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179
Claudia Dellavia, DDS, PhD1 Root coverage and soft tissue aug-
Giano Ricci, DDS2 mentation procedures are based
Letizia Pettinari, PhD3 on bilaminar techniques using a
Cristina Allievi, DDS, PhD4 pedicle flap to cover a subepithe-
Fabio Grizzi, PhD5 lial connective tissue autograft,1–4
Nicoletta Gagliano, PhD6 thus providing an additional blood
supply compared with free gin-
Since different clinical outcomes of periodontal bilaminar surgeries using the
palate or the maxillary tuberosity as connective tissue (CT) donor sites have gival grafts and resulting in more
been observed, tissues grafted with CT from the palate or the tuberosity 1 predictable functional and esthetic
year after surgical procedures for ridge augmentation were compared with outcomes.1,2,5 The graft thickness is
nongrafted tissues by using morphologic and molecular methods. Collagen an important factor for obtaining
content and matrix metalloproteinases 1 and 2 expression were similar in optimal esthetics: a 1.5- to 2-mm
tissues and cultured fibroblasts from the palate and tuberosity, although with
thickness is needed to maintain
interindividual differences. In contrast, differences in collagen cross-linking
and maturation in the tuberosity fibroblasts were observed, suggesting a vascularization, promote wound
possible role in determining hyperplastic responses in some patients. (Int J healing, and provide long-term
Periodontics Restorative Dent 2014;34:179–186. doi: 10.11607/prd.1929) graft stability.1,6,7 The main donor
site is the connective tissue (CT)
obtained from the palatal area be-
tween the first premolar and the
first molar, but its removal is tech-
1
Adjunct Professor, Department of Biomedical, Surgical and Dental Sciences,
Università degli Studi di Milano, Milan, Italy. nique sensitive, unpleasant for pa-
2Private Practice, Florence, Italy. tients, and sometimes limited in
3Postdoctoral Student, Department of Biomedical Sciences for Health, Faculty of
size.8 The maxillary tuberosity can
Medicine and Surgery, Università degli Studi di Milano, Milano, Italy.
4Postdoctoral Student, Department of Biomedical, Surgical and Dental Sciences,
be used as an alternative,5,6,8 al-
Faculty of Medicine and Surgery, Università degli Studi di Milano, Milano, Italy. lowing surgeons to collect thicker
5Biologist, Laboratories of Quantitative Medicine, IRCCS Istituto Clinico Humanitas,
grafts and, in concomitance with
Rozzano, Milan, Italy. other oral interventions, reduce
6Associate Professor, Department of Biomedical Sciences for Health,
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180
approximately 1 year after sur- second and first premolar where the surgery), and 9 months after the
gery.10 The gingival tissues grafted thickness of the mucosa is greater plastic surgery by insertion of a
with palatal mucosa showed a sig- (palatal donor site: n = 7; mean periodontal probe at a 90-degree
nificant initial shrinkage or contrac- age: 58.6 ± 6.1 years) and group B angle in the tissue using a calibrat-
tion but remained stable over time received a graft from the maxil- ed surgical stent retained by the
with a good esthetic result. In con- lary tuberosity (tuberosity donor adjacent teeth with a fixed refer-
trast, the gingival tissues grafted site: n = 7; mean age: 59.7 ± 8.3 ence point.
with tuberosity CT were dimension- years). All subjects were free from
ally stable in the first few months, periodontal disease and did not
then progressively tended to a hy- use hormonal contraceptives or Histochemistry and image
perplastic reaction and assumed hormonal replacement medica- analysis
a not esthetically pleasing white tions. All patients signed a consent
tissue patch appearance.8,10 Since form to participate in the experi- Tissue fragments were fixed in 4%
the mechanism responsible for a ment. The study was conducted in phosphate-buffered formalin and
different maturation of the grafted accordance with the ethical princi- routinely processed for paraffin
tissue using the two donor sites is ples of the World Medical Associa- embedding. Sections were stained
still unclear, this study aimed to (1) tion Declaration of Helsinki and the with hematoxylin-eosin to evaluate
compare the palatal and the max- local ethics committee. the tissue morphology and with
illary tuberosity mucosa in healthy Sirius red to specifically stain colla-
subjects, with particular attention gen and to calculate the area frac-
to collagen content and turnover- Surgical technique tion index (AA%).
