The Regulation Of Substrate Topography And Stiffness On The Paracrine Properties Of Mesenchymal Stem Cells

Mesenchymal stem cells(MSCs)were widely used in tissue engineering and cell therapy due to their ability to secrete varieties of soluble factors(immunosuppressive factors,trophic factors,etc.)and exosomes to resolve inflammation,stimulate angiogenesis and activate intrinsic repairing cells.Extensive efforts have been made to activate the paracrine property of MSCs over the past few years.Physical properties of substrates,including topography,stiffness,mechanical rheological properties,roughness,etc,have been reported to regulate the migration,proliferation and differentiation of MSCs,while there's only a few researches studying the effects of those parameters on the paracrine properties of MSCs.In the present work,substrates topography and stiffness were choosed to investigate how these two parameters regulates the paracrine properties of MSCs.Further efforts were also made to reveal the underlying mechanisms.Firstly,polycaprolactone(PCL)substrates with flat and grooved surfaces fabricated by soft photolithography and melt-casting methods were used as a model to explore the effects of substrate topography on the paracrine function of rat adipose-derived mesenchymal stem cells(r ASCs).r ASCs cultured on grooved substrates expressed higher levels of repairing-related genes,and produced more exosomes compared with r ASCs cultured on flat substrates.Exosomes produced by r ASCs cultured on grooved substrates contained higher levels of pro-angiogenic related micro RNAs and growth factors,which could further promote the chemotactic migration and tube formation of human umbilical vein endothelial cells(HUVEC).This present study also suggested that the higher expression of Alix might be involved in mediating the increased production of exosomes by r ASCs cultured on grooved substrates.Secondly,polyacrylamide(PAAm)hydrogels with different stiffness were used as a model to explore the effects of substrate stiffness on the paracrine function of human bone-derived mesenchymal stem cells(h MSCs).Similarily,h MSCs cultured on soft substrates(0.5 k Pa)expressed significantly higher levels of immunomodulatory and trophic factors,and secreted more PGE2 compared with h MSCs cultured on rigid substrates(200 k Pa).The enhanced paracrine function of h MSCs was confirmed by the M2 phenotypic polarization observed in macrophages,as well as the accelerated chemotactic migration and tube formation observed in HUVEC after treatment with conditioned media(CM)collected from h MSCs cultured on soft substrates.The inhibited secretion of immunosuppressive and trophic factors by h MSCs cultured on rigid substrates could be largely rescued by treatment with F-actin,myosin or ROCK inhibitor,which indicated the cytoskeletal tension and actin polymerization playing a key role in the regulating of substrate stiffness on the paracrine behaviors of MSCs.The present study elucidated the effects of substrate topography and stiffness on the paracrine properties of MSCs,and explored the potential underlying regulatory mechanisms,which provided not only new platforms to induce and regulate the paracrine properities of MSCs but also the theoretical basis and foundation for the design of new pro-repairing tissue-engineering scaffolds.