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DOI: ./JPER.-
ORIGINAL ARTICLE
The effect of smoking on clinical and biochemical early
healing outcomes of coronally advanced flap with
connective tissue graft: Prospective cohort study
Duygu Taş1,2Şivge Kurgan1Zeliha Güney3Muhittin A. Serdar4
Dimitris N. Tatakis5
Department of Periodontology, Faculty of
Dentistry, Ankara University, Ankara,
Turkey
Graduate School of Health Sciences,
Ankara University, Ankara, Turkey
Department of Periodontology, Faculty
of Dentistry, Ankara Medipol University,
Ankara, Turkey
Department of Medical Biochemistry,
School of Medicine, Acıbadem University,
Ankara, Turkey
Division of Periodontology, College of
Dentistry, The Ohio State University,
Columbus, Ohio, USA
Correspondence
Sivge Kurgan, Department of
Periodontology, School of Dentistry,
Ankara University, -Cankaya,
Ankara – Turkey.
Email: sivgeakgun@gmail.com
Funding information
TUBITAK project program,
Grant/Award Number: S
Abstract
Background: This study aimed to determine the effects of smoking on early
(≤ months) clinical outcomes and relevant molecular biomarkers following root
coverage surgery.
Methods: Eighteen smokers and nonsmokers, status biochemically verified,
with RT gingival recession defects were recruited and completed study proce-
dures. All patients received coronally advanced flap plus connective tissue graft.
Baseline and month recession depth (RD), recession width (RW), keratinized
tissue width (KTW), clinical attachment level (CAL), and gingival phenotype
(GP) were recorded. Root coverage (RC) percentage and complete root coverage
(CRC) were calculated. Recipient (gingival crevicular fluid) and donor (wound
fluid) site VEGF-A, HIF-α, -OHdG, and ANG levels were determined.
Results: There were no significant intergroup differences for any baseline
or postoperative clinical parameters (P>.), except for whole mouth gin-
gival index (increased in nonsmokers at months; P<.). Compared to
baseline, RD, RW, CAL, KTW, and GP significantly improved postoperatively,
without significant intergroup differences. There were no significant intergroup
differences for RC (smokers =%, nonsmokers =%, P=.), CRC (smok-
ers =%, nonsmokers =%, P=.), and CAL gain (P=.). The
four biomarker levels significantly increased postoperatively (day ; P≤.)
in both groups and returned to baseline (day ) without significant inter-
group differences (P>.). Similarly, donor site parameters were not different
between groups. Strong correlations, consistent over time, were found between
biomarkers implicated in angiogenesis (VEGF-A, HIF-α, and ANG).
Conclusions: The early ( month) clinical and molecular changes after root cov-
erage surgery utilizing a coronally advanced flap plus connective tissue graft are
similar between smokers and nonsmokers.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf ofAmerican Academy of Periodontology.
J Periodontol. ;–. wileyonlinelibrary.com/journal/jper 1
2TA .
KEYWORDS
-hydroxy-deoxyguanosine, angiogenin, gingival recession, hypoxia inducible factor-𝛼,
smoking, vascular endothelial growth factor
1 INTRODUCTION
Gingival recession (GR) is defined as the “apical migra-
tion of the gingival margin beyond the enamel-cementum
junction.”GR, which can be found in every popula-
tion at any age, often causes patient complaints such
as aesthetic concerns, dentinal hypersensitivity, and root
caries.Among the various root coverage procedures typ-
ically used, connective tissue graft (CTG) with coronally
advanced flap (CAF) has been determined as the gold
standard., However, many factors have been identified
that affect the success of various GR surgical treatment
approaches, including patient-related factors, such as
smoking.,, Smoking, in particular, affects relevant gin-
gival crevicular fluid (GCF) molecular components,, and
gingival immune response elements,and impairs wound
healing.
Wound healing is a complex process that involves coor-
dinated interactions between diverse systems, and molec-
ular events considered critical for optimal outcomes.
