Department of Industrial Engineering, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
Health Sciences and Technologies - Interdepartmental Center for Industrial Research (CIRI-HST), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
J Microsc. 2020 Mar;277(3):160-169. doi: 10.1111/jmi.12827. Epub 2019 Aug 2.
The regeneration of injured tendons and ligaments is challenging because the scaffolds needs proper mechanical properties and a biomimetic morphology. In particular, the morphological arrangement of scaffolds is a key point to drive the cells growth to properly regenerate the collagen extracellular matrix. Electrospinning is a promising technique to produce hierarchically structured nanofibrous scaffolds able to guide cells in the regeneration of the injured tissue. Moreover, the dynamic stretching in bioreactors of electrospun scaffolds had demonstrated to speed up cell shape modifications in vitro. The aim of the present study was to combine different imaging techniques such as high-resolution X-ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to investigate if hierarchically structured poly (L-lactic acid) and collagen electrospun scaffolds can induce morphological modifications in human fibroblasts, while cultured in static and dynamic conditions. After 7 days of parallel cultures, the results assessed that fibroblasts had proliferated on the external nanofibrous sheath of the static scaffolds, elongating themselves circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts growth on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds instead, allowed a physiological distribution of the fibroblasts along the nanofibre direction. Inside the dynamic scaffolds, cells appeared thinner compared with the static counterpart. This study had demonstrated that hierarchically structured electrospun scaffolds can induce different fibroblasts morphological modifications during static and dynamic conditions, modifying their shape in the direction of the applied loads. LAY DESCRIPTION: To enhance the regeneration of injured tendons and ligaments cells need to growth on dedicated structures (scaffolds) with mechanical properties and a fibrous morphology similar to the natural tissue. In particular, the morphological organisation of scaffolds is fundamental in leading cells to colonise them, regenerating the collagen extracellular matrix. Electrospinning is a promising technique to produce fibres with a similar to the human collagen fibres, suitable to design complex scaffolds able to guide cells in the reconstruction of the natural tissue. Moreover, it is well established that the cyclic stretching of these scaffolds inside dedicated systems called bioreactors, can speed up cells growth and their shape modification. The aim of the present study was to investigate how hierarchically structured electrospun scaffolds, made of resorbable material such as poly(L-lactic acid) and collagen, could induce morphological changes in human fibroblasts, while cultured during static and dynamic conditions. These scaffolds were composed by an external electrospun membrane that grouped inside it a ring-shaped bundle, made of axially aligned nanofibres, resembling the morphological arrangement of tendon and ligament tissue. After 7 days of parallel cultures, the scaffolds were investigated using the following imaging techniques: (i) high-resolution X-ray tomography (XCT); (ii) scanning electron microscopy (SEM); (iii) fluorescence microscopy and (iv) histology. The results showed that fibroblasts were able to grow on the external nanofibrous sheath of the static scaffolds, by elongating themselves along their circumference. The dynamic cultures revealed instead a preferential axial orientation of fibroblasts grown on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds allowed an axial distribution of the fibroblasts along the nanofibres direction. This study has demonstrated that the electrospun hierarchically structured scaffolds investigated can modify the fibroblasts morphology both in static and dynamic conditions, in relation with the direction of the applied loads.
受伤的肌腱和韧带的再生具有挑战性，因为支架需要适当的机械性能和仿生形态。特别是，支架的形态排列是驱动细胞生长以正确再生胶原细胞外基质的关键。静电纺丝是一种很有前途的技术，可以生产出具有分级结构的纳米纤维支架，能够引导细胞在受伤组织中再生。此外，在生物反应器中对静电纺丝支架进行动态拉伸已被证明可以加速细胞体外形状的改变。本研究的目的是结合高分辨率 X 射线断层扫描（XCT）、扫描电子显微镜（SEM）、荧光显微镜和组织学等不同成像技术，研究聚 L-乳酸和胶原静电纺丝支架是否能在静态和动态条件下诱导人成纤维细胞发生形态学改变。经过 7 天的平行培养，结果评估表明，成纤维细胞在静态支架的外部纳米纤维鞘上增殖，沿圆周方向自身伸长。动态培养显示，成纤维细胞在外部鞘上的生长具有优先的轴向取向。而在分级支架内部的取向纳米纤维束则允许成纤维细胞在纳米纤维方向上沿生理方向分布。在动态支架内，细胞看起来比静态支架薄。本研究表明，分级结构的静电纺丝支架可以在静态和动态条件下诱导成纤维细胞发生不同的形态改变，改变其在施加负载方向上的形状。
为了增强受伤肌腱和韧带的再生，细胞需要生长在具有机械性能和类似天然组织纤维形态的专用结构（支架）上。特别是，支架的形态组织对于引导细胞定植、再生胶原细胞外基质至关重要。静电纺丝是一种很有前途的技术，可以生产出类似于人胶原蛋白纤维的纤维，适合设计复杂的支架，能够引导细胞在天然组织的重建中。此外，已经证实，在称为生物反应器的专用系统中对这些支架进行周期性拉伸，可以加速细胞生长和形状改变。本研究的目的是研究由可吸收材料（如聚 L-乳酸和胶原）制成的分级静电纺丝支架如何在静态和动态条件下诱导人成纤维细胞发生形态变化。这些支架由一个外部静电纺丝膜组成，该膜内有一个环形束，由轴向排列的纳米纤维组成，类似于肌腱和韧带组织的形态排列。经过 7 天的平行培养后，使用以下成像技术对支架进行了研究：（i）高分辨率 X 射线断层扫描（XCT）；（ii）扫描电子显微镜（SEM）；（iii）荧光显微镜和（iv）组织学。结果表明，成纤维细胞能够在静态支架的外部纳米纤维鞘上生长，通过沿圆周方向自身伸长。而动态培养则显示，成纤维细胞在外部鞘上的生长具有优先的轴向取向。分层支架内的取向纳米纤维束允许成纤维细胞沿纳米纤维方向轴向分布。本研究表明，在所研究的静电纺丝分级结构支架中，无论是在静态还是动态条件下，都可以改变成纤维细胞的形态，这与施加的负载方向有关。
