The effects of freeze-thaw cycle and application of proteolysis inhibitors and cryopreservants on biomechanical properties of articular cartilage

The Effects of Freeze-Thaw Cycle and Application of Proteolysis Inhibitors and Cryopreservants on Biomechanical
Properties of Articular Cartilage
+1Hirviniemi, M; 1Lammi, MJ; 1Jurvelin, JS; 1,2Töyräs, J +1University of Eastern Finland, Kuopio, 2Kuopio University Hospital, Kuopio, Finland INTRODUCTION
at room temperature, a biomechanical stress-test was performed with the For practical reasons, articular cartilage (AC) samples are often frozen for further analyses. This fact has raised questions, whether biological After the second biomechanical stress-relaxation test, the water and biomechanical properties of AC change during a freeze-thaw cycle.
content of the samples in groups 2, 3 and 4 was analyzed. The samples The effects of freezing and thawing on articular cartilage have been were cut vertically in half, and subchondral bone was removed with a studied by several research groups (e.g. [1, 2]). Many of these studies, razor blade. The wet weight of each sample (half of the articular however, have focused on detection of glycosaminoglycan (GAG) loss, cartilage plug) was measured. Subsequently, the samples were freeze- which may happen due to possible collagen network damage during dried (Christ Alpha 1 -2, B Braun Biotech Inc., Allentown, USA). After thawing, and enzymatic degradation of proteoglycan (PG) core protein lyophilization (18 - 68 h), the dry weight of the samples was measured.
Young’s modulus determined in indentation geometry was calculated PGs have significant effect on cartilage permeability and equilibrium from the equilibrium response. Dynamic modulus was determined from modulus. Thus, the loss of PGs has significant effect on cartilage peak-to-peak values, of the second compressive step, using the functional properties. Zheng et al. [1] reported a significant GAG loss stress/strain information. [5] Statistical analyses were conducted using due to freezing without cryoprotective agents (CPAs).
the SPSS Statistics software (SPSS Inc. Chicago, USA). A Wilcoxon The aim of this study was to investigate the effects of freezing and signed ranks test was performed to test differences between parameter thawing on biomechanical properties of AC. The effectiveness of values before and after the freeze-thaw cycle.
protecting cryopreserved cartilage was our main interest. For thispurpose, a biomechanical stress-relaxation test was conducted before 21- 24 h freezing, and again after 18 h storage at room temperature.
There were no significant differences in water content between thegroups (mean values 77.7-78.1 %). A slight trend towards lower dynamic modulus was seen in all sample groups (Table 1), but it was Intact, mature bovine knee joints (age 18 to 24 months) were obtained statistically significant only in samples of group 3, which were kept in from a local slaughterhouse (Atria Oyj, Kuopio, Finland). Osteochondral the presence of enzyme inhibitors and antibiotics. A larger amount of disk (d = 25.4 mm) was prepared from each patella (PAT) within 12 h samples is needed to find out whether this unexpected finding will post mortem. Four 6 mm diameter plugs were then detached from each remain, since the equilibrium modulus in the same group was slightly disk (n = 9) using a biopsy punch (Figure 1).
Table 1. Mean values ± SD of biomechanical parameters of the
samples before and after the freeze-thaw cycle.

Figure 1. Sample preparation
1 = Control group; 2 = Samples in PBS bath; 3 = Samples in PBS bath with enzyme inhibitors The samples were divided into four groups: control group (1) and and antibiotics; 4 = Samples in PBS bath with enzyme inhibitors and antibiotics, freezing in 30% DMSO; BF = Before freezing; AF = After freezing; three experimental groups (2, 3 and 4). Each plug in the groups 1, 3 and4 was immersed in 5 ml of Dulbecco’s phosphate buffered saline (PBS, DISCUSSION
pH = 7.4, Euroclone, Siziano, Italy) containing inhibitors of proteolytic In this study we investigated the effects of freeze-thaw cycle on articular enzymes (5 mM benzamide and 5 mM EDTA) and antibiotics (100 U/ml cartilage mechanical properties. Furthermore, the effectiveness of the penicillin and 100 mg/ml streptomycin). Each plug in the group 2 was use of CPAs and inhibitors of proteolytic enzymes was studied.
immersed in 5 ml of PBS without enzyme inhibitors or antibiotics.
Based on our results, a freeze-thaw cycle does not appear to Subsequently, the thicknesses of the cartilage plugs were measured significantly affect the biomechanical properties of articular cartilage.
using a stereomicroscope (Nikon SMZ-10, Nikon Co., Tokyo, Japan) Zheng et al. [1] reported a significant GAG loss caused by freezing and thawing on articular cartilage without the use of DMSO during the Biomechanical properties of the samples were measured using a freezing. Such a loss can be expected to affect also the biomechanical custom made computer controlled material testing device [3]. A properties of the AC remarkably, which was not observed in this study.
stepwise stress-relaxation indentation (three 5% compressions with 30 This difference in the results could be due to the small number of min relaxation time) was done with a plane-ended indenter (d = 1.01) at samples (n = 9) used in our study. The only statistically significant room temperature. At all times (also during biomechanical testing and difference was a slight decrease in the dynamic moduli in samples freezing) the samples were kept in same solution bath in groups 1, 2 and immersed in PBS containing proteolytic inhibitors and antibiotics.
3. Before freezing, samples in group 4 were immersed into 30% Logically, this group should have shown the smallest differences due to dimethylsulfoxide (DMSO)/saline solution bath, containing same freez-thaw cycle. Thus, collection of larger amount of samples and their concentration of enzyme inhibitors and antibiotics as the PBS solution, further biochemical and quantitative histology analyses, which are used for at least 1 h 30 min [4]. This was done to protect cartilage from to evaluate the proteoglycan content and integrity of the collagen possible damages caused by ice formation during freezing [1].
network, are needed to be able to evaluate the effects of freeze-thaw In the group 1 (control), the water content analysis of the samples cycle on the articular cartilage structure.
was performed immediately after biomechanical analyses, while thesamples in groups 2, 3 and 4 were frozen for 21-24 h at -21°C. Freezing REFERENCES
was followed by thawing and storage at room temperature (22-33°C) for [1] Zheng, M. et al., Magn Reson Imaging 27, 648-655, 2008; [2] Laour, 18 h. After approximately 1 h of thawing, the samples in group 4 were L. et al., Cryobiology 54, 36-43, 2007; [3] Töyräs, J., et al., Phys Med moved back to PBS saline containing 5 mM benzamide, 5 mM EDTA Biol 44, 2723 – 2733, 1999 [4] Mukherjee, B.S., et al., Osteoarthritis and 100 U/ml penicillin and 100 mg/ml streptomycin. After 18 h storage Cartilage, 16, 1379-1386, 2008 [5] Hayes, W. C., et al., J Biomech 5,541-551, 1972 Poster No. 2141 • ORS 2011 Annual Meeting


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