Influenza della topografia delle fibre di gelatina sulla selezione delle sottopopolazioni neuronali

Several biomaterials, both of natural and synthetic origin, are used in nerve tissue engineering and repair to bridge the gap of severed nerves. The aim of this study was to understand the influence of gelatin electrospun nano or microfiber topography, in combination with different growth factors, on axon growth, cell migration and selection of different neuron subpopulations. Gelatin was chosen because of its biocompatibility, mechanical and degradation properties. Electrospun fibres were chosen because they mimic the natural extracellular matrix (ECM) and can provide a substrate for cellular adhesion and activation of signalling pathway. It has been recently shown that biomaterial topographical characteristics (i.e. groves, crinkles, cavities, pillars) can significantly affect cell adhesion, proliferation, migration and axonal outgrowth. In order to understand if the different fibre topography, alone or in combination with different growth factors, may affect axon growth, cell migration and neuronal selection, dissociated neonatal rat dorsal root ganglion (DRG) cultures were used as experimental model. DRG were cultured on electrospun gelatine nanofibres (300 nm diameter size) or microfibers (1300 nm diameter size), with or without different growth factors (NGF, VEGF, GDNF, BDNF and NT3). To quantify the number of neurites, the area occupied by neurites, the number of Schwann cells and the length of neuritis, dissociated DRG neurons were drop-cultured in gel (with or without growth factors) on different gelatin fibres and analysed at different time points after seeding, by immunocytochemistry for Schwann cells and neurons. Data show that fibre topography affects axon growth, but not Schwann cell migration. There are different types of neurons within DRG that are specialized in different perceptual modalities because they express exclusive types of ion channels and respond to distinctive sets of stimuli. These neurons are classified into major subtypes, including i) proprioceptors that sense body position, ii) low threshold mechanoreceptors that sense touch, pressure and vibration, iii) thermoceptors that respond to cold or warm temperatures, iv) nociceptors that respond to pain-inducing stimuli, and v) pruriceptors that respond to itch-inducing compounds. Proprioceptors are characterized by the expression of TrkC (neurotrophin 3/NT-3 receptor), mechanoreceptors by the expression of TrkB (brain-derived growth factor/BDNF receptor) or Ret (glial-derived growth factor/GDNF receptor). Nociceptors, termoceptors, pruriceptors, and C-fiber mechanoreceptors are characterized by the expression of TrkA (nerve-derived growth factor/NGF receptor). In order to understand if the different fibre topography, alone or in combination with different growth factors, may result in neuronal selection, dissociated DRG neurons seeded on nanofibres, microfibers and control condition, with or without growth factors, were analysed by quantitative real time PCR analysis for the expression of markers specific of the different neuron subpopulations. Intriguingly, these data show that the combined effect of different fibre topography and growth factors strongly affect the expression of some specific markers of the different neuron subpopulation, suggesting that fibre topography may result in neuron selection.