Darryl D. D’Lima,1 Arnie Bergula, 1 Peter C. Chen, 1 Clifford W. Colwell, 2 and Martin
Lotz3
Age Related Differences in Chondrocyte Viability and Biosynthetic Response to
Mechanical Injury
Reference: D’Lima, D. D., Bergula, A., Chen, P. C., Colwell, C. W., Lotz, M., “Age
Related Differences in Chondrocyte Viability and Biosynthetic Response to
Mechanical Injury,” Tissue Engineered Medical Products (TEMPs), ASTM STP 1452,
G. L. Picciolo and E. Schutte, Eds., ASTM International, West Conshohocken, PA, 2003.
Abstract: Mechanical trauma has been shown to cause chondrocyte death. The response
of the surviving cells has not been fully characterized especially with regards to aging.
This study investigates the response to injury in aging chondrocytes. Human articular
chondrocytes from younger and older donor knees were cultured in agarose gel disks for
three weeks. Disks were submitted to a brief 30% compressive insult (injured), or
cultured in IL-1beta (IL-1), or served as controls. Glycosaminoglycan biosynthesis was
measured by radiolabeled sulfate (35SO4) uptake 48 hours after injury. Chondrocytes
from the older group synthesized less glycosaminoglycan as measured by 35SO4 uptake.
This ranged from a 22% to 61% reduction relative to the younger group. After injury, a
further decline in glycosaminoglycan synthesis was noted in both older and younger
groups. However, the decline in glycosaminoglycan synthesis was more marked in the
older group. While mechanical injury results in chondrocyte death, the surviving cells
exhibit the effect of injury by reduced biosynthesis and increased loss of matrix. This
suggests that the impact of mechanical injury may progress beyond the traumatic event.
With age, fewer cells may survive with a further decrease in biosynthetic response. This
has implications in the repair response and may provide insights in the development of
chondroprotective measures.
Keywords: Chondrocyte, cartilage, injury, aging, viability, biosynthesis,
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 &
Education, 11025 N. Torrey Pines Road, Suite 140, La Jolla, CA, 92037.
2
Director, Scripps Clinic Center for Orthopaedic Research & 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, MSM161, La Jolla, CA, 92037.
Introduction
Aging is one of the most significant risk factors associated with cartilage degeneration
and osteoarthritis. Studies have shown significant cellular, biochemical, and
biomechanical changes in articular cartilage with aging[1-5]. Matrix synthesis has also
been found to be reduced in cartilage explants from older subjects in rabbits, horses, and
humans[6-10]. However, the cellular responses of aging cartilage to injury have not been
clearly defined. This study compared deoxyribose nucleic acid (DNA) and
glycosaminoglycan synthesis in chondrocytes from younger and older donors and
evaluated the response to injury.
Methods
Agarose-Chondrocyte Constructs
Cartilage that appeared normal to visual inspection was harvested from the articular
cartilage of knee joints (femoral condyles and tibial plateaus) of fresh cadaver donors
(within 72 hours of death). The cartilage was diced and digested with pronase and
collagenase to release the chondrocytes. Chondrocytes were resuspended at a density of
2 million cells/mL and mixed with an equal volume of 6% low gelling temperature
agarose (Fluka 05073, Sigma-Aldrich, Milwaukee, WI). This final suspension (1 million
cells/mL in 3% agarose) was gelled at 4º C for 20 minutes in a custom chamber to form a
2 mm thick slab. Cylindrical disks 5 mm in diameter and 2 mm thick were cored from
this gel slab using a dermal punch. Approximately 40 explants were obtained from each
donor. Each cell-agarose disk construct was then cultured in DMEM (Dulbecco’s
modified Eagle’s medium) supplemented with 10% fetal calf serum for 21 days before
being subjected to injury. Each explant was cultured in 1 mL of media, with media
changed twice a week.
Experimental Design
Donors were divided into two groups: younger and older. The younger group (n = 5)
contained donors who were less than 35 years of age at the time of death. The older
group contained donors (n = 5) who were greater than 50 years of age at the time of death
and who did not demonstrate significant evidence of degeneration or osteoarthritis (no
full thickness lesion). Within each group, explants were divided into control, injury, and
IL-1 experimental subgroups. The injury subgroup was subjected to transient mechanical
injury (see below). The IL-1 subgroup was stimulated with 10 ng/mL of IL-1beta
(interleukin-1 beta). The control subgroup was not subjected to injury or IL-1beta
stimulation.
Mechanical Injury
Cell-agarose constructs from the injury group were placed inside the injury chamber
and subjected to a 30% unconfined compressive strain for 500 msec using an
electromagnetic actuator with a resolution of 1 micron (SMAC, Carlsbad, CA). The
magnitude and duration of loading was chosen from previous experiments that produced
a consistent cell death of about 30%. Cell-agarose constructs from the control group
were placed in the same chamber but were not subjected to mechanical compression.
Biochemical Analysis
After injury, all groups were incubated for 48 hours in the presence of [35S]-sodium
sulfate (35SO4) at a concentration of 20 microcuries/mL (Perkin Elmer, Boston, MA). At
the end of 48 hours, the level of 35SO4 newly incorporated in the cell-agarose constructs
was measured. Cell-agarose constructs were washed five times in ice-cold phosphatebuffered saline (10 minutes per wash), blotted dry, then digested at room temperature for
30 minutes by the addition of Buffer RLT (10 microL per mg, Qiagen, Valencia, CA).
