Study On Biological Characteristics Of Human Nucleus Pulposus Decellularized Extracellular Matrix Scaffold With Different Degrees Of Degeneration

Objective:In recent years,the stem cell-based tissue engineering strategy of intervertebral disc nucleus pulposus for repairing degenerative nucleus pulposus has been the focus of research.Seed cells and scaffolds are the core components of tissue engineering.Nucleus Pulposus Mesenchymal Stem Cells(NPMSCs)have the characteristics of Mesenchymal Stem Cells(MSCs)and the function of multidirectional differentiation.Compared with other sources of MSCs,the adaptability to the intervertebral disc microenvironment has been a unique advantage.Scaffolds provide three-dimensional spatial structure and bioactive substances for seed cells to survive,which makes up for the deficiency of simple stem cell transplantation and injection.Nucleus Pulposus DecellularizedExtracellular Matrix(NP-dECM)scaffold can simulate the natural intervertebral disc microenvironment and facilitate the adhesion,proliferation and differentiation of seed cells.Studies have shown that the repair effect of NP-dECM scaffolds is related to a variety of factors,among which the degree of degeneration of the source of nucleus pulposus tissue is one of the important factors affecting the repair effect.By comparing the biological characteristics of human NP-dECM scaffolds with different degrees of degeneration(Ⅱ,Ⅲ,Ⅳ),this study explored and optimized the preparation process of human NP-dECM scaffolds,and selected the NP-dECM scaffolds with the best biological characteristics,laying a foundation for the subsequent preparation of composite hydrogel scaffolds for repairing degenerative intervertebral discs.The results provide theoretical basis for clinical treatment of related diseases of Intervertebral Disc Degeneration(IDD).Methods:1.Extraction,culture and identification of human NPMSCs: Type Ⅱ collagenase digestion method was used to extract human NPMSCs.The adherence,cell morphology and growth of NPMSCs were observed under the microscope.Cell surface markers CD73,CD90,CD105,CD34,CD45 and HLA-DR were detected by flow cytometry.Adipogenic,osteoblastic and chondrogenic differentiation were induced by induction differentiation medium,and the differentiation ability of NPMSCs in three lines was observed to determine that they were the seed cells needed for this study.2.Preparation and identification of human NP-dECM scaffolds with different degrees of degeneration: According to the inclusion and exclusion criteria,the human nucleus pulpose tissues removed during the operation were divided into groups,control group: Group Ⅱ-C,group Ⅲ-C,group Ⅳ-C;Acellular groups: Group Ⅱ-D,group Ⅲ-D,group Ⅳ-D.Human NP-dECM scaffolds with different degrees of degeneration were prepared by freeze-thaw cycle,Triton X-100,Sodium Dodecyl Sulfate(SDS),and nuclease.The DNA content in nucleus pulposus tissue before and after decellularity was detected,Hematein Eosin,HE)staining,4’,6-Diamidino-2-Phenylindole(DAPI)staining and DNA gel electrophoresis were used to determine the cell removal effect.The glycosaminoglycan(GAG)and Hydroxyproline(HYP)contents in the nucleus pulposus tissue before and after decellular treatment were quantitatively determined,and the retention of extracellular matrix components after decellular treatment was evaluated by Alessin blue staining and type Ⅱ collagen immunohistochemistry.The microstructures of scaffolds were observed by scanning electron microscopy(SEM),and the pores of scaffolds were evaluated.The above experiments were used to evaluate the effect of decellularization of human nucleus pulposus tissue with different degrees of degeneration.3.In vitro compatibility of human NP-dECM scaffolds with different degrees of degeneration: Human NPMSCs were cultured with the leach solution of human NP-dECM scaffolds with different degrees of degeneration,and the cytotoxicity of the leach solution of scaffolds with different degrees of degeneration was evaluated by CCK-8 method.Human NPMSCs were inoculated on human NP-dECM scaffolds with different degrees of degeneration for 3D culture in vitro,and survival was evaluated by live/dead cell staining.Results:1.Human