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

Abstract

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.

Iatrogenic cartilage injury associated with the use of stainless‐steel cannulas and silicone‐guarded cannulas for canine stifle arthroscopy

“…Reasons for continued IACI with the guarded cannula could be displacement of the silicone ET on the cannula resulting in steel exposure within the joint space; that the silicone barrier used was thin, allowing IACI; or cadaveric articular cartilage is soft due to a freeze-thaw cycle and, therefore, becomes excessively susceptible to IACI. 27 While no guard displacement was noted in the present study, displacement was commonly encountered in the pilot surgeries during surgical methodology development. Ensuring that the guard covers the entire cannula shaft is vital in preventing guard collapse and displacement as the cannula is pushed into the joint during joint entry.…”

“…The unchanged incidence of IACI provides evidence that, although the silicone makes intra‐articular collisions feel softer, IACI can still occur secondary to intra‐articular cannula manipulation even when a silicone guard is used. Reasons for continued IACI with the guarded cannula could be displacement of the silicone ET on the cannula resulting in steel exposure within the joint space; that the silicone barrier used was thin, allowing IACI; or cadaveric articular cartilage is soft due to a freeze–thaw cycle and, therefore, becomes excessively susceptible to IACI . While no guard displacement was noted in the present study, displacement was commonly encountered in the pilot surgeries during surgical methodology development.…”

“…The cadavers included in the current study had no evidence of intra‐articular pathology. However freeze–thaw cycles decrease the shear storage modulus, shear loss modulus, and elastic modulus of canine femoral condylar cartilage, as compared to fresh cartilage samples . The compromised micromechanical properties of articular cartilage after freeze–thaw cycle may cause canine femoral cartilage to be more susceptible to indentation and damage.…”

Synchrotron MicroCT Reveals the Potential of the Dual Contrast Technique for Quantitative Assessment of Human Articular Cartilage Composition

“…Furthermore, the samples were frozen and thawed three times before the synchrotron microCT experiment, which may have caused some degeneration. However, this degeneration can be assumed to be similar between the samples and with no major effect on biomechanical properties …”

“…However, this degeneration can be assumed to be similar between the samples and with no major effect on biomechanical properties. [43][44][45] In vivo, the diffusion of an anionic contrast agent reaches its maximum concentration in the cartilage at 30-60 min after the injection 7 being a result of physiological clearance, while it takes longer for cationic contrast agents (hours; currently the only in vivo report is with rabbit knee joints). 46 The diffusion time and in vitro conditions of the contrast agents are limitations of this laboratory study and for the clinical applicability.…”