A MicroCT in situ protocol to reveal the structure-function relation in electrospun hierarchical scaffolds for tendon/ligament regeneration

Electrospun PLLA/collagen bundles (SB) and hierarchical structures (EHS) are under research as bioinspired tendon-ligament scaffolds [1]. Understanding how the hierarchical structure evolves with increasing strain is of the utmost importance to relate scaffold design and function. With this aim, a microCT in situ uniaxial loading protocol was developed. Firstly, to define reference mechanical characteristics, 4 SB and 4 EHS were tested with a monotonic ramp to failure at 0.33 %/s of strain rate. Then, 3 SB and 3 EHS underwent the microCT in situ test. Samples were submerged in PBS for two minutes before clamping for tensioning in the microCT apparatus (Skyscan 1172, Bruker, Belgium). After microCT scan at the minimum strain allowed by the load cell sensitivity (0.45 Newton, corresponding to 2% strain for SB and 0% strain for EHS), progressive strains were imposed (SB: 3-45-7-9%; EHS: 1.5-3-5-7%). Each strain was followed by 15 minutes stress-relaxation, to exhaust viscous phenomena, before each microCT acquisition. SB were scanned at a voxel size of 13 µm, with no filter and an x-ray tube voltage of 40 kV. Scanning protocol for EHS was the same, except for a higher spatial resolution (9 µm) to discriminate between different internal hierarchical levels. The region of interest (ROI) selection and morphometric analysis were performed using SkyScan CT-Analyser software. ROI extended for 6 mm in length, 2 mm below the superior fixed clamp, to avoid metal artefacts. The microCT in-situ test reported strain-load curves consistent with reference mechanical characterizations, confirming that the procedure did not alter the scaffold original mechanics, allowing the study of the evolution of their internal structure. Bundle tortuosity decreased and alignment along the mechanical axis increased with strain for EHS, while not for SB, probably because SB extensively overpassed the yielding point during the in-situ test. Contraption with increasing strain was common for both kinds of samples, as expected, but corresponded to a decrease in porosity only for SB (intra-bundle), while the presence of voids increased for EHS (inter-bundle). The microCT in situ mechanical test successfully allowed investigation, for the first time, of the relation between microstructure and mechanics of electrospun scaffolds. This protocol will be fundamental for future Digital Volume Correlation studies, for obtaining unpreceded information to design the future generations of scaffolds for tissue engineering.