| Part 1 Preparation,related characterization and in vitro biological performance and optimization of GelMa hydrogelResearch objectivesPrepare and synthesize GelMa hydrogel,and its related characterization,preliminary detection of physical and chemical properties and biological properties,to determine its optimal parameters.MethodsDifferent concentrations of GelMa were prepared,and the swelling rate test,elastic modulus evaluation,in vitro collagenase degradation test,in vivo hydrogel degradation test in rats,and electron microscope observation of the characterization of the hydrogel were performed.Observe the effect of different UV cross-linking time on the activity of rat bone marrow mesenchymal stem cells BMSCs in the hydrogel.ResultsThe 24h swelling rate of GelMa hydrogel at the concentration of 3%,5%,7%and 10%is 20.1±0.6,15.7±0.8,13.2±0.9 and 8.3±0.6,respectively,and the elastic modulus is 3.3 ±2.6kPa,respectively,7.6±3.3kPa,15.9±4.4 kPa,25.0±6.6kPa,and the complete degradation time was 3d,7d,14d and 21d,respectively.The degradation time of hydrogel planted on the back of SD rats is about 20 days.Scanning electron microscope observation of the section of the hydrogel shows that the inside of the hydrogel is a smooth,porous three-dimensional grid structure.The shorter the UV cross-linking time,the better the cell viability and the stronger the cell proliferation ability.ConclusionThe pore size of GelMa hydrogel is suitable for the osteogenic environment.The elastic modulus of the hydrogel is directly proportional to the concentration of the hydrogel.The swelling rate and degradation rate of the hydrogel are inversely proportional to the concentration.The degradation rate of hydrogel in vivo and in vitro is basically the same.The longer the cross-linking time of the UV lamp,the greater the impact on the biological behavior of cells.GelMa with a concentration of 10%is suitable for the osteogenesis of rat bone marrow mesenchymal stem cells within 3 0S of UV light cross-linking time.Part 2 The effect of GelMa hydrogel loaded with nano-silicate and growth factor SDF-1 ? and its role in repairing skull bone defectsResearch objectivesAutologous bone transplantation is the gold standard for the treatment of bone defects.Limited by the limited bone mass of the donor site and various morbidities,such as infection and pain.Injectable hydrogels with osteogenic potential have the advantage of being minimally invasive and filling irregular bone defects.Nano silicate(SN)has good osteoinductive properties,slow release of growth factors and enhanced hydrogel injectability.Stromal cell-derived factor 1 ?(SDF-1 ?)as an osteoinductive factor also has the ability to recruit stem cells.Therefore,we have designed a simple,cell-free,fast-injectable GelMa-SN-SDF-1 a hydrogel,a composite hydrogel that recruits stem cells,has good osteoinductivity,and has a slow-release effect on growth factors.And verify its osteogenesis effect in the skull defect model.MethodsSN and SDF-1 ? are introduced into the prepolymer to synthesize gelatin-methacryloyl(GelMa)prepolymer to improve injectability,controlled release performance,osteogenic ability and stem cell homing.Analyze the injectability of the hydrogel of different types of needles,observe the internal structure and porosity of the scanning electron microscope,and test the compressive modulus of the hydrogel sample for swelling rate test and degradation ability test.A rat SDF-1 ? ELISA kit was used to determine the concentration of released SDF-1?.Analyze cell viability,proliferation,proliferation and migration.Alizarin Red S staining,quantitative analysis of bone protein expression.On the 8mm skull defect model in SD rats,the osteogenesis effect in vivo was further analyzed by Micro-CT,H&E staining and Goldner-Masson trichrome staining.ResultsGelMa-SN-SDF-1? shows excellent injectability through various types of needles at room temperature.The loaded SDF-1 ? showed a long-term controlled release pattern and effectively stimulated the migration and homing of MSCs.GelMa-SN-SDF-1? hydrogel can promote cell proliferation,migration,expression