Identify Key Biomarkers Associated With Intervertebral Disc Degeneration And The Experimental Study Of The Repair Of Degenerated Intervertebral Disc With Transplantation Of TGF-?3 Scaffold

BackgroundLow back pain(LBP)is one of the major health problems of developed country and a major cause of disability.LBP is related to Intervertebral disc degeneration(IDD).The height of the intervertebral disc(IVD)is reduced by degeneration,altering the mechanical properties of the affected spinal segments.This process accelerates the degeneration of adjacent segments and other CRISTA structures,such as facet joints,ligaments,and muscles.IDD affects not only IVD,but also surrounding tissues such as muscles and ligaments,and the ability of the column to cope with normal physiological load in daily life.In the long run,IDD can lead to lumbar spinal stenosis and subsequent nerve tissue compression.Lumbar spinal stenosis is a major cause of lower back pain and neurogenic claudication,especially in the elderly.In view of the improvement of social living standards and the development of medical and health undertakings,the elderly population is increasing year by year.Elderly patients often also combined with osteoporosis,cardio-cerebral vascular and other chronic diseases,making the treatment of such patients with LBP and IDD-related diseases more difficult.At present,the treatment of IDD includes conservative treatment such as drug therapy,physical therapy and invasive treatment such as discectomy,column fusion and disc replacement,but these methods can not restore the original structure and function of IVD.In recent years,the rapid development of the bioinformatics has brought the opportunity to study IDD,which integrates the methods of statistics,mathematics,informatics and computer science to solve the problem properly.Bioinformatics's main areas of application include:Gene Discovery,prediction of gene expression,sequence alignment,protein structure alignment,prediction of protein structure,prediction of protein interaction,and so on.By searching public databases,we can find and verify the potential genes and signal pathways related to IDD,so as to understand the pathogenesis of IDD from the genetic level,and provide new ideas and research directions for the prevention and treatment of IDD.In recent years,cell-based tissue engineering has been proved to be a promising treatment for IDD.Tissue Engineering implants corresponding to the structure and function of IVD have been constructed by tissue engineering method,the utility model can replace the degenerated IVD and retain its mechanical motion range,so that the degenerated IVD structure can be reconstructed and repaired,and the function of IVD can be restored to achieve the goal of IDD treatment.Fiber ring tissue engineering is a kind of repairing method aiming at the regeneration and repair of fiber ring.After screening,a new type of composite scaffold,namely acellular fibrous ring Matrix/chitosan Hydrogel,was used as the scaffold material for the repair of fibrous ring injury because of its good Biocompatibility,degradability and mechanical properties,it can carry and release appropriate growth factors continuously to meet the demand of growth factors in different stages of the new annulus tissue,and finally achieve the goal of repairing the damaged IVD.Prat ?Identify key biomarkers associated with intervertebral disc degenerationObjective:The objective of this study was to identify key biomarkers included genes and signaling pathways associated with intervertebral disc degeneration(IDD)based on GSE56081 and GSE124272 datasets,and this study will provide new ideas for the study of the mechanism of IDD.Methods:The GSE56081 and GSE124272 datasets were downloaded from Gene Expression Omnibus(GEO)database,and the data from GSE56081 and GSE124272 datasets were preprocessed,and the differentially expressed genes between IDD group and Healthy group were screened utilizing the classical Bayesian method provided by the limma package.Then,the disease-related genes were searched in the Comparative Toxicogenomics Database(CTD)database,and the disease-related genes were intersected with differentially expressed genes,and the overlapped genes were redefined as the disease-related differentially expressed genes.In addition,the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on disease-related differentially expressed genes by the DAVID online tool.Moreover,the protein-protein interaction(PPI)network analysis of disease-related differentially expressed genes was carried out using the STRING database,and the nodes with high degrees were used as the hub proteins.Then the significant modules were identified by MCODE plug-in in Cytoscape software,and the KEGG pathway enrichment analysis was performed on these significant modules.In addition,the hub proteins obtained from PPI network were intersected with the genes in significant modules,and the overlapped genes were redefined as the key genes,and these genes were further verified in the GSE124272 dataset,and the marker genes were obtained.The receiver operating characteristic curve(ROC)was conducted on the marker genes.Finally,the Gene Set Enrichment Analysis(GSEA)was performed on these marker genes to obtain the important signaling pathways.Results:Totally,1184 differentially expressed genes between IDD group and Healthy group were screened.Then the 2295 disease-related genes were searched in the CTD database,and the 2295 disease-related genes were intersected with the 1184 differentially expressed genes,and the 142 overlapped genes were redefined as the disease-related differentially expressed genes.The GO and KEGG pathway enrichment analysis shown that the 142 disease-related differentially expressed genes were enriched in 130 GO-biological process(BPs),18 GO-cellular component(CCs)and 28 GO-molecular function(MFs),total 27 KEGG pathways were also involved,and the 142 disease-related differentially expressed genes were mainly involved in the TNF signaling pathway and HTLV-I infection.Additionally,the PPI network analysis shown that there were 223 