Experimental Research Of Combine Mesenchymal Stem Cells And Self-assembly Peptide Scaffold For Spinal Cord Injury Treatment

Part 1Experimental research of transplant mesenchymal stem cells to treat spinal cordinjuryObjective: To investigate the expressions of neurotrophins in MSCs in vitro, and thetherapeutic effects of MSCs transplantation for SCI.Methods: MSCs were isolated from marrow of rats, and purified by continuous culture.The cells were characterized by the expression of CD34, CD90, CD44 and CD45 by flowcytometry. The expression of BDNF and NGF were detected in MSCs using RT -PCR andELISA. SCI model was established by modified Allen's method. MSCs were injected intothe epicenter of injured spinal cord in one week. Locomotive function of SCI rats wasgraded with BBB score weekly, and in four weeks injured spinal cord was taken, theprotein level of BDNF and NGF was detected by ELISA, and NF200, GFAPimmunofluorescent staining were performed in frozen sections of injured spinal cord.Results: Isolated cells were adherent to culture flask and were spindle-shaped, and werepositive for CD90 and CD44, negative for CD34 and CD45 by flow cytometry. MSCsexpressed BDNF and NGF mRNA, and expressed the protein. The rats treated with MSCstransplantation showed improvement in functional outcome, the BBB scorces were higherthan the control, the protein levels of BDNF and NGF in spinal cord were higher than thecontrol. And immunofluorescent staining show smaller cysts and more regenerated axonsin spinal cord tn transplantation rats.Conclusions: MSCs could expressed BDNF and NGF, and could improve functionaloutcome in SCI rats. neurotrophic factor, nerve growth factorPart 2Experimental study of Peptide implantation in situ of spinal cord injuried rats for SCItreatmentObjective: To design Peptide-Amphiphile C₁₆H₃₁O-A₃G₄D₂IKVAV, investigate 3-Dconstruction of self-assembly gel and study the therapeutic effects of its implantation forSCI treatment in rats.Methods: Self-assembly of PA C₁₆H₃₁O-A₃G₄D₂IKVAV was improved by simulated bodyfluid and detected under TEM. SCI rats models were established by modified Allen'smethod, peptide solution was injected into injuried site. BBB test was performed everyweek postoperatively in 6 weeks, spinal cord samples were collected 3d, 1w, 2w, 3w, 4wand 6w after SCI. the expression of GFAP was detected by Real-time PCR. Spinal cordsample of injuried sites were collected again and observed after immunofluorescentstaining with NF200 and GFAP.Results: Self-assembly of peptide solution into 3-D gel could be trigged by SBF, andpresented as nanofiber under TEM, with 7-8nm in diameter and big gaps between the fibers.BBB scores in peptide implantation group were higher than controls in the 5th and 6thweeks after surgery, Real-time quantitative fluorescent PCR demonstrated the expression ofGFAP in the injured sites was weaker than controls. Immunofluorescent staining showedthe low expression of GFAP and many axon regenerations after peptide transplantation.Conclusion: PA C₁₆H₃₁O-A₃G₄D₂IKVAV could self-assembly into 3-D gel, GFAPformation could be inhibited and axon regeneration could be improved after itstransplantation into rats. Objective: To construct tissue-engineered neural grafts with Self-assembled PA 3-D gel asscaffold material and MSC.Methods: Self-assembly of peptide solution was trigged by MSCs suspension, 3-D gelcombined with MSCs was formed, cells distribution was observed under microscope.Live/dead cell staining was performed with Calcein-AM PI after 1d, 7d and 21d culture.Living condition of cells in the gel was observed. The gel combined with cells was culturedand detected by CCK-8 method to determine the cell proliferation. The expressions ofBDNF and NGF in the medium were detected by ELISA.Results: Cell suspension trigged peptide solution to self-assembly into 3-D gel, the cellswere kept alive in the gel. Live/dead cell staining presented most MSCs were still aliveafter 21 days culture. Cell proliferation test demonstrated MSCs proliferation was prettywell, and there's no significant difference with 2-D culture. ELISA showed BDNF andNGF were generated and secreted by MSCs in the gal.Conclusion: peptide solution self-assembly was trigged by MSCs suspension successfully;tissue-engineered material was constructed and presented good activity in vitro experiment.