| BackgroundWith the aging of the population and the change of lifestyle,the incidence of intervertebral disc herniation has increased significantly in our contury,which becoming a major public health problem.Intervertebral disc herniation can lead to pain,sensation disorders,decreased muscle strength,and even lead to paraplegia in severe cases,seriously affecting the life quality of patients.In the pathological process of disc herniation,annulus fibrosus injury is the direct cause of herniation of nucleus pulposus,which leads to a series of cascade reactions such as release of inflammatory factors and nerve compression.Therefore,how to properly repair annulus fibrosus injury is the key problem in the treatment of disc herniation.At present,annulus fibrosus repair methods used in clinical treatment mainly include suturing,sealing and other methods.However,the long-term clinical effects of these methods are still dissatisfactory.Tissue engineering technology is a good method for tissue repair or reconstructioin.However,because cognitive deficiencies of the heterogeneity of annulus fibrosus cell populations and matrix compositions,the reconstruction of annulus fibrosus by tissue engineering still has many problems which limit the clinical transformation of this technology.Firstly,because the deficient understanding of annulus fibrosus cells populations,it is difficult to accurately reconstruct the cell components of annulus fibrosus.Second,because of the annulus fibrosus mainly contain two kinds of matrix,fibroid and chondroid matrix,so it is difficult to match the two different matrices by the scaffold material contained a single component.In addition,how to induce the differentiation of the seed cells correctly in the repair aera is also a difficult problem in annulus fibrosus reconstructioin by tissue engineering.Therefore,how to make the annulus fibrosus repair materials to match the heterogeneity of annulus fibrosus cells and matrix remains to be further studied.ObjectiveThis study aimed to decode the annulus fibrosus cell atlas by single-cell RNA sequencing and lineage tracing,to propose a new annulus fibrosus repair strategy by tissue engineering.And to verify the repair effect and clinical prospect of this new strategy in annulus fibrosus injury animal model.Methods1.Histomorphometric methods and immunohistochemical staining for relevant markers were used to analyze and compare the morphology and amount of annulus fibrosus cells in clinical annulus fibrosus samples with different degrees of degeneration,which preliminarily analyzed the heterogeneity of human annulus fibrosus cells.2.Single-cell RNA sequencing technology was used to decode the atlas of mouse annulus fibrosus cell populations,and the function and characteristics of popularions were analyzed.Further,immunostaining of the marker genes was performed to identify the cell populatoins found by single-cell sequencing at tissue level.3.Lepr-cre lineage cells were used to trace the migration and differentiation pathway of AF stem cells.4.The composite hydrogel based on SFMA and HAMA was produced.To obtain the ability to stimulate differentiation,the composite hydrogels were combined with a fibrochondrocyte-inducing supplement(FCI),which combined the main components of fibrogenic medium and chondrogenic medium.The characterization and inductive effect of the composite hydrogels were tested in vitro.5.The AF stem cells loaded composite hydrogel was used to repair the mice AF defect,and the repair effect was evaluated from multiple levels.Results1.AF cells can be mainly divided into two parts by histological morphology,spindle cells and round cells.Compared with the moderate degeneration,the spindle cells were decreased and round cells were increased in serious degeneration samples,besides the length of AF cells were decreased generally.In addition,the AF cell population which expressed Mmp13 were significantly increased in the serious degeneration AF samples compared with the moderate degeneration samples.2.Single-celled sequencing revealed annulus fibrosus cells in mice can be divided into10 subgroups: AF stem cells,chondrocyte-like AF progenitor cells,fibroblast-like AF progenitor cells,prechondrocyte-like AF cells,prefibroblast-like AF cells,immature chondrocyte-like AF cells,immature fibroblast-like AF cells,mature chondrocyte-like AF cells,mature fibroblast-like AF cells,and fibrochondrocyte-like AF cells.AF stem cells were located in the stem cell niches at the interface between annulus fibrosus and vertebral bone.The fibroblast-like AF cells were located in the outer AF,and the chondrocyte-like AF cells were located in the inner layer of AF.3.Based on the single-cell sequencing data and lineage tracing,we found that LepR-cre lineage cells can be used for tracing the migration of AF stem cells.The tracing results showed that the migration of LepR-cre lineage cells from the stem cell niche to the AF could be detected at postnatal 2 weeks,and the amount of LepR-cre lineage cells were increased in the AF with the age of mice.4.Based on single-cell sequencing data and histological verification,we found a population of annulus fibrosus cells expressing both fibroid and chondroid markers,located in the middle layer of the annulus fibrosus,which was named fibrochondrocyte-like AF cell according to the cell function.5.In order to match the fibroid and chondroid matrix of the AF,SFMA and HAMA were selected and combined with the FCI to prepare SFMA/HAMA/FCI composite hydrogel.And it was found that the hydrogel has good mechanical stability,cellular compatibility,and drug-controlled release effect.The bidirectional induction effect of the hydrogel was verified in vitro.6.AFSC-loaded SFMA/HAMA/FCI composite hydrogel was used to repair AF defects in mice,lamellar structure of AF was partially restored,the histological score of AF injury was significantly reduced,and the area of type II collagen was significantly increased in the repaired AF.Conclusion1.Human AF cells are heterogeneous and the populations are variable in the samples from different grades of intervertebral disc degeneration.2.The mouse annulus fibrosus can be divided into two main differentiation directions:fibrogenic direction and chondrogenic direction.The differentiation process can be divided into five stages,AF stem cells,progenitor cells,pre-AF cells,immature cells,and mature cells.3.AF stem cells locate in the intervertebral disc stem cell niches and migrate into the AF along the transition zone between vertebral body and annulus fibrosus.4.A new population of AF is identified,fibrochondrocyte-like AF cell,which can secrete both fibroid and chondroid matrix,located in the middle layers of AF.5.The SFMA/HAMA/FCI composite hydrogel can induce the fibrogenic and chondrogenic of AF stem cells concurrently,and restore the fibroid and chondroid matrix of AF effectively in mouse AF defects.In conclusion,this study decoded the atlas of mouse AF cells by single cell sequencing technology,providing a cytological basis for AF repair,based on which a new strategy for annulus fibrosus repair was proposed.That is,used AF stem cells loaded composite hydrogels and further added the bidirectional induction factor to reconstruct the AF defect.By the experiment of AF repair in mice,we verified that the new strategy can correctly initially reconstruct the heterogeneity of AF cells and matrices of AF and provide a new potential solution to the clinical problem of AF repair. |
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