Among several investigated biomarkers, vascular endothe-
lial growth factor (VEGF), hypoxia inducible factor-𝛼
(HIF-α), -hydroxy-deoxyguanosine (-OHdG), and
angiogenin (ANG), have been implicated in wound heal-
ing, angiogenesis, and vascularization, and are released
from cells participating in wound healing and tissue
remodeling. Especially in the early wound healing period
( to days), angiogenesis is the most active. For this rea-
son, VEGF, ANG, and HIF-α, which play important roles
in response to hypoxia and angiogenesis, were chosen as
target molecules in the current study. Of these molecules,
only VEGF has been studied in relation to root coverage
procedures.,
Although studies have evaluated the impact of smok-
ing on root coverage outcomes of CAF alone,,
and with CTG procedures,,– the underlying mech-
anisms have not been fully understood. Of the afore-
mentioned studies, only two included biochemical ver-
ification of smoking status,, an important consider-
ation for studies on smokers given that self-reported
smoking status may lead to significant bias (% to
%).
The aim of the present study was to determine the
effects of smoking on the clinical (root coverage and donor
site healing) and molecular (levels of VEGF-A, HIF-α,
-OHdG, and ANG) early healing outcomes of CAF+CTG.
2 MATERIALS AND METHODS
2.1 Study population and design
Systemically healthy patients diagnosed with recession
type (RT) GR defects were included in this prospec-
tive cohort study (Figure ). Recruitment occurred between
February and December in the Ankara Univer-
sity Faculty of Dentistry, Department of Periodontology.
The protocol and consent for this study were approved
by the Ankara University Ethics Committee on Human
Research (#/; April , ); the study was
conducted according to the Declaration of Helsinki, as
revised in . All participants provided written informed
consent.
Each participant contributed only one single recession
site to the study. Inclusion criteria were assessed after
radiographic and full-mouth clinical periodontal examina-
tion, including probing depth (PD), plaque index (PI),
gingival index (GI), bleeding on probing (BOP), clinical
attachment level (CAL), recession depth (RD), recession
width (RW), and keratinized tissue width (KTW). Par-
ticipants had to have ≥ natural teeth (excluding third
molars) and RT GR.
Patients with systemic diseases, for example diabetes
mellitus, rheumatoid arthritis, obesity, cancer, and severe
cognitive or psychiatric disorders, and pregnant and lactat-
ing women were excluded. Patients having oral appliances
or prostheses with any contact with the hard palate,
active orthodontic treatment (fixed or removable appli-
ances), cleft palate, and history of any surgical procedure at
either the palatal donor site or the recipient site were also
excluded.
Patients were treated with CAF +CTG for root coverage,
and follow-up visits were conducted on days and and
at months postoperatively.
2.2 Clinical parameters
All clinical parameters were recorded preoperatively
(baseline) and days and months postoperatively, by
a sole blinded examiner (DT). For each patient, a custom
plastic occlusal stent was fabricated to standardize the
mesiodistal location of midfacial measurements and used
to record clinical parameters, which were measured using
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TA . 3
FIGURE 1 CONSORT flowchart of the study. CAF, coronally advanced flap; CTG, connective tissue graft.
a calibrated UNC- periodontal probe*and recorded to the
nearest . mm. More specifically, the recorded parameters
and their assessment criteria for this study are as follows.
. Gingival phenotype (GP): A standard periodontal probe
was placed in the gingival sulcus, and GP was consid-
ered “thin” if the probe was visible/shining through
the gingival marginal tissue and “thick” if the probe
was not visible
. Recession depth (RD): Distance from the cemen-
toenamel junction (CEJ) to the gingival margin
(GM)
. Recession width (RW): Distance between mesial and
distal GMs, on a horizontal line tangential to the CEJ
. Probing depth (PD): Distance from the GM to the
bottom of the sulcus
. Clinical attachment level (CAL): Calculated as RD+PD,
while CAL gain was calculated as the difference
between postoperative and baseline CAL
. Keratinized tissue width (KTW): Distance from the GM
to the mucogingival junction
. Bleeding on probing (BOP): Presence or absence at the
treatment site
. Gingival Index and Plaque Index (GI and PI): Measured
on three surfaces, mesial, distal, and midfacial
. Root coverage percentage (RC %): Calculated as
described by Silva et al., namely, as (preoperative RD
−postoperative RD) ×/preoperative RD
. Complete root coverage (CRC): percent of sites present-
ing with % RC
GP, RD, PD, CAL, and KTW were measured on the
midfacial surface of the treatment site.
