Mechanical and Biological Evaluation of Melt-Electrowritten Polycaprolactone Scaffolds for Acetabular Labrum Restoration

Abstract

Repair or reconstruction of a degenerated or injured acetabular labrum is essential to the stability and health of the hip joint. Current methods for restoration fail to reproduce the structure and mechanical properties of the labrum. In this study, we characterized the structure and tensile mechanical properties of melt-electrowritten polycaprolactone scaffolds of varying architectures and assessed the labrum cell compatibility of selected graft candidates. Cell compatibility was assessed using immunofluorescence of the actin skeleton. First, labrum explants were co-cultured with scaffold specimen to investigate the scaffold compatibility with primary cells. Second, effects of pore size on pre-cultured seeded labrum cells were studied. Third, cell compatibility under dynamic stretching was examined. Grid-like structures showed favorable tensile properties with decreasing fibre spacing. Young's moduli ranging from 2.33 ± 0.34 to 13.36 ± 2.59 MPa were measured across all structures. Primary labrum cells were able to migrate from co-cultured labrum tissue specimens into the scaffold and grow in vitro. Incorporation of small-diameter-fibre and interfibre spacing improved cell distribution and cell spreading, whereas mechanical properties were only marginally affected. Wave-patterned constructs reproduced the non-linear elastic behaviour of native labrum tissue and, therefore, allowed for physiological cyclic tensile strain but showed decreased cell compatibility under dynamic loading. In conclusion, melt-electrowritten polycaprolactone scaffolds are promising candidates for labral grafts; however, further development is required to improve both the mechanical and biological compatibility.

Keywords: acetabular labrum; biofabrication; melt electrowriting.

Figure 1

(a) CAD model of the custom-built MEW setup consisting of a melt head, XYZ axes, and a collector platform. (b–d) Top view of the manufactured PCL microfibre structures with 1000 µm, 500 µm, and 250 µm interfibre spacing. (e) Multiscale approach with defined grids with an interfibre spacing of 250 µm and a fine mesh of fibres alternating the grids. (f) Top view on waved structures for improved hyperelastic behaviour.

Figure 2

(a) The entire spectrum acquired using ATR-FTIR with two different focus areas marked and shown in (b,c). (b) Spectrum focusing on the characteristic absorption peaks around C-O, OC-O, and C-O-C bonds. (c) Spectrum focusing on the characteristic absorption peaks of methylene groups and ester carbonyl groups.

Figure 3

(a) Workflow for the preparation of test specimens for tensile tests, consisting of a custom-made blade holder for parallel cuts at a defined distance. (b) Exemplary stress–strain curve with yield point (red), linear region (green), and ultimate strength (blue). (c–f) Young’s modulus, yield strain, yield stress, and ultimate stress for each sample type. (g) Exemplary stress–strain curve for the stepwise cyclic test performed on structures with straight fibres. (h) Yield strain from each sample type derived from cyclic testing. (i) Tensile curves for the comparison of structures composed of waved against straight fibres. (j) Stress–strain curves from cyclic testing of waved structures up to 10% strain, well in the hyperelastic region. (k) Development of the modulus over the course of 3600 cycles during cyclic testing of waved samples.

Figure 4

(a,b) SEM image of structures for labrum cell experiments with and without layers of fine fibres intermitting a defined grid of 250 µm interfibre spacing. (c,d) Confocal microscopy images of labrum cells (red: f-actin, blue: cell nuclei) growing on a grid-like scaffold without (c) and with (d) fibres of smaller diameter and smaller spacing incorporated. The scaffolds were seeded with cells and cultured for 2 days. In contrast with the grid-like scaffold, the multiscale scaffold (d) facilitates homogenous cell distribution in space and cell spreading. On both scaffolds however, high cell-material interaction can be observed, as indicated by the elongated/spread-out cell morphology. (e) Schematic representation of the co-culture experiment with a labrum piece sitting on top of a PCL scaffold with a porous silicone ring around. (f,g) Confocal microscopy images of labrum cells (red: f-actin, blue: cell nuclei) growing on a grid-like scaffold without (f) and with (g) fibres of smaller diameter and smaller spacing incorporated. The scaffolds were co-cultured with bovine labrum tissue specimen for 6 weeks. Both scaffold architectures allowed for cell migration and cell growth.

Figure 5

(a) Scanning electron microscopy image showing the architecture of wave-patterned scaffolds used for static and dynamic cell culture. (b–e) Fluorescent widefield microscopy images of actin-stained cells on wave-patterned scaffolds following long-term physiological stretching or static fixation (non-stretched control).