Fetal calf serum (fcs)

Human gingiva was harvested during the extraction of impacted wisdom teeth from young and healthy patients who stayed anonymous and had given informed and written consent. A total of three strains of fibroblasts were established by explant cultures. Cells growing out from the gingiva explants were further expanded and stored in liquid nitrogen at low passage. Fibroblasts expanded for fewer than 10 passages were used for the experiments. Approval was obtained from the Ethics Committee of the Medical University of Vienna (EK NR 631/2007). A total of three strains of fibroblasts were established by explant cultures and fewer than 10 passages were used for the experiments. Gingival fibroblasts were grown and supplemented with 1% antibiotics (Sigma Aldrich, St. Louis, MO, USA) and 10% fetal calf serum (Bio&Sell GmbH, Nuremberg, Germany). The cells were exposed to the respective treatments for another 24 h under standard conditions at 37 °C, 5% CO₂ and 95% humidity.

The oral squamous cell carcinoma cell line HSC2, originally obtained from the Health Science Research Resources Bank (Sennan, Japan) and cultivated in growth Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA), 10% fetal calf serum (Bio&Sell GmbH, Nuremberg, Germany), and 1% antibiotic-antimycotic (Sigma Aldrich, St.Louis, MO, USA). HSC2 cells were seeded at a concentration of 2.5 × 10⁵ cells/cm² onto culture dishes one day prior to stimulation. Primary oral epithelial cells were taken from the epithelial layer of human gingiva harvested from the extracted third molars of patients who had given informed and written consent. The Ethics Committee of the Medical University of Vienna (EK NR 631/2007) approved the protocol. Primary cells were cultivated in a keratinocyte growth medium (PromoCell, Heidelberg, Germany) and seeded at a concentration of 4.0 × 10⁵ cells/cm² onto culture dishes one day prior to stimulation. In the basic setting, HSC2 and primary epithelial cells were treated overnight with 10 ng/mL TNFα and 10 ng/mL IL-1β (both ProSpec, Ness-Ziona, Israel) with and without 300 µg/mL Emdogain^® (EMD; Straumann Group, Basel, Switzerland) or with serum-free medium alone at 37 °C, 5% CO₂, and 95% humidity before analysis.

All chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA). HO inhibitors [tin protoporphyrin (SnPP), zinc protoporphyrin (ZnPP), and chromium mesoporphyrin (CrMP)] were obtained from Frontier Scientific (Logan, UT, USA). Fetal calf serum was purchased from Bio & Sell (Nürnberg, Germany).

Human gingiva was harvested from extracted wisdom teeth from patients who had given informed and written consent. An approval was obtained from the Ethics Committee of the Medical University of Vienna (EK NR 631/2007). A total of three strains of fibroblasts were established by explant cultures and fewer than 10 passages were used for the experiments. Gingival fibroblasts were grown and supplemented with 1% antibiotics (Sigma-Aldrich, St. Louis, MO, USA.) and 10% fetal calf serum (Bio&Sell GmbH, Nuremberg, Germany). The cells were seeded at 30,000 cells/cm² onto culture dishes one day prior to stimulation. Cells were exposed to 30% lysates of PRF prepared at 210 g, 650 g and 1500 g for another 24 h under standard conditions at 37 °C, 5% CO₂ and 95% humidity. To examine the influence of TGF-β signaling, the inhibitor of TGF-β receptor type I kinase, SB431542 (Calbiochem, Merck, Billerica, MA, USA), was used at 10 µM.

Human gingiva was harvested from extracted wisdom teeth from patients who had given informed and written consent. Approval was obtained from the Ethics Committee of the Medical University of Vienna (EK NR 631/2007). A total of three strains of fibroblasts were established by explant cultures, and fewer than 10 passages were used for the experiments. Gingival fibroblasts and RAW264.7 macrophage-like cells (LGC Standards, Wesel, Germany) were grown and supplemented with 1% antibiotics (Sigma–Aldrich, St. Louis, MO, USA) and 10% fetal calf serum (Bio&Sell GmbH, Nuremberg, Germany). The cells were seeded at 30,000 cells/cm² onto culture dishes one day prior to being stimulated. Cells were exposed to 30% PRF and UBC lysates for required time (overnight for the gene and protein expression, 1 h for the immunofluorescence experiment, 30 min for the WB analysis) under standard conditions at 37 °C, 5% CO₂ and 95% humidity. Cells were treated with 10 ng/mL TGF-β1 (Cell Signaling Technology Europe, B.V., Frankfurt am Main, Germany) or 100 ng/mL LPS from Escherichia coli 0111: B41 (Sigma Aldrich, St. Louis, MO, USA), 10 µg/mL poly (1:C) HMW (InvivoGen, Toulouse, France), and 10 µg/mL FSL-1 (InvivoGen, Toulouse, France) in the indicated experiments. After overnight treatments (~16 h), gene and protein modulation were analyzed.

Tissue samples of human gingiva were harvested from the extracted third molars of patients who had given informed and written consent. Prior to sample attainment, the Ethics Committee of the Medical University of Vienna (EK NR 631/2007) approved the protocol. In total, three strains of fibroblasts were established through explant cultures and less than 10 passages were used for the experiments. Cells were cultured in a humidified atmosphere at 37 °C in a growth medium consisting of DMEM, 10% fetal calf serum (Bio&Sell GmbH, Nuremberg, Germany), 1% of 10,000 units penicillin, and 10 mg of streptomycin/mL (Sigma Aldrich, St. Louis, MO). Cells were seeded at a concentration of 30,000 cells/cm² onto culture dishes one day prior to stimulation. The stimulation was done by cell exposure to the supernatants and the lysates of the allografts. To examine the influence of TGF-β signaling, the inhibitor of TGF-β receptor type I kinase SB431542 (Calbiochem, Merck, Billerica, MA, USA) was used at 10 µM. Gene expression analysis and immunostainings were done as indicated. To identify the influence of proteases, the protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) was solved in 50 mL of serum-free DMEM and used for preparing the supernatant of OraGRAFT^® Prime. Gene expression analysis was done as indicated.

Tissue samples of three independent donors were harvested from extracted third molars, and gingival fibroblasts were prepared after approval of the Ethical Committee of the Medical University of Vienna (EK Nr. 631/2007). Cells were cultured in a humidified atmosphere at 37 °C in growth medium consisting of DMEM, 10% fetal calf serum (Bio&Sell GmbH, Nuremberg, Germany) and 1% antibiotics (Sigma Aldrich, St. Louis, MO, USA). Cells were plated in growth medium at 30,000 cells/cm² into culture dishes. The following day, fibroblasts were exposed to the supernatants and the lysates of the collagen membranes. SB431542, a TGF-β receptor I kinase inhibitor, was used at 10 µM (Calbiochem, Merck Millipore, Billerica, MA, USA). The TGF-β neutralizing pan-specific polyclonal rabbit IgG AB-100-NA (R&D Systems, Minneapolis, MN, USA) was used at 20 µg/mL. Cell conditioned medium was harvested after 24 h, centrifuged and stored frozen until subjected to immunoassay. Gene expression analysis and immunostainings were performed as indicated. Fibroblasts expanded for less than 10 passages were used for the experiments.