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Technical Briefs

# Effects of Refrigeration and Freezing on the Electromechanical and Biomechanical Properties of Articular Cartilage

[+] Author and Article Information

Biomaterials and Cartilage Laboratory, École Polytechnique de Montréal, P. O. Box 6079, Station Centre-ville, Montréal, QC H3C 3A7, Canadaadele.changoor@polymtl.ca

Biomaterials and Cartilage Laboratory, École Polytechnique de Montréal, P. O. Box 6079, Station Centre-ville, Montréal, QC H3C 3A7, Canadaliah.fereydoonzad@polymtl.ca

Alex Yaroshinsky

2345 St. Andrews Way, P.O. Box 546, San Francisco, CA 95249alexy@celladon.net

Michael D. Buschmann1

Biomaterials and Cartilage Laboratory, École Polytechnique de Montréal, P. O. Box 6079, Station Centre-ville, Montréal, QC H3C 3A7, Canadamichael.buschmann@polymtl.ca

1

Corresponding author.

J Biomech Eng 132(6), 064502 (Apr 26, 2010) (6 pages) doi:10.1115/1.4000991 History: Received November 30, 2009; Revised January 10, 2010; Posted January 13, 2010; Published April 26, 2010; Online April 26, 2010

## Abstract

In vitro electromechanical and biomechanical testing of articular cartilage provide critical information about the structure and function of this tissue. Difficulties obtaining fresh tissue and lengthy experimental testing procedures often necessitate a storage protocol, which may adversely affect the functional properties of cartilage. The effects of storage at either $4°C$ for periods of 6 days and 12 days, or during a single freeze-thaw cycle at $−20°C$ were examined in young bovine cartilage. Non-destructive electromechanical measurements and unconfined compression testing on 3 mm diameter disks were used to assess cartilage properties, including the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability $(k)$. Cartilage disks were also examined histologically. Compared with controls, significant decreases in SPI (to $32.3±5.5%$ of control values, $p<0.001$), Ef (to $3.1±41.3%$ of control values, $p=0.046$), Em (to $6.4±8.5%$ of control values, $p<0.0001$), and an increase in $k$ (to $2676.7±2562.0%$ of control values, $p=0.004$) were observed at day 12 of refrigeration at $4°C$, but no significant changes were detected at day 6. A trend toward detecting a decrease in SPI (to $94.2±6.2%$ of control values, $p=0.083$) was identified following a single freeze-thaw cycle, but no detectable changes were observed for any biomechanical parameters. All numbers are $mean±95%$ confidence interval. These results indicate that fresh cartilage can be stored in a humid chamber at $4°C$ for a maximum of 6 days with no detrimental effects to cartilage electromechanical and biomechanical properties, while one freeze-thaw cycle produces minimal deterioration of biomechanical and electromechanical properties. A comparison to literature suggested that particular attention should be paid to the manner in which specimens are thawed after freezing, specifically by minimizing thawing time at higher temperatures.

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## Figures

Figure 1

Schematic of the experimental design. (a) Medial femoral condyle used for the refrigeration experiment. (b) Lateral femoral condyle used for the freezing experiment. Four regions per condyle were marked with small diameter pins (×). Black dots (●) represent sites where electromechanical measurements were made (eight around each pin). Subsequently, osteochondral cores were harvested from the medial femoral condyle on day 1 for control sites (open circles, ○) and after storage at 4°C for 6 days (squares, ◻) and 12 days (diamonds, ◇), and from the lateral femoral condyle at day 1 for control sites (open circles, ○) and at day 7 (triangles, △) after a single freeze-thaw cycle at −20°C.

Figure 2

Sequential electromechanical measurements (mean±standard deviation). (a) Sites (n=20) on the medial femoral condyle where SPI was obtained prior to storage and at two time points during refrigeration at 4°C. A statistically significant decrease (p<0.001) between days 1 and 12 was detected using a repeated measures ANOVA and Tukey’s post-hoc test. (b) Sites (n=23) on the lateral femoral condyle where SPI was obtained before and after storage at −20°C. Repeated measures ANOVA identified a tendency (p=0.083) for SPI to decrease following 7 days of frozen storage. Note that technical issues during osteochondral core extraction for biomechanical testing reduced the number of sites available for sequential electromechanical testing by one.

Figure 3

(a) Cartilage thickness, (b) matrix modulus, (c) fibril modulus, (d) permeability, and (e) SPI measurements at sites subsequently removed for biomechanical testing. All values are mean±standard deviation. The stars ( ∗) indicate statistically significant differences between day 12 and day 1 controls obtained using t-tests.

Figure 4

Representative Safranin-O/Fast Green stained sections from samples stored at 4°C. ((a) and (d)) Day 1, ((b) and (e)) day 6, and ((c) and (f)) day 12. In (a)–(c) the scale bar is 1 mm. In (d)–(f) the scale bar is 250 μm.

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