Transforming growth factor β3

Fibrinogen and Fresh frozen plasma solutions (FFP) were obtained from Iranian Blood Transfusion Organization. FFP was thawed in water bath at 37°C. Then 15 mL of it was mixed with 10 mL of calcium glucuronate (mid 5:3 Ratio). The obtained solution was incubated for 1 h at 37°C and subsequently centrifuged at 2200 rpm for 10 min. The supernatant was collected as thrombin. Fibrinogen and thrombin solutions were prepared for use as cell culture. To this end, the adipose tissue-derived stem cells at a concentration of 5×10⁶ cells per ml were dissolved within thrombin and subsequently fibrinogen was added to them. Then the scaffolds in chondrogenic medium containing DMEM-high glucose along with 50 μg/mL ascorbate 2- phosphate, 1% insulin-transferin-selenium (Sigma–Aldrich, St. Louis, MO, USA), dexamethasone 100 nM (Sigma–Aldrich, St. Louis, MO, USA), 50 mg/mL FBS, 5 μg/mL linoleic acid (Sigma–Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin and 10 ng/ml transforming growth factor-β₃ (Sigma–Aldrich, St. Louis, MO, USA) was placed in the incubator (37°C, 5% CO2, 99% humidity) for 14 days.

To induce adipogenic differentiation, confluent cells were cultured in medium supplemented with 1 × 10⁻⁶ M dexamethasone, 0.02 mg/mL indomethacin and 10 μg/ mL insulin (Sigma-Aldrich). After 12 days, cell differentiation into lipid-laden adipocytes was confirmed by oil red O staining (Sigma-Aldrich). For chondrogenic differentiation, cells were incubated for 1 hour at 5 × 10³ cells/ μL in 10 μL of culture medium to achieve conditions for micromass formation. Cells were cultured in medium supplemented with 10⁷ M dexamethasone, 50 μg/mL ascorbic acid, and 10 ng/mL of transforming growth factor-β3 (Sigma-Aldrich) for 7 days, and chondrogenic differentiation was assessed by Safranin O staining (Merck, Darmstadt, Germany). To induce osteogenic differentiation, adherent cells were grown at 3 × 10⁴ cells/cm² in culture medium with 10⁻⁷ M dexamethasone, 50 g/mL ascorbic acid, and 10 mM β-glycerophosphate (Sigma-Aldrich). After 21 days of culture, calcium deposits were detected by Alizarin Red staining (Sigma-Aldrich).