*UNC Color-Coded Probe, Hu-Friedy, Chicago, IL, USA.
2.3 Examiner calibration
Prior to the study, the examiner was calibrated for clinical
parameter and photographic measurements. Ten volun-
teers were assessed twice with a hour period in between,
with the second set performed blinded with respect to
the initial one. Calibration was accepted if the agreement
between repeated clinical measurements was ≥%. Ten
photographs were taken at distinct moments from patients
not participating in the study; these measures were submit-
ted to an intraclass correlation (ICC) test and training was
considered complete when the ICC value exceeded ..
2.4 Treatment procedures
Before surgery, all patients received oral hygiene instruc-
tions. Surgery was performed only when ≥% of all sites
were plaque free and the treatment site had a PI value of
. Defects were treated by a sole experienced periodon-
tist (SK). After establishment of local anesthesia (articaine
mg/mL + mcg/mL epinephrine), exposed root sur-
faces (CEJ to just below the GM) were instrumented with
periodontal curettes, EDTA %†was applied for min, fol-
lowed by rinsing for s with sterile saline. The surgical
approach was as described by Cairo et al. After local anes-
thesia, two opposing oblique relaxing incisions were made
extending beyond the mucogingival junction. An intrasul-
cular incision was made at the buccal aspect of the involved
tooth. After the papillae were dissected split-thickness,
a full-thickness flap was raised up to the mucogingival
junction. Then, a partial-thickness flap extending beyond
the mucogingival junction was raised to allow passive
†EDTA Gel %, Biodinamica, Ibiporã, Portugal.
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4TA .
coronal displacement of the flap. The papillae adjacent
to the involved tooth were de-epithelialized. The exposed
buccal and interproximal areas were debrided using a
sharp curette. Grafts were obtained using the extraoral
de-epithelialization approach and harvested between the
canine and first molar area, with a thickness of . to mm.
Grafts were positioned on the root surface slightly apical
to the CEJ and stabilized using a cross-suspended suture.‡
Subsequently, the flap was positioned to mm coronal to
the CEJ and stabilized with suspensory sutures. Finally,
interrupted sutures closed the vertical releasing incisions.
Periodontal dressing was not applied.
2.5 Postoperative protocol
Patients were instructed to avoid mechanical trauma,
chewing, brushing, and flossing in the surgical field for
weeks. Plaque control was achieved with prescribed
antimicrobial rinse (chlorhexidine .%, twice daily, for
weeks) and pain control with prescribed analgesic
(ibuprofen) as needed. Although patients were informed
of the negative effects of smoking on procedure outcomes,
no specific smoking cessation instructions were given.
Sutures were removed days after surgery.
2.6 Donor site evaluation
The following parameters were assessed regarding the
palatal donor site.
Early bleeding was recorded after the donor site was
sutured and pressure was applied with sterile gauze for
min. Hemostasis was confirmed when no active bleeding
was observed.
Late bleeding was recognized as any bleeding from the
palate reported by the patient during the postoperative
period.
2.7 Patient-reported outcomes
Postoperative pain was recorded by the patients postop-
eratively (at and days, and at months), using a
visual analog scale ( =no pain, =extreme pain), after
application of air spray for s.
2.8 Smoking status determination
Patient smoking history was confirmed biochemically,
through salivary cotinine analysis. Unstimulated saliva
‡Propylene -, GOLNIT, Kiev, Ukraine.
samples were collected during morning hours (: to
: a.m.). Patients were asked to thoroughly rinse their
mouth with distilled water and then instructed to sit
comfortably and spit into a plastic tube ( min). Col-
lected samples were then centrifuged ( ×g;min)
and supernatants transferred into Eppendorf tubes and
stored at −◦C until analysis. Salivary cotinine analysis
was performed by liquid chromatography–mass spectrom-
etry (LC-MS/MS)§. Patients were classified as smokers
if they reported smoking ≥ cigarettes daily for ≥ years
and if salivary cotinine levels were >. mg/L (based on
receiving operator characteristic analysis of prior results
obtained with the in-house LC-MS/MS detection method),
whereas nonsmokers were individuals who had never
smoked in their lifetime.
