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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., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Previous studies have shown that aggregated alpha-synuclein (α-s) protein, a key pathological marker of Parkinson's disease (PD), can propagate between cells, thus participating in disease progression. This prion-like propagation has been widely studied using in vivo and in vitro models, including rodent and human cell cultures. In this study, our focus was on temporal assessment of functional changes during α-s aggregation and propagation in human induced pluripotent stem cell (hiPSC)-derived neuronal cultures and in engineered networks. Here, we report an engineered circular tripartite human neuronal network model in a microfluidic chip integrated with microelectrode arrays (MEAs) as a platform to study functional markers during α-s aggregation and propagation. We observed progressive aggregation of α-s in conventional neuronal cultures and in the exposed (proximal) compartments of circular tripartite networks following exposure to preformed α-s fibrils (PFF). Furthermore, aggregated forms propagated to distal compartments of the circular tripartite networks through axonal transport. We observed impacts of α-s aggregation on both the structure and function of neuronal cells, such as in presynaptic proteins, mitochondrial motility, calcium oscillations and neuronal activity. The model enabled an assessment of the early, middle, and late phases of α-s aggregation and its propagation during a 13-day follow-up period. While our temporal analysis suggested a complex interplay of structural and functional changes during the in vitro propagation of α-s aggregates, further investigation is required to elucidate the underlying mechanisms. Taken together, this study demonstrates the technical potential of our introduced model for conducting in-depth analyses for revealing such mechanisms., (© 2024. The Author(s).)