92
Braz Dent J (2002) 13(2): 92-96
L.I. Yugoshi et al.
ISSN 0103-6440
Histometric Study of Socket Healing after Tooth
Extraction in Rats Treated with Diclofenac
Luciana Ibara YUGOSHI
Miguel Angel SALA
Luiz Guilherme BRENTEGANI
Teresa Lúcia LAMANO CARVALHO
Department of Morphology, Stomatology and Physiology, Faculty of Dentistry of Ribeirão Preto,
University of São Paulo, Ribeirão Preto, SP, Brazil
The purpose of the present study was to investigate if diclofenac administration interferes with the time course of alveolar wound
healing in rats. Forty-two Wistar rats were used, 21 rats received 10 mg/kg/day of diclofenac one day before and 4 days after extraction
of the right maxillary incisors and 21 rats received saline. The animals were sacrificed 7, 14 and 21 days after tooth extraction.
Progressive new bone formation and a decrease in the volume fraction of blood clot and connective tissue from 1 to 3 weeks after tooth
extraction was quantified using the histometric point-counting method. Diclofenac treatment caused a significant delay in new bone
formation in association with an impairment of blood clot remission/organization.
Key Words: diclofenac, socket healing, tooth extraction.
INTRODUCTION
Diclofenac is a non-steroidal anti-inflammatory
drug (NSAID) presenting significant analgesic and
anti-inflammatory activities. Drugs of this type are
commonly used in dental clinics to relieve pain after
tooth extraction. There is evidence, however, that the
NSAIDs may delay the repair of damaged tissues, such
as the skin (1), uterine musculature (2), cartilage (3),
fractured bone (4-6) and new bone formation after
tooth extraction (7). Studies carried out on rodents have
shown that NSAIDs may disturb not only bone healing
but also normal bone growth. Several NSAIDs, including diclofenac, seem to cause a significant inhibition of
femoral growth and a delay in parietal bone healing (8).
Ibuprofen and indomethacin may also retard the healing of long bone fractures (4,6,9). The same degree of
inhibition of femoral healing was found regardless of
whether indomethacin was given orally or locally applied to rats (5). The inhibitory action of indomethacin
on parietal wound healing, however, seems to be weaker
than that of the steroidal anti-inflammatory drug (SAID)
dexamethasone (10). A marked impairment of alveolar
wound healing following prednisolone (a steroidal antiinflammatory drug) treatment was observed in rats (11).
The aim of the present study was to evaluate the
effect of diclofenac sodium on the alveolar healing
following tooth extraction in rats by morphometric
analysis.
MATERIAL AND METHODS
Male Wistar rats, about 70 days old, (200-210 g
body weight) were anesthetized with an intraperitoneal
injection of 2,2,2-tribromoethanol (25 mg/100 g body
weight; Sigma-Aldrich, St. Louis, MO, USA). The
maxillary right incisors were extracted with a forceps
after disconnection of the surrounding gingiva and
luxation with an enamel hatchet with a cutting edge.
Immediately after surgery, the gingival tissues were
sutured with monofilament (Johnson & Johnson, New
Brunswick, NJ, USA) and a single intramuscular dose
of antibiotic (0.2 ml/rat; Pentabiótico Veterinário,
Wyeth, São Bernardo do Campo, SP, Brazil) was given.
The animals of the treated groups (n=21) received intraperitoneal injections of diclofenac (10 mg/
Correspondence: Dr. Miguel A. Sala, Departamento de Morfologia, Estomatologia e Fisiologia, Laboratório de Patologia, Faculdade de Odontologia
de Ribeirão Preto, Universidade de São Paulo, 14040-904 Ribeirão Preto, SP, Brasil. Fax: +55-16-633-0999. e-mail: masdmatt@forp.usp.br
Braz Dent J 13(2) 2002
93
Effect of diclofenac on tooth socket healing in rats
kg/day; Novartis, São Paulo, SP, Brazil) from
the day before surgery until 4 days after tooth
extraction. Control rats (n=21) received the same
volume of saline intraperitoneally.
The animals were sacrificed by decapitation 1, 2 and 3 weeks postoperatively (n=7 per
group), their mandibles were removed, and heads
were immersed in 10% formalin for 48 hours.
After fixation, the maxilla was dissected and
divided along the median sagittal plane. The
right halves were cut tangentially to the distal
surface of the molars, decalcified, and processed
for paraffin embedding. Longitudinal semiserial
6-µm thick sections, cut at intervals of 60-µm,
were stained with hematoxylin and eosin.
Histometric Analysis
Table 1. Volume fraction (%) of blood clot in the apical, middle and
cervical alveolar thirds of control and diclofenac treated rats from 1 to 3
weeks following tooth extraction.