2.9 Biological fluid sample collection
From the recipient site, GCF samples were obtained preop-
eratively (day of surgery), and wound fluid (WF) samples
were obtained on postoperative days and . After iso-
lation of the area to prevent saliva contamination, and
gentle air-drying of the tooth surface, samples were col-
lected using standardized paper strips** carefully inserted
in the sulcus orifice (extra sulcular technique), to avoid
mechanical tissue stimulation. Four sequential samples
( s each) were obtained from each site and were pooled
for analyses.
From the donor site, WF samples were obtained on post-
operative day , using the paper strips, after the wound
area was gently air-dried and cotton rolls were placed in
the buccal sulcus to prevent saliva contamination. Paper
strips were placed sequentially in the middle of the wound
area for s. Four strips were collected and pooled
from the same wound. Blood-contaminated strips were
discarded. Collected fluid volume was measured using a
calibrated instrument†† and the paper strips were immedi-
ately placed in coded polypropylene microcentrifuge tubes
andstoredat–
◦C until further analysis.
2.10 Biochemical parameters
Pooled strips were eluted as previously detailed. Eluted
samples were analyzed for VEGF-A, HIF-α,-OHdG,and
ANG using commercial ELISA kits,‡‡ following manu-
facturer’s instructions. Concentrations were determined
§TSQ Quantum Access MAX Triple Quadrupole Mass Spectrometer,
Thermo Fisher Scientific, Waltham, MA, USA.
** Periopaper, Oraflow, Hewlett, NY, USA.
†† Periotron R , Oraflow, Hewlett, NY, USA.
‡‡ ELISA Cloud Immunoassay, Cloud-Clone Corp., Houston, TX, USA.
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TA . 5
based on the respective assay standard curve. Intra- and
inter-assay coefficients of variation were <% for all
parameters.
All samples were analyzed in duplicate, and the aver-
age used in subsequent calculations. Obtained values were
corrected for the original GCF/WF volume and results
were expressed as picogram (pg)/site (HIF-α,ANG)or
femtogram (fg)/site (-OHhdG, VEGF-A).
2.11 Power analysis and data
management
Sample size determination was performed a priori. For
two groups, with effect size of . to detect a minimum
clinically significant difference in root coverage of mm
(primary outcome) using α-error =., and power =%,
sample size per group was n=. Considering the possi-
bility of dropouts and incomplete data, the study recruited
patients ( per group).
All analyses were performed using statistics software.§§
Data normality was tested by the Shapiro–Wilk test prior
to further analysis. Student’s t-tests and Mann–Whitney
U-tests were performed to compare smokers and non-
smokers. Chi-square and Fisher exact chi-square tests were
applied for categorical variables. Friedman and repeated
measures analysis of variance were performed for depen-
dent variables to compare baseline, and day, and
month data. Parametric data are reported as mean ±SD
and nonparametric data are reported as median (interquar-
tile range). The Spearman test was applied for the corre-
lation analysis. Linear regression models were performed
to evaluate the existence of any significant difference
regarding, HIF-α, ANG, -OHdG, and VEGF-A between
smoking status, time (baseline, day , and day ), and
the interaction between smoking status and time. Analysis
with the Bonferroni test was completed to compare multi-
ple scores at each time point. All tests were performed at a
significance level of α=..
3RESULTS
3.1 Study population
Forty patients ( smokers, nonsmokers) met the inclu-
sion criteria and were recruited in the study. Of those, four
patients (two smokers, two nonsmokers) failed to attend
follow-up visits and were excluded. Therefore, smok-
ers ( females), aged . ±. years, and nonsmokers
§§ SPSS for Windows v., IBM SPSS Inc., New York, NY, USA.
( females), aged . ±. years, completed all study
procedures. There were no significant differences between
the groups regarding age or sex (P>.). Salivary coti-
nine levels were . ±. mg/L and . ±. mg/L
for smokers and nonsmokers, respectively (P<.). All
smokers reported smoking ≥ and ≤ cigarettes/day.
