I
Journal of the International
A
P Academy of Periodontology
The official journal of the International Academy of Periodontology
Volume 16 Number 2
Published by
The International Academy of Periodontology
April 2014
I
A
P
Volume 16
Number 2
April 2014
ISSN 1466–2094
Journal
of the
International Academy of
Periodontology
EDITORIAL BOARD
Mark R Patters
Editor
Memphis, TN, USA
Andrea B Patters
Associate Editor
Sultan Al Mubarak
Riyadh, Saudi Arabia
P Mark Bartold
Adelaide, SA, Australia
Michael Bral
New York, NY, USA
Nadine Brodala
Chapel Hill, NC, USA
Cai-Fang Cao
Beijing, People’s Republic of China
Daniel Etienne
Paris, France
Ahmed Gamal
Cairo, Egypt
Vincent J Iacono
Stony Brook, NY, USA
Zucchelli’s Technique or Tunnel Technique with Subepithelial
Connective Tissue Graft for Treatment of Multiple Gingival Recessions
Chanchal Bherwani, Anita Kulloli, Rahul Kathariya, Sharath Shetty,
Priyanka Agrawal, Dnyaneshwari Gujar and Ankit Desai 34
Tooth Loss Assessment during Periodontal Maintenance in Erratic
versus Complete Compliance in a Periodontal Private Practice in Shiraz,
Iran: A 10-Year Retrospective Study
Amir Haji Mohammad Taghi Seirafi, Reyhaneh Ebrahimi, Ali Golkari,
Hengameh Khosropanah and Ahmad Soolari 43
Treatment of Amalgam Tattoo with a Subepithelial Connective Tissue
Graft and Acellular Dermal Matrix
Vivek Thumbigere-Math and Deborah K. Johnson 50
Gingival Crevicular Fluid Bone Morphogenetic Protein-2 Release Profile
Following the Use of Modified Perforated Membrane Barriers in Localized
Intrabony Defects: A Randomized Clinical Trial
Ahmed Y. Gamal, Mohamed Aziz, Salama M.H., Vincent J. Iacono
55
Isao Ishikawa
Tokyo, Japan
Georges Krygier
Paris, France
Hamdy Nassar
Cairo, Egypt
Rok Schara
Ljubljana, Slovenia
Uros Skaleric
Ljubljana, Slovenia
Shogo Takashiba
Okayama, Japan
Thomas E Van Dyke
Boston, MA, USA
Warwick Duncan
Dunedin, New Zealand
Nicola Zitzmann
Basel, Switzerland
The Journal of the International Academy of Periodontology is the official journal of the International Academy of Periodontology
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Produced in Great Britain by Dennis Barber Limited, Lowestoft, Suffolk
Journal of the International Academy of Periodontology 2014 16/2: 34–42
Zucchelli’s Technique or Tunnel Technique
with Subepithelial Connective Tissue Graft
for Treatment of Multiple Gingival Recessions
Chanchal Bherwani, Anita Kulloli, Rahul Kathariya, Sharath
Shetty, Priyanka Agrawal, Dnyaneshwari Gujar and Ankit Desai
Department of Periodontics and Oral Implantology, Dr. D. Y
Patil Dental College and Hospital, Dr. D. Y Patil Vidyapeeth
(Deemed University), Pune-411018, Maharashtra, India
Abstract
Background: Gingival recession is both unpleasant and unesthetic. Meeting the esthetic
and functional demands of patients with multiple gingival recessions remains a major
therapeutic challenge. We compared the clinical effectiveness of Zucchelli’s technique
and tunnel technique with subepithelial connective tissue graft (SECTG) for multiple
gingival recessions.
Methods: Twenty systemically and periodontally healthy subjects having 75 recession
defects (Miller’s class I or II, 39 test and 36 control sites) were included. After initial
nonsurgical therapy, test sites were treated with Zucchelli’s technique and control sites
with tunnel technique with SECTG. Plaque index, bleeding index, pocket depth, recession depth, clinical attachment level, and keratinized gingiva height were evaluated at
baseline, 3 and 6 months post-surgery.
Results: The mean root coverage was 89.33% ± 14.47% and 80.00% ± 15.39% in the
test and control groups respectively, with no significant difference between groups.
Statistically significant root coverage was obtained for 82.50% ± 23.72% and 71.40%
± 20.93% of defects in the test and control groups, respectively.
Conclusion: Zucchelli’s technique is effective for the treatment of multiple adjacent
recessions in terms of both root coverage and keratinized tissue gain, irrespective of the
number of defects. Moreover, this technique does not require an additional surgical site
as required in the gold standard SECTG.
Key words: Multiple gingival recessions, Zucchelli’s technique, connective
tissue graft, envelope technique
Introduction
Gingival recession is defined as the apical displacement
of the gingival margin in relation to the cementoenamel
junction (CEJ, Glossary of Periodontology Terms, AAP,
2001). It is a common occurrence in individuals with
poor oral hygiene as well as those with good oral hygiene, and it usually affects multiple teeth simultaneously.
Occurrence in the anterior regions of the mouth leads
to compromised esthetics. Therefore, many patients
request cosmetic correction (Marmar et al., 2009) and
Correspondence to: Dr. Rahul Kathariya, Department of Periodontics and Oral Implantology, Dr. D. Y. Patil Dental College
and Hospital, Dr. D. Y. Patil Vidyapeeth (Deemed University),
Pune-411018, Maharashtra, India. Tel: +918983370741.
E-mail: rkathariya@gmail.com
© International Academy of Periodontology
meeting their esthetic and functional demands remains a
major therapeutic challenge (Philipe et al., 2009). Several
surgical approaches for covering exposed root surfaces,
including free gingival graft placement (Miller, 1985),
the coronally advanced flap (CAF; Harris et al., 1995),
subepithelial connective tissue graft (SECTG) placement
(Langer and Langer, 1985; Paoloantonio et al., 1997),
the Langer and Langer technique (Langer and Langer,
1985) and guided tissue regeneration (Pini et al., 1996)
have been proposed in the last few decades.
The CAF is the first choice of surgical technique in
cases with adequate keratinized tissue apical to the defect.
It results in optimum root coverage, good color blending
with respect to adjacent soft tissues, and good recovery of
original soft tissue morphology. In most cases, SECTG is
used in combination with CAFs. However, it necessitates
vertical incisions on the buccal gingiva, which hampers
Bherwani et al.: Zucchelli technique or SECTG for multiple root coverage
blood supply and early esthetic recovery. To avoid these
incisions on the recipient site, the envelope technique
was advocated. The advantage of this procedure is the
fast early healing that results from the absence of these
external incisions (Zabalegui et al., 1999).
Subepithelial connective tissue graft placement
reportedly shows increased predictability of total root
coverage and is regarded as the standard approach for
the management of multiple gingival recessions (Langer
and Langer, 1985). Chambrone et al. (2008) reported a
systematic review that included 23 clinical trials on Miller’s
class I and II recession defects treated with SECTG with
at least 10 participants per group. The authors concluded
that SECTG provided significant root coverage, clinical
attachment and keratinized tissue gain, and stated that
SECTG is considered the “gold standard” procedure
in the treatment of recession-type defects. The same
authors, in their consecutive Cochrane systematic reviews
in 2009 and 2010, stated that cases where both root coverage and keratinized tissue gain are expected, the use
of SECTG seems to be ideal. Dembowska et al. (2007)
stated that connective tissue grafts (CTGs) in combination with tunnel surgical techniques in the treatment of
multiple adjacent gingival recessions resulted in significant
root coverage of both class I and class II recessions, and
increased keratinized gingival width.
It is important to note that the use of grafts in procedures involving root coverage gingival augmentation
and aesthetics are always associated with complications.
Harris et al. (2005) evaluated the incidence and severity of complications that occur after connective tissue
grafts for root coverage or gingival augmentation (n =
500). The authors evaluated certain factors that could
influence the rate of complications, including age, sex
of patient, smoking status, purpose of the graft (i.e., for
root coverage or for gingival augmentation), size of the
recipient area, and location of the defect being treated.
Complications evaluated included pain, bleeding, infection and swelling. The authors concluded that none of
the factors evaluated in this study were associated with
a statistically significant increase in the rate or intensity
of complications, and the incidence and severity of
complications seemed to be clinically acceptable.
In 2000, Zucchelli and De Sanctis demonstrated
promising results with a new surgical approach (Zucchelli’s technique; modification of the CAF) to treat
multiple recession defects affecting adjacent teeth. To
our knowledge, no study has compared the clinical effectiveness of Zucchelli’s technique with that of techniques that use SECTGs for the treatment of multiple
recession defects. This study compared the clinical
effectiveness of Zucchelli’s technique with that of the
tunnel technique with SECTG placement for the treatment of multiple gingival recessions affecting adjacent
teeth in the esthetic areas of the mouth.
35
Materials and methods
This study included 20 age- and sex-matched subjects
(18 to 55 years) who were systemically and periodontally
healthy and had a minimum of two recession (Miller’s
class I or II) defects affecting adjacent teeth in the
esthetic areas of the maxilla. Subjects were recruited
from the outpatient section of the Department of Periodontology & Oral Implantology, Dr. D. Y. Patil Dental
College & Hospital, Pimpri, Pune. The study design was
approved by the Institute’s Scientific and Ethical Committee. Written informed consent was obtained from
subjects who voluntarily agreed to participate after a
detailed explanation of the study was provided to them.
Affected teeth included those between 15 (maxillary 2nd
right premolar) and 25 (2nd left premolar). All subjects
demonstrated acceptable oral hygiene. Ten participants
were allocated to each group (n = 20), which comprised
a total of 75 recession defects. The power of the study
was calculated based on comparing means of our two
study groups, and was 80% at a confidence interval of
95% with a sample size of 10 per group. Participants
were randomized into each group based on a computergenerated list. The test site included 39 defects, which
were treated by Zucchelli’s technique, and 36 control
sites, which were treated by the tunnel technique with
SECTG placement. The control sites were selected in
subjects with medium to deep palatal vaults so that adequate graft material could be obtained. Exclusion criteria
included the following: a history of prolonged use of
antibiotics, steroids, immunosuppressive agents, aspirin,
anticoagulants, or other medications that influence the
periodontium; systemic diseases, such as diabetes, hypertension, HIV, cancer, and metabolic bone diseases;
radiation therapy and immunosuppressive therapy;
tobacco consumption; unacceptable oral hygiene; faulty
tooth brushing technique; labially positioned teeth; teeth
with prominent roots; and pregnancy.
Before surgery, a planned case history was recorded,
followed by a complete periodontal evaluation. A
complete haemogram was also obtained. Scores of the
plaque index (Silness and Loe, 1964) and bleeding index
(Loe and Silness, 1963) were calculated. Recession depth
(RD) was measured from the CEJ to the most apical
extension of the gingival margin. Probing depth (PD)
was measured from the gingival margin to the base of
the gingival sulcus. Keratinized gingiva height (KGH)
was measured from the gingival margin to the mucogingival junction. Recession depth, PD, and KGH were
measured using a William’s graduated periodontal probe.
All the above-mentioned parameters were recorded on
the standardized chart at baseline and 3 and 6 months
after surgery. Following initial examination, all subjects
received oral prophylaxis and oral hygiene instructions.
A coronally directed roll brushing technique was advised
for teeth with recession defects in order to minimize
36
Journal of the International Academy of Periodontology (2014) 16/2
brushing trauma to the gingival margin. Surgical treatment was scheduled once the patient demonstrated
adequate supragingival plaque control (Zucchelli and
De Sanctis, 2000).
To ensure adequate intra-clinician reproducibility, a
previously trained clinician (CB) performed all surgeries
in both groups, and all pre- and post-treatment clinical parameters and analyses were recorded by another
examiner (AK), who was blinded to the type of surgery
done. The examiner was considered calibrated once statistically significant correlation for RD, PD, and KGH
were found and statistically non-significant differences
between their duplicate measurements were obtained.
Surgical procedure
For the test group, local anesthesia was induced, following which the exposed root surfaces were planed
with a combination of hand instruments and burs to
eliminate any surface irregularities. The exposed surfaces
were conditioned with tetracycline HCI solution (100
mg/ml) for 4 minutes with a light pressure burnishing
technique as described previously (Tolga et al., 2005) following which the root surfaces were thoroughly rinsed.
A modified envelope flap (Zucchelli’s technique) was
used for the test subjects in this study. Horizontal incisions comprised oblique submarginal incisions placed
in the interdental areas with the blade parallel to the
tooth’s long axis in order to dissect the surgical papillae
in a split thickness manner. These incisions continued
with the intrasulcular incision around the defects.
