Bovine fibrinogen type 1 s

Multipotent stromal cells were suspended in 10,000 KIU/mL aprotinin solution (Nordic Pharma) containing 19 mg/mL sodium chloride and 100 mg/mL bovine fibrinogen type I-S (60–85% protein, 10% sodium citrate, and 15% sodium chloride; Sigma-Aldrich), which was combined 1:1 with 5 U/mL thrombin in 40 mM CaCl₂ (pH 7) and transferred by syringe to recesses in custom designed flexible silicone strips. These strips were then incubated at 37°C for 45 min to allow cross-linking to complete, producing dog-boned shaped constructs with final concentrations of 50 mg/mL fibrinogen, 2.5 U/mL thrombin, 5,000 KIU/mL aprotinin, 17 mg/mL sodium chloride, 20 mM CaCl₂, and 10 × 10⁶ cells/mL. The constructs were then loaded into bioreactors containing differentiation medium (DM), consisting of DMEM supplemented with 100 U/mL penicillin/streptomycin (both from Gibco), 100 mg/mL sodium pyruvate, 40 mg/mL l-proline, 50 mg/mL l-ascorbic acid-2-phosphate, 1.5 mg/mL bovine serum albumin, 1× insulin–transferrin–selenium, and 100 nM dexamethasone (all Sigma-Aldrich). For studies requiring chondro-inductive stimuli, constructs were culture in chondrogenic medium [DM supplemented with 10 ng/mL of transforming growth factor-β3 (TGF-β3, ProSpec-Tany TechnoGene, Ltd., Israel)]. Media changes were performed twice weekly.

Phosphate buffered saline (PBS) (Biolot, St. Petersburg, Russia); 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrasolium bromide (MTT) and Mowiol (Sigma-Aldrich, St. Louis, MO, USA); 96% ethanol (Chimmed, Moscow, Russia); Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA); fetal bovine serum (FBS) (Gibco, Waltham, MA, USA); 0.9% saline (PanEco, Moscow, Russia); dimethyl sulfoxide (DMSO) (Amreso, Solon, OH, USA); 0.02% EDTA; 0.05% trypsin solutions (Gibco, Waltham, MA, USA); and gentamycin (PanEco, Moscow, Russia) were used for the experiments with cell cultures. BCA protein assay kit, lysozyme (Thermo Fisher, Waltham, MA, USA), BSA, and bovine fibrinogen (type I-S) (Sigma-Aldrich, Burlington, MA, USA). Blood collection tubes (Vacuette, Greiner Bio-One, Kremsmünster, Austria).