Effects Of Platelet Lysate Combined With Domestic Porous Tantalum On Osteoblast Function Under Dynamic Mechanical Stimulation

Objectives A Rotary Cell Culture System（RCCS）was established to investigate the effects of platelet lysate（PL）combined with domestically produced porous tantalum on osteoblast-related protein expression and osteoblast differentiation under dynamic mechanical stimulation.Methods 1 The morphology of porous tantalum was observed under microscope and macroscopically.2 The platelet lysate was prepared by repeated freeze-thaw method,the domestic porous tantalum was modified with PL,and the growth state of the cell-porous tantalum composite was observed.3 CCK-8 method to screen the best volume fraction of PL.4 Experimental grouping: MG63 cell group（group A）,MG63 cell + porous tantalum group（group B）,MG63 cell + porous tantalum + dynamic culture group（group C）,MG63 cell + platelet lysate + porous tantalum group（group D）,MG63 cells + platelet lysate + porous tantalum + dynamic culture group（group E）.5 ELISA method was used to detect the expression of osteoblasts in each group.6 Western blot method was used to detect the expression of osteogenic factor.7 Detection of alkaline phosphatase activity and calcium ion content in cells of each group.8 Detect the content of lactic acid and glucose in dynamic group and static group.Results 1 The porous tantalum metal material is a disc-shaped metal with a diameter of about 1.5cm and a thickness of about 0.2cm.The appearance is gray,with a metallic luster,and the surface is uneven.Many small particles can be seen.Under the scanning electron microscope,small particles can be seen.The honeycomb-shaped microporous structure,the pores are connected to each other,the pore size is uniform,and there is also a microporous structure in the pore wall.The surface of porous tantalum modified by PL is more rough and uneven,and there are medium deposits in the pores.The deposits are well combined with porous tantalum and distributed evenly.2 Observation by inverted phase contrast microscope shows that the cells grow steadily in the porous tantalum sheet.The cells are in good condition and full of shape.The prosthetic foot sticks around the porous tantalum.As the culture time increases,the number of cells gradually increases and adheres and fuse in the porous tantalum pore And gathered into a cell mass.Scanning electron microscopy showed that the cells and porous tantalum metal were closely adhered,and the surrounding cell matrix was secreted.The cells protruded and overlapped the pseudopods,and some cells and pseudopods penetrated into the pores.The porous tantalum modified by platelet lysate is more tightly bound to the cells,the pseudopodia of the cells are stretched more fully,and the extracellular matrix is secreted more vigorously.Fluorescence microscopy observed the complex of phalloidin staining,showing the microfilament structure of the cell.The fluorescence images overlapped with the porous tantalum,indicating that the microfilament structure and nucleus penetrated into the pore,indicating that the cell and porous tantalum co-growth and penetrated into the pore.3 After culturing for 1,2,and 3 days,the number of cell proliferation in the PL group was more than that in the blank group,and the difference was statistically significant（P<0.05）.On the fourth day,the number of cells in the 7% and 10% concentration groups was significantly higher than that in the other experimental groups（P<0.05）.It shows that PL can promote the proliferation of MG63 cells,and 7% volume fraction of platelet lysate is the best volume fraction.4 ELISA method was used to detect differences in the expression levels of COL-1,RUNX2,and OPN in the culture supernatant.On the third and fifth days of culture,there was no difference in the expression levels of groups A and B（P>0.05）.On the seventh day of culture,B The secretory expression of group A was more than that of group A（P<0.05）,the expression of group D was more than that of groups B and C（P<0.05）,and the expression of group E was more than that of groups D and C.The difference was statistically significant（P<0.05）.5 Western blot method was used to detect the expression difference of COL-1,RUNX2 and OPN protein in cells.Each group was cultured for 3 days.There was no statistically significant difference between the three proteins in groups A,B and C（P>0.05）.On the 7th day of culture,the protein expression of group E was higher than that of the first 4 groups,the difference was statistically significant（P<0.05）,the results are basically consistent with the ELISA results.6 The results of ALP activity and Ca²⁺content showed that the ALP activity and Ca²⁺content of group E were higher than those of the first 4 groups,and the ALP activity and Ca²⁺content of group D were higher than those of groups B and C.The difference was statistically significant（P<0.05）.7 The lactic acid content of the dynamic culture group and the static culture group increased with the increase of the culture time,and the difference between the groups was statistically significant（P<0.05）.The content of lactic acid in the static culture group was significantly higher than that in the dynamic culture group at 3 and 5 days（P<0.01）.There was no difference in the 7-day lactic acid content between the two groups（P>0.01）.The glucose content in the dynamic culture group and static culture group decreased with the increase of time at 3 days,5 days and 7 days（P<0.05）.The glucose content in the dynamic culture group on the 3rd,5th and 7th days was significantly more than that in the static culture group at the same time,the difference was statistically significant（P<0.01）.Conclusions Platelet lysate combined with domestic porous tantalum under dynamic mechanical stimulation can promote the expression of osteoblast COL-1,RUNX2,OPN factors,can increase osteoblast ALP activity and calcium ion content,stimulate the expression and mineralization of osteoblast-related factors and It can improve cell growth environment,increase glucose utilization rate and reduce lactic acid accumulation.Figure 21;Table 14;Reference 131...