The Effect Of Injectable Hydrogel Loaded Exosomes On The Osteogenic Ability Of Adipose-derived Stem Cells

Objective: Exosomes are nano-cellular vesicles with cellular communication function and play an important role in various physiological and pathological activities,such as immune regulation,tumorigenesis,angiogenesis and tissue healing.Hydrogel is a hydrophilic polymer,or macromolecules,copolymer in aqueous solution of the expansion with a three-dimensional network structure,it can absorb the water several times volumes compared to their own weight of material,above all,it able to provide a suitable micro-environment which similar to the extracellular matrix,so as to promote the osteoblast migration,adhesion,proliferation and differentiation,and effective to deliver nutrients and growth factors.In particular,the development of cell load of water gel for cell therapy has opened up new possibilities.In this study,the osteogenic capacity of exosomes derived from human adipose-derived stem cells in a temperature-sensitive hydrogel containing chitosan,nano-hydroxyapatite,collagen and odium β-glycerophosphate/n HAC/ chitosan was studied by comparison.Methods: 1.Extraction,cultivation and identification ADSCs in vitro.2.Extraction,cultivation and identification ADSCs derived exosomes in vitro.3.Preparation of injectable thermosensitive hydrogels containing chitosan,nano-hydroxyapatite,gelatin and sodium β-glycerophosphate(CS/n HAC/β-GP).4.The co-culture of ADSCs-Exo,cells and hydrogels was achieved by setting up a control experiment.5.Observed by SEM,using Western Blotting method,ALP and other indicators to verify exosomes induced by ADSCs which could increase the osteogenic capacity of ADSCs on hydrogels.Results: 1.The morphology of exosomes can be observed by transmission electron microscopy,which is scattered and distributed,presenting low-density electron particles with a diameter between 30-130 nm.Western blot analysis indicated that the expressions of specific proteins CD9 and CD63 were significant,which confirmed that the extracted protein was exosomes.2.Scanning electron microscopy showed that the hydrogel was a three-dimensional porous structure with pore sizes ranging from 10 um to nearly 300 um.3.In vitro,ALP expression was significantly higher in the exosome group than in the control group.4.On the 21 st day,in vitro,comparing with the control group,the expression of ALP was significantly elevated in the exosome group.Exosomes significantly promoted the proliferation of ADSCs in CS/n HAC/β-GP hydrogels,and exosomes significantly promoted osteogenic differentiation.Conclusion: Our new material consisting of exosome derived from ADSCs and CS/n HAC/β-GP hydrogel provides a new treatment for bone tissue engineering and shows good potential for repairing bone defects.