HIERARCHICAL ELECTROSPUN BIOINSPIRED SCAFFOLDS CAN MODIFY FIBROBLASTS MORPHOLOGY IN STATIC AND DYNAMIC CULTURE

Tissue engineering of injured tendons and ligaments is challenging since the scaffolds need to have a biomimetic hierarchical morphology and nonlinear mechanical properties [1]. The morphological arrangement of scaffolds is mandatory to drive cell growth and regenerate the collagen extracellular matrix. Electrospinning is the most promising technique to produce hierarchically structured nanofibrous scaffolds suitable to regenerate injured tendons and ligaments [1]. Moreover, the dynamic stimulation of electrospun scaffolds in bioreactors has demonstrated to speed up cell shape modification in vitro [2]. The aim of the present study was to combine different imaging techniques such as high-resolution X-ray computed tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to document the modifications in human fibroblasts morphology while cultured on hierarchically structured poly(L-lactic acid)/Collagen (PLLA/Coll) electrospun scaffolds, in static and dynamic conditions. In order to reproduce the morphology of tendon and ligament fibrils and fascicles [3], a PLLA/Coll-75/25 blend was electrospun on a high- speed rotating drum collector, producing mats of aligned nanofibers. Mats were cut in strips and wrapped up the drum, obtaining bundles of axially aligned nanofibers. To reproduce the structure of a whole tendon or ligament [3], each bundle was pulled out from the drum, obtaining a ring-shaped structure. Each bundle was fixed in a custom-made rotating electrospinning machine, and covered with an electrospun epitenon/epiligament-like sheath [4]. Finally, the scaffolds were crosslinked in a solution of EDC/NHS [5]. The scaffolds were then cultured with human fibroblasts for 7 days in static and dynamic conditions, using a commercial bioreactor (MCB1, CellScale, Waterloo, Canada). The imaging techniques employed assessed that cells had proliferated on the external nanofibrous sheath of the static scaffolds, elongating their cytoplasm circumferentially on the scaffolds. The dynamic cultures revealed a preferential axial orientation of fibroblasts grown on the external sheath. The aligned nanofiber bundles inside the hierarchical scaffolds permitted a physiological distribution of the fibroblasts along the nanofiber direction. Inside the dynamic scaffolds, cells appeared more elongated compared to the static counterpart. This study demonstrated that hierarchically structured electrospun scaffolds can induce fibroblasts morphological modifications both in static and dynamic conditions of culture. These preliminary results seem to confirm the suitability of these scaffolds for tendon and ligament tissue regeneration. Moreover, future optimizations of the imaging workflows implemented in this study, will be useful to define correlative microscopy protocols for the investigation of electrospun materials.