Each surgical papilla was displaced with respect to the
anatomic papilla by the oblique submarginal interdental
incisions. In particular, the surgical papillae mesial to the
flap midline were displaced apically and distally, while
the papillae distal to the midline were displaced more
apically and mesially. The envelope flap was raised with
a split-full-split approach in the corono-apical direction; the surgical papillae were raised in a split thickness
manner, the gingival tissue apical to the root exposure
was raised in a full thickness manner to ensure adequate
thickness for root coverage, and the most apical portion
of the flap was elevated in a split thickness manner to
facilitate coronal flap displacement. Of the exposed root
surfaces, those that exhibited loss of clinical attachment
level (CAL; recession + gingival sulcus) were subjected
to mechanical curettage, whereas those in areas of bone
dehiscence were not instrumented to avoid damage to
any connective tissue fibers still inserted in the cementum. The remaining anatomic interdental papillae were
de-epithelialized to create the connective tissue beds to
which the surgical papillae would be sutured. A sharp
dissection into the vestibular lining mucosa was performed to eliminate muscle tension. Adequate coronal
displacement of the flap is facilitated by the elimination
of lip and muscle tension in the apical portion. During
coronal advancement, each surgical papilla was rotated
towards the end of the flap to finally reside at the center
of the interproximal area. Flap mobilization was considered adequate when the marginal flap portion could
passively reach coronally to the CEJ at each single tooth
and remain stable even without sutures. The buccal flap
was coronally repositioned without tension and precisely
adapted on the root surfaces. Each surgical papilla was
stabilized over the interdental connective tissue bed
and sling sutures were placed using 5-0 mersilk nonabsorbable sutures. [Ethicon; Johnson and Johnson PVT
LTD., Jharmajri, H.P., India] A periodontal dressing was
applied to protect the surgical area from mechanical
injury during the initial healing phase (Zucchelli and
De Sanctis, 2000)
For the control group, local anesthesia was induced,
following which a tunnel was created under the buccal
aspect of the gingival tissue. A sulcular partial thickness
incision was placed at each recession area, undermining
the tissue far beyond the mucogingival junction (MGJ)
to ensure adequate relaxation of the pedicle flap and
create an area for the connective tissue graft (CTG).
The partial dissection was extended laterally through
the papillae between the treated teeth without severing them. This incision was also extended 3 to 5 mm
mesially and distally to the area of the CTG. Great
care was taken when going through the MGJ to avoid
perforation of the flap.
Following induction of local anesthesia, a free
SECTG was harvested from the palate (premolar to
molar) using the trap door technique (Harris, 1992).
Transmucosal probing was used to ensure adequate connective tissue thickness, and a horizontal split thickness
incision was placed approximately 4 mm from the palatal
gingival margin and extended according to the mesiodistal width of the recipient site. Vertical incisions were
then placed at either end of the first incision to facilitate
access to the underlying connective tissue. The exposed
connective tissue was harvested using a scalpel and a
periosteal elevator to obtain a 1.5 to 2 mm thick graft.
The flap was then repositioned to completely cover the
donor site and sutured. The SECTG was immediately
placed over the prepared recipient site and secured in
place. The tissue flap was coronally repositioned over
the graft and secured at the level of the CEJ using
interdental 5-0 mersilk nonabsorbable sutures. A periodontal dressing was applied to protect the surgical area
from mechanical injury during the initial healing phase
(Wennström and Zucchelli, 1996)
Patients were given postoperative instructions and
prescribed antibiotics (amoxicillin, 500 mg thrice a day for
7 days; Marmar and Hom, 2009) and analgesics. A 0.2%
chlorhexidine rinse was prescribed for the early healing
phase. Sutures were removed 2 weeks after surgery. The
buccal flap usually heals without any visible surgical signs
Bherwani et al.: Zucchelli technique or SECTG for multiple root coverage
by the end of 2 postoperative weeks (Zucchelli and De
Sanctis, 2000). Oral prophylaxis was performed at regular
intervals, i.e., 1, 3, and 5 weeks after suture removal and
every 3 months thereafter until the final follow-up. All subjects were evaluated at 3 and 6 months to record the plaque
scores, bleeding scores, RD, PD, KGH, and root coverage
[Figures 1-3 (test group), Figures 4-6 (control group)]. No
patient exhibited postoperative complications.
Figure 1: Test group at baseline.
37
Statistical analysis
Results are expressed as mean ± SD for each parameter.
Data were analyzed using Student’s t-test for paired and
unpaired observations to assess changes within and
between groups (p < 0.05 was considered statistically
significant). All analyses were performed using SPSS
software version 16.10 (SPSS Inc., IBM, Chicago, USA).
Figure 4: Control group at baseline.
Figure 2: Test group at 3 months.
Figure 5: Control group at 3 months.
Figure 3: Test Group at 6 months.
Figure 6: Control group at 6 months.
38
Journal of the International Academy of Periodontology (2014) 16/2
Results
Mean plaque index scores significantly decreased after
surgery compared with those at baseline in both groups.
Mean scores in the test group decreased by 0.43 ± 0.25
after 3 months and 0.68 ± 0.24 after 6 months, whereas
those in the control group decreased by 0.42 ± 0.19 after
3 months and 0.69 ± 0.21 after 6 months (p < 0.05 for
all; Table 1) There were no significant differences in the
decrease in mean plaque index scores between the two
groups during both time intervals (Table 4).
Mean bleeding index scores significantly decreased
after surgery compared with those at baseline in both
groups. Mean scores in the test group decreased by 0.30
± 0.32 after 3 months and 0.44 ± 0.25 after 6 months,
whereas those in the control group decreased by 0.46
± 0.43 after 3 months and 0.85 ± 0.44 after 6 months
(p < 0.05 for all; Table 1). There were no significant differences in the decrease in mean bleeding index scores
between the two groups during both time intervals
(Table 4).
In the test group, the mean PD decreased by 0.05
± 0.22 mm at 3 months compared with baseline PD
(not significant). In the control group, the mean PD
decreased by 0.14 ± 0.35 mm at 3 months compared
with baseline PD; this decrease was statistically significant (p < 0.05; Table 2). At 6 months, the test and control groups exhibited mean decreases of 0.08 mm (not
significant; p > 0.05) and 0.11 ± 0.31 mm (significant; p
< 0.05), respectively (Table 2). There were no significant
differences in the decrease in mean PD between the two
groups during both time intervals (Table 4).
Table 1. Comparison between plaque scores and bleeding scores (mean ± standard deviation) at 3 and 6 months with those at baseline in the test (Zucchelli’s technique) and control
(tunnel technique with subepithelial connective tissue graft) groups.
(n = 10)
Test
Control
Mean ± SD
p value
Mean ± SD
p value
Plaque
scores
Baseline
3 months
6 months
1.01 ± 0.47
0.58 ± 0.29
0.33 ± 0.32
p < 0.01
p < 0.001
1.47 ± 0.44
0.69 ± 0.15
0.420 ± 0.20
p < 0.001
p < 0.001
Bleeding
scores
Baseline
3 months
6 months
0.80 ± 0.42
0.54 ± 0.38
0.36 ± 0.32
p < 0.01
p < 0.001
1.16 ± 0.59
0.64 ± 0.29
0.31 ± 0.25
p < 0.01
p < 0.001
Table 2. Comparison between pocket depth, recession depth, clinical attachment level and keratinized
tissue gain (mean ± standard deviation) at 3 and 6 months with values at baseline in the test (Zucchelli’s technique) and control (tunnel technique with subepithelial connective tissue graft) groups.
Parameters
Time
Interval
Test (n = 39)
Control (n = 36)
Mean ± SD
(mm)
p value
Mean ± SD
(mm)
p value
Pocket depth
Baseline
3 months
6 months
1.08 ± 0.27
1.03 ± 0.16
1.00 ± 0.00
NS
NS
1.17 ± 0.38
1.03 ± 0.17
1.06 ± 0.23
p < 0.05
p < 0.05
Recession
depth
Baseline
3 months
6 months
2.03 ± 0.81
0.54 ± 0.82
0.10 ± 0.31
p < 0.001
p < 0.001
2.22 ± 0.72
0.89 ± 0.71
0.22 ± 0.42
p < 0.001
p < 0.001
Clinical
attachment
level
Baseline
3 months
6 months
3.08 ± 0.81
1.56 ± 0.88
1.18 ± 0.45
p < 0.001
p < 0.001
3.42 ± 0.73
1.92 ± 0.73
1.31 ± 0.47
p < 0.001
p < 0.001
Keratinized
tissue gain
Baseline
3 months
6 months
4.74 ± 1.35
5.03 ± 1.14
5.31 ± 1.08
p < 0.05
p < 0.001
5.08 ± 1.34
5.20 ± 1.21
5.42 ± 1.27
NS
p < 0.001
Bherwani et al.: Zucchelli technique or SECTG for multiple root coverage
39
Table 3. Comparison of root coverage and number of patients with
complete root coverage (mean ± standard deviation) between test (Zucchelli’s technique) and control (tunnel technique with subepithelial
connective tissue graft) groups.
(n = 10)
Group
Mean ± SD
p value
Mean root
coverage (%)
Test
Control
89.33 ± 14.47
80.00 ± 15.39
NS
Proportion of sites
exhibiting complete root
coverage (%)
Test
Control
82.50 ± 23.72
71.40 ± 20.93
NS
Table 4. Comparison of all parameters measured at baseline, 3 months, and 6 months between the test
(Zucchelli’s technique) and control (tunnel technique with subepithelial connective tissue graft) groups.
Parameters
Test
(n = 39)
Control
(n = 36)
Recession
depth (mm)
Probing depth
(mm)
Clinical
attachment
level (mm)
Keratinized
tissue gain
(mm)
Plaque scores
Bleeding
scores
Baseline
3 months
6 months
Mean ± SD
p value
Mean ± SD
p value
Mean ± SD
p value
3 and 6
months
Test
Control
2.03 ± 0.81
2.22 ± 0.72
NS
0.54 ± 0.82
0.89 ± 0.71
NS
0.10 ± 0.31
0.22 ± 0.42
NS
p < 0.05
p < 0.001
Test
1.08 ± 0.27
NS
NS
NS
NS
p < 0.01
p < 0.001
NS
p < 0.001
p < 0.01
NS
p < 0.01
p < 0.001
NS
NS
p < 0.05
Control
1.17 ± 0.38
Test
3.08 ± 0.81
Control
3.42 ± 0.73
Test
4.74 ± 1.35
Control
5.08 ± 1.34
Test
1.01 ± 0.47
Control
1.47 ± 0.44
Test
0.80 ± 0.42
Control
1.16 ± 0.59
1.03 ± 0.16
NS
1.03 ± 0.17
1.00 ± 0.00
NS
1.56 ± 0.88
NS
1.92 ± 0.73
1.18 ± 0.45
NS
5.03 ± 1.135
NS
5.20 ± 1.21
NS
0.69 ± 0.15
0.50 ± 0.53
1.16 ± 0.59
At three months, the mean RD decreased by 1.49
± 0.56 mm in the test group and 1.33 ± 0.59 mm in
the control group (Figures 2 and 5, respectively) when
compared with baseline (Figures 1 and 4, respectively);
which were statistically significant (p < 0.001; Table 2).
At 6 months, the test and control groups exhibited
significant mean recession depth reduction of 1.93 ±
0.77 mm and 2.0 ± 0.72 mm, respectively (p < 0.001 for
both groups; Table 2, Figures 3 and 6). However, there
were no significant differences in the decrease in mean
RD between the two groups during both time intervals
(Table 4).
Both the test and control groups exhibited significant
mean CAL gains of 1.52 ± 0.60 mm and 1.5 ± 0.56
mm, respectively, at 3 months and 1.89 ± 0.79 mm and
1.31 ± 0.47
5.31 ± 1.08
NS
0.58 ± 0.28
NS
1.06 ± 0.23
5.42 ± 1.27
0.33 ± 0.32
NS
NS
0.42 ± 0.20
0.36 ± 0.32
0.31 ± 0.25
2.11 ± 0.70 mm, respectively, at 6 months (p < 0.001;
Table 2). There were no significant differences in mean
CAL gain between the two groups during both time
intervals (Table 4).
