Hierarchical electrospun bioinspired scaffolds can modify fibroblasts morphology in static and dynamic culture

none10siTissue engineering of injured tendons and ligaments requires that scaffolds have a biomimetic hierarchical structure and biomimetic mechanical properties [1]. The morphology of scaffolds is fundamental to drive cell growth and regenerate the extracellular matrix. Electrospinning is a promising technique to produce nanofibrous scaffolds suitable to regenerate injured tendons and ligaments [1]. The use of a bioreactor with dynamic simulation of a tendon/ligament environment to investigate changes in fibroblast morphology, when cultured on electrospun scaffolds, has shown to speed up cell proliferation, changes in cell morphology and the production of extracellular matrix in vitro [2]. The aim of the study was to combine different imaging techniques such as high-resolution x-ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to evaluate the changes in human fibroblasts shape when cultured on electrospun hierarchical poly(L-lactic acid)/Collagen (PLLA/Coll) scaffolds, in static and dynamic conditions. In order to reproduce the morphology of tendon and ligament fibrils and fascicles [3], a PLLA/Coll blend was electrospun on a high-speed rotating drum collector, producing mats of aligned nanofibers. Mats were cut in stripes and wrapped up on the drum, obtaining ring-shaped bundles of axially aligned nanofibers [4]. To reproduce the structure of a whole tendon or ligament [3], each bundle was covered with an electrospun epitenon/epiligament-like sheath [5]. The scaffolds were crosslinked with a previously developed procedure [6]. The scaffolds were then cultured with human fibroblasts for 7 days in static and dynamic conditions (5% strain, 3600 cycles for 2 session), using a commercial bioreactor (MCB1, CellScale, Canada). At the end of the culture the scaffolds were investigated by means a XCT, SEM, fluorescent microscopy and histological investigation. The imaging techniques showed that cells have proliferated on the nanofibrous sheath of the static specimens, elongating their body circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts grown on the external sheath. The aligned nanofiber bundles inside the hierarchical scaffolds allowed a biomimetic distribution of the fibroblasts along the nanofiber direction. These results confirm the suitability of these scaffolds for tendon and ligament tissue regeneration. Moreover, future optimization of the imaging sequences implemented in this study, will be useful to define in vitro correlative microscopy protocols for the investigation of electrospun materials.noneA.Sensini,L.Cristofolini,A.Zucchelli,M.L.Focarete,C.Gualandi,A.DeMori,A.P.Kao, M. Roldo, G. Blunn, G. TozziA.Sensini,L.Cristofolini,A.Zucchelli,M.L.Focarete,C.Gualandi,A.DeMori,A.P.Kao, M. Roldo, G. Blunn, G. Tozz