Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds.

Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.