Mechanobiological Mechanism Of Repairing Injured Peripheral Nerves With LIPUS And IPSCs-NCSCs

The incidence of peripheral nerve injury is estimated between 13 and 23 per100,000 persons per year in the developed countries and results in partial or total loss of movement,sensory,and autonomic function.Peripheral nerve injuries,especially severe injury,often face poor nerve regeneration and incompletely functional recovery even after surgical nerve repair.So far,autologous nerve transplantation is still the"gold standard"of nerve transplantation.However,autologous nerve transplantation is limited due to limited autogenous nerve origin and new trauma.Meanwhile,it is very difficult to prepare nerve grafts that are perfectly comparable to autologous nerves.Therefore,the combination of growth factors,nutritional factors and physical stimulation methods to improve the repair of peripheral nerve injury is a new option.The previous study of our group found that low intensity ultrasound stimulation(LIPUS)with intensity of 500 mW/cm~2 could promote the proliferation of induced pluripotent stem cells-derived neural crest stem cells(iPSCs-NCSCs),enhance the cell viability and promote i PSCs-NCSCs differentiation into neurons and Schwann cells.And in vivo studies have also found that LIPUS combined with iPSCs-NCSCs treatment can repair the injury of sciatic nerve in rat with 10 mm injury.However,the molecular mechanisms involved in the LIPUS treatment of iPSCs-NCSCs proliferation and differentiation are still unclear.The first aim of this study is to understand the effect of LIPUS on gene expression of iPSCs-NCSCs by high-throughput sequencing.Secondly,the signal pathways of the proliferation and differentiation of iPSCs-NCSCs by treating with LIPUS were also investaged in this work.Finally,according to the results of differentially expressed genes of iPSCs-NCSCs after LIPUS treatment,to investigate whether the protein encoded by growth differentiation factor 5(GDF5)could promote the repair of peripheral nerve injury by LIPUS combined with iPSCs-NCSCs.The main contents and results of this paper are as follows:(1)Effects of LIPUS stimulation on gene expression of iPSCs-NCSCs by gene expression profiling.iPSCs-NCSCs were treated with LIPUS with intensity of 500 mW/cm~2 and then analyzed by Illumina HiSeq 2000 sequencing platform for high-throughput sequencing.The differentially expressed genes of iPSCs-NCSCs after LIPUS treatment were obtained.Gene function annotation and enrichment,pathway annotation and enrichment analysis were performed on these differentially expressed genes.Finally,some differentially expressed genes were quantified by quantitative polymerase chain reaction(PCR)to verify the accuracy of the sequencing results.The results showed that LIPUS treatment could significantly affect the gene expression of iPSCs-NCSCs in basic medium culture condition or neuronal differentiation medium culture condition.It was found that expression of 76 genes of iPSCs-NCSCs cultured in basic medium and 21genes of iPSCs-NCSCs cultured in neuronal differentiation medium were significantly changed by LIPUS treatment.Gene Ontology(GO)annotation analysis showed that the significant differentially expressed genes produced by LIPUS treatment were annotated to 47 and 40 gene functions under the culture conditions of basic medium and neuron differentiation culture medium respectively.Significant enrichment analysis showed that the differentially expressed genes of LIPUS treatment were also enriched with biological processes(BP)and molecular functions(MF)to perform gene function.The Kyoto Encyclopedia of Genes and Genomes(KEGG)notes found that the differentially expressed genes after LIPUS treatment were annotated to 326 KEGG pathways in basic medium culture condition.The differentially expressed genes after LIPUS treatment were annotated to 323 KEGG pathways in neuronal differentiation medium culture condition.And the quantitative analysis showed that the expression levels of these selected differentially expressed genes were consistent with the results of sequencing.(2)Mechanical signal transduction pathway of LIPUS promoting iPSCs-NCSCs proliferation and differentiation.iPSCs-NCSCs were treated by LIPUS with intensity of 500 mW/cm~2 and FAK inhibitor or ERK1/2 inhibitor.The effects of LIPUS and FAK inhibitor or ERK1/2inhibitor on the proliferation,differentiation and skeletal arrangement of iPSCs-NCSCs were examined.And then the effects of LIPUS on FAK-ERK signaling pathway were also investigated.The results showed that LIPUS promotes the proliferation and differentiation of iPSCs-NCSCs,and this promoting effect is affected by FAK and ERK inhibitors.LIPUS can promote the F-actin expression of iPSCs-NCSCs,and this effect is inhibited by the use of FAK or ERK inhibitors.LIPUS can activate both FAK and ERK phosphorylation of iPSCs-NCSCs,and the addition of the corresponding inhibitor treatment inhibits the expression of phosphorylated FAK and phosphorylated ERK.Adding inhibitors to treatment while applying LIPUS treatment can mitigate this inhibition.These results show that LIPUS treatment can activate the FAK-ERK signal pathway of iPSCs-NCSCs,and this pathway is likely to be directly or indirectly involved in the regulation of LIPUS promoting the proliferation,differentiation,and cytoskeleton rearrangement of iPSCs-NCSCs.(3)Effects of LIPUS combined with iPSCs-NCSCs,PFTBA,and GDF5 on repair of peripheral nerve injury.The above-mentioned high-throughput sequencing showed that LIPUS could significantly elevate the expression level of GDF5 in iPSCs-NCSCs.Based on this finding,perfluorotributylamine(PFTBA)with strong dissolution capacity and the GDF5 protein were loaded on allogeneic acellular nerve scaffold.The effects of iPSCs-NCSCs,iPSCs-NCSCs+PFTBA,iPSCs-NCSCs+GDF5,and iPSCs-NCSCs+PFTBA+GDF5 on the repair of sciatic nerve injury were investigated by establishing the rat sciatic nerve injury model.In vitro experiment results showed that PFTBA could promote the iPSCs-NCSCs viability under 5%hypoxia culture condition,and the LIPUS could not further improve the promoting effect of PFTBA.The addition of GDF5 can promote the differentiation of iPSCs-NCSCs,and with the extension of processing time,the promotion of differentiation was enhanced.In vivo studies have shown that LIPUS+iPSCs-NCSCs+PFTBA+GDF5 have the best effect of repairing sciatic nerve injury.In summary,LIPUS treatment can affect the gene expression of iPSCS-NCSCS and produce genes with significantly differentially expressed.LIPUS may regulate the proliferation,differentiation,and cytoskeleton morphological changes of iPSCs-NCSCs through the FAK-ERK signal pathway.LIPUS treatment of allogeneic decellularized nerve conduit containing iPSCs-NCSCs+PFTBA+GDF5 has a good effect on the repair of sciatic nerve injury.The results of this paper not only reveal the mechanism of LIPUS on cell behavior,but also provide a theoretical basis for the application and development of LIPUS in peripheral nerve tissue repair.