R. Periodontia - Dezembro 2010 - Volume 20 - Número 04
THE INFLUENCE OF LOW DOSE OF DOXYCYCLINE ON
INDUCED PERIODONTITIS ASSOCIATED WITH OCCLUSAL
OVERLOAD
A influência de baixas doses de doxicilina sobre a periodontite induzida associada à sobrecarga
oclusal
Fabiana Cavallini1, Fernanda Faria Lenzi de Lemos2, Carolina de Carlo Gomes e Santos2, Daniela Martins de Souza3, Debora
Pallos4, Lucilene Hernandes Ricardo4
RESUMO
A degradação dos tecidos de suporte periodontal é o
principal evento biológico da doença periodontal. O objetivo
deste estudo foi avaliar a influencia do uso sistêmico de
baixas doses de doxiciclina (BDD) sobre o suporte ósseo
periodontal (SOP) e a perda de inserção (PI) na periodontite
induzida associada à sobrecarga oclusal em ratas. Neste
estudo foram utilizadas 25 ratas Wistar pesando 250g,
divididas em 5 grupos (n=5): GC (controle), GL (ligadura);
GO (sobrecarga oclusal); GLO (ligadura e sobrecarga
oclusal); GLOD (ligadura sobrecarga oclusal e doxiciclina). A
periodontite foi induzida pela inserção de uma ligadura ao
redor do 1º molar inferior (1MI) permanecendo por 28 dias.
Para que o 1MI ficasse em sobrecarga oclusal, as superfícies
oclusais do 2º e 3º molares superiores foram desgastadas. Os
animais do grupo GLOD receberam 0,14 mg de doxiciclina
por via oral diariamente por todo o período experimental.
Os animais foram sacrificados e as hemimandíbulas direitas
foram analisadas por meio de radiografias digitais quanto ao
SOP (%) e as hemi-mandíbulas direitas foram processadas
para a análise histopatológica da PI (mm). Os valores médios
foram submetidos à análise de variância (ANOVA/Tukey) ao
nível de 5%. Os resultados para SOP em GC (55.49±4.86)
e GO (60.03±3.37) foram iguais entre si e diferentes de GL
(46.73±4.80), GLO (51.69±2.84) e GLOD (57.76±4.50)
(p=0.001). Considerando a PI, GC (0.11±0.07) and GO
(0.12±0.10mm) foram semelhantes entre si e diferentes
de GL (0.41±0.09), GLO (0.58±0.12) e GLOD (0.54±0.12)
(p=0.0001). Em conclusão, o uso de BDD pareceu não
modular a redução de SOP ou PI em ratas com periodontite
induzida e sobrecarga oclusal.
UNITERMOS: doxiciclina, oclusão dentária, periodontia. R Periodontia 2010; 20:73-79.
1
MSc in Periodontology, Department of Dentistry, University of Taubaté, Taubaté, SP, Brazil
2
Dentistry graduate student, Department of Dentistry, University of Taubaté, Taubaté, SP, Brazil
3
PhD, Professor, Department of Dentistry of Pindamonhangaba, Pindamonhangaba, SP, Brazil
4
PhD, Professor, Department of Dentistry, University of Taubaté, Taubaté, SP, Brazil
INTRODUCTION
Periodontal disease is a multifactor inflammatory
disorder in the gingival crevice associated with
increased periodontal pathogen colonization. The
periodontium is functionally adaptable to resist and
support the pressure of masticatory forces. Some
studies have sought to identify whether smoking,
occlusal overload, diabetes and other events (Kinane,
2001; Nociti et al. 2000), such as environmental and
host risk factors, are involved with in the onset and
progression of periodontal disease. Occlusal overload
associated with periodontal disease may produce
irreversible changes in periodontal structures, leading
to the rupture and attachment loss of collagen fibers
and increased periodontal pocket depth (Carranza
2004).
Nogueira-Filho studied an experimental model
based on the occlusal trauma as a risk factor in
periodontal disease and found significant changes at
the periodontal ligament space in the furcation area of
lower molars submitted to occlusal overload, ligature
and nicotine use. The onset of periodontal disease
occurs near the gingival crevice and initially involves
Recebimento: 07/10/10 - Correção: 04/11/10 - Aceite: 29/11/10
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the protective periodontium. This injury progresses towards
the destruction of the supporting periodontium, which is
characterized by bone resorption, connective attachment loss
and apical migration of the junctional epithelium (NogueiraFilho et al. 2004).
