DPSCs Seeded In Acellular Nerve Grafts Processed By Myroilysin Improve Nerve Regeneration

Background:The repair of peripheral nerve defects is a major problem for neuroscience.In recent years,constructing scaffolds with good biocompatibility property has become a hot topic for research in repairing of peripheral nerve injury.The use of scaffold can bridge the nerve endings,guide axial growth of axons,avoid the formation of neuromas,and provide a relatively isolated microenvironment for nerve regeneration.This study aims to use Myroilysin to manufacture a novel acellular xenogeneic nerve scaffold,when using rabbit dental pulp precursor cells with neural differentiation potency as seeding cells to compound together with this scaffold,we can obtain a new tissue engineered nerve graft that can repair and regenerate peripheral nerves.Chapter 1 Nerve scaffolds preparation,structure observation and mechanical properties evaluation of Myroilysin acellular xenogeneicOBJECTIVE:To prepare Myroilysin acellular xenogeneic nerve scaffold and observe and evaluate its structure and mechanical mechanics.METHODS:Wistar rat sciatic nerves were harvested and decellularized using Myroilysin and Sondell’s chemical methods respectively.Macroscopic and light microscope observations of histological sections were performed by HE and Masson staining.The decellularization effect of nerves,the neural basement membrane and collagen structure were observed under electron microscope.The surface properties of the nerve scaffolds prepared by different methods were observed by scanning electron microscopy.The limit Load,toughness,elastic modulus,ultimate stress,limit strain,and fracture power were compared in vitro between the ANG and natural rat nerve using a Wintest mechanical test.RESULTS:The myroilysin enzymatically prepared nerves swelled significantly compared to Sondell’s chemically prepare nerves.Histological observations revealed that the acellular nerves of two methods exhibited disappeared axonal and axonal structures.The basal membranes of the nerves were wavy and the collagen fibers were arranged in an orderly manner.Scanning electron microscopy showed that the longitudinally gill-like structure of the nerve scaffold obtained by the Myroilysin enzymatic method was more pronounced.Wintest mechanical analysis showed that the Myroilysin enzymatic decellularized nerve scaffolds had no statistically significant differences(P>0.05)between mechanical load and mechanical properties,such as the limit Load,elastic modulus,toughness,ultimate stress,ultimate strain,and fracture power.Conclusion:Myroilysin enzymatic method and Sondell chemical method have similar decellularization effects.The neural scaffolds prepared by these methods have little influence on the mechanical mechanics of the nerve scaffolds,meeting the mechanical requirements for nerve scaffold implantation;and the more apparent gutter-like structure of Myroilysin decellularized nerve scaffold facilitates the growth of nerve cells.We conclude that Myroilysin decellularized nerve scaffold is a type of material worthy of consideration.Chapter 2 Culture,identification and neural differentiation of rabbit dental pulp precursor cellsOBJECTIVE:To cultivate dental pulp precursor cells and investigate their feasibility as tissue engineered nerve graft seeding cells.METHODS:Healthy dental pulp tissue of three-month-old pure New Zealand white rabbits and premolars of 12-15 year-old patients for clinical orthodontic extraction were recruited.Primary cultures were performed according to the Gronthos enzyme digestion method to obtain dental pulp cells,and then these cells were passaged.Cells of the third generation were subjected to flow cytometry and immunofluorescence staining to identify mesenchymal stem cell markers and peripheral Schwann cell markers.These obtained rabbit dental pulp precursor cells were induced into the nerve direction and the induced cells were identified.Results:Highly proliferating cells were obtained.The flow cytometric detection of human dental pulp precursor cells were CD29+,CD106+,CD44+,CD73+,CD90+/CD34-,CD45-,CD271-,CD133-,CD31-.Cultured rabbit dental pulp precursor cells had GFAP,MBP,P75,S-100 positive cells.Immunofluorescence of neural-like cells obtained after induction of neural direction in rabbit pulp precursor cells showed that markers such as GFAP,MBP,P75,S-100 were positive.PCR and WB results showed that the expression of GFAP and MBP on the surface of the cells increased.Conclusion:Dental pulp precursor cells are found in the cells obtained in this study.Neuronal-like cells that could secret neurotropin with neural surface markers are obtained when the pulp precursor cells are induced into the nerve direction;in the pulp precursor cells obtained