A double-blind randomized clinical trial of subgingival minocycline for chronic periodontitis.

Abstract

The purpose of this study was to evaluate the presence of A. actinomycetemcomitans, P. gingivalis, P. intermedia, E. corrodens and F. nucleatum in 30 subjects with chronic periodontitis treated by scaling and root planing (SRP) plus minocycline (test group) during 12 months with regular trimester maintenance care. Additionally, we evaluated whether the beneficial effects of the therapy on the microbial flora persisted for 24 months. The test group (n = 15) and the control group [SRP plus placebo (n = 15)] were randomly assigned. After SRP, subjects received minocycline or placebo at the baseline, and at 3, 6, and 9 months at all sites with a periodontal pocket depth (PD) of >or= 6 mm. Moreover, two homologous teeth, initially PD >or= 6 mm, were clinically and microbially monitored by PCR at the baseline, and at 3, 6, 9, 12 and 24 months. Differences in mean PD values between groups were analyzed by Student's t-test (P < 0.05). The results for bacterial frequencies showed no significant differences between groups (Fisher's Exact test, P < 0.05) or between time-points (Friedman test, P < 0.05). We failed to detect any differences between groups related to the presence of target pathogens for 12 months. The effects of both therapies on the microbial flora did not persist for 24 months. The group without supportive periodontal therapy showed an improvement in the pattern of pathogens with either of the therapies.

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Abstract:The purpose of this study was to evaluate
the presence of A. actinomycetemcomitans, P. gingivalis,
P. intermedia, E. corrodens and F. nucleatum in 30
subjects with chronic periodontitis treated by scaling
and root planing (SRP) plus minocycline (test group)
during 12 months with regular trimester maintenance
care. Additionally, we evaluated whether the beneficial
effects of the therapy on the microbial flora persisted
for 24 months. The test group (n = 15) and the control
group [SRP plus placebo (n = 15)] were randomly
assigned. After SRP, subjects received minocycline or
placebo at the baseline, and at 3, 6, and 9 months at
all sites with a periodontal pocket depth (PD) of ≥6 mm.
Moreover, two homologous teeth, initially PD ≥ 6 mm,
were clinically and microbially monitored by PCR at
the baseline, and at 3, 6, 9, 12 and 24 months. Differences
in mean PD values between groups were analyzed by
Student’s t-test (P < 0.05). The results for bacterial
frequencies showed no significant differences between
groups (Fisher’s Exact test, P< 0.05) or between time-
points (Friedman test, P< 0.05). We failed to detect any
differences between groups related to the presence of
target pathogens for 12 months. The effects of both
therapies on the microbial flora did not persist for 24
months. The group without supportive periodontal
therapy showed an improvement in the pattern of
pathogens with either of the therapies. (J. Oral Sci. 50,
259-265, 2008)
Keywords: minocycline hydrochloride; bacteria;
periodontitis; polymerase chain reaction.
Introduction
It has been estimated that about 500 species of bacteria
inhabit the human oral cavity (1,2,3). While the majority
of these organisms are commensals, a subset are likely to
be opportunistic pathogens that can cause systemic disease.
Consequently, it is important to know that microorganisms
are present in the oral cavity for the diagnosis and treatment
of systemic as well as oral diseases. Recently (4), on the
basis of the full 16S rRNA sequences for all cultivable and
yet uncultivated species that colonize the oral cavity, over
600 species have been characterized.
