See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/331261736

Histologic and Histomorphometric Analyses of De-epithelialized Free

Gingival Graft in Humans

Article  in  The International journal of periodontics & restorative dentistry · March 2019

DOI: 10.11607/prd.3544

CITATIONS READS

0 1,052

4 authors:

Emilio Azar Mariana Rojas

Universidad de Buenos Aires Sapienza University of Rome
2 PUBLICATIONS   0 CITATIONS    19 PUBLICATIONS   23 CITATIONS   

SEE PROFILE SEE PROFILE

Patricia Mandalunis Nelson Carranza

Universidad de Buenos Aires Instituto Carranza
111 PUBLICATIONS   597 CITATIONS    24 PUBLICATIONS   157 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Alveolar dimensional changes View project

Histologic and Histomorphometric Analyses of De-epithelialized Free Gingival Graft in Humans View project

All content following this page was uploaded by Mariana Rojas on 22 February 2019.

The user has requested enhancement of the downloaded file.

The International Journal of Periodontics & Restorative Dentistry

© 2019 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.
NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
 221

Histologic and Histomorphometric Analyses of

De-epithelialized Free Gingival Graft in Humans

Emilio L. Azar, DDS1 Different techniques for harvesting

Mariana A. Rojas, DDS1 connective tissue graft (CTG) have
Mandalunis Patricia, BSc, PhD2 been described in the literature,1–11
Nelson Carranza, DDS, MS, PhD3 and the evaluation of the palatal
thickness is critical. In some clini-
cal situations, the tissue is not thick
A graft’s histologic composition depends on the harvesting technique enough for the primary flap and the
used, and different connective tissue–harvesting procedures have been graft, resulting in a greater risk of
described in the literature. Some authors suggest the submucosal tissue incorporated fatty and glandular tis-
not be incorporated into the graft because it may interfere with the graft
sues in the graft9,12; it has been sug-
revascularization. In those cases, the de-epithelialized gingival graft (DGG) is
obtained with a superficial harvesting technique that leaves the deep portion gested that this tissue be removed
of the submucosa and the periosteum excluded from the graft. The aim of because it can interfere with the
this case series was to histologically and histomorphometrically evaluate graft’s revascularization.13 Ouhayoun
the tissue obtained with this technique. The findings demonstrated that the et al,14 in a histologic and biochemi-
DGG was mainly composed of connective tissue, and adipose tissue was in cal study in humans, suggested that
minimal proportions. However, epithelium was found in all of the samples.
the deep portion of the connec-
Int J Periodontics Restorative Dent 2019;39:221–226. doi: 10.11607/prd.3544
tive tissue from the palate will not
induce keratinization. According to
Zucchelli et al,9 traditional CTG har-
vesting techniques are not recom-
mended if the palatal soft tissue is
not sufficiently thick because of the
risk of primary flap necrosis and/or
the inadequacy of the graft (due to
the presence of a fatty and glandular
tissue instead of a desirable connec-
tive tissue). In such cases, a de-epi-
thelialized free gingival graft (DGG)
technique was recommended. In the
Department of Periodontics, University of Buenos Aires, Buenos Aires, Argentina.
1
same study, the authors compared
Department of Histology and Embriology, University of Buenos Aires,
2
CTG with DGG for the treatment of
Buenos Aires, Argentina.
3Private Practice, Carranza Institute, Buenos Aires; Department of Periodontics, gingival recessions and observed a
University of Buenos Aires, Buenos Aires, Argentina. greater increase in gingival thickness
at the buccal aspects of the patients
Correspondence to: Dr Mariana Andrea Rojas, Department of Periodontics,
University of Buenos Aires, Marcelo T de Alvear 2142 C1122AAH CABA,
treated with DGG, even though no
Buenos Aires, Argentina. Email: rojasmarianaandrea@gmail.com differences were found in the thick-
 Submitted August 10, 2017; accepted October 10, 2017.
ness of the graft at the time of su-
 ©2019 by Quintessence Publishing Co Inc. turing between the two treatment