3.2 Clinical periodontal measurements
Periodontal clinical parameters at the recipient (treat-
ment) site over time are reported in Table . For the
clinical periodontal parameters of PI, GI, BOP, and PD
of the treated site, no significant intra- or intergroup dif-
ferences were observed at any time point (P>.). For
CAL and gingival thickness (phenotype), there was no
significant intergroup difference at any time point, but sig-
nificant intragroup differences were found between pre-
and postoperative values for both groups (P<. for
both smokers and nonsmokers). CAL gain (baseline to
months) was not significantly different between groups
(P=.; Table ).
There were no significant differences between smoker
and nonsmoker groups in RD, RW, and KTW at baseline
and month measurements (P>.) (Table ). In both
groups, RD and RW decreased significantly at months
compared with baseline (P<.). KTW increased sig-
nificantly at months in both smoker and nonsmoker
groups (P<.). There were no significant differences
for change (difference between baseline and months) in
RD and KTW between groups (P=. and P=.,
respectively).
RC and CRC were higher for nonsmokers than for
smokers, although the differences were not statistically
significant (P=. and P=., respectively).
No significant correlations were found between coti-
nine levels and any of the clinical parameters (data not
shown).
3.3 Donor site clinical parameters
There was no significant difference between groups
regarding early or late postsurgical bleeding (P=. and
P=., respectively) (Table ).
3.4 Patient-reported outcomes
There was no significant difference between groups, at
any postoperative time point, regarding postoperative pain
(P≥.) (Table ).
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6TA .
TABLE 1 Recipient site clinical parameters over time.
Parameters Smokers (n=18) Nonsmokers (n=18) P
Gingival phenotype (thin/thick)
Baseline / / .
months /*/*.
RD (mm)
Baseline . (.–.) . (.–.) .
months (–)*. (–)*.
∆Baseline- months . (.–.) . (.–.) .
PD (mm)
Baseline . ±. . ±. .
Day . ±. . ±. .
months . ±. . ±. .
CAL (mm)
Baseline . ±. . ±. .
months . ±.*. ±.*.
∆Baseline- months . ±. . ±. .
RW (mm)
Baseline . (.–.) . (.–.) .
months . (.–.)*. (.–.)*.
KGW (mm)
Baseline . ±. . ±. .
months . ±.*. ±.*.
∆Baseline- months . ±. . ±. .
BOP (%)
Baseline . (.–.) . (.–.) .
Day . (.–.) . (.–.) .
months . (.–.) . (.–.) .
PI
Baseline . (.–.) . (.–.) .
Day . (.–.) . (.–.) .
months . (.–.) . (.–.) .
GI
Baseline . (.–.) . (.–.) .
Day . (.–.) . (.–.) .
months . (.–.) . (.–.) .
RC (%)
months . ±. . ±. .
CRC (yes/no; %)
months /; /; .
Note: Reported values are mean ±SD, median (interquartile range), or frequency (n/n).
Abbreviations: BOP, bleeding on probing; CAL, clinical attachment level; CRC, complete root coverage; GI, gingival index; KGW, keratinized gingivawidth;PD,
probing depth; PI, plaque index; RC, root coverage; RD, recession depth; RW, recession width.
*Significant change over time in within-group comparisons, P<..
3.5 Biochemical parameters
For the recipient site, the results of the GCF/WF sample
biochemical analyses are presented in Figure . There were
no significant differences in the analyzed biochemical
parameters (VEGF, HIF-α, ANG, and -OHdG) between
the smoker and nonsmoker groups (P≥.).
For the four assessed biomarkers, the effect of time was
statistically significant in both groups (P<.), with day
levels being significantly higher than baseline (P≤.)
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TA . 7
TABLE 2 Donor site clinical and biochemical parameters.
Parameters Smokers (n=18) Nonsmokers (n=18) P
Early bleeding . (.–.) . (.–.) .
Late bleeding . (.–.) . (.–.) .
Postoperative pain
Day . (.–.) . (.–.) .
Day . (.–.) . (.–.) .
months . (.–.) . (.–.) .
