Abstract
Background: Osteochondral grafts, used to treat chondral and
osteochondral defects, require high insertional forces that may affect the
viability of chondrocytes in the graft. The objectives of this study were to
(1) measure the loading impact during insertion of osteochondral grafts, (2)
evaluate the effect of insertional loading on chondrocyte viability, and (3)
assess this effect on chondrocyte apoptosis and activation of caspase-3.
Methods: The distal parts of twelve fresh femora from six adult
human cadavers were harvested within seventy-two hours after the death of the
donor. From each femur, four 15-mm-diameter cylindrical osteochondral grafts
were isolated; two of these grafts (a total of twenty-four grafts in the
study) were transplanted with standard impact insertion into recipient sockets
in the other condyle of the ipsilateral femur. The other two grafts served as
unloaded controls. Loads were measured during the insertion of ten of the
twenty-four transplanted grafts. Full-thickness cartilage disks were then
removed from the grafts, incubated for up to forty-eight hours, and analyzed
for cell viability, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP
nick end labeling)-positive reactivity, and caspase-3 activation, each as a
function of the depth from the articular surface.
Results: The insertion of an osteochondral graft was characterized,
on the average (and standard deviation), by 10 ± 4 impacts, each
generating 2.4 ± 0.9 kN of load and 13.3 ± 4.9 MPa of stress for
a duration of 0.57 ± 0.13 ms with a 0.62 ± 0.25 N·s
impulse. Impact insertion increased cell death in the superficial 500 µm to
21% at one hour (p < 0.001) and 47% at forty-eight hours (p < 0.001) and
also increased cell death in deeper layers at forty-eight hours. Some cell
death was due to apoptosis, as indicated by an increase in caspase-3
activation at eight hours (p < 0.01) and TUNEL-positive cells at
forty-eight hours (p < 0.05) in the superficial 500 µm of impacted
cartilage.
Conclusions: Impact insertion of osteochondral grafts generates
damaging loads that cause chondrocyte death, particularly in the superficial
zone, mainly as a result of apoptosis mediated by the activation of
caspases.
Clinical Relevance: Chondrocyte death that occurs during impact
insertion of osteochondral grafts may lead to compromised function.
Understanding the mechanisms and consequences of such impact loading may
provide insights into potential therapeutic interventions, or lead to changes
in the insertion technique, to decrease the cell injury associated with impact
loading.
Osteochondral grafting is sometimes used as a method of treating focal
chondral and osteochondral defects in the femoral condyles and
elsewhere1-5.
Impaction forces are used to insert the osteochondral donor tissue into the
recipient site and to generate a secure
fit6. The effect of
such impaction forces on the viability of the chondrocytes that reside within
the cartilage tissue of the implant has not been studied previously, either in
vivo or in situ, to our knowledge.
Trauma can damage articular cartilage and its chondrocytes. Traumatic
impact on joints in vivo results in chondrocyte death both in
humans7-9
and in experimental
animals10,11.
In vitro, biomechanical factors associated with mechanically induced
chondrocyte death include the loading
rate12-14
and
duration15,16,
contact
stress17-23,
strain
rate24-26,
and overall compression
level21,27.
The duration of an impact can affect the cartilage and chondrocyte response.
Slowly applied loads may initially allow tissue fluid to support the load but
then gradually transfer load to the
matrix28. In
contrast, suddenly applied loads may not allow time for fluid movement and
thus may cause variable degrees of tissue deformation depending on mechanical
constraints9,18.
In classic mechanics, the intensity of an impact is described by the impulse,
defined as the product of load and time, with units of
N·s29.
Unlike accidental or experimental trauma, insertional loading of osteochondral
grafts typically involves multiple impacts rather than a single impact. The
biomechanical characteristics of insertional impacts have not been described
previously, to our knowledge.
The process by which excessive mechanical loading of cartilage can lead to
chondrocyte death may involve
apoptosis21,30-32.
Apoptosis occurs through an ordered sequence of cellular events,
characteristically including activation of a cascade of cysteine-dependent
aspartate-specific proteases
(caspases)11,33.
The regulated degradation of nuclear DNA by a caspase-activated
deoxyribonuclease (CAD) is a hallmark of
apoptosis34. In the
typical sequence of cellular events during apoptosis, caspase activation is an
early event35,
while DNA fragmentation is a late event that occurs in
days21,36.
