Construction Of Tissue - Engineered Nerve In Vitro Based On PLGA Membrane And Necl - 1 Protein

BACKGROUND:Currently the treatment of peripheral nerve defects is not very good, tissue engineering nerve repair peripheral nerve defects get a lot of attention. As seed cells, Schwann cells (SCs) were commonly used, but its effects at tissue engineering nerve field was not good enough for its difficulty adhesion on scaffolds. How to increase the adhesion between cells and the carrier, and increase the number of cells on a support, it is expected to improve its effectiveness.OBJECTIVE:To study the effect of Necl-1 protein on SCs in vitro, biological characteristics of SCs and the feasibility of Necl-1 to improve the adhesion, proliferation and neurotrophic factors (GDNF, BDNF and CNTF) of SCs were observed.METHODS:RSC96 Schwann cells cultured in vitro. Effect of different concentrations of Necl-1 on proliferation of SCs was measured by MTT assay. Three preferred concentration (25μg/ml,50μg/ml, 100μg/ml) were selected, and cultured in groups. In order to observe the changes in the biological characteristics of SCs, the cell FDA/PI fluorescence staining, Haematoxylin and Eosin staining, S-100b immunohistochemical determination, MTT cell proliferation assay and gene expression of GDNF, BDNF, CNTF, S-100b detected by cell RNA extraction and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) were measured, and further observed on day 2,4,6 respectively.RESULTS:MTT cell toxicity test results show when Necl-1 concentration was 50μg/ml, the proliferation of SCs had a good role in promoting. But FDA/PI staining indicated that there was no difference among the control group and Necl-1 groups. Although no morphological differences showed among the groups of HE staining, cell density of each Necl-1 group was larger than the control group, and reached a maximum in N2 (50μg/ml) concentration. In the Cell proliferation assay, we found that each Necl-1 group OD value was higher than those in the control group at 2d,4d,6d respectively. When Necl-1 was at a concentration of 50μg/ml, relatively OD value was higher than others at 4,6 days respectively. Through the S-100b immunohistochemical staining, we found that S-100b expression had significantly increased in each Necl-1 group compared with the control group, and Necl-1 at a concentration of 50μg/ml expression ofS-100b reached its maximum. In terms of gene expression, the expression of GDNF, BDNF, CNTF, S-100b in each Necl-1 group were significantly upregulated compared with the control group. CNTF, GDNF, S-100b expressed strongest in Necl-1 at a concentration of 50μg/ml when SCs were cultured at 4d,6d. And BDNF expression in each group showed no significance at 2d, but 25μg/ml group showed a more significant role in promoting at 4d,50μg/ml group had a stronger role in promoting at 6d.CONCLUSION:Experimental results show that the appropriate concentration of SCs Necl-1 can promote the proliferation and adhesion of SCs, and can maintain its morphology, enhanced expression of GDNF, BDNF and CNTF and other neurotrophic factors for SCs involved in peripheral nerve repair provides a more appropriate micro environment.Our study indicates that Necl-1 is a more stable preparation for possible nerve tissue engineering.BACKGROUND:Polylactic acid-glycolic acid copolymer (PLGA,a lactic acid and glycolic acid random polymerization of two monomers, is a function degradable organic polymer, which is biocompatible, non-toxic, well into the capsule and film-forming properties.As PLGA widely used in tissue engineering, combined with the proliferation of adhesion Necl-1 protein for SCs and its specific anchorage effect. This is a new idea for the preparation of nerve tissue engineering.OBJECTIVE:By tissue factor binding protein engineering technology, to provide a new model of peripheral nerve repairing, testing related biological characteristics of SCs, SCs inquiry after intervention by Necl-1 protein in the PLGA membrane material proliferation and adhesion situation.METHODS:The PLGA membrane was evenly coated with Necl-1 protein. SCs were seeded on PLGA membrane, different concentration of Necl-1 protein (25μg/ml,50μg/ml, 100μg/ml) were added to the corresponding groups. MTT cell proliferation was determined at three time points(2,4,6d) to understand the impact of PLGA joint Necl-1 protein on the proliferation and adhesion of SCs. Morphological characteristic changes were measured by FDA/PI staining and hematoxylin-eosin staining. S-100b expression evels by immunohistochemistry test for detection of specific proteins of SCs. Gene expression of GDNF, BDNF, CNTF, S-100b was detected by RT-PCR. Cell growth and morphology on PLGA membrane were observed using Scanning electron microscopy (SEM).RESULTS:SCs can adhere and grow well in PLGA membrane. The MTT cell toxicity test show that it had a better role in promoting Schwann cells proliferation in each concentration of Necl-1 group than in the control group in 2d,4d and 6d, particularly in the N2 (50μg/ml). The cell proliferation assay showed that OD value of each group was higher compared with the two-dimensional non-PLGA membrane in the same period while it had a best role in promoting Schwann cells proliferation in the concentrations of Nl and N2 in 4d and 6d. The H&E staining also showed cells in each concentration of Necl-1 groups clusters grow significantly in 4d and 6d. Through FDA/PI fluorescence staining, we found that cell morphology did not change and the proportion of living and dead cells had no obvious difference in each concentration of Necl-1 groups and the control group. By immunohistochemical staining of Schwann cells, we found the S-100b expression in the concentration of N2 (50μg/ml) was obviously higher than these in others. The gene expression quantity of GDNF, BDNF, CNTF and S-100b in each concentration of Necl-1 group had a significant increase compared with these in the control group and their expression level was increased significantly with a longer culture time while it’s highest in the concentration of 50μg/ml group. Scanning electron microscopy (SEM) showed Schwann cells can be more stable adhesion and growth in the stent surface spreading in PLGA membrane at 2,4,6d, some cells can grow which was embed in intramembrane. With a longer time, the adhesion cells increased significantly on the stents. At the same time the amount of cells in the N2 (50μg/ml) group was significantly higher than it in the control group and there were a large number of synapse formation in the cell surface in N2(50μg/ml) group.CONCLUSION:Necl-1 protein binding in vitro tissue engineering nerve is more stable and favorable. Necl-1 protein is conducive to promoting the growth of SCs and adhesion on PLGA membrane material, of which has a positive meaning to maintain their phenotype and function. Building a PLGA and Necl-1 protein in vitro neural tissue it is feasible to further animal model for nerve defects mentioned basis and reference.