Myoblast seeding in a collagen matrix evaluated in vitro

Collagens may be used as biomaterials for soft tissue reconstruction, e.g., the abdominal wall. We previously developed a biocompatible dermal sheep collagen (DSC), which in an abdominal wall reconstruction model showed controlled biodegradation and functioned as a matrix for in-growth of fibroblasts but not of muscle. It was hypothesized that regeneration of muscle via DSC may be possible by seeding of muscle cells. Using a syringe, mouse C2C12 myoblasts were seeded in DSC disks and incubated in methylcellulose-based growth medium, changed at 24 h into differentiation medium. An estimated 85% of the cells were well distributed, especially in the top half of the DSC disks. Some 15% of the cells ended up on top. At 4 h, all cells showed a spherical morphology, sometimes with clear adhesion plaques. At 24 h, cells on the top started to form a "capsule" with well-spread cells. Underneath the capsule, of the remaining 85% of the cells, approximately 30% showed adhesion and spreading on/in between collagen bundles. At day 3 after the addition of differentiation medium, the spread cells showed first indications of myotube formation. At day 7, myotube formation had proceeded, while extracellular matrix, i.e., collagen and elastin, had been deposited. This study shows that myoblast seeding into DSC is feasible, resulting in a reasonable cell distribution and survival of 45% of the cells. The surviving cells are able to differentiate into myotubes and form an extracellular matrix.