Clinical Oral Investigations

https://doi.org/10.1007/s00784-018-2470-6

ORIGINAL ARTICLE

Acellular dermal matrix allograft versus free gingival graft: a histological

evaluation and split-mouth randomized clinical trial
Daniel Romeu Benchimol de Resende 1 & Sebastião Luiz Aguiar Greghi 2 & Aline Franco Siqueira 1 &
César Augusto Magalhães Benfatti 3 & Carla Andreotti Damante 2 & Mariana Schutzer Ragghianti Zangrando 2

Received: 25 October 2017 / Accepted: 23 April 2018

# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Objectives This split-mouth controlled randomized clinical trial evaluated clinical and histological results of acellular dermal
matrix allograft (ADM) compared to autogenous free gingival graft (FGG) for keratinized tissue augmentation.
Material and methods Twenty-five patients with the absence or deficiency of keratinized tissue (50 sites) were treated with FGG
(control group) and ADM (test group). Clinical parameters included keratinized tissue width (KTW) (primary outcome), soft
tissue thickness (TT), recession depth (RD), probing depth (PD), and clinical attachment level (CAL). Esthetic perception was
evaluated by patients and by a calibrated periodontist using visual analog scale (VAS). Histological analysis included biopsies of
five different patients from both test and control sites for each evaluation period (n = 25). The analysis included percentage of
connective tissue components, epithelial luminal to basal surface ratio, tissue maturation, and presence of elastic fibers. Data were
evaluated by ANOVA complemented by Tukey’s tests (p < 0.05).
Results After 6 months, PD and CAL demonstrated no differences between groups. ADM presented higher RD compared to
FGG in all periods. Mean tissue shrinkage for control and test groups was 12.41 versus 55.7%. TT was inferior for ADM group
compared to FGG. Esthetics perception by professional evaluation showed superior results for ADM. Histomorphometric
analysis demonstrated higher percentage of cellularity, blood vessels, and epithelial luminal to basal surface ratio for FGG group.
ADM group presented higher percentage of collagen fibers and inflammatory infiltrate.
Conclusions Both treatments resulted in improvement of clinical parameters, except for RD. ADM group presented more tissue
shrinkage and delayed healing, confirmed histologically, but superior professional esthetic perception.
Clinical relevance This study added important clinical and histological data to contribute in the decision-making process between
indication of FGG or ADM.

Keywords Gingival recession . Mucogingival surgery . Grafts . Acellular dermal . Gingiva

Introduction

Autogenous free gingival graft (FGG) has been employed in

Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00784-018-2470-6) contains supplementary
material, which is available to authorized users.
* Mariana Schutzer Ragghianti Zangrando
mariana@fob.usp.br
1
Bauru, Brazil
2
Department of Prosthodontics and Periodontics, Bauru School of
Dentistry, University of Sao Paulo, Al. Octávio Pinheiro Brisolla
9-75, Bauru, SP 17012-901, Brazil
3
Department of Dentistry, Federal University of Santa Catarina,
UFSC, Campus Reitor João David Ferreira Lima,
Florianópolis, SC s/n-88040-900, Brazil
periodontal surgery to increase width of attached gingiva
around teeth since 1963 [1, 2]. Considering the challenges of
soft tissue augmentation procedures in periodontal and im-
plant surgical procedures, FGG has been indicated for increas-
ing keratinized tissue around teeth and implants in esthetic
zones [3]. Despite the high success rate, some disadvantages
are related to FGG such as two surgical sites (recipient and
donor); pain and discomfort in donor site healing process,
anesthetic appearance, and limited source of donor tissue
[4–6].
Since the 80s, tissue substitutes were introduced as an al-
ternative to autogenous grafts in achieving increased width of

