Fibroblast growth factor 2 (fgf2)

Basic fibroblast growth factor, also known as FGF-2, was loaded onto the scaffolds and the release profiles of FGF-2 were evaluated to determine the release kinetics of the scaffolds. FGF-2 (ProSpec-Tany TechnoGene, Rehovot, Israel) was mixed with distilled water to achieve a concentration of 500 ng/mL. The various scaffolds were then immersed in the FGF-2 solution for 2 h, for loading of FGF-2. After which, the scaffolds were immersed in medium at 37 °C for various durations to evaluate release profiles of FGF-2 using an enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA). The concentration was quantified using absorbances measured using a spectrophotometer. Finally, influence of the FGF-2-loaded scaffolds on cell proliferation and osteogenesis was also evaluated.

JB6 P⁺ cells were plated at 500 cells/well (or otherwise indicated) in a 24-well plate in 200 µL of MEM media with 10% FBS in 0.33% agar overlaid onto 350 µL of MEM media with 10% FBS in 0.5% agar. FGF2 (Prospec, CYT-386) was incubated with the cells at 0.5 ng/mL and compared to untreated controls. Soft agar plates were left at room temperature for 30 minutes then incubated at 37 °C.
MCF-10A cells were seeded at 750 cells/well (or otherwise indicated) in a 24-well plate in 200 µL of DMEM/Ham’s F12, 5% HS, and 0.33% agar overlaid onto 350 µL of DMEM/Ham’s F12, 5% HS, and 0.5% agar. FGF2 (prospec, CYT-386) was incubated with the cells at 20 ng/mL and compared to untreated controls. Soft agar plates were left at room temperature for 30 minutes before 200 µL of MCF-10A growth media was gently added to each well and then stored at 37 °C. Every 3–4 days, the growth media was removed from each well and replenished with 200 µL of MCF-10A growth media.
After two weeks, JB6 P⁺ and MCF-10A soft agar plates were fixed in 70% ethanol (EtOH) and stained with 150 µL of 0.01% crystal violet. Colonies were visually counted and used to calculate the percent of colony formation from the number of cells plated ([Colonies counted × 100]/number of cells plated).

PE HH17 explants and HH24 embryonic epicardial primary cultures were treated for 24 h with Bmp2, Bmp4, Fgf2, Fgf8 and thymosin beta 4 (Prospec, East Brunswick, NJ, USA), respectively, as reported by Hinkel et al.²⁹ . Tissue explants were collected and processed according for qPCR and/or immunohistochemistry. Each experimental condition was carried out in isolated tissues from at least 30 embryos. In all cases, 3–5 independent biological replicates were analyzed.