The purpose of periodontal therapy is to control
bacterial infection and to control the inflammatory process
using mechanical or medication treatment and directly or
indirectly modulate the host response. Tetracyclines are
proven antimicrobials that also present inhibition properties
in relation to host matrix metalloproteinase (MMP) activity.
Therefore, this medication has been used with mechanical
treatment in combined therapy for periodontal disease.
Doxycycline has been well studied, since it is the tetracycline
most capable of inhibiting collagenase activity (Ryan 1996).
Bezerra described the inhibition of bone resorption in rats
submitted to low doses of doxycycline in ligature-induced
periodontitis and observed that the process was related to
the dose administered. This decrease was marked by lower
numbers of osteoclasts and inflammatory cells in the groups
tested when compared to the control group. Histological
analysis also showed the integrity of the alveolar bone crest
in doxycycline-treated rats (Bezerra et al. 2002). In a review of
the literature, Preshaw et al. reported that long-term usage of
low doses of doxycycline were related to the improvement of
clinical parameters without bacterial resistance to tetracycline.
The ability of doxycycline to modulate the collagenase activity
has been studied as a new protocol in periodontal therapy.
Ashley also reported this modulation in a 12 week LDD therapy
protocol combined with scaling and root planning mechanical
therapy for periodontal disease. In this study, a reduction in
collagenase activity was observed at the gingival crevice for
all the treated rats after four weeks (Ashley 1999). Karimbux
et al. also observed the modulation of collagenase activity on
the connective insertion (Karimbux et al. 1998). A short-term
administration period of LDD was sufficient to reveal the
inhibitory effect in the host response at the periodontium
(Bezerra et al. 2002). However it is not knew if this inhibitory
effect can be effective on periodontal breakdown when this
process is associated with occlusal overload. The aim of this
study was to evaluate the effect of LDD on the periodontal
bone support reduction and attachment loss in female rats
with induced periodontitis associated to occlusal overload.
MATERIALS AND METHODS
Experimental design
This study was performed according to ethical guidelines
and it was submitted and approved by the Ethics Committee
in Animal Research of UNITAU under protocol number
010/2005. Twenty-five female rats (Wistar, albinus Rattus
novergicus), ten weeks-old, weighing approximately 250
g each were housed in temperature-controlled rooms and
received water and food ad libitum. They were randomly
divided into five groups: group CG, control; group LG, ligated;
group OG, presenting occlusal overload; group LOG: ligated
and presenting occlusal overload; and group LODG, ligated,
presenting occlusal overload and medicated with 0.14mg of
doxycycline.
Periodontitis induction and occlusal overload
Periodontitis was induced by the placement of cotton
thread around the first lower molar of each rat in LG and LOG
groups for 28 days. To insert the ligature, the rats anesthetized
with a general analgesic drug solution (xylazine hydrochloride,
Anasedan, Vetbrands laboratory, Jacareí, SP, Brazil) and
a muscle relaxant (ketamine base, Dopalen, Vetbrands
laboratory) in a 1 to 0.5 ml ratio, using 0.1 ml per 100 g of rat
body weight (Ricardo et al. 2006). To obtain occlusal overload
at the first lower molar, the second and third upper maxillary
molars were flattened along their occlusal surfaces without
pulp exposure in OG, LOG and ODG groups.
Drug treatment
During the periodontal disease induction period daily
doses of 0.14mg of doxycycline (0.7 mg/dl concentration) were
orally administered to the rats from group LODG once a day.
Radiographic analysis of periodontal bone support
(PBS)
After 28 days the rats were killed and the hemi-mandibles
were removed and fixed in 10% formol solution. The right
hemi-mandibles were submitted to radiographic analysis and
the left hemi-mandibles were processed for histopathological
analysis. The samples were radiographically analyzed using the
Digital RVG ® Intraoral Radiographic System (Radiovisiography,
Trophy Radiology Inc., Marietta, GA, USA) and the images
were captured with an Adopted load apparatus (CCD sensor).