WA (mm )
IP . (.–.)a. (.–.)a.
Day . (.–.)b. (.–.)c.
Day . (.–.) . (.–.) .
months . (.–.) . (.–.) .
VEGF (fg/site)
Day . (.–.) . (.–.) .
HIF-α(pg/site)
Day . (.–.) . (.–.) .
ANG (pg/site)
Day . (.–.) . (.–.) .
-OHdG (fg/site)
Day . (.–.) . (.–.) .
Note: Reported values are median (interquartile range).
Abbreviations: -OHdG, -Hydroxy-deoxyguanosine; ANG, angiogenin; HIF-α, hypoxia inducible factor- alpha; IP, immediate postoperatively; VEGF, vascular
endothelial growth factor; WA, wound area.
aDay significantly different than day and months in both smokers and nonsmokers (P<.).
bDay significantly different than day and months in smokers (respectively; P=., P<.).
cDay significantly different than day and months in nonsmokers (respectively; P=., P<.).
or day levels (P≤.), and no significant differences
between baseline and day (P>.). There was no sig-
nificant interaction between time and smoking status for
any of the biochemical parameters (P≥.).
For the donor site, WF biochemical parameter
results are presented in Table . There were no sig-
nificant intergroup differences for any of the analyzed
biomarkers (VEGF, HIF-α, ANG, and -OHdG) at day
(P≥.).
3.6 Correlation analyses
The correlation analysis of biochemical parameters is
reported in Table . The results indicate that GCF base-
line VEGF was significantly positively correlated with both
baseline HIF-αand baseline ANG (P<.) and baseline
HIF-αwas also significantly positively correlated with
baseline ANG (P<.). No significant correlations were
found between molecular markers and clinical parameters
(data not shown).
The recipient site results were consistent over time, with
VEGF values being significantly positively correlated with
corresponding HIF-αand ANG values on day (P<.)
and day (P<.), and HIF-αlevels being significantly
positively correlated with ANG levels on both day and day
(both P<.) (Table ).
When the values from all three time points (baseline,
day , and day ) were analyzed together (n= per
parameter), the correlations between VEGF, HIF-α,and
ANG remained significant (r≥., P<.), while
correlations between -OHdG and VEGF (r=.), HIF-
α(r=.), and ANG (.) also became significant
(P<. for all three).
At the donor site (day ), the WF findings were sim-
ilar to the recipient site GCF results, with VEGF being
significantly positively correlated with HIF-αand ANG
(P=. and P=., respectively), and HIF-α
also being significantly positively correlated with ANG
(P<.) (Table ).
4DISCUSSION
The present study aimed to determine the clinical (root
coverage and donor site healing) and molecular (levels of
VEGF-A, HIF-α, -OHdG, and ANG) outcomes during
the early healing ( days to months postoperatively) of
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8TA .
FIGURE 2 Biomarker levels at recipient site over time, showing gingival crevicular and wound fluid levels of (A) vascular endothelial
growth factor (VEGF), (B) hypoxia inducible factor -𝛼(HIF-α), (C) angiogenin (ANG), and (D) -hydroxy-deoxyguanosine (-OHdG), in
smokers (red) and nonsmokers (black), preoperatively (baseline) and postoperatively (days and ). Box-and-whisker plots with the median
(horizontal line), interquartile range (box), and outlier (circles) values are shown. Day is significantly higher than baseline (VEGF, HIF-a,
ANG: P≤.; -OHdG: P=.) and day (all four molecules: P=.) in nonsmokers. Day is significantly higher than baseline
(VEGF, HIF-a, ANG: P<.; -OHdG: P=.) and day (VEGF, HIF-a, ANG: P<.; -OHdG: P=.) in smokers.
CAF +CTG in smokers and nonsmokers. There were no
statistically significant differences in clinical or molecular
parameters between smokers and nonsmokers during this
early healing period. The results of this study indicate that
on postoperative days and there is a strongly correlated
molecular response of factors implicated in angiogenesis
and wound healing (VEGF-A, HIF-α, and ANG). This
response, which was evident in both the recipient and
the donor site, and consistent between the two surgi-
cal sites, provides new insights regarding the molecular
mechanisms operating during the early healing after this
common surgical approach for root coverage. These novel
findings should help guide future research on the molecu-
lar aspects of wound healing following periodontal plastic
surgery procedures.
