High-resolution x-ray tomographic morphological characterisation of electrospun nanofibrous bundles for tendon and ligament regeneration and replacement.

Repair of ligaments and tendons requires scaffolds mimicking the spatial organisation of collagen in the natural tissue. Electrospinning is a promising technique to produce nanofibres of both resorbable and biostable polymers with desired structural and morphological features. The aim of this study was to perform high-resolution x-ray tomography (XCT) scans of bundles of Nylon6.6, pure PLLA and PLLA-Collagen blends, where the nanofibres were meant to have a predominant direction. Characterisation was carried out via a dedicated methodology to firmly hold the specimen during the scan and a workflow to quantify the directionality of the nanofibres in the bundle. XCT scans with 0.4 and 1.0 μm voxel size were successfully collected for all bundle compositions. Better image quality was achieved for those bundles formed by thicker nanofibres (i.e. 0.59 μm for pure PLLA), whereas partial volume effect was more pronounced for thinner nanofibres (i.e. 0.26 μm for Nylon6.6). As expected, the nanofibres had a predominant orientation along the axis of the bundles (more than 20% of the nanofibres within 3° and more than 60% within 18° from the bundle axis), with a Gaussian-like dispersion in the other directions. The directionality assessment was validated by comparison against a similar analysis performed on SEM images: the XCT analysis overestimated the amount of nanofibres very close to the bundle axis, especially for the materials with thinnest nanofibres, but adequately identified the amount of nanofibres within 12°. LAY DESCRIPTION: Repair of ligaments and tendons requires dedicated materials (scaffolds) mimicking the spatial organisation of the collagen (the main material composing such natural tissue). Electrospinning is a promising technique that allows production of fibres with nanometric dimension using high voltage to stretch very tiny drops of polymeric solutions. Electrospinning allows processing both polymers that can be resorbed by the host tissue, and nonresorbable ones, to obtain the desired structural and morphological features by arranging the nanofibres in bundles. The aim of this study was to perform high-resolution x-ray computed tomography (XCT) scans of bundles, where the nanofibres were meant to have a predominant direction. The investigation included bundles of different compositions: a biostable polymer (Nylon) and bioresorbable ones (pure Poly-L-lactic acid (PLLA) and PLLA-Collagen blends). The electrospun bundles were produced using a validated method (Sensini et al 2017: https://doi.org/10.1088/1758-5090/aa6204). To this end, we developed a dedicated methodology to scan such small specimens, and a workflow to quantify the directionality of the nanofibres in the bundle. For all the compositions, XCT scans with extremely high resolution (i.e. down to 0.4 μm) were successfully collected. As expected, better images were obtained for those bundles where the nanofibres were thicker than the scanning resolution (i.e. 0.59 μm for pure PLLA). The images of the thinnest nanofibres (i.e. 0.26 μm for Nylon) were poorer because the fibre diameter was smaller than the resolution (partial volume effect). The nanofibres had a predominant orientation along the axis of the bundles (more than 60% of the nanofibres were within 18° from the bundle axis). The nanofibres had a Gaussian-like dispersion in the other directions. As this is the first time that XCT is used to quantify the directionality of this kind of bundles, the directionality assessment was further validated by comparison against a similar analysis performed on SEM images. Overall, this study has demonstrated the usefulness and reliability of using high-resolution x-ray computed tomography (XCT) scans to investigate the morphology of polymeric scaffolds made of electrospun nanofibres.