| BACKGROUND: Bone and cartilage regenration are now still lack of effective technical means,which is plagued by clinicians.Actively exploring has important clinical significance to promote bone and cartilage regeneration technology.Researches show that stem cells can promote bone and cartilage regeneration,but directly transplanting stem cells might be related with problems including immune rejection,ethical issues and tumorigenic risk.Recent studies found that stem cells play function by paracrine mechanism,among which exosomes(Exosomes,Exos)played an important role.Exos are nano-sized vesicles secreted by stem cells,which plays an important role in transferring the protein,mRNA,microRNA and other important functional materials.It has been reported that stem cells have the function of promoting the regeneration of damaged tissue.The transplantation of exos can avoid the risk of direct transplantation of stem cells,and it is sasy for the preserving and transporting of exos,showing a broad clinical application prospects.The biological carrier is another problem for the application of stem cells and exos,it is important to make exos accumulated effectively in the injury site.The hydrogel is polymer with crosslinked structure dispersed in water,commonly used hydrogels such as hyaluronic acid,gelatin and fibrin glue.Although hydrogel has excellent biocompatibility and physical properties similar to human tissue,its problem is the lack of firm adhesion integration.In order to solve this problem,we introduce Phototriggered Imine Crosslink(PIC)hydrogel as an active substance carrier,the hydrogel can form strong integration by chemical crosslinking with tissue and active materials.This research applies PIC hydrogel as the carrier of exosomes and other active substances,evaluating the regeneration of cartilage and bone necrosis,to provide new strategies for the treatment of cartilage injuries and bone necrosis.METHODS:1.Using PIC hydrogel as biological carrier,loaded with exosomes derived from bone marrow mesenchymal stem cells(BMSC-Exos),exosomes derived from chondrocytes(AC-Exos)and exosomes derived from induced pluripotent stem cell derived mesenchymal stem cells(iPS-MSC-Exos),with New Zealand rabbit cartilage defect as the animal model.The in vivo expriments evaluate the effects of cartilage regeneration through general view evaluation,ICRS cartilage repair score,histological staining and immunohistochemical staining;the in virto expriments evaluate the influence of exosomes on proliferation and migration of BMSCs and AC,and the influence of exosomes to maintain the phenotype of chondrocytes;2.constructing the photosensitive group 2-nitrobenzyl alcohol(o-Nitrobenzyl alcohol,NB)modified hyaluronic acid(HA-NB)/platelet rich plasma(PRP)composite hydrogel(HNPRP),the in vitro expriments evaluate growth factor release ability,rheological characteristics,and tissue adhesion;the in vivo experiment evaluate the effects of cartilage repairing and tissue integration performance;3.Construction of SD rats steroid induced necrosis of the femoral head model,intravenous injection of iPS-MSC-Exos,microCT,angiography,histological staining and immunohistochemical staining were performed to evaluate the function,observe the role of iPS-MSC-Exos in promoting angiogenesis through in vitro experiments,analyze the effect of iPS-MSC-Exos on the PI3K/Akt signaling pathway.RESULTS: 1.The effects of BMSC-Exos and AC-Exos to repair cartilage injury are different:(1)ICRS cartilage repair score,HE staining,safranin O/ fast green staining showed that both exos generate tissue repair;(2)the in vivo results showed that the repaired tissue by BMSC-Exos is mainly fibrous tissue,and AC-Exos promotes regeneration of hyaline cartilage;(3)the in vitro results show that the BMSC-Exos lead to the dedifferentiation of chondrocytes.While AC-Exos can maintain normal phenotype of chondrocytes in long term;2.iPS-MSC-Exos loaded within PIC hydrogel could promote cartilage regeneration:(1)the results of ICRS cartilage repair score,HE staining,safranin O/ fast green staining showed that iPS-MSC-Exos can promote the repair of cartilage defect,immunohistochemical staining results showed that the newly formed tissue are mainly hyaline cartilage;(2)in vitro results showed that iPS-MSC-Exos could significantly promote the proliferation and migration of BMSCs and AC;3.HNPRP hydrogel can significantly promote cartilage repair:(1)in vitro results showed that the cartilage is adhesive to the HNPRP hydrogel tightly,and sustained release of growth factors can be detected to the surrounding environment;(2)the in vivo results showed that HNPRP hydrogels promote hyaline cartilage regeneration and close integration;(3)the results show that the HNPRP hydrogel show perfect effect of repairing cartilage injuries.4.Intravenous iniection of iPS-MSC-Exos can effectively prevent steroid induced necrosis of the femoral head:(1)the in vivo results showed that iPS-MSC-Exos can effectively prevent the progression of bone necrosis;(2)the in vitro results showed that iPS-MSC-Exos can activate PI3K/Akt pathway of vascular endothelial cells,significantly enhanced the proliferation,migration and tube formation abilities;(3)iPS-MSC-Exos may activate the PI3K/Akt signaling pathway of vascular endothelial cells to promote angiogenesis,improve blood supply of necrotic site,so as to prevent femoral head necrosis.CONCLUTION:1.IPS-MSC-Exos has the function of promoting the regeneration of cartilage;2.PIC hydrogel is a suitable carrier for biological active substances such as stem cells,exos and PRP;3.iPS-MSC-Exos/PIC hydrogel can provied solid integration,and show perfect effect of cartilage regeneration;4.HNPRP can provide sustained-release of bioactive factor and strong integration,achieve perfect cartilage regeneration,provide a new strategy for the clinical application of PRP to repair tissue damage;5.Exos from different cell type can generate different repairing effects of cartilage.BMSC-Exos mainly generate fibrous tissue,while AC-Exos generate mainly hyaline cartilage.The results suggest that exos selection can directly affect the cartilage repair effect.6,iPS-MSC-Exos can effectively prevent steroid induced necrosis of the femoral head,the mechanism is through activation of PI3K/Akt signaling pathway in vascular endothelial cells to promote angiogenesis,improve blood supply,so as to prevent necrosis of femoral head.1.Exosomes from different cell sources have different effects of cartilage repair.Although BMSC-Exos promoted the repairing of cartilage defects,the regenerated tissue was mainly fibrous tissue,suggesting that the selection of cells directly affects tissue repairing effects of exosomes;2.iPS-MSC-Exos has a significant role in promoting the regeneration and repairing of cartilage defects,showing a broad application prospects;3.HNPRP realized the sustained-releasing capacity of PRP,and made the PRP to be firmly adhered to the tissue,achieving the perfect effects of cartilage repairing,this new method provides a new strategy for the clinical application of PRP to repair tissue damage;4.PIC hydrogel can effectively loaded with exosomes,PRP and other active substances,showing good cartilage repair effect and excellent tissue integration performance,which is an ideal biological carrier to repair cartilage damage;5.iPS-MSC-Exos can effectively prevent steroid induced necrosis of the femoral head,the mechanism of which is through the activation of PI3K/Akt signaling pathway in vascular endothelial cells to promote angiogenesis,thereby improving the blood supply of bone necrosis,promoting bone regeneration. |
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