Gdf 5

C2C12 and murine mesenchymal cells (C3H10T1/2) were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% FBS and antibiotics. The cells were passaged before reaching confluence and used within 10 passages. To induce tenocyte differentiation, C2C12 cells were cultured in serum‐free AIM‐V medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with GDFs (500 ng·mL⁻¹ of GDF‐5, ‐6, ‐7 and 10, 100, 500 ng·mL⁻¹ of myostatin; R&D Systems, Minneapolis, MN, USA). The medium was replaced every 2 days. To analyze signaling pathways, cells were treated with 500 ng·mL⁻¹ of myostatin and 10 μmol·L⁻¹ of inhibitors for ALK (SB431542), p38MAPK (SB203580), and MEK1 (PD98059) (Abcam, Cambridge, MA, USA) for 5 days.

With regard to the differentiation experiment, TSPCs were cultured in 6-well plates (50,000 cells/well). Osteogenic, adipogenic, chondrogenic, and tenogenic differentiation were induced by a corresponding differentiation medium. The osteogenic differentiation medium contained a growth medium supplemented with 10 nM dexamethasone (Sigma-Aldrich), 5 mM β-glycerophosphate (APEXBIO), and 0.05 mM l-ascorbic acid 2-phosphate (Sigma-Aldrich). The adipogenic differentiation medium consisted of a growth medium supplemented with with 500 μM isobutyl-methylxanthine, 60 μM indomethacin (Sigma-Aldrich), 0.5 μM hydrocortisone, and 10 μM insulin (Sigma-Aldrich). The chondrogenic differentiation medium was a Gibco StemPro chondrogenic differentiation kit (Thermo Fisher Scientific). The tenogenic differentiation medium contained a growth medium supplemented with 10 ng/ml TGF-β1 (Peprotech), 10 ng/ml GDF-5 (R&D System), 0.05 mM l-ascorbic acid 2-phosphate. After a two-week induction, Alizarin Red S, Oil Red O, Toluidine Blue, Sirius Red and Masson’s trichrome stainings were applied to evaluate osteogenic, adipogenic, and tenogenic differentiation capacity, respectively.

With regard to the differentiation experiment, TDSCs were cultured in 6-well plates (50,000 cells/well). Osteogenic, adipogenic, and chondrogenic were induced by a corresponding differentiation medium. The osteogenic differentiation medium contained a growth medium supplemented with 10 nM dexamethasone (Sigma-Aldrich), 5 mM β-glycerophosphate (APEXBIO), and 0.05 mM l-ascorbic acid 2-phosphate (Sigma-Aldrich). The adipogenic differentiation medium consisted of a growth medium supplemented with 500 μM isobutyl-methylxanthine, 60 μM indomethacin (Sigma-Aldrich), 0.5 μM hydrocortisone, and 10 μM insulin (Sigma-Aldrich). The chondrogenic differentiation medium was a Gibco StemPro chondrogenic differentiation kit (Thermo Fisher Scientific). The tenogenic differentiation medium contained a growth medium supplemented with 10 ng/ml TGF-β1 (Peprotech), 10 ng/ml GDF-5 (R&D System), and 0.05 mM l-ascorbic acid 2-phosphate. After a 2 week induction, Alizarin Red S, Oil Red O, and Alcian Blue were applied to evaluate osteogenic, adipogenic, tendon stem cell differentiation capacity, respectively (21 (link)).