Runx2/Cbfa1-genetically engineered skeletal myoblasts mineralize collagen scaffolds in vitro

Genetic engineering of progenitor and stem cells is an attractive approach to address cell sourcing limitations associated with tissue engineering applications. Bone tissue engineering represents a promising strategy to repair large bone defects, but has been limited in part by the availability of a sustained, mineralizing cell source. This study examined the in vitro mineralization potential of primary skeletal myoblasts genetically engineered to overexpress Runx2/Cbfa1, an osteoblastic transcriptional regulator essential to bone formation. These cells were viable at the periphery of 3D fibrous collagen scaffolds for 6 weeks of static culture. Exogenous Runx2 expression induced osteogenic differentiation and repressed myogenesis in these constructs relative to controls. Runx2-modified cells deposited significant amounts of mineralized matrix and hydroxyapatite, as determined by microcomputed tomography, histological analysis, and Fourier transform infrared spectroscopy, whereas scaffolds seeded with control cells exhibited no mineralized regions. Although mineralization by Runx2-engineered cells was confined to the periphery of the construct, colocalizing with cell viability, it was sufficient to increase the compressive modulus of constructs 30-fold relative to controls. This work demonstrates that Runx2 overexpression in skeletal myoblasts may address current obstacles of bone tissue engineering by providing a potent cell source for in vitro mineralization and construct maturation. Additionally, the use of genetic engineering methods to express downstream control factors and transcriptional regulators, in contrast to soluble signaling molecules, represents a robust strategy to enhance cellular activities for tissue engineering applications.