related pathways; and (2) analyze
the morphology of sites augment- For ridge augmentation, the epi-
ed with subepithelial CT grafted thelium and the adipose tissue Cell cultures
from either the palate or tuber- were removed with a no. 15 blade
osity, along with the ability to re- from a 3.5-mm-thick graft, a split- Gingival fibroblasts were obtained
model extracellular matrix (ECM) in thickness flap was raised on the from hard palate and tuberos-
cultured fibroblasts obtained from buccal aspect of the surgical area ity biopsy specimens and from the
the same grafted tissues to under- without the use of vertical inci- grafted tissue, as previously de-
stand the mechanisms responsible sions, and the CT graft was kept scribed.11
for different clinical outcomes. in position with a combination of
vertical and horizontal mattress
and single 5-0 vycril sutures. One Real-time RT-PCR
Method and materials year after surgery, an epithelium-
CT biopsy specimen of all grafted Total RNA was isolated by Tri-
Experimental model sites was obtained for histologic Reagent. mRNA levels of collagen
and molecular analyses, as were bi- type I and type III (COL-I, COL-III),
Fourteen healthy women aged 49 opsy specimens from healthy pala- long lysyl hydroxylase 2 (LH2b),
to 68 years with a Class I defect who tal mucosa and maxillary tuberosity and matrix metalloproteinase 1
underwent surgical procedures for from nongrafted oral sites for com- and 2 (MMP-1, MMP-2) were as-
ridge augmentation were divided parisons. A clinical measurement sessed. GAPDH was used as a
in two groups: group A received of the grafted site was carried out housekeeping gene, and gene ex-
a graft from the palatal mucosa presurgery, after 1 month, after 1 pression levels were normalized on
taken from the palate between the year (concomitantly with plastic its expression.
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181
a b c
Fig 1 Microphotographs showing palatal (a, c, e) and tuber maxillae (b, d, f) mucosa. (a
and b) Hematoxylin-eosin staining; original magnification ×10. (c and d) Sirius red staining;
original magnification ×10. (e and f) Polarized light; original magnification ×10.
d e f
COL-I, COL-III, and MMP-1 protein For morphologic (AA%) and mo- Nongrafted palatal and
levels secreted by palate and tu- lecular data, descriptive statistics maxillary tuberosity mucosa
berosity fibroblasts were assessed (mean and SD) were computed for
by slot blot.12 the CT from the palatal mucosa, Morphologic analysis
the tuberosity, and the grafted mu- All specimens from the nongrafted
cosa for groups A and B. Compari- palatal and maxillary tuberosity mu-
SDS-zymography sons between the two nongrafted cosa showed a healthy structure,
sites were performed by Wilcoxon without any inflammation or acan-
Concentrated culture media were signed-rank test for paired samples thosis, and normal epithelial crests
analyzed by SDS-zymography, as and between grafted tissues from (Figs 1a and 1b). The CT in the pala-
previously described.12 different donor sites by Wilcoxon tal lamina propria was loose and
sum-rank test for unpaired sam- highly vascularized (Fig 1c), but in
ples. A level of significance of 5% the tuberosity was more dense and
(P ≤ .05) was used. poorly vascularized (Fig 1d). In both
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182
10 20
8 16
6 12
4 8
2 4
0 0
Palate Tuberosity Palate Tuberosity
200,000
60,000
150,000
40,000
100,000
50,000 20,000
0 0
Palate Tuberosity Palate Tuberosity
4.0 1,200
LH2b/COL-I mRNA %
1,000
3.0
800
2.0 600
400
1.0
200
0 0
Palate Tuberosity Palate Tuberosity
Fig 2 COL-I and COL-III mRNA and protein levels, LH2b gene expression, and the LH2b/COL-I mRNA ratio in palate and tuberos-
ity human fibroblasts. Immunoreactive bands revealed by the Amplified Opti-4CN substrate were scanned densitometrically. Data were
obtained from confluent human gingival fibroblasts used between the fourth and fifth passage and cultured in duplicate for 72 hours for
molecular evaluations. Changes in mRNA were normalized on GAPDH gene expression. Values are means ± SDs for duplicate samples.
sites, collagen fiber bundles ran in that collagen content (AA%) was Molecular analysis
all directions, as confirmed by anal- similar in the CT of the tuberosity COL-I and COL-III gene expression
ysis from a polarized microscope (80.91% ± 8.77%) compared with tended to decrease in the tuberosi-
(Figs 1e and 1f). Image analysis of the palate (75.57% ± 7.68%). ty compared with palate fibroblasts
Sirius red-stained sections revealed (P < .05 and P > .05, respectively)
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183
4 10
8
3
6
2
4
1
2
0 0
Palate Tuberosity Palate Tuberosity
Densitometric units
250,000
Densitometric units
500,000
200,000
400,000
150,000
300,000
100,000
200,000
100,000 50,000
0 0
Palate Tuberosity Palate Tuberosity
Fig 3 MMP-1 and MMP-2 gene expression, MMP-1 protein levels, and MMP-2 activity in cultured palate and tuberosity human fibroblasts
and in their cell culture supernatants. MMP-1 protein levels secreted by palate and tuberosity fibroblasts were assessed by slot blot using
a monoclonal antibody to MMP-1. Immunoreactive bands revealed by the Amplified Opti-4CN substrate were scanned densitometrically.
