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
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
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.  reported a significant GAG loss
stress/strain information.  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
= 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
= 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,
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.
 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 . 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
= 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 . 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 .
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
was followed by thawing and storage at room temperature (22-33°C) for
 Zheng, M. et al.
, Magn Reson Imaging
27, 648-655, 2008;  Laour,
18 h. After approximately 1 h of thawing, the samples in group 4 were
L. et al.
54, 36-43, 2007;  Töyräs, J., et al.
, Phys Med
moved back to PBS saline containing 5 mM benzamide, 5 mM EDTA
44, 2723 – 2733, 1999  Mukherjee, B.S., et al.
and 100 U/ml penicillin and 100 mg/ml streptomycin. After 18 h storage
16, 1379-1386, 2008  Hayes, W. C., et al.
, J Biomech
Poster No. 2141 • ORS 2011 Annual Meeting
• Project 95.001 "Onderzoek naar de oorzaken en het trombose risico van de tot nu toe onbegrepen vormen van APC-resistentie" • Project 97.003 "Karakterisatie van bloedplaatjes exosomen" • Project 98.001 "Effects on hemostasis and fibrinolysis of prostagens used in second and third generation oral contraceptives" • Project 99.001 "Verlengde werkingsduur en ef
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