The Study Of Pretreated Exosomes For Promoting Osteoporotic Osseointegration And Diabetic Wound Healing

PART1 Overexpressing MiR-20 a Promoted the Osseointegration of Porous Titanium Alloy by Enhancing Osteogenesis Via Targeting BAMBIBackground: Patients with osteoporosis have a high risk of implant loosening due to poor osteointegration,possibly leading to implant failure,implant revision and refracture.RNA interference therapy is an emerging epigenetic treatment,and we found that mi R-20 a could enhance osteogenesis.Moreover,exosomes(Exos)derived from bone marrow mesenchymal stem cells(h BM-MSCs)were utilized as nanoscale carriers for the protection and transportation of mi R-20a(Exo-20a).In this study,we intended to determine whether Exos overexpressing mi R-20 a could exert a superior effect on osteoporotic bone defects and the underlying mechanism.Methods: For evaluating the effect of Exo-20 a on osteogenesis,in vitro and in vivo studies were performed.In vitro,we first showed that mi R-20 a was upregulated in the osteogenic process and overexpressed Exos with mi R-20 a by the transfection method.Then,the proliferation,migration,and osteogenic differentiation abilities of h BM-MSCs were detected by treated with Exo-20 a by CCK-8 assays,alkaline phosphatase staining and alizarin red staining,q RT-PCR and western blot.In vivo,we established an osteoporotic bone defect model and evaluated the effect of Exo-20 a on bone formation by Micro CT,sequential fluorescent labelling,and histological analysis.To further explore the mechanism,we applied a bioinformatics method to identify the potential target of mi R-20 a.Results: In vitro,Exo-20 a was successfully established and proved to promote the migration and osteogenesis of h BM-MSCs.In vivo,Exo-20 a promoted osteointegration in an osteoporotic rat model.To further elucidate the related mechanism,we proved that Exo-20 a could enhance osteogenesis by targeting BAMBI.Conclusions: Collectively,the in vitro and in vivo results confirmed that MSC-derived Exo-20 a therapy effectively promoted osteoporotic porous titanium alloy osseointegration via pro-osteogenic effects by targeting BAMBI.PART2 Melatonin-stimulated MSC-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway.Background: After surgery,wound recovery in diabetic patients may be disrupted due to delayed inflammation,which can lead to undesired consequences,and there is currently a lack of effective measures to address this issue.Mesenchymal stem cell(MSC)-derived exosomes(Exo)have been proven to be an appropriate candidate for diabetic wound healing through anti-inflammatory effects.In this study,we investigated whether exosomes pretreated with melatonin(MT)derived from MSCs(MT-Exo)could exert superior effects on diabetic wound healing,and we attempted to elucidate the underlying mechanism governing this effect.Methods: For the evaluation of the anti-inflammatory effect of MT-Exo,in vitro and in vivo studies were performed.For in vitro research,we detected the levels of inflammation-related factors,such as IL-1β,TNF-α,and IL-10 via ELISA and the relative gene expression of the IL-1β,TNF-α,IL-10,Arg-1,and i NOS via q RT-PCR and investigated the expression of PTEN,AKT and p-AKT by Western blotting.For in vivo study,we established air pouch model and streptozotocin(STZ)-treated diabetic wound model and evaluated the effect of MT-Exo by flow cytometry,optical imaging,H&E staining,Masson trichrome staining,immunohistochemical staining of and immunofluorescence and the relative expression of α-SMA and Collagen I and III via qRT-PCR.Results: MT-Exo significantly suppressed the pro-inflammatory factors IL-1β and TNF-α and reduced the relative gene expression of IL-1β,TNF-α and i NOS,while promoting the anti-inflammatory factor IL-10 along with increasing the relative expression of IL-10 and Arg-1,compared with that of the PBS,LPS and the Exo groups in vitro.This effect was achieved by increasing the ratio of M2 polarization to M1 polarization through increasing the expression of PTEN and inhibiting the phosphorylation of AKT.Similarly,MT-Exo significantly promoted the healing of diabetic wounds by inhibiting inflammation,thereby further facilitating angiogenesis and collagen synthesis in vivo.Conclusions: Melatonin-pretreated MSC-derived exosomes could promote diabetic wound healing by suppressing the inflammatory response,which was achieved by increasing the ratio of M2 polarization to M1 polarization through activating the PTEN/AKT signalling pathway,and the pretreatment of MT proved to be a promising method for the treating diabetic wound healing.