Experimental Study On Tissue Engineer Nerve Graft Constructed By AECM And Induced BMSCs

ObjectiveBone Mesenchymal Stem Cells(BMSCs),residing in bone marrow,Which is multipotent stem cell that can differentiate to many types. Rencent experimental studies have confirmed that BMSCs can differential to neurons and glial cells. its can be harvested easily by amplification in vitro and vivo. as a result,its can be used to construct the production of tissue enginnering. Acellular Extracellular Matrix (AECM)is three dimensional mesh stent which mainly composed by vessel of basilar membrane and coated by epineurium. As a bioactive nerve graft,it own natural construction,lower immunogenicity. The purpose of this study is to establish a technical platform which used for BMSCs culture, purify, amplify in vitro,Obtain cells used for tissue enginnering. it also suggest differentiatal mechanism of BMSCs induced by antioxygen revulsivum. Testify the possibility of BMSCs co-culture with AECM to construct the graft used in tissue enginnering in vitro. Provide experimental support for clinic work and the further study of peripheral nerve tissue engineering.Materials and Methods1.Experimental animals15 Wistar rats weighed100-120g .25 Wistar rats weighed 180-200g, supplied by China Medical University.2.BMSCs culture, purify, amply ,induce ,identify and co-culture with AECM in vitro.Rinse the medullary cavity of bones and harvest the rinse solution which contains 10%FBS low glucose DMEM. Put it into culture flask for culture. change the new DMEM after 24 hours. When more about 80% cells touch eachother, serial subcultivation at the ratio of 1:2.Harvest the purified cells for identification by use of CD 44,CD34 antibody. The cells induced by DMEM,which contains 1mmol / Lβ-ME,and co-cultured with AECM in vitro.3. Observed index and detected methodsObserving the morphology of BMSCs,chromotropic acid 2R-brilliant green staining of graft. Immunohistochemistry stain of graft, Immunocytochemical stain ofinduced cells.Results1.The triple substitution of BMSCs are purified, The ninth substitution auto-differentiated conspicuously suggest the 3th-7th substitutions are fit to use for tissue enginnering.β-ME can inducing BMSCs differential to neurons and glial cells.2. AECM filled with induced BMSCs which differentiated again and both of them affinited better, co-cultured graft can be used in tissue enginnering.Conclusions1.Purified BMSCs can be harvest by adherent culture method. 3th-7th substitutions are fit to use for tissue enginnering. They can be used for drug screening, cytotoxicity test.2.BMSCs can differentiate to neural-like cells and glial-like cells byβ-ME.Induced cells display NSE and GFAP positive,but far more to real neural cells and glial cells. Suggests us the differentiation of BMSCs is complicated,poly-step and a result of multiple factors.3.Co-culture Induced cells and AECM. The cells display S-100 positive. The microenvironment provide by AECM can induces the cells differentiat.