| Intervertebral disc degenerative disease is a common orthopedic disease that seriously affects the life quality of the patients.The symptoms include the pain of the neck,back,waist and the sensory or motor dysfunctions of the limbs.Although most patients can get relieved by conservative treatments,still many patients have to be treated with operations.In the process of the operations,the annulus fibrosus should be incised in order to remove the degenerative and protruded intervertebral disc.In the past,the methods of repairing annulus fibrosus include direct suture and closure of the defect sites.Animal experiments and limited clinical applications showed that these methods could partially achieve the immediate closure and physical repair of the annulus fibrosus defects.By these methods,the integrity and tightness of the intervertebral disc could be restored in a certain degree.However,neither the entire healing between the defects with the surrounding tissue nor the regeneration of the annulus fibrosus and intervertebral disc could be realized.Therefore,the repair effects were limited.The existence of annulus fibrosus defects and poor healing of the annulus fibrosus are both important factors of the reprotrusion of the nucleus pulposus.In recent years,tissue engineering and regenerative medicine technology have been applied in the field of annulus fibrosus repair.By constructing tissue engineering scaffolds in accordance with the structural characteristics of the annulus fibrosus,cells with potential ability of proliferation and differentiation could be delivered to the defect sites.Growing of these cells could repair the annulus fibrosus defects,which could create suitable environment for regeneration of the annulus fibrosus and intervertebral disc.In this process,coordination of the scaffolds and the cells should be achieved in order to improve the growth,proliferation and differentiation of the cells and improve the results of the repair.Previous studies have shown that using of stem cells with multi-directional differentiation potential could achieve desired results in the repair of tendon,cartilage and bone.Then introduction of stem cells into annulus fibrosus and intervertebral disc repair also revealed that the stem cells can repair the degenerative or injured annulus fibrosus.Meanwhile,many difficulties were still existed in this field such as poor local growth conditions,low cell proliferation efficiency and unclear differentiation trend.In order to achieve better repair effects,one of the current attempts is to improve the structure of tissue engineering scaffolds and simulate the specific structure of the annulus fibrosus by controlling the spatial structure properties such as fibers orientations and pore shapes.By these methods,the suitable microenviroment could be achieved and the proliferation and differentiation of the stem cells could be improved.In previous researches,our group used electrospinning technology to construct nanofiber scaffolds.By improving the spinning method,aligned nanofibrous scaffolds were fabricated,which could simulate the anisotropy of the annulus fibrosus.With the oriented arranging fibrous,the tensile resistance of the scaffold was improved obviously.So,the scaffold achieved enough mechanical properties to repair the annulus fibrosus.Co-culturing of the scaffolds and the bone mesenchymal stem cells indicates excellent biocompatibility of the scaffolds and the cells could adhere,grow and proliferate well in the scaffolds.In addition,three-dimensional porous scaffold could be obtained by homogenization and freeze-drying using random nanofiber scaffold.The porous structure was similar to the inner annulus fibrosus.After cross-linking of the aligned nanofibrous scaffold and the porous scaffold,composite scaffold was constructed.This kind of composite scaffold could be used to repair the defect model of annulus fibrosus in animals by loading the bone mesenchymal stem cells.In these experiments,we also observed that the bone mesenchymal stem cells cultivating in different scaffolds showed discrepancy in differentiation.When cultivated on the aligned nanofibrous scaffold,the stem cells extended and distributed along the direction of the fibers,showing a long spindle shape consistent with the morphological characteristics of outer annulus fibrosus cells.At the same time,the expression of phenotypic molecule mRNA was also consistent with that of outer annulus fibrosus cells.On the other hand,the cells in three-dimensional porous scaffold grew into the pores and showed the colony-like manner.Also,the morphology of the cells was round,similar to the characteristics of the inner annulus fibrosus cells.Then the phenotypic molecule mRNA was also consisitent with the inner annulus fibrosus cells.These phenomenons indicated that the surface structures of nanofiber scaffolds could regulate the differentiation of stem cells.It could provide available references for further construction of tissue engineer scaffold by analyzing and researching this regulating,exploring the possible mechanism and searching for involved factors.In this study,electrospinning nanofiber scaffolds with different surface structures were constructed to simulate the outer or inner annulus fibrosus respectively.Bone mesenchymal stem cells were cultivated on the scaffolds,then high-throughput sequencing was applied to analyzed the differential expression of genes in order to determine the effects of the scaffolds on the cells.Then the expression levels of phenotypic molecule mRNA of annulus fibrosus cells in different scaffolds were examined and compared in order to evaluate the effects of the scaffold structures on the differentiation of the cells and explore its possible mechanisms.Finally,the Aligned nanofibrous scaffold with the surface structure nearly to the anatomical characteristics of the outer annulus fibrosus was applied to repair the outer annulus fibrosus defect of lumbar interverbal disc in rabbits,and the repair effect was evaluated.The results of this study showed that,(1)All the three electrospinning scaffolds could provide suitable growth environments for the bone mesenchymal stem cells.The results of high-throughput sequencing showed that there were many expressing differences of the genes after cultivation of the cells on the scaffolds comparing with the control group.These differences suggested that the scaffolds could affect the biological behavior of the stem cells and result the differences at the transcriptional level.