Reconstruction of a Hard Connective Tissue Utilizing a Pressed Silk Sheet and Type-I Collagen as the Scaffold for Fibroblasts

A pressed silk sheet is a new biomaterial composed of a network of numerous cocoon filaments and having excellent mechanical strength and shape stability compared to a cotton-gauze sheet. To reconstruct a hard connective tissue using the silk sheet and type-I collagen as the scaffold for fibroblasts, three different three-dimensional floating culture systems were designed. “On sheet” system: fibroblasts were seeded on the silk sheet coated with collagen and the cell-attached sheet was cultured. “In gel” system: fibroblasts and the silk sheet were co-embedded in a collagen gel and the gel was cultured. “On vitrigel” system: fibroblasts were seeded on both sides of a collagen vitrigel involving the silk sheet and the vitrigel was cultured. The fibroblasts in all culture systems grew and formed disk-shaped connective tissue models involving the silk sheet by 14 days of culture. The “on sheet” and “on vitrigel” models retained a maximum elastic load of about 23 kgf and an ultimate tensile load of about 3.6 kgf, which were almost the same as for the individual silk sheet. However, the “in gel” system showed a low value for the tensile load. Cell damage following application of mechanical stress was lowest in the “on vitrigel” system. These data demonstrated the advantage of the “on vitrigel” system in reconstructing hard connective tissues. Such a novel culture method would contribute to a regenerative medicine for the failure of ligaments, tendons, and other connective tissues.