After big loos of substances of peripheral nerves, in order to connect proximal with distal stump, it is possible to use, in alternative to autologous grafting, different kind of conduits. The chitosan conduit and the muscle in vein technique showed very good results in pre clinical and clinical settings. We compared in this study the efficacy of empty chitosan conduit versus chitosan conduit enriched with fresh muscle fibbers (MIT) to improve peripheral nerve regeneration.

The median nerve of rat was repaired by means of empty chitosan conduit or MIT (nerve gam 6mm, conduit length 10 mm). As control group we used auto grafting technique. We performed analysis at short term (7,14,28 days) and at long term (12 weeks) in order to register bimolecular modification (quantitative real time PCRand western blot), morphological modification (optic and electronic microscope) and functional changing (grasping test).

Bimolecular analysis showed that muscle fibbers produced and released Neuregulin1, needed for regeneration and activity of Schwann cells. Otherwise also the autograft product Neuregulin1, instead no production was observed in empty conduit. So muscle fibbers compensate this fact. Morphological analysis showed that the first myelinc fibbers appear in MIT after 14 days, but not in empty tube.

The results of our work are very interesting because can merge the easiness of the implantation of chitosan tube and the efficacy of fresh muscle fibbers, as previously demonstrated by muscle in vein technique.

From a clinical point of view this procedure could be an alternative to auto grafting that is nowadays the gold standard for nerve repair, but present soma disadvantages.

Aims: The combination of microsurgical techniques with engineering of pseudo-nerves has recently seen an increased employment for the repair of peripheral nerve defects. Over the last ten years, we have investigated a particular type of bio-engineered nerve guide, the muscle-vein-combined tube, which is made by filling a vein with skeletal muscle. However, the basic mechanism underlying the effectiveness of this surgical technique are still unclear and yet an experimental study on its efficacy on functional recovery compared to traditional nerve autografts is still lacking in the literature. The aim of the present study was thus to fill this gap.

Methods: In rats, 10-mm-long median nerve defects were repaired using either traditional autografts or fresh muscle-vein-combined bioengineered scaffolds. Posttraumatic nerve recovery was assessed by grasping test. The samples were collected at different times after surgery: 5, 15, 30 days and 6 months. Analysis was carried out by light and electron microscopy. In addition, reverse transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of mRNAs coding for glial markers as well as glial growth factor (NRG1) and its receptors (erbB2 and erbB3).

Results: Results showed that both types of nerve repair techniques led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Rats operated on by traditional nerve autografts performed better in the grasping test. Biomolecular analysis by RT-PCR demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3.

Conclusions: Our results confirmed that use of muscle-vein-combined tissue-engineered conduits is a good approach for bridging peripheral nerve defects in selected cases when traditional autografts are not employable and disclosed one of the basic biological mechanism that support the effectiveness of this surgical technique. Our experience also suggested that the rat forelimb experimental model is particularly appropriate for the study of microsurgical reconstruction of major mixed nerve trunks. Furthermore, since the forelimb model is less compromising for the animal, it should be preferred to the hindlimb model for many research purposes.

Aims: Biosynthetic scaffolds made of degradable bio-materials enriched with cultured cells holds promise for peripheral nerve repair after complex traumatic injuries. In the perspective of future transplantation applications, the aim of this study was to investigate how cultures of olfactory ensheathing cells (OECs), in particular neonatal olfactory ensheathing cells (NOBECs), grow up in vitro on degradable polymeric films made with polycap-rolactone matrices and multi-block polyesterurethane respectively. In addition, since several transplantation studies use green fluorescent protein (GFP) positive cells so that they can be easily located in the receiving tissues, the cDNA encoding for GFP was cloned in expression vector and transfected in NOBECs.

Methods: To characterize NOBECs we employed electron microscopy, immunohistochemistry, RT-PCR and western blotting analyses. Moreover the proliferative ratio of NOBECs and the ability of the cells to migrate in a three dimensional environment were evaluated under basal and experimental culture conditions. Finally, the GFP-positive NOBEC were seeded on two types of synthetic films and their behaviour was analyzed to determine cell adhesion, survival and proliferation.

Results: We examined the expression of glial markers and NRG1/ErbB system in the NOBEC cell line at RNA and protein level. Results showed that NOBECs express both glial markers (GFAP and S-100), ErbB receptors (ErbB1, ErbB2 and ErB3) and different isoforms of NRG1. NOBECs exhibited a remarkable proliferation activity and a high basal migration activity. GFP positive NOBECs showed no significant difference in their behaviour as compared to untransfected parental cells. Finally, both normal and GFP-NOBECs showed good cell adhesion, survival and proliferation properties when seeded on both films employed in this study.

Conclusion: Taken together, results of our study showed that the glial cell line has similar biochemical properties as primary cultures of OECs. Moreover, we showed that NOBECs survive, proliferate and migrate on two different types of synthetic films that were prepared in the perspective of build up nerve scaffolds. Therefore, our results indicated that the NOBECs produce growth-promoting proteins and possess regeneration-promoting capabilities that make them a potentially good transplant material to enhance axonal regeneration inside synthetic tubes used to bridge nerve lesion with substance loss.