attached gingiva [7]. The acellular dermal matrix (ADM) con-
sists of an allogeneic freeze-dried connective tissue matrix,
without epithelium and cellular components, containing types
I- and III- collagen bundles and elastic fibers [8, 9]. This ma-
trix is obtained from human tissue banks by a standardized and
controlled industrialized procedure [8, 9]. The ADM allograft
is considered a bioactive scaffold for migration of fibroblasts,
epithelial, and endothelial cells and could consistently inte-
grate into the host tissue [8, 9]. Structural integrity of the
material is maintained, and revascularization process depends
on vascular channels of recipient site [10]. Advantages of
ADM compared to FGG are single surgical site, less post-
operative pain and discomfort, better esthetics, and blending
with the surrounding tissue [6, 11]. However, clinical out-
comes revealed that FGG was more effective and predictable
than ADM in increasing attached keratinized tissue [6, 11].
ADM presented considerable shrinkage and inconsistent qual-
ity of gained attached tissue [6, 11]. Otherwise, ADM revealed
superior esthetic results compared to autogenous graft [6, 11].
There are few studies comparing clinical and histological
results of FGG and ADM for keratinized tissue augmentation
[6, 11–13]. According to a meta-analysis [13], only two ran-
domized clinical trials [6, 14] compared ADM versus FGG.
Results demonstrated no statistically significant differences
between groups and high levels of heterogeneity among stud-
ies [13]. Most ADM studies included a small sample size that
lacked sufficient statistical power to draw conclusions regard-
ing the efficacy of ADM [13]. A recent published study [11]
evaluated and compared, only by clinical parameters, the ef-
fectiveness of FGG and ADM in the ability to increase at-
tached gingiva. Thus, the present split-mouth controlled and
randomized clinical trial, with a substantial sample, aimed to
evaluate clinical and histological outcomes of ADM com-
pared to autogenous FGG for keratinized tissue augmentation.
Material and methods
Clinical study
This study was approved by The Ethics Committee in
Research of Bauru School of Dentistry- University of Sao
Paulo (USP-0110-1999). Clinical trials.gov registration:
NCT03251001. The procedures were explained to patients
who signed a consent form according to the research terms.
Patients were treated at the Periodontics Clinic of Bauru
School of Dentistry (University of Sao Paulo) and presented
the following inclusion criteria: absence or deficiency of
keratinized tissue (KTW < 1 mm) in two homologous
contralateral sites of inferior premolars, vital tooth or with
adequate endodontic treatment, root surfaces without caries,
good oral hygiene (PI < 20%), and absence of active
periodontal disease. Exclusion criteria were allergy to
Clin Oral Invest
penicillin, systemic disease that impedes surgical procedure,
smokers, pregnancy, and intake of medications that cause
gingival enlargement.
Clinical parameters were collected at baseline and after 30,
60, 90,120, 150, and 180 days after surgeries. Site-related
outcome variables were measured at the mid-buccal point of
the treated tooth, using an acrylic occlusal guide and an end-
odontic stop, to standardize the positioning of periodontal
probe during examination. Measurement between endodontic
stop and tip of periodontal probe was made with a digital
caliper. Clinical parameters included primary outcome:
keratinized tissue width (KTW), measured from gingival mar-
gin to mucogingival junction; and secondary outcomes: prob-
ing depth (PD), clinical attachment level (CAL), and recession
depth (RD). Soft tissue thickness (TT) was evaluated using an
ultrasound device (SDM, Krupp Medizintechnik GmbH -
Germany) positioned 1 mm apical to gingival margin. The
percentage of tissue shrinkage was calculated based on stan-
dardized initial width of ADM (AlloDerm®, BioHorizons,
Birminghan - USA) and FGG (5 mm) and mean value of
KTW after 6 months, according to the formula:
Percentage of soft tissue shrinkage
ð5−final KTWÞ  100%
¼
5
All clinical parameters were evaluated by a calibrated ex-
aminer (AFS) (intraclass correlation coefficient = 0.98)
blinded to test and control groups.
Esthetic perception of clinical outcomes was evaluated by
patients and by an experienced calibrated periodontist
(SLAG) (intraclass correlation coefficient = 0.95) using a hor-
izontal visual analog scale (VAS) after 6 months. This out-
come was assessed using a 10-point scale of which, 0 was
unsatisfactory esthetics and 10 was fully satisfactory esthetics.
All patients were treated by the same operator (DRBR).
Randomization was performed by a computer-generated ran-
dom sequence to choose FGG or ADM (AlloDerm®,
BioHorizons, Birminghan - USA) for right recipient site. If
the right side received FGG, the left side of the same patient
received ADM and vice versa. Allocation concealment was
completed by sequentially numbered, opaque, sealed enve-
lopes. Immediately after recipient site preparation, the enve-
lope was opened, and treatment assignment (ADM or FGG)
was communicated to the operator (DRBR).
Non-surgical periodontal therapy included procedures of
scaling and root planing and oral hygiene instructions.
Grafting surgical procedures were performed when plaque
and bleeding index were up to 20% of site. Patients were
maintained within these thresholds during the study.
Both surgeries on test (ADM) and control (FGG) groups
were performed in the same day. The technique performed in
this study was introduced by Sullivan and Atkins (1968) [2],
Clin Oral Invest
and the dimensions of the FGG and ADM were standardized,
being 5 mm of occlusal-apical height and 10 mm of mesial-
distal length. FGG thickness was between 1.5 and 2 mm [15],
while ADM presented a standardized thickness of 2 mm.
The recipient site was anesthetized with 2% mepivacaine
(1:100.000 epinephrine). A horizontal incision was made with
#15C scalpel blade along with gingival margin. The standard-
ized dimensions of FGG and ADM (Fig. 1) determined the
extension of recipient site. Two vertical incisions were made,
and a partial thickness flap was designed to provide a firm and
immobile periosteal bed. The raised partial thickness flap was
excised. Muscle and unattached connective tissue fibers were
thoroughly scraped with a scalpel to prevent graft mobility.
Test group received ADM allografts, which were rehydrated
in sterile saline for at least 10 min. The grafts were stabilized
on periosteal bed with the epithelium side facing upward the
vestibule. The technique of ADM allograft was performed
according to the manufacturer’s instructions. Autogenous
FGG was harvested with #15C scalpel blade from hard palate
at the same side randomly selected to receive the FGG. Donor
area was sutured with 4–0 silk sutures and protected with
periodontal dressing (Coe Pak, GC America Inc., Alsip
USA). Both grafts, FGG and ADM, were placed and stabi-
lized with simple interrupted 5–0 vicryl sutures at recipient
site coronal border and horizontal or periosteal anchorage su-
tures over the graft.
Fig. 1 Clinical views of FGG and
ADM sites. a, b preoperative
view. c FGG Graft. d ADM. e
FGG stabilization. f ADM
stabilization
Antibiotics (amoxicillin, 500 mg capsules for 7 days) and
analgesics (dipyrone 500 mg pills for 3 days) were prescribed.
Continuous rinsing with 0.12% chlorhexidine solution, twice a
day, for 4 weeks was instructed, starting 24 h after surgery.
Patients were instructed to abstain from regular oral hygiene
regimen in surgical sites during 15 days. Sutures were removed
7 days post-surgery. Patients were controlled weekly to monitor
healing and plaque index until 1 month post-surgical and after
40 days, 2, 3, 4, 5, and 6 months. Localized supragingival
scaling and oral hygiene reinforcement were performed accord-
ing to individual needs at each visit.
Histological evaluation
Histological analysis was performed after 10, 20, 40, 60, and
180 days after surgical procedures. Each evaluation period
included biopsies of five different patients from both test and
control sites. Soft tissue biopsies were harvested under local
anesthesia, from apical-mesial portion of the healed graft to
alveolar mucosa. The biopsies were rectangular (4 mm ×
3 mm) comprising partial thickness of tissue. Biopsy wound
was left to heal by secondary intention and it was protected
with periodontal dressing. All specimens were fixed in 10%
neutral buffered formalin solution for further descriptive his-
tological analyses. Following dehydration, specimens were
embedded in paraffin and serially sectioned in 5-μm thick

