Osteoblastic differentiation on hydrogels fabricated from Ca 2+ -responsive self-assembling peptides functionalized with bioactive peptides.

We recently developed an amphiphilic peptide, E1Y9 (Ac-E-YEYKYEYKY-NH2 ), that self-assembles into nanofibers and forms a hydrogel in the presence of Ca2+ ion. Four E1Y9-derivatives (E1Y9-ALK, E1Y9-DGR, E1Y9-PRG and E1Y9-RGD) were designed as conjugates of E1Y9 with bioactive peptide sequences named as ALK (ALKRQGRTLYGF), DGR (DGRDSVAYG), PRG (PRGDSGYRGDS) and RGD (RGDS), respectively, and stimulated osteoblast cells growth as well as differentiation. In this study, E1Y9/E1Y9-derivative mixed hydrogels were constructed to serve as scaffolds for osteoblastic differentiation of MC3T3-E1 cells. E1Y9 and E1Y9-derivatives co-assembled into networked nanofibers and formed hydrogels in response to Ca2+ ion. The pre-osteoblast cell line MC3T3-E1 was cultured and differentiated on mixed hydrogels. An E1Y9/E1Y9-ALK mixed hydrogel exhibited the highest cell proliferation and differentiation activity among the peptide hydrogels. The peptide sequence ALK promoted expression of RUNX2 and osteopontin, a key transcription factor and bone tissue matrix protein, respectively, during the differentiation stage. During the later stage, localization of RUNX2 and osteopontin was regulated in the cytosol and extracellularly, respectively, indicating that the E1Y9/E1Y9-ALK mixed hydrogel controlled the differentiation of MC3T3-E1 cells. Thus, the E1Y9/E1Y9-ALK mixed hydrogel developed in this study showed potential for the culture and regulation of differentiation of osteoblast cells for bone regeneration.