The mean KGH gain at 3 months was 0.29 ± 0.69
mm in the test group (significant; p < 0.05) and 0.12 ±
0.42 mm in the control group (not significant; Table 2,
Figures 2 and 4, respectively). The mean KGH gain at
6 months was 0.57 ± 0.50 mm and 0.34 ± 0.77 mm in
the test and control groups, respectively (Figures 3 and
6); both were statistically significant (p < 0.001; Table
2). However, there were no differences in mean KGH
gain between the two groups during both time intervals
(Table 4).
40
Journal of the International Academy of Periodontology (2014) 16/2
The mean percentage of root coverage was calculated using the following formula:
% root coverage = 100 × [Baseline RD − Postoperative
RD]/Baseline RD
When compared from baseline the mean root coverage at 3 months was 89.33% ± 14.47% in the test group
and 80.00% ± 15.39% in the control group. The proportion of defects that exhibited complete root coverage
was 82.50% ± 23.72% in the test group and 71.40% ±
20.93% in the control group (Table 3). There were no
statistically significant differences in either parameter
between the two groups (Table 4).
Discussion
The treatment of gingival recession is becoming an
important therapeutic issue from the viewpoint of esthetics. Improving esthetics during smiling or function
is becoming the main aim of root coverage procedures.
Gingival recession frequently affects groups of adjacent
teeth. In order to minimize the number of surgeries and
optimize the esthetic results, all the defects should be
simultaneously treated (Zucchelli and De Sanctis, 2000).
Multiple adjacent recession defects are a therapeutic challenge considering that several defects must be treated in
a single surgical session to minimize patient discomfort.
The CAF and the supraperiosteal envelope flap, along
with its modification, the so-called tunnel technique, are
most commonly employed for the treatment of multiple
recessions (Jung et al., 2008). The premolars and molars
are the most common sites of involvement (Loe et al.,
1992; Serino et al., 1994). However, Serino et al. (1994),
after 12 years of longitudinal evaluation, reported that
in subjects aged 18-29 years the incisors and maxillary
canines were the most frequently affected by recession.
Therefore, incisors, canines and premolars were selected
for the present study (Wennström and Zucchelli, 1996).
Cigarette smoking may affect the short-term outcome
of root coverage procedures and should be carefully
considered when planning periodontal plastic surgery
(Luiz and Leandro, 2006). Therefore, our study included
only nonsmokers. All root surfaces in our study were
conditioned with tetracycline HCL in accordance with a
report by Isik et al. (2000) indicating that a 50-150 mg/
ml tetracycline HCL solution resulted in a statistically
significant opening of dentinal tubules.
Among the various treatment modalities, variations
of SECTG procedures demonstrate high predictability
with a high percentage of root coverage and a low
complication rate. Root coverage achieved with SECTG
procedures remains stable over the long term. Therefore, SECTG procedures are used as a “gold standard”
for the evaluation of the safety and efficacy of new
root coverage procedures (Jung et al., 2008). However,
SECTG is most commonly used in combination with
CAFs, which necessitate buccal vertical incisions and
consequently retard early esthetic results. Therefore, the
envelope (tunnel) technique, which results in quick early
healing by eliminating the need for vertical incisions, was
advocated (Zabalegui et al., 1999).
To our knowledge, no studies have evaluated the
prevalence of single versus multiple recessions in patients with esthetic demands. Very little data regarding
the treatment of multiple recession defects are available,
and no data comparing the two procedures employed
in this study are available. Moreover, there are less data
on the use of SECTG procedures for the treatment of
multiple recession defects. “Lack of popularity may be
attributed to increased patient discomfort caused by the
harvesting of large grafts from the palate. Furthermore,
larger grafts impair the vascular exchange between the
covering flap and the underlying recipient bed, thus
increasing the risk of flap dehiscence and causing unesthetic graft exposure” as stated by Zucchelli et al. in his
classical study in 2009. Therefore, we aimed to elucidate
the effectiveness of Zucchelli’s technique in this study
using SECTG procedures as the control.
The importance of tooth brushing technique for the
long-term maintenance of clinical outcomes achieved
by root coverage procedures has been demonstrated.
Patients in this study were instructed and motivated
to perform a coronally directed roll technique to minimize toothbrush trauma and achieve optimal plaque
control (Wennström and Zucchelli, 1996). Because of
this constant motivation, plaque and bleeding scores
significantly decreased over the follow-up period in
both groups. This is in accordance with the study of
Wenstrom and Zucchelli (1996), where it was indicated
that an altered nontraumatic toothbrushing technique
was crucial for achieving successful outcomes of root
coverage procedures.
In the present study, mean PD and RD significantly
decreased while mean CAL and KGH significantly
increased 6 months after surgery in both the test and
control groups. Furthermore, statistically significant root
coverage was obtained in both groups, and the proportion of defects with complete root coverage was also
statistically significant in both groups. With regard to
the test group, all these outcomes were similar to those
reported in 1-year and 5-year studies (the latter was a
continuation of the former) by Zucchelli and De Sanctis
(2000) in another study by Zucchelli and De Sanctis
(2005). However, the outcomes in these studies were
evaluated after a longer follow-up period of (minimum
1 year). Therefore, our study showed results within 6
months when compared to these studies, which were
followed for 1 to 5 years.
With regard to the control group, there is no concrete data available concerning an increase in CAL and
a decrease in PD and RD associated with the tunnel
Bherwani et al.: Zucchelli technique or SECTG for multiple root coverage
technique with SECTG placement for the treatment of
multiple recession defects. However, it is interesting to
note that there was no significant difference in any of
the parameters evaluated 6 months after surgery between
the test group and the control group in the present study,
although the mean percentage of root coverage and the
number of patients with complete root coverage were
slightly higher in the test group than in the control group.
When comparing two different techniques, a split
mouth study design would have been ideal (Zucchelli
technique on one side and SEGTG technique on the
other. However, tissue shrinkage is different with different techniques. Also, different people have different
wound healing potential and it would compromise the
overall esthetics in such esthetic-oriented studies. Thus
we avoided split mouth design and used a parallel design
in our study. This has been mentioned as one of the
limitations of our study.
The fact that the coronally advanced procedure
resulted in an increased apicocoronal gingival height
may be explained by several events taking place during
healing and maturation of the marginal tissue. First,
there is a tendency of the mucogingival line to regain
its genetically defined position following coronal dislocation during the flap procedure, and second, it cannot
be excluded that granulation tissue derived from the
periodontal ligament tissue may have contributed to the
increased gingival dimensions.
Taken together, the present study demonstrated that
the proposed modification of the CAF, i.e., Zucchelli’s
technique, is effective for the management of multiple
recession defects affecting adjacent teeth in the esthetic
regions of the mouth. This new modification does not
involve a palatal donor site and has been demonstrated
to be a safe and predictable approach (Zucchelli and
De Sanctis, 2000). Multiple gingival recessions involving
teeth in the esthetic areas of the mouth have been successfully treated using this technique (Zucchelli and De
Sanctis, 2000). In addition, root coverage and esthetic
outcomes have been reported to be well maintained
in the long term (5 years) in patients using a correct,
non-traumatic, toothbrushing technique (Zucchelli
and De Sanctis, 2005) The presumed advantage of this
technique is the use of a flap without vertical releasing
incisions, which could otherwise damage the lateral
blood supply to the flap and result in unesthetic visible
scars (keloids; Joly et al., 2007).
On the other hand, procedures involving SECTG
placement require autogenous grafts, which results in
the creation of a second wound site, longer chair time,
higher possibility of tissue morbidity, and intra- and/
or postoperative discomfort, all of which can lower
patient acceptance (Terrence et al., 2006). Another
possible explanation for the improved results in our
study may be the strict entry criteria. Only Miller’s class
41
I and II defects with no deep cervical abrasion or root
demineralization were included. Yet another explanation
could be the design of the envelope flap, which involves
extension of the flap to one tooth mesial and distal to
the affected teeth. This influences the soft tissue margins of the neighboring teeth, thus resulting in a more
harmonious, scalloped, knife-edged outline of all teeth
belonging to the quadrant jaw.
Limitations of our study include its short-term
follow-up period (6 months), unlike the previous studies (Zucchelli and De Sanctis, 2000 and 2005; Zucchelli et al., 2009). A longer period of evaluation may
be necessary in future clinical trials to appreciate the
clinical effectiveness of this technique and to evaluate
its long-term benefits. Also, our study included Miller’s
class I and II recession defects with an average depth
of 2 mm. Moreover, we used a parallel design of study;
in comparative clinical trials a split-mouth design would
have been more appropriate to evaluate the response to
different techniques in the same patient.
Conclusion
Both the techniques employed for the treatment of
multiple recession defects in this study demonstrated
effective results in terms of both root coverage and
increase in KGH. Root coverage could be achieved
irrespective of the number of recessions and the presence or absence of a secondary surgical intervention.
However, the advantages of Zucchelli’s technique
(modification of the CAF) overpower the advantages
of the tunnel technique with SECTG placement. The
former technique makes treatment easy for both the
clinician and the patient being treated (Zucchelli and De
Sanctis, 2000). Further long-term, multi-center clinical
trials with split-mouth designs comparing Zucchelli’s
technique with different techniques and analyzing the
histology of the attachment achieved are warranted to
provide conclusive evidence.
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Journal of the International Academy of Periodontology 2014 16/2: 43–49
Tooth Loss Assessment during Periodontal
Maintenance in Erratic versus Complete
Compliance in a Periodontal Private Practice
in Shiraz, Iran: A 10-Year Retrospective Study
Amir Haji Mohammad Taghi Seirafi1, Reyhaneh Ebrahimi2,
Ali Golkari3, Hengameh Khosropanah2 and Ahmad Soolari4
Dental Student, School of Dental Medicine; 2Department of
Community Dentistry; 3Department of Periodontology, School
of Dental Medicine, Shiraz, Iran; 4Private Practice, Silver
Spring, Maryland, United States
1
Abstract
Background: Several studies have demonstrated the efficacy of periodontal maintenance
(PM), but there are conflicting data regarding tooth loss following patient compliance.
Method: Seventy-two periodontal patients (52 women, 20 men), 86% of whom had
been diagnosed with chronic moderate to severe periodontitis, were included in this
retrospective study. Clinical variables such as tooth loss, bleeding on probing (BOP),
plaque index and probing depth were collected from patients after 10 years of PM. The
periodontal status of regular compliers (RCs) and erratic compliers (ECs) were compared
in a private practice.
Results: The statistical analysis showed that clinical variables were not significant between RCs and ECs except for BOP (p = 0.038). During PM, 24 teeth (a mean of 1.5
teeth per participant) were lost in the RC group, and 80 teeth (a mean of 1.43 teeth per
participant) were lost in the EC group. Molars were the most frequently lost teeth and
canines the least. In general, those patients with less BOP lost fewer teeth (p = 0.002)
and attended more recall visits (p = 0.001).
Conclusions: In the present sample, RCs and ECs did not show significant differences
in rates of tooth loss. However, a significant difference between RCs and ECs in regard
to BOP was observed at the final examination (p = 0.038). There was also a strong
relationship between BOP and recall visits: the patients with less BOP attended more
recall visits (p = 0.001).
Key words: Maintenance, compliance, periodontitis, tooth loss
Introduction
Supportive periodontal treatment is the phase of periodontal therapy during which periodontal disease and
conditions are monitored and etiological factors are
reduced or eliminated. Periodontal maintenance (PM)
is known to have a significant impact on periodontal
prognosis and eventual tooth survival (American Academy of Periodontology, 2003).
The efficacy of PM and patient compliance has
been evaluated by several retrospective and prospective cohort studies, and those studies demonstrated
Correspondence to: Dr. Ahmad Soolari, 11616 Toulone Dr.
Potomac, MD, USA 20854. Telephone: +1 301-384-5407.
Fax: +1 240-845-1087. E-mail: asoolari@gmail.com
© International Academy of Periodontology
that periodontal patients who comply with regular PM
have less attachment loss and lose fewer teeth compared
to patients who fail to receive PM following active
periodontal therapy (Hirschfeld and Wasserman, 1978;
McFall, 1982; Costa et al., 2011, 2012).
In some studies, different reasons were given by
noncompliant patients for abandoning PM. Stressful life events were reported to decrease compliance
(Becker et al., 1988). Mendoza et al. (1991) have shown
that regular visits to general dentists, cost, and lack of a
perceived need for periodontal treatment were the main
stated reasons for noncompliance. Wilson et al. (1993)
suggested several ways to improve compliance, such as
setting early appointments, providing reminders, and
informing patients about PM.
