| Objective: Exosomes are nano-scale extracellular vesicles secreted by cells and new carriers for the transfer of genetic material between cells.They play a key role in many pathological and physiological processes such as tumor markers,cancer treatment,and tissue regeneration.Stem cells from human exfoliated deciduous teeth(SHED)are a type of stem cells with high proliferation,pluripotent differentiation ability and easy access.The extracellular vesicles such as exosomes derived from SHED have not been studied.Therefore,in this study,exosomes derived from human exfoliated deciduous teeth stem cells(SHED-Exos)were used to investigate the osteogenic effect of periodontitis mice in vivo and to regulate osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro,they provide reference for the study of exosomes derived from human exfoliated deciduous teeth stem cells for osteogenic differentiation.Methods: 1.Exosomes collection: SHED culture from the 4th to 7th generation,collect cell culture medium,extract SHED-Exos by ultracentrifugation with 30% sucrose cushion,and observe the morphological characteristics of SHED-Exos using transmission electron microscope(TEM),the surface marker CD63 was identified by western blot;2.BMSCs culture and biological characteristics testing(1)Bone marrow mesenchymal primary cells were taken from femur and tibia of 10-month-old mice and passaged to the first and second passage(P1-P2)to observe cell morphology and multi-directional differentiation ability;(2)BMSCs were co-cultured with PBS,SHED-Exos(1 ?g/ml)and SHED-CM and the proliferation of BMSCs in each group was detected using cell counting kit-8(CCK-8).We used 5 ?M camptothecin to induce early apoptosis in BMSCs,after different processing,then used flow cytometry to detect apoptosis;(3)In order to detect the osteogenesis level,BMSCs,PBS,SHED-Exos,and SHED-CM were added to the osteogenesis induction medium to culture BMSCs respectively.After 3 days of osteogenesis induction,quantitative real time polymerase chain reaction(q RT-PCR)was used to detect the expression of ALP and Runx2 osteogenic genes;After 7 days of osteogenesis induction,p-Nitrophenyl Phosphate(p-NPP)was used to detect alkaline phosphatase(ALP)activity;After 14 days of osteogenesis induction,the expression of Runx2 and Osx osteogenic genes were detected by q RT-PCR,the formation of mineralized nodules was observed with alizarin red staining,and cetylpyridinium chloride solution was used for quantification,and use western blot to detect differences in Runx2 protein translation levels;(4)Add PBS or SHED-Exos or SHED-CM to the adipogenesis induction medium,and then detect the expression level of the adipogenesis gene peroxisome proliferator activated receptor ? by q RT-PCR,and observe the formation of lipid droplets under a microscope and quantify the lipid droplets;(5)BMSCs were stimulated with 1 ?g/ml lipopolysaccharide(LPS)for 24 hours to induce the expression of inflammation-related genes.At the same time,PBS,SHED-Exos(10 ?g/ml or 1 ?g/ml),SHED-CM were co-cultured with BMSCs for 24 hours,and the chang es of the expression levels of pro-inflammatory factor interleukin 6(IL-6)and tumor necrosis factor(TNF-?) were detected by q RT-PCR.3.Establishment of periodontitis model: 10-month-old mice were selected,and periodontal ligation was performed around the first molar of the mouse with 5-0 silk ligatures.After 14 days,the ligatures were removed,and the buccal and palatal areas of the first molar were injected phosphate buffer solution(PBS)or SHED or SHED-Exos at the same time,once a week,two weeks later,the maxillary was taken,scanned by micro computed tomography(Micro CT),and 3D reconstruction was performed to analyze the bone regeneration of buccal and palatal bone of the first molar.And the distance were measured from the cementum-enamel junction(CEJ)to the alveolar bone cone(ABC)in the mesial and distal regions,and the neonatal bone quality were observed by HE and Masson staining through histological sections;Results: 1.SHED cells were cultured to P4-P7 generation.The cells showed spindle-shaped morphology with uniform size.SHED-Exos were extracted through a method with high speed centrifugation.The exosomes were observed by TEM and were about 100 nm diameter spherical-like membrane structure,western blot showed positive expression of exosomes CD63;2.To further explore the biological regulatory effects of exosomes on bone marrow mesenchymal stem cells / stromal cells,we isolated and cultured b one marrow mesenchymal stem cells from the tibia and femur bone marrow from 10-monthold mice.Cells cultured to P1-P2 generation were spindle-shaped for subsequent experiments.SHED-Exos can promote BMSCs proliferation,but not inhibit early apoptosis.After 3 days of osteogenesis induction,SHED-Exos had no obvious induction effect on the osteogenic genes A LP and Runx2;After 7 days osteogenesis induction,SHED-Exos enhanced alkaline phosphatase activity and gradually exerted osteogenesis;After 14 days osteogenesis induction,SHEDExos enhanced the formation of mineralized nodules,increase d Runx2 gene expression and protein expression levels,and significantly increase d Osx gene expression,and promoted BMSCs to differentiate into osteoblasts.Therefore,SHED-Exos can specifically promote BMSCs osteogenesis differentiation;After 14 days of induced adipogenesis differentiation,oil red O staining and quantitative results showed that SHED-Exos attenuated lipid droplets formation ability of BMSCs.q RT-PCR results showed that SHED-Exos inhibited the expression of adipogenesis genes PPAR ? m RNA expression;After 1 ?g/ml lipopolysaccharide(LPS)induction for 24 hours,q RT-PCR results showed that 1 ?g/ml SHED-Exos could inhibit the m RNA expression of BMSCs IL-6 and TNF-?,while high concentrations of exosomes(10 ?g/ml SHED-Exos)promoted IL-6 and TNF-? expression.3.In order to investigate the in vivo osteogenesis of bone loss by exosomes,silk ligatures were used to induce periodontitis around the maxillary first molar of 10-month-old mice.After 14 days of ligation,obvious bone loss were visible on the buccal and palatal sides of the first molar.At the same time,local injection of PBS or SHED or SHED-Exos in the periodontal buccal and palatal side of the first molar.After 2 weeks,Micro CT analysis of hard tissue volume and histological staining(HE and Masson staining)showed that SHED-Exos can significantly promote bone regeneration after periodontitis,the exosomes group can achieve osteogenesis similar to that of source cell therapy,and at the same time,obviously periodontal ligament formation can be observed;Conclusion: The above experiments prove that exosomes derived from exfoliated deciduous teeth stem cells can be directed to promote the osteogenic differentiation ability and inhibit the adipogenesis differentiation ability of bone marrow mesenchymal stem cells;at the same time,exosomes promoted the cell proliferation ability.In addition,exosomes can inhibit the up-regulation of LPS-induced inflammatory factors.In animal models,we verified again with the periodontitis model that exosomes derived from exfoliated deciduous teeth stem cells promote alveolar bone repair and regeneration.Therefore,exosomes derived from exfoliated deciduous teeth stem cells are expected to become a new treatment stra tegy to promote bone regeneration. |
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