| Objective:Construct graphene oxide/polylactic acid（GO/PLLA）composite scaffold combining graphene oxide（GO）and polylactic acid（PLLA）,test its biocompatibility,and further explore its ability to proliferate human dental pulp stem cells（hDPSCs）,adhesion,migration and osteogenic differentiation.Methods:（1）The GO/PLLA with GO mass fractions of 0.15%,0.20%,0.25%were obtained by combining GO of different masses with the same amount of PLLA,and the surface morphology of the composite stent was detected by scanning electron scanning microscope.Use Fourier transform infrared spectroscopy to analyze and detect the composition of the stent.（2）After the GO/PLLA was fully disinfected,it was immersed in a complete medium containing 10%FBS,and placed in a cell culture environment at 37°C and 5%CO2 for48 h.The OD value at 450 nm wavelength was detected by CCK-8 method on 1,3,and5 days to detect the biocompatibility of the stent.In addition,the cells were directly seeded on the scaffold,and the effect of GO/PLLA on the morphology of hDPSCs was observed by live-dead fluorescence staining after 3 days.（3）hDPSCs was seeded on GO/PLLA.After 12 h,the cytoskeleton was stained and observed under a fluorescent inverted microscope to verify the adhesion effect of cells on the surface of GO/PLLA.（4）CCK-8 method was used to detect the effects of GO/PLLA with different mass fractions on the proliferation ability of hDPSCs.（5）The effect of the scaffold on cell migration was detected by the scratch experiment:hDPSCs was cultured with carrier extract without FBS,and photographed under a microscope every 6 h,and the scratch area was statistically analyzed.（6）The GO/PLLA is soaked in the osteogenic induction liquid,and the obtained GO/PLLA osteogenic induction liquid is used for the induction of cell mineralization.Alizarin red S staining was performed 14 days after induction;alkaline phosphatase staining and ALP activity were performed on 7 and 14 days respectively.（7）After culturing the cells with GO/PLLA extract for 7 and 14 days,the expression of hDPSCs osteogenic differentiation-related genes（RUNX2,ALP,COL1 and OCN）was detected,and the osteogenic differentiation ability of the cells was analyzed.Results:（1）GO was dispersed in the PLLA solution to obtain GO/PLLA composite scaffolds of different concentrations,which appear as white flocculent solids.Scanning electron microscopy showed that PLLA was in the shape of a corrugated sheet,and GO adhered to its surface in the form of small balls.Fourier transform infrared spectroscopy analysis showed that a new chemical bond was formed,indicating the combination of the two.（2）After culturing the cells with GO/PLLA extract,the proliferation ability of hDPSCs was not significantly different from that of the control group.Live-dead fluorescent staining revealed that after GO/PLLA culture,hDPSCs was in the shape of a long spindle,with elongated protrusions,clear outline,uniform cytoplasm,and the nucleus was located in the center of the cell,similar in shape to the negative control group without significant difference.（3）Cells seeded on GO/PLLA were stained with cytoskeleton.Fluorescence inverted microscope showed that hDPSCs can adhere to the surface of GO/PLLA,and the cells are long spindle or polygonal,with cell protrusions and uniform cytoplasm.The nucleus is round and located in the center of the cell.（4）When the GO mass fraction in the GO/PLLA composite scaffold is 0.15%,the cell proliferation ability is enhanced,and there is a statistical difference higher than 0.20%and 0.25%GO/PLLA;while the GO mass fraction is 0.20%,0.25%At this time,the cell proliferation rate has a downward trend.（5）The results of the scratch experiment showed that the migration rate of 0.15%GO/PLLA cells was higher than that of the control group,and the other two groups were lower than the control group.（6）After culturing hDPSCs with GO/PLLA for 14 days,Alizarin Red S staining showed the formation of calcified nodules.The alkaline phosphatase staining was positive on the7th and 14th day,and the ALP activity was enhanced.（7）The expression of osteogenic differentiation-related genes（RUNX2,ALP and COL1）increased to varying degrees,but there was no significant difference in the expression of OCN.Conclusion:（1）GO and PLLA are successfully combined,and GO/PLLA has good biocompatibility to hDPSCs.Cells can adhere to the surface of the scaffold and maintain its normal growth morphology.（2）When the GO concentration is 0.15%,GO/PLLA can promote the proliferation ability and migration speed of hDPSCs.（3）GO/PLLA can promote the osteogenic differentiation of hDPSCs. |
PDF Full Text Request