Search Results (3)

Axon polarization is a fundamental process in neuronal development, providing the structural basis for directional signaling in neural circuits. Precise control of axon specification is, thus, essential for the bottom-up construction of neuronal networks with defined architecture and connectivity. Although neurite length and elongation timing have both been implicated as determinants of axonal fate, their relative contributions have remained unresolved due to technical limitations in manipulating these factors independently in conventional culture systems. Here, we developed a constructive neuroengineering platform based on modifiable agarose gel microstructures that enables dynamic, in situ control of neurite outgrowth length and timing during neuronal cultivation. This approach allowed us to directly address whether axon polarization depends primarily on neurite length or the order of neurite extension. Using a single-neurite elongation paradigm, we quantitatively defined two length thresholds for axon specification: a critical length of 43.3 μm, corresponding to a 50% probability of axonal differentiation, and a definitive length of 95.4 μm, beyond which axonal fate was reliably established. In experiments involving simultaneous or sequential elongation of two neurites, we observed that neurite length—not elongation order—consistently predicted axonal identity, even when a second neurite was introduced after the first had already begun to grow. The presence of a competing neurite modestly elevated the effective critical length, suggesting inhibitory interactions that modulate length thresholds. These findings provide the first direct experimental confirmation that neurite length is the primary determinant of axon polarization and demonstrate the utility of constructive microfabrication approaches for dissecting fundamental principles of neuronal polarity. Our platform establishes a powerful experimental foundation for future efforts to achieve complete control over axon and dendrite orientation during the engineered construction of functional neuronal circuits. Full article

Approximately 185,000 people in the United states experience limb loss each year. There is a need for an intuitive neural interface that can offer high-fidelity control signals to optimize the advanced functionality of prosthetic devices. Regenerative peripheral nerve interface (RPNI) is a pioneering advancement in neuroengineering that combines surgical techniques with biocompatible materials to create an interface for individuals with limb loss. RPNIs are surgically constructed from autologous muscle grafts that are neurotized by the residual peripheral nerves of an individual with limb loss. RPNIs amplify neural signals and demonstrate long term stability. In this narrative review, the terms “Regenerative Peripheral Nerve Interface (RPNI)” and “RPNI surgery” are used interchangeably to refer to the same surgical and biological construct. This narrative review specifically focuses on RPNIs as a targeted approach to enhance prosthetic control through surgically created nerve–muscle interfaces. This area of research offers a promising solution to overcome the limitations of existing prosthetic control systems and could help improve the quality of life for people suffering from limb loss. It allows for multi-channel control and bidirectional communication, while enhancing the functionality of prosthetics through improved sensory feedback. RPNI surgery holds significant promise for improving the quality of life for individuals with limb loss by providing a more intuitive and responsive prosthetic experience. Full article

Constructing stable and flexible neuronal networks with multi-neurite wiring is essential for the in vitro modeling of brain function, connectivity, and neuroplasticity. However, most existing neuroengineering platforms rely on static microfabrication techniques, which limit the ability to dynamically control circuit architecture during cultivation. In this study, we developed a modifiable agarose gel-based platform that enables real-time microstructure fabrication using an infrared (IR) laser system under live-cell conditions. This approach allows for the stepwise construction of directional neurite paths, including sequential microchannel formation, cell chamber fabrication, and controlled neurite–neurite crossings. To support long-term neuronal health and network integrity in agarose microstructures, we incorporated direct glial co-culture into the system. A comparative analysis showed that co-culture significantly enhanced neuronal adhesion, neurite outgrowth, and survival over several weeks. The feeder layer configuration provided localized trophic support while maintaining a clear separation between glial and neuronal populations. Dynamic wiring experiments further confirmed the platform’s precision and compatibility. Neurites extended through newly fabricated channels and crossed pre-existing neurites without morphological damage, even when laser fabrication occurred after initial outgrowth. Time-lapse imaging showed a temporary growth cone stalling at crossing points, followed by successful elongation in all tested samples. Furthermore, the direct laser irradiation of extending neurites during microstructure modification did not visibly impair neurite elongation, suggesting minimal morphological damage under the applied conditions. However, potential effects on molecular signaling and electrophysiological function remain to be evaluated in future studies. Together, these findings establish a powerful, flexible system for constructive neuroengineering. The platform supports long-term culture, real-time modification, and multidirectional wiring, offering new opportunities for studying neural development, synaptic integration, and regeneration in vitro. Full article