Introduction: The barnacles bond two different materials using secretion which was constructed of multiprotein complex in water[1]. It revealed that amino acids sequence constructed by 20 residues (RRKYSGILGDLIQVAVIRYY: R-Y) plays an important role in retaining secretion strength and forms stable β-sheet structure. R-Y peptide was formed self-assembling nanofiber structure because of existence of inter molecular interaction. These self-assembling peptide were conjugated with cell adhesive motif derived from cell adhesive protein fibronectin[2]. Fibronectin has the RGDS sequence from the extra-cellular matrix protein as a model bioactive ligand. This sequence interacts with cell surface integrin receptors, plays a critical role in cell adhesion, behavior and signaling and is the minimum motif required for cell adhesion on a surface.
We designed bioactive self-assembling peptide R-Y-RGDS (RRKYSGILGDLIQVAVIRYYGRGDS) was synthesized by using Fmoc chemistry. R-Y and R-Y-RGDS peptides were immobilized tissue culture plate (TCP) and carried out cell attachment activity test. R-Y and R-Y-RGDS peptides were formed haydrogel formation in cell culture medium. Then we were evaluated by differentiation activity of hMSC for osteogenesis encapsulation within hydrogels.
Materials and Methods: R-Y and R-Y-RGDS peptide were synthesized by manually Fmoc solid-phase peptide synthesis method. All peptides were characterized by HPLC, MALDI-TOF-MS and amino-acid analysis. These peptides were applied onto the cell culture plate and evaluated by X-ray photoelectron spectroscopy (XPS), FT-IR, and water contact angle measurements. The cell-attachment activity was assessed by L929 and hMSC cell onto two types peptide-immobilized TCP. Differentiation of encapsulated hMSC within hydrogels towards osteogenesis was evaluated by ALP activity assay.
Results and Discussion: Results of FT-IR measurement suggested that R-Y and R-Y-RGDS peptides take β-sheet structure. This fact indicates that these peptides were taken nanofiber with intermolecular interaction structure on TCP and RGDS sequence displayed on its surface. R-Y immobilized TCP showed higher cell attachment activity than no-coat substrate in the early stage. Many cells were attached and spread onto R-Y-RGDS-immobilized TCP, integrin receptor recognized RGDS sequence displayed on surface of peptide nanofiber. R-Y-RGDS was easily taken solgel transition at various pH solution.
When hMSC within hydrogels were cultured statically in osteoblast inducer media, as expected, the 2D culture level of the ALP expression is already about 3 time higher in the 3D culture level in the hMSC cells.
Conclusions: These synthetic peptides have cell adhesion and cell spreading activity. And then, β-sheet peptide of R-Y-RGDS could built up hierarchically organized nanofiber and coating materials. R-Y-RGDS was easily taken solgel transition at various pH solution and several types of cell were encapsuled within hydrogels. These peptide materials can be applied for tissue engineering 3D scaffold.
This work was supported in part by Grant-in-Aid for Scientific Research (C) and MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2010-2014.
References:
[1] M. Nakano, J-R. Shen, and K. Kamino, Biomacromolecules, 8, 1830-1835 (2007)
[2] H. Yokoi, T. Kinoshita, S. Zhang, Proc. Natl. Acad. Sci. USA., 102, 8414-8419 (2005).