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

在组织工程和创伤修复应用中，支架材料被用来为细胞生长和组织形成提供机械支撑。这些支架可以被修饰，以便它们还向细胞提供特定的生物信号，以便控制或促进组织的形成或再生。细胞黏附肽已被引入支架材料以增强细胞黏附或允许生物特异性细胞黏附。研究表明，具有Arg-Gly-Asp(RGD)序列的寡肽可能作为细胞膜上细胞黏附受体的配体，该序列与纤维连接蛋白、玻璃连结蛋白、胶原和纤维蛋白原的细胞黏附活性有关。在本工作中，为了提高精氨酸-甘氨酸-天冬氨酸-丝氨酸多肽对组织工程支架的细胞黏附活性，我们试图稳定Suc…的折叠构象通过设计在转角部分具有Arg-Gly-Asp-Ser序列的ss-发夹多肽来获得更多的h多肽。这些序列分别为Ala-Glu-Ala-Gla-Glu-Ala-Lys-Ala-Lys(EAK8)、(Ala-Glu-Ala-Ala-Lys-Ala-Lys)_2(EAK16)或(Arg-Ala-Arg-Ala-Asp-Ala-Asp-Ala)_2(RAD 16)。用核磁共振、MALDI-TOF-MS、氨基酸分析和元素分析对其结构进行了表征。用L929成纤维细胞对多肽固定化底物的细胞黏附实验检测了这些多肽的细胞黏附活性，L929细胞黏附在多肽固定化细胞培养皿上，表明这些多肽固定化细胞培养皿的细胞黏附活性存在差异。这些结果表明，设计的RGDS序列通常与细胞的整合素受体相互作用。EAK16RGDS和RAD 16RGDS固定化聚苯乙烯盘具有最高的铺展活性。由于ss-发夹结构的形成导致的构象约束太强，不能稳定Arg-Gly-Asp-Ser部分的所需构象，其中Arg-Gly-Asp-Ser序列与为形成ss-发夹结构而设计的序列直接相连。为了提高Arg-Gly-Asp-Ser序列和形成单链结构的序列之间的活性，设计间隔序列是可取的。可以相对容易地制备组织工程3D支架。较少

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