| description abstract | Abstract. Nonanimal models (NAMs) provide an important platform for studying musculoskeletal tissue formation under controlled conditions while reducing reliance on vertebrate animal models. In this study, we advanced a simple, scaffold-free three-dimensional (3D) NAM system to guide the self-assembly of murine C3H/10T1/2 mesenchymal stem cells (MSCs) and C2C12 myoblast progenitor cells into neotendon and neomuscle structures. Custom 3D-printed molds and biologically inert agarose were used to form nonadherent wells that promoted high cell density and directed cell–cell adhesion without exogenous extracellular matrix (ECM) or biomaterial scaffolds. Transforming growth factor (TGF)β2 treatment enhanced actin cytoskeleton alignment in neotendons, with initial collagen fibril formation observed by day 7. C2C12 myoblasts exhibited progressive actin alignment, myotube formation, and desmin production by day 14. A custom bioreactor was used to apply cyclic tensile loading to the neotendons early in their development. Co-cultures of C3H/10T1/2 MSCs and C2C12 myoblasts formed cohesive structures, with aligned cytoskeletal organization and desmin distribution throughout, suggesting potential interactions at the developing myotendinous junction. This scaffold-free NAM system enables the evaluation of key biochemical and mechanical cues that regulate early musculoskeletal tissue formation in vitro. By recapitulating features of the embryonic environment, this approach refines current in vitro methods and establishes a simple, versatile platform to ultimately reduce the need for vertebrate animal models in developmental studies. | |