The negative effect of smoking on root coverage proce-
dures, and especially for CAF +CTG, has been reported
in several studies and systematic reviews.,,,, The
present study failed to identify any statistically signifi-
cant differences in clinical outcomes between smokers
and nonsmokers during the early healing period (≤
months), despite the fact that both RC and CRC were
lower in smokers, and the fact that smoking status was
biochemically verified. This discrepancy is not unique to
the present study; other investigators have reported no
statistically significant effect of smoking on root cover-
age at early time points, but by the month follow-up
there were differences., Similarly, in the case of guided
tissue regeneration therapy for intrabony defects, the neg-
ative effects of smoking, in terms of tissue and CAL gain
(%), were not evident until months postoperatively.
Collectively, the available evidence, in conjunction with
the reported trends at months (current study), suggests
that the planned long-term follow-up of the present study
population should help settle this apparent discrepancy.
The average root coverage outcomes obtained in the
present study (% for nonsmokers and % for smokers)
are consistent with or better than the results of several
other studies employing the same surgical approach for
treatment of GR defects.,, Also consistent with earlier
studies is the finding that CAF +CTG resulted in KTW
increases and biotype modification (change from thin to
thick), without significant differences between smokers
and nonsmokers.,,
With respect to the donor site healing outcomes,
that is, postoperative bleeding, pain, and wound area,
there were no differences between smokers and non-
smokers. This contrasts with studies that have reported
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TA . 9
TABLE 3 Correlations between biochemical parameters.
Recipient site
Parameters VEGF HIF-1α8-OHdG
Baseline
HIF-α0.588** ––
-OHdG . . –
ANG 0.751** 0.781** .
Day
HIF-α0.876** ––
-OHdG –. . –
ANG 0.813** 0.833** .
Day
HIF-α0.450*––
-OHdG . 0.396*–
ANG 0.685** 0.726** .
Donor site
Day
HIF-α0.546*
-OHdG . –.
ANG 0.388*0.703** .
Note: Biochemical parameters were determined from gingival crevicular fluid
(recipient site) or wound fluid (donor site) samples obtained at the spec-
ified time points. Spearman’s correlation test (n=, per parameter, per
time-point).
Abbreviations: -OHdG, -Hydroxy-deoxyguanosine; ANG, angiogenin; HIF-
α, hypoxia inducible factor- alpha; VEGF, vascular endothelial growth factor.
*P<..
**P<..
decreased bleeding, increased pain, and delayed
epithelialization in smokers. The disagreement between
these earlier studies and the present one may be related to
the reduced thickness (≤ mm) of the grafts harvested in
the current study, as well as the much smaller wound size
(∼% smaller on average) in the present study.
The superiority of CAF +CTG as a root coverage modal-
ity has been attributed, at least in part, to the additional
blood supply provided by the overlying flap. Despite
the significance of revascularization for the proper heal-
ing of CTGs, there are very few studies on the molecular
mechanisms involved. The present study demonstrated
that VEGF-A, HIF-α, and ANG, three interconnected
molecules implicated in angiogenesis and wound heal-
ing, are significantly elevated at days postoperatively
and return to baseline levels by day , with their lev-
els being highly correlated with each other. In addition,
the day increased levels coincide with the reported most
active period of CTG revascularization. This finding is
in agreement with the reported increased ANG expression
that correlates with the increase of neovascularization in
human burn wounds.
The highly correlated levels of ANG, VEGF, and HIF-
αduring CTG healing, for both recipient and donor sites,
are consistent with the known biological functions of these
molecules. HIF-α, which plays a critical role in response
to hypoxic conditions, to which the harvested gingival graft
tissues are exposed until revascularization is established,
has been shown to stimulate ANG and VEGF production
in periodontal fibroblasts., In addition, ANG and VEGF
are two main molecular factors associated with endothe-
lial cell functions. ANG is necessary for angiogenesis,
and the coordinated expression of VEGF and HIF-αis
necessary for neovascularization. In our study, HIF-α,
VEGF, and ANG values were correlated with each other in
both recipient and donor sites, consistent with reported in
vitro coordinated responses for these molecules, obtained
with periodontal ligament fibroblasts. Smoking lev-
els in moderate smokers in our study group did not
affect these biological markers, which have an impor-
tant role in wound healing. Long-term studies in heavy
smokers may be more appropriate for evaluating these
results.
