Modified Method To Obtain Muscle-derived Stem Cells (MDSCs) And Construct Muscle-derived Artificial Nerve Conduit To Repair Mice Sciatic Nerve Defect

Background:Peripheral nerve injury(PNI)is a common lesion that occurs during accidents and surgeries,and over a half million patients suffer PNI worldwide.Although peripheral nerves,in contrast to nerves of the central nervous system,can regenerate following a complete transection,the functional recovery rate(about 40%-50%in the clinic[1,2])is not favorable.Transplantation of autologous nerve segments is the current gold standard for long-gap PNI treatment;however,the donor site defect and subsequent neuroma formation limit application in the clinic[3].The application of tissue-engineered autologous nerve conduits to bridge nerve defects has emerged as an alternative strategy[4].In previous research,our team applied the epimysium of the external oblique muscle to prepare nerve conduits containing lumens and successfully used microsurgical techniques to bridge repair long-gap sciatic nerve defects,providing an easily obtained and highly biocompatible option for PNI treatment[5].In further research,we considered adding seed cells to promote the effectiveness of epimysium conduits(EMCs)and exploring the role of muscle-derived cells in nerve repair.Stem/progenitor cells isolated from murine and human skeletal muscles by various methods give rise to progeny cells with neuronal and glial phenotypes.Muscle-derived stem cells(MDSCs)were our first choice of seed cells attributed to the strong capacity of self-renewal,multipotent differentiation and immune-privileged behavior,which were reported to successfully promote PNI recovery.According to reported studies,MDSCs have the potential to undergo multi-lineage differentiation,such as myogenic,neural,osteogenic,and hematopoietic lineages[6-8].In transplantation studies,grafted MDSCs demonstrated the ability of multilineage differentiation and growth factor(such as IGF、NGF、b-FGF、VEGF)secretion in vivo and promote tissue regeneration[9-12].We assumed that combining EMCs with MDSCs would increase the efficiency of EMCs,and the homologous conduit could provide a more suitable microenvironment for MDSCs.To confirm this hypothesis,we obtained and proliferated MDSCs from green fluorescent protein(GFP)-carrying mice and combined them with EMCs from wild-type mice to build a muscle-derived artificial nerve conduit.The results indicated the multilineage potential of MDSCs in vivo,which could promote peripheral nerve axon outgrowth,remyelination and functional recovery.The prupose of study1.To explore and acquire the seed cells of artificial muscle-derived nerve conduit.2.To combine the epimysiumconduit with seed cells to build the nerve conduit and repair the sciatic nerve defect.Evaluate the nerve function and histology after surgery.3.To analysis the specific mechanism of MDSCs promoting peripheral nerve regeneration.Methods and results1.Modify the traditional preplate culture method and explore the better way to obtain MDSCsMethod:According to the previous studies,we tried traditional preplate method and find out the efficiency is unsatisfied.Thus,we bring out the modified preplate method through changing the culture medium and microenvironment Then we compared two methods on morphology,growth curve and specific markers.Result:The modified method could reduce the duration of preplating allowing more efficient MDSCs collection process.Following repeated of preplate,expression of specific markers increased.2.Evaluate the neurogenic capacity of different cell groups from skeletal muscle tissue.Method:According to different time of adhering,we applied modified method to separate diverse cell groups from muscle tissue.Then we induced those cells groups into neural lineage and evaluate their neurogenic capacity through morphology,immunofluorescence and Real-time PCR.Result:The late adhering cells obtained from modified preplate method showed stronger neurogenic potential,which could be the better seed cell candidate in following research.3.Build artificial nerve conduits to repair the defect of mice sciatic nerve.Method:Removed 3mm-long tissue of unilateral sciatic nerve,forming a 5mm-long defect.Constructed a 7mm-long conduit with 0.7mm inner diameter which was then used to bridge the nerve defect through microsurgery.Subsequently,injected the mixture of Matrigel and MDSCs into lumen of conduit to support the structure.As for the control group,injected Matrigel without MDSCsResult:Built muscle-derived artificial nerve conduit and repaird sciatic nerve defect successfully.Postoperative conditions of animals were stable.4.To assess the therapeutic efficiency of muscle-derived artifial nerve conduit.Method:We measured the sciatic nerve index(SFI)every two weeks after surgery as well as the recovery of sensory through Von Frey test.The histological assessments including immunofluorescence,Masson trichrome staining,toluidine blue staining and transmission eletron microscope were performed in 4 and 8 weeks.Result:Both groups presented regeneration in 8 weeks.The experimental group showed better SFI,sensory recovery and the atrophy of gastrocnemius was less severe comparing to control group.In histological tests,the thickness of myelin and the diameter of myelinated axons were both of larger numbers in experimental group comparing to control group.The experimental group also showed higher expression of myelin related protein in Western-blot test.5.To clarify the mechanism of MDSCs promoting nerve regenerationMethod:In order to track the fate of MDSCs in vivo,we grafted GFP-positive MDSCs into wild type mice.According to the expression of GFP in 4 and 8 weeks,we confirmed the differentiation of grafted MDSCsResult:Grafted MDSCs demonstrated myogenice,neurogenic and angiogenesis capabilities in vivo which could promote the regeneration of injured sciatic nerve.Conclusion:The modified preplate method is an efficient and convenient tool to obtain pure MDSCs which could separate cell populations according to the differences in morphology,cellular physiology,markers expression and differentiation potential.MDSCs as a promising seed cell could promote the maturation,remyelination and vascularization of new born axons,which contributed to the motor and sensory recovery.