| BackgroundDecellularized extracellular matrix(dECM)derived from tissue or mesenchymal cells can promote nerve repair and regeneration and is a potential material for treating spinal cord injury(SCI).Dental pulp stem cells(DPSCs),originating from neural crest,are mesenchymal stem cells that can secrete a variety of neurotrophic factors and form rich extracellular matrix.However,there are no studies on extracellular matrix from dental pulp stem cell sheets and its therapeutic effect on spinal cord injury.Objective1.To construct and characterize the decellularized extracellular matrix from dental pulp stem cell sheets.2.To construct a T10 spinal cord hemisection model,and to analyze the therapeutic effect of DPSC dECM on SCI.3.To investigate the effects of DPSC dECM on migration and differentiation of NSCs.Methods1.DPSCs were isolated and cultured,and their biological characteristics were identified.The cultured DPSC sheets were treated with decellularized protocol of 0.5%TritonX-100 5 min,0.5%TritonX-100 10 min,0.5%TritonX-100 30 min,1%TritonX-100 5 min,1%TritonX-100 10 min or 1%TritonX-100 30min.Then,H&E staining,scanning electron microscopy,type Ⅰ collagen immunofluorescence staining,DNA quantitative analysis and CCK8 cytotoxicity test were conducted.2.The spinal cord injury model of T10 hemisection spinal cord injury rats were established.The experiment was divided into three groups:DPSC sheet group,DPSC dECM group and control group.BBB scale was performed on days 7,14,21 and 28 after transplantation,and hind limb footprint analysis was performed on day 28 after transplantation.H&E staining was performed on day 7 and 28 after transplantation,and immunofluorescence staining was performed on day 28 day for nerve filament protein(NF-200)and glial fibrillary acidic protein(GFAP).3.In vitro transwell experiment was used to study the effects of DPSC sheet and DPSC dECM on NSCs migration,and the effects of DPSC sheet and DPSC dECM on NSCs differentiation were studied by co-culture and real-time quantitative PCR(qRTPCR).Results1.The DPSCs isolated and cultured in the laboratory showed good proliferation ability by CCK8 assay,clonal formation ability by monoclonal assay,multidirectional differentiation ability by osteogenic,lipogenic and neurogenic induction experiments,and mesenchymal origin by flow cytometry.H&E staining,scanning electron microscopy and type I collagen immunofluorescence staining showed that DPSC dECM in 1%TritonX-100 5 min group could remove the cell structure and basically preserve the structure and components of extracellular matrix.DNA quantitative results showed that 1%TritonX-100 5 min DPSC dECM could remove cell components,and CCK8 method proved that 1%TritonX-100 5 min DPSC dECM had no toxic effect on NSCs.2.H&E staining results of spinal cord tissue at day 7 and 28 showed that the tissue cavity area in the DPSC sheet group and the DPSC dECM group was decreased compared with the control group,but there was no statistical difference between the two groups.On day 28,immunofluorescence staining of spinal cord NF-200 and GFAP showed that the fluorescence signal intensity of NF-200 in the DPSC sheet group and the DPSC dECM group was higher than that in the control group.Hind limb motor function score and hind limb footprint analysis showed that both DPSC sheet group and DPSC dECM group could promote the recovery of hind limb motor function after SCI.3.Transwell results showed that the number of NSCs migration cells in the DPSC sheet group and the DPSC dECM group was higher than that in the control group.QrtPCR results showed that Nefl expression of NSCs in the DPSC sheet group and the DPSC dECM group increased after co-culture.Conclusion1.The cells isolated and cultured were DPSCs derived from mesenchymal and capable of clonal formation and multidirectional differentiation.1%TritonX-100 5 min was the best condition for the preparation of DPSC dECM,which could remove the cell structure and components of it,and basically retain the structure and components of extracellular matrix,without cytotoxicity,and could be used for subsequent experimental studies.2.Both DPSC dECM and DPSC sheet can promote nerve repair and regeneration after spinal cord injury,and promote the recovery of hind limb motor function in rats.3.Both DPSC dECM and DPSC sheet can promote the migration of NSCs and promote the differentiation of NSCs. |
