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

在组织工程和伤口愈合应用中，支架材料被用来为细胞生长和组织形成提供机械支持。这些支架可以经过修饰，从而向细胞提供特定的生物信号，以控制或促进组织的形成或再生。细胞粘附肽已被纳入支架以增强细胞粘附或允许生物特异性细胞粘附。结果表明，具有Arg-Gly-Asp （RGD）序列的寡肽与纤维连接蛋白、玻璃体连接蛋白、胶原蛋白和纤维蛋白原的细胞附着活性有关，可作为细胞膜上细胞附着受体的配体。研究还表明，纤维连接蛋白的细胞附着活性位点及其相关肽形成紧密折叠的构象。为了提高Arg-Gly-Asp-Ser肽在组织工程支架上的细胞附着活性，我们试图通过设计具有Arg-Gly-Asp-Ser序列的ss-hairpin肽来稳定这类h肽的折叠构象。这些序列Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys (EAK8), (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_2 （EAK16）或（Arg-Ala-Arg-Ala-Asp-Ala-Asp-Ala）_2 （RAD 16）被选为具有高ss-链形成倾向的典型序列。采用液相法和固相法合成含ss-sheet模型肽的RGDS。所有肽均通过NMR，MALDI-TOF MS，氨基酸分析和元素分析进行了表征。采用L929成纤维细胞对肽固定化底物的细胞附着实验，检测了这些肽的细胞附着活性。L929细胞附着在肽固定化细胞培养皿上，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，设计的肽的RGDS序列通常与细胞的整合素受体相互作用。EAK16RGDS和RAD 16RGDS固定化聚苯乙烯培养皿在肽固定化聚苯乙烯培养皿中扩散活性最高。对于Arg-Gly-Asp-Ser序列与设计形成ss-hairpin结构的序列直接相连的肽，由于ss-hairpin结构的形成造成的构象约束太强，无法稳定Arg-Gly-Asp-Ser部分所需的构象。设计一个间隔序列可以提高Arg-Gly-Asp-Ser序列与形成ss-链结构的序列之间的活性。制备组织工程3D支架相对容易。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