Human recombinant bmp 2

Human mesenchymal stem cells (hMSCs, Lonza) were expanded in standard flasks in complete mesenchymal stem cell growth media (Low glucose Dulbecco’s Modified Eagle Medium; 10% MSC Fetal bovine serum; 1% Penicillin-Streptomycin; 1% L-glutamine; Invitrogen, Carlsbad, CA) at 37°C and 5% CO₂. After expansion to passage 6, hMSCs were seeded onto 8 mm diameter (4 mm thick) scaffold specimens. A total of 7.5 ×10⁴ cells were seeded onto the scaffold disc via a previously described static seeding method.^{32 (link), 41 (link)} MSC seeded scaffolds were subsequently cultured at 37°C and 5% CO₂ for up to 56 days. CGCaP scaffolds were cultured in one of three media formulations throughout: (1) complete MSC growth media (CGCaP Growth); (2) complete MSC growth media supplemented with 100 ng/mL BMP2 (CGCaP BMP2); or (3), complete MSC osteogenic media (CGCaP Osteo). Human recombinant BMP-2 was obtained from ProSpec (Israel). Osteogenic media contained 50μM ascorbic acid, 0.1μM Dexamethasone, and 10mM β-glycerophosphate added to growth media. MSC-seeded CG scaffolds were cultured in standard MSC growth media (CG Growth) as a control. The chosen dose of BMP-2 (100ng/mL) was based on previous studies by our group that documented a pro-osteogenic effect in CG scaffolds.^{32 (link), 34 , 44} All cell seeded scaffold groups were fed their respective media every 3 days.

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.