Gingival Phenotype Changes After Different Periodontal Plastic Surgical Techniques.
Phase: NA
Conditions: Gingival Recession
Sponsor: G. d'Annunzio University

GINGIVAL THICKNESS CHANGES AFTER USE OF PLATELET RICH FIBRIN 
MEMBRANE COMPARED TO SUB EPITHELIAL CONNECTIVE TISSUE GRAFT AND 
CORONALLY ADVANCED FLAP ALONE: A CONTROLLED AND RANDOMIZED 
CLINICAL TRIAL  
NCT03712852
08/02/2021

Study Protocol
This is a prospective, randomized and controlled clinical trial comparing the effects of
three surgical treatment modalities for Miller’s class I and II recessions on gingival
phenotype: CAF with L-PRF (CAF+L-PRF group), CAF with SCTG (CAF+SCTG group) and
CAF alone (CAF group). Clinical parameters were evaluated at baseline and 6 months
after surgery. This study is registered at ClinicalTrials.gov as NCT03712852 from
October 19th, 2018. First participants were enrolled in November 2018 and final data
from last participants were collected in July 2019. Figure 1 shows the CONSORT flow
diagram.
Patients’ inclusion criteria
1)to be systemically healthy;  2)not having taken any medications related to
periodontal status in the previous 6 months; 3)not being pregnant/lactating; 4)to be
never-smoker/former-smoker≥10yrs; 5)to have a full-mouth plaque score (FMPS)25  and
full-mouth bleeding score (FMBS)26 <20% at surgery, 6)to have ≥20 teeth without
dental mobility, 7) not having periapical lesions at experimental sites, 8) to have at
least one Miller’s class I or II recession; each patient participated with a single
recession. When more than one GR 
was present and treated, the deepest one only was included in the analysis. Patient’s
concern for GR progression, aesthetics and dentinal hypersensitivity were the main
indications for the surgical procedure. The patients signed a consent form approved by
the ethical committee of the G. D’Annunzio University after having received
comprehensive information. The study is in accordance with the declaration of Helsinki
of 1975, as revised in 2013.

Sample Size 
The primary outcome of the study was GT gain at 6 months. According to a previous
study20 a sample size of 16 patients per group was calculated to detect at the 6-months
follow-up a minimum difference of 0.3 mm in GT between the groups, with an expected
standard deviation of 0.3 mm, an α set at 0.05, and a power of 0.80. To compensate
possible dropouts, 20 patients for group were recruited. 
Randomization and Blinding protocol
The trial director was responsible for randomly assigning patients to treatment after
enrollment and was not involved with the clinical interventions or the study
measurements. A computer-generated table1§  was used to make the random
assignment, known only to the trial director. An opaque envelope concealed group
allocation and was opened just before the intervention surgery. Matching between
group and treatment was performed by a figure extraneous to the experimentation,
responsible even for keeping and breaking the blinding, and known only to him.
A blood draw, needed for the CAF+L-PRF treatment, was done to all patients. Patients
and examiners were masked to group membership; clinical examiners were blinded to
each other.  The study analyst was also blind to group membership. The analyst
received the data by groups labeled as A and B and returned two 90%CIs for the
differences (A minus B and vice versa). The blind was not broken until after study
completion and the correct difference retained.
§ R Core Team (2019) Vienna, Austria

Pre-Surgical Treatment
Participants were instructed with adequate oral hygiene methods. It was suggested the
use of an electric toothbrush with pressure control2‖ and extra-soft head3¶. Instructions
on the optimal use of dental floss, and/or interdental brush were given. All patients
underwent professional supragingival scaling, and they were strictly monitored about
the maintenance of periodontal health. 
Clinical Measurement
All clinical parameters were measured by the same investigator (PS) at baseline and at
6-month. GT was measured 1 mm apical to the sulcus depth, using a #15 endodontic
reamer4#  that was inserted at the mid-buccal site of each tooth. The reamer was
inserted perpendicularly to the gingival surface until the hard tissue was reached.  A
silicon disk stop was moved to contact the soft tissue surface, and this position was
mantained by a cyanoacrylate adhesive drop. Once the reamer was gently removed,
the GT was calculated measuring the distances from the reamer’s tip to the silicon disk
with a caliper5** accurate to the nearest 0.1 mm. Other measurements were accurately
recorded to the nearest millimeter with a periodontal probe6††. GR was measured from
the cement-enamel junction (CEJ) to the mid-buccal point of the gingival margin. KT
resulted from the distance of the mid-buccal site of the gingival margin to the
mucogingival junction. PD and CAL were measured as the distance between the bottom
of the pocket and the gingival margin and CEJ, respectively. 
Surgical Technique
All surgical procedures (figure 2) were performed by a single expert clinician (MP).
‖ Oral-B Pro 6000 Cross Action; Procter & Gamble Italia SPA Gattatico (RE)
¶ Oral B Sensitive EBS17; Procter & Gamble Italia SPA Gattatico (RE)
# Mani, Reamer lenght 25 mm, size 15, Utsonomiya shi, Japan
** Mitutoyo , Model CD-6’’ B , Andover, U.K.
†† XP 23/UNC15, Hu-Friedy MFG-Co, Inc., Chicago, IL, USA

