Construction Of BMP-2 And TGF-?1 Loading Microspheres Complex With Biomaterial Jawbone Scaffold

In order to avoid the shortcomings of low activities of existing bone replacement materials and to achieve the purpose of repairing a large area of bone defect,the growth factor-loaded nano-microspheres prepared and biomaterials that are in close to the extracellular matrix of bone cells were combined together to construct a security three-dimensional porous scaffold with bone inductivity.1.The results of chitosan microspheres prepared by ion gel method show that when preparing the solution,under the condition of 1 mg/m L of chitosan solution,1mg/m L sodium tripolyphosphate solution,chitosan and sodium tripolyphosphate quality ratio of 5:1 ~ 6:1 as well as chitosan solution p H value of 5.0 ~ 6.0,chitosan microspheres distributed in uniform particle size,of which average is about 150 ~ 350 nm,could be obtained.The blank chitosan microsphere was used to adsorb BMP-2growth factor,and the adsorption amount was measured by ELISA kit and enzyme labeling instrument.According to the adsorption curve derived,the adsorption equilibrium was reached at 100 min,and the curve fitting showed that the adsorption model was consistent with the second-order adsorption kinetics.The BMP-2 loading rate of chitosan microspheres against was 7.2206±0.0024 ng/mg,and the encapsulation rate was(92.06±0.02)%.2.The optimal conditions of emulsion cross-linking method for the preparation of chitosan microspheres,of which the average particle size was distributed at about150 ~ 350 nm,were determined as following: 1000 RPM stirring speed,2% quality percentage of acetic acid concentration,10 mg/m L chitosan concentration,oil-water ratio(v/v)for 4:1,1m L cross-inking agent,about 30 ? cross-linking temperature and the 1.2 g span-80.According to the curve fitting,the adsorption equilibrium was reached at 100 min,and the adsorption model conforms to the second-order adsorption kinetics.The drug-loading rate was(3.83±0.02)ng/mg and theencapsulation rate was(0.8972±0.0037).3.In the co-adsorption experiment,the adsorption of TGF-1 on chitosan microspheres which prepared by ionic gel method reached an equilibrium state within6 hours,meanwhile,the drug loading rate was(5.03±0.02)ng/mg,and the adsorption rate was(92.42±0.02)%.Within 2 hours,the adsorption of BMP-2 reached an equilibrium state,and the drug loading rate was(2.89±0.02)ng/mg,as well as the adsorption rate was(92.33±0.02)%.According to the curve fitting,the chitosan microspheres exhibited a second-order kinetic adsorption on TGF-1 and BMP-2.4.Sodium alginate/sodium hyaluronate/collagen scaffold was constructed with fixed mass ratio and different solid contents of biological hydrogels solution by free-drying method.FT-IR spectrometer was used to characterize the chemical structure of the scaffold.The three-dimensional porous structure of the scaffold was observed by SEM,and the result showed that pore size of the scaffold was about100-250 ?m.With the increase of scaffold material concentration,the moisture content of the scaffolds decreased,as indicated by measurement using freeze-drying method.When measured by drainage method,the porosity of the scaffolds was above90%.The tensile strength of the scaffold measured by the tensile machine increased with the increase of the material concentration of the scaffold,while the elongation at break decreased.Appropriate biomaterial scaffolds were selected and inoculated with ADSCs culture,and the CCK-8 test results were used to to test the cytotoxicity.It was found that the growth of cells on the scaffold was not significantly different from that of the blank control group.SEM observation showed that the cells adhered well to the scaffold,indicating that the biomaterial scaffold had a good cell compatibility.5.The chitosan microspheres loaded with TGF-1 and BMP-2 combined with sodium alginate/hyaluronate/collagen scaffold was constructed.ADSCs was used for bone induction,and all the three osteogenic indicators(ALP activity quantification,osteocalcin expression and VK mineralized nodule staining identification)showed that the porous scaffold combined with growth factor-loaded microspheres could effectively and orientatedly induce bone formation.Additionally,results of CCK-8method,HE staining and microscope observation showed that no significantdifferences were presented between the scaffold groups and the control groups,indicating that the composite scaffold showed no cytotoxicity.Therefore,the growth factor-loaded microspheres combined with scaffold has a strong bone repair ability and potential value in bone tissue engineering.