Enhanced Osteogenic Potential of Knockout C2C12 Cells on BMP-2 Releasing Silk Scaffolds.

The CRISPR/Cas9 mechanism offers promising therapeutic approaches for bone regeneration by stimulating or suppressing critical signaling pathways. In this study, we aimed to increase the activity of BMP-2 signaling through knockout of , thereby establishing a synergistic effect on the osteogenic activity of cells in the presence of BMP-2. Since is an antagonist expressed in skeletal tissues and binds to subunits of bone morphogenetic proteins (BMPs) to inhibit osteogenic differentiation, here expression was knocked out using the CRISPR/Cas9 system. In accordance with this purpose, C2C12 (mouse myoblast) cells were transfected with CRISPR/Cas9 plasmids. Transfection was achieved with Lipofectamine and confirmed with intense fluorescent signals in microscopic images and deletion in target sequence in Sanger sequencing analysis. Thus, knockout cells were identified as a new cell source for tissue engineering studies. Then, the transfected cells were seeded on highly porous silk scaffolds bearing BMP-2-loaded silk nanoparticles (30 ng BMP-2/mg silk nanoparticle) in the size of 288 ± 62 nm. BMP-2 is released from the scaffolds in a controlled manner for up to 60 days. The knockout of by CRISPR/Cas9 was found to synergistically promote osteogenic differentiation in the presence of BMP-2 through increased and expression and mineralization. Gene editing of and BMP-2 increased almost 2-fold expression and almost 3-fold expression compared to the control group. Moreover, transfected cells produced extracellular matrix (ECM) containing collagen fibers on the scaffolds and mineral-like structures were formed on the fibers. In addition, mineralization characterized by intense Alizarin red staining was detected in transfected cells cultured in the presence of BMP-2, while the other groups did not exhibit any mineralized areas. As has been demonstrated in this study, the CRISPR/Cas9 mechanism has great potential for obtaining new cell sources to be used in tissue engineering studies.