Changes in mRNA were normalized on GAPDH gene expression. Values are means ± SDs for duplicate samples.
(Fig 2). COL-I and COL-III protein ity compared to palate fibroblasts. Grafted tissue
levels were similar in fibroblast su- MMP-1 and MMP-2 mRNA levels
pernatants (Fig 2), although they tended to decrease in tuberos- Clinical outcomes
increased, respectively, in 57% ity compared to palate fibroblasts All cases were treated in the maxil-
and 66% of tuberosity fibroblast (P > .05) (Fig 3). MMP-1 protein lev- la: 65% in the anterior region, 25%
supernatants. The overall LH2b els decreased in 50% of tuberosity in the premolar area, and 10% in
mRNA levels tended to increase in fibroblast supernatants, compared the molar area. At surgery all de-
the tuberosity fibroblasts and were with palate fibroblasts, but the fects were completely filled, and a
upregulated in 54% of samples mean levels were similar in both primary intention healing occurred.
(Fig 2), although with a wide in- palate and tuberosity fibroblast After 1 year, the sites grafted with
terindividual variation. The over- supernatants (Fig 3), possibly due CT from the palate did not show
all LH2b/COL-I mRNA ratio in the to interindividual variations. A simi- any hyperplastic reaction and
tuberosity fibroblasts resulted in lar pattern was observed for the some displayed a reduction in vol-
four-fold increased (Fig 2), and mean MMP-2 activity analyzed by ume (Fig 4a). In contrast, the sites
was increased in 69% of tuberos- SDS-zymography (Fig 3). grafted with CT from the maxillary
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184
a b
1.0 100
0.5 50
0 0
Normal Hyperplastic Normal Hyperplastic
response response
c d
Fig 4 Postsurgical outcomes in patients receiving a graft from (a) the palatal or (b) the tuberosity mucosa. A morphology consistent with a
hyperplastic response is evident in group B. LH2b gene expression (c) and the LH2b/COL-I mRNA ratio (d) in fibroblasts obtained from graft-
ed tissue with good esthetic results or after hyperplastic reaction. LH2b/COL-I mRNA ratio is expressed as a percentage compared to palate
fibroblasts (100%). Changes in mRNA levels were normalized on GAPDH gene expression. Values are means ± SDs for duplicate samples.
After 9 mo after
Presurgery 1-mo postsurgery 1-y postsurgery plastic surgery plastic surgery
Graft donor site (mm) (mm) (mm) (mm) (mm)
Palate (group A) 2.0 ± 0.2 5.5 ± 0.3 4.9 ± 0.6 4.7 ± 0.4 4.7 ± 0.3
Tuberosity (group B) 2.1 ± 0.2 5.8 ± 0.2 6.8 ± 1.1 5.6 ± 0.8 6.4 ± 1.0
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185
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186
it is advisable to thin it down to a 8. Jung UW, Um YJ, Choi SH. Histologic 18. Seguier S, Godeau G, Brousse N. Immu-
thickness of 3 mm, even if the de- observation of soft tissue acquired from nohistological and morphometric analy-
maxillary tuberosity area for root cover- sis of intra-epithelial lymphocytes and
fect to be treated is 5 mm deep, to age. J Periodontol 2008;79:934–940. Langerhans cells in healthy and diseased
avoid a fibrotic response that can 9. Studer SP, Allen EP, Rees TC, Kouba A. human gingival tissues. Arch Oral Biol
The thickness of masticatory mucosa in 2000;45:441–452.
lead to an unesthetic result. the human hard palate and tuberosity as 19. Ejeil AL, Gaultier F, Igondjo-Tchen S,
potential donor sites for ridge augmen- et al. Are cytokines linked to collagen
tation procedures. J Periodontol 1997; breakdown during periodontal disease
68:145–151. progression? J Periodontol 2003;74:
Acknowledgments 10. Arcidiacono A, Ricci G. Clinical and 196–201.
