A Comparison Of The Long Peripheral Nerve Repair Capability Between Bone Marrow Stromal Cells And Adipose-Derived Stem Cells

The regeneration and function recovery of peripheral nerve is one of the hottest issues in the field of neurosurgery. In recent year, rapid developmental tissue engineering technology brings new hope to resolve peripheral nerve regeneration. The key point of tissue engineering nerve is seed cells, scaffold and neurontrophic factor. Then, how to choice seed cells and scaffold is still an unsolved question. Adipose tissue-derived stem cells and bone marrow stromal cells are two kinds of multipotential adult stem cell and can be differentiated into Schwann-like cells in vitro. Some studies have demonstrated the potential for using cultured ADSCs and BMSCs in peripheral nerve regeneration.In the present study, we first reveal that acellular allogenic nerve grafts, recombining with ADSCs and BMSCs respectively, repaired a 5cm extend peripheral nerve gap in dog and further compare ADSCs with BMSCs to peripheral nerve regeneration effect in order to select ideal seed cells for tissue engineering nerve. This study explored that acellular nerve allografts implanted with autologous ADSCs and TGF-β1 was feasible in repairing a 9 cm large-gap peripheral nerve defect. There are four parts in this experiment as follows:1. Isolation, culture and characterization of seed cellsIn order to establish a method for isolation and culture of Adipose tissue-derived stem cells and bone marrow stromal cells in vitro and explore their biological chracteristics, we excised inguinal fat pads with sterile technique, followed by digestion with I type collagenase, then cultured in DMEM with 10 % fetal bovine serum and assay its growth curve. BMSCs were isolated adopting percoll density gradient centrifugation, then cultured in DMEM with 10 % fetal bovine serum and assay its growth curve. To verify the multipotential differentiati on of mesenchymal characteristics of ADSCs and BMSCs, cells were subjected to differentiation in conditions known to induce adipogenic and osteogenic lineages and used for phenotypic analysis by flow cytometry. The results showed that differentiated ADSCs and BMSCs were stained with Oil Red O and Von kossa to detect fat and calcium deposits, the same as the cultured ADSCs and BMSCs in this study were positive for CD29, CD90 and negative for CD80 and CD45. 2. Schwann cells differentiation of ADSCs and BMSCs in vitroTo observe induced differentiation capability of ADSCs and BMSCs into schwann-like cells in vitro. ADSCs and BMSCs cells from three passages in the same animal were induced by chemical-inducing and co-culture. The undifferentiated control group was set up.The morphology, immunocytochemistry, western blot and RT-PCR were used to identify cells type. Results: Morphous of Schwann cells were observed in both inducing groups but control groups. The expressing of protein and mRNA for glail fibrillary acidic protein (GFAP) and S-100 were similar to the morphological results.3. Prepation of acellular peripheral nerve graftIn order to prepare acellular peripheral nerve graft and estimate its biocompatibility, we prepare allograft nerve by hypotension, freeze-thawed at -80℃repeatedly, NaOH spallation using fresh dog sciatic nerve, then observe characters of its structure under light and electron microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). Lastly we evaluated biocompatibility of acellular allograft nerve and found that SCs,epineurium , medullary sheath and axon of nerve fibers was eliminated but three dimensional scaffold constructed by basement membrane tube of SCs, extracellular matrix of epineurium and nerve perineurium were reserved . Biocompatibility assay suggests us acellular nerve xeno-graft cytotoxicity was first grade and SD no obvious immunologic rejection reaction can be deteced in rat subdermal implantation test.4. Tissue engineering nerve was constructed in vitro and study of peripheral nerve repairwe prepare tissue engineering nerve with TGFβ1 and ADSCs or BMSCs combined in collagen gel and then inject acellular nerve scaffold. Grafting length were divided into 5 cm and 9cm groups.5 cm groups including , A group: recombinant TGFβ1 and ADSCs nerve graft; B group: recombinant TGFβ1 and BMSCs nerve graft; C group: recombinant nerve graft without cell; D group: 5 cm autologous nerve graft. 9 cm groups including, A group: recombinant TGFβ1 and ADSCs nerve graft; B group: recombinant nerve graft without cell; C group: 9 cm autologous nerve graft. We use electron microscope, immunohistochemistry, image analysis, electrophysiology, FluoroGold retrograde tracing to evaluate nerve regeneration. Our results suggest that 5cm nerve repair effect in A group, B group and autologous nerve graft are similar and better than C groups; A group and B group are not significant deviation; C group is the worst. In 9cm group, A group is similar to C group, B group is inferior A group and C group.In conclusion, acellular nerve allografts respectively implanted with ADSCs and BMSCs successfully repaired 5cm extended peripheral nerve gap. Moreover, they are similar result with autologous nerve grafts. Meanwhile, recombinant TGFβ1 and ADSCs nerve graft can repair 9cm large extended peripheral nerve gap too. Hence two kinds of stem cells all possess potential clinic application value in peripheral nerve regeneration. Taking into consideration material harvesting, cell proliferation and differentiation capability, adipose-derived stem cells may be a better seed cells.