López-Serrano Cristina, Côté-Paradis Yeva, Habenstein Birgit, Loquet Antoine, Le Coz Cédric, Ruel Jean, Laroche Gaétan, Durrieu Marie-Christine
Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, Pessac 33600, France.
Laboratoire d'Ingénierie de Surface, Centre de Recherche sur les Matériaux Avancés, Département de Génie des Mines, de la Métallurgie et des Matériaux, Université Laval, Québec, QC G1 V 0A6, Canada.
ACS Appl Mater Interfaces. 2024 Jul 31;16(30):39165-39180. doi: 10.1021/acsami.4c10755. Epub 2024 Jul 23.
Methods for promoting and controlling the differentiation of human mesenchymal stem cells (hMSCs) in vitro before in vivo transplantation are crucial for the advancement of tissue engineering and regenerative medicine. In this study, we developed poly(ethylene glycol) diacrylate (PEGDA) hydrogels with tunable mechanical properties, including elasticity and viscoelasticity, coupled with bioactivity achieved through the immobilization of a mixture of RGD and a mimetic peptide of the BMP-2 protein. Despite the key relevance of hydrogel mechanical properties for cell culture, a standard for its characterization has not been proposed, and comparisons between studies are challenging due to the different techniques employed. Here, a comprehensive approach was employed to characterize the elasticity and viscoelasticity of these hydrogels, integrating compression testing, rheology, and atomic force microscopy (AFM) microindentation. Distinct mechanical behaviors were observed across different PEGDA compositions, and some consistent trends across multiple techniques were identified. Using a photoactivated cross-linker, we controlled the functionalization density independently of the mechanical properties. X-ray photoelectrin spectroscopy and fluorescence microscopy were employed to evaluate the functionalization density of the materials before the culturing of hMSCs on them. The cells cultured on all functionalized hydrogels expressed an early osteoblast marker (Runx2) after 2 weeks, even in the absence of a differentiation-inducing medium compared to our controls. Additionally, after only 1 week of culture with osteogenic differentiation medium, cells showed accelerated differentiation, with clear morphological differences observed among cells in the different conditions. Notably, cells on stiff but stress-relaxing hydrogels exhibited an overexpression of the osteocyte marker E11. This suggests that the combination of the functionalization procedure with the mechanical properties of the hydrogel provides a potent approach to promoting the osteogenic differentiation of hMSCs.
在体内移植前促进和控制人间充质干细胞（hMSCs）体外分化的方法对于组织工程和再生医学的发展至关重要。在本研究中，我们开发了具有可调机械性能（包括弹性和粘弹性）的聚（乙二醇）二丙烯酸酯（PEGDA）水凝胶，并通过固定RGD和BMP-2蛋白模拟肽的混合物实现了生物活性。尽管水凝胶机械性能对细胞培养至关重要，但尚未提出其表征标准，并且由于采用的技术不同，各研究之间的比较具有挑战性。在此，我们采用了一种综合方法来表征这些水凝胶的弹性和粘弹性，整合了压缩测试、流变学和原子力显微镜（AFM）微压痕技术。在不同的PEGDA组成中观察到了不同的机械行为，并确定了多种技术之间的一些一致趋势。使用光活化交联剂，我们独立于机械性能控制功能化密度。在hMSCs接种到材料上之前，采用X射线光电子能谱和荧光显微镜来评估材料的功能化密度。与我们的对照相比，即使在没有分化诱导培养基的情况下，在所有功能化水凝胶上培养的细胞在2周后都表达了早期成骨细胞标志物（Runx2）。此外，在用成骨分化培养基培养仅1周后，细胞显示出加速分化，在不同条件下的细胞之间观察到明显的形态差异。值得注意的是，在坚硬但应力松弛的水凝胶上的细胞表现出骨细胞标志物E11的过表达。这表明功能化程序与水凝胶机械性能的结合提供了一种促进hMSCs成骨分化的有效方法。