NPMSCs meet the identification criteria set by the International Society for Stem Cell TherapyIsolated and cultured human NPMSCs were adherent to the wall,and the cells showed short spindle shape and spindle shape.The third-generation NPMSCs showed high expression of CD73,CD90,CD105 and low expression of CD34,CD45,HLA-DR and other surface markers.The results of three lines of differentiation proved that the NPMSCs had the ability of adipogenic,osteogenic and chondrogenic differentiation.2.Characteristics of human nucleus pulposus tissue with different degrees of degenerationWith the increase of degeneration degree,the appearance of human nucleus pulposus tissue gradually deepened color,texture gradually hardened,and the DNA and GAG contents in human nucleus pulposus tissue showed a decreasing trend,which were in group Ⅱ-C > Group Ⅲ-C > Group Ⅳ-C.The variation trend of HYP content was slightly different in DNA and GAG,group Ⅲ-C > Group Ⅱ-C > Group Ⅳ-C.3.Identification of NP-dECM scaffolds with different degrees of degenerationThe results of HE staining and DAPI staining showed that a small amount of nuclei remained after decellularity treatment in the nucleus pulposus with three degrees of degeneration.The DNA quantitative results showed that the DNA removal rates of groups Ⅱ-D,Ⅲ-D and Ⅳ-D were 81.63%,87.20% and 87.95%,respectively.DNA gel electrophoresis did not detect DNA fragments remaining after decellularization in each group.The quantitative and qualitative detection results of GAG and HYP showed that the glycosaminoglycan and type Ⅱ collagen of extracellular matrix were well retained.GAG retention was in the order of group Ⅱ-D > Group Ⅲ-D > group Ⅳ-D from high to low.The content of HYP in groups Ⅱ-D and Ⅲ-D was higher than that in groupⅣ-D.The results of scanning electron microscopy showed that the tissues of groupsⅡ-D,Ⅲ-D and Ⅳ-D were loose and porous after decellularity treatment,and the pores increased and enlarged compared with those before decellularity treatment.However,the size and depth of the pore size of scaffolds with different degrees of degeneration were inconsistent,and the arrangement was irregular.4.In vitro compatibility of human NP-dECM scaffolds with different degrees of degenerationThe results of CCK-8 showed that groups Ⅱ-D,Ⅲ-D and Ⅳ-D showed good cytocompatibility and no obvious cytotoxicity.Human NPMSCs were inoculated on human NP-dECM scaffolds with different degrees of degeneration for in vitro three-dimensional culture,and live/dead cell staining was performed on the 28 th day.It was found that the number of live cells in group Ⅱ-D was the largest,distributed on the surface and inside of the scaffolds,and very few dead cells.The number of cells in group Ⅲ-D was lower than that in group Ⅱ-D,and the number of living cells was similar to that of dead cells.The cells were mainly distributed on the edge and surface of the scaffold.The number of cells in group Ⅳ-D scaffolds was less than that in the first two groups,and the distribution was sparse.The living cells with green fluorescence and dead cells with more red fluorescence could be seen,and the cells were mainly distributed on the edge and surface of the NP-dECM scaffolds.Conclusion:1.With the increase of the degree of degeneration,the contents of DNA,glycosaminoglycan and type Ⅱ collagen in human nucleus pulposus tissue generally showed a downward trend.2.In this study,the decellulated scheme of 5 freeze-thawed-cycles,2%Triton X-100,1%SDS combined with nuclease can successfully decellulated human nucleus pulposus tissue.The prepared NP-dECM scaffolds can be removed most of the cells and nucleic acid and other antigenic substances,while the extracellular matrix components and structure are well retained,with certain porosity and good compatibility.3.The retention of extracellular matrix components and the growth of human NPMSCs on scaffolds after acellular treatment were comprehensively evaluated.Human nucleus pulposus tissue with degree Ⅱ degeneration was the most suitable choice for preparing extracellular matrix scaffolds,followed by human nucleus pulposus tissue with degree Ⅲdegeneration,and finally human nucleus pulposus tissue with degree Ⅳ degeneration.