Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

Neurons are mechanosensitive cells. The role of mechanical force in the process of neurite initiation, elongation and sprouting; nerve fasciculation; and neuron maturation continues to attract considerable interest among scientists. Force is an endogenous signal that stimulates all these processes in vivo. The axon is able to sense force, generate force and, ultimately, transduce the force in a signal for growth. This opens up fascinating scenarios. How are forces generated and sensed in vivo? Which molecular mechanisms are responsible for this mechanotransduction signal? Can we exploit exogenously applied forces to mimic and control this process? How can these extremely low forces be generated in vivo in a non-invasive manner? Can these methodologies for force generation be used in regenerative therapies? This review addresses these questions, providing a general overview of current knowledge on the applications of exogenous forces to manipulate axonal outgrowth, with a special focus on forces whose magnitude is similar to those generated in vivo. We also review the principal methodologies for applying these forces, providing new inspiration and insights into the potential of this approach for future regenerative therapies.

Magnetosomes are membrane-enclosed iron oxide crystals biosynthesized by magnetotactic bacteria. As the biomineralization of bacterial magnetosomes can be genetically controlled, they have become promising nanomaterials for bionanotechnological applications. In the present paper, we explore a novel application of magnetosomes as nanotool for manipulating axonal outgrowth via stretch-growth (SG). SG refers to the process of stimulation of axonal outgrowth through the application of mechanical forces. Thanks to their superior magnetic properties, magnetosomes have been used to magnetize mouse hippocampal neurons in order to stretch axons under the application of magnetic fields. We found that magnetosomes are avidly internalized by cells. They adhere to the cell membrane, are quickly internalized, and slowly degrade after a few days from the internalization process. Our data show that bacterial magnetosomes are more efficient than synthetic iron oxide nanoparticles in stimulating axonal outgrowth via SG.

Mechanical stimulation modulates neural development and neuronal activity. In a previous study, we proposed magnetic “nano-pulling” as a tool to generate active forces. By loading neural cells with magnetic nanoparticles (MNPs), a precise force vector is remotely generated through static magnetic fields. In the present study, human neural stem cells (NSCs) were subjected to a standard differentiation protocol, in the presence or absence of nano-pulling. Under mechanical stimulation, we found an increase in the length of the neural processes which showed an enrichment in microtubules, endoplasmic reticulum, and mitochondria. A stimulation lasting up to 52 days induced a strong remodelling at the level of synapse density and a re-organization of the neuronal network, halving the time required for the maturation of neural precursors into neurons. We then injected the MNP-loaded NSCs into mouse spinal cord slices, demonstrating that nano-pulling stimulates the elongation of the NPC processes and modulates their orientation even in an ex vivo model system. To the best of our knowledge, this is the first evidence showing that active mechanical stimuli can guide the outgrowth of NSCs transplanted into the spinal cord tissue. Our findings suggest that MNPs play an important role in neuronal maturation which could be applied in regenerative medicine.

Spinal cord injury (SCI) is an extremely invalidating condition with a severe physical and psychological impact. Resolutive cures are still lacking, due to its complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCI. Standard SC organotypic cultures are maintained for up to 2 or 3 weeks in vitro . Here, we describe an optimized protocol for long-term maintenance for up to three months (90 days). The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a GFP reporter were transplanted into mouse SC-slices. 30 days after the transplant, cells still show GFP expression, and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an e x vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments. SUMMARY: In this paper, we provide a reproducible method to generate and maintain long–term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.