce a periapical re-
action (20).
Thirty days after the surgical procedures, the teeth were radio-
graphed as described previously.
Radiographic Evaluation
The two sets of radiographs were evaluated, by three trained
examiners, by analyzing the integrity of the lamina dura, the
presence or absence of root resorption, and areas of periapical bone
resorption.
Statistical Analysis
The results were analyzed statistically by the Mann-Whitney
nonparametric test by using the software GMC7.7 (http://www-
.forp.usp.br/restauradora/gmc/gmc.html).
RESULTS
The 20 roots of group 1 (LPS) had radiographically visible
periapical lesions with a loss of lamina dura integrity and extensive
circumscribed areas of periapical bone resorption (Fig. 1).
The lamina dura was intact and there were no areas of periapical
bone resorption in 17 roots of group 2 (LPS plus calcium hydrox-
ide) (Fig. 2). Only slight thickening of the periodontal ligament
occurred in the remaining three roots at the apical level.
There was slight thickening of the periodontal ligament at the
apical level in only 1 of 10 roots of group 3 (saline). The other nine
roots were normal (Fig. 3).
There were radiographically visible periapical lesions in all 10
roots of group 4 (experimental lesion) with a loss of integrity of the
lamina dura and extensive diffuse areas of bone resorption (Fig. 4).
There was no root resorption in any of the groups.
The loss of integrity of the lamina dura and the presence of
periapical bone resorption were statistically similar (p ? 0.05) in
groups 1 and 4 and in groups 2 and 3 (group 1 ? group 4 ? group
2 ? group 3).
FIG 1. Radiograph, 30 days after filling root canals with LPS (group
1), showing extensive circumscript radiolucent areas.
FIG 2. Radiograph, 30 days after filling root canals with LPS plus
calcium hydroxide (group 2), showing normal apical and periapical
tissues.
FIG 3. Radiograph, 30 days after filling root canals with saline (group
3), showing normal apical and periapical tissues.
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DISCUSSION
Despite the fact that the role of bacteria in the etiology of
periapical lesions has already been proven, little research has been
performed to evaluate the isolated effect of LPS on apical and
periapical tissues (9–12). The results of this study, using dog teeth,
are in agreement with those of Dwyer and Torabinejad (9), using
cat teeth, Dahle ´n et al. (10), using monkey teeth, and Pitts et al.
(11) and Mattison et al. (12), who also used dog teeth. We also
found that after 30 days there were radiographically visible peri-
apical lesions in 20 roots with loss of integrity of the lamina dura
and extensive circumscribed areas of bone resorption (group 1),
similar to the findings of Mattison et al. (12), and diffuse areas of
resorption in group 4. The fact that after 30 days the lesions of
group 4 were not circumscribed may be because the lesions were
caused by bacteria, their products and subproducts, such as hyal-
uronidase, collagenase, and indole, which act in the dissociation of
fibers and the collagen matrix leading to a diffuse lesion. In group
1, LPS adhered irreversibly to mineralized tissues (10), causing
more localized extensive periapical bone resorption.
Even after their death, Gram-negative bacteria release endo-
toxin. Thus, from a clinical viewpoint, the use of medication that
leads only to the death of bacteria for the treatment of teeth with
pulp necrosis and chronic periapical lesion is not sufficient, but
medication must also inactivate bacterial endotoxin. In 1993,
Safavi and Nichols (16) reported that calcium hydroxide hydro-
lyzed lipid A in vitro, and in 1994, they concluded that after lipid
A hydrolysis, this highly toxic agent releases free-hydroxy fatty
acids that are nontoxic (17).
Furthering this line of research, Barthel et al. (3) and Olsen et al.
(18) evaluated, in vitro, the capacity of neutralization of endotoxin
by calcium hydroxide and reported that LPS was inhibited by
calcium hydroxide. Using dog teeth in vivo, Silva et al. (19)
reported histopathologically that calcium hydroxide inactivates the
toxic effects of bacterial endotoxin.
In this study, 30 days after filling the root canals of dog teeth
with a high concentration of LPS plus calcium hydroxide (group
2), the lamina dura was intact and there was no bone resorption. In
most cases, there was no thickening of the ligament. These results
were statistically similar to the teeth filled with saline (group 3).
Comparison of these results with the current literature is not
possible because there are no other in vivo studies. The results of
group 3, in which saline was used, were similar to those reported
by others (9–12) with an intact lamina dura and absence of a
periapical lesion.
This radiographic evaluation of the effect of LPS combined with
calcium hydroxide shows that calcium hydroxide detoxifies the
bacterial endotoxin LPS in vivo and thus should be the medication
of choice for intracanal dressings in teeth with pulp necrosis and a
periapical lesion.
Drs. Nelson-Filho, Silva, and Assed are professors, Department of Pedi-
atric Dentistry, School of Dentistry of Ribeira ˜o Preto, University of Sa ˜o Paulo,
Ribeira ˜o Preto, SP, Brazil. Dr. Leonardo is professor, Department of End-
odontics, School of Dentistry of Araraquara, University of the State of Sa ˜o
Paulo, Araraquara, SP, Brazil. Address requests for reprints to Professor
Paulo Nelson-Filho, Rua Piauı ´, 426, Sumarezinho, 14055-040 Ribeira ˜o Preto,
SP, Brazil.
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FIG 4. Radiograph, 30 days after the experimental induction of
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696 Nelson-Filho et al.Journal of Endodontics