Comparative Of The Therapeutic Effect And Mechanism Of Exosomes Derived From Dental Pulp Stem Cells Of Deciduous And Permanent Teeth In The Repair Of Bone Defects

Objective:In recent years,cell-free replacement therapy based on "exosomes" has great potential in immunity,tissue homeostasis,cancer and neurodegenerative diseases.Exosomes contain bioactive molecules such as lipids,nucleic acids,and proteins,which confer different biological functions on exosomes.Therefore,this study aimed to explore whether paracrine-derived exosome proteins derived from Dental pulp stem cells(DPSCs)and Stem cells from human exfoliated deciduous teeth(SHED)are effective in regulating osteogenic differentiation in apical inflammatory bone defect,which provides new enlightenment for the treatment of clinical diseases.Methods:1.Collection of DPSCs and SHED P4-P7 cell supernatants:When cell growth density was around 70%or 90%,the basal medium was replaced after washing with PBS,and the culture supernatants were collected for 24-48 hours.The exosomes were separated by differential centrifugation and exosome isolation reagent.The morphological characteristics were observed by Transmission Electron Microscope(TEM),and the specific labeled proteins were detected by Western blot(Wb).2.The isolated exosomes were analyzed by Proteomic analysis.3.Modulation of biglycan(BGN)on osteogenic differentiation of inflammatory periodontal ligament stem cells(IPDLSC):(1)The proliferation capacity of IPDLSC and PDLSC was detected by cell counting kit-8(CCK8)assay;(2)The osteogenic differentiation ability of IPDLSC and PDLSC was stained by Alizarin Red S and semi-quantitatively analysis.The gene levels of osteogenic factors:runt-related transcription factor 2(RUNX2)、Osteocalcin(OCN)、Bone Morphogenetic Protein 4(BMP-4)were detected by RT-qPCR;(3)The regulatory effect of Biglycan on the osteogenic differentiation of IPDLSC was screened by Western blot for the optimal effective concentration and duration of BGN,and the expression level of relevant proteins of the BMP-4/Smad signaling pathway was detected;(4)IPDLSC were co-cultured with 1 μg/mL DPSC-Exos,10 μg/mL DPSC-Exos,1μg/mL SHED-Exos or 10 μg/mL SHED-Exos for 24 hours.The expression levels of BGN and BMP-4/Smad signaling pathway were detected.4.The animal model of apical inflammatory bone defect was established,and the hydrogel carrying exosomes was placed on the bone defect to suture the wound.After 2 weeks,the material was fixed,and the three-dimensional reconstruction was performed by Micro-computed Tomography(Micro-CT)to analyze the bone of the alveolar bone.Histological sections were then performed,tissue morphology was observed by HE&Masson staining,and expression levels of relevant antibodies were observed by immunohistochemical staining.Results:1.DPSC-Exos and SHED-Exos are extracellular vesicles produced by DPSC and SHED paracrine,both of them have a bilayer membrane structure with a diameter of less than 150 nm,which conforms to the morphological characteristics of exosomes.And CD63,CD81,TSG101 were also positive expressed.2.There are differences in protein expression between DPSC-Exos and SHED-Exos.Among them,Pyruvate kinase M(PKM),Biglycan(BGN),Calreticulin,and Eukaryotic translation initiation factor 5A-1(EIF5A-1)were significantly up-regulated in SHED-Exos.3.Compared with PDLSC,IPDLSC have higher proliferative ability and lower osteogenic differentiation ability in vitro.In vitro cell experiments,human recombinant BGN proteins were co-cultured with IPDLSC.It verified that BGN proteins in DPSC-Exos and SHED-Exos may excite the BMP-4/Smad signaling pathway for osteogenesis.Low dose(1 μg/mL)SHED-Exos had the same effect as high dose(10μg/mL)DPSC-Exos,while high dose(10 μg/mL)SHED-Exos had more significant effect on osteogenesis.4.The animal model was successfully established.There are more fresh bone tissue in the bone defect of H-DPSC-Exos and H-SHED-Exos groups.BGN and BMP-4 were positive expressed in the bone defect area,which confirmed that DPSC-Exos and SHED-Exos had a certain therapeutic effect.Conclusion:The results of this study demonstrated that exosomes derived from DPSC and SHED promoted osteogenic repair of apical inflammatory bone defect.Among them,BGN is an important regulator,which riched in SHED-Exos and may promote osteogenic healing by activating the BMP-4/Smad signaling pathway.