Periodontal therapy is directed primarily towards
reducing the number of pathogenic microorganisms in
contact with periodontal tissues, because the role of
microorganisms in the development and pathogenesis of
periodontal disease is well established (5). It has been
shown that scaling and root planing is an effective
mechanical approach for reducing bacterial colonization
to sub-threshold values and also for controlling the
progression of periodontal disease. However, thorough
subgingival scaling and root planing is technically
demanding, as access to and visibility of the area are
required, so that complete subgingival plaque and calculus
removal is rarely achieved and sometimes ineffective (6),
and may not eradicate species that can reach epithelial cells
Journal of Oral Science, Vol. 50, No. 3, 259-265, 2008
Original
Correspondence to Dr. José Roberto Cortelli, Rua Professor
Nelson Freire Campelo, 343 Jardim Eulália, Taubaté, SP - Cep
12010-700 Brazil
Tel: +55-12-3631-2373
Fax: +55-12-3632-2947
E-mail: jrcortelli@uol.com.br
A double-blind randomized clinical trial of subgingival
minocycline for chronic periodontitis
José R. Cortelli1), Davi R. Aquino1), Sheila C. Cortelli1), Jonas Carvalho-Filho2),
Caio V. G. Roman-Torres1)and Fernando O. Costa3)
1)Department of Dentistry, Periodontics Research Division, University of Taubaté, Taubaté, SP, Brazil
2)Institute of Basic Sciences, Molecular Biology Division, University of Taubaté, Taubaté, SP, Brazil
3)Department of Dentistry, Periodontics Research Division, Minas Gerais Federal University, Belo Horizonte,
MG, Brazil
(Received 27 November 2007 and accepted 6 June 2008)

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and subepithelial connective tissues of the periodontium
(7).
Microencapsulated minocycline hydrochloride in
conjunction with scaling and root planing seems to provide
significantly greater probing depth reduction than scaling
and root planing alone (8,9). Previously, our group
conducted a clinical and microbiological short-term study
(from baseline to 120 days), to evaluate the presence of
Actinobacillus actinomycetemcomitans, Porphyromonas
gingivalis, Prevotella intermedia, Tannerella forsythia,
Campylobacter rectus, Eikenella corrodens and
Fusobacterium nucleatum and the clinical response to
scaling and root planing associated with subgingival
minocycline, in 36 subjects with chronic periodontitis
(10). The results indicated that scaling and root planing
plus subgingival minocycline was as effective as scaling
and root planing plus placebo in reducing both the
prevalence of target periodontal pathogens and probing
depth. Later, we conducted another study focusing only
on clinical data, but applying a longitudinal design (baseline,
3, 6, 9, 12 and 24 months). In the later investigation, the
clinical response to scaling and root planing with the use
of locally delivered minocycline microspheres was
monitored in 26 individuals with chronic periodontitis
(11). We observed that scaling and root planing plus
minocycline achieved a higher reduction of periodontal
pocket depth in 9 and 12 months than scaling and root
planing plus placebo.
Supported by our previous results, we hypothesized
whether the profile of longitudinal clinical response to
scaling and root planing plus minocycline could be related
to subgingival microorganisms. Thus, the first purpose of
this double-blind randomized clinical and microbial trial
was to evaluate the presence of A. actinomycetemcomitans,
P. gingivalis, P. intermedia, E. corrodensand F. nucleatum
in subjects with chronic periodontitis treated with
minocycline adjunctively to scaling and root planing during
12 months with trimester maintenance care visits. In
addition, we had the opportunity to evaluate whether the
beneficial effects of the test therapy on microbial flora
persisted for 24 months.
Materials and Methods
Study design and clinical procedures
In 2002, a total of 59 subjects were recruited to participate
in this double-blind randomized clinical and microbial
study, all of whom were diagnosed as having advanced
chronic periodontitis. However, 11 subjects left the study,
and 18 did not comply with the study criteria; therefore,
30 subjects remained to the end of the study in 2005.
The study population received regular supportive
periodontal therapy, at intervals of three months, for a
total of 12 months. During this period, general dental
treatment was offered to all participants. Thereafter, all the
subjects were reminded about their periodontal
appointments every trimester in the second year. However,
since dental treatment had finished, some patients who had
no dental pain or required no esthetic procedures considered
that no further visits to the dental clinic were justified. After
24 months had passed, we were finally able to recruit
only 30 subjects, aged 25 to 69 years (mean age 46.5 ±
12.2 years) out of the initial 59 who had received only
supportive periodontal therapy during the first year. This
unique condition offered us the opportunity to evaluate the
study population with or without supportive periodontal
maintenance care.
Data and personal information related to the medical and
dental histories of the subjects were obtained by
questionnaire. All subjects provided written informed
consent, and the study was reviewed and approved by the
institutional review board at the University of Taubaté, São
Paulo, Brazil (protocol 013/02).
Subjects were excluded if they (1) had a chronic medical
disease or condition; (2) required antibiotic premedication
prior to dental treatment; (3) had taken antibiotics within
6 months prior to the clinical examination; or (4) had
received periodontal therapy within the previous 6 months.
