Bone enhancing effect of titanium-binding proteins isolated from bovine bone and implanted into rat calvaria with titanium scaffold

Based on our previous finding that a chromatography with titanium beads selectively binds phosphoproteins, including caseins, phosvitin and dentin phosphoproteins, we investigated whether bone phosphoproteins also bind to titanium. Bovine bone matrix proteins were extracted with 2 M urea/PBS after demineralization. The 2 M urea extract was directly applied to the titanium chromatography column as reported. The chromatogram showed an initial large peak at breakthrough position (non-binding fraction) and a smaller second peak eluted later (titanium-binding fraction). Both peaks were analyzed by SDS polyacrylamide gel electrophoresis. Stains-all staining which preferentially identifies phospho-proteins revealed that the first peak contained no positively stained band, while the second peak showed 4 or 5 distinctive bands indicative of bone phosphoproteins. To investigate the biological functions of the titanium-binding bone proteins (TiBP), we implanted them into calvaria of rats, combined with titanium web (TW), a highly porous titanium scaffold of thin titanium-fibers. Bone TiBP induced significantly enhanced bone formation, and new bone appeared connected directly to titanium fibers, accompanied by active blood vessel formations. Control TW alone did not induce bone formation within the titanium framework. These results demonstrate that the bone titanium-binding proteins include phosphoproteins which enhance bone formation when implanted into bone with titanium.