sections. Each section was individually stained with either
hematoxylin and eosin (H&E) stain and Verhoeff-van
Gieson stain for elastic fibers. Descriptive morphologic anal-
ysis and histomorphometric analysis consisted in evaluation
of density and volume (percentage) of fibroblasts, collagen
fibers, blood vessels and inflammatory cells, epithelial lumi-
nal to basal surface ratio, maturation of grafted tissues, and
presence of elastic fibers.
A sample size calculation suggested that a minimum of 21
patients were needed to demonstrate a 1-mm difference in the
TW levels between study groups after treatment (85% power,
α of 0.05, standard deviation of 1.1) [11]. Considering a drop-
out rate, to achieve at least 21 evaluable patients, a total sam-
ple of 25 patients was enrolled in this study. Clinical data and
histomorphometric analysis were evaluated by ANOVA
complemented by Tukey’s tests (p < 0.05).
Results
In total, 25 patients, 12 males and 13 females (mean age =
42.5 years old), participated in this study. However, three par-
ticipants were excluded at clinical evaluation; two were absent
from control visits and one moved to another state. All
Fig. 2 Postoperative outcomes of
surgical sites. FGG outcome after
30, 90, and 180 days (a, c, e) and
ADM outcome after 30, 90, and
180 days (b, d, f)
Clin Oral Invest
patients healed without serious complications following both
treatments. Mild pain and/or swelling were experienced by
some patients. None of them presented allograft or autogenous
graft necrosis during the follow-up period. After 10 days,
FGG group presented reduction in inflammatory features, a
smooth surface, color varying between pale pink to red, and
complete re-epithelization. After 30 days, grafts were
completely healed with initial formation of gingival stippling
(Fig. 2). From 60 to 180 days, a progressive maturation was
observed with FGG grafts demonstrating palatal mucosa char-
acteristics and clinical gain of soft tissue thickness. ADM
grafts presented no epithelization after 10 days. The external
surface of ADM graft was covered with a white-to-gray tissue
layer, firmly attached to recipient site. After 30 days, the new-
ly formed tissue was red with an irregular surface and an initial
process of epithelization without keratinization. After 60 days,
tissue surfaces were more uniform with color varying between
pale pink to red. Tissue maturation was observed after
150 days when gingival color, texture, thickness, and contour
were similar to adjacent tissues. Clinical characteristics during
healing phase indicated a different and delayed pattern of ap-
proximately 10 to 20 days in ADM group compared to FGG.
At the end of the study (6 months postoperatively), there has
been no mobility of the newly-gained tissue in both groups.
 Clin Oral Invest

Table 1 Clinical parameters for FGG and ADM in different periods of evaluation (n = 22)

Clinical parameters RD (mm) CAL (mm) PD (mm) KTW (mm) TT (mm)

