| objective : in the correlation studies about treating bone defects,Lithium has been founds with osteogenic effects in recent years.The purpose of this study is to analyze the effect of lithium on the proliferation of cells and the effect of promoting osteogenesis in vitro cell culture model,and explore the optimum concentration of its function.combine lithium and polypeptide hydrogelto in this concentration,to explore the effect of this culture on the osteogenic differentiation of cells,and if it is better than using lithium alone,in order to provides a new way for the clinical oral implant or treatment for bone defect.Methods: MC3T3-E1 cultured in vitro for 8days.Then,use CCK-8 assay to record cells multiplication and draw the corresponding growth curve,so that provide reference for the follow-up experiment.The concentration of Li Cl is divided into 5 groups:0mmol/L、5mmol/L 、 10mmol/L 、 20mmol/L and 40mmol/L,and act on cells respectively,observe the effect of cells proliferation rate in 3 days,test the effect of osteogenesis of cells on every concentration through detecting the ALP activity after 5days,select the optimal concentration for cells proliferation and osteogenesis to be the following experimental concentration.Dissolve rada16-Ⅰ dry powder in deionized water,fully mix both of them to be well-distributed by sonic oscillation,then asepticly filter it,lining up to the polypeptide aqueous solution rada16-Ⅰ of 1%(W/V),adding a small amount of PBS solution into it to make the polypeptide solution transform into a gelatinous substance voluntarily,then,dry and make the SEM scanning sample to observation morphology of the hydrogel.choose the optimum concentration of the previous part,adding rada16-Ⅰ that loaded the optimum concentration Li Cl,the optimum concentration of Li Cl without rada16-Ⅰ,and no intervention to the cells separately,extract m RNA after 4 days,real-time PCR was used to detection of the expression of specific gene OSX and Runx2 for cells osteogenesis,analyse if there is obvious difference between the combined effect of Li Cl and rada16-Ⅰ and with the treatment of Li Cl alone.Results:The attached cells are fibroblast like in shape.Cell growth curve,roughly“S”shaped,showed a quick proliferation in 3-6 days after inoculation,.In contrast of cells multiplication capacity by CCK-8 assay after treating cells with different concentration of Li Cl,the amount of cells in low concentration 5mmol/L and 10mmol/L group were increased in comparison with the control group,which the amount of 5mmol/L cells was the most.The cells proliferation in concentration of 20mmol/L and 40mmol/L was limited,and the cells morphology had been changed,which the cells proliferation was obviously limited in concentrtion of 40mmol/L.ALP activity showed that concentration of 5mmol/ L has higher ALP activity.There is no significant difference in ALP activity between the concentrtion of 10mmol/L and the untreated group.The ALP activity of 20mmol/L and 40mmol/L is both decreased.The self-assembled polypeptide hydrogel,triggered by PBS,showing colourless and ability to maintain a certain form,had a low level fluxility.Through the SEM,the internal structure of the hydrogel had a porous structure.Real-time PCR was performced to detect the general conditions of OSX and Runx2 of cells treated by different intervention modes.The result showed that Li Cl,whether loaded on rada16-Ⅰ or not,had a higher expression of OSX and Runx2 genes than that of the compared group,further more,the gene expression of rada16-Ⅰ loaded of Li Cl is higher than use Li Cl alone,the differences of the gene expression are statistically significant.Conclusion:Li Cl has the ability to promote cells proliferation and osteogenesis,and the polypeptide hydrogel,as a scaffold loaded with Li Cl,can do it better. |
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