Strategies that enable hydrogel substrates to support cell attachment typically incorporate either entire extracellular matrix proteins or synthetic peptide fragments such as the RGD (arginine–glycine–aspartic acid) motif. Previous studies have carefully analysed how material characteristics can affect single cell morphologies. However, the influence of substrate stiffness and ligand presentation on the spatial organisation of human mesenchymal stem cells (hMSCs) have not yet been examined. In this study, we assessed how hMSCs organise themselves on soft (E = 7.4 – 11.2 kPa) and stiff (E = 27.3 – 36.8 kPa) poly(ethylene glycol) (PEG) hydrogels with varying concentrations of RGD (0.05 – 2.5 mM). Our results indicate that hMSCs seeded on soft hydrogels clustered with reduced cell attachment and spreading area, irrespective of RGD concentration and isoform. On stiff hydrogels, in contrast, cells spread with high spatial coverage for RGD concentrations of 0.5 mM or higher. In conclusion, we identified that an interplay of hydrogel stiffness and the availability of cell attachment motifs are important factors in regulating hMSC organisation on PEG hydrogels. Understanding how cells initially interact and colonize the surface of this material is a fundamental prerequisite for the design of controlled platforms for tissue engineering and mechanobiology studies.

使水凝胶基质支持细胞附着的策略通常包含完整的细胞外基质蛋白或合成的肽片段，例如RGD（精氨酸-甘氨酸-天冬氨酸）基序。先前的研究已经仔细分析了材料特性如何影响单细胞形态。但是，尚未研究基质刚度和配体呈递对人间充质干细胞（hMSCs）空间组织的影响。在这项研究中，我们评估了hMSC如何在软（E = 7.4 – 11.2 kPa）和硬（E= 27.3 – 36.8 kPa）的聚（乙二醇）（PEG）水凝胶，其RGD浓度不同（0.05 – 2.5 mM）。我们的结果表明，无论RGD浓度和亚型如何，接种在软水凝胶上的hMSC均具有减少的细胞附着和散布面积。相反，在刚性水凝胶上，RGD浓度为0.5 mM或更高时，细胞以高空间覆盖率扩散。总之，我们发现水凝胶刚度和细胞附着基序的相互作用是调节PEG水凝胶上hMSC组织的重要因素。了解细胞最初是如何相互作用并在这种材料的表面定殖的，这是设计用于组织工程和力学生物学研究的受控平台的基本前提。