Volume 39, Number 2, 2019

© 2019 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.
NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
222

Fig 1 (left)  Multiple Miller Class I gingival recession defects at baseline. Surgical design
was performed according to Zucchelli multiple recession-type defects treatment tech-
nique.15

Fig 2 (below)  (a) Graft before de-epithelialization. (b) De-epithelializing of the graft with a
15c blade. (c) Graft after de-epithelialization. The thickness obtained was 0.8 mm.

a b c

Fig 3  The graft was divided for optimiza- Fig 4  Flap elevation. Each portion of the Fig 5  Grafts were secured to the flap with
tion16 and sample collection was performed graft was placed in each recession defect. horizontal mattress sutures.
(2 mm to the distal portion of the graft).

groups. Zucchelli et al speculated
that the differences in the quality of
the connective tissues used in the
two techniques were responsible
for this increase in gingival thickness
because DGG allows the incorpo-
ration of the portion of connective
tissue closest to the epithelium into
the graft. This tissue is dense, firmer,
more stable, and presumably more
suitable for root coverage.12 There-
fore, the DGG is a harvesting tech-
nique designed to leave the deep
portion of the submucosa and the
periosteum excluded from the graft.
The authors believe that the
proportion of adipose tissue ob-
tained with this technique is minimal,
but until the present and to the best
of the authors’ knowledge, there
has not been a histologic study in
humans that describes the compo-
sition of the CTG harvested with
this technique only, and the need
is critical. Therefore, the purpose of
this case series was to histologically
and histomorphometrically evaluate
the characteristics of DGG in its final
stage of preparation, immediately
before its application.
Materials and Methods
Five healthy adults were selected
from a pool of patients referred to
the Department of Periodontology,
School of Dentistry, University of
Buenos Aires for various procedures
that required CTG. Two cases of
biotype thickening in implants and
three cases of root coverage in pa-
tients, with multiple adjacent Miller
Class I gingival recession defects,
were treated (Figs 1 to 9).15,16
Surgical Procedure:
Sample Collection
All grafts were taken from the max-
illary palatal area, ranging from the
canine to the first molar, and samples
were taken from between the second

The International Journal of Periodontics & Restorative Dentistry

© 2019 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.
NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
 223

premolar and first molar. The surgical

technique was performed following
the description by Zucchelli et al9:

“. . . Two horizontal (the coronal inci-

sion was performed 1–1.5 mm
apical to the soft tissue margin
of the adjacent teeth) and two
vertical incisions were traced Fig 6  The flap was advanced coronally and Fig 7  Surgical palatal wound immediately
to delimitate the area to be sutured (sling sutures). after removal of the graft. The bleeding
was minimal.
grafted. Along the coronal
incision, the blade was oriented
almost perpendicular to the
bone plate and once an ade-
quate soft tissue was obtained,
it was rotated in order to be
almost parallel to the superficial
surface. The thickness of the
graft was maintained uniform
at approximately 1.5 mm,
while proceeding apically with Fig 8  Donor area at 1 week postoperative. Fig 9  Complete root coverage was
observed in all treated recession defects at
the blade. The graft was de- 6 months.
epithelialized with a 15c blade.”

