Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin

After approval by the Institutional Review Board of the Bauru School of Dentistry, University of São Paulo, #055/2011, three systemically and periodontally healthy individuals (28, 38 and 50 years-old, 3 females) were selected in Periodontics Clinics, all of whom needed root coverage in areas with scarce keratinized mucosa. They had no signs of gingival inflammation, no bleeding on probing or critical probing depth. All individuals were submitted to anamnesis, periodontal clinical examination and radiographic exams. The inclusion criteria were individuals with deficient keratinized mucosa, both in quantity and quality, without systemic complications that could contraindicate the surgical procedures, and that provided written informed consent to participate in the study.

The palatal mucosa specimens, sized 3x3 mm, were immediately obtained when an epithelium-connective tissue graft was removed from the palatal donor site on the premolars area during free gingival graft surgery [21]. After four months, specimens from gingival graft were obtained from the marginal area, during a second surgical procedure for root coverage. Fibroblasts were isolated by the explant technique as previously described [8‐11]. Briefly, the specimens were processed immediately in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum (FBS; Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA). The epithelial layer was removed and the specimens were minced as small as possible. Tissue fragments of palatal mucosa or gingival graft were plated separately in Petri dishes and covered with DMEM supplemented with 20% FBS and antibiotics (600 μL/mL penicillin, 300 μL/mL gentamicin sulfate and 100 μL/mL amphotericin B). The explants were placed in 25 cm² flasks and incubated at 37°C in a humidified atmosphere of 5% CO₂. The medium (DMEM 10% FBS) was changed every 2–3 days and cultures were maintained until fibroblasts reached confluence. After confluence, the fibroblasts were harvested and used between the fourth and eighth passages [8‐11].

The cells were seeded in 24 well plates at a density of 2×10⁴ cells/well and incubated at 37°C in a humidified atmosphere of 5% CO₂ during 18 hours in DMEM 1% FBS. After overnight adhesion, DMEM containing 1 μg/mL of Pg LPS (Invivogen, San Diego, CA, USA) or Ec LPS (Invivogen) was added to the wells for 24 or 48 h. Non-stimulated cells were used as controls. Both Pg LPS and Ec LPS were ultrapure and purchased from Invivogen.

Cells cultured from the palatal mucosa and gingival graft were characterized as fibroblasts by their morphology and staining with fibroblast surface protein (FSP; ab 11333, Abcam, Cambridge, UK) by means of immunostaining [11]. Cells were seeded in an 8-well plate with 1×10⁴ cells per well and incubated at 37°C with 5% CO₂ during 18 hours to adhere in subconfluence. Subsequently, the wells were divided into groups (control, Pg LPS, Ec LPS and negative control, which was performed without the primary antibody) in duplicate, to cell stimulation. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour. Coverslips were mounted with mounting medium VECTASHIELD Hard-Set Mounting Medium containing 49,6-diamidino-2-Phenylindole (DAPI; H-1500, Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscope laser scanning (TCS model, SPE, Leica Mannheim, Germany).

Cell viability was analyzed from enzymatic activity with a colorimetric assay MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Briefly, cells were washed with 1× PBS to remove culture medium and MTT (5 mg/mL) was added to the groups (control, Pg LPS and Ec LPS) or cell-free wells (blank). The plate was incubated during 4 hours at 37°C with 5% CO₂. After incubation, the plate was centrifuged (200 g during 7 minutes at 21°C) and MTT solution was removed to add dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO). The optical density of the wells was determined using a plate reader (Fluostar Optima, BMG Labtech, Ortenberg, Germany) in the wavelength of 570 nm.