(2)The aligned nanofibrous scaffold could promote the differentiation of the bone mesenchymal stem cells into outer annulus fibrosus cells.And Mkx was the regulatory factor involved in this process.(3)The Three-dimensional porous nanofibrous scaffold could promote the differentiation of the BMSCs into inner annulus fibrosus cells,and Integrin β1 was the regulatory factor involved in this process.(4)The aligned nanofibrous scaffold-cell complex could effectively repair the defect of outer annulus fibrosus of lumbar vertebrae in rabbits.Part Ⅰ Analysis of the Effects of Nanofibers Scaffolds Surface Structure on Bone Mesenchymal Stem Cells Based on High-Throughput SequencingObjective:To construct electrospinning scaffolds with different surface structures and inoculated with bone mesenchymal stem cells(BMSCs).Evaluate the effects of the scaffolds on the cells using high-throughput sequencing and analyze the different expression of the genes.Methods:Using electrospinning technology to fabricate nanofibers scaffolds with different surface structures as Random Nanofibrous Scaffold(RNS),Aligned Nanofibrous Scaffold(ANS),and Three-dimensional Porous Nanofibrous Scaffold(3-DPS).BMSCs of SD rat were extracted and cultivated on the surfaces of the scaffolds.After cultivation,the proliferation of the cells on each scaffold was detected by CCK-8 method and compared.The surface structures of the scaffolds were observed by scanning electron microscope(SEM).Also,the morphology of the BMSCs was determined by SEM.Histological staining was used to observe the growing of the cells on the scaffolds.After co-culture of cells on the scaffolds,high-throughput sequencing was performed and BMSCs cultured without scaffolds were used as the control group.Different expressings of the genes in each group were analyzed to research the effects of the scaffolds on the cells.Also,the compare between ANS and RNS was applied to determined the effect of fiber orientation on the cells.Results:Comparing results of the fiber diameters and angles between fibers indicated that there were no significant differences in the diameters of the fibers of RNS(1.22±0.17μm)and ANS(1.12±0.16μm),while the values of the angles between the fibers of RNS distributed more dispersedly,while the angles of ANS were mainly distributed between 15-35°.3-DPS was a spatial structure scaffold,in which the fibers stretching in three-dimensional space,and quasi-circular pores were formed between the fibers.BMSCs grew well after cultivated on each scaffold and infiltrated into the inner part of the scaffold.On the 3rd day of cultivation,the OD value of 3-DPS group(1.343±0.034)was higher than all of other groups(P<0.0001).And the value of ANS group(1.245±0.029)was higher than RNS group(1.193±0.020)and Control group(1.158±0.024)(P<0.01).On the 7th day,the value of 3-DPS group(1.779±0.034)was higher than those of all other groups.And the value of RNS group(1.663±0.025)and ANS group(1.685±0.032)were higher than the Control group(P<0.0001).The cells of RNS and ANS extended along the fibers and had the morphological characteristics of the outer annulus fibrosus cells,while the cells in 3-DPS grew in the pores and had the morphological characteristics of inner annulus fibrosus cells.HE staining showed that BMSCs could grow well on the surfaces of the scaffolds and infiltrate into the inner part.The results of high-throughput sequencing showed that there were differences at the level of transcription between every group with the group of control.And there were also differences between ANS and RNS.The results of enrichment analysis showed that compared with the control group,there were significant gene enrichments in extracellular matrix composition and function,tendinous tissue development,collagen fiber connection and other functions.Also,analysis of differential protein coding genes suggested that PI3K-Akt,focal adhesion,extracellular matrix receptor and other signal pathway related genes were enriched.There was difference in the expression level of Mkx(Mohawk)between either ANS with the control group or RNS with the control group.Also,there was difference between these two groups.There was also difference of expression level of Integrin β1 between 3-DPS with the control group.Conclusion:The surface structures of RNS,ANS and 3-DPS were different.The structures of ANS was similar to the outer annulus fibrosus,while the 3-DPS was similar to the inner annulus fibrosus.All the three scaffolds provided suitable growing environments for BMSCs.The high-throughput sequencing results showed that nanofiber scaffolds could affect the genes expression of BMSCs.And the effects involved many aspects of biological processes of the cells and were related to a variety of pathways.Mkx(Mohawk)and Integrin β1 are related to the effect of ANS and 3-DPS surface structure to BMSCs.Part Ⅱ Effect and Mechanism of Surface Structure of Nanofibrous Annulus Fibrosus Scaffolds on Bone Mesenchymal Stem Cells Differentiation into Annulus Fibrosus CellsObjective:(1)To discuss the effect of ANS and RNS on differentiation of BMSCs into outer annulus fibrosus cells and compare the difference between the two scaffolds and analyze the effect of Mkx in this process.