44
Journal of the International Academy of Periodontology (2014) 16/2
Many studies have reported low rates of regular compliance and adherence to PM (Hirschfeld and Wasserman,
1978; Nabers et al., 1989; Wilson et al., 1993; Soolari and
Rokn, 2003), but some longitudinal studies have provided
more encouraging information concerning compliance
with maintenance appointments for periods ranging up
to 34 years post-treatment (Becker et al., 1984; Lindhe and
Nyman, 1984; Goldman et al., 1986). Wilson et al. (1984)
reported on 961 treated patients who were provided the
opportunity to receive maintenance care over an 8-year
period in a private practice. Only 16% of the patients
complied with the suggested maintenance intervals, 34%
never returned for recall appointments, and the remainder
were erratic in complying. The authors also pointed out
that, in some clinical trials involving periodontal surgery,
the proportion of non-compliers ranged from 11% to
45%. In another study, Soolari and Rokn (2003) evaluated
the degree of compliance of 519 patients who had completed active periodontal treatment up to 7 years. They
reported an overall rate of complete compliance of 3.3%.
Female patients complied better than male patients, and
patients who had received surgery complied better with
PM than patients who had received only scaling and root
planing. In a prospective study conducted by Lorentz et
al. (2009) in Brazil, a total of 250 individuals diagnosed
with chronic moderate to advanced periodontitis and who
had finished active periodontal treatment were incorporated into a PM therapy program. During the 12-month
monitoring period, which featured quarterly recalls, 150
patients were classified as regular compliers (RCs; 60%)
and 62 were non-compliers (24.8%). Among the 150 RCs,
only 20 subjects (13.3%) showed periodontal progression.
With regard to tooth loss, several studies support the
benefit of PM in terms of tooth survival, prevention of
periodontal disease recurrence, and prevention of periodontal disease progression in treated patients (Wilson
et al., 1987, 1993; Soolari, 2002; Costa et al., 2012). In
general, the majority of patients who are compliant with
PM will keep their teeth over a longer period of time.
In a study conducted by Wilson et al. (1987), tooth loss
in erratic compliers (ECs) and in complete compliers
over a 5-year period after active periodontal treatment
was compared. Their results showed that the ECs lost
an average of 0.06 teeth per patient per year and the
complete compliers lost 0 teeth. Checchi et al. (2002)
reported the efficacy of periodontal therapy and PM in
preventing tooth loss in 92 patients over a period of 7
years. The results demonstrated that irregular compliers were at a 5.6 times greater risk of tooth loss than
regularly compliant patients.
Chambrone et al. (2010) assessed the factors influencing tooth loss during long-term PM among 13
retrospective studies. They reported that age, smoking,
and initial prognosis were found to be associated with
tooth loss during PM. In a 3-year follow-up study in
Brazil (Costa et al., 2012), it was shown that RCs presented a lower progression of periodontitis and tooth
loss compared to patients who complied only irregularly.
Moreover, important risk variables such as smoking and
diabetes influenced periodontal status. However, studies
conducted by other groups have suggested that tooth
survival in noncompliant patients is not significantly
different from that in patients with complete compliance after active treatment is performed (McGuire and
Nunn, 1996; Konig et al., 2001), although it should be
remembered that the definitions of noncompliance and
compliance used by various studies may differ.
Konig et al. (2001) conducted a 10-year retrospective
study to determine whether compliant and noncompliant patients with moderate to severe periodontitis had
comparable periodontal conditions during supportive
periodontal therapy. The results indicated that both
groups had similar periodontal conditions at the outset,
but noncompliant patients responded less favorably
to maintenance. McGuire and Nunn (1996) evaluated
the survival rate of periodontally compromised dentitions and investigated the relationship between commonly measured clinical parameters and actual tooth
survival. The results indicated that compliance did not
significantly affect tooth survival. Therefore, it is still
questionable whether a tooth in a completely compliant
patient has an improved survival when compared to a
tooth in an EC. Meanwhile, populations with varying
periodontal status with PM have been reported in the
literature, and there are conflicting data regarding tooth
loss following patient compliance. Hence, the purpose
of the present study was to determine and compare the
periodontal status, especially tooth loss, between RCs
and ECs under PM after a 10-year monitoring period in
a periodontal private practice in an Iranian population.
Materials and methods
Study population
A list of 295 patients in a cohort study from patient
records of a periodontal private practice who were
surgically treated between March 2002 and March 2003
(Shiraz, Iran) was compiled. All the participants had
provided written informed consent, and the study was
approved by the research committee of Shiraz Dental
School.
The study inclusion criteria were as follows: 1) diagnosis with moderate or moderate to severe chronic
periodontitis (Armitage, 1999); 2) good general heath; 3)
presence of ≥ 14 teeth. Subjects were excluded from the
study if they: 1) were pregnant; 2) showed debilitating
disease; 3) presented with drug-induced gingival hyperplasia; 4) had uncontrolled diabetes; 5) presented with
aggressive periodontitis; 6) had used systemic antibiotics
within the previous 4 months; 7) received regenerative
procedures during treatment.
Seirafi et al.: Tooth Loss during Periodontal Maintenance
Clinical examination
All patients remained in a PM program at 3- or 6-month
intervals. All clinical measurements were evaluated at
baseline and final examination in a new chart. All examinations were performed with a manual periodontal
probe (Hu-Friedy, Chicago, IL). Pocket depths (PDs)
were measured at four sites per tooth (three facial sites
and one lingual site). All PDs that were ≥ 5 mm were
recorded and considered as critical.
At the final maintenance visit, a periodontal examination was performed. Data regarding tooth loss, plaque
index (PI; Silness and Loe, 1964), pocket depth (PD)
and bleeding on probing (BOP) via the gingival bleeding
index of Ainamo and Bay (1975) were recorded on a new
chart so that the examiner (M. Seirafi) was blinded as to
which group each patient fell into (RC or EC) to prevent
bias in measurements. Tooth loss was determined from
chartings done at the initial and final examinations.
Because some of the patients (three patients) did not
know the cause of tooth loss, such teeth were counted
as lost due to periodontal causes (McFall, 1982).
Treatment
All patients were initially treated with full-mouth scaling
and root planing by one periodontist (M. Seirafi) with
an ultrasonic device (Cavitron) and hand instruments
(curettes, Hu-Friedy); these procedures were repeated
if necessary during the maintenance period (American
Academy of Periodontology, 2000).
To achieve optimal plaque control, patients also
received oral hygiene instructions. Tooth brushing
(Bass method) was demonstrated in the patient’s mouth
while he or she observed with a hand mirror; then the
demonstration was repeated with dental floss and other
interdental cleaners according to patient need. All the
patients had undergone mucoperiosteal flap surgery,
with or without osseous procedures (osteoplasty, ostectomy) and occlusal adjustment as appropriate. All
the patients after active periodontal treatment had continued in a maintenance program. All the participants
were classified into one of two groups (Miyamoto et
al., 2006). Regular compliers attended at least 70% of
the expected visits, and ECs failed to attend more than
30% of expected visits. In other words, ECs attended
no more than 6 appointments during the 10-year recall
period, and RCs attended at least 14 appointments; any
patients who attended between 7 and 13 appointments
were excluded from analyses.
Statistical analyses
Statistical analyses were conducted using a statistical
software package (SPSS version 20, SPSS Inc, Chicago,
IL). Differences between clinical parameters of RCs
and ECs, such as number of teeth at initial and 10-year
visit, BOP, PD ≥ 5 mm, and PI, were evaluated using
45
the Mann-Whitney U test and the Spearman correlation
when appropriate. Initial evaluation of the categorical changes in these clinical parameters over time was
conducted using the chi-square test of independence.
Post-power calculations of our study were performed
between clinical parameters. Power was calculated at ≥
87% (NCSS-PASS 2004). This value was considered
acceptable. Results were considered significant if a p
value < 5% was attained.
Outcome variables
The main purpose of the study was to compare tooth
loss between RCs and ECs after 10 years; therefore, the
primary outcome was changes in tooth loss between the
two groups after PM. Secondary outcomes included
differences between groups for changes in PD, PI, and
BOP, as well as the frequency of recall visits.
Results
Seventy-two patients (52 women, 20 men) were identified who met all the criteria for participation. The
patients’ ages ranged from 30 to 78 years (mean age
51.30 ± 10.24 years). The characteristics of the sample
by patient age and frequency at the final examination are
presented in Table 1. During the 10-year maintenance
program, 21 patients (29.16%) were classified as RCs and
51 patients (70.84%) were characterized as ECs (Table 2).
Table 1. Distribution of patients by age at final exam
Patient age (y) No. of patients
30 – 35
36 – 41
42 – 47
48 – 53
54 – 59
60 – 65
66 – 71
≥ 72
5
7
11
21
12
10
5
1
% of patients
6.94
9.72
15.27
29.16
16.66
13.88
6.94
1.38
Table 2. Comparison of numbers of patients and
teeth lost between regular compliers (RCs) and erratic
compliers (ECs) and by sex
Group
Sex
RC
RC
EC
EC
Female
Male
Female
Male
No. of patients (%) Teeth lost (%)
17 (23.61)
4 (5.55)
35 (48.61)
16 (22.22)
18 (17.30)
6 (5.77)
49 (47.12)
31 (29.81)
46
Journal of the International Academy of Periodontology (2014) 16/2
With regard to tooth loss, 24 teeth (23.07%) were lost
by RCs, compared to 80 teeth (76.93%) lost by ECs. No
significant difference in tooth loss was observed between
the two groups. Table 3 shows the distribution of tooth
loss with respect to tooth type in both arches. Sixty-four
teeth in the maxilla and 40 teeth in the mandible were
lost over the 10-year period. Molars were lost most often
and canines the least often. None of the lost teeth was
extracted before PM, but during PM three patients lost
five teeth to unknown causes. Two of them were in the
EC group (2 teeth lost by each patient) and one patient
belonged to the RC group (lost one tooth).
The periodontal variables of the patients are presented in Table 4. The mean recall visit interval for the
RC group was 6.31 months and for the EC group it
was 3.16 years. The mean number of recall appointments attended was 3.70 ± 1.55 (range 2 - 6) for the
EC group and 18 ± 3.52 (range, 14 - 26) for the RC
group. Summary statistics were calculated for clinical
parameters in both groups, such as number of teeth at
initial and reevaluation visits, recall frequency, BOP, PI,
and percentage of sites with PD ≥ 5 mm. There were
no statistically significant differences between RCs and
ECs with respect to gender, number of teeth at initial
and final examinations, PI, or number of sites with PD
≥ 5 mm. However, a significant difference between
RCs and ECs in regard to BOP was observed at the
final examination (p = 0.038). There was also a strong
relationship between BOP and recall visits: the patients
with less BOP attended more recall visits (p = 0.001).
Table 3. Number and types of teeth lost in both arches
Maxilla
Tooth no.
Tooth no.
Mandible
7
2
31
7
5
3
30
5
5
4
29
4
1
5
28
0
3
6
27
0
4
7
26
2
4
8
25
2
2
9
24
2
1
10
23
3
0
11
22
1
4
12
21
0
10
13
20
6
10
14
19
4
8
15
18
4
Table 4. Periodontal clinical variables of regularly compliant (RC) and erratically compliant (EC) patients
at the final examination
Variable
Regular compliers
Age
PD ≥ 5 mm (%)
No. of teeth at initial exam
No. of teeth at final exam
PI
BOP (%)
Tooth loss
No. of recalls
Erratic compliers
Mean ± SD
Range
Mean ± SD
Range
p value
53.69 ± 11.80
2.31 ± 4.90
25.63 ± 3.46
24.13 ± 4.59
1.11 ± 0.48
24.14 ± 21.63
1.50 ± 1.71
18.69 ± 3.52
44 – 78
0 – 19
15 – 28
10 – 28
0.37 – 2.17
0 – 90
0–5
14 – 26
50.63 ± 9.77
2.09 ± 3.55
26.27 ± 2.14
24.84 ± 3.93
1.23 ± 0.35
28.64 ± 13.94
1.43 ± 2.34
3.70 ± 1.55
30 – 79
0 – 16
19 – 28
8 – 28
0.45 – 2.62
4 – 87.50
0 – 14
2–6
NS
NS
NS
NS
NS
0.038*
NS
< 0.001*
NS, not significant; *significant; BOP, bleeding on probing; PD, pocket depth; PI, plaque index; SD, standard deviation
Table 5. Spearman correlation (Spearman’s rho) between bleeding on probing (BOP%) and tooth
loss, and BOP% and number of recall visits
Correlations
Spearman’s rho
N = 72
Recall visits
BOP%
Tooth loss
Recall visits
Correlation coefficient
p value (2-tailed)
1.000
_
-0.377
0.001
-0.067
0.574
BOP%
Correlation coefficient
p value (2-tailed)
-0.377
0.001
1.000
_
0.358
0.002
Tooth loss
Correlation coefficient
p value (2-tailed)
-0.067
0.574
0.358
0.002
1.000
_
Seirafi et al.: Tooth Loss during Periodontal Maintenance
Moreover, a greater likelihood for older patients to comply with suggested maintenance was also seen, although
this difference was not statistically significant (Table 4).
