Darryl D. D’Lima,1, 3 Nicholai Steklov, 1 Arnie Bergula, 1 Peter C. Chen, 1 Clifford W.
Colwell, 2 and Martin Lotz3
Cartilage Mechanical Properties after Injury
Reference: D’Lima, D. D., Steklov, N., Bergula, A., Chen, P. C., Lotz, M., and Colwell,
C. W., “Cartilage Mechanical Properties after Injury,” Tissue Engineered Medical
Products (TEMPs), ASTM STP 1452, G. L. Picciolo and E. Schutte, Eds., ASTM
International, West Conshohocken, PA, 2003.
Abstract: Cartilage injury often results in matrix degradation and in secondary
osteoarthritis. This study was designed to correlate cell death and matrix degradation
with biomechanical properties of cartilage. Full-thickness mature bovine femoral
articular cartilage was harvested as 5 mm diameter cylindrical disk explants. Explants
were divided into three groups: control, injury, and IL-1. The injury group was subjected
to mechanical compression of 40% strain for five minutes. The IL-1 group was cultured
in media containing 10 ng/mL of IL-1beta. The control group was not injured or exposed
to IL-1beta. Chondrocyte viability, glycosaminoglycan release in media, and equilibrium
creep were measured ten days after injury. A reduction in cell viability was seen after
injury. A significant increase in glycosaminoglycan release and in equilibrium creep
was detected in injured explants and in explants exposed to IL-1beta. A correlation was
also found between the equilibrium creep and glycosaminoglycan content after injury and
IL-1beta stimulation.
Keywords: Chondrocyte, cartilage, injury, viability, biomechanics, biomechanical
properties, elastic modulus, glycosaminoglycan, trauma, cartilage repair, cartilage
degeneration, cartilage lesion.
1
Director, Research Associates, and Senior Bioengineer, respectively, Orthopaedic
Research Laboratories, Scripps Clinic Center for Orthopaedic Research and
Education, 11025 N. Torrey Pines Road, Suite 140, La Jolla, CA 92037.
2
Director, Scripps Clinic Center for Orthopaedic Research and Education, 11025 N.
Torrey Pines Road, Suite 140, La Jolla, CA 92037.
3
Professor, Division of Arthritis Research, Scripps Research Institute, 10550 N. Torrey
Pines Road, MEM161, La Jolla CA 92037.
Introduction
It is well recognized that impact loads can cause cartilage injury resulting in matrix
degradation and in subsequent loss of cartilage tissue. The relationship between injury
and matrix degeneration is readily apparent when the injury involves the mechanical
disruption of the cartilage surface. However, cartilage degradation can also occur in the
absence of gross matrix damage. Cell death has been reported after cartilage injury[1-4]
and can occur in the form of apoptosis[5-9]. A correlation between chondrocyte
apoptosis and osteoarthritis has been proposed[10-12]. Chondrocyte apoptosis has also
been detected at significantly higher levels in osteoarthritic cartilage relative to normal
cartilage in human subjects[12]. However, the link between injury, apoptosis, matrix
loss, and loss of mechanical integrity has not been clearly established. The goal of this
study was to demonstrate that injury-induced chondrocyte death and glycosaminoglycan
release correlated with changes in the mechanical properties of cartilage over time.
Methods
Cartilage Explants
Full-thickness cartilage explants, 5 mm in diameter, were harvested from the
weight-bearing region of the femoral condyles of mature fresh bovine joints using a
dermal punch. The explants were cultured at 37°C, 5% C02, and 100% humidity in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf
serum. Explants were cultured in media for 48 hours before injury to allow
glycosaminoglycan release levels to stabilize.
Experimental Design
Explants were divided into three groups: control, injury, and IL-1. The injury group
was subjected to mechanical injury as described below. The IL-1 group was stimulated
with 10 ng/mL of IL-1beta (interleukin-1 beta). IL-1beta is known to cause matrix
degradation. The control group was not subjected to injury or to IL-1beta stimulation. A
single experiment used cartilage (n = 4 to 6 explants in each group) harvested from two
bovine joints. Each experiment was repeated separately four times.
