Special Poster Session 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

Freezing-Induced Interlamellar Mechanical Changes in Degenerated Annulus Fibrosus (115412)

Manmeet S Dhiman 1 2 , Mohammed A Salaam 1 2 , Taylor J Bader 2 3 , Paul T Salo 2 4 , David A Hart 2 4 5 , Ganesh Swamy 2 4 , Neil A Duncan 2 6
  1. Biomedical Engineering, University of Calgary, Calgary, Alberta, Canada
  2. McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, Alberta, Canada
  3. Medical Sciences, University of Calgary, Calgary, Alberta, Canada
  4. Surgery, Cumming School of Medicine , University of Calgary, Calgary, AB, Canada
  5. Kinesiology, University of Calgary, Calgary, AB, Canada
  6. Civil Engineering, University of Calgary, Calgary, AB, Canada

INTRODUCTION

A comprehensive assessment of the biomechanical properties of human annulus fibrosus (AF) is important to better understand lumbar intervertebral disc (IVD) degeneration. However, the majority of the mechanical testing is performed using frozen tissue as this makes testing convenient [1], [2], and as there is an assumption that frozen AF adequately represents the mechanical properties of fresh AF [3]. The impact of freezing on the interlamellar properties of the degenerated AF has not been fully explored. We hypothesize that freezing will decrease the interlamellar properties of the degenerated AF tissue compared to the fresh tissue.

METHODS

We tested three cohorts: First, paired AF samples from young bovine tails served as healthy, non-degenerated control tissues. Second, we tested unpaired fresh and frozen degenerated human AF samples. Finally, we tested paired degenerated human AF samples that were large enough to be divided into two equal segments. Fresh human AF tissue from patients undergoing surgery for degenerated discs were collected. All fresh human tissue was tested within 1-hour post-surgery, while fresh bovine tissue was tested within 4 hours post-mortem. All frozen tissue was placed in the freezer at -20°C for 3 weeks before testing. Frozen tissue was thawed for 1 hour until they reached room temperature before testing. A peel test was performed to measure the interlamellar mechanical properties of the AF [4]. A two-tailed paired sample t-test was used to evaluate differences between fresh and frozen paired groups, while a two-tailed unpaired t-test was applied to compare fresh and frozen unpaired degenerated human samples.

RESULTS

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There were no significant differences for any parameter between fresh and frozen tissue in the paired bovine comparison. However, frozen samples from the unpaired degenerated human group showed a significant decrease in all interlamellar parameters compared to the fresh tissue. In the paired human degenerated samples, the frozen tissue had statistically reduced Standard Deviation of the Peel Region compared to fresh tissue and showed a decreasing trend for Peel Stiffness, Peel Strength, and Peel Toughness.

DISCUSSION

The decreased interlamellar parameters for the frozen degenerated samples (both unpaired and paired) showed that freezing at -20°C increases the AF susceptibility to delamination compared to the immediate testing of fresh tissue. However, this was not observed with the healthy bovine samples, possibly due to the decreased integrity between the lamellae in the degenerating AF [5]. Ice crystals may form and induce further damage to interlamellar cross-bridges, increasing the ease of delamination. However, histological inspection is required to confirm this hypothesis. When performing mechanical tests on tissue samples, careful consideration must be given to the techniques used for freezing, the specific pathology of the tissue, and the test methodology to ensure accurate results for fresh and frozen samples. It is possible that existing literature values of AF mechanical properties in degenerative AF are not accurate, and lower than in vivo values.  The mechanical changes in AF tissue resulting from degeneration may be best tested as fresh as possible for insights into potential mechanical differences in patients with different clinical phenotypes.

  1. E. C. Bass, N. A. Duncan, J. S. Hariharan, J. Dusick, U. H. Bueff, and J. C. Lotz, “Frozen Storage Affects the Compressive Creep Behavior of the Porcine Intervertebral Disc,” Spine, vol. 22, no. 24, p. 2867, Dec. 1997
  2. J. L. Calvo-Gallego, M. S. Commisso, J. Domínguez, E. Tanaka, and J. Martínez-Reina, “Effect of freezing storage time on the elastic and viscous properties of the porcine TMJ disc,” Journal of the Mechanical Behavior of Biomedical Materials, vol. 71, pp. 314–319, Jul. 2017, doi: 10.1016/j.jmbbm.2017.03.035.
  3. M. Hongo et al., “Effect of multiple freeze–thaw cycles on intervertebral dynamic motion characteristics in the porcine lumbar spine,” Journal of Biomechanics, vol. 41, no. 4, pp. 916–920, Jan. 2008, doi: 10.1016/j.jbiomech.2007.11.003.
  4. D. E. Gregory, W. C. Bae, R. L. Sah, and K. Masuda, “Anular delamination strength of human lumbar intervertebral disc,” Eur Spine J, vol. 21, no. 9, pp. 1716–1723, Sep. 2012, doi: 10.1007/s00586-012-2308-x.
  5. D. E. Gregory, W. C. Bae, R. L. Sah, and K. Masuda, “Disc degeneration reduces the delamination strength of the annulus fibrosus in the rabbit annular disc puncture model,” The Spine Journal, vol. 14, no. 7, pp. 1265–1271, Jul. 2014, doi: 10.1016/j.spinee.2013.07.489.