Research Of Bone Tissue Engineering Based On Stem Cells-Derived Exosomes

BACKGROUND: Bone tissue engineering is the important technical method to promote bone regeneration and repair bone defects,seed cells and carrier materials are the important components.It has been suggested that exosomes secreted by stem cells can play a role in promoting tissue regeneration which similar to stem cells,and also can avoid the risks of immune response and genovariation of stem cell transplantation.However,no reports have thusfar described the application of exosomes in bone defects repair.There is a challenge to construct the carrier material with good biocompatibility,tissue adhesion and integration.Photocuring hydrogel showed certain advantages in these aspects,but traditional photocuring hydrogel is difficult to achieve high adhesion and integration,the free radicals and other side effects also be produced during preparation.The effects and mechanisms of exosomes in bone regeneration have been evaluated through a series of experiments;phototriggered imine crosslink(PIC)hydrogel with good operability,biocompatibility,tissue adhesion and integration has also been constructed and the carrier properties of bioactive compounds were evaluated.OBJECTIVE: Explore the effects and mechanisms of exosomes secreted by stem cells in promoting bone regeneration and bone defects repair;construct ideal hydrogel carrier material and evaluate the application potential in tissue engineering.METHODS: hiPSCs were induced into MSCs and then the exosomes were separated and collection,the effects of exosomes on promoting skin wound healing and bone regeneration were evaluated in vivo experiments;the effects of exosomes on the proliferation,migration,osteogenesis,and osteogenic-related pathway of BMSCs were evaluated in vitro.Via grafting the photo-aldehyde-generated functional group NB to hyaluronic acid(HA),hydrogel could be constructed between HA-NB with diverse amino-bearing macromolecules via imine ligation,the gel-forming property,cytotoxicity,tissue adhesion,performances of exosomes loading and controlled-release of PIC hydrogel were evaluated in vitro;biodegradability,tissue adhesion and integration,and the function of PIC hydrogel in bone repair were evaluated in vivo.RESULTS:(1)Exosomes can be extracted with the methods of ultrafiltration combined with ultracentrifigation.hiPS-MSC-Exos with the characteristics of exosomes by the results of TEM,particle size and markers;(2)hiPS-MSC-Exos play a important role in promoting skin wound healing and bone regeneration: new tissue growth into skin defects and obvious vascularization were ovserved by histological staining and immunohistochemistry,new bone growth into defects were observed by micro-CT,hiPS-MSC-Exos promote bone mineral deposits and mineralization were ovserved by fluorescence labeling,hiPS-MSC-Exos promote the expression of osteogenesis markers by the results of immunohistochemistry;(3)hiPS-MSC-Exos promote bone regeneration by activation of PI3K/Akt pathway in BMSCs: hiPS-MSC-Exos can promote the proliferation and migration of BMSCs by the results of CCK8 and scratch assay,hiPS-MSC-Exos can promote the osteogenesis of BMSCs by the results of ALP activity assay,hiPS-MSC-Exos can activate the PI3K/Akt pathway by the results of gene expression profile chip technology and bioinformatics analysis,the osteogenesis effects of hiPS-MSC-Exos were inhibited when the PI3K/Akt pathway was blocked;(4)PIC hydrogel can be serve as good carriers in bone tissue engineering: PIC hydrogel with good gel-forming property,biocompatibility,and tissue adhesion were observed by the results of rheological test,CCK8,and tensile testing.PIC hydrogel with good controlled-release effectiveness by the uptake results of exosomes.PIC hydrogel with good biodegradability and tissue integration by the results of histological staining.PIC hydrogel carrying bioactive compounds can play a role in promoting bone regeneration by the results of micro-CT and histological staining.CONCLUTION:(1)hiPS-MSC-Exos play a important role in promoting skin wound healing and bone regeneration and bone defects repair;(2)hiPS-MSC-Exos promote bone regeneration by activation of PI3K/Akt pathway in BMSCs;(3)PIC hydrogel with good operability,biocompatibility,biodegradability,tissue adhesion and integration,can be serve as good carriers of stem cells or exosomes,having a broad application prospect.