The radiographs were obtained with a Gendex 765DC®
Intraoral X-ray Unit (Gendex, Dentsply Internacional, USA)
apparatus, using 0.08 seconds of exposure time and a focal
distance of thirty cm from the sensor. Points were established
for periodontal bone support measurements: the most
coronal part of the distal cusp, the top of the bone crest or
bone defect and the distal root apex (Fig 1). These points were
considered linear measurements to determine PBS (Klausen,
1989). The measurements were expressed as a percentage
of the proportional values obtained.
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Histological analysis of attachment loss (AL)
The samples were processed for histological analysis
by decalcification in 17% EDTA solution and were then
embedded in paraffin with their vestibular surface towards the
Fig 1 - The top of the bone crest or bone defect and the distal root apex .
cutting plane. Mesial to distal sections of 6 μm thickness were
made and were stained with hematoxilin-eosin to evaluate the
connective attachment loss at the distal surface of the lower
first molar. The cement-enamel junction and the insertion
of the first periodontal ligament fibers at the root cement
were chosen as reference points to the linear measurement
of AL. Histological descriptive analysis was made in every
group. Histometric and radiographic analysis were performed
using the Image Tool software for images (Windows, version
2.0,UTHSCSA, San Antonio,TX,USA). All measurements were
made blind to the group assignment of the rats
Statistical analysis
The mean values of PBS and AL of groups were analyzes
by variance analysis. The correlation of the variable was
analyzed by the Pearson correlation test. The analysis was
realized using the Biostat software version 5.0, both at a 5%
significance level.
Fig 2 - Periodontal ligament fibers were inserted at the root cementum along its extension
Fig 4 - Besides bone loss and the increase in periodontal space, some sections showed the
presence of epithelium at the top of the furcation, which means that attachment loss occurred in
this region in the LOG and LODG groups
Fig. 3 - The furcation area of the first molar was filled with bone tissue and periodontal ligament
with uniform dimensions were observed at the top of the furcation.
Fig 5 - PBS and AL revealed a weak negative correlation between themselves.
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RESULTS
the AL results also showed statistically significant differences
among the groups (p = 0.0001), and homogeneous group
formation was observed in two groups with similar mean
values in witch was not inserted the ligature (Tab 1). PBS and
AL revealed a weak negative correlation between themselves
(Fig 5).
Descriptive histological analysis
Histological analysis of the fragments from the control
group revealed the periodontium presenting a normal
appearance, with a triangle papilla at the interdental
space between the first and the second molars covered by
stratified squamous nonkeratinized epithelium. Periodontal
ligament fibers were inserted at the root cementum along
its extension (Fig. 2). The furcation area of the first molar
was filled with bone tissue and periodontal ligament with
uniform dimensions were observed at the top of the furcation
(Fig. 3). The interdental spaces from the LG, LOG and LODG
groups were filled with gingival papilla in a concave shape and
covered by stratified squamous nonkeratinized epithelium. In
these sections the epithelium was observed apically to the
cementoenamel junction and over the root cement, showing
that the attachment loss of the ligament fibrils had occurred
on the cementum in this region (Fig.3) An increase in the
periodontal ligament area was observed in the furcation
region of the first molar in the LG group. Besides bone loss
and the increase in periodontal space, some sections showed
the presence of epithelium at the top of the furcation, which
means that attachment loss occurred in this region in the
LOG and LODG groups (Fig 4).
DISCUSSION
The modulator effect of LDD on alveolar bone support
reduction and connective attachment loss that occurs in
periodontitis induced by ligature procedure associated with
occlusal overload in female rats was the main objective
of this study. The use of animal models has been an
important recourse to control the confounding variables
normally presented in human studies. Klausen B has
already commented about the importance of overcoming
limitations, difficulties and interference factors in human
studies. Some authors have also affirmed that the use of rats
in experimental studies is favorable to evaluate the influence
of certain risk factors concerning the onset and progression
of the periodontal disease (Nociti et al. 2001; Benatti et al.
