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Technical Brief

Effects of Cryopreservation on the Depth-Dependent Elastic Modulus in Articular Cartilage and Implications for Osteochondral Grafting

[+] Author and Article Information
David Kahn

Department of Physics and Center for
Biomedical Research,
Oakland University,
2200 N Squirrel Road,
Rochester, MI 48309
e-mail: djkahn@oakland.edu

Clifford Les

Department of Physics,
Oakland University,
2200 N Squirrel Road,
Rochester, MI 48309
e-mail: les@oakland.edu

Yang Xia

Department of Physics and Center for
Biomedical Research,
Oakland University,
2200 N Squirrel Road,
Rochester, MI 48309
e-mail: xia@oakland.edu

Manuscript received August 22, 2014; final manuscript received November 10, 2014; accepted manuscript posted November 20, 2014; published online March 6, 2015. Assoc. Editor: Ram Devireddy.

J Biomech Eng 137(5), 054502 (May 01, 2015) (6 pages) Paper No: BIO-14-1413; doi: 10.1115/1.4029182 History: Received August 22, 2014; Revised November 10, 2014; Online March 06, 2015

Cryopreservation of articular cartilage is often used in storage of experimental samples and osteochondral grafts, but the depth-dependence and concentration of glycosaminoglycan (GAG) are significantly altered when cryogenically stored without a cryoprotectant, which will reduce cartilage stiffness and affect osteochondral graft function and long-term viability. This study investigates our ability to detect changes due to cryopreservation in the depth-dependent elastic modulus of osteochondral samples. Using a direct-visualization method requiring minimal histological alterations, unconfined stepwise stress relaxation tests were performed on four fresh (never frozen) and three cryopreserved (−20 °C) canine humeral head osteochondral slices 125 ± 5 μm thick. Applied force was measured and tissue images were taken at the end of each relaxation phase using a 4× objective. Intratissue displacements were calculated by tracking chondrocytes through consecutive images for various intratissue depths. The depth-dependent elastic modulus was compared between fresh and cryopreserved tissue for same-depth ranges using analysis of variance (ANOVA) with Tukey post-test with a 95% confidence interval. Cryopreservation was found to significantly alter the force–displacement profile and reduce the depth-dependent modulus of articular cartilage. Excessive collagen fiber folding occurred at 40–60% relative depth, producing a “black line” in cryopreserved tissue. Force–displacement curves exhibited elongated toe-region in cryopreserved tissue while fresh tissue had nonmeasurable toe-region. Statistical analysis showed significant reduction in the elastic modulus and GAG concentration throughout the tissue between same-depth ranges. This method of cryopreservation significantly reduces the depth-dependent modulus of canine humeral osteochondral samples.

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Figures

Grahic Jump Location
Fig. 1

A representative diagram of the experimental setup (not to scale). Not indicated is the clear glass window (directly below the tissue cassette) or the 154 mM bathing solution (surrounding the tissue). The direction of compression is along the axial direction (horizontal in the figure).

Grahic Jump Location
Fig. 2

Representative images used in the calculation of chondrocyte displacement and elastic modulus for (a) fresh tissue and (b) cryopreserved tissue. AS indicates the articular surface and TM indicates the tidemark. The white circles connected by arrows show the displacements of chondrocytes through several strains (%). The thick white arrows indicate the formation of a black line.

Grahic Jump Location
Fig. 3

Chondrocyte force–displacement curves for (a) fresh and (b) cryopreserved tissue at several relative depths (%). Each curve is illustrative of one chondrocyte from one sample tracked through consecutive images. Y-axis scales differ due to the significant reduction of cryopreserved tissue stiffness. Error bars include load sensor error, multimeter error, and force fluctuations at equilibrium.

Grahic Jump Location
Fig. 4

Depth-dependent profiles of elastic modulus and GAG concentration. Depth is plotted on a relative scale where 0–10% indicates the SZ and all other percentage ranges indicate relative intratissue depth. Values and error bars are mean ± S.D. and modulus is fit exponentially through 80% relative depth as (a) Ef = 2.57 + 0.25e0.45x and (b) Ec = 0.90 + 0.02e0.71x, where x is relative depth, and Ef and Ec are the elastic modulus of fresh and cryopreserved cartilage, respectively.

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