The Role Of Low Dose Ultrashortwave In Repairing Rat Sciatic Nerve Lesions By Using Acellular Allogenic Nerve Scaffold Implanted With Bone Mesenchymal Stem Cells

ObjectiveUltrashortwave therapy can resolve inflammatory states and reduce swelling promote vasodilation,ameliorating blood circle and nutrition of nerve and peripheral tissue,and intensifying tissue metabolizability and nerve system function.Studies have shown that through the acellular transplants combination ultrashortwave therapy,the repair of rat sciatic nerve injury in accelerated nerve growth played a big role in speed. With the development of tissue engineering,homogeneous acellular nerve transplant gained well development,it was used as bioactive surrogate of nerve,having better bionic property and exhibiting excellent histocompatibility,may offer fitting microenvironment for axonal regeneration,so as to facilitate and induce effective regeneration of the injured nerve.Meanwhiel,with the study to bone marrow stromal cells(BMSCs) differentiate into Schwann cells deepening gradually,it was proved that BMSCs can be easily accessible,capable of rapid expansion in culture, immunologically inert,capable of long-term survival.So BMSCs were used as 'seed cells' to promote nerve regeneration.The objective of this experiment is observe the role of low dose ultrashortwave in repairing rats sciatic nerve lesions using acellular allogenic nerve scaffold implanted with bone mesenchymal stem cells.The results will provide experimental basis for clinical nerve injury repairment and rehabilitation.Materials and Methods1.Preparing of acellular transplantsWistar rats were anaesthetized and the sciatic nerves were took out,then the cerves were prepared to acellular transplants via chemical extracted method.2.Isolation and culture of BMSCs and identificationMale Wistar rats were used at the age of 25-30 days,BMSCs were isolated from the femurs and tibia.Adherence method was used to culture BMSCs to the three passage 3,then were storaged for transplantation.Rat BMSCs within 3-5 passages were used to verify the multi-potential differentiation capacity.When cells were grown to at least 80%confluence,they were cultured in osteogenic and adipogenic the induction medium.Two weeks later,the cells were stained with Alizarin Red and Oil-Red O.3.Experimental animals and groupForty-eight adult male Wistar rats(200-250 g),divided into Dulbecco's Modified Eagle's Medium(DMEM) control groups(n=16),BMSC-treated groups(n=16) and BMSC-treated ultrashortwave(USW) therapy groups(n=16),and four animals in each group were sacrificed at 2,4,8 and 12 weeks after surgery.The animals were supplied by China Medical University.4.Implanting acellular nerve alloscaffold and Surgical ProcedureBMSCs in suspension with a density of 1×10~6 cell/ml were injected into the prepared acellular nerve scaffold of BMSC groups and USW groups,from both stumps using a sample micro-injector under a dissecting microscope.Following injection of BMSCs,scaffold were maintained in DMEM complex culture medium supplemented with 10%FCS in a 5%CO2 incubator at 37℃for 72h until the experiments were performed.Both proximal and distal nerve stumps were anchored into the graft with 9-0 nylon microsutures.5.Low dose ultrashortwave(USW) therapyAfter suturing 24 hours,the sixteen rats of USW groups were treated by an USW device.(1) Observe the morphology and construction of AECM by employing the light microscope and electron microscope.(2) Observe the morphological changes of BMSCs with phase-contrast microscopic during the process of culture.(3) The sciatic function index(SFI) was used to evaluate the function of sciatic nerve regeneration.(4) Observe the mean number of nerve fiber,myelin sheath depth and axon average diameter in regenerated nerve with toluidine blue staining.(5) Observe the expression of S-100,Glial fibrillary acidic protein(GFAP) and Vascular endothelial growth factor(VEGF) in regenerated nerve with immunofluorescence staining analysis.(6) Detect the expression of S-100 and VEGF mRNA in regenerated nerve with RT-PCR.6.Statistical Analysis.Results1.AECM were obtained by chemical extracted method and reserved structural integrity of vessel of basilar membrane and 3D- stereochemical structure.2.Phase-contrast microscopic observation showed the morphological changes BMSCs,BMSCs exhibited the small rounded,spindle-shaped or fibroblast-like morphology at day 5,and at dayl2 cells have reached 90%confluence.3.Two weeks later,BMSCs were stained by Alizarin Red and showed a extensive calcification deposition;the accumulation of intracellular lipid droplets was detected by Oil Red O at day 14.4.Typical walking tracts obtained from normal and the other three groups at 2,4, 8 and 12 weeks post-operation.The SFI value of ultrashortwave therapy group had statistical significance at 12 weeks compared with the BMSC-treated group and the control group.5.The result of toluidine blue stain and transmission electron microscopy are that the mean number of nerve fiber,myelin sheath depth and axon average diameter in the transverse sections at the distal portions of the three experiment groups was different, and the structure of regenerate nerve in USW therapy group was more intact.6.Immunofluorescence indicated that the positive expression of S-100 and GFAP are increasing gradually in regenerated nerve with the time growth,the positive expression is most in regenerated nerve.The positive expression of VEGF increased to peak,then decreasd gradually.7.The S-100 mRNA level were rising gradually with the growth of time.After 4 weeks of transplantation,the expression of VEGF mRNA were at highest level,then dropped gradually.Compaired with BMSCs group and DMEM group,the expression of the S-100 mRNA and VEGF mRNA level were at highest level in USW group.Conclusions1.The acellular nerve alloscaffold with implantating BMSCs could provide a favorable environment for the growth and myelination of regenerating axonscompared to acellular nerves alone,ultrashortwave therapy were beeter than BMSCc group.2.The expression of S-100 and GFAP in graft testified that acellular allogenic nerve scaffold were able to promote BMSCs differentiate into Schwann cells or to promote Schwann cells' proliferation and migration,but continuous ultrashortwave therapy was able to accelerate the process.3.The expression of VEGFmRNA and protein in graft testified that acellular allogenic nerve scaffold were able to conclude that increased angiogenesis in transplant and enhanced Schwann cell proliferation,provid more nutritive substance for nerve regeneration.