Active caspase-3, an executioner caspase, facilitates the assembly of CAD into
its active form, leading to the production of DNA
fragments37. In
human cartilage disks subjected to 30% compression, signs of apoptosis may
appear as early as six hours after injury and the percentage of cells
undergoing apoptosis may continue to increase for up to seven days after
injury21.
We hypothesized that the impact loading used to implant osteochondral
grafts leads to chondrocyte death due in part to apoptosis mediated by
activation of caspases. The specific aims of this study were to (1)
characterize the impact load during the insertion of osteochondral grafts, (2)
evaluate the effect of insertional loading on cell viability, and (3) assess
this effect on apoptosis and activation of caspase-3.
Osteochondral Harvest and Grafting
All donor tissue was obtained from University of California-San Diego
Lifesharing (San Diego, California). The distal parts of twelve fresh human
femora were removed en bloc from six human cadavers, four male and two female,
with a mean age (and standard deviation) at the time of death of 53 ± 4
years (range, forty-six to fifty-six years). The specimens were obtained under
sterile conditions within seventy-two hours after the donor's death and were
stored at 4°C in serum-free culture medium for up to seventy-two hours.
Only normal articular cartilage (grade 1A according to the classification
system of Noyes and
Stabler38) was used
in the study. A surgeon who was experienced with the osteochondral grafting
procedure performed the experimental harvest and implant procedures.
A set of four cylindrical osteochondral grafts, 15 mm in diameter with 10
mm of subchondral bone (Fig. 1,
B), was harvested from each femur with use of
osteochondral allograft instruments (Arthrex, Naples, Florida) under
continuous irrigation with lactated Ringer solution in order to minimize
thermal damage to the cartilage. The subchondral bone was subjected to pulse
lavage with lactated Ringer solution to remove marrow elements. From each set
of four grafts, two grafts (one from the trochlea and one from the condyle;
Fig. 1, A) were
transplanted (designated as "loaded") to two recipient sites in
the other condyle of the ipsilateral knee. The other two grafts (one from the
condyle and one from the trochlea) were not implanted or impacted and were
used as controls (designated as "unloaded"). In each pair of donor
knees, medial grafts from one knee were transplanted to the lateral side of
that knee, and lateral grafts from the other knee were grafted to the medial
side of that knee. Thus, a total of twenty-four grafts from twelve femora from
six cadavers were transplanted into recipient sockets.
The grafts were inserted with use of a tamp until their cartilage surface
was flush with that of the surrounding host cartilage. Of the twenty-four
grafts, ten from six femora from three donors were inserted with a
load-cell-instrumented tamp (surface area, 7.3
cm2)6
(Fig. 1, C) to allow
load measurement during graft insertion. (Load was not measured during
insertion of every graft because the instrumented tamp was not always
available, and sufficient data were obtained from the ten grafts.) For each
impact, the loading force was taken as the peak, the magnitude of the load
impulse was calculated as the area under the force-time curve, and the
duration was considered to be the interval between half-maximum loads
(Fig. 1, D). The load
history, the characteristics of each impact, the total number of impaction
taps, and the tap number at which the peak impact was maximal were recorded
for each inserted graft (Fig. 1,
E). The loading force was normalized to graft area to
estimate compressive stress.
Cartilage Retrieval
After insertion of the osteochondral grafts, cartilage disks were removed
from each transplanted graft and from each control graft for subsequent
analysis. The retrieved grafts were placed in Dulbecco modified Eagle medium
supplemented with 2 mM L-glutamine, 100 U/mL penicillin, and 50 µg/mL
gentamicin at 5% CO2/95% O2 at 37°C. Full-thickness
cartilage was removed from the subchondral bone with a scalpel and then was
cut with a dermal punch to yield six, seven, or eight 3-mm-diameter
full-thickness disks from each graft.
Quantification of Cell Death
Six disks from each graft were incubated in Dulbecco modified Eagle medium,
with a culture duration of one, four, or forty-eight hours (two disks each).
