Perfusion Cell Culture Induces Oxygen Diffusion and Osteogenic Activity in Multi-Channeled Critical-Size Silk Fibroin Scaffolds with Pre-Osteoblasts: Experimental and Finite Element Modeling Study

PURPOSE: The development of 3D-scaffolds to treat critical-size bone defects in orthopedic and reconstructive surgery remains a challenge due to low oxygen diffusion causing cell death in the core of the scaffolds. Perfusion cell culture and fluid-conducting hollow channels might be critically important to enhance oxygen diffusion. Therefore, we aimed to test whether perfusion cell culture synergistically improves oxygen diffusion and osteogenic activity in multi-channeled 3D-porous silk fibroin scaffolds with pre-osteoblasts using experiments and finite element modeling. METHODS: Pre-osteoblasts were cultured on scaffolds without or with channels (diameter: 0.5 or 1 mm) up to 21 days in perfusion or static bioreactors. The scaffolds' physicomechanical properties, oxygen diffusion, and pre-osteoblast activity were evaluated. RESULTS: Channeling did not change the scaffolds' porous structure, biodegradation rate, or compressive modulus, but decreased compressive strength. Finite element modeling showed that all scaffolds remained resilient under 2%-compressive strain, which is below their yield stress. Channeled scaffolds demonstrated homogeneous oxygen diffusion and cell distribution in static and perfusion bioreactors. Experimentally, channeling increased gene expression of Fgf2, Ki67, Runx2, and Ocn, and oxygen concentration in a static bioreactor. In a perfusion bioreactor, channeling increased cell number, collagen deposition, and matrix mineralization. CONCLUSION: Perfusion cell culture in combination with multi-channeling of 3D-porous silk fibroin scaffolds with pre-osteoblasts significantly enhanced oxygen diffusion, cell infiltration, and osteogenic activity without affecting the physicomechanical properties of the scaffolds, which may help to further improve and overcome insufficient oxygen diffusion and cell activity in the core of critical-size 3D-scaffolds for bone defects in vivo.