in this study we could find cells functionally sirnilar to certain nerve cells.There is feasibility in using GFAP,MBP,P75,and S-100 positive dental pulp precursor cells as seed cells for peripheral nerve tissue engineering to obtain neural grafts with more neurotrophic function.Chapter 3 Biological properties evaluation of Myroilysin acellular xenograftOBJECTIVE:To evaluate the biological properties of Myroilysin acellular xenogeneic nerve scaffolds,to explore the biocompatibility of dental pulp precursor cells when compounding with nerve scaffolds,and to provide a theoretical basis for the construction of new artificial nerves for clinical use in the future.METHODS:Two types of acellular xenogeneic nerve scaffold leaching solution was prepared and co-cultured with rabbit dental pulp precursor cells.The cytocompatibility of the scaffold was evaluated by CCK-8 cell proliferation assay.Type I collagenase was used to cleave natural nerves,Sondell acellular xenogeneic nerves and Myroilysin acellular xenogeneic nerves,so as to compare the collagen stability of the three types of nerves and to evaluate their degradation rates.Rabbit dental pulp precursor cells were implanted into Myroilysin and Sondell acellular xenograft nerve scaffolds.The EdU test was performed to observe the arrangement pattern of cells on the scaffold.The rabbit dental pulp precursor cells were co-cultured with Myroilysin and Sondell acellular xenogeneic nerve leaching solution.The qRT-PCR and Western blotting analysis of the scaffold leaching solution on the expression of neural surface markers and neurotrophic factors in the dental pulp precursor cells were analyzed at mRNA and protein levels.The composite rabbit dental pulp precursor cells and acellular xenogeneic nerve grafts were implanted into three-month-old pure New Zealand white rabbit sciatic nerve defect models.After 30 days and 90 days,the sciatic nerve and its dominance in the skeletal muscle of the rabbit sciatic nerve defect model were analyzed.RESULTS:After co-cultivation of dental pulp precursor cells with acellular nerve leaching solution,the cell proliferation activity in Myroilysin acellular xenogeneic nerve leaching solution was higher than that of the control group and Sondell group.That showed good cell compatibility of Myroilysin acellular xenogeneic nerve;but in the Sondell group,the cell proliferation rate was significantly decreased and that showed a significant cytotoxicity of this scaffold.The rate of degradation of the two acellular xenogeneic nerve scaffolds was the same as that of natural nerves.Myroilysin acellular xenogeneic nerve scaffolds adhered to the dental pulp precursor cells and exhibited a more obvious directional growth pattern,but no proliferation cells were found in the Sondell acellular xenogeneic nerve scafold.After co-cultivation of dental pulp precursor cells with Myroilysin acellular nerve leaching solution,the surface markers MBP and S-100 were up regulated at mRNA and protein levels;beta III Tubulin,CD90,NeuN and neurotrophins BDNF and NGF were up-regulated at mRNA levels.The composite rabbit dental pulp precursor cells and acellular xenogeneic nerve grafts were implanted into the three-month-old pure New Zealand white rabbit sciatic nerve defect model.After 30 days,in the Myroilysin compound dental pulp precursor cells group,S-100 positive cells were detected by EdU test,proliferating cells increased,and CD31-positive proliferating cells penetrated deeply into the scaffold.After 90 days,the difference in the autologous nerve grafts group and the Myroilysin complex pulp precursor cell group decreased(P>0.05)in the composite action potential amplitude(compose muscular action potential,CMAP)and nerve conduction velocity(NCV),and there was no significant difference in the recovery rate of muscle wet weight(P>0.05).Conclusion:Myroilysin acellular xenogeneic nerve scaffold has good cell compatibility and degradation rate in vitro.It can promote the neural induction of dental pulp precursor cells,and its porous longitudinal gutter-like structure can exert terrain-inducing effect and its residual extracellular matrix components of peripheral nerves have certain biological induction effect,which can make the dental pulp precursor cells adhere to them and induce much more directional growth.After implantation of composite rabbit dental pulp precursor cells and acellular xenogeneic nerve grafts into the three-month-old pure New Zealand white rabbit sciatic nerve defect models,its repair effect is close to autologous nerve transplantation.The dental pulp precursor cells have good adaptability with the extracellular matrix of the Myroilysin acellular xenogeneic nerve scaffold and can be one of the new choices of tissue engineered nerve grafts.