Recombinant human tgf β1

Human bone marrow-derived mesenchymal stem cells were purchased from Lonza (Walkersville, MD). Cells from multiple lots, originating from separate donors, were combined to account for any donor-specific responses. The MSCs were cultured at 37°C and 5% CO₂ in a complete MSC growth medium, consisting of low-glucose DMEM with 10 vol% MSC-qualified, USDA-approved fetal bovine serum and 1 vol% antibiotic-antimycotic (Thermo Fisher, Waltham, MA), fed every three days and used at passage 6.
Selected CG and CGcyclo scaffolds were incubated in 5 ng/mL human recombinant BMP-2 or human recombinant TGF-β1 (ProSpec-Tany, Ness-Ziona, Israel) for 1 h in sterile PBS in the same manner as previously described. The scaffolds were then moved into 5% FBS MSC Media for 2 h as cells were lifted and centrifuged in preparation for seeding using a previously established static method [38 (link)]. Scaffolds were partially dried with Kimwipes and seeded with 1.0 × 10⁵ MSCs per 24 μL medium (one 10 μL drop on opposite sides of the scaffold) in six well plates, with six scaffolds per well. Cells were allowed to attach for 1 h before submerging in 6 mL of low-serum MSC media, containing 5% FBS, per well. Cell-seeded scaffolds were cultured at 37°C and 5% CO₂ and fed every three days with the low-serum media.