In this study, we use non-linear imaging microscopy to characterize the structural properties of porous collagen-GAG scaffolds (CGS) seeded with human umbilical vein endothelial cells (HUVECs), as well as human mesenchymal stem cells (hMSCs), a co-culture previously reported to form vessel-like structures inside CGS. The evolution of the resulting tissue construct was monitored over 10 days via simultaneous two- and three-photon excited fluorescence microscopy. Time-lapsed 2- and 3-photon excited fluorescence imaging was utilized to monitor the temporal evolution of the vascular-like structures up to 100 µm inside the scaffold up to 10 days post-seeding. 3D polarization-dependent second harmonic generation (PSHG) was utilized to monitor collagen-based scaffold remodeling and determine collagen fibril orientation up to 200 µm inside the scaffold. We demonstrate that polarization-dependent second harmonic generation can provide a novel way to quantify the reorganization of the collagen architecture in CGS simultaneously with key biomechanical interactions between seeded cells and CGS that regulate the formation of vessel-like structures inside 3D tissue constructs. A comparison between samples at different days in vitro revealed that gradually, the scaffolds developed an orthogonal net-like architecture, previously found in real skin.

中文翻译：

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极化二次谐波产生对细胞接种胶原蛋白支架中胶原蛋白重构的三维表征

在这项研究中，我们使用非线性成像显微镜来表征植入人脐静脉内皮细胞（HUVEC）以及人间充质干细胞（hMSCs）的多孔胶原GAG支架（CGS）的结构特性以前曾报道过在CGS内部形成血管样结构。通过同时的两光子和三光子激发荧光显微镜，在10天内监测所得组织构建体的演变。利用延时的2光子和3光子激发荧光成像来监测支架内部高达100 µm的类血管结构在播种后长达10天的时间演变。利用3D偏振相关的二次谐波生成（PSHG）来监视基于胶原的支架重塑，并确定支架内部高达200 µm的胶原原纤维取向。我们证明极化相关的二次谐波的产生可以提供一种新颖的方式来量化CGS中胶原结构的重组，同时还可以调节种子细胞与CGS之间的关键生物力学相互作用，从而调节3D组织构造内部血管样结构的形成。在体外不同天的样品之间的比较显示，逐渐地，支架形成了以前在真实皮肤中发现的正交网状结构。