Collagen Simulation Peptide And Its Effect On Fibroblasts Adhere To Research

Collagens, characterized by a unique triple-helical structure, are the predominant component of extracellular matrices (ECMs). Collagens not only maintain structural integrity of tissues and organs, but also regulate a number of biological events. The specific functions of collagens are generally triggered by specific interactions of collagen-binding molecules with certain structures displayed on the collagen triple helices. Thus, synthetic triple-helical peptides that mimic the structure of native collagens have been used to investigate the individual collagen-cell interactions. It will provide theoretical basis for collagens as bioactive materials and artificial collagen-like biomaterial exploitation. Collagen mimetic peptide (CMP), which simulated the characteristics of collagen structure, provides a new method for the study of the structure and function of collagen.In this study, three CMPs were designed. They were CMP21which incorporating the triple-helical repeat sequence Pro-Hyp-Gly, CMP9which incorporating the integrin-binding sequence GFOGER and CMP27which containing above two kinds of sequence. The triple helix structures and thermal stability of type Ⅰ collagen and CMPs were studied. Different effects of these CMPs on fibroblast adhesion were revealed. The correlation of triple-helical conformation with the cellular recognition of GFOGER in an integrin-specific manner was revealed, too. The main contents of this work were as follows:1. Triple-helical structures and thermal stability of CMPsThe triple helix structures and thermal stability of CMPs were characterized by CD spectroscopy. The results verified that CMP21and CMP27self-assemble into a stable triple-helical conformation in solution, and its triple helix was similar to that observed in native type Ⅰ collagen. Conversely, CMP9showed no evidence of assembly into triple helical structure. Thermal denaturation process of triple helix was a continuously conformational process. Increasing temperature could loosen the triple helix. As the temperature increased further, the triple helix could unfold to a denatured monomer state. In contrast to native collagen, the thermal denaturation process of CMP21and CMP27was reversible in certain conditions.2. Cytotoxicity of CMPsTo evaluate cytotoxicity of CMPs in vitro, fibroblast cells (L929) were cultured. It is demonstrated that all CMPs had no obvious effect on the cell viability (as deterimined by CCK-8) or morphology of L929cells. The result was indicative of non-cytotoxic responses, furthermore, CMP27promoted cell proliferation to some extent.3. Fibroblast adhesion on physically adsorbed CMPsBiological assays, including cell adhesion, competitive inhibition, and fluorescent staining, revealed that physically adsorbed CMPs had cell adhesion activity. Briefly, all CMPs promoted proliferation and attachment of L929to the coated surface. And the cells adhesion morphology and proliferation rate of L929were good. Compared with others, CMP27significantly promoted the cell adhesion and spreading of L929with plenty of stress fibers and parapodium, which was comparable to that observed on type I collagen. Therefore, the triple-helical sequence and the integrin-binding sequence, working in a cooperating way, showed to be important for cell adhesion.4. Fibroblast adhesion on CMP-conjugated chitosan membranesCMP9or CMP27was conjugated onto the maleimidobenzoyloxy (MB)-chitosan membranes, and further characterized by H-NMR and XPS. Cell adhesion assay showed that L929cell adhesion on the CMP-chitosan membranes depended on the amount of chitosan, and that3ng/mm2and6ng/mm2of chitosan membrane were effective for promoting cell spreading. The CMP27-chitosan membranes induce the strongest cell attachment and cell spreading. Meanwhile, well-grown stress fibers and microvilli were observed. The results indicated that the triple-helical sequence and the integrin-binding sequence work in a cooperating way to promote integrin α2β1-targeted cell adhesion.To sum up, the CMP which incorporating the triple-helical sequence and integrin-binding sequence is effective for improving the bioactivity of cell adhesion. CMPs may be applied as a surface modification agent to promote α2β1specific cell ashesion. Moreover, this study provided new insights in designing other peptide-based bioactivity materials, and provided research foundation for developing artificial collagen-like biological materials.