以亲和力层粘连蛋白作为3D神经干细胞培养系统的工程水凝胶,Biomaterials Science

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以亲和力层粘连蛋白作为3D神经干细胞培养系统的工程水凝胶
Biomaterials Science ( IF 5.8 ) Pub Date : 2019-10-09 , DOI: 10.1039/c9bm00348g
Daniela Barros _{1,

2,

3,

4,

5} , Eduardo Conde-Sousa _{1,

2,

3,

4,

5} , Andreia M. Gonçalves _{1,

2,

3,

4,

5} , Woojin M. Han _{6,

7,

8,

9,

10} , Andrés J. García _{6,

7,

8,

9,

10} , Isabel F. Amaral _{1,

2,

3,

4,

5} , Ana P. Pêgo _{1,

2,

3,

4,

5}

Affiliation

已经探索了将层粘连蛋白掺入生物或合成水凝胶以概括神经干细胞（NSC）壁ni的动态性质和生物学复杂性。但是，目前在三维（3D）矩阵中研究层粘连蛋白固定化的策略并没有解决影响细胞-基质相互作用的关键方面，这是固定化时对层粘连蛋白构象和方向的控制。这是保持蛋白质生物活性的关键特征。在这项工作中，我们探索了一种基于亲和力的方法来介导层粘连蛋白的位点选择性固定化为可降解的合成水凝胶。具体而言，将四臂马来酰亚胺终止的聚（乙二醇）（PEG-4MAL）大分子单体用单PEG化的重组人N末端凝集素（NtA）域进行功能化，促进层粘连蛋白的高亲和力结合。使用不同的NtA浓度（10、50和100μM）来研究NtA密度对层粘连蛋白掺入，水凝胶生物物理特性和生物学结果的影响。层粘连蛋白在所有测试条件下均有效掺入（层粘连蛋白掺入率> 95％），并且开发的水凝胶显示出机械性能（平均储能模量（在NSC增殖和神经突分支和延伸的优选值内，G '）的范围为187至256 Pa）。与未修饰的水凝胶和含有物理包埋的层粘连蛋白的水凝胶相比，亲和结合层粘连蛋白PEG-4MAL水凝胶可更好地保持层粘连蛋白的生物活性，这种作用取决于NtA的浓度。与后者相比，掺有层粘连蛋白的10μMNtA功能化PEG-4MAL凝胶证明了这一点，后者支持增强的人NSC增殖和神经突延伸。总的来说，这项工作凸显了拟议的工程基质作为建立人工NSC壁ches的定义3D平台以及支持人NSC移植的细胞外基质模拟微环境的潜力。

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Engineering hydrogels with affinity-bound laminin as 3D neural stem cell culture systems

Laminin incorporation into biological or synthetic hydrogels has been explored to recapitulate the dynamic nature and biological complexity of neural stem cell (NSC) niches. However, the strategies currently explored for laminin immobilization within three-dimensional (3D) matrices do not address a critical aspect influencing cell–matrix interactions, which is the control over laminin conformation and orientation upon immobilization. This is a key feature for the preservation of the protein bioactivity. In this work, we explored an affinity-based approach to mediate the site-selective immobilization of laminin into a degradable synthetic hydrogel. Specifically, a four-arm maleimide terminated poly(ethylene glycol) (PEG-4MAL) macromer was functionalized with a mono-PEGylated recombinant human N-terminal agrin (NtA) domain, to promote high affinity binding of laminin. Different NtA concentrations (10, 50 and 100 μM) were used to investigate the impact of NtA density on laminin incorporation, hydrogel biophysical properties, and biological outcome. Laminin was efficiently incorporated for all the conditions tested (laminin incorporation >95%), and the developed hydrogels revealed mechanical properties (average storage modulus (G′) ranging from 187 to 256 Pa) within the values preferred for NSC proliferation and neurite branching and extension. Affinity-bound laminin PEG-4MAL hydrogels better preserve laminin bioactivity, compared to unmodified hydrogels and hydrogels containing physically entrapped laminin, this effect being dependent on NtA concentration. This was evidenced by the 10 μM NtA-functionalized PEG-4MAL gels incorporating laminin that support enhanced human NSC proliferation and neurite extension, compared to the latter. Overall, this work highlights the potential of the proposed engineered matrices to be used as defined 3D platforms for the establishment of artificial NSC niches and as extracellular matrix-mimetic microenvironments to support human NSC transplantation.

更新日期：2019-10-09

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