Cellular Uptake Mechanism Of Mesenchymal Stem Cells-derived Extracellular Vesicles And The Role In Intervertebral Disc Repairing

Objectives:Stem cells-derived extracellular vesicles(EVs)are the efficient approaches for treating intervertebral disc degeneration(IDD).Usually,EVs are internalized by the receptor cells and then release the bio-active molecules,affecting the phenotype and function of the receptor cells.Previous studies have indicated that EVs could retard the progression of IDD and ameliorate the cell injury.It shows a great potential of EVs in intervertebral disc regeneration.However,the underlying mechanisms of EVs in treating IDD,as well as the factors that affect the therapeutic efficiency still remain unclear.This study is intended to investigate the effects of EVs on nucleus pulposus(NP)cells and the internalized mechanism of EVs.Moreover,the engineered EVs with improved uptake rate are constructed and utilized to optimize the therapeutic effects on IDD.Methods: Immunoblotting and immunofluorescence are used to investigate the EVs effects of MSCs on the NP cell pyroptosis in a cell co-culture model.To reveal the mechanism of EVs therapeutic effects,interfering si RNA is used to identify the functional EVs protein which mediates the anti-pyroptosis effect.Besides,the endocytosis pathway in NP cells is predicted by the transcriptome sequencing and bioinformatics analysis.The specific effects of endocytosis inhibition are evaluated based on immunofluorescence and flow cytometry.Lastly,the effects of engineered EVs aimed to activate the endocytosis are testified in the two-dimensional cell culture,three-dimensional cell culture and disc culture model.Results: The results of immunoblotting and immunofluorescence reveals that MSC-EVs significantly inhibit the pyroptosis of NP cells.Knockdown of peroxiredoxin 2 in EVs impairs the therapeutic effect on NP cell pyroptosis.The effects of EVs on inflammation pre-treated NP cells are significantly weakened.Based on the results of immunofluorescent staining and flow cytometry,the EVs uptake rate in inflammation pre-treated NP cells decreases significantly.Among this,the Cavin-2 mediated Caveolae-associated endocytosis is impaired in NP cells.Cavin-2 modified technique could increase the uptake rate of the engineered EVs in NP cells.Besides,engineered EVs could inhibit the inflammationinduced cell pyroptosis in three-dimension hydrogel culture model and this effect is better than naive EVs.In the disc culture model,engineered EVs decreases the histological grades of disc tissues and that is much lower than IDD group and naive EVs group.Conclusion: EVs delivers anti-oxidant proteins to ameliorate the pyroptosis of NP cell upon the inflammatory stimulus.The cellular uptake of EVs is impaired due to the dysfunction of NP cell,which results in the decreased therapeutic efficiency.Among this,the culprit of impaired EVs uptake is the dysfunction of Cavin-2 mediated endocytosis.The impaired endocytosis affects the therapeutic effect of EVs on NP cells.Cavin-2 modified engineered EVs could restore the impaired uptake mechanism of NP cells and then increase the EVs uptake rate,ultimately optimizing the therapeutic effect on IDD.In all,engineered EVs present a promising effect and have great potentials in IDD repairing.