Experimental Study Of PTH1-34-loaded BMSCs-derived Exosomes In The Treatment Of Osteoporosis

Background: Osteoporosis（OP）is a common chronic disease characterized by decreased bone mass,destruction of bone microstructure and increased bone fragility.At present,there are some problems in the treatment of OP,such as poor curative effect,obvious side effects,inability to improve the proliferation and differentiation of osteoblasts,difficulty in reversing the process of OP,which limit patients’ compliance and disease treatment effect.Other scholars explored the feasibility of stem cell transplantation for OP based on tissue engineering technology.The results showed that although stem cell transplantation can improve the bone formation ability of OP mice,there are still many problems such as immune rejection,increased risk of cell malignant transformation,stem cell homing disorder and so on.Therefore,it is urgent to find a treatment method that can promote bone tissue regeneration and minimize the potential harm caused by long-term drug exposure.Exosomes（EXOs）,as a natural biological origin and complex endogenous nano-carrier,can participate in the information exchange between cells in the process of bone reconstruction through receptor-mediated endocytosis.Compared with ordinary nano-carriers,EXOs can combine the size of nano-particles with the advantages of non-cytotoxicity,low immunogenicity and high drug loading,which opens up a new way for the transport of anti-osteoporosis drugs.Methods: In this study,relying on the excellent self-loading characteristics of EXOs,the osteogenic drug-human parathyroid hormone（PTH1-34）was loaded into EXOs derived from bone marrow mesenchymal stem cells（BMSCs）by saponin,and the unique endogenous complex of EXOs-PTH was prepared.The morphology of EXOs was detected by transmission electron microscope observation,nanoparticle tracer analysis（NTA）and Western blot.Cell proliferation test（CCK-8）,alkaline phosphatase staining（ALP）,alizarin red staining（ARS）and Western blot were used to detect the effects of EXOs-PTH on the proliferation,differentiation and osteogenic differentiation-related protein expression of MC3T3-E1 cells.Subsequently,we applied EXOs-PTH to the osteoporosis mouse model by intravenous injection.After 8weeks of drug treatment,the mice were killed and samples were taken.The therapeutic effect of drugs on osteoporosis mice was evaluated by micro-CT scanning analysis,histological and immunological section staining observation and serum bone metabolism index detection.Results: The primary BMSCs were successfully extracted,and their multi-directional differentiation potential was identified by osteogenesis,adipogenesis and chondrogenesis induced differentiation experiments.Under transmission electron microscope,EXOs and EXOs-PTH showed typical vesicle-like and disk-like structures.The exocrine membrane structure was complete and uniform with a diameter distribution of about 100 nm,and the EXOs-specific protein markers CD9 and Tsg101 were positive expressed,while negative marker Calnexin was not expressed.The particle size of EXOs-PTH is slightly higher than that of EXOs,but generally speaking,there is no significant difference in the particle size distribution of EXOs after drug loading.In vitro experiment,CCK-8 found that intermittent stimulation of MC3T3-E1 with low concentration of EXOs-PTH could promote cell proliferation.The results of alkaline phosphatase staining and alizarin red staining showed that EXOs-PTH could significantly promote the osteogenic differentiation of MC3T3-E1,and the early osteogenic marker ALP and the late osteogenic evaluation index calcium nodule size and number were significantly higher than those of PTH1-34 alone.The osteogenic effect of EXOs-PTH was also confirmed in western blot experiment.After EXOs-PTH stimulated MC3T3-E1,the expression of osteogenic related proteins OCN,Runx2,Osterix,BMP2 in cells was significantly up-regulated.In vivo experiments,EXOs-PTH can enhance the therapeutic effect of PTH1-34,improve the bioavailability of drugs,and show a strong ability to promote bone formation.The results of micro-CT and histological staining showed that EXOs-PTH could restore the lost bone mass of osteoporosis mice and promote bone tissue regeneration.The bone density,the number and thickness of trabeculae of osteoporosis mice treated with EXOs-PTH were better than those of other experimental groups,and the fat accumulation in bone marrow cavity of osteoporosis mice was significantly reduced.Immunofluorescence and immunohistochemical staining showed that the fluorescence intensity of OCN and the positive expression of Runx2 in bone tissue of mice in EXOs-PTH group were significantly higher than those in each experimental group.In addition,the results of serum ELISA showed that EXOs-PTH could significantly improve the efficiency of bone turnover and promote the process of bone remodeling in osteoporosis mice,and the levels of ALP,OCN,OPN and Runx2 in serum were significantly increased.Conclusions: To sum up,we successfully constructed a drug delivery system loaded with PTH1-34 with exosomes derived from bone marrow mesenchymal stem cells as carriers,and preliminarily explored its role in promoting osteoblast proliferation and differentiation,and evaluated its therapeutic effect in osteoporosis.