INM-Leibniz Institute for New Materials , Campus D2 2, 66123 Saarbrücken. Germany.
Max-Planck-Institut für Polymerforschung , Ackermannweg 10, 55128 Mainz, Germany.
Biomacromolecules. 2017 Mar 13;18(3):906-913. doi: 10.1021/acs.biomac.6b01784. Epub 2017 Feb 17.
Biomaterials for cell culture allowing simple and quantitative presentation of instructive cues enable rationalization of the interplay between cells and their surrounding microenvironment. Poly(acrylamide) (PAAm) hydrogels are popular 2D-model substrates for this purpose. However, quantitative and reproducible biofunctionalization of PAAm hydrogels with multiple ligands in a trustable, controlled, and independent fashion is not trivial. Here, we describe a method for bifunctional modification of PAAm hydrogels with thiol- and amine- containing biomolecules with controlled densities in an independent, orthogonal manner. We developed copolymer networks of AAm with 9% acrylic acid and 2% N-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide. The covalent binding of thiol- and amine-containing chromophores at tunable concentrations was demonstrated and quantified by UV spectroscopy. The morphology, mechanical properties, and homogeneity of the copolymerized hydrogels were characterized by scanning electron microscopy, dynamic mechanical analysis, and confocal microscopy studies. Our copolymer hydrogels were bifunctionalized with polylysine and a laminin-mimetic peptide using the specific chemistries. We analyzed the effect of binding protocol of the two components in the maturation of cultured postmitotic cortical neurons. Our substrates supported neuronal attachment, proliferation, and neuronal differentiation. We found that neurons cultured on our hydrogels bifunctionalized with ligand-specific chemistries in a sequential fashion exhibited higher maturation at comparable culture times than using a simultaneous bifunctionalization strategy, displaying a higher number of neurites, branches, and dendritic filopodia. These results demonstrate the relevance of quantitative and optimized coupling chemistries for the performance of simple biomaterials and with sensitive cell types.
用于细胞培养的生物材料，允许简单且定量地呈现指导线索，从而使细胞与其周围微环境的相互作用合理化。聚（丙烯酰胺）（PAAm）水凝胶是为此目的的流行的 2D 模型底物。然而，以可信赖、可控和独立的方式，对 PAAm 水凝胶进行多种配体的定量和可重复的生物功能化并非易事。在这里，我们描述了一种在独立、正交的方式下，用含硫醇和胺的生物分子对 PAAm 水凝胶进行双功能修饰的方法，其密度可控。我们开发了丙烯酰胺与 9%丙烯酸和 2%N-（4-（5-（甲基磺酰基）-1,3,4-噁二唑-2-基）苯基）丙烯酰胺的共聚网络。通过紫外光谱证明并定量了含硫醇和胺的生色团在可调浓度下的共价结合。扫描电子显微镜、动态力学分析和共聚焦显微镜研究表征了共聚水凝胶的形态、力学性能和均一性。我们使用特定的化学方法，将聚赖氨酸和层粘连蛋白模拟肽双功能化我们的共聚物水凝胶。我们分析了两种成分在培养的有丝分裂后皮质神经元成熟过程中的结合方案对其的影响。我们的基质支持神经元附着、增殖和神经元分化。我们发现，在用配体特异性化学物质依次双功能化的水凝胶上培养的神经元在可比的培养时间内表现出更高的成熟度，其神经突、分支和树突丝状伪足的数量更多。这些结果表明，对于简单的生物材料和敏感的细胞类型，定量和优化的偶联化学具有相关性。
