Effect And The Possible Machanism Of LIPUS-stimulated Human Dental Follicle Stem Cell-derived Exosomes On The Proliferation And Differentiation Of HDFSCs

Objective:This study aimed to reveal the role of exosomes derived from human dental follicle stem cells(hDFSCs)in proliferation and differentiation of hDFSCs through cell molecular biology techniques.Low-intensity pulsed ultrasound(LIPUS)was given as a stimulator,and its effect on the characteristics of exosomes was evaluated as well as the possible mechanisms.Methods:1.Human dental follicle stem cells were isolated from dental sac tissue of impacted teeth of patients aged 10-14.The ability of osteogenic and adipogenic differentiation was evaluated by alizarin red staining and oil red O staining.The surface markers of hDFSCs were assessed by flow cytometry.2.The exosomes(con-Exo and LIPUS-Exo)of hDFSCs were isolated by ultracentrifugation.Western blot(WB)was performed to identify the surface markers CD9,CD63,TSG101 of exosomes.Transmission electron microscope(TEM)was used to observe the morphology,and nanoparticle tracking analysis(NTA)was applied to detect the concentration and diameter distribution of exosomes.Exosomes were stained by PHK26 to examine endocytosis and the distribution of exosomes.3.The functional experiment was divided into three groups:(1)control;(2)con-Exo;(3)LIPUS-Exo.The CCK8 test measured the effect on the hDFSCs proliferation ability induced by exosomes.After 7 days induction of osteogenic differentiation,alkaline phosphatase(ALP)staining,ALP activity,and osteogenic gene expression were detected.Calcium nodule mineralization was analyzed by alizarin red staining after 21 days of induction.4.The mechanism investigation: the osteogenesis experiment was divided into 6 groups:(1)control-0?M GW4869;(2)control-10?M GW4869;(3)control-20?M GW4869;(4)LIPUS-0?M GW4869;(5)LIPUS-10?M GW4869;(6)LIPUS-20?M GW4869.First,hDFSCs were treated with different concentrations(0?M,10?M,20?M)of GW4869,an inhibitor of exosome secretion.Then,group(4)-(6)were treated with LIPUS during 7 days of osteogenesis.After 7 days of induction,ALP staining and RT-PCR were performed to evaluate the role of LIPUS in the release or characteristics of exosomes.The inflammatory inhibition experiment was divided into 5 groups:(1)control;(2)LPS;(3)LIPUS;(4)LIPUS+LPS;(5)LIPUS+LPS GW4869.hDFSCs were treated by GW4869,and LPS was added to stimulate the secretion of inflammatory factors.The expression of inflammatory factors was detected by RT-PCR after LIPUS treatment.The production experiment was divided into 2 groups:(1)GW4869;(2)GW4869+LIPUS.hDFSCs were treated by GW4869,and LIPUS was applied to group(2)for 30 minutes.WB detection was used to detect the expression of exosome secretion-related proteins.Results:1.The primary hDFSCs were successfully isolated from dental sac tissue of impacted teeth of patients aged 10-14,and they have osteogenesis and adipogenesis potentials.The results of flow cytometry showed that hDFSCs expressed CD90,CD105,CD146,but did not express CD31 or CD45.2.The exosomal marker proteins CD9,CD63,and TSG101 were detected in con-Exo and LIPUS-Exo derived from hDFSCs,and the expression of these proteins in LIPUS-Exo was higher than con-Exo.TEM analysis showed that con-Exo and LIPUS-Exo were round,membranebound vesicles ranging from 30-100 nm in diameter,with no apparent differences in morphology between con-Exo and LIPUS-Exo.NTA showed that the size of exosomes mainly ranged from 70-180 nm,with a mean size of 130 nm,with no distinguishing differences between the con-Exo and LIPUS-Exo.Exosomes uptake by hDFSCs were confirmed by fluorescence microscopy,indicating that the PHK26-labeled exosomes had been taken up and transferred into the cytoplasmic compartment.3.The result of CCK8 test proved that the proliferation ability of hDFSCs was enhanced after con-Exo and LIPUS-Exo treatment.After 7 days of osteogenic differentiation,ALP staining was deeper in exosomes treated groups than in control group.Meanwhile,exosomes could strengthen the ALP activity of hDFSCs and increase ALP,Runx2 and Col-1 gene expression.After 21 days of osteogenic differentiation,alizarin red staining was deeper in exosome treated groups than in control group.The effect of LIPUS-Exo was stronger than con-Exo.4.The results of ALP staining,RT-PCR and WB showed that GW4869 could significantly block the osteogenic differentiation of hDFSCs in a concentration-dependent manner and reduce the expression of exosome secretion-related proteins.When treated with LIPUS,ALP staining was deeper,osteogenic gene expression was higher,and the expression of exosome secretion-related proteins was higher than control groups.The expression of IL-8 increased after GW4869 inhibited exosome secretion,while the expression of IL-8 decreased after LIPUS treatment.Conclusion:In conclusion,this study indicated that hDFSCs-derived exosomes could uptake by hDFSCs.These exosomes could promote the ability of proliferation and osteogenic differentiation of hDFSCs,and LIPUS was proved to enhance these abilities.Besides,the effects of LIPUS may be partly attributed to the increased expression of exosome secretion-related proteins of hDFSCs,which promoted the production of hDFSC-derived exosomes,and the inhibition of inflammation.LIPUS-stimulated hDFSC-derived exosomes could provide a potential treatment for periodontal regeneration.