Time/groups FGG ADM FGG ADM FGG ADM FGG ADM FGG ADM
Baseline
Mean ± s.d. 2.98 ± 1.19a 2.84 ± 1.29a 5.82 ± 1.46a 4.79 ± 1.37a 2.09 ± 0.84a 1.95 ± 0.46a 0.79 ± 0.7a 0.79 ± 0.68a 0.85 ± 0.22a 0.75 ± 1.48a
Range 0.3–4.85 1–5.21 2.83–8.62 2.3–7.77 0.3–4.38 1.6–2.96 0–2.2 0–1.78 0.6–1.4 0.6–1.1
Time 0
Mean ± s.d. – – – – – – 5.0 ± 0.0b 5.0 ± 0.0b – –
30 days
Mean ± s.d. 3.06 ± 1.36a 3.49 ± 1.36c 4.87 ± 1.41b 5.08 ± 1.57b 1.81 ± 0.42b 1.59 ± 0.59b 4.37 ± 0.46c 3.39 ± 0.96d 2.41 ± 0.29b 1.92 ± 0.39d
Range 0.92–5.91 1.6–5.61 2.93–8.0 2.7–8.17 1.04–2.5 0.41–2.85 3.25–5.0 2.03–5.0 1.9–3.4 1.2–2.5
60 days
Mean ± s.d. 3.03 ± 1.36a 3.31 ± 1.28bc 4.84 ± 1.36b 4.89 ± 1.41b 1.80 ± 0.38b 1.58 ± 0.37b 4.37 ± 0.47c 3.11 ± 0.83d 2.22 ± 0.29c 1.68 ± 0.39e
Range 0.92–5.91 1.5–5.57 2.75–7.91 2.63–7.62 1.08–2.45 0.84–2.27 3.3–4.97 1.62–4.99 1.6–3.1 0.9–2.2
90 days
Mean ± s.d. 3.03 ± 1.37a 3.3 ± 1.29b 4.8 ± 1.37b 4.85 ± 1.31b 1.77 ± 0.45b 1.54 ± 0.36b 4.35 ± 0.45c 2.47 ± 0.49e 2.04 ± 0.26d 1.34 ± 0.34f
Range 0.92–5.91 1.5–5.55 2.84–8.04 2.8–7.1 1.11–2.61 0.84–2.23 3.3–4.95 1.62–3.39 1.3–2.5 0.9–2.2
120 days
Mean ± s.d. 3.03 ± 1.37a 3.2 ± 1.34b 4.54 ± 1.43b 4.53 ± 1.37b 1.51 ± 0.4c 1.33 ± 0.44c 4.34 ± 0.46c 2.21 ± 0.61e 2.04 ± 0.26d 1.24 ± 0.27f
Range 0.88–5.91 1.29–5.35 1.93–7.57 2.38–7.01 0.88–2.46 0.55–2.2 3.22–4.95 1.01–3.22 1.3–2.5 0.9–1.9
150 days
Mean ± s.d. 3.03 ± 1.37a 3.2 ± 1.34b 4.49 ± 1.42b 4.45 ± 1.29b 1.47 ± 0.4c 1.26 ± 0.43c 4.34 ± 0.46c 2.19 ± 0.64e 2.04 ± 0.26d 1.17 ± 0.23f
Range 0.88–5.91 1.3–5.34 1.91–7.51 2.45–6.91 0.85–2.47 0.39–2.22 3.2–4.95 1.04–3.34 1.3–2.5 0.9–1.6
180 days
Mean ± s.d. 2.99 ± 1.36a 3.2 ± 1.34b 4.48 ± 1.42b 4.46 ± 1.3b 1.49 ± 0.4c 1.25 ± 0.43c 4.38 ± 0.47c 2.21 ± 0.66e 2.04 ± 0.26d 1.17 ± 0.23f
Range 0.84–5.9 1.3–5.34 1.93–7.5 2.44–6.91 0.9–2.48 0.39–2.18 3.21–5.0 1.04–3.34 1.3–2.5 0.9–1.6

For each evaluated parameter, different lowercase letters represent statistical difference.
RD recession depth, CAL clinical attachment level, PD probing depth, KTW keratinized tissue width, TT soft tissue thickness, FGG free gingival graft, ADM acellular dermal matrix

