Abstract The arginine–glycine–aspartic acid (RGD) motif plays a critical role in cell-extracellular matrix (ECM) interactions through focal adhesion, which may orchestrate various aspects of cellular behaviors for development and maintenance of tissues. Here, we prepared novel thermosensitive hydrogel systems comprising methoxy polyethylene glycol-b-polycaprolactone (MP) and RGD-conjugated MP (MP-RGD) with various RGD contents (MP/MP-RGD) based on our recently patented technology. The conjugation of RGD to MP through biomolecular nucleophilic substitution was qualitatively and quantitatively assessed using 1H nuclear magnetic resonance (NMR) spectroscopy and the Sakaguchi assay, respectively. The sol–gel–sol transition with increasing temperature was confirmed using viscoelastic measurements together with tilting tests. In vitro experiments using bone marrow-derived mesenchymal stem cells (BMSCs) revealed that MP/MP-RGD hydrogels significantly enhanced expression of integrins (α2, α5, and β1) and facilitated formation of focal adhesions (increased expression of FAK, focal adhesion kinase) that lead to cytoskeletal reorganization through integrin-mediated cell signaling. Real-time polymerase chain reaction (PCR) array also showed upregulation of signaling genes involved in the receptor tyrosine kinase (RTK) signaling cascade such as mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) for growth factors and phosphoinositide 3-kinase (PI3K) and AKT (protein kinase B) for survival factors. This suggests that extensive crosstalk pathways exist between integrins and RTKs, which was confirmed through western blotting. Consequently, MP/MP-RGD hydrogels showed a significantly increased BMSC proliferation rate with increasing RGD content of the samples.

中文翻译：

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新型 RGD 共轭热敏 mPEG-PCL 复合水凝胶的制备及其对粘附依赖性细胞行为影响的体外研究

摘要 精氨酸-甘氨酸-天冬氨酸 (RGD) 基序通过黏着斑在细胞 - 细胞外基质 (ECM) 相互作用中发挥关键作用，可协调组织发育和维持的细胞行为的各个方面。在这里，我们基于我们最近的专利技术制备了新型热敏水凝胶系统，包括甲氧基聚乙二醇-b-聚己内酯 (MP) 和 RGD 共轭 MP (MP-RGD)，具有各种 RGD 含量 (MP/MP-RGD)。RGD 通过生物分子亲核取代与 MP 的结合分别使用 1 H 核磁共振 (NMR) 光谱和 Sakaguchi 测定进行定性和定量评估。使用粘弹性测量和倾斜试验证实了随着温度升高的溶胶-凝胶-溶胶转变。使用骨髓间充质干细胞 (BMSCs) 进行的体外实验表明，MP/MP-RGD 水凝胶显着增强了整联蛋白（α2、α5 和 β1）的表达并促进了粘着斑的形成（FAK、粘着斑激酶的表达增加） ) 通过整合素介导的细胞信号传导导致细胞骨架重组。实时聚合酶链反应 (PCR) 阵列还显示参与受体酪氨酸激酶 (RTK) 信号级联反应的信号基因上调，例如丝裂原活化蛋白激酶 (MAPK) 和细胞外信号调节激酶 (ERK) 的生长因子和磷酸肌醇 3-激酶 (PI3K) 和 AKT（蛋白激酶 B）用于生存因子。这表明整合素和 RTK 之间存在广泛的串扰通路，这通过蛋白质印迹得到证实。最后，