Collagen-Like Polypeptide Poly(Pro-Hyp-Gly) Conjugated with Fibronectin-Derived Peptides Enhances the Cell Adhesion, Migration and Stratification

摘要

Animal-derived collagens are used as a scaffold for tissue regeneration. However, the collagens have several drawbacks, such as nonspecific cell adhesion, low thermal stability, residual antigenicity, and contamination of pathogenic substances. To overcome these problems, we reported the chemical synthesis of the collagen-like polypeptide, poly(prolyl-hydroxyprolyl-glycyl) (poly(PHG)), which forms a collagen-like triple-helical structure and has biodegradability and biocompatibility. Here, we designed a new scaffold where fibronectin-derived RGD, Gly-Arg-Gly-Asp-Ser (GRGDS) or cydo(Arg-Gly-Asp-D-Phe-Lys) (cRGDfK), and Pro-His-Ser-Arg-Asn (PHSRN) peptides were simultaneously conjugated on poly(PHG). We assessed NIH3T3 cell adhesion and migration and stratification of immortalized rabbit corneal epithelial (RCE) cells on the scaffold. Cell adhesion was enhanced with increasing in the amount of the conjugated RGD. Cell adhesion activity of conjugated RGD were significantly higher than bovine type I atelocollagen but lower than bovine fibronectin. Cell adhesion activity of cRGDfK-conjugated poly(PHG) was higher than that of GRGDS below the conjugation ration of 150 nmol/mg. This result suggests that cRGDfK is suitable for interaction with cell surface receptor, integrin, because RGD sequence is located in a loop region of fibronectin. Interestingly, simultaneous conjugation of GRGDS and PHSRN synergis-tically enhanced cell migration. Scaffolds containing almost equal amounts of GRGDS and PHSRN showed significantly higher cell migration activity than bovine type I atelocollagen. Addition of free GRGDS completely inhibited cell migration on the scaffold, whereas addition of free PHSRN partially inhibited cell migration. These results suggest that GRGDS plays a definitive role, and PHSRN plays an additional role, in cell migration. Conjugation of GRGDS resulted in the same level of stratification of RCE cells compared with bovine type I atelocollagen and bovine fibronectin. Because the simultaneous conjugation of GRGDS and PHSRN on poly(PHG) enhances cell adhesion, migration and stratification, it may be a useful scaffold for tissue regeneration.