The de-epithelization of the

graft was performed using a surgical
microscope (×10 magnification), uti-
lizing the reflection of light as a ref-
erence to determine the complete
removal of the epithelium. After
epithelial removal, the thickness of
the grafts was reduced to approxi-
mately 0.8 mm (Fig 2). The histologic
sample was obtained by removing a
2-mm-wide segment from the most
distal end of the graft (Fig 3).16 All
samples were immediately fixed in
10% formalin for histologic analysis.
Histologic Processing
Histologic samples were processed
at the Department of Histology,
University of Buenos Aires. Sections
measuring 7 μm thick were prepared
and stained with hematoxylin-eosin.
Sample Analysis
All sections were first scanned with a
microscope (Axio Lab.A1, ZEISS) us-
ing a digital microscopy system (ZEN
blue edition 2011, ZEISS). Digital
JPEG images were obtained. When
the sample size was larger than the
microscope’s field of vision, a series
of microphotographs were obtained
without altering the magnification
and focus.
Tissue-composition analysis was
performed on the microphotograph
with image analysis software (Image
Pro Plus, Media Cybernetics), outlin-
ing the following areas:
• Connective tissue proper
area (CT/TA [%]): Fraction of
total area corresponding to
connective tissue proper area
• Adipose tissue area (AT/TA
[%]): Fraction of total area
corresponding to adipose
tissue area
• Vascular tissue area (VT/TA
[%]): Fraction of total area
corresponding to vascular
tissue area
• Epithelial tissue area (ET/
TA [%]): Fraction of total area
corresponding to epithelial
tissue area

Volume 39, Number 2, 2019

© 2019 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.
NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
224

Table 1  Composition of the Grafts

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Median Range
CT (% of TA) 89.17 89.33 86.53 78.66 92.20 89.17 78.66–92.20
AT (% of TA) 1.11 4.70 0.00 4.08 0.85 1.11 0.00–4.70
VT (% of TA) 1.50 2.73 1.01 11.24 2.59 2.73 1.01–11.24
ET (% of TA) 8.22 3.23 12.46 6.01 4.36 6.01 3.23–12.46
CT = connective tissue; TA = total area; AT = adipose tissue; VT = vascular tissue: ET = epithelial tissue.

Fig 10 (left)  Sample 2. Results

(a) Predominantly dense
connective tissue was
observed with some Histomorphometric results are pre-
epithelial and adipose sented in Table 1. The complete
tissues (H&E stain; original
magnification ×25).
sample and representative sections
(b) Apical portion. Note are shown in Figs 10 and 11. The
the epithelium still present. most notable feature of the samples
Dense connective tissue
with some blood vessels and was their homogeneity (Fig 10b).
adipocytes were observed The samples were composed by
(H&E stain; original
magnification ×100).
dense connective tissue (CT, me-
dian 89.17% of the total area) with
Fig 11 (right)  Sample 4. minimal amount of adipose tissue
(a) Dense connective
tissue. Minimal amount of (AT, median 1.11% of the total area).
epithelium and absence Vascular tissue (VT) was found in
of adipose tissue was
observed. (H&E stain;
minimal proportions (< 3% of the to-
original magnification ×25). tal area) in all samples except one,
(b) Central portion. Dense where VT was observed in 11.24% of
connective tissue was
observed. Note the absence the total area (Fig 11). Remnants of
of epithelium (H&E stain; epithelial tissue (ET) were found in
original magnification
×100).
different proportions among all the
samples (median 6.01% of the total
area, Figs 10 and 11).
All of the grafts were adequate
in size and volume for their intended
applications, and they produced
clinically successful results. The
a a
surgical procedures were toler-
ated well, without complications.
The healing patterns of the palatal
wounds were similar for all treated
cases. An example of the healing
response at 1-week postsurgery
is shown in Fig 8. The level of dis-
comfort and pain reported by the
b b patients was described as minimal.

The International Journal of Periodontics & Restorative Dentistry

© 2019 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.
NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.