ELISA was performed following the manufacturer’s instructions to determine the protein levels of IL-6 (R&D System, Minneapolis, MN, EUA), IL-8/CXCL8 (R&D System, Minneapolis, MN, EUA), MIP-1α/CCL3 (R&D System, Minneapolis, MN, EUA), TGF-β (eBioscience, San Diego, CA, USA), VEGF (PeproTech, London, UK) and CXCL16 (PeproTech, London, UK). Briefly, plates of 96 wells were incubated with capture antibody anti-IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-β, VEGF or CXCL16 diluted in 1x PBS buffer. After blocking for 1 hour to avoid non-specific binding, 100 μl of standard IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-β, VEGF and CXCL16 and culture supernatants were placed. The cytokines were detected by horseradish peroxidase-labeled monoclonal antibody to each target after addition of 100 μl anti-human IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-β, VEGF and CXCL16 biotinylated antibodies were placed in each well and incubated for 2 hours at room temperature. The microplate was washed to remove unbound enzyme-labeled antibodies. The amount of horseradish peroxidase bound to each well was determined by the addition of 100 μl substrate solution. The reaction was stopped by the addition of 100 μl of 1 M sulfuric acid, and the plates were read at 450 nm (Synergy™ MX Monochromator Based Multi-Mode Microplate Reader, BioTek Instruments, Inc, Winooski, VT, USA). The concentrations of IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-β, VEGF and CXCL16 were determined by interpolation from a standard curve and presented as pg/mL for duplicate assays of duplicate samples of each of the tested conditions.

Comparisons for viability through MTT among samples were performed using One-way ANOVA test followed by Tukey test. ELISA data normality was verified by the Kolmogorov-Smirnov method. The statistical differences were determined by three-way ANOVA followed by Tukey test. Statistical analyses were performed with STATISTICA (version 11.0; StatSoft Inc). P values < 0.05 were considered significant. The results were expressed as mean and one standard deviation of the mean.

Cellular staining with a fibroblast surface marker (FSP) was performed to confirm the fibroblastic phenotype. Fibroblasts were analyzed by immunofluorescence in all groups (control, Pg LPS, Ec LPS and negative control). Positive staining was observed for all groups (Figure 1), with Pg LPS (Figure 1C) and Ec LPS (Figure 1B) groups being slightly higher. For the negative control group (Figure 1D), which did not receive primary antibody, no signal was detected. This result confirms the characterization of the cultured cells as fibroblasts.

Cell viability was evaluated after 24 and 48 h, in the control (non-stimulated) and testing groups (stimulated with Pg LPS and Ec LPS). There was no difference in cells viability with the stimuli used after 24 and 48 h (Figure 2).

When the targets of this study were compared between the two subpopulations of fibroblasts, IL-6 secretion was statistically significant after 24 and 48 h of Pg LPS stimulation (Figure 3A and B) by both subtypes of fibroblasts (from the palatal mucosa and gingival graft in marginal area), and the difference was greater with 48 h (Figure 3B). On the other hand, the secretion after Ec LPS stimulation was only statistically significant after 48 h, by both subtypes of fibroblasts (Figure 3D). Additionally, IL-6 secretion was greater by non-stimulated fibroblasts from the marginal gingival graft (Figure 3).

Fibroblasts from the palatal mucosa secreted more IL-8/CXCL8 than fibroblasts from the marginal gingival graft when stimulated by Pg LPS with a statistically significant difference at 48 h (Figure 4A and B). Also, regarding this chemokine, fibroblasts from the palatal mucosa showed an increase at both 24 h and 48 h when stimulated by Ec LPS (Figure 4C and D), which was not observed by cells from the marginal gingival graft.

In this study, MIP-1α/CCL3 was observed to be constitutively secreted by both fibroblasts subtypes (Figure 5). Moreover, there was no statistically significant difference in MIP-1α/CCL3 secretion between unstimulated fibroblasts (control group) and fibroblasts stimulated with Pg or Ec LPS from the palatal mucosa (Figure 5). However, statistically significant difference was observed in MIP-1α/CCL3 secretion after 48 h when fibroblasts from the marginal gingival graft were stimulated with Pg LPS (Figure 5B) and after 24 h when challenged by Ec LPS (Figure 5C).

When TGF-β secretion was evaluated, fibroblasts from the palatal mucosa showed no detectable levels even in the presence of Pg LPS (Figure 6A and B) or Ec LPS (Figure 6C and D). On the contrary, fibroblasts from the marginal gingival graft showed a constitutive TGF-β secretion and there was no statistically significant difference between stimulated and unstimulated cells, both at 24 h and 48 h (Figure 6). VEGF and CXCL16 secretion was not detected either on fibroblasts supernatant cultured from the palatal mucosa neither from the marginal gingival tissue, regardless stimulation by Pg or Ec LPS.