Mechanical Injury
To simulate mechanical injury, bovine cartilage explants were subjected to 40%
compressive strain in radially unconfined compression for five minutes. The injurious
compression fell within the range of strain values previously reported to induce
apoptosis[6,8]. Explants from the control or IL-1 groups were not mechanically injured.
After injury, explants were cultured for ten days before analysis.
Cell Viability
Cell viability was quantitated by staining with Calcein-AM[13]. Calcein-AM is an
uncharged nonfluorescent esterase substrate which diffuses freely into cells. Viable cells
containing esterase activity convert Calcein-AM to Calcein, which is a charged,
fluorescent green stain, and only retained by cells with an intact plasma membrane.
Propidum iodide was used as a counterstain as it reacts with nuclear DNA to produce a
red fluorescence. Since propidium iodide is not cell membrane permeable, it cannot stain
cells with an intact cell membrane. Cell viability was quantified as the percentage of
fluorescent green cells in the explant section at 48 hours after injury.
Glycosaminoglycan Assay
The concentration of glycosaminoglycans released into the media was measured
using 1,9-Dimethylene Blue (DMMB) to monitor spectrophotometric changes which
occur during the formation of the sulfated GAG dye complex[14]. Explants were
digested in papain and collagenase, and glycosaminoglycan concentration was
determined at ten days after injury.
Mechanical Properties
The elastic modulus of the solid phase was measured on day ten after injury by
allowing the explants to creep under a constant load. Explants were placed in a creep
chamber and were preloaded with a small tare load. An additional 120 gm of constant
load was added and the creep monitored until equilibrium (slope of creep against time
was less than 1x10-6). The equilibrium creep is directly related to the load and to the
elastic modulus (Es) of the cartilage matrix[15]. For the same constant load, the
equilibrium creep is inversely related to Es. The confined compression creep test is more
widely accepted and was initially chosen to measure the aggregate modulus of the
explants. However, injury often changed the shape and the size of the explant, making it
difficult to consistently fit the explant in the confined compression chamber.
Statistical Analysis
Multifactor ANOVA (Analysis of Variance) was used to test differences in results
between experimental groups. Neuman-Keuls pairwise contrasts was used to test for
individual pairwise differences. Linear regression was used to test the association
between glycosaminoglycan content and creep. A p value < 0.05 was used to denote
statistical significance.
Results
Cell Viability
Figure 1 displays the mean chondrocyte viability (with standard deviation bars) for
the different groups, ten days after injury. Cell viability was significantly reduced in the
injury group (p < 0.05) compared with the control and IL-1 group.
Percentage of viable cells
Figure 1 – Mean Chondrocyte Viability at Ten Days after Injury (n = 24, in 4 separate
experiments).
Glycosaminoglycan Release and Content
The concentration of glycosaminoglycan in culture media from control explants
was less than 10 microgm/mL per mg of explant (over ten days of culture). This more
than doubled after injury (19.9 microgm/mL/mg, Figure 2, p < 0.01). An even higher
release of glycosaminoglycan was detected in explants treated with IL-1beta (p < 0.001).
A corresponding decrease was noted in glycosaminoglycan content in injury and
in IL-1beta explants after ten days in culture (Figure 3). This links the
glycosaminoglycan released in media with the glycosaminoglycan content of the explant.
Mechanical Properties
Figure 4 shows the mean equilibrium creep after mechanical testing in unconfined
compression with standard deviation bars. At ten days after injury mean equilibrium
creep was significantly greater in the injured explants and in the explants cultured in IL1, relative to control (p<0.01). Since the equilibrium creep inversely relates to Es, this
suggests a proportionate reduction in Es.
A negative correlation was also found between glycosaminoglycan content and
equilibrium creep in unconfined compression (Figure 5, R2 = 0.3, p = 0.03). This finding
links the biomechanical properties with the biochemical changes during the process of
matrix degradation.
Microgm/mg/mL
Microgm/mL of digested explant
Figure 2 – Mean Glycosaminoglycan Released in Media Over Ten Days Post Injury
(n = 24, in 4 separate experiments).
