C. Audisio

Chapter 11: Tissue engineering of peripheral nerves.

Tissue engineering of peripheral nerves has seen an increasing interest over the last years and, similarly to many other fields of regenerative medicine, great expectations have risen within the general public to its potential clinical application in the treatment of damaged nerves. However, in spite of the scientific advancements, applications to the patients is still very limited and it appears that to optimize the strategy for the tissue engineering of the peripheral nerves in the clinical view, researchers have to strive for a new level of innovation which will bring together (in a multitranslational approach) the main pillars of tissue engineering: namely (1) microsurgery, (2) cell and tissue transplantation, (3) material science, and (4) gene transfer. This review paper provides an overview of these four key approaches to peripheral nerve tissue engineering. While some of these issues will also be specifically addressed in other papers in this special issue on peripheral nerve regeneration of the International Review of Neurobiology, in this paper we will focus on an example of successful translational research in tissue engineering, namely nerve reconstruction by muscle-vein-combined nerve scaffolds.

Employment of the mouse median nerve model for the experimental assessment of peripheral nerve regeneration.

The experimental investigation of nerve regeneration after microsurgical repair is usually carried out in rats, rather than mice, because of the larger sized peripheral nerves. Today however, the availability of genetically modified mice makes the use of this laboratory animal very intriguing for investigating nerve regeneration at a molecular level. In this study we aimed to provide a standardization of the experimental model based on microsurgical direct repair, by 12/0 suture, of the left median nerve in adult male mice. Postoperative recovery was regularly assessed by the grasping test. At day-75 postoperative, regenerated median nerve fibers were analyzed by design-based quantitative morphology and electron microscopy. Yet, sections were immuno-labelled using two axonal antibodies commonly employed for rat nerve fibers. Results indicated that functional recovery begun at day-15 and progressively increased reaching values not significantly different from normal by day-50. Quantitative morphology showed that, at day-75, the number of regenerated nerve fibers was not significantly different in comparison to controls. In contrast, differences were detected in fiber density, mean axon and fiber diameter and myelin thickness which were all significantly lower than controls. Immunohistochemistry showed that axonal markers commonly used for rat nerves studies are effective also for mouse nerves. Similar to the rat, the mouse median nerve model is superior to sciatic nerve model for the minimal impact on animal well-being and the effectiveness of the grasping test for motor function evaluation. The main limitation is the small nerve size which requires advanced microsurgical skills for performing 12/0 epineurial suturing.

Poly(ester urethane) Guides for Peripheral Nerve Regeneration.

A biocompatible and elastomeric PU was synthesized from low-molecular-weight PCL as macrodiol, CMD as chain extender and HDI as chain linker for applications in the field of peripheral nerve repair. PU cast films supported in vitro attachment and proliferation of NOBEC. The in vitro adhesion and proliferation of S5Y5 neuroblastoma cells on the inner surface of uncoated, gelatin- and PL-coated PU guides were compared. Due to their superior in vitro performance, PL-coated PU guides were tested in vivo for the repair of 1.8 cm-long defects in rat sciatic nerves. The progressive regeneration was confirmed by EMG and histological analysis showing the presence of regenerating fibers in the distal stumps.

Morphological and biomolecular characterization of the neonatal olfactory bulb ensheathing cell line

Cell transplantation therapy has raised a great interest in the perspective of its employment for nerve tissue repair. Among the various cell populations proposed, olfactory ensheathing glial cells have raised great interest over recent years, especially in the perspective of their employment for neural repair because of their homing capacity in both central and peripheral nervous system. This paper is aimed to provide an in vitro characterization of the NOBEC (neonatal olfactory bulb ensheathing cell) line that was obtained from primary cells dissociated from rat neonatal olfactory bulb (OB) and immortalized by retroviral transduction of SV40 large T antigen. Light and electron microscopy investigation showed that NOBECs are a homogeneous cell population both at structural and ultrastructural level. RT-PCR, Western blotting and immunocytochemistry showed that NOBECs express the glial markers S100, GFAP (Glial Fibrillar Acid Protein) and p75NGFR as well as NRG1 (neuregulin-1) and ErbB1-2-3 receptors; while they are negative for ErbB4. Yet, NOBECs exhibit a high proliferation and migration basal activity and can be transducted with vectors carrying GFP (green fluorescent protein) and NRG1 cDNA. Functional stimulation by means of NRG1-III-beta3 overexpression through viral transduction induced a significant increase in cell proliferation rate while it had no effect on cell migration. Altogether, these results show that NOBEC cell line retain glial features both morphologically and functionally, responding to the NRG1/ErbB-mediated gliotrophic stimulus, and represents thus a good tool for in vitro assays of glial cell manipulation and for in vivo experimental studies of glial cell transplantation in the central and peripheral nervous system.

ErbB receptors modulation in different types of peripheral nerve regeneration

ErbBs are a family of receptors involved in the trophic maintenance of Schwann cells. Little is known about their expression changes during peripheral nerve regeneration. The aim of this study was thus to investigate variations in ErbBs after end-to-end and end-to-side nerve regeneration in the rat median nerve model. Expression of ErbBs was assessed at 7, 14, and 28 days postoperatively by real-time PCR. Results showed that expression of ErbB1 and ErbB4 mRNAs was downregulated, whereas ErbB3 mRNA was upregulated. No significant changes in ErbB2 mRNA were detected. Our results suggest that ErbBs changes are involved in the molecular response to peripheral nerve injuries.

Specificity of the second messenger pathways involved in basic fibroblast growth factor‐induced survival and neurite growth in chick ciliary ganglion neurons

Basic fibroblast growth factor (bFGF) exerts multiple neurotrophic actions on cultured neurons from the ciliary ganglion of chick embryo, among them promotion of neuronal survival and of neurite outgrowth. To understand the specificity of the signal transduction cascades involved in the control of these processes, we used pharmacological inhibitors of the three main effectors known to act downstream of the bFGF receptor (FGFR): phospholipase Cγ (PLCγ), mitogen‐activated protein kinase (MAPK), and phosphatidylinositol 3‐kinase (PI3‐K). Neuronal survival was assessed at 24 and 48 hr; neurite growth was analyzed both on dissociated neurons and on explants of whole ganglia. Our data show that only the PI3‐K pathway is involved in the survival‐promoting effect of bFGF; on the other hand, all three effectors converge on the enhancement of neurite outgrowth, both on isolated neurons and in whole ganglia.

Neuregulin1 administration increases axonal elongation in dissociated primary sensory neuron cultures

Neuregulin1 is a family of growth and differentiation factors involved in various functions of both peripheral and central nervous system including the regenerative processes that underlie regeneration of damaged peripheral nerves. In the present study we tested in vitro the effect of Neuregulin1 administration on dissociated rat dorsal root ganglion (DRG). Activity of neuregulin1 was compared to the activity of nerve growth factor in the same in vitro experimental model. Results showed that neurite outgrowth is enhanced by the addition of both neuregulin1 and nerve growth factor to the culture medium. While neuregulin1 was responsible for the growth of longer neurites, DRG neurons incubated with nerve growth factor showed shorter and more branched axons. Using enzyme-linked immunosorbent assay we also showed that the release of nerve growth factor, but not of brain derived neurotrophic factor is improved in DRG neuron treated with neuregulin1. On the other hand, the assay with growth factor blocking antibody, showed that effects exerted by neuregulin1 on neurite outgrowth is only partially due to the release of nerve growth factor. Taken together the results of this study provide a better understanding on the role of neuregulin1 in sensory neurons.