The root surface was accurately treated using Gracey curettes. First, two horizontal
incisions were performed mesial and distal to the GR at a distance from the tip of the
anatomical papillae equal to the depth of the recession plus 1 mm. Secondly, two
oblique incisions were made from the end of the two horizontal incisions to the alveolar
mucosa. The achieved trapezoidal-shaped flap was elevated using a split-full-split
approach27 until the CEJ of the tooth could be passively covered by the marginal portion
of the flap. The anatomic papillae coronal to the horizontal incisions were disepithelized
to supply a connective tissue surface to which the flap’s surgical papillae were sutured.
In L-PRF+CAF and CAF+SCTG groups, the L-PRF grafts and the SCTGs were placed over
the exposed root surface at the CEJ level. The grafts were stabilized by sutures and
covered by the ﬂap that was coronally positioned and sutured about 1-2 mm over the
CEJ in a tension-free position. 
L-PRF Preparation
The Choukroun et al. 28  protocol was applied to produce L-PRF immediately before
surgery. From each patient of both groups, to avoid unblinding, 30 ml of blood was
collected in three 10-ml sterile tubes without anticoagulant, and it was quickly
centrifuged7‡‡ at 3,000 revolutions/minute for 10 minutes.
The fibrin clot (L-PRF) was collected and squeezed in the L-PRF Box8§§ to obtain 2
membranes: each membrane was turned in on itself, and two membranes placed one
over the other (quadruple L-PRF layer) represented the L-PRF graft.
Connective Tissue Graft Preparation 
The connective tissue graft was harvested from the palatal area on the opposite side of
the gingival defect according to Zucchelli et al12. Briefly, two horizontal and two vertical
‡‡ IntraSpin™, Intra-Lock System Europa SpA, Salerno, Italy
§§ Xpression™ Fabrication Kit, Intra-Lock System Europa SpA, Salerno, Italy

incisions delimitated the donor area. The graft was separated from the underlying
tissues by the scalpel’s blade oriented parallel to the palatal surface to obtain an about
2 mm thick graft. Then, the graft was de-epithelialized by a 15c blade and the fatty
tissue was eliminated until obtaining a graft with a thickness of about 1,5 mm.
measured by a standard caliper9‖‖ .
Postoperative Care
 All patients received 2 g/day amoxicillin+clavulanic acid10¶¶ for 6 days for post-
operative infection prevention; 400 mg of oral ibuprofen11##, twice daily, controlled the
pain; 0.12% chlorhexidine12*** rinses, twice daily for 3 weeks, were prescribed to the
patients. Sutures were removed after 14 days. Only 2-4 weeks after sutures removal,
respectively, cautious brushing by a soft toothbrush and interdental brushing were
recommended; meantime, the patients used a 1% chlorhexidine gel13††† twice daily.
Weekly supragingival professional hygiene and motivational reinforcement were
administered to the patients for 6 weeks.
Statistical Analysis
Statistical software R 3.5.1 was used to analyze the clinical data. Descriptive statistics
of GT, KT, CAL, REC, and PPD were planned, expressed as observed means ± SDs and
‖‖ Mitutoyo , Model CD-6’’ B , Andover, U.K.
¶¶ Augmentin, SmithKline Beecham, Milan, Italy
## Nurofen Express 400 mg, Reckitt Benckiser Group, Slough, Berkshire, UK
*** Dentosan 0.12 Trattamento Mese, Johnson & Johnson, Pomezia, Italy
††† Corsodyl Dental gel, GlaxoSmithKline Consumer Healthcare S.p.A. - Baranzate, Italy

95% confidence intervals. Multiple univariate analyses for each variable were
performed. GT was analyzed by non-parametric Cliff's delta tests to assess group
dominance and by a Heteroscedastic ANOVA with Games-Howell posthoc tests. A
sensitivities analysis with different types of robust analyses (M-estimators and High
Breakdown LTS Estimators, both with Huber’s, Hampel’s and Biweight’s loss functions)
was conducted to get an effect-size estimate by means of Bootstrap Bias Corrected and
accelerated (BCa) 95% Confidence Intervals. All the outcomes were analyzed by
posthoc Nemenyi's tests; CAL with a Moderated Regression (Treatment by Baseline
values) too, while their interaction probed by the Johnson-Neyman technique.
RESULTS
Study Population

Sixty patients (twenty-seven women), between 18 and 47 years (mean 32.4 ± 5.0
years), were enrolled in this study after having been visited at the Unit of
Periodontology of the “G. D’Annunzio” University. All 60 patients completed the trial
fitting with specifications, and no postoperative complications were detected.
Experimental groups were balanced by age and gender (p>0.05). 
Clinical Outcomes
A single recession from each patient was evaluated. Out of 60 recessions examined, 35
were classified as Miller Class I (10 incisors, 13 canines and 12 premolars), and 25 were
classified as Class II (7 incisors, 10 canines and 8 premolars). 
The results obtained in this study are summarized in Tables 1 and 2, and in Fig. 3 and 4. 
GT significantly increased from baseline in all experimental groups. 
CAF + SCTG and CAF + L-PRF groups showed a significantly greater GT increase (0.99 ±
0.02 and 0.92 ± 0.52 respectively) as compared to the CAF group (0.31 ± 0.10) while
their direct comparison was inconclusive (NS).
KT showed a significant increase from baseline to the 6 months follow-up in the
CAF+SCTG group, while the same result was not observed in CAF+L-PRF and CAF
groups. In particular, KT has more than doubled in the CAF+SCTG group, and this
increase was significantly greater when compared to CAF+L-PRF and CAF groups; KT
increase was not significantly different between the latter two groups.
GR significantly decreased and CAL significantly improved in each group from baseline
to the 6 months follow-up, without significant differences.
PD showed limited changes in the experimental period without significant differences
among groups, the only exception being CAF+SCTG and CAF groups comparison
(p = 0.012).
Figure 3 shows pairwise group comparisons for all clinical parameters and the Johnson-
Neyman technique probing the interaction between treatment and CAL baseline in the

CAL gain regression for the CAF+PRF and CAF-SCTG group comparison.