(2)To evaluate the effect of 3-DPS on the differentiation of BMSCs into inner annulus fibrosus cells and analyze the role of Integrin β1.Methods:BMSCs were inculated on each scaffold and cultivated.The morphology of the cells and cytoskeletons were observed after staining with phalloidine and DAPI.Then,(1)qPCR was used to detect the expression levels of phenotypic molecules(Col I、Scx、Col II、Sox 9、Bgn、Tnc、Dcn)mRNA of annulus fibrosus cells in ANS and RNS groups.The group of BMSCs cultivated without scaffold was used as control.The differentiation of the cells was determined and compared between the groups.si RNA was constructed to inhibit the effect of Mkx.Then the effect of Mkx on the cell differentiation was explored by examining the expression level change of phenotypic molecules(Col I,Scx,Bgn,Tnc and Dcn)mRNA in RNS and ANS.(2)The expression levels of phenotypic molecules(Col II,Sox-9 and Aggrecan)mRNA in groups of 3-DPS,ANS,RNS in chondrogenic induction culture were detected by qPCR,and the group of BMSCs cultivated without scaffold was used as control.The differentiation of BMSCs into inner annulus fibrosus was compared in groups.Then lentivirus and adenovirus vectors were constructed to inhibit or upregulate the effects of Integrin β1.Then the expression level changes of phenotypic molecules mRNA in 3-DPS were examinated.By these methods the effect of Integrin β1 in this process was discussed.Results:Observation of staining showed that the shape of BMSCs was long fusiform with polar distribution of the cytoskeleton,which accorded with the morphological characteristics of outer annulus fibrosus cells in ANS and RNS,while the cells in 3-DPS were similar to the morphological characteristics of the inner annulus fibrosus cells as polygonal,non-polar.The results of qPCR showed that the expression levels of Col I and Scx mRNA of BMSCs in ANS and RNS were higher than the control group(P<0.05),also the expression levels of Bgn,Tnc and Dcn mRNA were higher than the control group(P<0.05).At the same time,the expression levels in the ANS group were higher than the RNS group.After inhibition of Mkx,the expression levels of Col I,Scx,Bgn,Tnc and Dcn mRNA decreased,while the expression levels of Col II and Sox 9 mRNA increased.The expression levels of Col II,Sox-9 and Aggrecan mRNA of BMSCs cultured in 3-DPS were higher than the Control group,RNS group and ANS group.When the effect of Integrin β1 was inhibited the expression levels of Col II,Sox-9 and Aggrecan mRNA decreased in 3-DPS,while when the effect of Integrin β1 was upregulated the expression levels of Col II,Sox-9 and Aggrecan mRNA increased accordingly.Conclusion:(1)ANS could promote the differentiation of BMSCs into outer annulus fibrosus cells and Mkx is the regulatory factor involved in this process.(2)3-DPS could promote the differentiation of BMSCs into inner annulus fibrosus cells,and Integrin β1 involved in this process.Part Ⅲ In vivo Experiment of Aligned Nanofibrous Scaffold for Repairing of Outer Annulus Fibrosus Defects of Lumbar Intervertebral Disc in RabbitsObjective:To establish the lumbar annulus fibrosus defect model in rabbits and observe the repair effect of ANS,which simulating the characteristics of outer annulus fibrosus,combined with autologous bone marrow.Methods:The defect model of outer annulus fibrosus of lumbar intervertebral disc was established in rabbits.Eighteen New Zealand rabbits were divided into 3 groups randomly,and there were 6 rabbits in each group.The group of control was only incised and exposed to the annulus fibrosus of the L5-6 intervertebral disc.Then an annular defect of 2 mm in diameter and 1 mm in depth was performed in the defect group at the outer annulus fibrosus of L5-6 intervertebral disc after exposure.While in the repair group,scaffolds of 3-DPS mixed with autologous bone marrow were planted in the defects.After operations,the examinations of DR were performed at the 4th and 8th week.Then the relative disc height index(rDHI)was examined and compared.At the 8th week after the operations,the animals were killed and samples were taken.Then the repair sites were examined by and mechanical examination and pathological examination.Results:On the 4th week after operation,the rDHI of the control group(1.03±0.09)was higher than the defect group(0.85±0.12)(P<0.05).And on the 8th week,the rDHI of the defect group(0.63±0.06)was lower than both of the control group(1.02±0.08)and the repair group(0.89±0.11)(P < 0.05).Also,the rDHI of the repair group was between the control group and the defect group,and there were significant differences with both of the other two groups(P < 0.05).The tensile mechanical test showed that the maximum tensile strength of the defect group(6.38±0.99 MPa)was the lowest,which was different from either of the control group(13.54±1.00 MPa)or the repair group(9.60±1.44MPa)(P < 0.05).And the maximum tensile strength of the repair group was lower than the control group(P < 0.05).Pathological examination showed that there was repair tissue formation in the repair group,but not obvious tissus formation in the defect group.Conclusion:The cell-scaffold composite of ANS as carrier can effectively repair the outer defect of lumbar intervertebral disc annulus fibrosus in rabbits. |
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