Spearman analysis (Spearman rho) showed significant
correlations between BOP and tooth loss and between
BOP and number of recall visits (Table 5).
Discussion
The present retrospective study was done with two
objectives in mind: (1) to assess tooth loss between RCs
and ECs over a 10-year period; and (2) to determine the
periodontal status of the patients after PM.
In this study, none of the periodontal clinical variables, especially tooth loss, were statistically significantly
different between RCs and ECs except for BOP. This
result is not in agreement with most studies regarding
PM (Mendoza et al., 1991; Wilson et al., 1993; Lorentz
et al., 2009; Costa et al., 2011, 2012), although, as mentioned earlier, the definitions of noncompliance with
PM may have differed among studies. With regard to
tooth loss, some other studies have shown an indirect
ratio between compliance and the number of teeth lost
(Chace and Low, 1993). However, data from our study
and some other studies suggest that no clear association exists between erratic compliance with PM and a
decreased incidence of tooth loss when completely
noncompliant patients are excluded from analyses (Miyamoto et al., 2006; Carnavale et al., 2007; Chambrone
and Chambrone, 2006). McGuire and Nunn (1996)
reported that compliance did not significantly affect
tooth survival. Chambrone and Chambrone (2006)
confirmed that the duration of PM and frequency of
recall visits was not associated with periodontal tooth
loss. Miyamoto et al. (2006) evaluated the relationship
between patient compliance and tooth loss. The results
showed that completely compliant patients were more
likely to experience tooth loss than patients with erratic
compliance. However, they also suggested that dentists’
decisions to extract teeth at PM visits may have resulted
in greater tooth loss in the compliant patients.
In another study, Miyamoto et al. (2010) stated
that tooth loss is occasionally referred to as the “true
endpoint characteristic” in dental studies and as the
landmark of tangible patient benefit. However, when
the accumulating evidence of dental implant treatment
or periodontal disease systemic health interactions influences the recommendation to extract a tooth, the validity
of those endpoint characteristics becomes questionable.
Data from the present study showed that the mean
tooth loss rates (MTLR) in RCs and ECs were 0.15
and 0.14, respectively. Meanwhile, several other studies
reported different rates of tooth loss in periodontal
patients during PM: 0.01 (Axelsson et al., 1991), 0.13
(McGuire, 1991), 0.16 (Goldman et al., 1986), 0.24
(Becker et al., 1984), and 0.28 (Checchi et al,. 2002).
47
Matuliene et al. (2010) reported loss rates of 0.13 and
0.30 for RCs and ECs, respectively. One reason for the
variations in these numbers may be the distribution
of disease severity within each study population. The
MTLR in this study is similar to that seen in other studies
(Goldman et al., 1986; McGuire, 1991; Matuliene et al.,
2010). No similar studies of Iranian patients have been
conducted, so no data are available for comparison in
the Iranian population.
The relationship between compliance and common
clinical variables such as BOP, PI and PD was another
point of discussion in this study. There is a general
consensus that complete compliance results in better
oral hygiene, as measured by these parameters. Lang
et al. (1990) showed that the absence of BOP in PM
is considered a good predictor of periodontal stability.
Joss et al. (1994) revealed that a frequency of 25% of
sites with BOP may be considered a limit among patients with progression of periodontitis. In this study,
BOP was significantly different between the RC and EC
groups. In other words, the patients with more recall
visits had less BOP. This finding is in agreement with
those of previous studies (Lang et al., 1990; Joss et al.,
1994; Miyamoto et al., 2006; Lorentz et al., 2009; Costa
et al., 2011, 2012).
In this study, a mean PI of 1.11 was seen in RCs,
while in ECs the mean PI was 1.23, but this difference
was not statistically significant. It is important to state
that efforts in oral hygiene motivation during PM have
proven to be relatively ineffective (Mendoza et al., 1991;
Faggion et al., 2007).
Another parameter evaluated in this study was PD.
At the final clinical examination in 72 patients, the
number of critical sites (i.e., PD ≥ 5 mm) in RCs and
ECs was not significantly different. However, if we had
chosen to define the progression of periodontitis based
on this parameter, we believe that changes in PD that
occurred between the intervals of recall visits might
not necessarily represent the actual loss of periodontal
insertion, especially because PD is more susceptible to
measurement error or because it simply reflects changes
in periodontal marginal inflammatory tissues (Costa et
al., 2007). Therefore, it has been suggested that clinical attachment level (CAL) should be used as the gold
standard for periodontal diagnosis in future studies, although many studies of larger groups have not used this
measurement for the sake of preserving simplicity and
to limit expenses (McGuire and Nunn, 1996; Konig et
al., 2001; Chambrone and Chambrone, 2006). Another
subject of this study is different degrees of compliance
with PM. Among the 295 patients originally treated, 78
(26%) never returned, about half were ECs, and only
23% were RCs. These data are in agreement with those
of Wilson et al. (1984, 1993) and other researchers
(Mendoza et al., 1991; Lorentz et al., 2009).
48
Journal of the International Academy of Periodontology (2014) 16/2
Most studies that have analyzed historic data have
limitations inherent to their retrospective nature, because
the treatment procedures provided were based on clinical judgment, the patient’s desires, prosthetic expediency
and financial considerations, rather than being allocated
randomly, as would be done in a randomized controlled
clinical trial (Miyamoto et al., 2010). In addition, the lack
of a parallel control group and standardization may have
affected the statistical analyses and results (Chambrone
and Chambrone, 2006; Lorentz et al., 2009). Hence, it
should be emphasized that, although the majority of the
studies on PM feature a retrospective design, long-term
prospective studies, although expensive and logistically
difficult, tend to produce more reliable results.
One limitation of the present study is the inclusion
of several clinical variables in a small patient sample.
Thus, the statistical power of this study is reduced. In
this sense, additional studies in large patient populations
are needed to validate these findings.
Conclusions
A long-term retrospective study of the relationship between patient compliance and clinical parameters such as
tooth loss, BOP, PI, and PD was performed. Based on
the results, regular compliance and erratic compliance
with PM did not produce significantly different effects
with respect to tooth loss. However, a significant difference between RCs and ECs in regard to BOP was
observed at the final examination (p = 0.038). There
was also a strong relationship between BOP and recall
visits: the patients with less BOP attended more recall
visits (p = 0.001).
Acknowledgments
This paper has been extracted from Mr. Amir Haji
Mohammad Taghi Seirafi’s thesis, which was conducted
under the supervision of Dr. Reyhaneh Ebrahimi and
the advice of Dr. Ali Golkari. This study was approved,
registered with ID# 8691001, and supported by the
International Branch of Shiraz University of Medical
Sciences. The authors thank Dr. Mohsen Seirafi, former
Assistant Professor, Shiraz Dental School, and Mrs.
Zahra Ghandi and Hurieh Seirafi for helping with preparation of the manuscript. We also thank Dr. Mehrdad
Vosughi, Assistant Professor, Shiraz Dental School, for
his technical assistance. The authors report no conflicts
of interest related to this study.
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Checchi L, Montevecchi M, Gatto MR and Trombelli
L. Retrospective study of tooth loss in 92 treated
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Costa FO, Miranda Cota LO, Costa JE and Pordeus
IA. Periodontal disease progression among young
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Costa FO, Miranda Cota LO, Lages EJ, et al. Progression of periodontitis in a sample of regular and
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Costa FO, Miranda Cota LO, Lages EJ, et al. Periodontal risk
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Faggion CM Jr, Petersilka G, Lang DE, Gerss J and
Flemming TF. Prognostic model for tooth survival
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Goldman MJ, Ross IF and Goteiner D. Effect of periodontal therapy on patients maintained for 15 years
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Journal of the International Academy of Periodontology 2014 16/2: 50–54
Treatment of Amalgam Tattoo with a
Subepithelial Connective Tissue Graft and
Acellular Dermal Matrix
Vivek Thumbigere-Math and Deborah K. Johnson
Division of Periodontology, University of Minnesota School of
Dentistry, Minneapolis, MN
Abstract
A 54-year-old female was referred for management of a large amalgam tattoo involving
the alveolar mucosa between teeth #6 and #9. The lesion had been present for over 20
years following endodontic treatment of teeth #7 and #8. A two-stage surgical approach
was used to remove the pigmentation, beginning with removal of amalgam fragments
from the underlying bone and placement of a subepithelial connective tissue graft and
acellular dermal matrix to increase soft tissue thickness subadjacent to the amalgam.
Following 7 weeks of healing, gingivoplasty was performed to remove the overlying
pigmented tissue. At the 21-month follow-up appointment, the patient exhibited naturally
appearing soft tissue with no evidence of amalgam tattoo.
Key words: Amalgam tattoo; graft; connective tissue; acellular dermal matrix;
pigmentation; tattoo
Introduction
Amalgam tattoo is an unintended sequela of dental
treatment. Amalgam tattoo results from inadvertent
deposition of dental amalgam within the oral mucosa
or alveolar bone during dental procedures. Over time,
metallic particles from dental amalgam leach into the
soft tissue, causing discoloration (Buchner and Hansen,
1980; Harrison et al., 1977).
Clinically, amalgam tattoos appear as blue-black or
blue-gray asymptomatic pigmentation, most commonly
involving the gingival surfaces (Buchner and Hansen,
1980; Neville, 2008). Radiographically, they appear as
radiopaque particles at the site of the lesion, but in
many cases these particles are too small or too diffuse
to be identified (Neville, 2008). Microscopic examination reveals dark solid fragments or numerous fine
granules dispersed along collagen bundles and around
blood vessels, frequently surrounded by inflammatory
infiltrate (Buchner and Hansen, 1980; Harrison et al.,
1977; Neville, 2008).
Correspondence to: Vivek Thumbigere-Math, BDS, PhD, Adjunct Assistant Professor, Division of Periodontology, Developmental and Surgical Science, University of Minnesota School
of Dentistry, 515 Delaware Street SE, Minneapolis, MN 55455.
Tel: 612-625-6155/Fax: 612-626-3076. Email: thumb002@umn.edu
© International Academy of Periodontology
Amalgam tattoos often do not require treatment,
as the mercury present in dental amalgam is not in a
free state and does not pose a health hazard. However,
amalgam tattoos in an esthetic region can be of cosmetic concern, especially for patients with a high smile
line. Various techniques have been described to treat
amalgam tattoos depending on their size, location and
complexity (Griffin et al., 2005; Shah and Alster, 2002;
Shiloah et al., 1988). The management of large lesions
is challenging when there is limited availability of donor
tissue. This deficiency could be overcome by utilizing
allografts such as acellular dermal matrix. This case
report highlights a two-stage surgical procedure for the
management of large amalgam tattoos in the esthetic
zone utilizing an acellular dermal matrix.
Case Description
A 54-year-old Caucasian female was referred for management of a large amalgam tattoo involving the alveolar
mucosa between teeth #6 and #9. Her past medical
history was significant for epilepsy (last episode in
1995), chronic gastritis, herpetic stomatitis and restless
leg syndrome, which were controlled with medications.
Her present medication list included: esomeprazole,
ranitidine, sucralfate, valacyclovir and pramipexole.
Dental history revealed that the patient had had
traumatic fractures of teeth #7 and #8 over 20 years
ago. Following this incident, root canal therapy was
performed and crowns were placed. A few years later,
Thumbigere-Math and Johnson.:Treatment of Amalgam Tattoo
she underwent root-end resection of teeth #7 and #8,
subsequent to which she started noticing the pigmented
lesion. Eventually, teeth #7 and #8 were extracted.
Clinical examination revealed an 18 x 10 mm diffuse,
bluish-pigmented lesion in the alveolar mucosa between
teeth #6 and #9 (Figure 1A). The lesion had progressively darkened and enlarged until reaching the present
size. Although asymptomatic, the pigmentation was
esthetically unappealing to the patient.
