| Three-dimensional(3D)printing can be used to prepare tissue engineering scaffolds individually,which has broad application prospects in tissue engineering.However,compared with the cell scale(μm level),the distance between filaments(mm level)in traditional 3D printing scaffolds is often larger,and cells can only adhere and proliferate on a single filament.The gap between filaments is difficult for cells to cross,which seriously limits the migration space and proliferation ability of cells and restricts the potential of scaffolds to promote tissue regeneration.In order to overcome the shortcomings of traditional 3D printing scaffolds,we prepared a 3D printed biodegradable poly(glycerol-co-sebacic acid-co-L-lactic acid-co-polyethylene glycol)(PGSLP)scaffold,and two methods to construct gelatin with two structures were filled with 3D printed scaffolds.The effects of different microstructure of scaffolds on cell migration,proliferation and bone tissue regeneration were researched.The first method was to form the nanofibrous structured gelatin in 3D printed PGSLP scaffold by thermally induced phase separation(TIPS)technology,nanoscale structured gelatin/PGSLP composite scaffolds were constructed,named NGP scaffolds.The second method was to filled gelatin solution in 3D printed PGSLP scaffolds and freeze-drying directly,microscale structured gelatin/PGSLP composite scaffolds are formed,named MGP scaffolds.Vascularization is one of the most important challenges in the repair of large bone defects.In the process of bone tissue repair,vascularization and bone formation promote each other and play a synergistic role.In order to realize the rapid vascularization of scaffolds and accelerate the process of bone regeneration,deferoxamine(DFO),a small-molecule drug that induces angiogenic ability and osteogenic differentiation,was loaded into the MGP and NGP scaffolds(DFO@MGP and DFO@NGP scaffolds),respectively.The physical and chemical properties of the scaffolds were tested,and the potential of the two scaffolds in promoting angiogenesis and osteogenesis in the process of bone tissue regeneration and repair was evaluated by in vitro and in vivo experiments.The main research contents include the following aspects:(1)Firstly,PGSLP polymer was synthesized,and its structure was verified and analyzed.The results of FTIR and ~1H NMR indicated that PGSLP was successfully synthesized.Then,salt particles were used as thickener to improve the printability of PGSLP,the proportion of PGSLP and salt in 3D printing composite ink was explored.By observing the high-temperature conformability of composite ink with different proportions,when the mass ratio of PGSLP to salt was 1:2,the composite ink could maintain its shape to a certain extent under high temperature conditions.Subsequently,PGSLP and salt mixture of this ratio were selected as 3D printing ink to prepare PGSLP scaffold.(2)The physicochemical properties of MGP and NGP scaffolds were investigated.SEM images showed that PGSLP scaffolds were filled with microscale structured gelatin and nanoscale structured gelatin respectively.XPS results showed that DFO was loaded into the composite scaffolds.(3)Finally,the biological evaluation of DFO@MGP and DFO@NGP were carried out.In vitro experiments showed that,DFO@MGP and DFO@NGP scaffolds can promote the migration and tubular formation of human umbilical vein endothelial cells(HUVECs),enhance the formation of mineralized nodules of bone marrow mesenchymal stem cells(BMSCs)and the expression of osteogenic related genes during osteogenic differentiation.In vivo,the angiogenesis and bone repair ability of the scaffolds were evaluated by subcutaneous embedding in mice and critical skull defect models in rats.The results of subcutaneous embedding showed that MGP,NGP,DFO@MGP and DFO@NGP scaffolds all have excellent biocompatibility.DFO-loaded composite scaffold significantly promoted the formation of blood vessels.In the rat model of skull defect,DFO-loaded scaffolds significantly promoted bone regeneration,and the DFO@NGP scaffold showed a better repair effect than DFO@MGP scaffold.In summary,PGSLP,a thermosetting biomaterial with good elasticity,biocompatibility and biodegradability,which was independently developed by the research group,was selected as 3D printing bioink,thus enriching the 3D printing bio ink library.Through the combination of 3D printing scaffold and bionic nanofiber will help the migration and signal transduction of the cells inside the scaffold,accelerate the tissue repair process and provide a new idea for the preparation of bone repair scaffolds.Therefore,this kind of composite scaffold loaded with DFO with micro-and nano-fibrous structure is a potential material for bone defect repair and has a certain application prospect in bone tissue engineering. |