Histomorphologic Analysis After Ridge 20. Ejeil AL, Igondjo-Tchen S, Ghomrasseni
Augmentation Using Connective Tissue S, Pellat B, Godeau G, Gogly B. Expres-
The study was supported by FIRST-UNIMI
Obtained Either From the Tuber Maxillae sion of matrix metalloproteinases (MMPs)
(2007). The authors reported no conflicts of or From the Palate: A One-Year Evalua- and tissue inhibitors of metalloprotein-
interest related to this study. tion [in Italian]. [Proceedings of the XII In- ases (TIMPs) in healthy and diseased
ternational Congress SIDP, Firenze, Italy]. human gingiva. J Periodontol 2003;74:
Florence: SIDP, 2001:57–61. 188–195.
11. Gagliano N, Moscheni C, Dellavia C, et 21. Walker LC, Overstreet MA, Yeowell HN.
al. Morphological and molecular analy- Tissue-specific expression and regula-
References sis of idiopathic gingival fibromatosis: tion of the alternatively-spliced forms of
A case report. J Clin Periodontol 2005; lysyl hydroxylase 2 (LH2) in human kid-
1. Paolantonio M. Treatment of gingival 32:1116–1121. ney cells and skin fibroblasts. Matrix Biol
recessions by combined periodontal re- 12. Gagliano N, Moscheni C, Dellavia C, 2005;23:515–523.
generative technique, guided tissue re- et al. Effect of cyclosporin A on human 22. Van der Slot AJ, Zuurmond AM, van den
generation, and subpedicle connective gingival fibroblast collagen turnover in Bogaerdt AJ, et al. Increased formation
tissue graft. A comparative clinical study. relation to the development of gingival of pyridinoline cross-links due to higher
J Periodontol 2002;73:53–62. overgrowth: An in vitro study. Biomed telopeptide lysyl hydroxylase levels is
2. Oates TW, Robinson M, Gunsolley JC. Pharmacother 2004;58:231–238. a general fibrotic phenomenon. Matrix
Surgical therapies for the treatment of 13. Muller HP, Schaller N, Eger T, Heinecke Biol 2004;23:251–257.
gingival recession. A systematic review. A. Thickness of masticatory mucosa. 23. Kagan H. Characterization and regula-
Ann Periodontol 2003;8:303–320. J Clin Periodontol 2000;27:431–436. tion of lysyl oxidase. In: Mecham R (ed).
3. Langer B, Calagna L. The subepithelial 14. Lee YJ, Kwon YH, Park JB, et al. Epitheli- Biology and Regulation of Extracellular
connective tissue graft. J Prosthet Dent al thickness of the palatal mucosa: A his- Matrix: A Series. Regulation of Matrix
1980;44:363–367. tomorphometric study in Koreans. Anat Accumulation, vol 1. Orlando: Academ-
4. Langer B, Langer L. Subepithelial con- Rec 2010;293:1966–1970. ic, 1986:321–398.
nective tissue graft technique for root 15. Soehren SE, Allen AL, Cutright DE, Seib- 24. Sakai T, Gross J. Some properties of the
coverage. J Periodontol 1985;56:715–720. ert JS. Clinical and histologic studies of products of reaction of tadpole collage-
5. Hirsch A, Attal U, Chai E, Goultschin J, donor tissues utilized for free grafts of nase with collagen. Biochemistry 1967;
Boyan BD, Schwartz Z. Root coverage masticatory mucosa. J Periodontol 1973; 6:518–528.
and pocket reduction as combined sur- 44:727–741. 25. Woessner JF Jr. Matrix metalloproteinas-
gical procedures. J Periodontol 2001;72: 16. Muller HP, Heinecke A, Schaller N, Eger es and their inhibitors in connective tissue
1572–1579. T. Masticatory mucosa in subjects with remodeling. FASEB J 1991;5:2145–2154.
6. Song JE, Um YJ, Kim CS, et al. Thickness different periodontal phenotypes. J Clin 26. Phipps RP, Borrello MA, Blieden TM. Fi-
of posterior palatal masticatory mucosa: Periodontol 2000;27:621–626. broblast heterogeneity in the periodon-
The use of computerized tomography. 17. Seguier S, Godeau G, Brousse N. Col- tium and other tissues. J Periodontal Res
J Periodontol 2008;79:406–412. lagen fibers and inflammatory cells in 1997;32:159–165.
7. Wara-aswapati N, Pitiphat W, Chandra- healthy and diseased human gingival 27. Tipton DA, Stricklin GP, Dabbous MK.
pho N, Rattanayatikul C, Karimbux N. tissues: A comparative and quantitative Fibroblast heterogeneity in collageno-
Thickness of palatal masticatory mu- study by immunohistochemistry and au- lytic response to cyclosporine. J Cell Bio-
cosa associated with age. J Periodontol tomated image analysis. J Periodontol chem 1991;46:152–165.
2001;72:1407–1412. 2000;71:1079–1085.
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