Effects Of Exosomes Derived From Periodontal Ligament Stem Cells On Biological Characteristics To Inflammatory Periodontal Ligament Stem Cells

Objective: Exosomes were derived from culture supernatant of periodontal ligament stem cells(PDLSCs)in healthy and periodontal conditions,the effects with exosomes on proliferation and osteogenic differentiation of P-PDLSCs were observed in vitro,to research the effect of exosomes on periodontal tissue regeneration by preliminary study.Methods:(1)The obtained,cultured and identified of periodontal ligament stem cells:primary periodontal ligament cells from health and periodontitis people were cultured by enzymatic digestion and passaged routinely;then we cultured healthy and inflammatory periodontal ligament stem cells(H-PDLSCs and P-PDLSCs)with limited dilution method,calculated the ability of cell clone formation and identified phenotype molecules related to PDLSCs by flow cytometry.(2)Isolation and identification of exosomes: the H-PDLSCs and P-PDLSCs of the third generation(P3)were cultured in Exo free fetal bovine serum(Exo FBS)medium for 48 h,we collected the cell supernatant and extracted exosomes by ultracentrifugation.The exosomes were identified by Transmission Electron Microscopy(TEM),Western Blot(WB)and Nanoparticle Tracking Analysis(NTA).(3)Co-culture of cells and exosomes: H-Exo and P-Exo were put into P-PDLSCs respectively,and the phagocytosis of exosomes were detected by inverted fluorescence;P-PDLSCs were treated with H-Exo and P-EXO respectively,while the control were P-PDLSCs,CCK-8 and clone formation were used to test the cell proliferation with exosomes;the effect of exosomes on osteogenic ability was detected by ALP and alizarin red staining;and the osteogenic related genes and proteins(COL1,RUNX2 and ALP)were further checked by RT-PCR and WB.Results:(1)H-PDLSCs and P-PDLSCs were cultured by enzymatic digestion and limited dilution method,cells grew in whirlpool shape with long spindle shape;flow cytometry showed that the two groups of cells highly expressed mesenchymal stem cell phenotype;after clone formation culture,the clone formation rates of the two groups were 35.50% ± 1.32% and 22.04% ± 1.45% respectively,and the difference was statistically significant(P<0.05).(2)Two groups of exosomes were extracted by ultracentrifugation,TEM,WB and NTA were used to further verify the extracts were consistent with the characteristics of exosomes.(3)Inverted fluorescence microscope showed that exosomes could get into cells,indicating that cells could internalize with exosomes;CCK-8 confirmed that the proliferation ability of cells with exosomes were higher than control group,especially with H-Exo(P<0.05);the results of cell clone formation were coincidence;ALP and alizarin red staining found that H-Exo treated group possessed the strongest mineralized formation potential,followed by P-Exo.The expression of COL1,RUNX2 and ALP detected by RT-PCR,the expression rate gradually increased in control,P-Exo and H-Exo group(P < 0.05),which was further verified by WB.Conclusion: Exosomes derived from PDLSCs can promote the proliferation and osteogenic differentiation of P-PDLSCs which H-Exo is better than P-Exo,indicating that exosomes derived from PDLSCs can participate in periodontal tissue regeneration and repair by “cell-free” way.