Experimental Study Of The Effects And Mechanism Of Osteogenesis Induced By A Chitosan Nanoparticle Sustained-release System Loaded With BMP-2,VEGF And TGF-?

Objectives: To study the efficiency and quality of induced osteogenesis by double-sustained release system loaded with BMP-2,VEGF and TGF-? and explore the network regulation mechanisms of induced osteogenesis.Methods: The chitosan nanoparticle double-sustained release system loaded with BMP-2,VEGF or TGF-? were prepared by an ionic gelation method and then were encapsulated into the thermosensitive microporous hydrogel.At the first stage,mouse embryo osteoblast precursor cells(MC3T3-E1)were cultured in vitro,and BMP-2,VEGF and TGF-? were added to an osteogenic induction medium(OM)in different combinations to conduct experiments and compare results by factor group,with the OM serving as the control group.At the second stage,MC3T3-E1 cells were cultured in the OM with different chitosan nanoparticle sustained release systems loaded with different combinations of BMP-2,TGF-? and VEGF.Particle size,zeta potential,morphologies,drug loading efficiency,encapsulation efficiency and the release curve were analyzed.The osteogenesis effects were analyzed by ALP staining,alizarin red staining.RT-PCR was used to detect the expression of osteogenesis-related genes,and Western blot analysis was used to explore the mechanisms of osteogenic induction.Results: The chitosan nanoparticles had a regular spherical shape,with a particle size of(197±7.1)nm,a PDI of 0.21,the drug loading rate of(59.84±3.06)%,and the cumulative release rate of the chitosan nanoparticle double-sustained release system loaded with BMP-2 of(34.78±3.42)% for the first 48 h and(78.05±3.22)% on the 30 th day,which can significantly reduce the burst release phenomenon to reduce the concentration of growth factors and prolong the action time of growth factors.First stage study showed that the ALP staining was deeper and the osteogenesis data were higher for the applied BMP-2,VEGF and TGF-? combination than for the other groups after 3 days(P<0.05),the ALP staining and ARS staining of the applied BMP-2 and VEGF combination were deeper and the osteogenesis data were higher than those of the group with the BMP-2,VEGF and TGF-? combination and the other groups at 7 and 14 days(P<0.05).After optimizing the time-effect relationship of the combined application,when BMP-2,VEGF and TGF-? were added in the early stage and BMP-2 and VEGF were added in the late stage,the ALP staining and ARS staining of these groups were significantly deeper and the osteogenesis data were higher than the BMP-2 and VEGF combination group at 7 and 14 days(P<0.05).The expression of the RUNX2 gene in the optimized group with the combination of BMP-2,VEGF and TGF-? was also significantly higher than that in the other groups(P<0.05).At the second stage,ALP staining and alizarin red staining showed that the efficiency and quality of osteogenic induction from a combined application of a double-sustained release system loaded with BMP-2,a double-sustained release system loaded with VEGF and chitosan nanoparticles loaded with TGF-? was significantly enhanced(P<0.05),and similar results were found in the expression of the osteogenic-related genes RUNX2 and ALP(P<0.05).The osteogenic differentiation induced by a combined application with BMP-2,TGF-?1 and VEGF was mainly through the classical smad signaling pathway,with p38-MAPK and erk1/2-MAPK signaling pathway as assist influencing factors.Conclusions: The chitosan nanoparticle sustained-release system loading BMP-2,VEGF and TGF-? with an optimal time-effect relationship could significantly enhance the efficiency and quality of osteogenic induction,mainly through the classical BMP-receptors-smad signal pathway,with the p38 and erk1/2 signal pathways assisting,to promote osteogenic differentiation.