The Application Of A Novel Tissue Engineered Nerve Graft Built With Adipse Tissue-derived Stem Cells And Cell Derived Extracellular Matrix In Peripheral Nerve Regeneration

The commercial nerve graft is difficult to obtain satisfactory effect for repairing crucial nerve defect in clinical. A large number of studies have confirmed that the extracellular matrix (ECM) is of great significance to regulate cell behavior, both for stem cells or nerve regeneration, and can promote their migration, proliferation and differentiation. So, ECM microenvironment may also have good effect for peripheral nerve regeneration. Adipose-derived stem cells (ADSCs) can easily get from clinical liposuction and is widely used in tissue engineering and nerve regeneration research. However, most researches focus more on its ability of differentiation and paracrine. So fa, there are little researches on the effect and application of ADSCs-derived ECMObjective:This research involve in how to use cell-derived extracellular matrix to construct a new type of tissue engineered nerve graft and evaluate its effect in the repair of peripheral nerve defect.Method:1.The primary ADSCs were cultured by the method of enzyme digestion, and then tested the capacity of cell proliferation and Schwann cell differentiation through CCK8 and flow cytometry; 2. The concentration of neurotrophic factor in the conditioned medium were measured through ELISA. The Schwann cell proliferation was estimated by EdU method. The neurite outgrowth length of dorsal root ganglion neurons was measured through immunofluorescence; 3. The ADSCs-derived extracellular matrix was successfully prepared and tested the physicochemical characteristics through light microscope and electron microscope; 4. ADSCs-derived extracellular matrix modified chitosan tubes which are compound with ADSCs were used to construct a novel tissue engineered nerve graft and evaluate its effect on peripheral nerve defect repair through the gait analysis, electrophysiology, and histology and target organs evaluation.5. According to above method, stromal vascular fraction (SVF) was compared with ADSCs in peripheral nerve defect repair.Results:1. The SCs-like cells which differentiated from ADSCs were positive for immunofluorescent staining of S100, GFAP. and p75.2. After had indirect co-cultured with ADSCs. the EdU positive cells in co-culture group was the same as it in conditioned medium group and significantly higher than it in growth medium(.P<0.01); when DRG neurons direct or indirect co-cultured with ADSCs, its neurite was longest in direct co-cultured group (P<0.01).3. ADSCs-derived ECM was fabricated and observed that there was a large number of fibril matrix which interacted with each other, and there were a large number of micro-pores. Type I collagen, Type IV collagen, laminin, fibronectin were positive proved. Rat dorsal root ganglia and Schwann cells were growing well on the extracellular matrix and maintain normal cell phenotype.4. The novel tissue engineered nerve graft show significant higher sciatic function index (SFI), compounded motor active potentials (CMAPs), diameter of myelinated fibers, thickness of myelin sheath, and area of muscle fiber of gastrocnemius compared with ECM group and ADSCs group and close to the effect of autologous graft.5. Compared with ADSCs, SVF also can enhance the diameter of myelinated fibers, thickness of myelin sheath, and the recovery rate of muscle. It has similar results in functional assessment and histological evaluation between the two groups.Conclusion:1. ADSCs can differentiate to SCs-like cells.2. The paracrine of ADSCs promoted the proliferation of SCs and neurite outgrowth of DRG neurons and the cells-axon interaction may also play important role in neurite growth.3. ADSCs-derived ECM promoted the proliferation of SCs and neurite outgrowth of DRG which grew along the ECM orientation.4. A novel tissue engineered nerve graft combined with ADSCs-derived ECM and ADSCs promoted the function recovery of SD rats which was close to the effect of autologous graft.5. SVF can promote sciatic nerve defect regeneration which was similar to ADSCs. SVF can substitute ADSCs as a seed cells in the peripheral nerve regeneration.