The Enteric Nervous System is Essential for Functional Muscle Layer Formation in Small Bowel Bioengineering

Tissue engineering is emerging as a promising treatment for patients with Short Bowel Syndrome (SBS). However, an important challenge is to reach a functional muscle in vitro, which would be essential to ensure intestinal motility.In this study, we investigated the co-culture of rat Smooth Muscle Cells (SMCs) and Enteric Nervous System (ENS) cells in layered 3D collagen-based scaffolds. Different culture media and interaction modalities—SMCs alone, paracrine signaling, and direct contact—were tested.Our results showed that ENS cells play a critical role in guiding the growth and alignment of muscle fibers. Electron microscopy revealed muscle structures resembling native intestinal smooth muscle. We observed spontaneous contractile activity and identified synaptic connections between ENS cells and SMCs, confirming functional integration.These findings demonstrate that functional smooth muscle fibers can be generated in vitro through ENS-SMC co-culture in 3D scaffolds. The ENS is essential for forming a biologically active muscle layer, as it promotes plexus-like network formation and enables peristaltic-like activity. This approach lays the foundation for engineering a functional intestinal muscle layer that mimics native tissue both structurally and functionally and opens a path for constructing a bioengineered intestinal muscle layer in the future.