A female patient, 34 years old at the time of the first visit, was referred to us to correct her gingival esthetic problem caused by the formation of gingival/mucosal overgrowths. Gingival outgrowths localized in the second, third, and fourth quadrants, corresponding to the position of premolars and canine roots, were identified during an oral examination. Their consistency appeared hard and unmovable, and they presented a normal gingival color. The lesions were asymptomatic, and the morphology did not change over time (Figure 1).
The anamnestic investigation revealed a history of FGG surgery in 2004 (8 years before our first examination) in each site that was treated for gingival recessions; allergic reactions to nonsteroidal anti-inflammatory drugs, paracetamol, cephalosporin, penicillin and its derivatives, and macrolide antibiotics; absence of any systemic conditions affecting metabolism; and altered wound healing (cutaneous keloids and hypertrophic scars).
The hypothesis of exostosis formation was suggested by the following clinical observations: benign aspect, nodular and hyperplastic tissue, hard and stable on palpation, not symptomatic, and localized in canine and premolar areas involved in a surgical procedure that could have acted as a promoter event.
We decided to perform an excisional surgery, followed by a histologic and molecular evaluation. The Review Board of the Research Center in Oral Implantology of the Universita degli Studi di Milano in Milan, Italy, approved the treatment and research protocol before the first surgery.
All procedures were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The patient was informed about the formulated medical hypothesis and the need for surgical intervention to remove the lesions and analyze the specimen. The patient agreed to the surgery and signed a written informed consent form. The surgery was planned after a complete evaluation of the patient's systemic conditions that confirmed an absence of clinical contraindications.
All surgeries were performed in three different sessions by the same operator, with more than ten years of experience in the field of oral surgery (LF), and following the same protocol. The first surgical event involved a lesion localized in the second quadrant. Local anesthesia was performed with articaine 4% + epinephrine 1 : 100.000, voiding injection in the lesion area. The lesion was first isolated by incisions with a microblade following its external border; after that, the incision was continued above the periosteum, removing the tissue covering the exostosis. Then, with a 15C blade, a full-thickness incision was performed coronally to the lesion and the exostosis surface was exposed using a periosteal elevator. The full-thickness incision was extended in the mesiodistal direction in order to have a better view of the surgical site. Gingival fragments obtained from the overgrown gingiva were collected for histological and molecular characterization. A carbide bur, mounted on a rotary instrument and under sterile saline solution irrigation, was used to remove the bone structure in order to recreate a plane surface. To obtain a primary closure of the flap after the removal of the lesion, a partial thickness incision was performed in the free alveolar mucosa. The flap was then sutured with a 6/0 nonresorbable suture (ETHILON  Nylon Suture, Ethicon  , Cincinnati, OH, United States). After flap closure, an infiltration of 4 mg corticosteroid (BENTELAN 4 mg/2 mL, Defiante Farmaceutica S.A., Madeira, Portugal) was performed, to prevent the recurrence of overgrowths in the short term. The last surgical procedure was performed within 4 months of the first visit.
The follow-up visits revealed small relapses after 18 months from the surgical intervention that were asymptomatic and did not affect aesthetics and function. The recurrences observed in the second and fourth quadrants were linearly arranged along the incision of the periosteum that occurred during the surgery, while the lesion treated in the third quadrant showed a more evident relapse.
One gingival fragment was processed for histological analysis while another fragment was cultured for in vitro evaluations. Moreover, to characterize the pathological gingiva, a fragment from the healthy papilla was obtained from the same patient for comparison.
Immediately after surgery, each tissue fragment was fixed in 4% formalin in 0.1 M phosphate-buffered saline (PBS), pH 7.4, routinely dehydrated and paraffin-embedded. Serial sections of 5 mum were obtained. Sections were stained with freshly made hematoxylin-eosin (Sigma-Aldrich, St Louis, MO, United States) to evaluate cell and tissue morphology.
To evaluate fibrillary collagen, slides were deparaffinized and immersed for 30 minutes in saturated aqueous picric acid containing 0.1% Sirius Red F3BA (Sigma-Aldrich, St. Louis, MO, United States), a staining method that specifically marks collagen. The sections were observed under a light and polarized microscope (Nikon Eclipse E600, Nikon, Tokyo, Japan) and photographed with a calibrated digital camera (DXM1200, Nikon, Tokyo, Japan) at a 20x magnification.
