Garrido Claudia A, Garske Daniela S, Bucher Christian H, Amini Shahrouz, Duda Georg N, Schmidt-Bleek Katharina, Cipitria Amaia
Julius Wolff Institute for Biomechanics and Musculoskeletal Regeneration, Berlin Institute for Health at Charité - Universitätsmedizin Berlin, Berlin, Germany; Max Planck Institute for Colloids and Interfaces, Potsdam, Germany.
Julius Wolff Institute for Biomechanics and Musculoskeletal Regeneration, Berlin Institute for Health at Charité - Universitätsmedizin Berlin, Berlin, Germany.
Acta Biomater. 2025 Jul 29. doi: 10.1016/j.actbio.2025.07.063.
Tissue engineering involves assembling cells and mimicking the complex anisotropic architecture of biological tissues to perform specific functions. This study uses 3D alginate-based hydrogels with RGD binding motifs to explore the impact of anisotropic degradation of patterned hydrogels (two components: degradable (Deg) and non-degradable (noDeg)) compared to single-phase materials (one component: Deg or noDeg), on the potential of enhancing cell spreading, collective alignment, mechanotransduction and osteogenic differentiation of encapsulated human mesenchymal stromal cells (hMSCs). Spatial patterns of Deg and noDeg subregions are formed by photolithography: UV-triggered thiol-ene crosslinking with matrix metalloprotease (MMP) sensitive peptides form Deg phases, while non-UV exposed regions result in Diels-Alder spontaneous click crosslinking and noDeg phases. 3D patterns in hydrogel degradation enhance hMSC spreading and allow collective cell alignment in Deg areas, while cells remain rounded with no alignment in noDeg regions. In addition, we observe a boosted osteogenic differentiation when compared to single-phase materials, as mid osteogenic markers (OOsteocalcin) are expressed at day 14 in anisotropic gels, whereas in single-phase only early osteogenic markers are found (OOsterix). Mechanosensing pathways were evaluated using the expression and localization of YAP. Deg sections in patterned materials have an enhanced nuclear translocation and higher YAP expression compared to single-phase Deg materials and noDeg sections. This effect is lost and no patterns in YAP expression and localization emerge when using an MMP-scramble peptide or no-RGD materials. These findings demonstrate that 3D patterns in alginate hydrogel degradation guide hMSC spreading, collective alignment, enhance YAP nuclear translocation and osteogenic differentiation. Mimicking tissue anisotropy in 3D patterned hydrogels could have broad applications in biofabrication and tissue engineering. STATEMENT OF SIGNIFICANCE: Patterned materials integrate multiple characteristics within a single material, closely mimicking the anisotropy found in tissues. This research goes further by demonstrating how anisotropic degradation of cell-laden hydrogels leads to emerging patterns in mechanics. As a consequence, anisotropic hMSC morphology and collective alignment are observed in 3D patterned materials compared to single-phase counterparts. Additionally, we show enhanced and spatially guided hMSC osteogenic differentiation in patterned materials. Furthermore, anisotropic mechanosensing via YAP/TAZ signaling is shown to mediate this enhanced and spatially guided mechanotransduction and osteogenic differentiation. Finally, we explore how additional biochemical stimuli can further boost the spatially guided hMSC osteogenic differentiation. These findings advance our understanding of cell response in anisotropic microenvironments, with broad applications in biofabrication and tissue engineering.
组织工程涉及组装细胞并模拟生物组织复杂的各向异性结构以执行特定功能。本研究使用具有RGD结合基序的基于藻酸盐的3D水凝胶，以探究图案化水凝胶（两种成分：可降解（Deg）和不可降解（noDeg））与单相材料（一种成分：Deg或noDeg）的各向异性降解对增强封装的人间充质基质细胞（hMSC）的细胞铺展、集体排列、机械转导和成骨分化潜力的影响。Deg和noDeg子区域的空间图案通过光刻形成：紫外线触发的硫醇-烯交联与基质金属蛋白酶（MMP）敏感肽形成Deg相，而未暴露于紫外线的区域导致狄尔斯-阿尔德自发点击交联和noDeg相。水凝胶降解中的3D图案增强了hMSC的铺展，并使细胞在Deg区域集体排列，而在noDeg区域细胞保持圆形且无排列。此外，与单相材料相比，我们观察到成骨分化增强，因为在各向异性凝胶中第14天表达中期成骨标记物（骨钙素），而在单相材料中仅发现早期成骨标记物（osterix）。使用YAP的表达和定位评估机械传感途径。与单相Deg材料和noDeg部分相比，图案化材料中的Deg部分具有增强的核转位和更高的YAP表达。当使用MMP-乱序肽或无RGD材料时，这种效应消失，YAP表达和定位中没有出现图案。这些发现表明，藻酸盐水凝胶降解中的3D图案引导hMSC铺展、集体排列，增强YAP核转位和成骨分化。在3D图案化水凝胶中模拟组织各向异性在生物制造和组织工程中可能具有广泛应用。
图案化材料在单一材料中整合了多种特性，紧密模拟组织中发现的各向异性。本研究进一步证明了载细胞水凝胶的各向异性降解如何导致力学方面出现新的图案。因此，与单相材料相比，在3D图案化材料中观察到各向异性的hMSC形态和集体排列。此外，我们展示了图案化材料中hMSC成骨分化的增强和空间引导。此外，通过YAP/TAZ信号传导的各向异性机械传感被证明介导了这种增强的和空间引导的机械转导和成骨分化。最后，我们探索了额外的生化刺激如何进一步促进空间引导的hMSC成骨分化。这些发现推进了我们对各向异性微环境中细胞反应的理解，在生物制造和组织工程中有广泛应用。
