Cell attachment and spreading activity of mixed laminin peptide-chitosan membranes.

Laminins are a multifunctional molecule with numerous active sites that have been identified in short peptide sequences. Mixed peptide-conjugated chitosan membranes using laminin-derived active peptides have been previously demonstrated to be useful as a biomaterial for tissue engineering. In this study, two syndecan-binding peptides, AG73 (RKRLQVQLSIRT) and C16 (KAFDITYVRLKF), and three integrin-binding peptides, EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle, binding to integrin α2ß1), A99a (ALRGDN, binding to integrin αvß3), and A2G10 (SYWYRIEASRTG, binding to integrin α6ß1), were mixed in various combinations, conjugated to chitosan membranes, and evaluated for their cell attachment and spreading activities. The cell attachment and spreading activity of EF1zz, A99a, and A2G10 were enhanced by AG73. In contrast, C16 enhanced only the cell attachment and spreading activity of A99a and did not influence the activity of EF1zz and A2G10. As well as previous study, the AG73-chitosan membrane bound to only syndecan. On the other hand, the C16-chitosan membrane interacted with both syndecan and ß1 integrin. These data suggest that interaction of different receptors can cause synergistic effects. Therefore, AG73 is widely applicable as a synergistic agent for mixed peptide-matrices using several types of integrin-binding peptides. Additionally, the A2G10/AG73-chitosan membrane may be useful to investigate detailed biological functions of α6ß1 integrin, which is a major laminin-binding receptor. Using a combination of tissue-appropriate laminin-derived peptides, the mixed peptide-chitosan membranes may serve as functional biomaterials for tissue engineering.

Reconstitution of laminin-111 biological activity using multiple peptide coupled to chitosan scaffolds.

Laminin-111, a multifunctional matrix protein, has diverse biological functions. Previously, we have identified various biologically active sequences in laminin-111 by a systematic peptide screening. We also demonstrated that peptide-conjugated chitosan matrices enhance the biological functions of the active sequences and are useful as a scaffold. Here, we conjugated sixty biologically active laminin-111 peptides onto chitosan matrices. The twenty-nine peptide-chitosan matrices promoted various biological activities, including cell attachment, spreading, and neurite outgrowth. The biological activities of peptide-chitosan matrices depend on the peptide. These peptide-chitosan matrices are categorized into six groups depending on their biological activities. Next, we conjugated five active peptides, which showed strong cell attachment activity in the each group, onto a single chitosan matrix to mimic the multiple activities of laminin-111. The mixed peptides-chitosan matrix significantly promoted cell attachment and cell spreading over that observed with the individual peptides. We also demonstrated that a mixed peptides-chitosan matrix, using four neurite outgrowth-promoting peptides each from a different group, enhanced the activity. These data suggest that the mixed peptides synergistically induce laminin-like biological activities on a chitosan matrix. The active peptides-chitosan matrices described here have potential for use as biomaterial for tissue engineering and regeneration.

Cell surface receptor-specific scaffold requirements for adhesion to laminin-derived peptide-chitosan membranes.

Scaffolds are used for bioengineering to regulate cellular functions. Previously, we developed laminin-derived peptide-conjugated chitosan membranes for cell engineering. Here, we determined whether changes in the chitosan scaffold altered the cellular response. When an alphavbeta3 integrin-binding peptide A99a (ALRGDN) was conjugated on chitosan membranes of varying density (1.5-1500 ng/mm(2)), cell adhesion was altered depending on the amount of chitosan. 3 or 30 ng/mm(2) of the A99a-chitosan membrane effectively promoted cell attachment, cell spreading with well-organized actin stress fibers, phosphorylation of FAK Tyr397, and neurite outgrowth. In contrast, syndecan-binding peptide AG73 (RKRLQVQLSIRT) conjugated chitosan membranes density (1.5-1500 ng/mm(2)) promoted similar biological activities at all of the concentrations tested. These results suggest that integrin-mediated cell adhesion is sensitive to the scaffold condition. To improve the function of integrin-mediated biological activities on a large amount of scaffold, we designed an A99a/AG73 mixed peptide-chitosan membrane. The mixed peptide-chitosan membrane promoted the strongest biological activities at 150-1500 ng/mm(2) of chitosan membrane. We conclude that the A99a/AG73 mixed peptide-chitosan membrane effectively interacts with both integrins and syndecans and is a useful multi-functional biomaterial.

Mixed peptide-chitosan membranes to mimic the biological activities of a multifunctional laminin alpha1 chain LG4 module.

Laminin alpha1 chain LG4 module is multifunctional and interacts with syndecans and integrin alpha2beta1 via AG73 (RKRLQVQLSIRT) and EF-I (DYATLQLQEGRLHFMFDLG) sites, respectively. Here, we conjugated the AG73 and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle) peptides on a chitosan membrane in various ratios to develop an LG4 mimic biomaterial. The AG73-chitosan membrane promoted strong cell attachment with membrane ruffling and the EF1zz-chitosan membrane promoted integrin-mediated cell adhesion with well-organized actin stress fibers. When AG73 and EF1zz were conjugated on a chitosan membrane with 1:9 molar ratio, the mixed peptide-chitosan membrane promoted the strong cell attachment and neurite outgrowth similar to that on the recombinant LG4 protein. Well-organized actin stress fibers and vinculin accumulated focal contacts were observed in the cells attached on the AG73:EF1zz (molar ratio=1:9)-chitosan membrane. These results suggest that the mixed peptide-chitosan membrane interacts with both syndecans and integrin alpha2beta1 and mimics the cell adhesion of a multifunctional LG4 protein. The mixed peptide-chitosan approach has potential as a multifunctional biomaterial for cell and tissue engineering.