Hierarchical electrospun tendon‐ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions

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.