Disks from an individual graft were distributed evenly among the time points
to control for the potential regional variation in cartilage
properties39. At
each time point, disks were cut vertically in half. One half was stained with
phosphate-buffered saline solution containing calcein-AM and ethidium
homodimer-1 (LIVE/DEAD Viability/Cytotoxicity Kit; Molecular Probes, Eugene,
Oregon) for one hour at 4°C and rinsed twice in phosphate-buffered saline
solution for twenty minutes each. The samples were imaged with use of a
fluorescence microscope (Eclipse TE300; Nikon, Melville, New York), an arc
lamp, G-2A (for "dead" images; Nikon) or B-2A (for
"live" images; Nikon) filter cubes, a Plan Fluor 4×
objective lens (NA = 0.13; Nikon), and a SPOT RT camera (Diagnostic
Instruments, Sterling Heights, Michigan). Each disk was imaged at the cut
vertical surface. From these images, cell death was quantified as a function
of the depth from the articular surface, in bins of 100-µm thickness, to a
depth of 1500 µm. To achieve this, the articular surface was localized by
fitting a line to 5~10 cells at the surface, rotating the image and cells
such that the articular surface was horizontally positioned, and tabulating
the cells located at certain depths. Since there is a margin of cell death
near a lacerated surface (as a result of isolation with a dermal punch
circumferentially)40,41,
cell viability was analyzed in regions excluding the area within 100 µm of
the lacerated surfaces. Live and dead cells were counted with custom image
processing routines with use of MATLAB (The MathWorks, Natick,
Massachusetts)42.
Images were processed by spatial filtering (5 × 5 Laplacian of Gaussian
filter, standard deviation of ~1) to accentuate regions representative of
cells, filtering (2 × 2 median) to suppress noise, and thresholding to
identify and localize cells. This automated method of cell counting had
~90% sensitivity and specificity for both live and dead cells as
determined by manual counting of randomly cropped regions containing >100
cells for eight sample images with varying overall cell density, from the
lowest to the highest (data not shown). For a given region of interest, the
percentage of cell death was quantified as the number of dead cells divided by
the total number of cells (live and dead).
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling
(TUNEL)
Apoptosis was assessed forty-eight hours after graft insertion. For this
analysis, one cartilage disk from each of five inserted grafts and five
control grafts (from five donors) was incubated for forty-eight hours. That
time point was selected because the number of apoptotic cells increases with
time, especially from six to ninety-six hours after application of
load21,36.
After incubation, cartilage disks were placed in 10% buffered formalin for
twenty-four hours, embedded in paraffin, and cut into 3-µm-thick sections.
In situ detection of apoptosis was performed with use of an ApoAlert DNA
Fragmentation Assay Kit (Clontech, Mountain View, California).
Fluorescein-dUTP labels DNA strand breaks and allows direct detection of DNA
fragmentation by emitting a bright green signal when viewed with fluorescence
microscopy. Apoptosis was quantified as a function of the depth from the
articular surface by counting the number of TUNEL-positive cells and total
cells in three sequential bins of 500-µm thickness over a depth of 1500
µm in three to six sections for each sample and determining the percentage
of TUNEL-positive cells.
Immunohistochemical Analysis for Active Caspase-3
Caspase-3 activation was assessed eight hours after insertion of the graft.
For this analysis, one cartilage disk from each of four inserted grafts and
four control grafts (from four donors) was incubated for eight hours. That
time point was chosen because the number of activated caspase-3 cells
increases with time, from four to twenty-four hours after application of
load35,43,44.
Cartilage samples were fixed, embedded in paraffin, and cut into 3-µm
sections, as described above. The presence of active caspase-3 was determined
by immunohistochemical analysis with the VECTASTAIN avidin-biotin-peroxidase
complex (ABC) kit (Vector Laboratories, Burlingame, California). In accordance
with the manufacturer's instructions, rabbit polycolonal anti-caspase-3
antibody (1:66 dilution; R&D Systems, Minneapolis, Minnesota) was used as
the primary antibody at 4°C overnight. A biotinylated goat anti-rabbit
antibody (R&D Systems) was used as the secondary antibody. The reaction
was then visualized by diaminobenzidine (DAB) (Sigma, St. Louis, Missouri),
resulting in a brown color. Caspase-3 activation was quantified by the number
of DAB-positive cells and expressed as the percentage of total cells. It was
quantified as a function of the depth from the articular surface in three
sequential bins of 500-µm thickness over a depth of 1500 µm in three to
six sections for each sample, as described above for TUNEL staining.
