Mitogenic activity of FGF-2 in FGF-2-apatite composite layers formed on titanium-based substrates with different surface chemistry

摘要

Introduction: FGF-2-apatite composite layers were formed by coprecipitaion of FGF-2 with apatite on various substrates by immersing the substrates in supersaturated calcium phosphate solutions containing FGF-2. FGF-2 is a growth factor that enhances soft tissue healing and stimulates bone formation. The aim of this study was to determine the effects of different titanium-based implants on chemical composition of the composite layer and FGF-2 activity. Materials and Methods: Commercially available 3 different half pins for external fixation (A;Ti6Al4V, B;Ti, C;Ti6Al4V) were examined. In each group, 3 pins were used. The pins were immersed in a supersaturated calcium phosphate solution supplemented with 4.0 μg/mL FGF-2 at 37°C for 48 hours. The FGF-2-apatite composite layer formed on the pins was dissolved in a citric acid buffer. Subsequently, the amount of FGF-2 in the buffer was measured using the Bradford method while the amount of Ca and P were measured by an inductively coupled plasma atomic emission spectrometer. The mitogenic activity of FGF-2 in the buffer was evaluated by the cell proliferation assay using fibroblastic NIH3T3 cells. The above experiments were repeated four times. Finally, one of three pins in each group was observed, after sonication in a citric acid solution and rinse with ultrapure water, using a scanning electron microscope equipped with an energy dispersive electron probe X-ray (EDX) analyzer. Results: FGF-2-apatite composite layers were successfully formed on all the pins. The amounts of FGF-2 (A: 1.3±0.5mg, B: 1,5±0.7mg, C: 1.2±0.5mg), the Ca/P molar ratio (A: 1.69±0.03, B: 1.68±0.05, C: 1.68±0.03) and the total amount of Ca (A: 266±7mg, B: 257±13mg, C: 265±16mg) were equivalent among the 3 pin groups (p ＞ 0.05). Mitogenic activity of FGF-2 in the composite layer was increased in the order of pin C＜A＜B with a significant difference between C and B. Oxygen and phosphorus were detected on the surface layer of pin B while silicon was detected on the pin C by EDX. Discussion: Previously, we reported that the biological activity of FGF-2-apatite composite layer in vivo changed depending on the Ca/P molar ratio of apatite matrix that embedded FGF-2. In the present study, apatite matrices with the same Ca/P molar ratio were formed on the pins with different surface chemistry. The surface chemistry had a significant impact on biological activity of FGF-2 in vitro. Thus, process parameters for coprecipitation of molecules with apatite need to be optimized carefully depending on the substrate. This study also provides an important insight into the role of chemistry of surfaces and matrices that are in contact with a growth factor. Conclusion: FGF-2-apatite composite layers were formed on titanium-based pins with different surface chemistry. The layers were equivalent in FGF-2 content, Ca/P molar ratio and Ca content. However, the different surface chemistry gave different mitogenic activity of FGF-2 in the layer.