There are no other studies reporting on ANG or HIF-
αin root coverage or other periodontal flap procedures.
However, Morelli et al. reported, at postoperative week
, increases in ANG and VEGF levels in WF obtained
from autogenous free gingival grafts used for gingival
augmentation, a finding consistent with the present
study’s results. Two previous studies investigated GCF
VEGF levels in the course of root coverage procedures.
Kaval et al. examined baseline GCF VEGF levels in smok-
ers and nonsmokers that were treated with CAF alone and,
similar to the present study, found no differences between
smokers and nonsmokers. Dias et al. examined GCF
VEGF levels in nonsmokers treated with CAF +CTG or
with CAF +CTG +biologic (enamel matrix derivative)
and found increased VEGF levels only at days and only
in the group with the added biologic.
Regarding the molecular results specifically at the donor
site, the coordinated levels of proteins critical for angiogen-
esis and wound healing is consistent with findings from
palatal wound gene expression studies where, at day post
wounding, angiogenesis- and vasculature development-
related genes are the most significantly regulated.,
The coordinated and elevated expression of interleukin-
(IL-) in such wounds is congruent with the recently
recognized role of this cytokine in inducing angiogenesis
and stimulating expression of ANG, HIF-α, and VEGF.
The present study is not without limitations. The level
of smoking of the participants, who were considered
moderate smokers (smoking ≥ and ≤ cigarettes per
day), may have contributed to the resulting lack of sig-
nificant differences between smokers and nonsmokers.
Analyses of additional relevant biomarkers (e.g., IL-
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10 TA .
and epidermal growth factor) would have offered fur-
ther insights into the functional healing mechanisms. The
lack of analyses at earlier time points (e.g., day or
postoperatively) might have concealed other, yet to be
determined, relevant molecular changes. However, the
present study’s strengths include the biochemical verifica-
tion of smoking status, the combined molecular and clini-
cal assessments, the synchronous evaluation of donor and
recipient sites, and the novel focus on relevant molecular
markers.
5CONCLUSIONS
Within the limitations of the present study, the early
(≤ months) healing outcomes of CAF +CTG are not
statistically significantly different between smokers and
nonsmokers. The investigated molecular markers, ANG,
VEGF, and HIF-α, are significantly elevated during the
first postoperative week and return to baseline levels by
the fourth postoperative week, while their levels in both
the recipient and the donor sites are consistently highly
correlated, indicative of the strictly regulated processes
necessary to have proper wound healing.
All authors have made substantial contributions to study
conception and design. Duygu Ta and ivge Kurgan per-
formed data collection. Duygu Ta, ivge Kurgan, Zeliha
Güney, Muhittin A. Serdar, and Dimitris N. Tatakis con-
tributed to data analysis, data interpretation, drafting, and
critically revising the manuscript. All authors approved the
final version submitted for publication.
AUTHOR CONTRIBUTIONS
All authors have made substantial contributions to study
conception and design. Duygu Ta and ivge Kurgan per-
formed data collection. Duygu Ta, ivge Kurgan, Zeliha
Güney, Muhittin A. Serdar, and Dimitris N. Tatakis con-
tributed to data analysis, data interpretation, and draft-
ing and critically revising the manuscript. All authors
approved the final version submitted for publication.
ACKNOWLEDGMENTS
This study was supported by project number S of
TUBITAK project program, Ankara, Turkey.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
ORCID
Dimitris N. Tatakis https://orcid.org/---
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How to cite this article: Ta D, Kurgan , Güney
Z, Serdar MA, Tatakis DN. The effect of smoking on
clinical and biochemical early healing outcomes of
coronally advanced flap with connective tissue
graft: Prospective cohort study. J Periodontol.
;-. https://doi.org/./JPER.-
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