Abstract
Neurons form predefined connections and innervate target tissues through elongating axons, which are crucial for the development, maturation, and function of these tissues. However, innervation is often overlooked in tissue engineering (TE) applications. Here, multimaterial 3D bioprinting is used to develop a novel 3D axonal guidance structure in vitro. The approach uses the stiffness difference of acellular hyaluronic acid-based bioink printed as two alternating, parallel-aligned filaments. The structure has soft passages incorporated with guidance cues for axonal elongation while the stiff bioink acts as a structural support and contact guidance. The mechanical properties and viscosity differences of the bioinks are confirmed. Additionally, human pluripotent stem cell (hPSC) -derived neurons form a 3D neuronal network in the softer bioink supplemented with guidance cues whereas the stiffer restricts the network formation. Successful 3D multimaterial bioprinting of the axonal structure enables complete innervation by peripheral neurons via soft passages within 14 days of culture. This model provides a novel, stable, and long-term platform for studies of 3D innervation and axonal dynamics in health and disease.