Given the clinical appearance, past dental history,
and presence of radiopaque fragments consistent with
amalgam scatter, a biopsy to confirm the diagnosis of
Figure 1:
51
amalgam tattoo was considered unnecessary. After discussing the possible risks, including incomplete removal
of the pigmentation and scar formation, the patient
initially consented to a two-stage surgical treatment
plan. Following the administration of local anesthesia,
a sulcular incision was made on tooth #6 with a crestal
incision in the region of teeth #7 and #8. A vertical
releasing incision sparing the papilla was made on the
mesial surface of tooth #9, which extended beyond
the mucogingival junction. A second vertical releasing
incision was made on the distal surface of tooth #6
(one tooth surface beyond the pigmented area). A full
A
B
C
D
E
Figure 1: A) Initial presentation of the amalgam tattoo (18 x 10 mm) that resulted from root-end resective surgery
performed 20 years earlier. B) Subepithelial connective tissue graft placed over the recipient site after traces of
metallic fragments in the bone were removed. C) Because of the limited availability of donor tissue, acellular dermal
matrix was also placed over the subepithelial connective tissue graft to further thicken the underlying connective
tissue. D) Surgical site after the first grafting phase, closed with polytetrafluoroethylene (e-PTFE) sutures and 5-0
chromic gut sutures. E) Two weeks post-operative healing without any evidence of graft exposure or sloughing
52
Journal of the International Academy of Periodontology (2014) 16/2
thickness flap was reflected and any traces of metallic
fragments in the bone were removed using hand and
rotary instruments under copious irrigation. A 2 mm
thick subepithelial connective tissue graft harvested
from the left palate was placed over the recipient site
in the area of teeth #7 and #8 (Figure 1B). It was realized that the donor tissue was inadequate in relation to
the lesion size (the pigmentation extended through the
entire thickness of soft tissue from the epithelium to
the periosteum along the area of teeth #6 to #9). Upon
the patient’s objection to harvesting another connective
tissue graft from a contralateral site, an acellular dermal
matrix (AlloDerm, LifeCell Corporation, NJ) was used
over the subepithelial connective tissue graft to further
thicken the underlying connective tissue (Figure 1C).
Both grafts were secured separately with 5-0 chromic gut
sutures. A periosteal release was performed and coronal
advancement of the flap was obtained. Primary closure
was achieved with polytetrafluoroethylene (e-PTFE)
sutures (GORE-TEX, W.L. Gore) and 5-0 chromic gut
Figure 2:
sutures (Figure 1D). The patient was prescribed ibuprofen (600 mg) and acetaminophen (500 mg) with codeine
(5 mg) for pain management along with amoxicillin (500
mg) and a medrol dose pack (oral methylprednisolone
tapered in one week). The patient was instructed to
rinse twice daily with 0.12% chlorhexidine gluconate
for 2 weeks. The post-operative healing (Figure 1 E) was
uneventful, without any graft exposure or sloughing, and
the sutures were removed after 2 weeks.
Seven weeks after the first surgery, the patient returned to the clinic for the second phase of treatment
(Figure 2A). The soft tissue was evaluated to confirm
that the thickness had increased by 2-3 mm following
grafting. At this visit, gingivoplasty was performed using
a high-speed diamond bur under copious irrigation to
remove approximately 0.5 mm of overlying pigmented
tissue. The pigmented tissue was completely removed,
exposing the underlying graft (Figure 2B). Complete hemostasis was achieved and a layer of oxidized cellulose
(Surgicel, Ethicon, Inc., a Johnson & Johnson company;
NJ) was applied to the surgical site, over which cyanoacrylate gel was applied. The patient was again prescribed
ibuprofen (600 mg) for pain management.
Around 5 weeks later the entire surgical area was
covered by new epithelium. At the 10- and 21-month
follow-up appointments, there was no evidence of re-
A
B
C
D
Figure 2: A) Surgical site 7 weeks after grafting. B) Seven weeks after grafting, gingivoplasty was performed to
remove the overlying pigmented tissue. Note the underlying graft from the initial surgery is now exposed. C) At
the 10-month follow-up appointment, there is no evidence of residual pigmentation. D) Final restoration and
clinical appearance at 21 months after grafting. Note the color change between native tissue and grafted site.
Thumbigere-Math and Johnson.:Treatment of Amalgam Tattoo
sidual pigmentation (Figures 2C and 2D) and the patient
was pleased with the outcome. As expected, there was
some amount of thickness reduction during the first
year of grafting owing to the shrinkage of the acellular
dermal matrix; however, the thickness was stabilized
after one year.
Discussion
The incidence of amalgam tattoo has been reported to
be around 8% in previously surveyed samples (Buchner
and Hansen, 1980; Owens et al., 1992). Amalgam tattoos
can be of esthetic concern, especially when located in
the maxillary anterior region. Various techniques have
been described for the management of amalgam tattoos
depending on their size, location and complexity (Griffin
et al., 2005; Shah and Alster, 2002; Shiloah et al., 1988).
Small superficial lesions can be removed using rotary
instruments (round or diamond bur) in the form of a
localized gingivoplasty. However, large lesions require
advanced management. Kissel and Hanratty described
a two-stage surgical treatment in which a connective
tissue graft was placed deep to the pigmented area followed by gingivoplasty of the overlying tissue (Kissel
and Hanratty, 2002). Although this technique results in
a favorable outcome with minimal scarring and good
color match, the limitation in availability of donor tissue
can be disadvantageous. Shiloah et al. utilized an epithelialized free soft tissue graft to treat amalgam tattoos
(Shiloah et al., 1988). The epithelialized free soft tissue
graft was placed over the curetted bone in the maxillary
anterior region; however, this technique has a significant
risk for scarring and poor color match. Furthermore,
Griffin et al. utilized acellular dermal matrix as an onlay
graft over the completely excised amalgam tattoo (Griffin et al., 2005). In this study, the full thickness of the soft
tissue outlining the amalgam tattoo was excised before
the acellular dermal matrix was placed over the surgical
site. The authors suggest that acellular dermal matrix is a
viable option in treating large amalgam tattoos, which are
otherwise very difficult to treat with autogenous grafts.
However, previous studies have reported that uncovered
acellular dermal matrix may not increase the zone of
keratinized tissue as predictably as an autologous soft
tissue graft, which is of importance in the esthetic zone
(Harris, 2004; Yan et al,. 2006).
Shah et al. utilized an alexandrite laser to remove
amalgam tattoo on the buccal mucosa and gingiva over
the course of three treatments at 8-week intervals (Shah
and Alster, 2002). Similarly, Campbell and Deas used
ER,Cr:YSGG laser to remove pigmented tissue in a
single treatment (Campbell and Deas, 2009). Although
this technique is feasible, the use of lasers (Nd:YAG,
Er:YAG, and Nd:YLF) has been reported to trigger
the release of mercury vapor from mercury-containing
amalgam surfaces (Pioch and Matthias, 1998). Mercury
53
released into the oral cavity by laser ablation may elicit
an intense inflammatory response and may also play a
role in triggering oral neuropathy (Donetti et al., 2008;
Forsell et al., 1998) and lichen planus (Staines and Wray,
2007). Furthermore, when ablating relatively thin soft
tissues (e.g., facial gingival and alveolar mucosa) using
lasers without irrigation, there is an apparent risk of
irreversible bone damage due to the excessive heat
generated by lasers.
Alternatively, amalgam tattoos or pigmentations
in the high smile line area can be masked using a lip
repositioning technique (Jacobs and Jacobs, 2013).
This technique involves precise resection of maxillary
mucosal tissues with reattachment of the lip in a more
coronal position, resulting in limited lip elevation on
smiling and increased lip fullness.
Although several techniques have been described to
remove amalgam tattoos, the current report highlights
the significance of using acellular dermal matrix when
there is limitation in the availability of donor tissue. A
two-stage surgical approach can be used to remove amalgam tattoos, beginning with a subepithelial connective
tissue graft and acellular dermal matrix to increase tissue
thickness and allow removal of amalgam fragments in
bone, followed by gingivoplasty of the surface tissue. In
conclusion, clinicians need to be aware of various treatment strategies for amalgam tattoos in esthetic zones
that result in esthetically appealing outcomes.
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Acknowledgments
We thank Drs. James E. Hinrichs and Gary Cook for
their support of this project.
Journal of the International Academy of Periodontology 2014 16/2: 55–63
Gingival Crevicular Fluid Bone Morphogenetic
Protein-2 Release Profile Following the Use
of Modified Perforated Membrane Barriers in
Localized Intrabony Defects: A Randomized
Clinical Trial
Ahmed Y. Gamal1, Mohamed Aziz2, Salama M.H.2, Vincent J. Iacono3
Department of Periodontology, Faculty of Dental Medicine, Ain
Shams University, Cairo, Egypt; 2Department of Periodontology,
Faculty of Dental Medicine, Al Azhar University, Cairo, Egypt;
3
Department of Periodontology, School of Dental Medicine,
Stony Brook University, NY, USA
1
Abstract
Background: In guided tissue regenerative surgery, membrane perforations may serve as
a mechanism for the passage of cells and biologic mediators from the periosteum and
overlying gingival connective tissue into the periodontal defects. To test this assumption,
this study was designed to evaluate levels of bone morphogenetic protein-2 (BMP-2) in
gingival crevicular fluid (GCF) during the early stages of healing for sites treated with
modified perforated membranes (MPMs) as compared with occlusive membranes (OMs).
Methods: Fifteen non-smoking patients with severe chronic periodontitis participated in
this prospective, randomized and single-blinded clinical trial. Each patient contributed
two interproximal contralateral defects that were randomly assigned to either an experimental modified perforated membrane group (15 sites) or a control occlusive membrane
group (15 sites). Plaque index, gingival index, probing depth (PD), clinical attachment
level (CAL) and the relative intrabony depth of the defect (rIBD) were measured at baseline and reassessed at three, six and nine months after therapy. Gingival crevicular fluid
samples were collected on day 1 and 3, 7, 14, 21, and 30 days after therapy.
Results: The MPM-treated group showed a statistically significant improvement in PD reduction and clinical attachment gain compared to the OM control group. Similarly, rIBD
was significantly reduced in MPM-treated sites as compared with those of the OM group.
BMP-2 concentrations peaked in the MPM samples obtained during the early postoperative
period (days 1, 3 and 7) with a statistically significant difference compared with OM-treated
groups. BMP-2 levels decreased sharply in the samples obtained at days 14, 21 and 30
with non-significant higher levels in MPM samples as compared with those of OM sites.
Conclusion: Within the limits of the present study, one can conclude that MPM coverage of
periodontal defects is associated with a significant initial increase in GCF levels of BMP-2,
a factor that could improve the clinical outcomes of guided tissue regenerative surgery.
Key words: Periodontal regeneration, guided tissue membranes, bone morphogenetic protein, growth factors, periodontal pockets
Correspondence to: Vincent J. Iacono, D.M.D. Distinguished
Service Professor and Chair, Department of Periodontology,
Director of Postdoctoral Education, School of Dental Medicine,
Stony Brook University, Tel: 631-632-8895/8955, Fax: 631-6323113. E-mail: Vincent.Iacono@stonybrook.edu
© International Academy of Periodontology
56
Journal of the International Academy of Periodontology (2014) 16/2
Introduction
Although guided tissue regenerative therapies have great
potential, they remain unpredictable in their ability to
consistently produce acceptable outcomes in all situations (Cho et al., 1995). Perhaps the most important
factor that would negatively affect guided tissue regeneration (GTR) is periosteal isolation. This barrier effect
would deprive the wound area from the regenerative
potential of the periosteum, including progenitor cells
and biologic mediators. The periosteum has been shown
to have significant regenerative potential (Ishida et al.,
1996). Periosteal grafts were found to have the potential
to stimulate osteogenesis in periodontal defects by their
capacity to upregulate osteogenic factors (Ueno et al.,
2001; Gamal and Mailhot, 2008). In addition, periosteal
grafts were reported to contribute additional osteoprogenitor cells that would compensate for their relative
deficiency in the defects (Gamal et al., 2010; 2011).