Periodontal and radiographic examinations were done
to determine the periodontal status of all subjects, based
on the criteria described by Armitage (12), including
gingival redness, bleeding on probing, probing depth, and
clinical attachment level measurements. During periodontal
examination, one blinded, trained and calibrated examiner
measured full-mouth periodontal probing depth (PD) at
six sites per tooth, using a manual periodontal probe
(PQWBR - Hu Friedy Mfg. Inc. Chicago, IL, USA). We
analyzed the reproducibility of these measurements using
kappa-statistics, and the examiner was considered calibrated
when k ≥ 0.80.
Each subject received local anesthesia followed by full-
mouth scaling and root planing by a trained periodontist
both supra- and subgingivally according to individual
need. Before scaling and root planing (pre-experimental
period), subgingival plaque samples were obtained from
the selected sites (two non-molar homologous teeth
presenting PD ≥6 mm associated with bleeding on probing
and clinical attachment loss, without furcation involvement)
in each participant to establish the microbiological baseline
data.
At the end of scaling and root planing (baseline), two
groups were randomly allocated; one was a test group (n
= 15 subjects) that received locally delivered minocycline

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(Arestin™, OraPharma, Inc., Warmister, PA, USA) in all
sites PD ≥6 mm at this time and also at 3, 6, and 9 months,
and the other was a control group (n = 15 subjects) that
received placebo (polymer without minocycline -
Byofórmula, Farmácia de Manipulação, São José dos
Campos, SP, Brazil). Likewise, placebo polymer was
applied at all sites PD ≥ 6 mm at the same time-points,
i.e., at the baseline, 3, 6, and 9 months. Only one examiner
assigned participants to the test or control group and the
subjects were also blinded to minocycline or placebo
treatment. In addition, a trained examiner evaluated plaque
index (PI) (13) at the baseline and 3, 6, 9, 12 and 24
months.
The presence of A. actinomycetemcomitans, P. gingivalis,
P. intermedia, E. corrodens and F. nucleatum was
determined in samples from the selected periodontal
pockets of both the test and control groups at the baseline,
and 3, 6, 9, 12 and 24 months.
Microbial sampling and laboratory procedures
Subgingival plaque samples were obtained using sterile
paper points (Fine, Johnson and Johnson, New Brunswick,
NJ, USA) inserted to the depth of the pockets after removal
of supragingival plaque using a sterile curette. The paper
points were removed after 10 seconds, and immediately
placed in phosphate-buffered saline (pH 7.4) (Promega,
Madison, WI, USA). The bacterial cells were dispersed
by vortexing at maximal setting for 1 min and then
maintained at -20°C until laboratory processing.
In the laboratory, the bacterial suspensions were thawed,
centrifuged at 12,000 × g for 3 min, and the DNA was
extracted from the pellet (Instagene purification matrix,
BioRad Laboratories, Hercules, CA, USA). After
centrifugation to remove cell debris, the supernatant was
used for polymerase chain reaction (PCR) analysis. Briefly,
5 µl of the sample was added to 45 µl of reaction mixture
containing 5 µl of 10 × PCR buffer (Promega, Madison,
WI, USA), 1.25 unit of Taq DNA polymerase (Promega,
Madison, WI, USA), and 0.2 mm of each deoxy-
ribonucleotide (Pharmacia LKB, Piscataway, NJ, USA).
PCR amplification was performed using a thermal cycler
(Perkin Elmer Cetus, Wellesley, MA, USA) under standard
conditions.
PCR products were analyzed using 1.5% agarose gel
(Sigma, Dorset, UK) electrophoresis performed at 4 V/cm
in Tris-acetate EDTA buffer (Promega, Madison, WI,
USA). The gel was stained with 0.5 µg/ml ethidium
bromide (Amershan, Airlington Heights, USA) and
photographed under 300 nm ultraviolet light.
Statistical analysis
Mean values of periodontal probing depth and PI scores
between the test and the control groups were analyzed
statistically by Student’s ttest. Differences were considered
statistically significant at P < 0.05. The association of
frequencies of A. actinomycetemcomitans, P. gingivalis,
P. intermedia, E. corrodensand F. nucleatumbetween the
test and the control groups were analyzed using Fisher’s
exact test. Moreover, bacterial data analysis included
comparisons between groups by Mann-Whitney test and
also between time-points determined by Friedman test. All
analyses were performed using Statistical Package for
Social Sciences release 12.0 (SPSS Inc. Chicago, USA).
