Co-culturing Human Adipose Derived Stem Cells And Schwann Cells On Spider Silk - A New Approach In Nerve Regeneration After Peripheral Nerve Injury
Annika Resch, MD, Alexandra-Larissa Stetco, MSc, Tamara Weiss, PhD, Markus Kerbl, MD, Paul Liebmann, MD, Christine Radtke, MD, FEBOPRAS, MBA.
Medical University of Vienna, Vienna, Austria.
PURPOSE: Innovative options for nerve reconstruction after peripheral nerve injury are of great interest in plastic and reconstructive surgery. Treatment of nerve defect injuries by autologous nerve transplantation represents the gold standard when a tension free end-to-end-coaptation is not achievable. However, with regard to donor site morbidity, nerve availability is limited. Recently, many studies focused on finding valid alternatives. Nerve conduits made of biodegradable materials were developed to guide and redirect proximal nerve growth. These conduits can be seeded with cells to improve recovery. Schwann cells represent the key to nerve regeneration by producing extracellular matrix molecules, integrins and trophic factors. Since clinical use of isolated Swann cells is limited due to donor site morbidity and slow growth in vitro, adipose derived stem cells (ADSCs) have been identified as viable alternative. Compared with other stem cells, ADSCs can be harvested by less invasive procedures (e.g. liposuction) and cultured with a greater proliferation rate. As shown in many studies ADSCs provide the potential to differentiate into several functional cell types (e.g. adipocyte, osteoblast, chondrocyte and neural phenotypes) and are therefore of high interest for research purposes. Additionally, ADSCs secret multiple growth factors and cytokines, which might further support and enhance the regeneration process of injured nerve axons. The use of spider silk could provide an additional guidance tool to improve regeneration after peripheral nerve injury. With its biocompatibility, it doesn't need any modifications to its applications. Studies using stem cells isolated from rats seeded on spider silk showed good results concerning proliferation and regeneration rates. METHODS: Native spider silk harvested directly from Nephilia edulis was woven on a steel frame and sterilized by autoclaving. Human ADSCs were isolated from the lipoaspirat of healthy patients undergoing liposuction. Cells were characterized by immunostaining with monoclonal mouse and rabbit antibodies against CD90, CD44, CD34, CD45 as well as stro-1. Immunofluorescence showed positivity for CD90 and CD44, cells were negative for CD34, CD45 and stro-1. The human Schwann cells were isolated from the ischiadic nerve of an organ donor. After immunocytochemical staining cells were positive for anti-S100 in the immunofluorescence. After isolation and characterization 0,5 x 10^6 (50% ADSCs (passage 2), 50% Schwann cells (passage 1)) were seeded on spider silk. RESULTS: In our experiment, human Schwann cells and human ADSCs were seeded in co-culture on spider silk, in order to combine the benefits of the silk and the ADSCs regarding improved proliferations and differentiation. Results so far showed that cells started to attached on the silk and aligned along the silk fibers. Proliferation could be observed starting in the corners where the fibers cross each other slowly stretching out over the mesh. CONCLUSION: Silk as matrix for cell adhesion is of great interest for research on nerve regeneration. By seeding Schwann cells and ADSCs on the silk fibres regeneration and guidance of the healing nerve may be improved. Further experiments, control trials and analyses by characterization via immunofluorescence staining and ELISA for growth factor production are planned to prove significance of our findings.
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