Micromechanical properties of canine femoral articular cartilage following multiple freeze-thaw cycles

Tissue material properties are crucial to understanding their mechanical function, both in healthy and diseased states. However, in certain circumstances logistical limitations can prevent testing on fresh samples necessitating one or more freeze-thaw cycles. To date, the nature and extent to which the material properties of articular cartilage are altered by repetitive freezing have not been explored. Therefore, the aim of this study is to quantify how articular cartilage mechanical properties, measured by nanoindentation, are affected by multiple freeze-thaw cycles. Canine cartilage plugs (n = 11) from medial and lateral femoral condyles were submerged in phosphate buffered saline, stored at 3-5 °C and tested using nanoindentation within 12 h. Samples were then frozen at −20 °C and later thawed at 3-5 °C for 3 h before material properties were re-tested and samples re-frozen under the same conditions. This process was repeated for all 11 samples over three freeze-thaw cycles. Overall mean and standard deviation of shear storage modulus decreased from 1.76 ± 0.78 to 1.21 ± 0.77 MPa (p = 0.91), shear loss modulus from 0.42 ± 0.19 to 0.39 ± 0.17 MPa (p=0.70) and elastic modulus from 5.13 ± 2.28 to 3.52 ± 2.24 MPa (p = 0.20) between fresh and three freeze-thaw cycles respectively. The loss factor increased from 0.31 ± 0.38 to 0.71 ± 1.40 (p = 0.18) between fresh and three freeze-thaw cycles. Inter-sample variability spanned as much as 10.47 MPa across freezing cycles and this high-level of biological variability across samples likely explains why overall mean “whole-joint” trends do not reach statistical significance across the storage conditions tested. As a result multiple freeze-thaw cycles cannot be explicitly or statistically linked to mechanical changes within the cartilage. However, the changes in material properties observed herein may be sufficient in magnitude to impact on a variety of clinical and scientific studies of cartilage, and should be considered when planning experimental protocols.