Biggi Silvia, Bassani Giulia A, Vincoli Valentina, Peroni Daniele, Bonaldo Valerio, Biagiotti Marco, Belli Romina, Alessandrino Antonio, Biasini Emiliano, Freddi Giuliano
Dulbecco Telethon Laboratory of Prions and Amyloids, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Povo, TN, Italy.
Silk Biomaterials Srl, Via Cavour 2, 22074 Lomazzo, Co, Italy.
ACS Appl Bio Mater. 2020 Dec 21;3(12):8361-8374. doi: 10.1021/acsabm.0c00613. Epub 2020 Nov 10.
The degradation profile and the cytotoxicity of the degradation products of a silk fibroin (SF)-based nerve conduit (SilkBridge), with a complex three-layered wall architecture comprising both native and regenerated (electrospun) fibers, are reported. The bacterial protease type XIV from was used as a hydrolytic agent at three different enzyme/substrate ratios (1:8, 1:80, and 1:800 w/w) to account for the different susceptibility to degradation of the native and regenerated components. The incubation time was extended up to 91 days. At fixed time points, the remaining device, the insoluble debris, and the incubation buffers containing soluble degradation products were separated and analyzed. The electrospun fibers forming the inner and outer layers of the conduit wall were almost completely degraded within 10 days of incubation at an enzyme/substrate ratio of 1:80 w/w. The progression of degradation was highlighted by the emergence of zones of erosion and discontinuity along the electrospun fibers, weakening of the electrospun layers, and decrease in resistance to compressive stress. Native SF microfibers forming the middle layer of the conduit wall displayed a higher resistance to enzymatic degradation. When incubated at an enzyme/substrate ratio of 1:8 w/w, the weight decreased gradually over the incubation time as a consequence of fiber erosion and fragmentation. Analogously, the tensile properties markedly decreased. Both spectroscopic and thermal analyses confirmed the gradual increase in the crystalline character of the fibers. The incubation buffers containing the soluble degradation products were subjected to cytotoxicity testing with human HEK293 cells and mouse neuroblastoma N2a cells. No detrimental effects on cell viability were observed, suggesting that the degradation products do not retain any toxic property. Finally, the mass spectrometry analysis of degradation products showed that the SF polypeptides recovered in the incubation buffers were representative of the aminoacidic sequence of the fibroin light chain and of the highly repetitive fibroin heavy chain, indicating that virtually the entire sequence of the fibroin protein constituent of SilkBridge was degraded.
报道了一种基于丝素蛋白（SF）的神经导管（SilkBridge）的降解情况及其降解产物的细胞毒性，该导管具有复杂的三层壁结构，包含天然纤维和再生（电纺）纤维。使用来自的 XIV 型细菌蛋白酶作为水解剂，采用三种不同的酶/底物比例（1:8、1:80 和 1:800 w/w），以考虑天然成分和再生成分对降解的不同敏感性。孵育时间延长至 91 天。在固定时间点，将剩余的装置、不溶性碎片以及含有可溶性降解产物的孵育缓冲液分离并进行分析。在酶/底物比例为 1:80 w/w 的情况下孵育 10 天内，构成导管壁内层和外层的电纺纤维几乎完全降解。沿着电纺纤维出现的侵蚀和不连续区域、电纺层的弱化以及抗压应力的降低突出了降解的进程。构成导管壁中层的天然 SF 微纤维对酶促降解表现出更高的抗性。当以 1:8 w/w 的酶/底物比例孵育时，由于纤维侵蚀和破碎，重量在孵育过程中逐渐下降。类似地，拉伸性能也显著下降。光谱分析和热分析均证实纤维的结晶特性逐渐增加。对含有可溶性降解产物的孵育缓冲液用人 HEK293 细胞和小鼠神经母细胞瘤 N2a 细胞进行细胞毒性测试。未观察到对细胞活力的有害影响，表明降解产物不具有任何毒性。最后，降解产物的质谱分析表明，在孵育缓冲液中回收的 SF 多肽代表了丝素蛋白轻链和高度重复的丝素蛋白重链的氨基酸序列，这表明 SilkBridge 的丝素蛋白成分的几乎整个序列都被降解了。