Blood Clot
1 week
Control Treated
Apical
Median
25%
75%
Middle
Median
25%
75%
Cervical
Median
25%
75%
2 weeks
3 weeks
Control
Treated
Control
Treated
21.8
0.6
28.1
20.2
18.9
22.0
32.0*
26.8
38.4
10.0
5.6
15.2
24.4*
22.2
33.4
9.9
3.4
10.3
2.0
1.3
9.0
10.0*
8.7
20.5
4.0
3.5
4.9
13.2*
11.1
25.9
1.0
0.7
1.5
6.4*
1.8
7.9
3.0
2.2
8.5
14.7
0.7
20.1
1.7
1.0
3.5
8.8*
3.4
16.0
1.0
0.0
1.9
0.7
0.0
4.5
A light camera (Zeiss, Jena) was used to
measure the volume fraction of the alveolar
components by a differential point-counting Data are reported as median, 25% and 75% range. *Statistically different
method, as previously described (12). Micro- from the respective control (α = 0.05).
scopic images of the alveolus (final magnification
420X) were superimposed on sheets of paper with a
the relative volume of trabecular bone in parallel to a
grid containing 100 equidistant points. Four thousand
decrease in the amount of blood clot and connective
points lying on connective tissue and trabecular bone
tissue. In treated rats, however, the volume fraction of
were counted in the apical, middle and cervical thirds of
blood clot and connective tissue was greater, causing a
each alveolus. Data were analyzed statistically by the
smaller quantity of trabecular bone (Figure 1) .
non-parametric Mann-Whitney test (GMC Basic SoftHistometric data provided quantitative results
ware, version 7.5, FORP-USP, Ribeirão Preto, SP,
confirming the histologic findings (Tables 1-3).
Brazil).
Diclofenac treatment caused a significant decrease in
bone volume fraction which was more pronounced
RESULTS AND DISCUSSION
from the second week on. The relative amount of
connective tissue was smaller in the alveolus of treated
The different phases of alveolar wound healing,
rats by the end of the first week (compatible with a
observed by histological examination from 1 to 3 weeks
delay in blood clot remission and organization) but was
after dental extraction, closely resembled histologic
greater by the end of the third week (as a consequence
data regarding white rats (12-14). At the end of the first
of a delay in the osteogenic process). The volume
week, the alveolus of control animals was filled with
fraction of blood clot was always greater in the alveolar
immature connective tissue rich in fibroblasts and
socket of treated animals.
neoformed capillaries (granulation tissue). Newly
Blood clot formation is a fundamental step for
formed trabecular bone was observed mainly on the
the subsequent phases of tissue repair. The fibrin netinternal surfaces of the alveolar socket and remnants of
work allows invasion by fibroblasts, endothelial cells
the blood clot were observed mainly in the apical third.
and macrophages, which are present in the remaining
Compared to controls, treated rats seemed to present a
periodontal ligament. New fibroblasts, produced by
larger amount of blood clot in association with smaller
differentiation of adventitial cells and mitosis of preamounts of connective tissue and bone. From the secexisting fibroblasts under the influence of growth factor
ond week on, control and treated rats had an increase in
released by platelets, synthesize a delicate collagen
Braz Dent J 13(2) 2002
94
L.I. Yugoshi et al.
Table 2. Volume fraction (%) of connective tissue in the apical, middle and
cervical alveolar thirds of control and diclofenac treated rats from 1 to 3
weeks following tooth extraction.
Connective Tissue
1 week
2 weeks
Control Treated
Apical
Median
25%
75%
Middle
Median
25%
75%
Cervical
Median
25%
75%
3 weeks
Control
Treated
Control
Treated
68.4
67.0
72.7
61.2*
55.4
64.2
65.8
63.4
69.6
64.7
59.8
69.6
59.6
54.4
61.9
56.2
48.5
76.3
77.6
73.1
78.7
73.6
68.4
78.4
59.2
56.6
66.5
58.7
51.1
62.8
56.4
55.9
61.0
61.8*
59.2
68.5
84.0
81.6
86.6
82.0
72.6
88.0
63.5
60.2
70.2
64.9
57.2
70.6
60.1
50.6
63.5
77.9*
66.6
78.9
Data are reported as median, 25% and 75% range. *Statistically different
from the respective control (α = 0.05).
Table 3. Volume fraction (%) of bone trabeculae in the apical, middle and
cervical alveolar thirds of control and diclofenac treated rats from 1 to 3
weeks following tooth extraction.
