| Objectives To observe the effect of bone morphogenetic protein 7(Bone morphogenetic protein-7,BMP-7)combined with domestic porous tantalum on chondrogenic differentiation of Bone marrow mesenchymal stem cells(Bone marrow mesenchymal stem cells,BMSCs).Provide experimental basis for clinical cartilage defect repair.Methods 1 The surface characteristics of domestic porous tantalum scaffolds were observed by scanning electron microscopy.2 Bone marrow mesenchymal stem cells of 3-week-old male SD rats(provided by Beijing huafukang Biology)were isolated and cultured,and the cell morphology was observed by scanning.3 BMSCs were identified by flow cytometry.4 BMSCs and porous tantalum compound culture growth state observation group: experimental group cells and porous tantalum compound culture,control group cell culture alone.The morphological characteristics of BMSCs and domestic porous tantalum on the 3rd,5th and 7th day were observed by inverted phase contrast microscope.5 The growth of BMSCs and porous tantalum on the 5th day was observed by ghost pen cyclic peptide staining.6 The cell proliferation was detected by CCK-8 method on the 1st,3rd,5th,7th day of culture.7 BMP-7 intervention group: group A: BMSCs + chondrocyte inducer;group B: BMSCs + chondrocyte inducer + BMP-7;group C: BMSCs + chondrocyte inducer + porous tantalum;group D: BMSCs + chondrocyte inducer + porous tantalum + BMP-7.On the 3rd,5th and 7th day of induction,toluidine blue staining and type II collagen(Col-II)immunofluorescence staining were used to observe and identify the morphology of cells in groups A and B.8 On the 7th,14 th and 21 st day of induction,scanning electron microscopy was used to observe the composite culture of cells and domestic porous tantalum scaffold materials.9 On the 7th,14 th and 21 st day after induction,the level of type Ⅱ collagen,SRY type high mobility group protein(Sox-9),matrix metalloproteinase 13(Mmp-13)secreted by cells was detected by ELISA.10 Western blot was used to detect the expression of type Ⅱ collagen,SRY type high mobility group protein and matrix metalloproteinase 13 in the cells on the 7th,14 th and 21 st day after induction.11 On the 3rd and 7th day after induction,the changes of calcium ions in the chondrogenic differentiation of BMSCs were detected by laser confocal microscopy and fluorescence microscopy.Results 1 The porous tantalum scaffolds made in China have three-dimensional porous structure,which is similar to human cancellous bone.2 The newly inoculated BMSCs are of various types,and the cells are not easy to be classified.With the increase of passage times,the cells are evenly distributed,and the third generation BMSCs are long fusiform and whirlpool like dense growth.3 The expression rate of CD45 was 0.82%,CD29 and CD90 were positive,the positive rates were 96.91% and 93.55%,respectively.The identification results showed that the third generation of BMSCs was of high purity.4 The results of inverted phase contrast microscope showed that BMSCs grew around porous tantalum in the experimental group,and the number of cells around tantalum increased with the increase of culture time,which proved that tantalum had good biocompatibility.5 The results showed that the cells grew and proliferated well on and around the porous tantalum surface.6 CCK-8 method showed that the proliferation of the experimental group was slower than that of the control group(P < 0.05)on the 3rd and 5th day of culture,and there was no difference between the experimental group and the control group on the 1st and 7th day of culture(P > 0.05).It is suggested that the domestic porous tantalum scaffolds have good cell compatibility.7 Toluidine blue staining showed that BMSCs successfully induced cartilage differentiation.8 Immunofluorescence staining of type II collagen showed that BMSCs induced chondrogenic differentiation successfully,and the induction amounts of chondrocytes gradually increased with the prolongation of induction time.9 Scanning electron microscopy showed that the cells grew and proliferated well on the surface and inside of porous tantalum,with the increase of culture time,the cells gradually covered with porous tantalum.10 The results of ELISA showed that on the 7th,14 th and 21 st day of culture,the expression of type II collagen and SRY high mobility group protein increased gradually among groups A,B,C and D(P < 0.05).At 7 days,the expression of matrix metalloproteinase-13 decreased gradually among groups A,B,C and D(P < 0.05).At 14 days,matrix metalloproteinase-13 secretion of matrix in group A was highest compared with that in group B,group C and group D(P < 0.05),but there was no significant difference between groups B,C and D(P > 0.05).At 21 days,there was no significant difference among groups A,B,C and D(P > 0.05).11 Western blot assay showed that at 7,14 and 21 days after culture,the expression level of type II collagen and SRY high mobility group protein increased gradually in groups A,B,C and D(P < 0.05).At 7 days,the expression of matrix metalloproteinase-13 decreased gradually in groups A,B,C and D(P < 0.05).At 14 days,the expression of matrix metalloproteinase-13 was higher in group A than in groups C and D(P < 0.05),and higher in groups B and C than in group D(P < 0.05).At 21 days,the expression of matrix metalloproteinase-13 was higher in group A than in groups B,C and D(P < 0.05).No significant difference was found among groups B,C and D(P > 0.05).12 The results showed that the fluorescence intensity of group B was stronger than that of group A(P < 0.05),the fluorescence intensity of calcium ion in group D was stronger than that in group C(P < 0.05),indicating that BMP-7 group had better induction effect than BMP-7 group.Conclusions 1 Bone morphogenetic protein 7 combined with domestic porous tantalum can promote the differentiation of BMSCs into cartilage.2 Bone morphogenetic protein 7 combined with domestic porous tantalum can enhance the expression of Col-II and Sox-9 protein in induced chondrocytes,and increase the intracellular calcium concentration,which has a certain protective effect on the function and stability of chondrocytes.3 Bone morphogenetic protein 7 combined with domestic porous tantalum can reduce the production of chondrocyte Mmp-13 and ensure the stability of bone marrow mesenchymal stem cells to chondrocytes.It is suggested that chondrogenic differentiation of MSCs may be a functional cell for cartilage defect repair.Figure20;Table13;Reference 107... |
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