The recent isolation of gingival mesenchymal stem
cells (GMSCs) from gingival connective tissue has made
it reasonable to reevaluate the protocol of gingival connective tissue isolation in GTR procedures. They have
been shown to exhibit clonogenicity, self-renewal, and
multipotent differentiation capacities (Mitrano et al.,
2010; Tomar et al., 2010; Tang et al., 2011). These cells are
capable of immunomodulatory functions, specifically
suppressing peripheral blood lymphocyte proliferation
(Zhang et al., 2009). At the functional level, mesenchymal stem cells (MSCs) display chemotactic properties
similar to immune cells in response to tissue insult and
inflammation, thus exhibiting tropism for the sites of injury via production of anti-inflammatory cytokines and
anti-apoptotic molecules (Spaeth et al., 2008; Karp et al.,
2009; Nauta and Fibbe, 2007). These unique characteristics of MSCs make them attractive candidates for the
enhancement of periodontal tissue regeneration. Isolation of the wound area from this important source of
GMSCs through the use of traditional occlusive guided
tissue membranes may therefore limit the regenerative
potential of GTR procedures.
Macroscopically, based on its larger surface area
compared to that of the periodontal ligament, gingival
connective tissue is highly vascular. In addition, gingival
connective tissue represents the most abundant structural cell in periodontal tissue (Nanci and Bosshardt, 2006).
Although many researchers suggested that gingival connective tissue cells lacked the potential for regeneration
and occlusive GTR devices showed significantly greater
bone regeneration (Polimeni et al., 2004; Karring et al.,
1980; Nyman et al., 1980), other experimental studies
have reported that gingival connective tissue cells may
contribute to the regenerative process (Aukhil and Iglhaut, 1988; Aukhil et al., 1985; Aukhil et al., 1986; Bowers
and Donahue, 1988; Iglhaut et al., 1988). In vitro, both
gingival and periodontal ligament fibroblasts were found
to express mRNA for BMP-2 and BMP-4 (Ivanovski
et al., 2001). Both cell types were also found to express
hard tissue-associated proteins in osteogenic media and
were able to synthesize and break down the collagen
fibers and other proteins from the ground substance
(Lallier et al., 2005; Ivanovski et al., 2001; Bartold and
Narayanan, 2006).
Gamal and Iacono introduced a novel perforated
collagen membrane as a modality that could enable
participation of periosteal cells, gingival fibroblasts
and gingival stem cells in GTR procedures (Gamal and
Iacono, 2013). They demonstrated in a clinical study that
the use of a modified perforated membrane (MPM) improved clinical outcomes significantly more than those
observed with the use of occlusive membranes. The design of their study did not allow for the identification of
which component(s) of the periodontium contributed
to the positive results obtained. Clinical findings were
not validated by further analysis to identify the nature of
healing and whether gingival fibroblasts, GMSCs and/or
periosteal cells contributed toward the enhanced regenerative process. It has also been suggested that growth
and differentiation factors from cells in the periosteum
and gingiva could pass through the membrane perforations and augment regeneration. Bone morphogenetic
proteins (BMPs) are crucial differentiation factors in
bone formation and healing (Reddi, 1998; Bessa et al.,
2008; Kanakaris and Giannoudis, 2008; Yu et al., 2010;
Reddi, 2005; Chen et al., 2004). They possess very strong
osteoinductive activity, induce differentiation of mesenchymal cells into chondrogenic and osteogenic cells, and
promote osteoblast proliferation (Takiguchi et al., 1999;
King et al., 1997; Jung et al., 2003; Jepsen and Terheyden,
2002; Zhao et al., 2003). In this study we studied levels
of BMP-2, which is reported to be the most active
member of the BMP phenotypes. A direct correlation
could exist between the number of the available cells
and their released growth and differentiation factors.
To test this assumption, the objective of this study was
to evaluate levels of bone morphogenetic protein-2 in
GCF during the early stages of healing for sites treated
with MPMs as compared with those sites treated with
occlusive barrier membranes.
Materials and methods
Patient selection
Fifteen non-smoking patients (8 males and 7 females)
who were 31 to 51 years of age at the time of baseline
examination (mean age 33.8 ± 6.1 years) with severe
chronic periodontitis (Armitage, 1999) participated in
this prospective, split-mouth, randomized and singleblinded clinical trial. Since no previous data on GCF
BMP-2 levels following the use of MPM or OM are
available to provide data for sample size calculation,
post-hoc power analysis was performed for the clinical
Gamal et al .:BMP-2 levels around perforated membranes
part of the study. The sample size was 7 subjects in
each group at an alpha level of 0.05 (5%), and β level of
0.20 (20%). The obtained power was 81%. The subjects
were recruited consecutively from the list of patients
seeking periodontal treatment in the Department of
Periodontology of the Faculty of Dental Medicine, Al
Azhar University, Cairo, Egypt, between March 2012 and
November 2012. The criteria implemented for patient
inclusion were: 1) no systemic diseases which could influence the outcome of the therapy; 2) good compliance
with the plaque control instructions following initial
therapy; 3) teeth involved were all vital with no mobility;
4) each subject contributed matched pairs of 2- or 3-wall
intrabony interproximal defects around premolar or
molar teeth without furcation involvement; 5) selected
2- or 3-wall intrabony defects (IBD) measured from the
alveolar crest to the defect base in diagnostic periapical
radiographs of ≥ 4 mm; 6) selected probing depth (PD)
≥ 5 mm and clinical attachment loss (CAL) ≥ 4 mm at
the site of intraosseous defects 4 weeks following initial
cause-related therapy; 7) availability for the follow-up
and maintenance program; 8) absence of periodontal
treatment during the previous year; 9) absence of systemic medications that could affect healing or antibiotic
treatment during the previous 6 months; 10) absence of
a smoking habit; and 11) absence of occlusal interferences, mobility, open interproximal contacts (diastema,
flaring or both). Pregnant females were excluded from
participating in the study. Patients were also excluded
from the study if they demonstrated inadequate compliance with the oral hygiene maintenance schedule. The
experimental protocol was approved by the Ethical
Committee of Al Azhar University (OMD - 45 – 2012).
Research procedures were explained to all patients and
they agreed to participate in the study and signed the
appropriate informed consent form. This clinical trial
was registered under a clinical trial registration number:
NCT01860495.
Presurgical therapy and grouping
Initial cause-related therapy consisted of thorough full
mouth scaling and root planing performed in quadrants
under local anesthesia. This procedure was performed
using a combination of hand and ultrasonic instrumentation using a P10 tip. Patients were recalled every 3
days for three weeks and received detailed mechanical
plaque control instructions that consisted of brushing
with a soft toothbrush in a roll technique and flossing.
Supra-gingival plaque removal was performed whenever
necessary. Four weeks after initial therapy, a reevaluation
was performed to confirm the need for periodontal
surgery. Criteria used to indicate that surgery was required included the persistence of two interproximal
sites with PD ≥ 5 mm, CAL ≥ 4 mm, and interproximal
intrabony component of ≥ 4 mm. Baseline periodontal
57
disease status of the selected sites was determined by
clinical periodontal assessments, including plaque index
(PI; Silness and Loe, 1964), gingival index (GI; Loe and
Silness, 1963), probing depth (PD; Polson et al., 1980)
and clinical attachment level (CAL; Ramfjord, 1967)
as the distance from the bottom of the pocket to the
gingival margin and the cementoenamel junction (CEJ),
respectively. The clinical measurements were obtained
using a University of Michigan “O” probe with William’s
markings and the measurements were rounded up to the
nearest 0.5 mm. The deepest point of baseline defects
was included in the calculations. Routine diagnostic
non-standardized periapical views using intraoral size 2
dental films were recorded by the long cone paralleling
technique and holders using an x-ray unit operating at
70 kV, 10 mA, and 0.8-second exposure time. To avoid
the unstable alveolar crest level, the linear distances
from CEJ to the base of the bony defect, representing
the rIBD component level, were measured from digital
radiographs. Initial cause-related therapy and clinical
measurements were performed by a single experienced
calibrated examiner who was not involved in the study
in any other way (SMH). Intra-examiner reproducibility
was assessed with a calibration exercise performed on
two separate occasions, 48 hours apart. Calibration was
accepted if 90% of the recordings could be reproduced
within a difference of 1.0 mm.
For a patient to serve as his own control, the study
used a split-mouth design where two interproximal
contralateral defects were randomly (toss of a coin; the
coin was flipped each time by the same individual (ADR)
assigned immediately before surgery to either the MPM
group (15 sites) or the OM group (15 sites). All surgeries were performed by the same operator (AYG). The
surgical treatment phase was initiated only if the subject
had a full-mouth dental plaque score of less than one.
Surgical procedures were accomplished as described in
detail by Gamal and Iacono (2013). A mucoperiosteal
flap was elevated using intrasulcular incisions under local
anesthesia. Debridement of all inflammatory granulation tissue from the intrabony defect was performed
until a sound, healthy bone surface was obtained. The
teeth were thoroughly root planed. For MPM samples,
membrane perforations were prepared just before surgery using a custom-made 2 mm diameter pin and 2
mm perforated acrylic template with a coronal occlusive
rim of about 3 mm (Figure 1). Inter-perforation spaces
were determined to be not less than 2 mm in order to
avoid loss of membrane stiffness. Collagen membranes
were hydrated in sterile saline, trimmed according to the
template prepared for each defect, and adapted over the
defects in such a manner that the entire defect and ≥
2 to 3 mm of the surrounding alveolar bone was completely covered to avoid membrane collapse within the
defect. The membranes were extended supracrestally
58
Journal of the International Academy of Periodontology (2014) 16/2
Figure 1. Template for membrane round-hole-pattern perforations (right) and perforated
membrane (left).
1 mm below the CEJ to ensure optimum gingival connective tissue involvement in supracrestal wound healing. Collagen membranes were simply adapted in place
according to the surgical protocol of the manufacturer
without suturing. The mucoperiosteal flap was coronally
positioned covering the entire membrane and sutured
with a non-resorbable suture. No periodontal dressing was applied. In a separate visit, the selected OM
sites underwent occlusive membrane coverage of the
intrabony defects. All patients received oral and written postoperative instructions. Patients were prescribed
amoxicillin (500 mg) every 8 hours for 1 week. Subjects
with allergies to amoxicillin and derivatives were prescribed clindamycin (300 mg) every 8 hours.
Plaque control effort was supplemented by rinsing
with chlorhexidine (0.12% chlorhexidine hydrochloride) for one minute three times daily for 2 weeks. The
patients were instructed to refrain from tooth brushing
and interdental cleaning was avoided at the surgical areas
during this time. Sutures were removed 14 days postoperatively and recall appointments for observation of
any adverse tissue reaction and oral hygiene reinforcement were scheduled every second week during the
first 2 months after surgery. One month after surgery,
all patients were instructed to resume their normal
mechanical oral hygiene measures, which consisted of
brushing using a soft toothbrush with a roll technique
and flossing. Supportive periodontal maintenance including oral hygiene reinforcement and supragingival
scaling was performed during each recall appointment.
Clinical and radiographic measurements were reassessed
at 3, 6, and 9 months after surgery by a blinded calibrated
investigator (MA).
Gingival crevicular fluid sampling and
quantitative measurement of BMP-2
To avoid irritation, samples were obtained 1 day following surgery and after individuals had fasted overnight
and between 8:00 am and 10:00 am. Using micropipettes
(5 µL), GCF samples were collected (Sueda et al., 1969)
by a single examiner (MA) who was masked to the attribution of the sites to MPM or OM. Following the
isolation and drying of the selected site with cotton rolls,
a Fisher brand disposable micropipette was placed intrasulcularly at the mesio-facial line angle of the selected
site to a maximum depth of 2 mm below the margin.
Micropipettes were left in place until 5 µL of fluid
was collected. GCF samples were collected at day 1
and 3, 7, 14, 21 and 30 days after therapy and diluted
in saline solution (50 µL) for BMP-2 level evaluation.
Samples were labeled, carried in a dark container and
kept at -80º C until tested. BMP-2 in the GCF samples
was measured using a human BMP-2 enzyme-linked
immunosorbent assay (ELISA) kit according to the
manufacturer’s protocol. This assay uses an antibody
specific for human BMP-2 coated on a 96-well plate.
Data analysis
The primary efficacy parameter for the study was
gingival crevicular fluid BMP-2 level at 1, 3, 7, 14, 21
and 30 days. Secondary efficacy parameters included
clinical and radiographic measurements at 3, 6 and 9
months after surgery. Data were presented as mean
and standard deviation (SD) values. Data were explored
for normality using the Kolmogorov-Smirnov test and
the Shapiro-Wilk test. Data showed non-normal (nonparametric) distribution, so the Mann-Whitney U test
was used to compare between the two groups. The
Wilcoxon signed-rank test was used to study the changes
Gamal et al .:BMP-2 levels around perforated membranes
59
Table 1. The means ± standard deviation values of BMP-2 concentrations (pg/mL) in the groups treated with modified perforated membranes (MPM) and occlusive membranes (OM).
