Independent tuning of multiple biomaterial properties using protein engineering
Independent tuning of multiple biomaterial properties using protein engineering
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DOI：
10.1039/b808504h
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发表时间：
2009-01-01
期刊：
影响因子：
3.4
通讯作者：
Heilshorn, Sarah C.
中科院分区：
文献类型：
--
作者：
Straley, Karin S.;Heilshorn, Sarah C.
当前许多生物材料缺少的一个关键属性是独立调整多种生物材料特性而不同时影响其他材料参数的能力。由于众所周知细胞会对生物材料的初始弹性模量、降解率和细胞粘附性的变化做出反应，因此开发合成设计策略至关重要，该策略允许对每个单独的参数进行解耦定制，以便系统地优化细胞与支架的相互作用。我们开发了一种由化学交联的弹性蛋白样蛋白组成的仿生支架，旨在通过细胞粘附和细胞诱导的降解和重塑的结合来支持神经再生。这些工程蛋白的设计包括使神经元附着的细胞粘附序列以及对尿激酶纤溶酶原激活剂（uPA）（一种从生长的神经突尖端局部分泌的蛋白酶）裂解敏感的序列，以实现高度局部化和可调节的降解特性。这些工程蛋白质是使用重组技术生产的，并通过化学交联形成具有可控机械性能的高度膨胀的水凝胶。通过对三种其他方面相同的工程蛋白的化学特性进行 3% 的适度改变，我们可以修改 uPA 底物特异性，从而使蛋白酶降解半衰期发生两个数量级的可调变化。在高 uPA 暴露下，设计的支架表现出支架寿命的系统变化，从一天内完全降解到一周内没有明显降解。使用 PC-12 神经元样细胞系模型进行的体外研究表明，交联蛋白支持可调节的细胞粘附和神经元分化。增加蛋白质底物中存在的 RGD 肽的密度会导致细胞粘附增加和更广泛的神经突生长。这些工程蛋白提供了独立定制支架的力学、降解特性和细胞粘附性的能力，用于中枢神经系统再生的研究。
A key attribute missing from many current biomaterials is the ability to independently tune multiple biomaterial properties without simultaneously affecting other material parameters. Because cells are well known to respond to changes in the initial elastic modulus, degradation rate, and cell adhesivity of a biomaterial, it is critical to develop synthetic design strategies that allow decoupled tailoring of each individual parameter in order to systematically optimize cell-scaffold interactions. We present the development of a biomimetic scaffold composed of chemically crosslinked, elastin-like proteins designed to support neural regeneration through a combination of cell adhesion and cell-induced degradation and remodeling. The design of these engineered proteins includes cell adhesion sequences to enable neuronal attachment as well as sequences sensitive to cleavage by urokinase plasminogen activator (uPA), a protease locally secreted from the tips of growing neurites, to enable highly localized and tunable degradation properties. These engineered proteins are produced using recombinant techniques and chemically crosslinked into highly swollen hydrogels with controllable mechanical properties. Through a modest 3% change in the chemical identity of three otherwise identical engineered proteins, we can modify the uPA substrate specificity resulting in tunable changes in protease degradation half-life over two orders of magnitude. Under high uPA exposure, the designed scaffolds exhibit systematic variation of scaffold lifetime, from being fully degraded within a single day to showing no noticeable degradation within a full week. In vitro studies using the model PC-12 neuronal-like cell line show that the crosslinked proteins support tunable cell adhesion and neuronal differentiation. Increasing the density of RGD peptides present in the protein substrates leads to increased cell adhesion and more extensive neurite outgrowth. These engineered proteins offer the ability to independently tailor the mechanics, degradation properties, and cell adhesivity of scaffolds for the study of central nervous system regeneration.