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

Abstract

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 Noggin, thereby establishing a synergistic effect on the osteogenic activity of cells in the presence of BMP-2. Since Noggin is an antagonist expressed in skeletal tissues and binds to subunits of bone morphogenetic proteins (BMPs) to inhibit osteogenic differentiation, here Noggin 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, Noggin 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 Noggin by CRISPR/Cas9 was found to synergistically promote osteogenic differentiation in the presence of BMP-2 through increased Coll1A1 and Ocn expression and mineralization. Gene editing of Noggin and BMP-2 increased almost 2-fold Col1A1 expression and almost 3-fold Ocn 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.

Integrated 4D Analysis of Intramuscular Fat Deposition: Quantitative Proteomic and Transcriptomic Studies in Wannanhua Pig Longissimus Dorsi Muscle

“…β-oxidation is the main process of fatty acid oxidation, in which fatty acids are activated to generate fatty acyl-CoA; the activated fatty acyl-CoA enters the mitochondria and is oxidized, being the main source of biological energy for the body or tissues [ 34 ]. Studies have shown that ACADL, ACADM, and CPT2 participate in fatty acid metabolism in porcine skeletal muscle and are associated with IMF deposition and muscle development in pigs [ 35 , 36 ]. In the present study, the expression levels of ACADL, ACADM, and CPT2 in the LD240 group were significantly higher than those in the LD120 group.…”

Conditioned Medium of Intervertebral Disc Cells Inhibits Osteo-Genesis on Autologous Bone-Marrow-Derived Mesenchymal Stromal Cells and Osteoblasts

“…Non-fusion or failed fusion after surgery is a recurring concern [9], and identifying the underlying factors has been a focal point of research. One possible explanation is linked to the intricate role of bone morphogenetic protein (BMP) antagonists secreted by the IVD itself [10,11]. These antagonists have been associated with inhibiting osteogenesis in the presence of IVD cells [12,13].…”