| Intervertebral disc degenerative disease is a disease that seriously endangers human health.It can cause nerve compression and damage,and backache is the main cause of leading disability.The treatment of intervertebral disc degenerative diseases mainly includes functional exercise,medicine,physical therapy and operation,among which surgery is still the most effective treatment.Excision of herniation intervertebral disc tissue and intervertebral fusion surgery is a traditional surgical approach,but it is difficult to reconstruct the motion loading,resulting in poor functional recovery.Artificial intervertebral disc or artificial nucleus pulposus can partly restore the intervertebral disc height,thus restoring the dissection and motor function of the intervertebral disc and improving the quality of life of postoperative patients.However,the implantation of artificial intervertebral disc and nucleus pulposus material have some flaws,it may make implants prolapse or shift,which can lead to complications.Tissue engineering and regenerative medicine strategies in solving these problems reflected in the significant advantages,tissue engineering technology can produce similar to the human body in the bionic natural anulus fibrosus structure of collagen fibers,electrostatic spinning technology can be made close to the natural anatomic characteristics of tissue engineering anulus fibrosus,and can effectively promote the implant surrounding tissue regeneration,the implant and the surrounding tissue integrality,thus partially postoperative bone tissue integrity and adjacent anulus fibrosus.However,the above tissue engineering anulus fibrosus by electrostatic spinning technique is not perfect and can not be used in clinic.The main problems include It is difficult for the cells to grow into the scaffold,and it is difficult to induce the directional differentiation of the seed cells to the inner and outer anulus fibrosus cells,and it is difficult to regulate and control due to the unknown mechanism of differentiation.the insufficient strength of these materials such as polycaprolactone,the degradation rate of certain products is not controllable,and the mechanism of promoting tissue regeneration is not clear.in order to research the clinical practice of bionic material of tissue engineering,the early study indicate Poly(L-Lactide)/ Polycaprolactone,(PLLA/PCL)has excellent mechanical performance,effective surface activity and the degradation rate of the advantages of the regulation.In order to further study and optimize of scaffold materials,we improved the proportion of configuration and production methods to PLLA/PCL materials,and comparatively studied the three different structure of PLLA/PCL materials,optimized the conditions of cell growth and explored the possible mechanisms of different structural materials to promote tissue regeneration.In this study,we prepare the Aligned Nanoyarn/Nanofibrous Scaffold(AYS),A1 igned Nanofibrous Scaffold,(AFS)and Three dimensional porous nano fiber Scaffold(3 DPS)Three different structure of nanometer fiber material,then implanted the rat Bone marrow Mesenchymal Stem cells(BMSC)in vitro to the Three Scaffolds,detection of BMSC in Scaffold material adhesion,growth,proliferation and Cell activity;To vaccination in AYS,AFS,3 DPS materials into subcutaneous of rats,observe the capsule wall thickness,number of form foreign body giant cells,inflammatory factors examined gene expression level,evaluate the biocompatibility of the three kinds of materials;The results were compared with normal culture method,intermittent centrifugation method and bioreactor dynamic culture method to cultivate the effect of BMSC,observe cell survival,growth and distribution,and optimize in vitro culture method.Static culture in vitro BMSC,observe cell in three kinds of nano fiber scaffold morphology,adhesion,proliferation and differentiation of behavior,compare the relative phenotypes of the anulus fibrosus cells(COL2a1 COL1a1 Aggrecan,Sox-9)mRNA expression level and FAK signaling pathways related protein AKT,ERK1/2,JNK,P38 expression level,the evaluation of different structure of nanometer fiber scaffolds promote BMSC proliferation and differentiation effect,and the preliminary exploring the mechanism about different structural scaffold materials to promote BMSC proliferation and differentiation.We found that the three materials of AYS,AFS and 3DPS have good biocompatibility.Compared with the normal culture method and the intermittent centrifugation method,the bioreactor perfusion method can promote the growth of BMSC.different structure scaffolds,cell adhesion,proliferation,directional differentiation behavior of BMSC have significant differences,AYS materials,BMSC tend to differentiation of fibroblasts,3 DPS materials,BMSC tend to differentiate into cartilage cells;There were significant differences in Aggrecan and sox-9 gene expression in BMSCs in different structural scaffolds,and significant differences in FAK signaling pathways related protein AKT、P38 expression level.indicating that the Integrin-FAK signaling pathway may play a role in promoting proliferation and differentiation.This study focous on the influence about three scaffold material(AYS,AFS and 3 DPS)to cell behavior,and promote cell proliferation and differentiation of mechanism,and help to further optimize anulus fibrosus scaffold tissue