of osteogenic biomarkers and matrix mineralization.GelMa-SN-SDF-1? hydrogel filled the critical skull defect in rats,repairing 77%of the volume.The osteogenic effect is significantly better than GelMa,GelMa-SN,GelMa-SDF-1 ?.ConclusionGelMa-SN-SDF-1? hydrogel is a simple,rapid preparation of cell-free injectable hydrogel for stimulating bone regeneration.The GelMa hydrogel introduced by SN and SDF-1?can promote the osteogenesis of the hydrogel and guide the homing of MSC.GelMa-SN-SDF-1? injectable hydrogel shows excellent injectability,biocompatibility,and osteogenic ability both in vitro and in vivo.The loaded SDF-1? showed a controlled long-term release pattern.GelMa-SN-SDF-1 ?is effective in repairing rat skull defect models.Part 3 Application of GelMa hydrogel with mesoporous silica microspheres sequentially releasing SDF-1 a and IGF-1 in tibial bone defect andintervertebral bone fusionResearch objectivesMost biomimetic materials consist of three important components,scaffolds,cells,and growth factors.The requirements for the scaffold are basically less immune elimination response,good biological description,and the role of providing a climbing skeleton for cells.There are many kinds of growth factors in organisms,and they play an effect on bone induction at different time periods of bone regeneration.The time window of SDF-1 ? is Id?10d,which has osteoinductive effect and the function of recruiting stem cells to homing.IGF-1 exerts osteoinductive effect from 7d-20d.In the initial stage of bone regeneration,it plays an important role in the final healing effect.We use GelMa to release SDF-1?,and use mesoporous silica microspheres to further extend the release window of IGF-1 to 20 days.It is hoped that the sequential release of the two growth factors can be achieved in this way,and GelMa-SDF-1 ?-SiO2(IGF-1)is produced and injected to verify its osteogenesis effect on the rat tibia bone defect model and the tail vertebra fusion model.MethodsThe hydrogel's own degradable characteristics and pore structure can slowly release SDF-1 ? in the hydrogel,and the mesopores on the surface of the mesoporous silica microspheres can delay the flow of IGF-1 into the hydrogel.GelMa-SDF-1 ?-SiO2(IGF-1)uses mineralization staining analysis in vitro and quantitative analysis of bone protein expression.In vivo experiment is a rat tibial bone defect model.The tibial tubercle is rubbed upward to create a 6mm defect depth.Reach the bone marrow cavity.In the rat coccygeal fusion model,the C4/5 coccygeal disc was removed,and a PEEK tube was used to maintain a 3mm gap for bone fusion.The two models were further analyzed in vivo by Micro-CT.ResultsSDF-1 ? was released more than half of the time on the 3rd day,and the release reached 95.8%on the 15th day.IGF-1 released more than half on the 7th day,and the cumulative release on the 21st day reached 90.5%.GelMa-SDF-1 ?-SiO2(IGF-1)can promote the expression of osteogenic proteins and the mineralization of hydrogels.The repair area of GelMa-SDF-1 ?-SiO2(IGF-1)in the in vivo tibial bone defect model was larger than the other three groups.X-ray showed that the repair effect was good,and the bone mass BV/TV of micro-CT analysis reached 67.55%.In the caudal fusion model,X-rays showed that there was a new bone connection between the caudal vertebrae,and the intervertebral space was smaller than other experimental groups.The bone mass BV/TV of micro-CT analysis reached 80.72%.ConclusionGelMa hydrogel loaded with SDF-1 a can have a sustained release effect on the growth factor.Centrifugation allows IGF-1 to enter the mesoporous silica microspheres,and then blending the mesoporous silica microspheres with GelMa can release the growth factor for a longer time.GelMa-SDF-1 ?-SiO2(IGF-1)can sequentially release two growth factors,which can better promote osteoblast differentiation and osteogenic protein expression in vitro.GelMa-SDF-1 ?-SiO2(IGF-1)has a good osteogenesis effect in the tibia bone defect model and the coccygeal intervertebral fusion model in animals. |
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