Human gingival fibroblasts were obtained from a gingival fragment covering the exostosis and from a gingival fragment obtained from the patient's healthy gingiva. Tissue fragments were washed with sterile PBS, plated in T25 flasks, and incubated in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) at 37 C in a humidified atmosphere with 5% CO2. Fibroblasts that grew out from the explant were trypsinized (0.025% trypsin-0.01% EDTA) for secondary cultures and plated in T75 flasks, as previously described. Human gingival fibroblasts were used between the fourth and fifth passages. Molecular evaluations were performed in samples cultured for 24, 48, and 72 h. All evaluations were performed on duplicate cultures for each sample.
Cell morphology of cultured gingival fibroblasts was analyzed by phase-contrast microscopy using a Leica DM IL microscope. Cell viability was determined by Trypan blue exclusion. Growth curves were obtained after plating cells in 6-well plates (150,000 cells/well). After the attachment, the cells were counted after 24, 48, and 72 h.
Total RNA was isolated from cultured fibroblasts as previously described. Briefly, 1 mug of total RNA was reverse-transcribed in a 20 muL final volume of reaction mix (Bio-Rad, Segrate, Milan, Italy). Gene expression for the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), lysyl oxidase (LOX), and focal adhesion kinase (FAK) was assessed. GAPDH was used as internal control for normalization of the differences in the amount of total RNA in each sample. The following primers were used: GAPDH: sense CCCTTCATTGACCTCAACTACATG, antisense TGGGATTTCCATTGATGACAAGC, antisense GGAACCAGGATGACCAGATGTACC; TIMP-1: sense GGCTTCTGGCATCCTGTTGTTG, antisense AAGGTGGTCTGGTTGACTTCTGG; LOX: sense GGATACGGCACTGGCTACTT, antisense GACGCCTGGATGTAGTAGGG; and FAK: sense GTCTGCCTTCGCTTCACG, antisense GAATTTGTAACTGGAAGATGCAAG.
Amplification was performed in a 96-well plate in a final volume of 20 muL per well containing 10 muL of 1x SYBR Green Supermix (Bio-Rad, Italy), 2 muL of template, and 300 pmol of each primer, and each sample was analyzed in triplicate in an iQ5 thermal cycler (Bio-Rad, Italy) after 40 cycles. After determining the cycle threshold (Ct), gene expression levels relative to that of GAPDH were calculated by the 2DeltaCt method.
Slot blot analysis was used to assess type I collagen (COL-I), type III collagen (COL-III), and matrix metalloproteinase-1 (MMP-1) protein levels secreted by gingival fibroblasts in cell culture supernatants, as previously described. Briefly, protein content in cell culture media was determined by a colorimetric assay (DC Protein Assay, Bio-Rad, Italy); 100 mug of total proteins per sample (final volume of 200 muL of Tris-buffered saline (TBS)) was spotted onto a nitrocellulose membrane using a Bio-Dot SF apparatus (Bio-Rad, Italy). After blocking for 1 h with 5% skimmed milk in TBST (TBS containing 0.05% Tween-20), pH 8, membranes were incubated for 1 h at room temperature in a monoclonal antibody to COL-I (1:1000 in TBST) (Sigma, Italy), to COL-III (1:2000 in TBST) (Sigma, Italy) and to MMP-1 (1 mug/ml in TBST, Millipore). After washing with TBST, membranes were incubated in HRP-conjugated rabbit anti-mouse serum for 1 h (1 : 6000 in TBST to detect COL-I and COL-III or 1 : 40,000 in TBST to detect MMP-1, respectively) (Sigma, Italy). Immunoreactive bands were revealed by the Opti-4CN substrate or Amplified Opti-4CN substrate (Bio-Rad, Italy) and quantified by densitometric scanning (UVBand, Eppendorf, Italy).
Serum-free cell culture media were mixed 3 : 1 with sample buffer (containing 10% SDS), as previously described. Five mug of total protein per sample was run under nonreducing conditions without heat denaturation on 10% polyacrylamide gel (SDS-PAGE) copolymerized with 1 mg/mL of type I gelatin at 4 C. After SDS-PAGE, the gels were washed twice in a renaturing buffer (2.5% Triton X-100, Tris-HCl 50 mM) for 30 min each and incubated overnight in a substrate buffer at 37 C (Tris-HCl 50 mM, CaCl2 5 mM, NaN3 0.02%, pH 7.5). After staining the gels with Coomassie Brilliant Blue R-250, MMP gelatinolytic activity was detected as clear bands on a blue background. Bands were quantified by densitometric scanning (UVBand, Eppendorf, Italy).
Data, expressed by mean +- standard deviation (SD), were analyzed by t-test. p values less than 0.05 were considered significant.