Utilization Of Extracellular Vesicle And Small Molecular As Treatment For Bone Diseases

Objective: Bone multipotent stromal cell(BMSC)and TGF-β inhibitor are reported to treat diseases caused by dysfunction of bone regeneration and remodeling.However,BMSCs treatment may cause tumor formation and immune suppression.TGF-β inhibitor treatment is non-specific and has significant side effects.Therefore,this project is to explore whether BMSCs derived extracellular vesicle(EV)can stimulate bone regeneration and develop new a small molecule to achieve bone specificity and less side-effects.Method: The EV identification was carried out by electron microscope and western-blot.The EV metabolic process in osteoblasts was observed through confocal microscope.The influence on proliferation and function of osteoblasts were evaluated by q RT-PCR,western-blot and flow cytometry.RNA sequencing was used to scrutinize which miRNA in EV played the most important role in regulating osteoblasts function and maturation.The in vivo efficacy was evaluated by micro CT and tissue staining.The small molecule(Alendronate-TGF-β receptor 1 inhibitor,ALN-TβR1I)was synthesized through chemical modification.The in vitro efficacy was evaluated by western-blot and immunochemistry staining.The safety dose of the novel small molecule was confirmed by survival analysis.Result: EV entered osteoblast Golgi-apparatus in 4 hours after incubation.The Alizarin red staining,q RT-PCR and western-blot all indicated the pro-regeneration effect of EV.mi R-196 a was found to play the most important role in EV by RNA sequencing.The in vivo results demonstrated EV facilitated bone regeneration in mice bone defect.The in vitro efficacy of small molecule was demonstrated by immunofluorescent staining and western-blot.The histochemistry demonstrated the high bone affinity of ALN-TβR1I.The disease progression in mouse model was attenuated after the small molecule treatment.Conclusion: BMSCs regulated osteoblasts maturation and function through EV.The mi R-196 a in EV played an important role in EV function.Hydrogel-EV system promoted bone regeneration in mouse model.The small molecule can attenuate osteoarthritis and CamuratiEngelmann disease in mice.