Discussion
The aim of the present case series
was to analyze the histologic charac-
teristics of a completed DGG graft
immediately before it was applied
to the recipient site, describing and
quantifying its tissue components.
The histologic and histomorpho-
metric analyses of the graft harvest-
ed in humans using this technique
were not reported previously.
In a histologic study in fresh hu-
man cadavers, Bertl et al17 evaluated
the composition of the anterior and
posterior palatal mucosae and ob-
served that higher amounts of adi-
pose tissue and lower amounts of
dense connective tissue are found
in areas where CTGs are usually
harvested with split-flap techniques
than in more superficial areas where
de-epithelialized grafts are harvest-
ed. Another recent histologic and
immunohistochemical study18 also
described the composition of the
harvesting area of the palatal mu-
cosa. The authors reported less cel-
lular components with larger blood
vessels in deeper connective tissue
than on the surface. The results of
the present case series demon-
strated that a CTG obtained with
the DGG technique is mainly com-
posed of dense CT (more than 75%
of the total area in all the samples;
median 89.17% of the total area,
range 78.66% to 92.20%). It was also
observed that adipose tissue was
present in minimal amounts (median
1.11% of the total area, range 0.00%
to 4.70%), confirming the adequacy
of the DGG harvesting technique
towards this goal. Although previ-
ous studies13,14 suggest that CT can
© 2019 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.
NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
interfere with the revascularization
of the graft and impede keratiniza-
tion, the clinical relevance of that
finding remains to be confirmed.
Despite the efforts to carefully
remove the ET, the results show
that small remnants were present
in all samples in different propor-
tions (median 6.01% of the total
area, range 3.23% to 12.46%). It has
been suggested that presence of
the epithelium on the graft may re-
sult in complications, such as epithe-
lial cysts and edema.19,20 However,
several authors suggested leaving
residual epithelium on the graft and
did not report any problems.1,3 These
observations coincide with the pres-
ent results, since the inclusion of the
epithelium did not seem to affect
the clinical results of the present
cases. Harris, in his histologic study,12
arrived at similar conclusions.
When the samples were ob-
served after epithelium removal, it
was necessary to reduce the graft
thickness by almost 50%. Although
no attempts were made to harvest
and evaluate thicker grafts, the fact
that some residual fatty tissue could
be seen in some samples, together
with the information from published
histologic analyses of palatal tis-
sue,21 suggests that harvesting thick-
er grafts would only render larger
proportions of submucosal tissue.
Thus, DGG thickness is likely
limited by the available subepithe-
lial lamina propria, predictably ren-
dering grafts of less than 1 mm. If
thicker grafts are desired, it is likely
that fatty and glandular tissues
must be included in the graft. The
obtained graft thickness of approxi-
mately 0.8 mm has been success-
225
fully reported to be suitable for root
coverage procedures.9 However, it
remains to be established if DGGs
can be successfully applied to clini-
cal situations where more volume is
required, such as ridge augmenta-
tions or biotype thickening. In the
present study, three cases of root
coverage and two cases of biotype
thickening in implants were success-
fully treated.
The samples examined in this
study were obtained from the distal
ends of grafts, all corresponding to
a small area located between the
second premolar and the first molar.
Bertl et al17 showed that the thick-
ness of the ET and its lamina propria
were constant between the anterior
and posterior palate. It is probable,
then, that the observations reported
from these samples also apply to
grafts obtained from more anterior
and more posterior areas.
Finally, it must be considered
that short- and long-term success
has been extensively reported in
root-coverage and tissue biotype–
thickening procedures that utilized
traditional harvesting techniques,
obtaining CTGs as well as submuco-
sal (called “undesirable”) tissues.
Conclusions
Within the limitations of the present
five-case series, the DGG technique
was found to be simple and appli-
cable to different clinical situations,
with minimal morbidity and no post-
