Experimental Study On The Treatment Of Sciatic Nerve Injury By Differentiation Of BMSCs Neuroblasts Induced By Autologous I-PRF In Rats

Objective:To investigate the ability of bone marrow mesenchymal stem cells(BMSCs)in differentiation into neuroblasts induced by injectable platelet-rich fibrin(i-PRF)in vivo and in vitro,and the therapeutic effect of combination of them on sciatic nerve injury.Methods:BMSCs were isolated from tibias of SD neonatal rats aged 10-15 days and cultured until the 4th generation,which were later randomly divided into control group(group A)and experimental groups(group B,group C).Prepare i-PRF using modified low-speed centrifugation and take 0,100,200 ul i-PRF into each group of 12% FBS complete medium and co-culture with BMSCs.Reduced 50% i-PRF and serum concentration after 2 weeks.The morphological changes of cells were observed on the7 th,14th and 21 th days and the induction results were detected by immunohistochemistry and Western blot.Twenty-four adult SD rats were used to establish models of sciatic nerve injury by forceps crushing before randomly divided into four groups: group A with BMSCs suspension + i-PRF injection,group B with i-PRF injection,group C BMSCs suspension and group D saline.BBB functional scales were scored every week after operation.Two months after the operation,rats were killed by neck-lift and their sciatic nerve tissues were removed.The nerve repair was observed under transmission electron microscopy and HE staining,respectably.Western blot was used to detect the expression of related proteins.Results:The morphology of BMSCs began to change after 7 days of i-PRF induction,and synaptic growth and neuron-like cell morphology appeared 20 days later.Cell immunohistochemistry showed that Nestind protein expression was positive in the experimental group.Immunofluorescence staining showed that S-100 ? protein expression was positive in the experimental group with significant difference(P < 0.05).Curves of BBB function scales shown that A group > B group > C group > D group,and the difference was statistically significant(P < 0.05).HE staining showed that nerve fibers in group A were arranged neatly without defect or demyelination change;nerve fibers in group B were not clear and slightly swollen;nerve fibers in group C were partly disordered and demyelinated;nerve fibers in group D shown poor continuity,obvious demyelination change and partial defect of epineurium.Electron microscopic observation indicated that the structure of nerve fibers in group A was clear,the myelin sheath was intact,whose nucleus was dense;group B was slightly inferior;group C shown blurred nerve fibers with demyelinating;group D had disordered nerve fibers with significantly demyelinating and discontinuous defect of epineurium could also be seen.Western blot analysis showed that Nestind protein was expressed in all groups,and there was no significant difference in the expressions(P > 0.05);for N-cadherin and GFAP protein,BMSCs + i-PRF group had the highest content,followed by PRF group,;the BMSCs group had the lower content compared with PRF group;Nand blank group the lowest(P < 0.05).Conclusion:i-PRF can effectively induce the differentiation of BMSc into neuroblasts.The combination of i-PRF and i-PRF can improve the therapeutic effect of sciatic nerve injury in rats,providing a new idea for clinical treatment of peripheral nerve injury.