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 6-8microm 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.