Preparation of collagen/calcium phosphate multilayer sheet using enzymatic mineralization

The multilayer sheets (2–10 layers), which consisted of alternately cumulated collagen and calcium phosphate layers with the thickness of 6–8 μm in each layer, were prepared. The inorganic layer was mineralized by means of an alkaline phosphatase-catalyzed hydrolysis of water-soluble phosphate esters in the presence of calcium ions. The calcium phosphate, which was formed on the collagen, was assayed as a mixture of hydroxyapatite (main) and amorphous calcium phosphate. The multilayer sheets were not only strong mechanically but also semitransparent and flexible in a dry state. Furthermore, the collagen/calcium phosphate multilayer sheets did not swell in water to keep the original morphology. As a scaffold, the sheets having the calcium phosphate layer on the top supported the attachment and growth of L929 fibroblast cells. The enzymatic mineralization and the collagen/calcium phosphate composite sheets were discussed in conjunction with physicochemical and biological properties.