Results were considered significant at P < 0.05.
Results
Table 1 shows data related to the profile of periodontal
probing depth (PD) from the baseline until 24 months. At
the baseline the mean PD values for the test and control
groups did not differ. When PD values were compared, there
was a bigger reduction in the test group in the 9th (P =
0.0188) and in the 12th (P= 0.0470) months, however, no
significant differences were observed at 3, 6 and 24 months.
When we considered the mean PD values longitudinally,
in the test group we observed significant reductions of PD
from the baseline (A) to 3 months (C), from 6 (CD) to 9
Table 1 Mean PD values (in mm) comparing control and test groups in each time (expressed by P values) and
compared longitudinally (from baseline to 24 months)

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and 12 (D) months, and a significant increase of PD in the
24th (B) month. In the control group we also observed
significant reductions of PD from the baseline (A) to 3
months (C), no significant difference from 3 (C) until 12
months (C), and a significant increase of PD in the 24th
(B) month.
Fig. 1 Comparison of mean Plaque Index values between the control and test groups at the baseline and 3,
6, 9, 12 and 24 months.
* Statistically significant difference (P < 0.05); ‡ No statistically significant difference
Table 2 Frequencies of bacteria in test and control groups at baseline, 3, 6, 9, 12, and 24 months

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The PI showed a statistically significant reduction in the
test group (P < 0.05) from the baseline to 3 months. No
statistically significant difference was observed in PI
between 3 and 12 months; however, in the 24th month, a
significant increase was observed. In the control group, a
statistically significant reduction was observed at the
baseline and at 3, 6, 9 and 12 months, followed by an
increase in the 24th month (Fig. 1).
With regard to frequencies of bacteria (Table 2), the
results showed no significant differences either between
groups or between time-points (Table 3) from the baseline
to 24 months (Mann-Whitney test).
In the control group, differences among time-points
related to P. gingivalis were not observed. Similar results
were found for E. corrodens in the test group and also for
F. nucleatum in both groups. Mean values for A.
actinomycetemcomitansand P. intermediadecreased from
6 months onwards. For P. gingivalis in the test group and
E. corrodens in the control group, mean values started to
decrease earlier, from 3 months until 12 months. However,
in the 24th month these values increased, becoming similar
to those reached in the 3rd month.
Discussion
Since bacteria represent the most important etiologic
factor for periodontitis, the purpose of periodontal treatment
is to suppress or eliminate target species from supra- and
subgingival areas, allowing recolonization by beneficial
bacterial species. Thus, the anti-infection approach in the
active phase of periodontal treatment consists of a number
of measures to reduce the total bacterial load. Deep
periodontal pockets that remain after full-mouth scaling
and root planing and oral hygiene instructions are treated
by surgery or other treatment strategies, including the use
of systemic and/or local antibiotic therapy. Additionally,
the frequency of maintenance care is an essential factor
for long-term success of periodontal therapies, depending
on a patients’ susceptibility to periodontal disease. Intervals
ranging from two weeks to six months have been used by
clinicians (14).
Taking anti-infective periodontal therapy into con-
sideration, the first purpose of this study was to investigate
whether scaling and root planing accompanied by
subgingival use of minocycline could result in improved
microbial outcomes. In order to do this, we monitored data
related to the frequencies of A. actinomycetemcomitans,
P. gingivalis, P. intermedia, E. corrodensand F. nucleatum
during 12 months. In addition we re-evaluated the oc-
currence of these pathogens after 1 year without supportive
periodontal therapy. The decision to use a local drug
delivery system in combination with scaling and root
planing was supported by evidence from the dental literature
(5,8,9,10,11,15-17), indicating that as probing depth
increases, scaling and root planing alone becomes less
efficient.
The initiation and progression of periodontal disease is
attributed to the presence of elevated counts of pathogenic
bacteria within the gingival crevice. Likewise, periodontal
pathogens such as P. gingivalis, A. actinomycetemcomitans,
T. forsythia, Treponema denticola and E. corrodens have
been associated with chronic periodontitis (18). Many
researchers agree that the success of periodontal therapy
depends on the reduction of these microorganisms in
subgingival areas.
