Optimal micro-patterning of a collagen scaffold coordinates the induction of morphogenetic pathways in adult nerve regeneration

Introduction Nerve injury is a frequent event especially after traumatic lesion, affecting mainly young people. Various therapeutic approaches have been proposed for patients suffering from peripheral nerve injuries. Despite the significant increase in the understanding of the pathophysiology of nerve, results have been, so far, inconsistent, in terms of both quality as well as extent of nerve regeneration and re-innervation. The use of a conduit to reconnect the proximal and distal stumps of a transected nerve and induce peripheral nervous system (PNS) regeneration has been the subject of a large number of investigations. While it is quite established that the presence of a conduit between the transected stumps allows PNS regeneration [1], the micro-structural, mechanical and compositional features of the conduit, as any material inserted within, might significantly affect the quality of regeneration [2, 3]. We have previously developed a micro-patterned collagen scaffold (MPCS), by a spinning technique [4], with a peculiar radially aligned porosity of the tube wall, and predicted that its microstructure might play a significant role in the regulation of cellular and molecular mechanisms sustaining cell behaviour inside the scaffold and, in turn, improving distal induced regeneration. In this work the clinico-pathological impact of this MPCS was evaluated in the adult rat sciatic nerve. Neurophysiological, morphological and whole genome expression studies were conducted to assess the major pathways regulating nerve development and morphogenesis. Materials & Methods A complete and detailed description of the process adopted to produce MPCS has been reported [4]. Scaffolds are prepared using a collagen-based slurry; spinning of the slurry in a proper apparatus and rapid freezing of the sedimented suspension in liquid nitrogen; freeze-drying of the solidified suspension to obtain the final MPCS. Nerve regeneration in MPCS-implanted transected rats was evaluated in vivo over a 10-mm critical size defect in the adult rat sciatic nerve. Rats with transection of the sciatic nerve and implanted with either commercial collagen (NeuraGen®) or silicon conduits were used as controls. To evaluate the degree of regeneration and re-myelination after nerve transection, neurophysiological and morphological studies were performed. Together with sciatic nerves, the tibial plantar nerves at the paw were also investigated, to establish the process of re-innervation at the distal part. Whole gene expression (GE) profiling studies on injured sciatic nerve rats were conducted to investigate the molecular changes and the regulation of biological processes provided by our MPCS. Results & Discussion A damage, which determines a detachment of two nerve stumps, like a traumatic lesion, needs a device that creates an ideal micro-environment in order to facilitate axonal regeneration and re-myelination. Thus, we have developed a MPCS under the hypothesis that the micro-structure of the wall conduit could create a favourable micro-environment for full nerve regeneration after experimental transection. Neurophysiological and morphological studies suggested a faster functional nerve recovery of MPCS-implanted rats compared to rats treated with other conduits, as well as a prompt restoration of a functional vasculature with a physiological blood-nerve barrier. Moreover, in the MPCS, the long-term analysis showed a normal development of nerve, consisting of normal axon diameters and myelin thickness. Up-regulation of the myelin specific-gene observed at early time only on MPCS suggested the ability of our scaffold in providing clues for Schwann cell differentiation. Whole genome gene expression analyses further confirmed that the MPCS induces selective gene expression patterns and enhanced cells proliferation, motility and myelination. Conclusion Our findings provide remarkable molecular and ultra-structural evidence that the optimal micro-patterning of the proposed collagen scaffold plays a key role in turning the inner micro-environment into hospitable for development, rather than induced regeneration, of the proximal nerve stump. Within these optimal conditions, the transected nerve recapitulates in a coordinated fashion its innate programs of differentiation, which ultimately lead to progressive substitution of non-neuronal with neuronal tissues. We hypothesize that the radially aligned porosity of the MPCS promotes nerve regeneration by inducing morphogenetic stimuli and orchestrating cell behaviour towards a physiological regeneration mode.