Acellular Nerve Allografts Combined With Mesenchymal Stromal Cells To Repair Sciatic Nerve Defects

ObjectiveWith the development of microsurgery, though the repair following injury of peripheral nerve has achieved a great progress in the recent years, the results of the repair are still not satisfied. Even after severe nerve injuries, reaction of tumor or congenital deformity, the loss of nerve substance resulting in a significant gap between the nerve stamps could not be repaired by direct epineural suture. The regeneration and function recovery of peripheral nerve injury are focus issue in the field of neuroscience. The preferred approach to repair peripheral nerve defect is to bridge the two ends of the injuried nerve with a segment of autologous nerve graft in clinic; as a result, this approach incurs some donors site sense dysfunction and usually involves a prolonged operation time and so on. High polymer such as PDLLA and PLGA has been used to make into tissue engineering bio-artificial nerve, but these bio-artificial nerve lacks of active cells and extra cellular matrix and nerve nutrient factors, and they could not mimic the contracture of nerve which would influence the nerve regeneration. The objective in this study include: (1) To investigate the differentiation of adult rat bone marrow stromal cells (BMSCs) into Schwann-like cells, BMSCs were induced by acellular nerve allografts (ANA) in vitro. (2)To evaluate the effectiveness of acellular rat sciatic nerve cultured with bone marrow stromal cells to bridge sciatic nerve defect with tissue engineering technique in rats. Methods1. Takes a three weeks age SD rat, death from cervical, under aseptic conditions femur and tibia removed, the 5 ml syringe washed bone marrow cavity, centrifugal fluid collection, made of single-cell suspensions inoculation training. After phenotype characterization, the fifth generation of the rats' BMSCs were extracted, cultured and amplificated in vitro, and then induced by ANA homogenate.2. The change of cellular morphology was observed every 6h and the expression of S-100, glial fibrillary acidic protein (GFAP) were detected in the induced cells with immunocytochemical staining, flow cytometry. The change of GFAP and S-100 in the induced or uninduced cells were detected by RT-PCR. While the cell activity was detected by MTT method.3. 18 adult SD rats were randomly divided into 3 groups : experimental group, ANA group and control group, 6 rats in each group. A 15-mm-long nerve defect was made in the right sciatic nerve and then was bridge . The thigh bone marrow was taken out from animals of experimental group under sterile condition, and mesenchymal stromal cells were purified using adherence method, cultivated and proliferated, and then the ANA was cultured with the mesenchymal stromal cells to repair the nerve defects in experimental group and with ANA in the ANA group ;. Twelve weeks after operation, the experimental evaluation was made, including: Sciatic nerve function index (SFI); Gastrocnemius wet weight recovery rate(%);HE staining and S-100 immunohistochemistry staining, ect.Results1. The results showed that the phenotype of BMSCs were CD44~+, CD54~+, and CD34~-. About 64%±5% or 42%±4% positive cells in the induced cell were glial fibrillary acidic protein (GFAP) or S-100 immunopositive. By using RT-PCR and flow cytometer, expression of GFAP and S-100 were also detected much higher in induced group than in control group. The MTT detection showed that the number of active cells were maximum when 1.0mg/ml inductor was applied. 2. SFI and gastrocnemius wet weight recovery rate were higher in experimental group than in ANA group. The S-100 immunohistochemistry staining, the density, quantity and diameter of opaque nerve fibers as well as neural sheath thickness of experiment group were all better than those of ANA group .The amount of the tactile corpuscle in average aera was significantly higher in the experimental group than in ANA group (P<0. 05). Similar results with the control group (P>0. 05).Conclusion1. The results mentioned above display that ANA can induce adult rat BMSCs differentiating into Schwann-like cells.2. Acellular rat sciatic nerve cultured with BMSCs can repair nerve defects . BMSCs induced by ANA have the effect of Schwann cells.