Szkolar Laura, Guilbaud Jean-Baptiste, Miller Aline F, Gough Julie E, Saiani Alberto
School of Materials, The University of Manchester, Manchester, M13 9PL, UK; Manchester Institute of Biotechnology, The University of Manchester, Manchester, M13 9PL, UK.
J Pept Sci. 2014 Jul;20(7):578-84. doi: 10.1002/psc.2666. Epub 2014 Jun 12.
We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix. First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work. We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa). The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis. In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel. Human dermal fibroblasts were then used to exemplify the use of these materials. FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control. We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate. After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts. The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells.
我们研究了使用酶触发肽水凝胶来封装和培养细胞的可能性。基于近期关于使用嗜热菌蛋白酶（一种来自嗜热栖热放线菌的蛋白酶）对FEFK（F，苯丙氨酸；E，谷氨酸；K，赖氨酸）进行酶触发凝胶化的研究工作，我们研究了利用这种凝胶化触发机制将细胞封装在三维水凝胶基质中的可能性。首先，研究了在磷酸盐缓冲溶液中制备的酶触发水凝胶的特性，并与我们之前工作中在HPLC级水中制备的水凝胶的特性进行了比较。我们发现，使用磷酸盐缓冲溶液能够制备出具有非常高剪切模量（>1 MPa）的水凝胶。我们还研究了凝胶化动力学，结果表明该系统的力学性能与酶通过逆水解合成八肽的过程密切相关。在第二阶段，我们根据所获得的信息，开发了一种简便的细胞封装和水凝胶铺板方案。然后用人皮肤成纤维细胞来验证这些材料的用途。将在相同条件下制备且具有相同力学性能的FEFEFKFK八肽水凝胶用作对照。我们发现，两个系统之间未观察到显著差异，并且在第1天细胞数量减少后，细胞开始增殖。培养5天后，可以看到细胞开始呈现成纤维细胞典型的伸展形态。结果清楚地表明，所开发的方案将嗜热菌蛋白酶对细胞可能产生的潜在有害影响降至最低，并且这些酶触发水凝胶可用于细胞的三维封装和培养。
