Synthesis and Evaluation of Peptide-based Hybrid Materials for Tissue Engineering

在组织工程和伤口愈合应用中，支架材料用于为细胞生长和组织形成提供机械支撑。这些支架可以被修饰，使得它们还向细胞提供特定的生物信号，以控制或促进组织形成或再生。细胞粘附肽被掺入支架中以增强细胞粘附或允许生物特异性的细胞粘附，具有Arg-Gly-Asp（RGD）序列的寡肽可以作为细胞膜上细胞粘附受体的配体，该序列与纤维连接蛋白、玻连蛋白、胶原和纤维蛋白原的细胞粘附活性相关。为了提高Arg-Gly-Asp-Ser短肽与组织工程支架的细胞粘附活性，我们尝试稳定纤维连接蛋白的折叠构象， 关于我们 通过设计在转角部分具有Arg-Gly-Asp-Ser序列的ss-发夹肽，选择这些肽的序列Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys（EAK 8）、（Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys）_2（EAK 16）或（Arg-Ala-Arg-Ala-Asp-Ala-Asp-Ala）_2（RAD 16）作为具有高形成β-链倾向的典型序列。所有的肽通过NMR、MALDI-TOF MS、氨基酸分析和元素分析表征。通过使用L929成纤维细胞对肽固定化底物的细胞附着试验来检查这些肽的细胞附着活性。L929细胞附着于肽固定化细胞培养皿，表明这些肽固定化细胞培养皿的细胞附着活性存在差异。这些结果表明所设计的肽的RGDS序列通常与细胞的整联蛋白受体相互作用。EAK 16 RGDS和RAD 16 RGDS固定化的聚苯乙烯盘具有最高的铺展活性，对于Arg-Gly-Asp-Ser序列直接连接到设计用于形成ss-hairpin结构的序列的肽，由于ss-hairpin结构的形成所引起的构象约束太强，不能稳定Arg-Gly-Asp-Ser部分的所需构象。还表明，设计间隔序列对于提高Arg-Gly-Asp-Ser序列和形成ss链结构的序列之间的活性是理想的。可以相对容易地制备组织工程三维支架。少

In tissue engineering and wound-healing application, scaffold materials are utilized to provide a mechanical support for cell growth and tissue formation. These scaffold can be modified such that they also provide specific biologic signals to cell in order to control or facilitate tissue formation or regeneration. Cell adhesion peptides have been incorporated into scaffolds to enhance cell adhesion or to allow biospecific cell adhesion.It is shown that oligopeptides having the Arg-Gly-Asp (RGD) sequence, which is related to the cell-attachment activity of fibronectin, vitronectin, collagen, and fibrinogen, may act as the ligands for the cell-attachment receptors on the cell membranes. It is also shown that the cell-attachment activity site of fibronectin and its related peptides form compactly folded conformations.In this work, for improving the cell-attachment activity of the Arg-Gly-Asp-Ser peptides to tissue engineering scaffold, we tried to stabilize the folded conformations of suc … More h peptides by designing the ss-hairpin peptides, which have the Arg-Gly-Asp-Ser sequence at the turn portion. These sequence, Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys (EAK8), (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_2 (EAK16) or (Arg-Ala-Arg-Ala-Asp-Ala-Asp-Ala)_2 (RAD 16) of the peptides is selected as a typical sequence for having high propensity to form ss-strands.RGDS containing ss-sheet model peptides were synthesized using liquid and solid phase procedures. All peptides were characterized by NMR,MALDI-TOF MS, amino acid analysis, and elemental analysis. Cell-attachment activities of these peptides were examined by cell-attachment test using L929 fibroblast cell toward peptide-immobilized substrate.L929 cells attaching to the peptide-immobilized cell culture dishes, indicating that difference in the cell-attachment activity were found for these peptides-immobilized cell culture dishes. These results suggest that the RGDS sequence of designed peptides commonly interact with integrin receptor of the cell. EAK16RGDS and RAD 16RGDS immobilized polystyrene-dish has the highest spreading activity among the peptide-immobilized ones.The conformational constraint caused by the formation of ss-hairpin structure is too strong to stabilize the desired conformation at the Arg-Gly-Asp-Ser portion for the peptides in which the Arg-Gly-Asp-Ser sequence is directly linked to the sequences designed for forming ss-hairpin structure. It is also suggested that designing a spacer sequence is desirable for improving the activity between the Arg-Gly-Asp-Ser sequence and the sequence forming ss-strand structure. It was possible to prepare the tissue engineering 3D scaffold with comparative ease. Less