| Objectives To explore the effect of the combination of hyaluronic acid and domestic porous tantalum on the function of chondrocytes under three-dimensional dynamic culture,so as to provide an experimental basis for clinical cartilage injury and repair.Methods 1 The morphology of the domestic porous tantalum was observed by naked eye and electron microscope scanning.2 The knee joint chondrocyte culture of the primary and subculture rats.3 Type II collagen immune fluorescence,toluidine blue and alician blue staining were used to identify the chondrocytes and to observe the combined culture domestic porous tantalum by scanning electron microscope.4 The experiment consists of two parts: Firstly,CCK-8 and immunofluorescence staining are used to screen the concentration of hyaluronic acid(HA),and four groups are set up in the experiment: Group A:chondrocyte group(blank control group);Group B:Chondrocytes+HA100mg/L group;Group C:chondrocytes+HA200mg/L group;Group D:chondrocytes+HA300mg/L group;Secondly,to observe the effect of the compound hyaluronic acid and domestic porous tantalum on chondrocyte function in three-dimensional dynamic culture.The experiment is set up in five groups,Group A:cell static culture group;Group B: tantalum+HA+cell static;Group C:tantalum+HA+cell dynamic;Group D:tantalum+cell static;Group E: tantalum+Cell dynamics,ELISA and Western Blot experiments are used to detect the secretion and protein development by proteoglycan(Aggrecan,Agg),collagen type II(Collagen Type II,COL II)and SOX9(SRY-related HMG-box gene,9)at day-3,5 and 7.Results 1 The appearance of domestic porous tantalum is disc-shaped,gray-black,with rough surface,many small pores,the quality is hard,the thickness of about 3mm,the diameter of 15 mm,its micropore structure is uniformly distributed in the material by scanning electron microscope observation,and the pore diameter is 400~600?m,the pores are connected with each other,forming a three-dimensional structure.2 Under inverted phase contrast microscope,chondrocytes grew close to the wall,showing polygonal or irregular shape,full cytoplasm,and closely combined with porous tantalum under scanning electron microscope.3 The results of immunofluorescence of type ? collagen,toluidine blue and alison blue staining were all positive.4 CCK-8 method was used to detect cell proliferation.The results showed that there was no statistically significant difference in chondrocyte proliferation between different groups at the same time point(P>0.05),which indicated that HA had no effect on chondrocyte proliferation.The number of chondrocytes in the same group increased gradually from day 1 to day 5 and reached the peak in the fifth day,then decreased gradually from day 5 to day 7.5 Immunofluorescence staining method was used to detect the effect of different concentrations of HA on chondrocyte proteoglycan expression.The results showed that: Group A proteoglycan expression was lower than that of the other three groups,the difference was statistically significant(P<0.05),there was no statistically significant difference in the expression of proteoglycan in Group B,Group C,Group D(P>0.05)respectively,which indicated that HA can promote chondrocyte proteoglycan expression and was not affected by HA concentration.The expression levels of proteoglycans in Group B and Group C were both numerically higher than that in Group D,thus a medium with a HA concentration of 200mg/L was selected for ELISA and Western blot experiments.6 ELISA experiment results: At the same time point,Agg secretion B is higher than those in Group A and Group D,while lower than those in Group C and Group E,the difference was statistically significant(P<0.05).The Agg secretion in Group C and Group E was higher than thosein Group A and Group D,the difference is statistically significant(P<0.05),There was no statistically significant difference in Agg secretion between Group C and Group E,Group A and Group D(P> 0.05).At the same time,COL?secretion was higher in Group B than that in Group A and Group D,but lower than those in Group C and Group E,the difference was statistically significant(P<0.05),the COL? secretion of Group C and Group E was higher than those of Group A and Group D,the difference was statistically significant(P<0.05).There was no statistically significant difference in COL II secretion between Group C and Group E,Group A and Group D(P>0.05).At the same time,there was no statistically significant difference in SOX9 between the groups(P>0.05).The comparison within the group,the amount of secretion of Agg,COL?,and SOX9 in 5 days was higher than that in 3 and 7 days,the difference was statistically significant(P<0.05),however,the difference of the secretion amount of Agg,COL?,and SOX9 in 3 and 7 days was not statistically significant.No statistical significance(P>0.05).The secretion of Agg,COL-II and SOX9 gradually increased on the third day,reached the peak on the fifth day,and gradually decreased on the fifth to seventh days.7 Western Blot experiment results: There was no statistically significant difference in the Agg expression level between the three groups on day 3(P>0.05).On day 5,the Agg expression of Group B,Group C and Group E werw higher than that of Group A and Group D,the difference was statistically significant(P<0.05),There was no statistically significant difference in Agg expression between Group B,C and E,and between Group A and Group D(P> 0.05).On day 7,the Agg expression of Group B was higher than those of Group A and Group D,but lower than Group C and Group E was statistically significant(P<0.05).The Agg expression in Group C and Group E was higher than that in Group A and Group D,the difference was statistically significant(P<0.05),there was no statistically significant difference in Agg expression between Group C and Group E,between Group A and Group D(P>0.05).On day 3,there was no significant difference in the expression of COL? between the groups(P>0.05).On the day 5 and day 7,the expression of COL? in Group B was higher than those in Group A and Group D,but lower than that in Group C and Group E,the difference is statistically significant(P<0.05),The expression of COL? in Group C and Group E was higher than that in Group A and Group D,the difference was statistically significant(P<0.05).There was no statistically significant difference in the expression of COL? between Group C and E,Group A and Group D(P>0.05).At the same time point,there was no statistically significant difference in SOX9 between the groups(P>0.05).Conclusions 1 Hyaluronic acid combined with domestic porous tantalum can promote the secretion and protein expression of chondrocytes Agg and COL? in dynamic and static environments,and the dynamic environment is better than static one.2 The coculture of domestic porous tantalum and chondrocytes in a dynamic environment can promote the secretion and protein expression of Agg and COL?.3 SOX9 secretion and protein expression are not affected by dynamic and static culture environment and hyaluronic acid.Figure 15;Table 14;Reference 112... |
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