Clinical outcomes
Table 1 represents clinical parameters for FGG and ADM at
baseline, time 0 (surgery day) and 30, 60, 90, 120, 150, and
180 days after surgical procedures. Both groups presented an
increase in means of RD after 30 days, with a tendency to
decrease in subsequent periods. FGG group presents no sta-
tistical significant differences between periods, but ADM
showed statistically significant differences between 30 days
and the others, except for 60 days. ADM presented higher RD
compared to FGG in all periods (p < 0.05). The ANOVA test
demonstrated no statistically significant differences for CAL
means between groups (p > 0.05). After 30 days, CAL mean
reduced for FGG and increased for ADM. After this period,
this parameter presented a tendency to reduce (p > 0.05). No
statistically significant differences were observed between ex-
perimental groups for PD mean values. Both groups demon-
strated significant reduction of PD after 30 days and after
120 days. For KTW, time 0 represents the moment of the graft
stabilization and consequently the value of the KTW was
5 mm for both groups. The inclusion of this period enabled
the analysis of tissue shrinkage according to the previously
described formula. Mean tissue shrinkage was 12.41% ±
9.44 for control group and 55.7% ± 13.31 for test group
(p < 0.05). Considering graft dimensions immediately after
surgery (5 mm), a decrease in KTW means, in control group,
occurred until 30 days, a minor decrease occurred until
150 days, and a tendency to increase was observed after
180 days. In test group, a significant decrease of KTW was
observed after 30/60 days, and major decrease occurred after
90/120/150/180 days with statistical significant differences.
Statistical significant differences were observed for TT mean
values comparing experimental groups, with exception of
baseline values, and between periods of evaluation. Mean
thickness increased from 0.85 to 2.04 mm for control group
and 0.75 to 1.17 mm for test group. Considering different
periods of analysis, control and test groups presented increase
of TT after 30 days. After 60 days, mean TT reduced for both
groups but with minor values for ADM.
Esthetics perception of patients comparing groups (FGG
and ADM) using VAS demonstrated no statistical differences
(7.99 × 8.85). Professional evaluation showed superior results
for ADM (6.40 × 7.68; p < 0.05).
Histological analysis
Descriptive analysis after 10 days showed that FGG group
presented initial reorganization of epithelium with develop-
ment of epithelial ridges and para-keratinization. Connective
tissue presented inflammatory infiltrate with transition of
acute to chronic phase and high cellularity, fibroblasts, and
blood vessels (Fig. 3a). In ADM group, superficial portion
of matrix exhibited no epithelium and presence of marked
Clin Oral Invest
inflammatory neutrophilic infiltrate. Compared to FGG,
ADM group presented less vascularization, fibroblasts, and
collagen fiber organization with higher metabolic activity
and inflammatory infiltrate (Fig. 3d). After 20 days, FGG
group demonstrated an organized para-keratinized epithelium
with less cellularity and vascularization and higher organized
collagen fibers with discrete lymphoplasmocytic infiltrate
(Fig. 3b). ADM group presented non-keratinized epithelium
without epithelial ridges. The connective tissue presented less
cellularity and vascularization compared to 10 days. Collagen
fibers were disorganized and presence of giant cells was ob-
served. Inflammation phase is progressing to repair phase
(Fig. 3e). After 40 days, organized epithelium structures and
higher collagen fiber density could be observed in FGG group
with rare inflammatory infiltrate (Fig. 3c). In ADM group,
epithelial ridges and para-keratinization initiated with reduc-
tion of fibroblasts, blood vessels, and inflammatory infiltrate.
A deep organization of collagen fibers could be observed with
a more superficially reparative tissue (Fig. 3f). After 60 days,
control group was characterized by a mature tissue (Fig. 4a),
but ADM group presented superficial epithelial ridges with a
heterogeneous para-keratin layer. In ADM group, deeper con-
nective tissue was dense with parallel collagen fibers, but in
the superficial region still remained repair characteristics and
inflammatory infiltrate (Fig. 4c). After 180 days, FGG group
presented tissue organization, maturation, and rare inflamma-
tory cells (Fig. 4b). In ADM group, para-keratinized epithelial
tissue presented superficial and non-homogeneous epithelial
ridges. Dense and immature connective tissue was organized
with the presence of chronic inflammatory infiltrate (Fig. 4d).
The Verhoeff-van Gieson stain demonstrated absence of elas-
tic fibers in FGG group (Fig. 5a, b) with presence only in
ADM group in all periods (Fig. 5c). Elastic fibers appeared
in deeper areas and concentration decreased over time (Fig.
5d).
Table 2 represents histomorphometric analysis with per-
centage of fibroblasts, collagen fibers, blood vessels, inflam-
matory infiltrate, and mean value of epithelium luminal-to-
basal surface ratio. Percentage of fibroblasts reduced from
initial to final periods in both groups (p < 0.05). ANOVA dem-
onstrated differences in cellularity between both groups with
superior values for FGG (p < 0.05). A tendency of increase in
percentage of collagen fibers was observed for both groups
during experimental periods (p < 0.05). Higher percentage of
collagen fibers was observed for ADM (p < 0.05). Percentage
of blood vessels decreased during experimental periods for
both groups with higher values for control group. Percentage
of inflammatory infiltrate was higher for test group in all pe-
riods of evaluation (p < 0.05) and decreased during experi-
mental intervals. Epithelium luminal-to-basal surface ratio in-
creased in both groups during experimental periods (p < 0.05).
FGG group presented higher epithelial thickness compared to
ADM in all times (p < 0.05).
Clin Oral Invest
Fig. 3 Histological samples (hematoxylin and eosin stain-original mag-
nification × 10 and × 40-rectangular area) after 10, 20, and 40 days of
FGG (a, b, c) and ADM sites (d, e, f). ADM group presented profuse
inflammatory infiltrate (d) compared to (a). FGG group demonstrated an
organized para-keratinized epithelium (b, c) and ADM presented non-
keratinized epithelium without epithelial ridges (e). ADM group initiated
formation of epithelial ridges and para-keratinization (f). a (× 10): INF -
inflammatory infiltrate in a transition from acute to chronic phase; Black
arrows - epithelium para-keratinization. 3a (× 40): White arrows – blood
Discussion
The aim of this split-mouth controlled randomized clinical
trial was to evaluate clinical and histological outcomes of
vessels; Yellow arrows – fibroblasts. b (× 10): White arrows – epithelial
ridges; Black arrows – para-keratinization. b (× 40): Blue arrows –
lymphoplasmocytic cells. c (× 10 and × 40): White arrows – epithelial
ridges; Black arrows – blood vessels. d (× 10): INF – inflammatory infil-
trate (neuthrophils). d (× 40): Black arrows – blood vessels; Yellow ar-
rows – fibroblasts; Blue arrows – inflammatory cells. e (× 10): White
arrows – inflammatory infiltrate. e (× 40): Black arrows – blood vessels;
Blue arrows – lymphoplasmocytic cells. f (× 10): Black arrows – para-
keratinization; White arrows – epithelial ridges
ADM compared to autogenous FGG for keratinized tissue
augmentation. Both treatments resulted in improvement of
clinical parameters, with the exception of RD that remained
stable or slightly increased during the study. ADM group