Period
Group
MPM
OM
p - value
1 day
406.1 ± 122.6
43.5 ± 24.8
0.032*
3 days
223.9 ± 111.6
66.8 ± 8.7
0.038*
7 days
221.6 ± 43.4
14 days
143.6 ± 97.8
76.5 ± 3.1
0.197
21 days
193.9 ± 152.8
65.2 ± 3.2
0.199
30 days
92.9 ± 66.4
72 ± 14.8
81 ± 22.5
0.045*
0.886
*p ≤ 0.05 (Mann-Whitney U test)
Table 2. Pre-surgery clinical measurements (mean
± standard deviation).
Characteristics
MPM
(n = 13)
OM
(n = 13)
PD
5.8 ± 0.3
6.1 ± 0.4
CAL
4.3 ± 0.3
4.1 ± 0.3
rIBD
6.6 ± 0.4
6.7 ± 0.3
PI
0.3 ± 0.01
0.4 ± 0.03
GI
0.2 ± 0.02
0.2 ± 0.04
MPM, modified perforated membranes; OM,
occlusive membranes; PD, pocket depth (mm);
CAL, clinical attachment level (mm); rIBD, relative
intrabony defect depth (mm); PI, plaque index; GI,
gingival index. *p ≤ 0.05 (Mann-Whitney U test)
by time within each group. The significance level was
set at p ≤ 0.05. Statistical analysis was performed with
statistical software.
Results
During the course of the study, all patients experienced
uneventful postoperative healing in all of the experimental and control defects. All patients completed the study
and tolerated the surgical procedures well. No site had
to be eliminated and no cases of clinically opened flap
dehiscence or infection were detected. Minimal swelling of soft tissues surrounding the operated areas was
observed during the early days of healing. Nevertheless, membrane exposure was a common event in both
groups. It was observed at 2 to 3 weeks after surgery
with minimal inflammation in five of the OM-treated
sites and four of the MPM-treated sites. It was decided
to include their records in the data analyses. Two patients did not continue their follow-up visits for sample
collections because they relocated. As a result, 13 of 15
OM and MPM treated sites completed the study. Bony
wall treated defects were distributed as follows: MPM,
three predominately 2-wall and ten predominately 3-wall
defects; OM, four predominately 2-wall and nine predominately 3-wall defects.
Table 1 illustrates the mean ± standard deviation
(SD) values and results of Mann-Whitney U test of the
BMP-2 concentrations in the GCF collected from sites
treated by MPM and OM at different sampling times.
BMP-2 concentrations peaked in the MPM samples
obtained during the early postoperative days (days 1, 3
and 7) and were statistically significantly different than
those in OM samples. BMP-2 levels decreased gradually
in the samples obtained at days 14, 21 and 30 in both
groups. In spite of the higher levels of BMP-2 levels in
the MPM test group at 14, 21 and 30 days, there were
no significant differences between the two groups.
A summary of the defect characteristics 4 weeks
pre-surgically using the mean ± SD for the appropriate
clinical measurements for both groups is provided in
Table 2. No statistically significant differences were found
preoperatively between MPM and OM groups with
respect to soft and hard tissue measurements. All GI
and PI scores were within clinically healthy parameters.
The defects had deep PDs (5.8 ± 0.3 mm for the MPM
group and 6.1 ± 0.4 mm for the OM group), and were
associated with deep rIBD (6.6 ± 0.4 mm for MPM and
6.7 ± 0.3 mm for OM). Similarly, CAL was 4.3 ± 0.3 mm
and 4.1 ± 0.3 mm for MPM and OM sites, respectively.
Table 3 shows the mean defect characteristics of both
groups during different observation periods. The mean
PI and GI were initially low; they remained unchanged
by 3, 6 and 9 months for both groups. There were no
statistically significant differences between the initial
and 3-, 6- or 9-month values or between the groups (p
> 0.05). Target teeth were free of gingival inflammation
and plaque before surgery and at the end of the study.
Patients were kept under a strict maintenance program,
60
Journal of the International Academy of Periodontology (2014) 16/2
Table 3. Chronological changes in clinical parameters.
Characteristics
PD
3 months
6 months
9 months
3 months
6 months
MPM
2.6 ± 0.7
2.4 ± 0.4
2.3 ± 0.3*
1.8 ± 0.4
1.6 ± 0.6* 1.4 ± 0.4*
OM
3.1 ± 0.5
2.9 ± 0.6
3.7 ± 0.2
2.1 ± 0.5
2.5 ± 0.4
2.6 ± 0.5
0.094
0.078
0.024
0.093
0.035
0.024
p value
Characteristics
CAL
rIBD
6 months
PI
GI
9 months
3 months
6 months
9 months
3 months
6 months
9 months
MPM
3.3 ± 03* 3.4 ± 0.5* 3.2 ± 0.6*
0.6 ± 0.4
0.4 ± 0.3
0.6 ± 0.3
0.8 ± 0.4
0.6 ± 0.4
0.7 ± 0.3
OM
4.4 ± 0.5
4.6 ± 0.7
4.2 ± 0.4
0.8 ± 0.4
0.6 ± 0.5
0.7 ± 0.4
0.6 ± 0.3
0.9 ± 0.6
0.8 ± 0.5
0.033
0.037
0.041
0.67
0.45
0.36
0.65
0.56
0.36
p value
3 months
9 months
MPM, modified perforated membranes; OM, occlusive membranes; PD, pocket depth (mm); CAL, clinical attachment level (mm);
rIBD, relative intrabony defect depth (mm); PI, plaque index; GI, gingival index. *p ≤ 0.05 (Mann-Whitney U test)
Figure 2. Clinical and radiographic views of the initial and 9-month follow up for a modified
perforated membrane-treated deep intrabony defect related to the mesial root of a lower right
first molar. A) 7 mm initial probing pocket depth (PPD); B) 2 mm 9-month post-operative
PPD; C) initial 5 mm relative intrabony defect radiograph; D) 2 mm post-operative relative
intrabony defect radiograph
Gamal et al .:BMP-2 levels around perforated membranes
and the overall plaque accumulation was minimal. By
the end of the study, the MPM-treated group showed a
statistically significant improvement in PD reduction and
clinical attachment gain compared with the OM control
group. Similarly, rIBD appeared to be significantly reduced in MPM-treated sites compared with that of the
OM group (Figure 2).
Discussion
The main objective of using guided tissue membranes
is to prevent soft tissue invasion into the periodontal
defects through the use of occlusive stiff materials.
Small membrane perforations and wide inter-perforation
areas were suggested to keep the membrane rigid that
could be easily occluded by a blood clot, providing a
membrane that is mechanically obstructive for soft tissue invasion and at the same time biologically permeable
for cells and mediators through fibrin clot-occluded
perforations. Because the MPMs used in the present
study employed bovine collagen membranes, an accepted biomaterial that does not require preclinical
documentation, it was decided to initiate studies on the
clinical and biochemical values of membrane perforations. It has been decided to further evaluate the positive
clinical outcomes using animal models that will include
immunohistochemical staining of mesenchymal stem
cell markers to test our hypothesis that gingival and/or
periosteal mesenchymal stem cells may selectively pass,
along with gingival fibroblasts, through the perforated
membrane and enhance periodontal regeneration. Our
recent human trial reported improved clinical outcomes
of perforated membranes as a suggested way to enhance
the contribution of such cells in periodontal regeneration when compared with GTR procedures using OM
(Gamal and Iacono, 2013). The hypothesis was that if
the collagen is perforated, this could induce periodontal tissue regeneration in a dual direction; first in the
remaining periodontal structures below the membrane,
and secondly in the periosteum and gingival fibroblasts
with their associated mesenchymal stem cells above
the membrane. The present study is the first to evaluate the biologic effects of membrane perforations on
periodontal healing. Because a direct correlation could
exist between the number of cells and the available
released growth and differentiation factors, the level
of GCF BMP-2 following the use of perforated and
occlusive membranes could reflect the number of cells
releasing them.
In the present study, glass micropipettes with an
internal diameter of 1.1 mm were used for the collection of GCF samples, where the fluid collection takes
place through capillary action. Micropipette sample
collection seems to be ideal for evaluating the released
BMP-2 at different time periods because it provides
an undiluted sample of ‘‘native’’ GCF whose volume
61
can be accurately assessed. The use of filter paper was
avoided because of the possible non-specific attachment
of BMP-2 to filter paper fibers with associated false
level reduction. GCF flow, with its physical protective
effects of flushing the pocket, is considered an excellent
undispersed medium for evaluating the released BMP-2
at different time periods. The selection of the intrabony
defect type is another factor that helps in maintaining
BMP-2 for accurate evaluation for its availability and
release pattern. We decided to start GCF collection a
day after surgery because samples collected immediately
after surgeries were usually contaminated with blood.
In both the MPM and OM groups, plaque control was
optimal and mean gingival index scores were < 1. There
were no statistically significant differences in PD, CAL,
and rIBD. Therefore, the GCF flow rate was nearly consistent and BMP-2 release and subsequent containment
could be under the same circumstances.
Analysis of the GCF in the present study revealed
that BMP-2 levels were significantly higher at 1, 3 and
7 days after surgery in the MPM group as compared
with the OM group. Levels were markedly reduced at
14, 21 and 30 days in both groups, with non-significantly
higher levels for the MPM-treated group. These findings
are demonstrated in previous growth factors studies in
which guided tissue membranes appear to obstruct the
chemotactic effect of the growth factor on periosteal
pluripotential mesenchymal cells (Canalis et al., 2003;
Zhao et al., 2003). Mechanical injury was also found to
upregulate BMP-2, as well as BMP-2 signaling in human
cartilage explants (Dell’Accio et al., 2006). The initial
low BMP-2 level that was reported in the OM-treated
group suggests that occlusive membranes could act as
a barrier, reducing diffusion of biologic mediators into
the defect area. The higher initial BMP-2 level that was
found under perforated membranes could be attributed
to either direct gingival and periosteal released mediator
flow through membrane perforations, or cellular migration into the defect area through membrane perforations, with subsequent enhanced mediator availability.
These findings suggested that, during the early stages
of healing, occlusive membranes could alter the physiologic growth and differentiation factor levels at the
defect site, while membrane perforations could allow for
such levels to reach periodontal defects at a physiologic
level. Zhao et al. (2003) reported that BMP-2 decreased
mRNA levels of bone sialoprotein and type I collagen
dose-dependently (10-300 ng/mL). At low doses, up
to 100 ng/mL, BMP-2 had no effect on transcripts for
osteocalcin and osteopontin, whereas at 300 ng/mL,
BMP-2 greatly increased expression of these two genes.
These data reflect the diverse responses of periodontal
cells to BMP-2 and highlight the necessity to consider
the need to maintain physiologic mediator levels in
designing predictable regenerative therapies.
62
Journal of the International Academy of Periodontology (2014) 16/2
The significant reduction of BMP-2 levels at 14, 21
and 30 days is supported by preclinical studies that have
shown that bone formation initiated by rhBMP-2 is a
self-limiting process. This self-limiting process is caused
by several factors, including the presence of BMP inhibitors in the surrounding tissues and a negative feedback
mechanism that functions at the molecular level (Jortikka
et al., 1997; Gazzerro et al., 1998). The actions of BMPs
are tightly regulated by natural inhibitors, such as follistatin, matrix Gla protein (MGP) and noggin. These
BMP antagonists can bind to BMPs, thereby inhibit the
binding of BMPs to their signaling receptors. The nonsignificant differences in the levels of BMP-2 between
the two groups that were reported at 14, 21 and 30 days
could be attributed to partial disintegration of the occlusive membranes at these time periods, which allowed
for free passage of the growth factors.
Conclusions
Within the limits of the present study one can conclude that perforated collagen membrane coverage of
periodontal defects is associated with a significant initial
increase in GCF levels of BMP-2. This finding suggests
that occlusive membranes could act as a mechanical
barrier, reducing the amount of biologic mediators of
the surrounding overlying tissues from reaching the
defect. Further investigations are necessary with other
mediators of growth and differentiation to confirm
these data with larger sample sizes. Levels of bone regeneration may be evaluated by cone beam 3D dental
imaging devices for further confirmation of the clinical
perforation values. The suggested periosteal and gingival
mesenchymal stem cellular penetration into periodontal
defects through membrane perforations needs to be
further investigated.
Acknowledgments
The authors thank Dr. Khaled Kerra, Al Azhar University, for statistical evaluation of the results. The authors
report no conflicts of interest related to this study.
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