2003; Nociti et al. 2004). Klausen observed that the use of
animals of similar ages was an important detail for controlling
confounding factors when the cement-enamel junction and
alveolar bone crest are considered as anatomic references
(Klausen, 1991). Based on these statements the present study
considered these criteria and the rats presented similar age
and weigh for all the experimental groups.
The induction of periodontitis by the technique of ligature
placement has been used in studies on rats by numerous
Radiographic and morphometric analysis
Analysis of the PBS results showed statistically significant
differences among the groups studied (p = 0.001), concerning
LG as de lowest PBS and OG the highest. Other groups
showed themselves with similar mean PBS values. Analysis of
Tab 1
PBS and AL analysis for each group
PBS (%)
Mean / SD
AL (mm)
Mean / SD GC
55.49 ± 4.86
BC
0.11 ± 0.07
A
GL
46.73 ± 4.80
A
0.41 ± 0.09
B
GO
60.03 ± 3.37
C
0.12 ± 0.10
A
GLO
51.69 ± 2.84
AB
0.58 ± 0.12
B
GLOD
57.76 ± 4.50
BC
0.54 ± 0.12
B
p
0.001
0.0001
PBS= periodontal bone support; AL= attachment loss; GC= control group; GL= ligature group; GO= overload group; GLO= ligature and overload group; GLOD= ligature, overload and doxycyline;
same letters represent absent of statistical difference.
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authors (Klausen 1991; Nociti et al. 2001; Galvão et al. 2003;
Gaspersic et al. 2003). Changes in the periodontal tissues
have been evaluated by radiographic (Klausen et al. 1989),
morphometric (Gaspersic et al. 2003), histometric (Nociti et
al. 2001) and molecular biology methods (Karimbux et al.
2001). In all these studies, authors have observed changes in
the alveolar bone response and connective attachment loss
(Galvão et al. 2003; Lu et al. 1999; Fernandes et al. 2007). It is
important to emphasize that the use of one specific method
alone is unable to include all the aspects of periodontal
disease and, depending on the purpose of the study, the
use of combined analysis may be applicable (Klausen et al.
1989). Thus, the present study considered the use of two
combined methods that presented complementary aspects.
The ligature-induced periodontitis produced radiographic
alterations as PBS reduction and histological changes as
attachment loss. This combination allowed evaluating of the
influence of risk factors for a specific application and regarding
therapeutic procedure on the periodontal structures.
The presence of occlusal trauma as a risk factor in
periodontal disease has been widely reported in the literature.
In a literature review, Harrel affirmed that occlusal overload
might produce irreversible alterations when associated with
the dental biofilm (Harrel. 2003). These results were confirmed
in the present studied, since the characteristics observed in
OG were similar to CG, both for PBS or AL evaluation. The
radiographic evaluation showed that the mean PBS values
for the trauma group were higher than those found for the
control group. The interdental area between the first and the
second lower molars showed alterations that occurred due
to these adaptation and extrusion events. These alterations
were noted in alveolar bone crest when ligature was not
realized as in OG. Kuhr observed in their study that a period
of up to fifteen days post-ligature placement was necessary
to observe the tissue alterations that occurred during induced
periodontitis and that after this period, the alterations
remained stable up to a 60 day observation period (Kuhr et
al. 2004). In the present study a 28 day period of induction
was enough to evaluate alveolar bone loss in rats subjected
to ligature. Moreover, this same period was also enough to
permit occlusal adaptation in those rats that were subjected
to occlusal overload, thus leading to the extrusion of the
second and third lower molars.
The main characteristic observed in the histological
evaluation was that rats subjected to occlusal overload without
ligature did not present significant attachment loss compared
to the rats from the control group. This occurred because the
absence of attachment loss is directly related to the absence of
periodontal disease, which was only observed in rats subjected
to ligature placement. This histological characteristic explains
the scenarios (Gaspersic et al. 2003) in which risk factors may
aggravate the development of periodontal disease, such as
stress or trauma, but are unable to initiate periodontal disease
alone. The results of the correlation test confirmed this fact
when observation revealed that certain rats that presented
lower SOP values did not show high AL values.
