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CHONDROCYTE DEATH PRECEDES STRUCTURAL DAMAGE IN BLUNT IMPACT TRAUMA
+*Duda, G N.; *Loh, L; *Eilers, M; *Hoffmann, J E.; *Schaser, K
+*Research Laboratory, Trauma and Reconstructive Surgery. Charité, Campus Virchow-Klinikum, Humboldt University of Berlin, Augustenburger Platz 1, D-13353
Berlin, Germany, +49 30 450 59079, Fax: +49 30 450 59969, georg.duda@charite.de
INTRODUCTION
Joint impact trauma is frequently sustained through accidents which involve
falls from a height or direct blows to the joint. Despite careful early surgical
reconstruction, the development of post-traumatic osteoarthrosis is frequently
a late consequence of joint impact trauma and may lead to disability and
subsequent surgical intervention (i.e. joint arthroplasty, replacement). Links
between single traumatic events and osteoarthrosis have previously been
reported for various clinical cases1,2. However, the cellular pathways
underlying progressive cartilage destruction remain largely unknown.
Previous studies focused on cartilage damage at load levels sufficient to
fracture the underlying bone or lead to macroscopical fissures and fibrillation
of the cartilage layer3. It was the goal of this in vitro investigation to
determine and quantitatively analyze which impact trauma magnitude causes
the immediate cellular respectively structural damage to the cartilage layer.
Understanding the initial cellular damage may help to find the missing link
between the traumatic impact and structural damage of cartilage layers in
post-traumatic joint diseases such as trauma-induced osteoarthrosis.
METHODS
12 fresh porcine patellae were harvested from 6 Yucatan minipigs (12-14 m)
within 1h of sacrifice and placed in bovine serum at 37°C to conserve vitality.
During processing, the cartilage was kept moist. Each patellar surface was
divided into quadrants. Using a drop tower device, a mass was dropped from
various heights resulting in different impact energies (0.06, 0.1 and 0.2) onto
one of three quadrants while the remaining quadrant served as control. Impact
force and cartilage deformation were recorded during testing to verify
reproducibility. 4 patellae were stained with India ink to identify surface
disruptions, fixed, freeze fractured and prepared for electron microscopy.
From the remaining patellae, blocks of full-thickness cartilage were removed
from each quadrant. Each block was stained with propidium iodide (PI) as
well as with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT). 10µm cryo-sections were taken from each specimen and
photographed within 2 hours (light microscope, 400x). Each cartilage section
was divided into a deep, intermediate and superficial zone. Functional and
non-functional cells were counted by two indepedent observers. In the MTT
staining, death rate was expressed as a ratio of dead cells to total cell
population in each section with the PI staining serving as control. A Wilcoxon
test was chosen for statistical evaluation.
RESULTS
All impact energies produced a transient indentation on the articular surface,
without disruption or fissures of the surface (India ink). Electron microscopy
unveiled no discernable evidence of structural damage in all specimens and
layers (Fig.1). Equally intense staining signals under normal as well as
fluorescent light allowed to distinguish clearly between functional (MTT) and
non-functional cells (PI). Significant differences among the death rates for
different impact energies were seen for all specimens: Increasing impact
energies led to an increasing number of non-functional cells compared to the
total number of cells within the cartilage layer. This significance was valid for
both, the full thickness cartilage layer as well as for the intermediate and
superficial zones individually (Fig. 2, p < 0.05). In addition, statistical
comparisons unveiled a significantly increased death rate for the superficial
and intermediate cartilage layer compared to the deep one at larger energy
levels (energy > 0.1 J; Fig. 2).
DISCUSSION
In this study, impact trauma magnitudes were identified which led to
immediate cellular damage of the cartilage layer. The results suggest that prior
to structural destruction considerable damage is experienced at the cellular
level even at low energy trauma. These initial trauma-induced cellular
dysfunction may indeed act as a precondition for ongoing tissue damage. This
is of particular importance since sufficient synthesis of cartilage matrix
proteins is directly dependent on tissue viability.
These first results demonstrate that chondrocyte death precedes gross
structural damage in blunt cartilage impact trauma. Findings of the present
study should draw the attention of the orthopedic surgeon to the cellular level,
where chondrocyte viability is compromised in response to trauma, even when
the cartilage clinically appears untouched. Therefore, surgery should also be
directed towards preserving cartilage viability in order to minimize persistent
post-traumatic joint disorders, i.e. osteoarthosis. Furthermore, the unique
approach of this model is to artificially produce cellular damage without gross
structural alterations. Using this model isolated studies on cellular reactions
and post-traumatic changes without gross matrix damage could be performed.
Our data is limited to a non-human species and focuses on a direct posttraumatic phase, excluding regenerative as well as degenerative effects.
Further studies are needed to determine the initial biological response after
low impact trauma to illuminate the processes of post-traumatic joint
degeneration. Finally, an advanced understanding of cartilage damage
processes might in the long term help to intensify early clinical treatment and
thereby reduce the risk of post-traumatic osteoarthrosis.
Fig. 1: Cartilage after 0.2J impact with no signs of macroscopical damage
30
*
% dead
*
*
cells
*
20
10
0
deep
medium
superficial
control 0.06
0.1
0.2
energy [J]
Fig. 2: Death rate increases with impact energy (*p < 0.05)
ACKNOWLEDGEMENT
Gabriele Hardung for the histological and Gudrun Holland for the SEM
analysis; Klaus Dannenberg, for manufacturing the impact device.
REFERENCES
1
Felson, D.T. Epidem.Rev. 1988;10:1-28. 2Jaskulka, R.A. et al., J.Trauma,
1989, 29, 1565-70. 3Repo, R.U. and Finlay, J.B., JBJS, 1977, 59-A, 10681076.
Poster Session - Physical Effects on Cells - VALENCIA D
0646
46th Annual Meeting, Orthopaedic Research Society, March 12-15, 2000, Orlando, Florida
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