NT-3 functionalized multiscale bilayer scaffold for peripheral nerve regeneration with radial axonal branching

Damage to the peripheral nervous system significantly impacts both motor function and sensory perception. Artificial regenerative grafts promote neuronal regeneration after peripheral nerve injury (PNI), but they are limited by various factors. The primary goal of these grafts is to guide the nerve toward its distal target muscle. Nonetheless, this approach does not adequately address the regeneration of axonal branches from the primary nerve to adjacent muscle regions affected by the damage to the principal peripheral nerve. This study aims to develop a polycaprolactone-collagen peptide-based multiscale bilayer scaffold functionalized with a concentration gradient of NT-3 growth factor, facilitating the bidirectional regeneration (axial and radial) of fascicular axons to the distal ends as well as the nearby affected region to restore both motor and sensory functions. The bilayer scaffold consists of an electrospun nanofibrous layer and a 3D-printed porous layer featuring spatially distributed concentration gradients of neurotrophic factors. The nanofibrous layer mimics the extracellular matrix, enhancing neuronal cell adhesion, migration, and proliferation in vitro. Radial axonal branching influenced by NT3 was demonstrated in vitro through immunofluorescence and scanning electron microscopy techniques. The bilayer scaffold was wrapped around a crushed rat sciatic nerve to assess its efficacy. Functional and behavioral results indicated that NT-3 scaffolds promote bidirectional peripheral nerve regeneration, with the potential for radial axonal branching. The morphometric analysis and quantification of radial axonal branching rely solely on more controlled in vitro studies because of nonavailability of advanced preclinical neuroimaging modalities for evaluating radial axonal branching in peripheral nerves in vivo.