Salvi and Lang reviewed the host response modulation
involved in reducing tissues alterations that occurred during
periodontal disease and discussed the efficacy of LDD
therapeutic protocol use to modulate bone loss by means
of artificial inhibitors of MMP tissue enzymatic activity (Salvi
& Lang, 2004). The period of usage for this medication
has been studied in relation to the occurrence of bacterial
resistance (Ryan et al. 1996). When the procedure of ligature in
association with occlusal overload was compared with groups
that only received ligature, the periodontal alterations were
aggravated, although the mean values were not statistically
different. However, these results showed a tendency for higher
values in the groups that received LDD than in the group
in which medication was not administered, though these
values were statistically similar. The histological evaluation
showed that the characteristics observed in rats that received
daily doses of LDD presented a reduction in the cement
extension without the insertion of periodontal ligament
fibrils at molars that received ligature. These alterations were
also not statistically significant, thus, using this experimental
model, it was not possible to confirm the literature results
regarding the risk factor in relation to bone loss modulation
and attachment loss in experimentally induced periodontitis in
rats associated with occlusal overload. The use of this protocol
with a larger sample and the use of molecular biology, a more
sensitive method for evaluating the effects of different doses
of inflammatory mediators, may be a favorable recourse for
verifying these results and a theme for future studies to test
the efficacy of this therapeutic protocol.
CONCLUSIONS
Based on these results it was not possible to conclude
whether the use of LDD modulated periodontal bone support
reduction or attachment loss in induced periodontitis in
female rats.
ABSTRACT
Periodontium support degradation is the main biological
event in periodontal disease. The aim of this study was to
evaluate the influence of systemic use of a low-dose of
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doxycycline (LDD) on periodontal bone support (PBS) and
attachment loss (AL) in induced periodontitis associated
with occlusal overload in rats. This study used 25 female
Wistar rats weighing 250g, divided into 5 groups (n=5): CG
(control group), LG (ligature group); OG (occlusal overload);
LOG (ligature and occlusal overload); LOGD (ligature, occlusal
overload and doxycycline). Periodontitis was induced by
inserting a ligature around the first mandibular molar (1MDM)
for 28 days. In order to obtain occlusal overload at the 1MDM,
the occlusal surfaces of the second and third maxillary molars
(2MXM and 3MXM) were flattened. 0.14mg of doxycycline was
orally administered every day to the LOGD group rats during
the experimental period. The rats were sacrificed and the right
hemi-mandibles were analyzed by digital radiograph of the
PBS(%), whereas the left hemi-mandible was processed for
histopathological analysis of AL(mm). The mean values were
treated using ANOVA and the Tukey test at 5% level. Results
for PBS in the CG (55.49±4.86%) and OG (60.03±3.37%)
groups were similar to each other and statistically different
from the LG (46.73±4.80%), LOG (51.69±2.84%) and
LOGD (57.76±4.50%) groups (p=0.001). For AL, CG
(0.11±0.07mm) and OG (0.12±0.10mm) were similar to each
other and statistically different from LG (0.41±0.09mm), LOG
(0.58±0.12mm) and LOGD (0.54±0.12mm) (p=0.0001). In
conclusion, the use of LDD appears not to modulate PBS or
AL reduction in rats with periodontitis associated with occlusal
overload.
UNITERMS: doxycycline, dental occlusion, periodontics.
REFERENCES
1- Kinane DF. Causation and pathogenesis of periodontal disease.
Periodontology 2000 2001; 25: 8-20.
2- Nociti Jr HF, Nogueira-Filho GR, Primo MT, Machado MA, Tramontina
VA, Barros SP, Sallum EA. The influence of nicotine the bone loss
rate in ligature-induced periodontitis. A histometric study in rats. J
Periodontol 2000; 71(9):1460-4.
9- Preshaw PM, Hefti AF, Jepsen S, Etienne D, Walker C, Bradshaw
MH. Subantimicrobial dose doxycycline as adjunctive treatment for
periodontitis. J Clin Periodontol 2004; 31(9): 697-707.
10-Ashley RA. Clinical trials of a matrix metalloproteinase inhibitor in
human periodontal disease. SSD clinical research team. Ann N Y Acad
Sci 1999; 30(878): 335-46.
