Kang Marci K, Colombo John S, D'Souza Rena N, Hartgerink Jeffrey D
Departments of †Chemistry and §Bioengineering, Rice University , Houston, Texas 77005, United States.
Biomacromolecules. 2014 Jun 9;15(6):2004-11. doi: 10.1021/bm500075r. Epub 2014 May 12.
Here we report three new nanofibrous, self-assembling multidomain peptide (MDP) sequences and examine the effect of sequence on the morphology and expansion of encapsulated Stem cells from Human Exfoliated Deciduous teeth (SHED). We modified our previously reported set of serine-based MDPs, changing the serine residues in the amphiphilic region to threonine. The three new threonine-based sequences self-assemble into antiparallel β-sheet nanofibers, confirmed by CD and IR. AFM and negative-stained TEM show that the nanofibers formed by the new sequences are more curved than their serine-containing predecessors. Despite this change in nanofiber morphology, SEM illustrates that all three new sequences still form porous hydrogels. K(TL)2SLRG(TL)3KGRGDS, with a designed cleavage site, is able to be degraded by Matrix Metalloprotease 2. We then examine SHED cell response to these new sequences as well as their serine-based predecessors. We observe faster cell attachment and spreading in hydrogels formed by K2(SL)6K2GRGDS and K(SL)3RG(SL)3KGRGDS. By day 3, the SHEDs in all of the serine-based sequences exhibit a fibroblast-like morphology. Additionally, the SHED cells expand more rapidly in the serine-based gels while the cell number remains relatively constant in the threonine-based peptides. In hydrogels formed by K2(TL)6K2GRGDS and K(TL)2SLRG(TL)3KGRGDS, this low expansion rate is accompanied by changes in morphology where SHEDs exhibit a stellate morphology after 3 days in culture; however, by day 7 they appear more fibroblast-shaped. Throughout the duration of the experiment, the SHED cells encapsulated in the K2(TL)6K2 hydrogels remain rounded. These results suggest that the basic MDP structure easily accommodates modifications in sequence and, for SHED cells, the threonine-containing gels require the integrin-binding RGDS sequence for cell attachment to occur, while the serine-based gels are less selective and support an increase in cell number, regardless of the presence or absence of RGDS.
在此，我们报告了三种新的纳米纤维状、自组装多结构域肽（MDP）序列，并研究了序列对封装的人脱落乳牙干细胞（SHED）的形态和增殖的影响。我们对之前报道的一组基于丝氨酸的MDP进行了修饰，将两亲区域中的丝氨酸残基替换为苏氨酸。通过圆二色光谱（CD）和红外光谱（IR）证实，这三种新的基于苏氨酸的序列自组装成反平行β-折叠纳米纤维。原子力显微镜（AFM）和负染透射电子显微镜（TEM）显示，新序列形成的纳米纤维比含丝氨酸的前体更弯曲。尽管纳米纤维形态发生了这种变化，但扫描电子显微镜（SEM）表明，所有三种新序列仍能形成多孔水凝胶。具有设计切割位点的K(TL)2SLRG(TL)3KGRGDS能够被基质金属蛋白酶2降解。然后，我们研究了SHED细胞对这些新序列及其基于丝氨酸的前体的反应。我们观察到，在由K2(SL)6K2GRGDS和K(SL)3RG(SL)3KGRGDS形成的水凝胶中，细胞附着和铺展更快。到第3天，所有基于丝氨酸序列中的SHED细胞呈现成纤维细胞样形态。此外，SHED细胞在基于丝氨酸的凝胶中增殖更快，而在基于苏氨酸的肽中细胞数量保持相对恒定。在由K2(TL)6K2GRGDS和K(TL)2SLRG(TL)3KGRGDS形成的水凝胶中，这种低增殖率伴随着形态变化，即SHED细胞在培养3天后呈现星状形态；然而，到第7天它们看起来更呈成纤维细胞形状。在整个实验过程中，封装在K2(TL)6K2水凝胶中的SHED细胞保持圆形。这些结果表明，基本的MDP结构很容易容纳序列修饰，对于SHED细胞而言，含苏氨酸的凝胶需要整合素结合RGDS序列才能发生细胞附着，而基于丝氨酸的凝胶选择性较低，无论是否存在RGDS，都能支持细胞数量增加。