Fig. 4 Histological samples (hematoxylin and eosin stain- original mag-
nification × 10 and × 40- rectangular area) after 60 and 180 days of FGG
(a, b) and ADM sites (c, d). FGG group presented tissue organization and
rare inflammatory cells (a, b). ADM group presented superficial epithelial
ridges with a heterogeneous para-keratin layer (Fig. 4). In ADM group,
para-keratinized epithelial tissue presented superficial and non-
homogeneous epithelial ridges. Dense and immature connective tissue
was organized with the presence of chronic inflammatory infiltrate (c,
presented more tissue shrinkage and delayed healing com-
pared to FGG, but superior esthetic professional evaluation.
ADM completely integrated into recipient site with similar
histological characteristics of autogenous grafts, although
maturation was not completed until 6 months.
In this study, ADM group demonstrated greater shrinkage
than FGG (56 versus 12%). This ADM shrinkage was smaller
than the results of Wei et al [6] (71 versus 16%). The present
study shows a minor shrinkage for both groups when com-
pared to Agarwal [11] (76.6 versus 49.7%). Differently, a
study [16] reported ADM mean shrinkage of 10–15% after
4–6 weeks.
Differences in tissue behavior may be related to surgical
procedures considering diverse grafts sizes and thickness, lo-
cation, and characteristics of the recipient site [17, 18]. Studies
demonstrated that 1.5 mm-thickness FGG presented predict-
able results as minor shrinkage [2, 19], in accordance with the
outcomes presented in this study. Inferior gain of KTW
Clin Oral Invest
d). a (× 10): Black arrows – para-keratinization. a (× 40): White arrows
– Collagen fiber arrangement. b (× 40): Blue arrows – rare inflammatory
cells. White arrows – Collagen fiber arrangement. c (× 40): White arrows
– blood vessels organized perpendicularly to epithelium. Black arrows –
epithelial ridges. d (× 10): White arrows: Non- homogeneous epithelial
ridges; Black arrows – para-keratinized epithelium; INF – Chronic in-
flammatory infiltrate
(2.21 mm), observed in ADM group, could be related to its
standardized dimensions (5 mm × 10 mm) used in this study.
Other studies [20, 21] observed major KTW gains between 4
and 8 mm. Consequently, larger sizes of ADM grafts were
related to superior KTW gain [20, 21].
A significant reduction of PD was observed after both pro-
cedures, similar from results of other studies [22, 23]. This
reduction was more marked between 90 and 120 days and
remained stable until 180 days; these findings were also re-
ported by Ward et al. (1974) [24]. Thus, PD decrease can be
justified by the formation of an attached keratinized marginal
tissue and reestablishment of periodontium homeostasis.
Concomitantly to PD reduction, CAL presented a tendency
for improvement in both groups. This attachment gain could
be promoted not only by the collagen fibers increase in mar-
ginal area, but also due to the keratinized tissue attachment,
since KTW gain and immobility of grafts were evident for
both groups. However, our findings demonstrated inferior
Clin Oral Invest
Fig. 5 Histological samples of FGG (Verhoeff-van Gieson stain) after
10 days (original magnification × 40) and 180 days (original magnifica-
tion × 10) without presence of elastic fibers (a, b). ADM histological
samples (Verhoeff-van Gieson stain- original magnification × 10 and ×
KTW gain for FGG and ADM groups (3.58 vs. 1.41 mm)
when compared to Wei et al. [6](6.15 vs. 3.25 mm) and
Agarwal et al [11] (4.8 vs. 2.13 mm).
The RD remained stable or slightly increased during this
study. Similar outcomes were observed by Wei et al [6]. This
finding could be related to the position of graft/matrix along
with gingival margin, since surgical objective was to increase
attached gingiva and not root coverage. Another important
aspect was the short period of evaluation (6 months). A recent
publication [25] investigated creeping attachment over a
prolonged period of time up to 25 years. Agudio et al.
(2017) [25] related an ongoing coronal migration of the gin-
gival margin could be noted not only during the first phase of
follow-up (6 to 12 months), but through subsequent long-term
periods of time. This phenomenon could not be observed in
this sample probably due to its short-term evaluation
(6 months), and could be related to RD reduction along time.
Another important parameter was TT of the newly formed
tissue. This parameter is related to local susceptibility to gin-
gival recession development and ability to periodontal health
maintenance [26, 27]. FGG thickness was standardized
40- rectangular area) demonstrating presence of elastic fibers after 10 and
180 days (c, d). a (× 40) – CF – collagen fibers. b (× 10) – CF – collagen
fibers. c (× 10 and × 40) – Blue arrows – elastic fibers. d (× 10 and × 40) –
Blue arrows – elastic fibers
between 1.5 and 2 mm [15] to remain similar to ADM thick-
ness. Evident inferior thickness gain was observed for ADM
group. Clinically, a superficial gray to white layer of the ADM
was subsequently lost 10 to 20 days post-surgical. This fact
related to healing process of ADM can justify inferior TT
compared to FGG.
Generally, ADM presents similarity of gingival color, tex-
ture, thickness, and contour with the adjacent tissues com-
pared to FGG [6, 20, 21]. Autogenous grafts present distin-
guishable margins with similar characteristics to donor palatal
site [19]. In this study, only esthetics perception of a calibrated
professional showed superior results for ADM. However, this
subjective outcome depends immensely on each individual
opinion.
Clinical characteristics during healing phase indicated a
delayed pattern of approximately 10 to 20 days in ADM group
compared to FGG. These findings corroborate with studies
that also demonstrated delayed healing between 4 and 5 days
up to 15 days [6, 20]. The difference between these clinical
outcomes of o FGG and ADM could be explained by histo-
logical healing processes of both tissues. Histological data
 Clin Oral Invest

Epithelium luminal-to-basal surface ratio (μm)

also demonstrated the delayed maturation of ADM compared
to FGG, supported by our clinical data and other authors [6,
21]. For FGG, collagen maturation period varied between

1.47 ± 0.17fg

1.57 ± 0.15fg

1.94 ± 0.59g
1.32 ± 0.09f
30 days to 3 months [17], ADM apparently requires more than

0.0 ± 0.0e
ADM
6 months for complete healing [14]. Tissue maturation is rep-
resented by epithelial keratinization and functional orientation
of collagen fibers on connective tissue [28]. Significant differ-
ences were observed between experimental groups in relation
1.74 ± 0.28ab to the percentage of fibroblasts, collagen fibers, blood vessels,
and inflammatory infiltrate. These data were consistent with
1.92 ± 0.2bc