3- Carranza FA. Perda óssea e padrões de destruição óssea, In:
CARRANZA, N.T. (Org.). Carranza Periodontia Clínica. 9ª ed. Rio de
Janeiro: Guanabara Koogan, 2004. p. 316-330.
11-Klausen B, Evans RT, Sfintescu C. Two complementary methods of
assessing periodontal bone level in rats. Scand J Dent Res 1989;
97(6): 494-9.
4- Nogueira-FiLho GR, Froes-Neto EB, Casati MZ, Reis SRA, Tunes RS,
Tunes UR, Sallum EA, Nociti Jr FH, Sallum AW. Nicotine effects on
alveolar bone changes induced by occlusal trauma: a histometric study
in rats. J Periodontol 2004; 75(3): 348-52.
12-Klausen B. Microbiological and immunological aspects of experimental
periodontal disease in rats: a review article. J Periodontol 1991; 62(1):
59-73.
5- RYAN ME, RAMAMURTHY NS, GOLUB, L. M. Matrix metalloproteinases
and their inhibition in periodontal treatment. Curr Opin Periodontol
1996; 3(1):85-96.
6- Karimbux NY, Ramamurthy NS, Golub LM, Nishimura I. The expression
of collagen I and XII mRNAs in porphyromonas gingivalis – induced
periodontitis in rats: the effect of doxycycline and chemically modified
tetracycline. J Periodontol 1998; 69(1): 34-40.
7- Bezerra MM, Brito GAC, Ribeiro RA, Rocha FAC. Low-dose doxycycline
prevents inflammatory bone resorption in rats. Braz J Med Biol Res
2002; 35(5):613-6.
8- Ricardo LH, Balducci I, Vasconcelos LM, Carvalho YR. Periodontal
healing after root conditioning using ttc-hci or citric acid: a histological
study in rats. Cienc Odontol Bras 2006; 9 (2): 40-47.
13-Benatti BB, Nogueira-Filho GR, Diniz MC, Sallum EA, Sallum AW, Nociti
Jr FH. Stress may enhance nicotine effects on periodontal tissues. An
in vivo study in rats. J Periodont Res 2003; 38(3): 351-3.
14-Nociti Jr HF, Nogueira-Filho GR, Tramontina VA, Machado MAN, Barros
SP, Sallum EA, Sallum AW. Histometric evaluation of the effects of
nicotine administration on periodontal breakdown: an in vivo study.
J Periodontol 2001; 36(6): 361-6.
15-Galvão MP, Chapper A, Rosing CK, Ferreira MB, de Souza MA.
Methodological considerations on descriptive studies of induced
periodontal diseases in rats. Pesqui Odontol Bras 2003; 17(1): 56-62.
16-Gaspersic R, Stiblar-Martincic D, Osredkar J, Skalerci U. Influence of
subcutaneous administration of recombinant TNF- alpha on ligatureinduced periodontitis in rats. J Periodontal Res 2003; 38(2): 198-203.
17-Lu L-H, Lee K, Imoto S, Kyomen S, Tanne K. Histological and
78
revista perio dezembro 2010 - 4ª REV - 08-02-11.indd 78
8/2/2011 19:21:40
R. Periodontia - 20(4):73-79
histochemical quantification of root resorption incident to the
application of intrusive force to rat molars. Eu J Orthod 1999; 21(1):
57-63.
18-Fernandes MI, Gaio EJ, Oppermann RV, Rados PV, Rosing CK.
Comparison of histometric and morphometric analyses of bone
height in ligature-induced periodontitis in rats. Braz Oral Res 2007;
21:.216-221.
19-Kuhr A, Popa-Wagner A, Schmoll H, Schwahn C, Kocher T.
Observations on experimental periodontitis in rats. J Periodont Res
2004; 39(2): 101-6.
20-Harrel, S. K. Occlusal forces as a risk factor for periodontal disease.
Periodontology 2000 2003; 32:111-7.
21-Salvi GE, Lang NP. Host response modulation in the management of
periodontal diseases. J Clin Periodontol 2005; 32: 108-29.
Address
Fabiana Cavallini
Rua Independência, 743 Jardim Bela Vista
CEP: 09041-310 – Santo André - São Paulo
E-mail: [email protected]
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