| Purpose: 1.Verify whether h ADSC-EV can promote osteogenic differentiation 2.Explore the feasibility of using MSC-EV for surface modification of Ti materials;3.Develop a method for anchoring MSC-EV on metal implant surfaces;4.Clarify whether the Ti material modified by h ADSC-EV has better ability to promote bone regeneration,and provides a new idea for surface modification of clinical medical metal implants.Methods: 1.h ADSC-EV was prepared by ultracentrifugation and identified by SEM,particle tracking analysis and Western-blot.Human osteoblast-like cell MG63 was used as the target to test whether h ASC-EV could promote osteogenic differentiation by alizarin red staining and calcium quantitative detection;h ASC was treated by gene chip and bioinformatics analysis to screen the substance that may exert this biological effect.2.Bio-Ppy-Ti(Biotin-polypyrrole-titanium)was prepared by potentiostatic method,and the experimental groups were set up: Ti,Ppy-Ti and Bio-Ppy-Ti.The surface morphology,roughness and biotin loading amount were characterized by means of SEM,AFM and fluorescence imaging.The biotin-modified h ADSC-EV was incubated on the surface of BioPpy-Ti,and the residues of h ADSC-EV were compared by confocal observation on the 0th,7th and 14 th day.The EV residues on the surface of the materials were detected to determine the long-term effectiveness of anchoring.The biotin-modified h ADSC-EV was incubated on the surface of each group of materials and then treated by ultrasonic vibration.The EV residues on the surface of each material were observed by SEM and confocal microscopy,and the stability of the anchoring was tested.3.Set up experimental groups: Ti,Bio-Ppy-Ti,EV-Bio-Ppy-Ti.Human osteoblast-like cells MG63 were inoculated on different materials.The morphology and adhesion of human osteoblast-like cells at different time points(6h,12 h,24h)were compared by F-actin staining.Human osteoblast-like cells MG63 were inoculated on different materials and cultured for 14 days.After cultured on different materials,the cells were observed by q RT-PCR and immunofluorescence.The expression of osteogenesis-related genes(ALP,COL1,OCN)and protein(COL1,OCN)were observed.Human osteoblast-like cells MG63 were inoculated on different materials and cultured for 14 days.Subcutaneous implantation was performed in nude mice(Balb/c,female,8-9 weeks old).HE and immunohistochemical analysis were performed on skin tissue one month later.Results: 1.The total intracellular and extracellular calcium content of MG63 cells that cultured with 10?g/m L h ADSC-EV for 14 days was 1.1855±0.1860 mmol/L,while the blank control group was 0.1418±0.0120 mmol/L,and the negative control group(h DF-EV)was 0.1729±0.0370.The calcium content of the h ADSC-EV group was significantly higher than that of the blank control group and the negative control group.Five mi RNAs that regulate osteogenic differentiation were screened in the top 30 h ADSC-EV mi RNAs.2.Bio-Ppy coating was successfully polymerized on Ti surface by electrochemical polymerization.When the electrochemical polymerization conditions were 1.5v and 120 s,the biotin doping amount on the surface of Bio-Ppy-Ti was the highest.After biotinylated h ADSC-EV was incubated on Bio-Ppy-Ti for 7 days and 14 days,the residual amount of EV was 96.17% and 94.67% of the initial state,respectively.After the biotinylated h ADSC-EV was incubated to the materials in each group for 30 s of ultrasonic shock treatment,the EV residuals on the surface of Ti,Ppy-Ti and Bio-Ppy-Ti were 0.34%,0.67% and 62.74% of the initial state,respectively,and the Bio-Ppy-Ti group was significantly higher than the Ti group and Ppy-.Ti group.3.After MG63 cells were inoculated on the surface of different materials for 6 hours,the projected area of cells on the surface of Ti,Ppy-Ti and Bio-Ppy-Ti were 946.42±69.32?m2?841.69±147.98?m2 and 1361.26±163.14?m2,respectively.The Bio-Ppy-Ti group was significantly higher than the Ti group and the Ppy-Ti group.After MG63 cells were inoculated on different materials for 14 days,the expression levels of ALP gene,COL1 gene and OCN gene in EV-Bio-Ppy-Ti group were 1.27 times,1.68 times and 1.82 times higher than those in Ti group,respectively.One month after subcutaneous implantation,OCN positive cells were found in subcutaneous tissues of EV-Bio-Ppy-Ti group,but none of OCN positive cells were found in subcutaneous tissues of Ti group and Bio-Ppy-Ti group.Conclusions: 1.h ADSC-EV can promote osteogenic differentiation of osteoblast-like cells MG63 in vitro;h ADSC-EV is rich in a variety of micro RNAs that can regulate osteogenic differentiation.2.Bio-Ppy coatings can be successfully polymerized on Ti surface by electrochemical polymerization.Bio-Ppy-Ti surface has the largest amount of biotin doping when the electrochemical polymerization conditions are 1.5V and 120 s.After 7 and 14 days incubation of biotinylated h ADSC-EV on Bio-Ppy-Ti surface,there is no significant decrease in EV residue.After the biotinylated h ADSC-EV was incubated to the materials of each group for 30 s of ultrasonic shock treatment,the EV residue on the surface of bio-ppy-ti was 182 times and 91 times of that of Ti and ppy-ti,respectively.3.Ti modified by h ADSC-EV can promote the early adhesion and extension of osteoblasts,and can promote the expression of osteoblast-related genes and proteins in osteoblasts in vivo and in vitro. |
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