N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37 °C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3β (GSK-3β), which phosphorylates β-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of β-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of β-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing β-catenin nuclear translocation and the associated transcriptional activity of β-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.

N-钙黏着蛋白是一种跨膜蛋白，是粘附连接的主要成分，介导细胞-细胞相互作用和细胞内信号传导，对调节细胞行为和器官发育很重要。先前的研究已经鉴定出具有与N-钙粘着蛋白相似的生物活性的模拟肽，这种肽可以促进人间充质干细胞（hMSCs）的软骨形成。但是，分子机制仍然未知。在这项研究中，我们将N-钙粘蛋白模拟肽（HAVDI）与自组装KLD-12肽结合：所得肽能够自组装为在磷酸盐缓冲液（PBS）中被N-钙粘蛋白肽功能化的水凝胶。在37°C下。与对照水凝胶相比，hMSCs在这些水凝胶中的包囊显示了软骨形成标记基因的表达增强和富含蛋白聚糖和II型胶原的软骨特异性细胞外基质的沉积，并在软骨培养14天后使用了杂乱序列的肽。此外，western blot显示活性糖原合酶激酶3β（GSK-3β）的表达显着升高，该酶使β-catenin磷酸化并促进泛素介导的降解，而N-蛋白中β-catenin和LEF1的表达较低。钙粘蛋白肽水凝胶与对照。免疫荧光染色显示，N-钙粘蛋白模拟肽水凝胶中β-catenin的核定位显着减少。我们的发现表明，N-钙粘蛋白肽水凝胶可通过减少β-catenin核易位和相关的β-catenin/ LEF-1 / TCF复合体转录活性来抑制hMSC中的经典Wnt信号传导，从而增强hMSC的软骨形成。我们的仿生自组装肽水凝胶可以用作可定制的多功能三维培养平台，以研究生物功能化对干细胞行为的影响。