surgical complications. The histo-
logic results showed that this graft
could be described as a “predomi-
nantly dense CTG” since minimal
Volume 39, Number 2, 2019
226
amounts of adipose and epithelial
tissues were found. Implications of
these tissue remnants should be fur-
ther evaluated in larger size in long-
term clinical and histologic studies.
Acknowledgments
The authors declare no conflicts of interest.
References
  1. Langer B, Langer L. Subepithelial connec-
tive tissue graft technique for root cover-
age. J Periodontol 1985;56:715–720.
  2. Nelson SW. The subpedicle connective
tissue graft. A bilaminar reconstructive
procedure for the coverage of denuded
root surfaces. J Periodontol 1987;58:
95–102.
  3. Bruno JF. Connective tissue graft tech-
nique assuring wide root coverage. Int
J Periodontics Restorative Dent 1994;14:
127–137.
 4. Harris RJ. The connective tissue and
partial thickness double pedicle graft:
A predictable method of obtaining
root coverage. J Periodontol 1992;63:
477–486.
 5. Harris RJ. A comparison of two tech-
niques for obtaining a connective tissue
graft from the palate. Int J Periodontics
Restorative Dent 1997;17:261–271.
 6. Harris RJ. The connective tissue with
partial thickness double pedicle graft:
The results of 100 consecutively-treated
defects. J Periodontol 1994;65:448–461.
The International Journal of Periodontics & Restorative Dentistry
© 2019 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.
View publication stats NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
 7. Hürzeler MB, Weng D. A single inci-
sion technique to harvest subepithelial
connective tissue grafts from the pal-
ate. Int J Periodontics Restorative Dent
1999;19:279–287.
  8. Lorenzana ER, Allen EP. The single-inci-
sion palatal harvest technique: A strate-
gy for esthetics and patient comfort. Int
J Periodontics Restorative Dent 2000;
20:297–305.
 9. Zucchelli G, Mele M, Stefanini M, et al.
Patient morbidity and root coverage
outcome after subepithelial connec-
tive tissue and de-epithelialized grafts:
A comparative randomized-controlled
clinical trial. J Clin Periodontol 2010;37:
728–738.
10. Reino DM, Novaes AB Jr, Grisi MF, Maia
LP, de Souza SL. Palatal harvesting tech-
nique modification for better control of
the connective tissue graft dimensions.
Braz Dent J 2013;24:565–568.
11. Carranza N. Harvesting Connective Tis-
sue Grafts from the Palate. Buenos Aires:
Carranza Institute, 2014:10–14. Apple
iBooks. https://institutocarranzadotcom.
files.wordpress.com/2016/08/harvest-
connective-tissue.pdf. Accessed 22 June
2018.
12. Harris RJ. Histologic evaluation of con-
nective tissue grafts in humans. Int J
Periodontics Restorative Dent 2003;23:
575–583.
13. Gordon HP, Sullivan HC, Atkins JH. Free
autogenous gingival grafts. II. Supple-
mental findings—Histology of the graft
site. Periodontics 1968;6:130–133.
14. Ouhayoun JP, Sawaf MH, Gofflaux JC,
Etienne D, Forest N. Re-epithelialization
of a palatal connective tissue graft trans-
planted in a non-keratinized alveolar
mucosa: A histological and biochemical
study in humans. J Periodontal Res 1988;
23:127–133.
15. Zucchelli G, De Sanctis M. Treatment
of multiple recession-type defects in
patients with esthetic demands. J Peri-
odontol 2000;71:1506–1514.
16. Alberichi J, Carranza N. Optimization of
the subepithelial connective tissue graft
to treat adjacent multiple gingival reces-
sions. Clinical case presentation. Rev
Asoc Odontol Argent 2017;105:23–27.
17. Bertl K, Pifl M, Hirtler L, et al. Relative
composition of fibrous connective and
fatty/glandular tissue grafts depends
on the harvesting technique but not
the donor site of the hard palate. J Peri-
odontol 2015;86:1331–1339.
18. Perotto S, Romano F, Cricenti L, Gotti S,
Aimetti M. Vascularization and innerva-
tion of connective tissue grafts in the
treatment of gingival recessions: A his-
tologic and inmunohistochemical study.
Int J Periodontics Restorative Dent 2017;
37:551–558.
19. Harris RJ. Formation of a cyst-like area af-
ter a connective tissue graft for root cov-
erage. J Periodontol 2002;73:340–345.
20. Parashis AO, Tatakis DN. Subepithelial
connective tissue graft for root cover-
age: A case report of an unusual late
complication of epithelial origin. J Peri-
odontol 2007;78:2051–2056.
21. Cho KH, Yu SK, Lee MH, Lee DS, Kim
HJ. Histological assessment of the pala-
tal mucosa and greater palatine artery
with reference to subepithelial connec-
tive tissue grafting. Anat Cell Biol 2013;
46:171–176.