Controlled collagen crosslinking process in tissue-engineered fibroblast sheets for preventing scar contracture on the surface of lungs

摘要

For preventing the scar contracture of host tissue and adjusting the tensile strength of covering cell sheets, a controlled collagen crosslinking step process in the preparation of skin-fibroblast sheets for repairing wound was investigated by using β-aminopropionitrile (BAPN), a collagen crosslinking inhibitor, in the culture medium. Skin fibroblasts obtained from neonatal rats were cultured in medium with and without 0.25mm BAPN for 7days and seeded on temperature-responsive culture dishes. After the confluent cells were non-invasively harvested as a monolithic cell sheet, two cell sheets were transplanted to a lung-injury site of athymic rats, which was closed by neither fibrin glue nor suturing. Four weeks after the transplantation the animals were sacrificed and the lungs with the transplanted cell sheets were examined. Although the control cell sheet-transplanted lungs contracted the surrounding tissue, BAPN-treated cell sheet-transplanted lungs showed no contraction of the tissue. Collagen fibres of control cell sheets were more dense and thick than those of BAPN-treated cell sheets, where the crosslinking of collagen fibres was clearly inhibited. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that BAPN-treated cell sheets gave no β-chain band, indicating that the collagen crosslinkings of the fibroblast sheets were able to be controlled by BAPN. BAPN-treated fibroblast sheets promise to allow wound clefts to be repaired without scar contractures.