Preparation Of BMP-2 Protein Microspheres Embedded In Nanofiber Double Sustained Release Scaffold And Its Effect On Proliferation And Differentiation Of MC3T3-E1 Cells

Objective:The material characterization of the polycaprolactone composite nanofiber scaffold containing BMP-2 sustained-release microspheres and the double sustained release of BMP-2 growth factor were investigated by electrospinning technique.The proliferation of MC3T3-E1 cells was further evaluated.Differentiation influence.Method:(1)The chitosan nanospheres loaded with BMP-2 were prepared by desolvation method and electrostatic self-assembly technique.The morphology and particle size of the nanospheres were detected by SEM,TEM and nanometer particle size analyzer.ART-FTIR and CLSM were used.The composition of the microspheres was detected,and the encapsulation efficiency and protein release of BMP-2 protein microspheres were detected by BMP-2 Elisa Kit.The biological activity of BMP-2 was detected by ALP kit.(2)Polycaprolactone composite fiber scaffolds containing BMP-2 nanospheres were prepared by electrospinning technique.The morphology of the composite scaffolds was observed by SEM and TEM.The properties of the materials were tested by tensile strength and contact angle test.BMP-2 Elisa Kit was used to detect the sustained release of BMP-2 protein in scaffolds.CLSM was used to observe the growth of cells in scaffolds.CCK-8 method was used to detect cell proliferation and cytotoxicity of materials.ALP and RT-PCR were used.The method and alizarin red method were used to detect the formation of alkaline phosphatase,related genes and mineralized nodules during scaffold differentiation.Results:(1)SEM and TEM results show that the BMP-2/Chi microspheres have a relatively regular spherical shape,uniform dispersion,smooth surface and particle size less than 1μm.A characteristic peak of chitosan was observed in the infrared spectrum of BMP-2/Chi,indicating that the successful microspheres were encapsulated by chitosan.The results of CLSM,PDI and Zeta potential indicate that the microspheres formed by chitosan-coated BSA are stable and evenly distributed.There was no statistically significant difference in drug loading rates between the three groups.The ELISA results showed that BMP-2 in nanoparticles could be sustained for up to 168 hours,and the sustained release curve of BMP-2/Chi nanoparticles was smoother,which could achieve sustained release of BMP-2 The results of ALP showed that NMP-2 released by BMP-2/Chi nanosphere group had better biological activity than the blank control group,and it was statistically insignificant compared with the positive control group(2)The three groups of nanofiber scaffolds shown by SEM and TEM showed a three-dimensional network structure with random distribution of criss-crossing,in which the microsphere-containing scaffolds agglomerated and the diameter was slightly thickened,and the surface of the fibers had spherical protrusions and microspheres.Wrapped in a PCL holder.The tensile strength of the fiber group added to the nanospheres is lowered.The nano-microsphere fiber scaffolds in the contact angle experiment showed increased hydrophilicity.The ELISA results showed that the growth factor of the PCL/BNPs group was continuously released for 28 days,and the release amount was over 90%.Under the CLSM microscope,the cells in the PCL/BNPs group were well spread on the scaffold,and there was obvious protrusion of the pseudopod.The CCK-8 results showed an increase in the number of cells adhering to the surface of the PCL/BNPs group.The results of the alizarin red experiment showed that the mineralization of the PCL/NPs group and the PCL/BNPs group was more obvious,and more calcium salts were deposited,and the size and number of calcium nodules increased significantly.The results of ALP and RT-PCR showed that the ALP expression and related osteogenic genes COL1,OPN and RUNX in PCL/BNPs group were higher than other groups.Conclusion:In this experiment,a polycaprolactone composite fiber scaffold containing BMP-2 nano-sustained release microspheres was successfully prepared,in which BMP-2 growth factor can be sustainedly released in small doses,and MC3T3-E1 cells can grow,proliferate and form on the surface of the material.Bone differentiation,which provides new material options for the development of bone tissue engineering.