| Objective:The purpose of this study was to explore the physical properties,physical properties,and physical properties of silk fibroin(SF)-bone decellularized extracellular matrix(D-ECM)-hydroxyapatite(HA)composite tissue engineering scaffolds loaded with human urine-derived stem cells(USCs).Biocompatibility and stem cell growth and migration mode,and based on the traditional two-dimensional planar cell culture mode,try to explore the growth characteristics of stem cells in the three-dimensional culture mode of composite scaffolds..Methods:USCs were obtained by differential centrifugation combined with precipitation method,cell morphology at different proliferation stages was observed by inverted phase contrast microscope,surface markers of USCs were detected by flow cytometry,and USCs were induced to differentiate into osteogenic,chondrogenic and adipogenic cells.Complete the identification of urine-derived stem cells.Scaffold material selection and treatment The ternary solution process was used to extract the silk fibroin in the degummed silk and dialysis to purify it.The bone tissue decellularized extracellular matrix was obtained by a composite method of mechanical vibration and biological enzyme digestion.After adding a certain proportion of hydroxyapatite Composite scaffolds were prepared using freeze-drying techniques The physical properties were evaluated using the weighing method to measure the porosity of the scaffold at a constant temperature of 25℃,the swelling rate of the sample was measured using simulated body fluid,the elastic modulus was determined by the compression test of a universal chemical testing machine,and the degradation performance was evaluated by using trypsin,lysozyme and simulated body fluid.Finally,the urine-derived stem cells were loaded on the scaffold,and the microscopic morphology and biological activity of the loaded cells were evaluated by inverted phase contrast microscope,scanning electron microscope(SEM)and CCK-8 method,respectively.Combined with inverted phase contrast microscope,scanning electron microscope and fluorescence confocal microscope,the cell growth patterns and characteristics in the composite scaffold were observed and evaluated.Result:In the plane culture mode,the extracted cells were spindle-shaped or long-spindle-shaped.The results of flow cytometry showed that the expression of CD29,CD44,and CD73 was positive.The extracted cells could successfully complete the three-way differentiation of osteogenic,adipogenic and chondrogenic.Urine-derived stem cells The general appearance of the composite scaffold is sponge-like,and the microscopic appearance is a porous three-dimensional structure with high porosity and swelling ratio.It has better anti-degradation properties in a liquid environment The co-culture of cells and composite scaffolds showed that the biocompatibility of scaffolds was better than that of flat culture,which was conducive to cell adhesion and growth.In the three-dimensional culture mode of composite scaffolds,there were three modes of cell growth:one-dimensional,two-dimensional and three-dimensional,and the cell behavior was better than that of flat culture.It is more in line with the real situation in the body.Conelution:The extracted cells are identified as human urine-derived stem cells.The SF/D-ECM/HA composite scaffold has excellent physical properties and biocompatibility.The composite scaffold loaded with urine-derived stem cells can effectively simulte the three-dimensional culture of stem cells in vitro.The model is closer to the real situation in vivo than the two-dimensional planar culture. |
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