PPI interactions and 83 proteins in the PPI network,and the TIMP metallopeptidase inhibitor 1(TIMP1)with high degrees was found in the PPI network,and there was 1 significant module,and the genes matrix metallopeptidase 2(MMP2),collagen type I alpha 2 chain(COL1A2),SMAD family member 3(SMAD3),SMAD family member 2(SMAD2),transforming growth factor beta 1(TGFB1),collagen type ? alpha 1 chain(COL4A1),mitogen activad protein kinse 1(MAPK1),snail family transcriptional repressor 1(SNAI1),TIMP1,aggrecan(ACAN)in this significant module.The KEGG pathway analysis shown that the significant module was involved in 16 KEGG signaling pathways,and mainly involved in the colorectal cancer.Then the hub proteins obtained from PPI network were intersected with the 10 genes in significant modules,and the 9 overlapped genes were redefined as the key genes.In addition,the 9 key genes were verified in the GSE124272 dataset,and 1 gene(ACAN)was screened as marker gene.Moreover,the ROC analysis shown that the ROC area reached more than 0.8 in both data sets,showing good diagnostic efficacy.Finally,the GSEA analysis illustrated that the ACAN gene were involved in 10 positively correlated signaling pathways and 9 negative correlation signaling pathways,among which were significantly involved in the parkinsons disease positively correlated signaling pathway and olfactory transduction negative correlation signaling pathway.Conclusion:HTLV-I infection and TNF signaling pathway might play important role in the development of IDD.TIMP1 was likely to be related to the progression of IDD via HIF-1?signaling pathway to regulate the apoptosis of nucleus pulposus cells.COL1A2 may play a role in the process of IAA through Platelet activation,Focal adhesion,and pI3K-Akt signaling pathways.ACAN gene may be a potential therapeutic target for the diagnosis of IDD and plays an important role through parkinsons disease and olfactory transduction signaling pathways.This study provides an analysis of potential markers of IDD and functional annotation of disease-related differentially expressed genes,which provides an idea to explore the regulatory mechanism of biological behaviors such as invasion,proliferation and apoptosis of IDD,and may provide a new clue to explore the research of IDD.Part ?The experimental study of the repair of degenerated intervertebral disc with transplantation of TGF-P3 acellular annulus fibrosus matrix/chitosan hydrogel scaffoldObjective:The acellular annulus fibrosus matrix/chitosan hydrogel was used as the scaffold material,and the sustained-release effect was achieved by wrapping Transforming Growth Factor-?3(TGF-?3).The growth of annulus fibrosus cell on transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold was observed and the repair effect of transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold on degenerative annulus fibrosus was analyzed.This study will provide theoretical basis and practical basis for the selection of materials and cytokines for the construction of intervertebral disc annulus fibrosus tissue engineering scaffolds.Methods:Firstly,the annulus fibrosus cells with multi-directional differentiation potential were isolated and purified from annulus fibrosus tissue,and their self-renewal and multi-directional differentiation ability were detected.Then the transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold was prepared,and the content of TGF-?3,cell proliferation,and the gene expression of Collagen ?,Collagen ? and Aggrecan were detected.Finally,magnetic resonance imaging(MRI)and hematoxylin-eosin staining(HE staining)were used to observe the fibroring repair,in order to detect the in vivo IDD repair effect of TGF-?3 loaded acellular fibroring matrix/chitosan hydrogel.Results:From the electron microscope,we could clearly see the pore structure in a large range on the scaffold,which was a three-dimensional network structure.In addition,the content of TGF-?3 increased with time and reached the peak at the 7th day.On the 1th,3th,5th day,there was no significant difference in cell proliferation between the 3 groups,but on the 7th day,the cell proliferation of the transplantation of TGF-p3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold group was significantly higher than that of the other two groups.Moreover,the mRNA and protein expression levels of Collagen ?,Collagen ? and Aggrecan genes in the transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold group were significantly higher than those in the without transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold group.Additionally,the acellular fibrous ring Matrix/chitosan hydrogel loaded with TGF-?3 was implanted into the injured IVD by in Vivo experiments in rats.The results of MRI at 4,8 weeks and HE staining at 4 weeks were analyzed in the experimental group and the Control Group,the results showed that TGF-?3 loaded acellular fibrous ring Matrix/chitosan Hydrogel could significantly restore IDD in Vivo.Conclusion:The constructed transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold had intercommunication space,and showed that the transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold had good structural properties.In addition,the constructed transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold had no obvious inhibitory effect on the growth and proliferation of the isolated and cultured annulus fibrosus cells,and the annulus fibrosus cells could adhere,grow and proliferate on the surface and in vivo of the scaffold,which showed that the transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold had good cytocompatibility.Additionally,the constructed transplantation of TGF-?3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold was implanted into the animal body.After 4 and 8 weeks,it could relieve the stenosis of the intervertebral space and decrease the biomechanical properties.After 4 weeks HE staining indicated that the structure was close to normal which indicted that the transplantation of TGF-P3 acellular annulus fibrosus matrix/chitosan hydrogel scaffold had a good repair effect on the IDD.