| Objectives To study the effect of TGF-?1 and IGF-1 on the induction of domestic porous tantalum composite BMSCs into chondrocytes in SD rats,observe the secretion function of porous tantalum-BMSCs composite cells and the effect on the expression of cartilage related factors COL-II,AGG and COL-X,so as to explore the effect of domestic porous tantalum composite BMSCs on cartilage injury repair,in order to provide theoretical and experimental basis for clinical cartilage repair.Methods 1 The Morphological characteristics of domestic porous tantalum materials were observed under gross and scanning electron microscopy;2 Acquisition,culture and purification of BMSCs from SD rats;3 BMSCs identification: the 3rd generation BMSCs were induced by osteoblasts,chondroblasts and adipocytes for 14 days and stained with alizarin red,Alcian blue and oil red O respectively;4 Domestic porous tantalum was cocultured with the 3rd generation BMSCs,and CCK-8 was used to measure the cell proliferation of BMSCs at different time points;5 The 3rd generation BMSCs were cocultured with porous tantalum to induce chondrogenesis and observe cell grow by SEM and TEM;6 Experimental grouping: TGF-?1+BMSCs group(group a),TGF-?1+BMSCs+porous tantalum group(group b),TGF-?1+IGF-1+BMSCs group(group c),TGF-?1+IGF-1+BMSCs+porous tantalum group(group d);7 ELISA was used to detect the effects of COL-II,AGG,COL-X secretion of cells after domestic porous tantalum scaffolds combined with BMSCs after cartilage induction for 7d,14 d and 21d;8 QPCR was used to detect the effects of COL-II,AGG,COL-X mRNA expression on cell secretion after porous tantalum scaffold combined with BMSCs on 7th,14 th and 21 st day after cartilage induction of each component;9 Western-blot was used to detect the effects of COL-II,AGG,COL-X protein expression on cell secretion after domestic porous tantalum scaffold combined with BMSCs on 7th,14 th and 21 st day after cartilage induction of each component.Results 1 The domestic porous tantalum stent material has a diameter of about 15 mm,a thickness of about 3mm,a disk-shaped appearance,hard gray color,uneven surface,and honeycomb pores uniformly distributed on the stent surface.Under scanning electron microscope,the domestic porous tantalum stent has a large number of uniformly distributed micropore structures,and the interconnected pores present a three-dimensional porous structure,which is similar to the human cancellous bone structure.2 Primary BMSCs have fewer adherent cells,scattered distribution,spindle shape,irregular shape,and a small number of round cells.The number of subcultured cells gradually increased with the extension of culture time,and the cells were long spindle-shaped with uniform cell morphology.3 BMSCs identification results: 14 days after osteogenesis,chondrogenesis and lipogenesis induction,alizarin red staining,Alcian blue staining and oil red O staining were all positive.4 The results of CCK-8 showed that the cell growth of BMSCs group was superior to that of porous tantalum-BMSCs group on the 3rd and 5th days,and the difference was statistically significant(P<0.05).There was no significant difference in cell proliferation between the 1st day,7th day,9th day and 11 th day(P>0.05).5 Results of SEM and TEM: BMSCs after induction were embedded into the trabecular structure of porous tantalum,and the cells grew well.6 ELISA results showed that on the7 th,14th and 21 st day,there was no statistical difference in the secretion of COL-II,AGG and COL-X between group a and group b,and between group c and group d(P>0.05).However,the comparison between groups showed that the secretion of COL-II and AGG in group C and D were higher than those in group A and group B(P<0.05);the secretion of COL-X was lower than that of the former(P<0.05).The secretion of COL-II and AGG increased gradually(P<0.05)and the secretion of COL-X decreased gradually(P<0.05)with time prolonging.7 QPCR results showed that there was no statistical difference in the expression of COL-II,AGG,COL-X mRNA between group a and group b,group c and group d at three time points(P>0.05).However,the inter-group comparison showed that the expressions of COL-II and AGG mRNA in group C and group D were higher than the former(P<0.05);The expression of COL-X mRNA was lower than that of the former(P<0.05).8 Western-blot results showed that there was no significant difference in the expression of COL-II,AGG,COL-X protein between group a and group b,group c and group d at three time points(P>0.05).However,the inter-group comparison showed that the expressions of COL-II and AGG proteins in group C and group D were higher than the former(P<0.05);The expression of COL-X protein was lower than that of the latter(P<0.05);With the extension of time,the expression of COL-II and AGG protein in each group gradually increased,and the expression of COL-X protein gradually decreased(P<0.05).Conclusions Compound culture of chondrogenic cytokines IGF-1,TGF-?1 and domestic porous tantalum material can promote differentiation of BMSCs into chondrocytes and accelerate cartilage formation in SD rats.IGF-1/TGF-?1/BMSCs/ porous tantalum complex can make the cartilaginous factors COL-II,AGG genes and proteins highly expressed while COL-X genes and proteins are poorly expressed during cartilaginous process.So as to reduce chondrocyte dedifferentiation and promote chondrocyte differentiation.Figure 22;Table 17;Reference 96... |
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