已经研究了各种水凝胶用于髓核再生。然而,它们未能克服椎间盘退变过程中酸性环境的变化。因此,通过缀合Sa12b设计了一种新的功能化肽RAD/SA1,酸敏感离子通道的抑制剂,RADA16-I的C端。然后,评价了RAD/SA1的材料特性和生物相容性,以及变性人髓核间充质干细胞(hNPMSCs)的生物活性和机制。原子力显微镜(AFM)和扫描电子显微镜(SEM)证实RAD/SA1在酸性条件下自组装成三维(3D)纳米纤维水凝胶支架。对3D支架中培养的hNPMSCs的分析显示,RADA16-I和RAD/SA1均表现出可靠的附着和极低的细胞毒性,通过扫描电镜和细胞毒性试验验证,分别。结果还表明,与培养板和纯RADA16-I相比,RAD/SA1增加了hNPMSCs的增殖。定量逆转录聚合酶链反应,酶联免疫吸附测定,Western印迹显示I型胶原的表达下调,而胶原蛋白II,aggrecan,SOX-9上调。此外,Ca2+浓度测定和蛋白质印迹显示RAD/SA1通过Ca2+依赖的p-ERK信号通路抑制p-ERK的表达。因此,利用Sa12b短基序设计的功能性自组装肽纳米纤维水凝胶可作为良好的髓核组织工程支架。此外,RAD/SA1在轻度退变的髓核再生中显示出巨大的潜在应用。
Various hydrogels have been studied for nucleus pulposus regeneration. However, they failed to overcome the changes in the acidic environment during intervertebral disc degeneration. Therefore, a new functionalized peptide RAD/SA1 was designed by conjugating Sa12b, an inhibitor of acid-sensing ion channels, onto the C-terminus of RADA16-I. Then, the material characteristics and biocompatibility of RAD/SA1, and the bioactivities and mechanisms of degenerated human nucleus pulposus mesenchymal stem cells (hNPMSCs) were evaluated. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that RAD/SA1 self-assembling into three-dimensional (3D) nanofiber hydrogel scaffolds under acidic conditions. Analysis of the hNPMSCs cultured in the 3D scaffolds revealed that both RADA16-I and RAD/SA1 exhibited reliable attachment and extremely low cytotoxicity, which were verified by SEM and cytotoxicity assays, respectively. The results also showed that RAD/SA1 increased the proliferation of hNPMSCs compared to that in culture plates and pure RADA16-I. Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting demonstrated that the expression of collagen I was downregulated, while collagen II, aggrecan, and SOX-9 were upregulated. Furthermore, Ca2+ concentration measurement and western blotting showed that RAD/SA1 inhibited the expression of p-ERK through Ca2+-dependent p-ERK signaling pathways. Therefore, the functional self-assembling peptide nanofiber hydrogel designed with the short motif of Sa12b could be used as an excellent scaffold for nucleus pulposus tissue engineering. Moreover, RAD/SA1 exhibits great potential applications in the regeneration of mildly degenerated nucleus pulposus.