Study On Histology And MRNA Differential Expression Of Pulp Regeneration Induced By SDF-1

Objective: In this study,the dental plup regeneration was induced by the pulp revascularization and the implantation of SDF-1 hydrogel after the establishment of the no-vital root cannal model in mature dog teeth.Histological evaluation of the differences in tissue structure between new tissue and normal pulp tissue in root canals,and using RNA-seq technology to detect differences of two kinds of regenerated tissues mRNA expressed genes.To compare and analysis differences of two kinds of regenerated tissues from histological observations and mRNA gene expression level.To investigate the possible biological role and mechanism of SDF-1 in pulp regeneration.Methods: 1.Three canine aged 12-14 months were selected.16 teeth including 8 anterior teeth and 8 premolars were selected as experimental teeth,and 6 teeth including 3 anterior teeth and 3 premolars were selected as normal control teeth.The no-vital root cannal model were established in 16 teeth of experimental teeth in mature dog,according the different ways of the dental pulp regeneration divided into the SDF-1 group(group S)and dental pulp reascularization group(group B)with 8 teeth in each group,group S wasimplanted SDF-1 hydrogel into the root canal,group B was performed according to the clinical procedures of pulp revascularization,the normal control group(N group)retained intact teeth without any treatment.HE staining,MASSON staining and immunohistochemistry staining were used to evaluate histological structure differences of each group 3 months after pulp regeneration.2.RNA-seq technology was used to detect the gene expression and differential genes of mRNA in new tissues in root canals of the two regeneration methods,and the GO and KEGG pathways of the differential genes were further analyzed.Results: 1.HE staining: dental pulp reascularization group of root canal with new osteoid tissue and periodontal-like tissue,osteoid tissue from the apex inward along the root cannal wall,and even the entire root cannal,a large number of long oval bone-like cells were distributed evenly in the bone matrix,short columnar osteogenesis-like cells lined up for new bone edges,visible board of layered structure and the harvard system,periodontal-like tisuue of blood vessels in the connective tissue and fibroblasts content more,could be found in macrophages.In the root canal of SDF-1 implantation group,a large amount of loose connective tissue,cordlike large blood vessels and a large number of fibroblasts were found.A small amount of mineralized tissue grew along the root canal wall,and macrophages were seen.The formation of collagen fibers was observed in each group by MASSON staining.The collagen fibers in normal dental pulp tissue were thick and bunchy,the collagen fibers in SDF-1group were long and thin and interwoven into a network,and the collagen fibers in the pulp revascularization group were short and wavy.Immunohistochemical staining results showed that the macrophage marker CD68 was positively expressed in the SDF-1 group and the pulp revascularization group,while thenormal pulp was negatively expressed.2.In this project,94.65 gb of data were obtained by RNA-seq sequencing,and the average output of each sample was 10.52 gb.The number of detected genes was 17,775,the number of known genes was 17,038,and the predicted number of new genes was 737,among which 11909 differentially expressed genes were obtained.(1)There were 1381 differentially expressed genes in the comparation of revascularization group and SDF-1 group,among which 787 genes were up-regulated and 594 genes were down-regulated in the SDF-1group.(2)A total of 4751 differentially expressed genes were detected in the comparation of normal group and the revascularization group,including 2433up-regulated genes and 2318 down-regulated genes in group B.(3)A total of5777 differentially expressed genes were detected in the comparation normal group and SDF-1 group,among which 2695 genes in the SDF-1 group were up-regulated and 3082 genes in the SDF-1 group were down-regulated.3.Compared with the normal group,CXCR4,MMP9,OPN and POSTN genes were up-regulated in the SDF-1 group and the revascularization group.Odontoblast markers DSPP,DSP,MMP20 and nerve-related genes NES,SEMA5 A and UNC5 were down-regulated in the SDF-1 group and the revascularization group.Compared with SDF-1 group,OC gene and OPG gene were up-regulated in the revascularization group.Compared with the pulp revascularization group,OSM gene was up-regulated in the SDF-1 group.KEGG pathway analysis results suggest that SDF-1 may recruit stem cells to niche in the root canal to participate in the process of tissue regeneration by activating the PI3K/Akt pathway,and SDF-1 /CXCR4 axis may activate the JAK/STAT pathway to up-regulate PIM1 in order to inhibit apoptosis.Conclusion: In this study,RNA-seq technology was applied to analyze themRNA of newly formed tissues in the root canal induced by two methods of pulp revascularization and root canal SDF-1 implantation.Combined with histological results,the biological mechanism of pulp revascularization and root canal SDF-1 implantation in pulp regeneration was preliminarily revealed.The increase of CXCR4 gene,MMP9,OPN,POSTN might be related to the vascularization and ossification of the regenerated tissue,down-regulated genes such as DSPP,DSP,MMP20,NES,SEMA5 A and UNC5 might be the key genes for the differentiation of dentin and nerve,the downregulation of their expression in the new tissues of the two experimental groups in root canals indicated that the ability of dentine and neural differentiation was weak.The up-regulation of OC and OPG genes might promote the formation of more bone-like tissues in pulp revascularization,OSM might be a key regulatory gene for SDF-1 in more vascularized tissues.These results provided a scientific basis for further exploring the mechanism of pulp regeneration.