Research On PCL-HAp Coating Loaded With BMP-2 For Titanium Surface Modification And Osteogenesis Property In Vitro

Objective:In this experiment,we prepared a polycaprolactone-hydroxyapatite(PCL-HAp)coating on titanium surface loaded with bone morphogenetic protein-2(BMP-2),and combined it with osteoblasts(MC3T3-E1)Co-cultivation explored the effect of the coating on the proliferation,adhesion and osteogenic differentiation of MC3T3-E1 cells,which laid the foundation for the application of coating in clinical practice.Methods:1.HAp nanorods were prepared by hydrothermal method,and PCL-HAp coating was deposited on the surface of titanium implant by electrospinning effect,which was stirred overnight in BMP-2 solution to form PCL-HAp-BMP-2 coating.2.According to the different substances contained in the surface of titanium implants,they were divided into four groups: Ti,Ti-PCL,Ti-PCL-HAp and Ti-PCL-HAp-BMP-2.The surface morphology of the coating was observed by scanning electron microscope(SEM);The contact angle was measured by contact angle tester;The composition of the coating surface was analyzed by fourier infrared spectroscopy(FT-IR);And the drug loading rate and sustained release effect of Ti-PCL-HAp were measured by bull serum albumin(BSA).3.MC3T3-E1 cells were inoculated on the surface of the material,and the biocompatibility of the material was detected by living/dead cell staining;The proliferation ability of osteoblasts on the surface of the material was detected by CCK-8;The adhesion ability of cells on the surface of the material was detected by DAPI staining;The morphology of cells on the material surface was observed by SEM;The ALP activity was detected by alkaline phosphatase(ALP)staining;The extracellular matrix was analyzed by alizarin red S staining;The related osteogenic genes were detected by PCR.Results:1.SEM showed that the surface of the PCL-HAp coating was an irregular porous structure,the pores were interpenetrated with each other,and the pore size was different;The contact angle showed that the contact angle of Ti-PCL was the largest,and the Ti-PCL-HAp-BMP-2 was the smallest;FT-IR demonstrated that we successfully prepared PCL-HAp coating on Ti surface;And the load rate of BSA on the surface reached 83.42±1.35%,the release duration of BSA was longer than 180 h,the final release rate reached about 79%.2.The Live/dead cell staining showed that there were no obvious dead cells in each group.And the number of cells on the surface of the materials was Ti-PCL-HAp-BMP-2>Ti-PCL-HAp>Ti-PCL>Ti;The CCK-8 showed the absorbance were Ti-PCL-HAp-BMP-2>Ti-PCL-HAp>Ti-PCL>Ti at 1 d,3 d,5 d,7 d(P<0.05);The DAPI staining showed that after 4 h and 24 h,the number of cells attached to the material surface was Ti-PCL-HAp-BMP-2>Ti-PCL-HAp>Ti-PCL>Ti,but there was no significant difference in the number of cells between the Ti-PCL and Ti(P>0.05);The SEM showed that the cell morphology of Ti-PCL-HAp-BMP-2 was typical polygonal extension,and the pseudopodia have extended to the surface of other cells,while the cell morphology of Ti was not obvious.3.The ALP staining showed that the ALP in all groups increased in a time-dependent manner.On the 7 d,the expression of ALP in Ti-PCL-HAp-BMP-2coating was significantly higher than other three groups,but the difference between Ti and Ti-PCL was not obvious;The ALP activity of the Ti-PCL-HAp-BMP-2 was still different from that the control group at 14 d and 21 d,but there was no obvious difference between Ti-PCL-HAp and Ti-PCL-HAp-BMP-2.The alizarin red S staining showed the calcium nodules were Ti-PCL-HAp-BMP-2>Ti-PCL-HAp>Ti-PCL>Ti(P<0.05).4.Gene expression analysis of cells on the material surface showed time-dependent upregulation,and we found that RUNX2,ALP and COL1A1 gene expression on Ti-PCL-HAP-BMP-2 were significantly higher than those on control group(P<0.05).Conclusions:1.The PCL-HAp coating on titanium surface loaded with BMP-2 was successfully prepared.2.The PCL-HAp coating on titanium surface loaded with BMP-2 had good sustained-release performance.3.The PCL-HAp coating on titanium surface loaded with BMP-2 promoted cell proliferation and cell adhesion on the surface of implant,and improved the osteogenic properties of cells in vitro.