The Research Of Photo-crosslinked Hydrogel Loaded Human Urine-derived Stem Cell Exosomes For The Promotion Of Bone Repair

Purpose:Bone defects caused by trauma,infection,bone tumor resection,and other factors have severely affected the limb function of patients.Hydrogel-based growth factor delivery strategies have become a new research trend in the field of bone defect repair.The methacrylated Gelatin(GelMA)and methacrylated hyaluronic acid(HAMA)hydrogels can transiently form network structures by mild chemical photo-crosslinking,but these hydrogels lack mechanical properties.For this reason,appropriate addition of nanohydroxyapatite(nHAP)can improve the mechanical properties of the above hydrogels.Exosomes from human urine stem cells(USC-Exos)have been reported to have osteogenesispromoting effects.The aim of this study was to design a new GelMAHAMA/nHAP injectable hydrogel as a sustainable delivery system loaded with exosomes,where USC-Exos was encapsulated in the hydrogel and released slowly,in order to obtain longer-term biological effects of exosomes.Methods:1.The hydrogel precursors of G5H1,G5H1.5 and G5H2 were prepared separately,and their injectability and gelation properties were verified under blue light irradiation.The hydrogels were subjected to 1H NMR and FTIR to analyze the characteristic peaks and chemical bonds of each component,and the rheological properties of the precursor liquids were examined by rheometer,and the compressive strength of the hydrogels was measured by universal mechanics after photocrosslinking.The exosome slow release efficiency was measured.Based on the physicochemical properties and characterization results,the ideal USC-Exos encapsulation carriers were selected for further in vitvo and in vivo experiments;2.USC-Exos was isolated and extracted from cultures of human urinederived stem cells(USCs)and identified.The internalization of USC-Exos by the target cells was observed.To verify the cytobiocompatibility of the three groups of hydrogels,G5H1,G5H1.5 and G5H2.The USC-Exos@G5H2 composite hydrogels were co-cultured with rat BMSCs and examined for their effects on cell chemotaxis,alkaline phosphatase(ALP)expression,calcium nodule formation and relative expression of osteogenic-related genes in rat BMSCs,respectively,based on the physicochemical properties and characterization results of the selected G5H2 group hydrogels as USCExos encapsulation carriers.USC-Exos@G5H2 composite hydrogels were co-cultured with rat EPCs to examine their effects on cell migration and tubule formation in EPCs,and on the relative expression of angiogenic genes,respectively;3.Three groups of G5H1,G5H1.5 and G5H2 hydrogels were established in a rat subcutaneous embedding model,and the in vivo biocompatibility of the hydrogels was investigated by H&E staining.The G5H2 hydrogels loaded with DiR fluorescently labeled exosomes were implanted subcutaneously in rat skull,and the in vivo release of exosomes was examined.A rat cranial bone defect model was established,and the ability of USC-Exo@G5H2 composite hydrogel to promote bone defect repair was investigated by micro-computed tomography(Micro-CT),H&E staining,Masson trichrome(MT)staining,immunohistochemistry(IHC)staining and immunofluorescence(IF)staining,respectively.Results:1.The hydrogel precursors have fluidity properties and can be gelatinized into different shapes.Different groups of hydrogels have microscopic pore structures.The hybrid GelMA-HAMA hydrogels can form stable chemical bonds and retain their respective characteristic wave peaks.The storage modulus G’ and viscosity of the hydrogels increased with the increase of HAMA ratio at room temperature.There was no significant trend in the viscosity change between groups when the ambient temperature was varied from 30℃ to 40℃.The ultimate compressive modulus of G5H1,G5H1.5 and G5H2 hydrogels were 8.4±0.6 kPa,12.2±0.7 kPa and 21.1±1.6 kPa,respectively(p<0.05).The wet weight of the hydrogels in each group stabilized from the 12th hour and there was no difference between the groups(p>0.05).The exosome release behavior of all groups of hydrogels lasted more than 17 days,and there was no statistical difference between groups(p>0.05).The hydrogels of G5H2 group were chosen as the hydrogel carriers for encapsulating USC-Exos based on the rheological results and compressive strength results;2.In in vitro experiments,USC-Exos was isolated and extracted,with ellipsoidal shape and diameter about 30 nm-150 nm and could express exosome-specific markers.USC-Exos could be absorbed and internalized by rat BMSCs and EPCs.The three groups of hydrogels,G5H1,G5H1.5 and G5H2,had good cell biocompatibility,and the effects on cell proliferation There was no difference(p>0.05).USC-Exo@G5H2 composite hydrogel significantly promoted the expression of alkaline phosphatase and calcium nodule formation in rat BMSCs,and had a converging effect on BMSCs,and it significantly promoted the expression of osteogenesis-related genes(p<0.05).USC-Exo@G5H2 composite hydrogel significantly promoted the formation of tubules in rat EPCs and had a chemotactic and migratory effect on EPCs.It significantly promoted the expression of angiogenesisrelated genes(p<0.05);3.In vivo experiments,the H&E staining results of G5H1,G5H1.5 and G5H2 hydrogels embedded subcutaneously in rats showed no significant macrophage infiltration,no significant difference in fibrous capsule wall thickness(p>0.05),and collapse and degradation inside the hydrogels.the slow release efficiency of G5H2 hydrogels was significantly higher than that of the PBS solution group(p<0.05).the Micro-CT and histological staining semi-quantitative results suggested that USC-Exo@G5H2 composite hydrogel group had excellent pro-bone repair ability(p<0.05),and this group could promote the formation of H-type blood vessels(p<0.05).Conclusion:The drug delivery system of GelMA-HAMA/nHAP hydrogel with injectability was successfully prepared.The synthesized hydrogels possess promising physicochemical properties as well as sustained and effective release properties for USC-Exos,among which the G5H2 group hydrogels with the best mechanical properties were selected as the sustained delivery carriers for USC-Exos.Based on possessing excellent biocompatibility and mechanical properties,USC-Exo@G5H2 composite hydrogels can promote osteogenesis of rat BMSCs and angiogenic ability of EPCs in vitro,respectively,and promote bone defect repair and H-type vessels to be expressed in large amounts in bone regeneration regions in vivo,thus achieving in situ bone defect repair.As a result,the construction of USCExo@G5H2 composite hydrogel provides a new idea for the repair of irregular bone defects in clinical practice and has potential application prospects.