2.5 ± 0.63cd

3.0 ± 0.27d
1.19 ± 0.1a

studies that evaluated separately the two types of grafts [6,

FGG

17]. Noticeable differences in repair process can be justified

by origin of these tissues. Authors have demonstrated repair
process and cellular differentiation are dependent on the
4.95 ± 1.65fg

4.59 ± 0.37g

3.04 ± 1.06h
6.93 ± 1.16e

5.11 ± 0.18f
Inflammatory infiltrate (%)

transplanted connective tissue [29, 30]. FGG maintained

physical and biochemical characteristics that induced tissue
ADM

repair with similar characteristics of palatal tissue. ADM pre-

sented no cellularity and no inductive capacity. Matrix would
0.52 ± 0.19d
3.36 ± 0.28a

1.03 ± 0.76c
1.15 ± 0.3bc
2.5 ± 0.34b

be only a scaffold for cell migration from adjacent tissues,

justifying delayed healing and better blending with the sur-
FGG

rounding tissues [12, 31].

Wei et al. [12] also related presence of a larger inflamma-
1.86 ± 0.48abce
2.37 ± 0.57abe

1.56 ± 0.47bce

tory infiltrate in ADM group, suggesting a direct reaction to

2.69 ± 0.73a

1.21 ± 0.18c

the matrix or a normal response due to a longer healing period.

ADM

Similarities between histological observations of Wei et al.

Blood vessels (%)

[12] and the present study were observed, except for keratini-
Histomorphometric analysis for FGG and ADM in different periods of evaluation (n = 25)

5.31 ± 0.91d

2.95 ± 0.58e

2.16 ± 0.57e

1.92 ± 0.23e

zation pattern. After 6 months, these authors [12] described

2.0 ± 0.49e

non-keratinized or ortokeranized epithelium in ADM group

FGG

and our observations, parakeratinized epithelium. ADM grafts

presented exuberant amount of elastic fibers in initial periods
For each evaluated parameter, different lowercase letters represent statistical difference
90.02 ± 1.57g
79.45 ± 2.04e

82.97 ± 0.65f

84.84 ± 2.21f

85.49 ± 0.74f

with consequent reduction and predominance in deeper areas

[12, 32]. This characteristic suggested that ADM was incor-
ADM

porated into the tissue and not exfoliated or totally reabsorbed

Collagen fibers (%)

[21, 32].
A limitation of this study was the small period of evaluation
83.51 ± 0.56cd
79.76 ± 1.06b

84.39 ± 0.38d
75.27 ± 0.71a

82.73 ± 0.77c

(6 months); thus, a longer follow-up might be necessary to

assess the effect of creeping attachment. Another limitation
FGG

of this study was the lack of VAS for pain/discomfort analysis.

Since surgical procedures were performed on the same day at
FGG free gingival graft, ADM acellular dermal matrix
10.92 ± 0.38e

9.54 ± 0.28ef

5.71 ± 0.65g

both sites (test and group), patients could be confused and did
8.35 ± 1.5f

8.34 ± 0.5f

not provide reliable data regarding symptomatology.

ADM

According to a meta-analysis [13], ADM-based mucogingival

surgery can be successfully used to repair gingival recession
Fibroblasts (%)

14.77 ± 0.39b

13.14 ± 0.21d
16.04 ± 0.17a

13.96 ± 0.32c

defects and to increase keratinized gingiva. However, most

13.3 ± 0.49d

ADM studies included a small sample size that lacked sufficient

statistical power to draw conclusions regarding the efficacy of
FGG

ADM. This split-mouth randomized clinical trial added impor-

tant clinical and histological data in order to contribute in the
Histological analysis

decision-making process. Both treatments resulted in improve-

Mean ± s.d.

Mean ± s.d.

Mean ± s.d.

Mean ± s.d.

Mean ± s.d.