In the present study, P. gingivaliswas the most prevalent
bacterium at the baseline in both the test and the control
groups. This finding is in agreement with other studies
showing that P. gingivalis is one of the most important
pathogens in advanced periodontitis based on its high
prevalence in progressive periodontitis lesions (19,20),
and that this organism should be considered a key pathogen
in destructive periodontal disease. Indeed, previous data
from Brazilian and Chilean cohorts showed that P. gingivalis
was present in a higher proportion of periodontal pockets
Table 3 Mean bacteria values according to time-points in test and control groups

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in groups with advanced chronic periodontitis (21).
Another finding verified in our study was that in the 9th,
12th and 24th months, the most prevalent pathogen in the
test and control groups was E. corrodens. Ebersole et al.
(22) reported that E. corrodens was associated with sites
in periodontal pockets, whereas another study (23)
demonstrated a weak association with chronic periodontitis,
and a third study (24) showed that there was no significant
difference between active and inactive sites in the
distribution of E. corrodens. In 2002, Avila-Campos and
Velasques-Mendez (25) evaluated the prevalence of putative
periodontopathogens in periodontal patients and healthy
subjects and found that E. corrodensand F. nucleatumwere
not significantly associated with periodontal disease. Our
results are in contrast with their findings because we found
high prevalence of E. corrodensat both baseline and post-
therapy, despite the lack of maintenance care. This contrast
is especially reinforced by findings related to our control
group due to the observed increasing in E. corrodens
prevalence besides PD increasing.
In 1993, a study (15) reported that concentrations of P.
gingivalisand P. intermediawere lower after minocycline
ointment treatment than after scaling and root planing in
combination with placebo ointment at 2, 4, 6, and 12
weeks post-treatment, as well as the concentration of A.
actinomycetemcomitans at 6 and 9 weeks. Another study
(16) demonstrated a significant reduction in the prevalence
of periodontal pathogens over a 15-month period, but no
significant reduction was observed when local delivery of
minocycline was used. Three years later, a further study
(17) demonstrated significant differences in outcome for
several microbial species including P. gingivalis, P.
intermedia, C. rectus, T. denticola, E. corrodens, F.
nucleatum, and A. actinomycetemcomitans at various
follow-up points from month 1 to month 15 between
patients receiving adjunctive minocycline ointment and
nonsurgical scaling and root planing alone. There were
significantly greater reductions only in the P. gingivalis,
T. denticola, and C. rectus counts from the baseline, but
for the other microbial species the changes were not
significant. In general, both therapies allowed for benefits
to the microbial flora. However, in the present study when
we compared the presence of all target pathogens between
the two groups, we clearly observed no statistically
significant difference after comparisons from the baseline
to 24 months, i.e., with or without trimester supportive
periodontal therapy.
Haffajee et al. (26) reported that there appears to be a
consensus that treatment of the infectious component of
periodontal diseases achieves its maximum benefit from
3 to 6 months after therapy, and that any clinical and
microbiological benefits slowly reverse thereafter. Although
between-group differences were not reported, the microbial
reductions during the first year did not persist for 24
months.
In our study, except for A. actinomycetemcomitans(test
group) and P. intermedia (control group), all bacterial
species increased without supportive periodontal therapy
(Table 2). Probably these microbial profiles are related to
the observed increase in mean PD values (Table 1). Our
results confirm that lack of supportive periodontal therapy
results in a worsening of microbial and PD profiles. When
patients were asked why they failed to comply with 12-
month maintenance care, two principal reasons were cited:
the first was absence of pain related to periodontal disease,
and the other was related to financial costs mainly associated
with transport.
In conclusion, the data from this double-blind
randomized clinical and microbial trial failed to demonstrate
any difference between scaling and root planing, with or
without subgingival minocycline treatment, in terms of the
presence of target periodontal pathogens within 12 months.
The effects on microbial flora achieved by both therapies
did not persist for 24 months. Absence of supportive
periodontal therapy increased the subgingival presence
of pathogens with either of the therapies.
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Actinobacillus