Application Of HucMSCs-derived Exosomes/SDF-1/Hydrogel Scaffolds On Bone Defects

BackgroundCritical-sized bone defects are common and difficult to treat in clinic.The main reasons are the weak differentiation potential and small number of stem cells at the bone defects sites,which are respectively reflected in the "quality" and "quantity" of stem cells.Exogenous stem cell transplantation has shortcomings such as storage difficulties and immune rejection,and the enhancement of endogenous stem cell differentiation potential by the production of exosomes(Exos)through the paracrine mechanism of stem cells may be a new idea for the treatment of bone defects.Human umbilical cord mesenchymal stem cells(hucMSCs)are characterized by painless collection and rapid self-renewal.In the field of non-bone regeneration,previous studies have demonstrated that hucMscs-derived exosomes(hucMSC-Exos)can regulate the function of target cells in vivo.Recent studies have shown that hucMSC-Exos transplantation can effectively treat bone loss caused by various diseases.Stromal Cell-derived Factor 1(SDF-1)is a cytochemokine that can recruit bone marrow mesenchymal stem cells(BMSCs)to play a role in bone defects sites.The combination of hucMSC-Exos and SDF-1 can effectively solve the problem of "quality" and "quantity" of stem cells in bone defects sites.ObjectiveIn this study,hucMSC-Exos will be extracted and co-loaded with SDF-1 in photocuring GelMA hydrogel to explore its synergistic effect in bone repair and key proteins that play osteogenic roles,to provide a new idea for the treatment of critical-sized bone defects.MethodshucMSC-Exos were extracted,and the morphological characteristics,particle diameter and biomolecular markers of hucMSC-Exos were analyzed by transmission electron microscopy(TEM),NanoSight particle size analyzer and Western Blot analysis.The absorption of hucMSC-Exos was analyzed by immunofluorescence staining.The osteogenic differentiation of hucMSC-Exos was determined by ALP staining and alizarin red staining.Scanning electron microscope(SEM),Micro-CT,universal mechanical testing machine and rheometer were respectively used to detect the pore size,porosity,compression modulus and energy storage modulus of GelMA hydrogel.A mouse skull defect model was established and divided into Control group,Gel group,Gel/Exos group,Gel/SDF-1 group and Gel/Exos/SDF-1 group.The biomaterials were studied in vivo by Micro-CT,HE staining,Masson staining and immunohistochemical staining.While Transwell assay,ALP staining and alizarin red staining were used in vitro.The expression of IGFBP5 in exosomes and its role in osteogenesis were investigated by Western Blot and alizarin red staining.ResultshucMSC-Exos have round cupule-like structures with a diameter of 130.2±37.5nm and expressed CD9,CD81 and TSG101 positively.They can be ingested by BMSCs.ALP staining and alizarin red staining showed that it had obvious osteogenic differentiation.GelMA hydrogel is a porous layered network structure with porosity of 82-87%,compression modulus of 20,000-25000Pa,and energy storage modulus of 15000-20000Pa.In vivo experiments:Micro-CT results showed that the use of hucMSC-Exos/SDF-1/GelMA hydrogel could significantly promote the healing of bone defects,which was consistent with the results of HE staining,Masson staining and immunohistochemical staining.In vitro experiments:Transwell assay showed that SDF-1 had chemotaxis.ALP staining and alizarin red staining showed that the use of hucMSC-Exos/SDF-1/GelMA could significantly improve osteogenic differentiation,in which hucMSC-Exos played a leading role.Western Blot results showed that hucMSC-Exos positively expressed IGFBP5 protein,which promoted osteogenic differentiation.ConclusionIn this study,hucMSC-Exos and SDF-1 were co-loaded in GelMA hydrogel to give full play to the osteogenic differentiation ability of exosomes and the chemotaxis ability of SDF1,improving the "quality" and "quantity" of stem cells,which confirmed the synergistic effect of hucMSC-Exos and SDF-1 on bone defects repair.This study found that IGFBP5 may be an important protein in bone repair effect.