Figure 3 – Mean Glycosaminoglycan Concentration of Explant Ten Days after Injury
(n = 24, in 4 separate experiments).
Percent Creep at Equilibrium
Percent Creep at Equilibrium
Figure 4 – Mean Equilibrium Creep at Ten Days after Injury (n = 16, in 4 separate
experiments).
Glycosaminoglycan Content at Ten Days Post Injury
(microgm/mL)
Figure 5 – Linear Regression of Creep with Glycosaminoglycan Content
Discussion
Cartilage injury is a significant contributor to secondary osteoarthritis. Because the
inciting event can be easily identified, it is easier to document the progression of cartilage
degeneration leading to arthritis compared with primary osteoarthritis or more insidious
forms of secondary osteoarthritis. Cartilage injury models are therefore attractive in
providing insights in the processes of cartilage degeneration and arthritis. This in vitro
model of cartilage injury allows for carefully controlled evaluation of cellular,
biochemical, and biomechanical events that may be a significant part of the posttraumatic
degenerative process.
In this model, cell viability, matrix loss, and biomechanical strength were found to
correlate with each other after injury. Analysis of matrix constituents can serve as
valuable surrogate markers of cartilage damage. However, the primary function of
articular cartilage is biomechanical support. Hence, the evaluation of biomechanical
properties is more important in the context of cartilage degradation and repair. Previous
reports have found significant correlations between the elastic modulus of cartilage and
the biochemical composition such as glycosaminoglycan and collagen content[16,17].
Patwari et al. reported increased swelling of injured calf cartilage explants when placed in
hypotonic sodium chloride solution[18]. Cartilage swelling is an estimate of damage to
the collagen network. Loening et al. also reported a significant reduction in equilibrium
modulus of injured calf cartilage explants when tested in unconfined compression
immediately after injury[6]. The authors attributed this to damage to the collagen
network. The present study was also able to correlate the equilibrium creep (which relates
inversely to elastic modulus) with the glycosaminoglycan concentration of cartilage
explants. This further served to validate surrogate markers of cartilage injury (such as
glycosaminoglycan release in media).
This study demonstrated a pattern of cartilage degradation after injury similar to,
although less severe than, that seen with IL-1 stimulation. This provides indirect
evidence that the posttraumatic degradation may have a significant biochemical
component. Other studies have also suggested this by reporting MMP-3 (stromelysin-1)
release after similar injurious compression[18]. MMP-3 levels in synovial fluid have
been reported to rise after clinical joint injury[19]. MMP-3 is known to cause
degradation of glycosaminoglycan and collagen and therefore can affect both the
aggregate modulus and the tensile strength of cartilage.
In early osteoarthritis, there may sometimes be increased synthesis of
glycosaminoglycan[20]. However, this has been shown to be associated with decreased
retention of newly synthesized glycosaminoglycan in matrix[21]. This may be attributed
to decreased synthesis of link protein, which may have reduced the formation of large
aggregates[22].
The consequences of cell death are also clinically relevant. There is a constant
turnover of the macromolecules (predominantly glycosaminoglycan and type II collagen)
that constitute cartilage matrix. Chondrocytes are the sole source of the components of
these macromolecules and are therefore primarily responsible for the maintenance,
repair, and remodeling of matrix. In mature articular cartilage, a limited pool of
chondrocytes is available for this maintenance, and therefore any reduction in this finite
pool of cells can result in reduction of net synthesis (leading to progressive degradation).
Coupled with the initial matrix damage, a significant loss in cell viability can lead
to accelerated degradation. This opens a novel therapeutic approach through
cytoprotection. Preliminary studies suggest that chondrocyte apoptosis can be prevented
resulting in increased cell viability after injury[8,23]. Whether increased cell viability
results in enhanced matrix synthesis, reduction in cartilage degeneration, or improved
repair has yet to be demonstrated. A model of cartilage injury that permits accurate
analysis of the cellular, biochemical, and biomechanical properties of cartilage can be
extremely useful in evaluating such newer therapies.
Acknowledgments
This study was funded in part by OREF Grant #98-052, NIH Grant #AG07996-12,
the ALSAM Foundation, and the Skaggs Institute for Research.
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