title: Stimulation of neurite outgrowth in a human nerve scaffold designed for peripheral nerve reconstruction.
abstract: The clinical outcome of microsurgical reconstruction of large peripheral nerve lesions depends on the availability of suitable graft material. Allogenic nerve grafts are rejected by the immune system. Extracellular-matrix proteins, in contrast to the resident cells, are of low immunogenicity in allografts. Here, human tibial nerve segments were extracted with lysophosphatidyl choline. The obtained cell-free and myelin-free scaffold consisted of empty endoneural tubes with maintained extracellular matrix architecture. The nerve scaffold had mechanical properties comparable to intact nerve, making it suitable for microsurgical reconstruction. Sections of the nerve scaffold were tested as a substrate for the adhesion and neuronal differentiation of human neuroblastoma-derived LAN-5 cells. Nerve extraction removed laminin-2, an isoform of laminin important for peripheral nerve regeneration. Laminin-2 reloading of the nerve scaffold did not improve cell adhesion and axon growth. Chemical crosslinking of heparan sulfate, on the other hand, increased the percentage of adherent cells with outgrowing neurites from 48 to 85%. Combined laminin-2 reloading and heparan sulfate crosslinking reduced the percentage of neurite-forming cells to 22% of the number of adherent cells. Implantation of the nerve scaffold into the peritoneal cavity of mice was not cytotoxic, and neovascularization of the graft material was observed within weeks. In conclusion, extraction of human nerve with detergents revealed a biocompatible nerve scaffold supporting neuronal cell adhesion. Heparan sulfate crosslinking to the scaffold surface improved neurite outgrowth, presumably mediated by midkine, a member of the neurokine family of growth factors, which is secreted by neuroblastoma-derived cells and binds to heparan sulfate.
authors.affiliation: Research Laboratory for Calcium Metabolism, Department of Orthopedic Surgery, University of Zürich, Klinik Balgrist, Forchstrasse 340, 8008 Zürich, Switzerland. cdumont@balgrist.unizh.ch
authors.forename: Charles E
authors.initials: CE
authors.lastname: Dumont
authors.forename: Walter
authors.initials: W
authors.lastname: Born
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism; D001672:Biocompatible Materials / Q000737:chemistry*; D001681:Biological Assay; D002448:Cell Adhesion; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002474:Cell-Free System; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D007107:Immune System; D007797:Laminin / Q000737:chemistry / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000737:chemistry; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D009416:Nerve Regeneration; D016501:Neurites; D009474:Neurons / Q000378:metabolism*; D010525:Peripheral Nerves / Q000473:pathology*; D011107:Polylysine / Q000737:chemistry; D013314:Stress, Mechanical; D013718:Tensile Strength; D013977:Tibia / Q000294:innervation / Q000473:pathology; D013997:Time Factors
chemical_list: D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D006497:Heparan Sulfate; D007797:Laminin; D008244:Lysophosphatidylcholines; D011107:Polylysine