Mechanisms And Effects Of GDNF-modified Artificial Nerve On Sciatic Nerve Defects In Rats

Peripheral nerve defects are a common clinical condition that can result from mechanical, thermal, chemical, congenital or pathological etologies. Failure to repair these damaged nerves can result in loss of muscle function, impaired sensations, or painful neuropathies. Current surgical strategies for repair of critical nerves involve autograft transfers of normal nerves from uninjured parts of the body. However, this "gold standard" treatment is frequently limited by tissue availability, risk of disease spread, secondary deformities, and differences in tissue structure and size. One possible alternative to autologous nerve replacement is the development of engineered artificial nerve grafts consisting mainly of a scaffold and support cells.Despite advances in the field of tissue engineering, results to date with artificial nerve grafts have failed to equal nerve regeneration achieved with autologous nerve grafts. In order to enhance nerve regeneration and find an alternative to autografts, we modified Schwann cells to increase GDNF expression using a gene transfer technique. We then combined GDNF expressing Schwann cells with extracellular matrix gel and a biodegradable guidance conduit made of poly(DL-lactide-co-glycolide) (PLGA) to construct an artificial nerve complex and used this construct to bridge sciatic nerve lesions in adult Wistar rats. Our studies were focused on three key aspects of graft construction and function and the main results are indicated below.Part I: Characterization of the stability and biocompatibility of PLGA conduits.1) Artificial nerve conduits were successfully fabricated using two different copolymer ratios of PLGA(85:15 and 50:50)(lactide:glycolide).2) Degradation of PLGA(85:15) and PLGA(50:50) conduits were examined in vitro for 12 weeks in phosphate buffered saline (PBS) at 37℃, pH 7.4 and in vivo for up to 8 weeks following implantation into the rat dorsal muscle. A) PLGA conduits constructed with a 50:50 copolymer ratio exhibited greaterchanges in weight, water absorption, and morphology and more extensive degradation in vitro than conduits constructed with an 85:15 polymer ratio.B) Rates of degradation for both types of conduits were more rapid in vivo than in vitro.3) Biocompatibility of PLGA grafts were assessed by implanting PLGA into dorsal muscle, culturing Schwann cells in PLGA film and assessing integrity of conduits bridging sciatic nerve lesions. A) PLGA caused only minor nonspecific inflammatory reactions characterized by infiltration of small numbers of lymphocytes and fibroblasts at early time points. B) Schwann cells grew and proliferated well on PLGA films.C) Similar to silicone tube implanted control groups, PLGA(85:15) conduits provided a stable support for axon regeneration for up to 12 weeks, while PLGA(50:50) collapsed 4 weeks after transplantation.Conclusions: Biocompatibility and degradation rate assessments indicate that PLGA(85:15) conduits are suitable for facilitating nerve repair and are significantly more stable than conduits made with equal co-polymer ratios. Part II: Characterization, package, and transfer of recombinant retroviral vector (pLXSN-GDNF)1) The pLXSN-GDNF construct, (a gift of Ruan Huaizheng ) was characterized by DNA sequencing and compared to gene bank data to confirm expression of proper sequences. 2) PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes. Recombinant retrovirus particles were then harvested from culture media of G418 resistant transfected cells and analyzed using RT-PCR. Virus titers in supernatants were 104-105 CFU/ml. 3) Rat Schwann cells were then exposed to supernatants containing recombinant retrovirus particles. Transfected Schwann cells were identified by G418 resistance and PCR of genomic DNA. PCR and Western blot analysis showed that both GDNF RNA and protein were upregulated in transfected Schwann cells as compared to untransfected cells.Conclusions: Transfection with pLXSN-GDNF retr...