Trabecular Bone
1 week
Control Treated
Apical
Median
25%
75%
Middle
Median
25%
75%
Cervical
Median
25%
75%
2 weeks
Control
3 weeks
Treated
Control
Treated
9.5
8.2
11.4
6.9
6.6
8.2
24.2
17.9
27.2
6.8*
6.0
9.3
30.2
28.9
39.0
22.0*
18.6
25.2
17.4
16.9
22.0
12.8*
7.6
15.0
36.2
30.4
37.9
23.5*
23.3
27.2
42.7
37.8
43.0
31.5*
26.1
35.0
12.4
8.0
13.6
6.7
5.2
9.8
32.4
28.4
37.2
26.2
23.3
30.5
37.0
36.3
48.0
22.1*
19.3
28.6
Data are reported as median, 25% and 75% range. *Statistically different
from the respective control (α = 0.05).
Braz Dent J 13(2) 2002
matrix which, in addition to newly formed capillaries, is characteristic of granulation tissue.
The macrophages are essential for clot remission as this tissue undergoes maturation with the
progressive apposition of collagen and production of bone matrix by osteoblasts (15,16).
Despite the unequivocal evidence that
the NSAIDs may delay the recovery of damaged
tissues, the precise mechanism of this deleterious action is not fully understood and it is
generally accepted that different NSAIDs may
act differently (6). The inhibitory effect of indomethacin on tooth extraction wound healing
in rats has been attributed to a probable interference with osteoblast and osteoclast function (7).
Considering that the presence of optimal inflammation is essential for proper wound healing, it has been proposed that the NSAIDs
ibuprofen and diclofenac hinder rat skin repair
by virtue of depressing the inflammatory process (17). Lindholm and Tornkvist (4) suggested
that the inhibitory effect of ibuprofen on new
bone formation following tibial fracture in rats
may be explained as a direct effect on boneforming cells on endosteal surfaces. Comparing
the depressing effects of dexamethasone (representative of SAID) and indomethacin (representative of NSAID) on rat parietal bone healing,
further evidence has been provided that the
former has a greater deleterious effect, probably
due to interference with calcium metabolism
(increased bone resorption caused by reduction
of calcium absorption) which results in impairment of acid mucopolysaccharide removal and
delay of trabecular bone mineralization (10).
The weaker inhibitory effect of indomethacin,
on the other hand, seems to occur at an earlier
stage than that of bone mineralization. Altman
et al. (6) reported that both ibuprofen and indomethacin retarded femoral fracture healing in
rats, as determined by mechanical testing and
histologic analysis, and discussed their results
on the basis of the inhibitory effect of NSAIDs
on prostaglandin E-2 and local growth factor
production.
By the end of week 3, alveolar bone
healing in diclofenac-treated rats was hindered
not only by larger remnants of blood clot but
Effect of diclofenac on tooth socket healing in rats
95
Figure 1. Alveolar wound healing 7 days (I and II), 14 days (III and IV) and 21 days (V and VI) after tooth extraction. I, III and V –
controls; II, IV and VI – treated with diclofenac. c = connective tissue, b = bone, bc = blood clot. H&E; original magnification = 56X.
also by the persistence of a greater volume fraction of
connective tissue. The latter phenomenon may be caused
by an impaired collagen synthesis/degradation ratio, as
proposed by Abdalla et al. (2) to explain delayed repair
in experimentally damaged myometrium in rats. The
authors reported data showing that indomethacin may
inhibit collagenase output by macrophages in vitro, an
effect which is reversed by the addition of E2-prostaglandin to the culture.
Even though the animal used in this study was
the rat, the results revealed significant information
associated to a therapy widely used in clinical dentistry.
RESUMO
Yugoshi LI, Sala MA, Brentegani LG, Lamano Carvalho TL.
Estudo histométrico do reparo alveolar após extração dental em
ratos tratados com diclofenaco. Braz Dent J 2002;13(2):92-96.
O objetivo do presente trabalho foi investigar se a administração
de diclofenaco interfere no processo de reparo alveolar em ratos.
Braz Dent J 13(2) 2002
96
L.I. Yugoshi et al.
foram utilizados 42 ratos Wistar, dos quais 21 receberam 10mg/
kg/dia de diclofenaco e o restante solução salina. Os animais
foram submetidos a extração dos incisivos superiores direito, e
sacrificados 7, 14 e 21 dias após a avulsão dental. A formação
progressiva de osso e a redução do volume de coágulo sangüíneo
e tecido conjuntivo no período de 1 a 3 semanas após a extração
dental foram quantificadas através do método histométrico de
contagem de pontos. O tratamento com o diclofenaco causou
significante atraso na neo-formação óssea, associado a um atraso
na organização e remissão do coágulo sangüíneo.
Unitermos: diclofenaco, alvéolo dental, exodontia.
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Accepted October 16, 2001
Braz Dent J 13(2) 2002
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