The digest was homogenized with Ecoscint (3 mL per sample, National Diagnostics,
Manville, NJ) and was placed in a scintillation counter for measurement of total
radioactivity. 35SO4 incorporation was normalized to explant weight and expressed as
coulter counts per minute per mg explant. This served to quantify newly synthesized
glycosaminoglycans. Cell-agarose constructs were also incubated in media containing
tritiated thymidine (3H -Thymidine) at various time points during the pre-injury culture.
The level of 3H -Thymidine in the cell-agarose construct was measured in a similar
manner to that described for 35SO4. 3H -Thymidine levels served to quantitate DNA
synthesis rates.
Statistical Analysis
Multifactor ANOVA (Analysis of Variance) was used to test differences between
donor groups and experimental groups. Neuman-Keuls pairwise contrasts was used to
test for individual pairwise differences. A p value < 0.05 was assumed to denote
statistical significance.
Results
DNA Synthesis
Coulter Counts per Minute
A consistent decrease in 3H -Thymidine uptake was seen in older compared with
younger group over the three weeks in culture (Figure 1, p<0.001). Cell-agarose
constructs were incubated in media containing 3H -Thymidine for 48 hours at each time
point. Data shown are means with standard deviation bars from three separate
experiments (n = 6 explants in each experiment, from each donor). This suggests a
significant reduction in DNA synthesis with age even before injury.
Time in Culture
Figure 1 – DNA Synthesis Over Three Weeks
Glycosaminoglycan Synthesis
35
SO4 uptake compared to controls
Cell-agarose constructs from the older group also synthesized less glycosaminoglycan
as measured by 35SO4 uptake. This ranged from a 22% to 61% reduction relative to the
younger group, p<0.01. After injury, a further decline in glycosaminoglycan synthesis
was noted (Figure 2) in both older and younger groups. Data shown are means with
standard deviation bars from three separate experiments (n = 6 in each experiment).
Glycosaminoglycan synthesis is normalized to uninjured control constructs. However,
the decline in glycosaminoglycan synthesis after injury was more marked in the older
group compared to the younger group (p < 0.05). Both older and younger groups were
sensitive to IL-1 stimulation. This was primarily used as a positive control, however, it
of interest to note that IL-1 affected older chondrocytes to a greater degree (p<0.01).
Experimental Groups
Figure 2 – Glycosaminoglycan Synthesis After Injury
Discussion
A strong linear correlation exists between aging and the incidence of significant
cartilage degeneration and osteoarthritis. This has been attributed to reduced cellularity,
reduction in matrix synthesis, and altered matrix biochemical and biomechanical
properties. The response of older chondrocytes to injury, however, has not been fully
defined. The agarose gel culture system provides a controlled means of testing altered
cellular response by normalizing cell density and removing any effect of a pre-existing
altered matrix.
The reduced 3H-Thymidine uptake over three weeks in culture suggests less DNA
synthesis in older chondrocytes. The starting cell density was the same for younger and
older explants in this study (1 million cells per mL of agarose gel). Verbruggen et al.
reported a mean 13.6% decrease in DNA content in the first week of culture (human
chondrocytes cultured in 1.5% agarose) but could not correlate the reduced DNA content
with age[11]. DeGroot et al. have shown that the age-related decrease in
glycosaminoglycan synthesis was not related to a decrease in cell density[8]. However, a
possibility exists that cell density may have changed over time in the cell- agarose
constructs containing older chondrocytes. Therefore the 35SO4 uptake was compared
with controls for each group. This corrected for any systematic change in cell density
between groups before injury. A decrease in 35SO4 uptake was also noted in older
chondrocytes. This was consistent with previous reports of decreased glycosaminoglycan
synthesis with increasing age[8,11]. In fact, Verbruggen et al. reported an exponential
regression of 35SO4 uptake with age, which suggests that 35SO4 uptake using
chondrocytes from 45- and 69-year old subjects is 50% and 25%, respectively, of the
uptake using chondroyctes from a 20-year old subject[11].
Injury caused a reduction in glycosaminoglycan synthesis. This is consistent with the
cell death that has been reported in response to this form of injury[12-15]. Older
chondrocytes may also be more susceptible to cell death after injury, which can lead to
reduced glycosaminoglycan synthesis.
IL-1beta is a known inhibitor of glycosaminoglycan synthesis and was used as a
positive control. Glycosaminoglycan synthesis following IL-l stimulation was even
lower than that following injury. Again, older chondrocytes appeared to be more
susceptible to IL-1beta effects. Aging chondrocytes have a reduced capacity to assemble
a proteoglycan-rich matrix[10,11]. This has been attributed to an insufficient synthesis of
link protein necessary for the stabilizaton of aggregates. Therefore, matrix loss after
injury can be affected not only by a reduction in rate of newly synthesized
glycosaminoglycans but also by the decreased ability to retain these newly synthesized
glycosaminoglycans in matrix.
A recent study suggested an altered response to mechanical stimulation in
chondrocytes from osteoarthritic cartilage[4]. Chondrocytes from normal human
cartilage expressed higher levels of aggrecan mRNA and lower levels of matrix
metalloproteinase-3 (MMP3) after mechanical stimulation. However, chondrocytes from
osteoarthritic cartilage did not show these changes, which suggested altered
mechanotransduction and signalling. The older chondrocytes tested in this study were
obtained from donors without significant cartilage degneration. However, it is entirely
possible that some of the donors had early degeneration, and that the reduction in matrix
synthesis may be a precursor to or a consequence of early disease.
These data suggest that older cartilage may be more susceptible to the deleterious
effects of injury which may partly explain the increased incidence of cartilage lesions
with age. In addition, current surgical techniques aimed at cartilage repair that use
autologous cells or grafts may not be as successful in older patients since even the host
cartilage may demonstrate a compromised repair response.
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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