| Objective This study first aimed to form double TiO2 nanotube arrays as a backing material. Then, the carbonyldiimidazole (CDI) was applied as a crosslinking agent to couple the recombinant human bone morphogenetic protein-2 (rhBMP-2).Eventually, TiO2 nanotube-CDI-rhBMP-2 composite bioactive layers were synthesized via coupling polymerization. Mouse bone marrow stromal stem cells (BMSCs) were selected by co-culture with TiO2 nanotube-CDI-rhBMP-2 composite materials, to explore the effects of the composite layers on the adhesion, proliferation and differentiation of BMSCs, in order to provide experimental evidence for the biochemical modification of titanium implants.Material and methods Commercial pure titanium foils with purity of 99.99% were cut into diameter 12mm titanium discs by laser cutting machine. Silicon carbide sandpaper (800#,2000#,3000#,5000#,7000#) were used to polish the discs until the surface was progressively polished mirror-like. Anodic oxidation was applied for preparing double TiO2 nanotubes, and then the discs were treated with carbonyldiimidazole (CDI) for chemically grafted rhBMP-2, which were classified as the experimental group. The mechanical polishing discs were classified as blank control group, meanwhile, the negative control groups (A and B) were Anodic TiO2 nanotubes and TiO2 nanotube+ CDI. Field emission scanning electron microscopy (FESEM) and X-ray photoelectron spectroscopy (XPS) were utilized to detect the surface physicochemical properties. Moreover, Cell adhesion and spreading had also been tested.CCK-8 kit was carries out for detecting proliferation, and alkaline phosphatase (AKP) kit was applied for a kind of indirect determination method for cell differentiation of BMSCs.Results Two-step electrochemical anodization could be utilized for preparing double TiO2 nanotube arrays, which were good substrate material for graft biologically active proteins. FESEM showed that the surface of the blank control group was smooth with occasional scratches; the negative control group A formed uniform double nanotube arrays, the inner and outer tubes closely connected and the diameter was about 20nm and160nm, respectively; the double tubes in the negative control group B was blurred inside or outside; the surface of the experiment group was seen sporadic miliary particulate. XPS Confirmed that the surface of the specimen was composed of N elements, C elements and O elements, the surface of the experimental group was significantly higher in the peak of N elements than other three groups. The BMSCs on the surface of the blank control group were most fusiform shape, cell adhesion was limited; the cells on the surface of the control group A spread well, but cell junction was loose and less pseudopodia; for the control group B cell body spread in good condition, the shape of the cells was flat with fewer tentacles; the growth of BMSCs on the experimental group spread evenly, extensive and cell junction was close connections, meanwhile, some cell pseudopods extending into the nanotubes. Cell proliferation activity on the 1st day among different groups was not obvious, the experimental group (0.355±0.058), blank control group (0.326±0.019), negative control A group (0.338±0.022), negative control B group (0.314±0.061) (P>0.05). Cell proliferation activity on the 3rd and 5th day in the experimental group (3.295±0.153,3.823±0.058) were significantly higher than that in the blank control group (2.479±0.064,3.131±0.096) and negative control group A (2.715±0.075,3.371±0.047) and negative control group B(2.756±0.132,3.637±0.047) (P<0.01), respectively. Alkaline phosphatase activity on the 5th,7th and 11th day in the experimental group (0.0477±0.0287, 0.0615±0.0016,0.0667±0.0018) were better than that in the control group (0.0468±0.0094,0.0509±0.0018,0.0544±0.0015), negative control A group (0.0455±0.0092,0.0024±0.0575 0.0553±0.0014) and negative control B group (0.0464±0.0099,0.0568±0.0021,0.0593±0.0014) (P<0.01), respectively.Conclusion TiO2 nanotube arrays could be loaded with rhBMP-2 by biochemical methods and had good biocompatibility. The BMSCs co-culture with TiO2 nanotube-CDI-rhBMP-2 complex layer in vitro confirmed that the complex film could promote cellular adhesion, proliferation and differentiation. Therefore, it could be a novel method for improving the surface modification of titanium implants. |
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