Study Of Screening In Vitro For Nanofiber Scaffold Materials In Tissue Engineered Nerve

Part 1:Culture in vitro and identification of schwann cell line.Objective:comparing with normal schwann cells,we observed the cell morphous and regularity,and detected the purity and genetype of schwann cell line cultured in vitro, to prove the possibility of application of screening the scaffold materials for peripheral nerve.Methods:Culture the schwann cell line RSC96 conventionally,and observe the cell morphous and regularity;Evaluate the purity by immunofluorence staining of S-100; Detect the expression of gene S-100,GDNF,BDNF,NGF,CNTF,PMP22 of schwann cell line through RT-PCR.Result:Schwann cell line RSC96 expressed the typical cell morphous of schwann cell,with the fusiform shape and spindly bipolar.The growth regularity was similar with the normal schwann cells while the course was abbreviated.All cells were positive by immunofluorence staining,which demonstrate 100%purity,and the distribution of S-100 consistented with the regularity of normal schwann cells. RT-PCR proved the expresion ofgene S-100,GDNF,BDNF,NGF,CNTF,PMP22 of schwann cell line,which were the significant gene for normal schwann cells.Conclusion:Schwann cell line RSC96 has the similar cell morphous,growth regularity and genetype with normal schwann cells,with the 100%purity,high speed of proliferation and lower degeneration.So schwann cell line RSC96 is more suitable to make use of the screening scaffold materials in vitro compared with others. Part 2:Identification in vitro of nanofiber scaffold materials made of PLLA and co-polymer with PCL.Objective:to identify and compare different kinds of composition of PLLA, P(LLA-CL)(75:25) and P(LLA-CL)(50:50) and different preparing technology for nanofiber scaffold materials for peripheral nerve,through schwann cell line,and finally choose the best one for the animal test in vivo.Methods:Observe the microstructure of three kind of nanofiber scaffold materials by scanning electron microscopy,compared with microstrcucture of extracellular matrix. Evaluate the biocompatibility of them through proliferation test,attachment test and observing of cell morphous.At the same time find the regularity between the orientation of nanofiber and alignment of cells.Analyse the expression of gene S-100,GDNF,BDNF,NGF,CNTF,PMP22 of schwann cell line which was cocultured with nanofiber scaffold materials using Real Time PCR,compared with the gene expression of schwann cell line which was not cocultured.Result:The cocultured schwann cell line attached and proliferate well,which indicate the favorable biocompatibility of three kind of scaffold materials,in which P(LLA-CL)(75:25) was the best one.The result was relative to the fact that the microstructure of P(LLA-CL)(75:25) is more similar to ECM's,which can be identified by SEM.At the same time we found that the cells can align along the orientation of nanofiber.And also,three kind of scaffold materials gived no impact on gene expression of S-100,GDNF,BDNF,NGF,CNTF,PMP22 of schwann cells.Conclusion:Compared with PLLA and P(LLA-CL)(50:50),P(LLA-CL)(75:25) has more favourable biocompatibility,with mircostructure more similar with ECM,and also has no obvious impact on gene expression.Especially,the orientation of nanofibers can lead cells to align regularly.These results demonstrate that the oriented nanofiber scaffold materials made of P(LLA-CL)(75:25) can be used in animal test.