Fig. 5

Osteogenic differentiation of HUVEC spheroids after OCT4-30Kc19 and BMP4 treatment. (A) Schematics of the HUVEC spheroids generation procedures. After incubation with MNPs mixed medium, HUVEC were detached and then magnetized HUVECs were sorted by using static magnets. Then, MNP-incorporated HUVECs were seeded into the magnetic pin array platform to generate 3D HUVEC spheroids. (B) Quantitative polymerase chain reaction (qPCR) for osteogenic marker genes, COL I, OPN, and OCN. HUVEC spheroids were treated with OCT4-30Kc19 and BMP4 for 2 weeks in osteogenesis medium. PCR analysis showed elevation in osteogenic markers when HUVECs were treated with OCT4-30Kc19 and BMP4 (n = 3). ^*p < 0.05, ^***p < 0.001. (C) Morphology of the 3D cell spheroids after 2 weeks of osteogenic induction. The number of initial cell seeding was 100,000 cells per well of the magnetic pin-array platform, yielding HUVEC spheroids of around 1,000 μm in diameter. The size of the spheroids was remaining the same without a loss for 2 weeks of osteogenic induction. Scale bar, 200 μm. (D) Alizarin red s staining (ARS) showing calcium deposition in HUVEC spheroids. The sample treated both with OCT4-30Kc19 and BMP4 showed the highest calcium deposition