FIG. 4.

MSCs maintain the potential to become osteogenic after extraction from 3D fibrin gels. Micrographs represent MSCs grown on 2D TCPS (A, C) or in 3D fibrin gels (B, D) for 7 days of preculture and then extracted and maintained in growth media (A, B) or differentiated in osteogenic media (C, D) for 21 additional days in 2D culture. Cells were stained using the von Kossa method. Scale bar represents 200 μm. (E, F, G) MSCs were grown for periods of 1 (E), 7 (F), or 14 days (G) in either 2D or 3D environments, extracted using trypsin (2D) or nattokinase (3D) and subsequently replated in 2D cultures. These cultures were then subjected to either growth media or osteogenic induction media for up to 21 days. Total calcium levels were then quantified as an indication of osteogenic differentiation as described in the “Materials and Methods” section. These data show that the presence of soluble osteogenic supplements and prolonged culture times in these supplements generally enhance osteogenic differentiation of the MSCs, as expected. They also show that MSCs retrieved from 3D fibrin gels using nattokinase have no apparent deficits in osteogenesis. ***p≤0.001 for statistical significance. Color images available online at www.liebertpub.com/tec