Anisotropic ice-templated collagen scaffolds for soft tissue engineering: a mechanical characterisation toolkit

Collagen-based soft tissues are often anisotropic in structure, and in regenerative medicine, it is important that scaffolds are designed to closely mimic their architecture and mechanical behaviour. Existing testing standards are not directly applicable to anisotropic structures in physiologically relevant conditions. The challenge is therefore to systematically quantify mechanical anisotropy, nonlinear tensile behaviour, and both in vitro degradation and fatigue in a way appropriate for soft, porous, natural macromolecular structures. In this study, we fabricated collagen scaffolds with elongated porosity via directional freeze-drying and then chemically crosslinked them using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Compressive mechanical behaviour was monitored along two characteristic directions, and distinct stress-strain curves were observed. The elastic modulus was identified as 6.42 kPa and 1.02 kPa along the two directions. Nonlinear tensile behaviour was characterised using the tangent modulus, which varied from 25 kPa to 270 kPa before failure. Standardised methods for monitoring degradation at 37 $°C$ were developed. In vitro degradation was investigated by immersing scaffolds in deionised water for 60 days, during which the critical stress decreased by 30% in the first 30 days. Fatigue was studied by monitoring the stress-strain curves under 20% tensile strain for 1000 cycles. Scaffolds exhibited weaker mechanical integrity when loaded at 1.43 Hz. This study addresses the lack of standardised testing methods for anisotropic ice-templated collagen scaffolds by establishing a set of protocols to characterise the mechanical anisotropy, quantify tensile behaviour, and monitor scaffold degradation. This flexible characterisation toolkit can be adapted to specific sample treatments during tissue culture.