ment of TT and KTW parameters. ADM group demonstrated

Time/groups

more tissue shrinkage and delayed healing compared to FGG,

180 days
10 days

20 days

40 days

60 days
Table 2

but presented esthetic advantages. The matrix completely inte-

grated into the recipient site with similar histological
Clin Oral Invest
characteristics of autogenous grafts, although maturation was not
completed until 6 months.
Conclusion
In this study, ADM and FGG resulted in improvement of
clinical parameters with the exception of RD. ADM group
presented more tissue shrinkage and delayed healing, con-
firmed histologically, but superior professional esthetic per-
ception. More studies are necessary to evaluate long-term
outcomes.
Funding This study was supported by a grant (#99/09834-2) from
FAPESP (São Paulo Research Foundation).
The work was supported by the Department of Prosthodontics and
Periodontics of Bauru School of Dentistry- University of São Paulo,
Brazil.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the institu-
tional and/or national research committee and with the 1964 Helsinki
declaration and its later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from all individual
participants included in the study.
References
1. Björn H (1963) Free transplantation of gingiva propria. Odont Revy
14:323
2. Sullivan HC, Atkins JH (1968) Free autogenous gingival grafts. I.
Principles of successful grafting. Periodontics 6:121–129
3. Zuhr O, Bäumer D, Hürzeler M (2014) The addition of soft tissue
replacement grafts in plastic periodontal and implant surgery: crit-
ical elements in design and execution. J Clin Periodontol 41(Suppl.
15):S123–S142
4. Ouhayoun JP, Sawaf MH, Goffaux JC, Etienne D, Forest N (1988)
Re-epithelialization of a palatal connective tissue graft transplanted
in a non-keratinized alveolar mucosa: a histological and biochem-
ical study in humans. J Periodont Res 23:127–133
5. Hall WB, Lundergan WP (1993) Free gingival grafts. Current indi-
cations and techniques. Dent Clin N Am 37:227–242
6. Wei P, Laurell L, Geivelis M, Lingen MW, Maddalozzo D (2000)
Acellular dermal matrix allografts to achieve increased attached
gingiva. Part 1. A clinical study. J Periodontol 71:1297–1305
7. Bartolucci EG (1981) A clinical evaluation of freeze-dried homol-
ogous dura mater as a periodontal free graft material. Study in
humans. J Periodontol 52:354–361
8. Cummings LC, Kaldahl WB, Allen EP (2005) Histologic evalua-
tion of autogenous connective tissue and acellular dermal matrix
grafts in humans. J Periodontol 76:178–186
9. Scarano A, Barros RR, Iezzi G, Piattelli A, Novaes AB Jr (2009)
Acellular dermal matrix graft for gingival augmentation: a
preliminary clinical, histologic, and ultrastructural evaluation. J
Periodontol 80:253–259
10. Jhaveri HM, Chavan MS, Tomar GB, Deshmukh VL, Wani MR,
Miller PD Jr (2010) Acellular dermal matrix seeded with autolo-
gous gingival fibroblasts for the treatment of gingival recession: a
proof-of-concept study. J Periodontol 81:616–625
11. Agarwal C, Tarun Kumar AB, Mehta DS (2015) Comparative eval-
uation of free gingival graft and AlloDerm(®) in enhancing the
width of attached gingival: a clinical study. Contemp Clin Dent 6:
483–488
12. Wei P, Laurell L, Lingen MW, Geivelis M (2002) Acellular dermal
matrix allografts to achieve increased attached gingiva. Part 2. A
histological comparative study. J Periodontol 73:257–265
13. Gapski R, Parks CA, Wang H (2005) Acellular dermal matrix for
mucogingival surgery: a meta-analysis. J Periodontol 76:1814–
1822
14. Harris RJ (2001) Clinical evaluation of 3 techniques to augment
keratinized tissue without root coverage. J Periodontol 72:932–
938
15. Borghetti A, Gardella JP (1990) Thick gingival autograft for the
coverage of gingival recession: a clinical evaluation. Int J
Periodont Rest Dent 10:217–229
16. Callan DP, Silverstein LH (1998) Use of acellular dermal matrix for
increasing keratinized tissue around teeth and implants. Pract
Periodont Aesthet Dent 10:731–734
17. Caffesse RG, Carraro JJ, Carranza FA (1972) Free gingival grafts in
dogs, a clinical-histological study (Spanish). Rev Assoc Odont
Argent 60:465–470
18. Dordick B, Coslet JG, Seibert JS (1976) Clinical evaluation of free
autogenous gingival grafts placed on alveolar bone. J Periodontol
47:559–567
19. Soehren SE, Allen AL, Cutright DE, Seibert JS (1973) Clinical and
histologic studies of donor tissues utilized for free grafts of masti-
catory mucosa. J Periodontol 44:727–741
20. Shulman J (1996) Clinical evaluation of an acellular dermal allo-
graft for increasing the zone of attached gingival. Pract Periodontics
Aesthet Dent 8:201–208
21. Silverstein LH, Duarte F (1998) Use of an acellular dermal allograft
for soft-tissue augmentation. Dent Implantol Updat 9:61–64
22. Dorfman HS, Kennedy J, Bird WC (1982) Longitudinal evalu-
ation of free autogenous gingival grafts. J Periodontol 53:349–
353
23. Fagan F, Freeman E (1974) Clinical comparison of the free gingival
graft and partial thickness apically positioned flap. J Periodontol 45:
3–8
24. Ward VJ (1974) A clinical assessment of the use of the free gingival
graft for correcting localized recession associated with frenal pull. J
Periodontol 45:78–83
25. Agudio G, Chambrone L, Prato GP (2017) Biologic remodeling of
periodontal dimensions of areas treated with gingival augmentation
procedure (GAP). A 25-year follow-up observation. J Periodontol
88:634–642
26. Chambrone L, Tatakis DN (2016) Long-term outcomes of untreated
buccal gingival recessions: a systematic review and meta-analysis. J
Periodontol 87:796–808
27. Müller HP, Schaller N, Eger T, Heinecke A (2000) Thickness of
mastigatory mucosa. J Clin Periodontol 27:231–236
28. Oliver RC, Karring HL (1968) Microscopic evaluation of the
healing and revascularization of free gingival grafts. J Periodontol
3:84–95
29. Karring T, Lang NP, Löe H (1975) The role of gingival connective
tissue in determining epithelial differentiation. J Periodontol Res
10:1–11
30. Karring T, Östergaard E, Löe H (1971) Conservation of tissue spec-
ificity after heterotopic transplantation of gingival and alveolar mu-
cosa. J Periodontol Res 6:282–293

31. Paolantonio M, Dolci M, Esposito P, D'Archivio D, Lisanti L, Di
Luccio A et al (2002) Subpedicle acellular dermal matrix graft and
autogenous connective tissue graft in the treatment of gingival re-
cessions. A comparative 1 year clinical study. J Periodontol 73:
1299–1307
Clin Oral Invest
32. Harris JR (1998) Root coverage with a connective tissue with par-
tial thickness double pedicle graft and an acellular dermal matrix
graft: a clinical and histological evaluation of a case report. J
Periodontol 69:1305–1311