The Effects of Post-Adsorption of Plasma Proteins on the Osteogenic Potential of Osteoblastic Cells Grown on GDF-5- Functionalized Titanium Nanotopography

Surface functionalization of titanium (Ti) surfaces with growth factors has been developed aiming to stimulate bone tissue repair at the biomaterial-tissue interface. This study evaluated the effects of post-adsorption of major plasma proteins- albumin, fibrinogen and fibronectin on a Ti surface nanotopography functionalized with growth and differentiation factor-5 (GDF-5) on in vitro mineralization. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvarial bone and were grown, for periods of up to 14 days, on the following Ti disc surfaces: 1) Machined; 2) Machined+GDF-5 – machined surface functionalized with GDF-5 by simple adsorption; 3) Machined+Plasma Protein – machined surface with plasma protein solution incubated for 2 h; 4) Machined+GDF-5+Plasma Protein – machined surface functionalized with GDF-5 by simple adsorption, subsequently incubated with plasma protein solution for 2 h; 5) Nano – nanotopography obtained by etching with a mixture of H₂SO₄/H₂O₂ (1:1) for 4 h; 6) Nano+GDF-5; 7) Nano+Plasma Protein; 8) Nano+GDF-5+Plasma Protein. On the day before primary cell plating, nanotextured Ti surfaces were incubated (functionalized) overnight at 4 °C with 200 ng/mL human recombinant GDF-5. The post-adsorption of albumin, fibrinogen, fibronectin or a mixture containing these proteins was then carried out for 2 h. Comparisons were performed using a parametric test for independent data (ANOVA), followed by multiple comparison test where applicable. The level of significance was set at 5%. At 14 days mineralized bone-like nodule formation was detected and quantified by two methods; 1) morphological analysis – the percentage of the disc area occupied by Alizarin Red stained (ARS) nodules was determined using the Image Tool software; 2) biochemical analysis – the quantitation of mineralization was determined by ARS extraction. Results – 1) morphological analysis – the fibronectin post-adsorption showed that there were no differences among cultures. Regarding the fibrinogen post-adsorption, the only statistically significant difference was found between Machined and 4h+GDF-5+Fibrinogen groups. 2) biochemical analysis; the fibronectin post-adsorption showed only statistically significant difference between Machined and Machined+GDF-5+Fibrinogen groups. The analysis of fibrinogen post-adsorption showed that there were no differences among groups. In both analyzes, the groups with albumin significantly decreased the osteogenic potential of cultures when compared with the other experimental groups. The post-adsorption of three proteins together showed no statistical differences among groups in both analyzes. In conclusion, the albumin post-adsorption on GDF-5-functionalized Ti surfaces significantly decreased bone-like matrix production in vitro, however the additional presence of fibronectin and fibrinogen reverted such inhibitory effect.

References

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