engineering products,to foundate for the follow-up study.Part I Biocompatibility research of nanofibers/porous nanofibers tissue engineering scaffold materialsObjective:to evaluate the biocompatibility of the orienting nano-yarn(AYS)、orienting nano-fiber(AFS)and 3D porous nano-fiber scaffold(3DPS).Methods:Polylactic acid(PLLA),polygonate(PCL)and gelatin were prepared by using electrostatic spinning technology to prepare the orienting nano-yarn(AYS).Polylactic acid(PLLA)and gelatin mixed with conventional electrostatic spinning technology were used to fabricate non-regulated nanofibers by changing the receiving device and preparing the orientation nanofiber scaffold(AFS).The non-regulated nanofibers were then made into three-dimensional porous nanofiber scaffolds(3DPS)by homogenate/freeze-dried.The bone marrow mesenchymal stem cells(BMSC)were inoculated with the scaffold materials.After in vitro culture,the cell adhesion,growth,proliferation and activity were detected in scaffold materials,and biocompatibility was evaluated in vitro.Will three stenting subcutaneous of rats,at different time points out,by histological staining,immunohistochemical,measuring capsule wall thickness,number of form foreign body giant cells and inflammatory cytokines gene expression,evaluation of biocompatibility in vivo.Results:in vitro experiments showed that bone marrow mesenchymal stem cells can adhere,proliferate and grow well in three kinds of materials.In vivo experiments showed that the three materials had mild inflammatory response in the early stage(1-2 weeks).After 4 weeks,the infiltration of inflammatory cells decreased and the number of foreign giant cells gradually decreased.The expression of inflammatory cytokines was consistent with inflammatory cell infiltration.Conclusion:the experimental results in vivo confirmed that the scaffold material has good biocompatibility and It lays a foundation for further development of the application of anulus fibrosus tissue engineering scaffold materials.Part II The optimization of growing conditions of BMSC in scaffold materialsObjective: to compare the survival conditions of cell growth under three different culture conditions,such as normal culture,intermittent centrifugation and bioreactor dynamic culture,to optimize the culture method.Methods : rat bone marrow mesenchymal stem cells(BMSC)vaccination in orientation between orienting nano-fiber(AFS),the orientation of nanometer yarn(AYS),three-dimensional porous nanometer fiber(3DPS)scaffold materials,common culture were used respectively to measure the dynamic culture method,intermittent centrifugation and bioreactor were sent to culture.After 7 or 14 days,the stent was removed for the following test.HE staining observed the growth of cells in scaffold materials.DAPI detects cell distribution and activity.Measure the depth of the cells into scaffold materials.Results:The experiments showed that the perfusion culture method of the bioreactor was better than that of the scaffold.Conclusion: the dynamic culture method of bioreactor perfusion culture is more favorable to the cell growth and the cell survival is good.It is of guiding significance to further develop the application and clinical treatment of anulus fibrosus tissue engineering scaffolds.Part III The research about Influence and mechanism of different structure of nanofibers scaffolds to BMSC behaviorObjective : to compare the cell morphologyand adhesion,proliferation,differentiation and other behaviors of cells in different scaffold materials.The expression levels of mRNA of cells in different scaffolds(COL2a1,COL1a1,Aggrecan,sox-9)and FAK signaling pathways related proteins were compared.To clarify the effect of scaffold structure on cell behavior and clarify its possible mechanism.Methods:three scaffold materials were prepared by disinfection.Between in vitro rat bone marrow mesenchymal stem cells(BMSC)vaccination scaffold in three different structure,the static compare BMSC after 3 weeks after cell morphology in the three kinds of scaffolds as well as the differences between the adhesion,proliferation and differentiation,etc;cDNA was extracted from the cells,and the mRNA expression levels of the key Phenotypic molecules(COL2a1,COL1a1,Aggrecan,sox-9)were determined by qRT-PCR.Western blot was used to detect the expression of FAK related protein AKT,ERK1/2,JNK and P38.The effects of different structural scaffolds on cell behavior were compared.Results:The experiments show that the different structural scaffold for cell adhesion and proliferation,directional differentiation behavior has obvious effect,the orientation are consistent and dense structure of scaffold similar fibroblast cells form,type I collagen content is higher,and extracellular matrix cells more in the direction of fibroblasts,and in three-dimensional porous materials,irregular form cells form assumes the circular,and extracellular matrix type II collagen content is higher,the PCR determination of Aggrecan and Sox-9 gene expression significantly higher than AFS,AYS group,cells tend to differentiate into cartilage cells.There were significant differences in the expression levels of AKT and P38 of FAK signaling pathway.Conclusion: the morphology of different scaffolds can directly affect cell behavior.The scaffold structure regulation cell behavior may be by the mechanism of integrin-FAK signaling pathway. |
