Final Report Summary - MECAR (Magnetically Enhanced Controlled Axonal Regeneration) Although the exact mechanisms involved in the growth of axons is still incompletely understood, it is clear that force is a crucial factor for both axonal guidance and lengthening. This is backed by a growing body of evidence, indicating the importance of physical stimuli for neuronal growth and development. Results of published experimental studies indicate that forces, when carefully controlled, act as powerful stimulators of axonal lengthening. The current project developed, tested and evaluated a practical implementation of this concept. Specifically, it established a novel approach for the application of a controlled tensile force to neurons and axons in order to accelerate regeneration after peripheral nerve injury. Magnetic beads are used as a powerful tool to study the biological response evoked by tension. Traditional approaches for axon stretching commonly involve use of glass microneedles, which are used to attach and apply stretching force to growth cones [1]. Another approach is based on strain applied by the use of a micro-stepper motor, i.e. mechanical tension is applied on axons of neurons seeded on two adjacent membranes by pulling them away from each other [2]. The approach we proposed and used in the present study is based on the use of superparamagnetic iron oxide magnetic nanoparticles (MNP). MNP are paramagnetic, i.e. they do not exhibit -magnetic properties in the absence of an external magnetic field, but develop a mean magnetic moment when exposed to an external magnetic field can be localized to axons to stimulate axon elongation in the desired direction under the influence of an external magnetic field. The application of directionally orientated forces based on the use of MNP carries several advantages over the those obtained by traditional approaches: (i) possession of a high dynamic range, enabling controlled variation of the applied tension from 0.1 pN [3] to tens of nN [4], depending on size and number of particles and external magnetic fields; (ii) it enables performance of biological studies on a cell cultures or single cell studies; (iii) it can be used in any experimental configuration and is compatible with common cell cultures conditions; and (iv) its use can be translated to eventual in vivo use, because static magnetic fields have been in use in medical imaging and magnetic nanoparticles are used in various biomedical applications (Endorem®, Resovist®, Feraheme® are examples of MNP used on humans). We have extensively studied MNP in biological experiments, ranging from cell lines [5-7] to primary neurons [8-9] and animal models of nerve regeneration (unpublished), and have not observed any signs of cytotoxicity or inflammation at concentrations of up to 10 times higher than the working dose.The research group which I lead has demonstrated in a neuron-like cell line (PC12) that MNPs bonded to neurites can develop forces in the presence of magnets, and such forces can direct the neurite outgrowth along the direction imposed by the magnetic field (MARVENE, NanoScie+2008). The MECAR project translated this concept into a model of regeneration of peripheral nerve. Specifically, an organotypic model of a spinal cord slice co-cultured with a peripheral nerve explant was used to validate the hypothesis underpinning the project. In this model, a spinal cord slice and a sciatic nerve explant from neonatal mice (D4-5) are cultured for seven days. The sciatic nerve is placed in front of ventral roots (3 mm gap) to allow motor neurons to innervate the sciatic nerve. After few days of incubation, axons from motor neurons of the ventral horn of the spinal cord usually reach the sciatic nerve. The project used this model to prove that MNPs can selectively bind axons of spinal cord motor neurons and expedite the process of re-innervation of the sciatic nerve under the directional influence of an external magnetic field. Specifically, MNP-mediated stretching was found to stimulate both axonal sprouting and elongation.Surprisingly, although recent knowledge suggests that this “stretch growth” is perhaps the most remarkable axon growth mechanism of all, little or nothing is known about the molecular mechanisms evoked by the tension. Axonal elongation as a function of the applied tensile force has been investigated by Heidemann’s team. It has been found that neurites show a transient elongation interpreted as viscoelastic deformation, when the applied tension is less than 1 nN. In contrast, long-term extension, interpreted as growth is observed when the applied tension is above 1 nN [1]. Based on this concept, the approaches used in the past were methodologically unsound because of the low detection limit of the techniques and short temporal window for observations. Instead, we used MNP to explore the effect of sub pico-Newton forces on axonal growth induced by stretch. Surprisingly, we found that a mechanical tension of 0.15 pN (100.000 below the previously identified threshold) induced a 50% increase in the length of differentiated PC12 cells in a week. This differential increase was interpreted as stretch growth, as no neurite thinning was observed. More importantly, we observed an elongation rate of 0.2-0.3 µm/h/pN, which is the same elongation rate previously reported for both central and peripheral nervous system [1]. This finding supports the concept that there is no threshold required for stretch-induced growth, and that axonal elongation is driven by tension, irrespective of its origin, i.e. from the traction exerted by the growth cone, the mass body growth or external force application. This new knowledge serves to provide a “unified” model of axonal growth and to propose a new paradigm to repair “in loco” nerve lesions in humans.[1] Zheng J, Lamoureux P, Santiago V, Dennerll T, Buxbaum RE, Heidemann SR. Tensile regulation of axonal elongation and initiation, the Journal of Neuroscience (1991) 11:1117[2] Pfister BJ, Iwata A, Meaney DF, Smith DH. Extreme stretch growth of integrated axons. J Neurosci (2004) 24:7978-83[3] Riggio C, Calatayud M P, Giannaccini M, Sanz B, Torres T E, Fernandez-Pacheco R, Ripoli A, Ibarra M R, Dente L, Cuschieri A, Goya G F, Raffa V. The orientation of the neuronal growth process can be directed via magnetic nanoparticles under an applied magnetic field. Nanomedicine (2014) 13:S1549-9634[4] Fass JN, Odde DJ. Tensile force-dependent neurite elicitation via anti-beta 1 integrin antibody-coated magnetic beads. Biophys J (2003) 85:623-36[5] Calatayud P, Sanz B, Raffa V, Riggio C, Ibarra M R, Goya G F. Protein adsorption onto Fe3O4 nanoparticles with opposite surface charge and its impact on cell uptake. Biomaterials (2014) 35:6389-99[6] Calatayud MP, Riggio C, Raffa V, Sanz B, Torres TE, Ibarra MR, Hoskins C, Cuschieri A, Wang L., Pinkernelle J., Keilhoff G., Goya G.F. Neuronal cells loaded with PEI-coated Fe3O4 nanoparticles for magnetically guided nerve regeneration. Journal of Materials Chemistry. B (2013) 1: 3607 - 3616[7] Riggio C, Calatayud M P, Hoskins C, Pinkernelle J, Sanz B, Torres TE, Ibarra MR, Wang L, Keilhoff G, Goya GF; Raffa V, Cuschieri A. Poly-l-lysine-coated magnetic nanoparticles as intracellular actuators for neural guidance. International Journal of Nanomedicine (2012) 7:3155 [8] Riggio C, Nocentini S, Catalayud MP, Goya GF, Cuschieri A, Raffa V, del Río JA. Generation of magnetized olfactory ensheathing cells for regenerative studies in the central and peripheral nervous tissue. International Journal of Molecular Sciences (2013) 14:10852[9] Pinkernelle J, Raffa V et al. Growth factor choice is critical for successful functionalization of nanoparticles. Front Neurosci. (2015) 9:305
