A mineralization‐associated membrane protein plays a role in the biological functions of the peptide‐coated bovine hydroxyapatite

Abstract

Anorganic bovine mineral coated with a cell-binding peptide (P-15) is superior to anorganic bovine mineral alone in the treatment of periodontal osseous defects. However, the molecular interactions between P-15 and periodontal ligament fibroblasts remain unclear. We first compared the in vitro osteogenic activities between periodontal ligament fibroblasts cultured with anorganic bovine mineral alone and with the P-15/anorganic bovine mineral combination. We then harvested the periodontal ligament cell lysate, incubated it with various graft materials, and then washed it to remove unbound proteins. The bound proteins were eluted from graft materials and analyzed using electrophoresis, followed by mass spectrometry and then western blotting. Finally, a neutralizing antibody against one bound protein was added to the cell cultures to repeat the osteogenic assays to clarify its role in the in vitro effects of the P-15/anorganic bovine mineral combination. Cells treated with P-15/anorganic bovine mineral were more viable and showed greater osteogenic activities than cells treated with anorganic bovine mineral alone and the no-graft control. Annexin II, a mineralization-associated protein, bound to P-15/anorganic bovine mineral significantly more than to anorganic bovine mineral alone. The addition of neutralizing antibody for annexin II decreased the osteogenic activities of the P-15/anorganic bovine mineral combination. Annexin II of periodontal ligament fibroblasts interacted with the peptide of P-15, and was partially responsible for better in vitro osteogenesis in the P-15 graft.