3D Bioprinted Double Crosslinking Hydrogel Scaffold For Promoting Vascularization And Bone Regeneration

3D printing technology,has gained broad focus in the engineering field of bone tissue attributed to its superiority of constructing personalized complex tissues structurally.Currently,extensive research on the development of bone repair materials has been conducted based on 3D printing technology,but these scaffold materials are limited in clinical application due to insufficient vascularization in early process and weak osteogenic induction activity.Compared with traditional3D printing technology,3D bioprinting technology can accurately control the assembly of biomaterials,seed cells and growth factors in a specific space,and create a biological repair body of bionic natural tissue macros,microstructure and regenerative microenvironment,which is an ideal way to achieve the engineering construction of bone tissue.However,to improve the vascularization and bone regeneration performance of 3D bioprinted hydrogel scaffold,it is much necessary to develop adaptable bioink and couple vascularization and bone regeneration function in the process of bone repair,which is capable to facilitate the availability of scaffold materials structurally and functionally and enhance their application in bone repair.In this study,a novel biodegradable and bioprintable scaffold consisting of methacrylated gelatin（Gel MA）and methacrylated gellan gum（GGMA）was constructed by 3D bioprinting technology to enhance the mechanical properties of the hydrogel network through dual photo-crosslinking and ionic crosslinking.Moreover,the long-lasting and slow release of DFO was achieved by adding Ethosome（Eth）loaded with deferoxamine（DFO）to the bioink,a drug with both angiogenic and osteogenic activities.By preparing this drug-loaded hydrogel scaffold（Eth-DFO@Gel MA/GGMA）with vascular regenerative ability to induce bone tissue regeneration and repair,the problem of inadequate vascularization and osteogenic performance of 3D printed hydrogel scaffolds.Also,we investigated the mechanical,rheological,degradation and drug release properties of the scaffolds and evaluated the angiogenic and osteogenic properties of the Eth-DFO@Gel MA/GGMA scaffold.Through the gel-forming concentration and printing suitability test for Gel MA and GGMA,the phenomenon that 7%Gel MA/1%GGMA bioinks had the best gel-forming performance and printing suitability was discovered.The mechanical test results showed that the Gel MA hydrogel had poor mechanical properties due to the presence of single covalent cross-linking in the 3D network;with the increase of GGMA and Ca²⁺concentration,the maximum stress and Young’s modulus of composite Gel MA/GGMA hydrogel were significantly increased.Drug release assays in vitro revealed that sustained drug DFO release achieved on an Eth-DFO@Gel MA/GGMA hydrogel.In vitro proliferation assay displayed that the prepared scaffolds all have good cell compatibility and can well promote cell proliferation.In vitro scratch tests,Transwell migration and Matrigel tube-forming experiments manifested that the Eth-DFO@Gel MA/GGMA scaffold can significantly promote the recruitment,migration and tubular structure formation of human umbilical vein endothelial cells（HUVECs）.Further,angiogenesis-related gene expression assays indicated that DFO-loaded hydrogel scaffolds can upregulate the expression of angiogenesis-related genes.The evaluation of the facilitated bone differentiation properties of drug-loaded hydrogel scaffolds in vitro by alizarin red staining,alkaline phosphatase（ALP）staining and osteogenesis-related gene expression detection indicated that the Eth-DFO@Gel MA/GGMA scaffold significantly promoted expedited formation of the mineralization matrix and enhanced expression of ALP and osteogenesis-related genes.Subcutaneous embedding experiments in mouse demonstrated extraordinary biocompatibility of the Gel MA,Gel MA/GGMA and Eth-DFO@Gel MA/GGMA scaffolds.Experiments in the critical size skull defect model of rats showed that the Eth-DFO@Gel MA/GGMA scaffold bone repair was superior to that of Gel MA group and Gel MA/GGMA group.Histological staining and immunofluorescence staining further indicated that the Eth-DFO@Gel MA/GGMA scaffold had excellent angiogenesis and bone regeneration properties.In conclusion,GGMA/Gel MA bioink has excellent mechanical properties and printability,with the capability of realizing slow release of the drug by using Eth as a carrier of DFO,while the prepared Eth-DFO@Gel MA/GGMA scaffold is able to activate the HIF1-αsignaling pathway to promote angiogenesis and bone regeneration.Therefore,the drug-controlled release strategy based on the Gel MA/GGMA bioink and microcarrier can be used to construct a 3D printed composite hydrogel scaffold coupled to vascularization and osteogenic effects,providing a new idea for the treatment of bone defect repair.