Statistical Analysis
Pilot data from preliminary experiments demonstrated a 10% standard
deviation for nonviable cells in the superficial layer of cartilage. Thus, to
detect a 20% difference at a significance of a= 0.05 and a power (1
— ß) of 0.80, six donors were needed.
The effects of loading and of depth from the articular surface on cell
viability were assessed with repeated-measures two-way analysis of variance
and post hoc testing. The effects of loading and of depth from the articular
surface on apoptosis and caspase-3 activation were assessed with
repeated-measures analysis of variance and post hoc testing. Statistical
analyses were performed with Systat 10.2 (Point Richmond, California). Data
are expressed as the mean and standard deviation.
Biomechanics of Graft Insertion
The mean loading force of all ninety-five impacts was 2.5 ± 1.3 kN
(52% coefficient of variation), corresponding to a stress of 14.0 ± 7.3
MPa. The impact duration averaged 0.56 ± 0.18 ms, and the impulse
averaged 0.63 ± 0.30 N·s. The means and coefficients of
variation were similar when the analysis was performed in terms of grafts (ten
grafts from three donors; Fig.
2) instead of impacts: the loading force averaged 2.4 ± 0.9
kN (37% coeffvcient of variation), the corresponding stress averaged 13.3
± 4.9 MPa, the impact duration averaged 0.57 ± 0.13 ms, and the
impulse averaged 0.62 ± 0.25 N·s. On the average, the maximum
impact applied to each osteochondral graft was associated with a loading force
of 3.9 ± 1.9 kN (49% coefficient of variation;
Fig. 2), a stress of 22.1
± 10.8 MPa, an impact duration of 0.48 ± 0.12 ms, and an impulse
of 0.82 ± 0.20 N·s. An average of 10 ± 4 impacts were
required to insert an osteochondral graft into the recipient socket, with the
maximum impact being 8 ± 5 taps from the start (first impact),
corresponding to 83% ± 21% of the total number of impacts, or 3
± 2 taps from the end (last impact).
Analysis of Chondrocyte Death After Load
Loading diminished cell viability, especially near the articular surface
(Figs. 3 and
4). The average cell death in
the unloaded samples did not differ significantly (p = 0.46) among the three
time points after graft insertion (6% ± 2%, 6% ± 2%, and 7%
± 3% at one, four, and forty-eight hours, respectively). However, at
each time point, the application of load increased cell death (p < 0.001)
compared with that in the unloaded samples. One hour after insertional
loading, cell death was significantly increased (p < 0.001) in the
superficial 500 µm, with an average of 21% ± 4% cells dead compared
with 9% ± 2% in the same area of the unloaded sample
(Fig. 4). By forty-eight hours
after insertion, cell death not only had increased more than twofold in the
uppermost 500 µm (47% ± 11% compared with 7% ± 5% in the
unloaded samples), but it also had extended deeper into the tissue, to
~1000 µm (p < 0.001), where cell death averaged 38% ± 16%
compared with 7% ± 4% in the unloaded samples
(Fig. 4).
Quantification of Apoptosis by TUNEL Assay
Impaction loading also induced apoptosis in a depth-varying manner. At
forty-eight hours, the baseline percentage of cells detected as apoptotic in
the unloaded samples (five donors) averaged 9% ± 2%, while the loaded
samples showed a significantly higher level (p < 0.05) of apoptosis (26%
± 7%) (Fig. 5, A, B, and
C). Application of load increased cell apoptosis in the
uppermost 1500 µm (p < 0.001) when compared with the apoptosis in that
region in the unloaded samples. Most of these TUNEL-positive cells were found
in the top 500 µm, where apoptosis was increased threefold (from 5%
± 2% in the unloaded samples to 16% ± 7% in the loaded samples,
Fig. 6, A). The trend
continued through the depth of the cartilage but with lower levels of
positively-stained cells (2% ± 1% in the unloaded samples compared with
7% ± 1% in the loaded samples in the next 500-µm area and 1%
± 0.5% in the unloaded samples compared with 3 ± 1% in the
loaded samples in the area that was even 500 µm deeper).
Evaluation of Caspase-3 Activation After Load
Immunohistochemical analysis of the cartilage disks (from four donors) at
eight hours (Fig. 5, D, E, and
F) showed active caspase-3 in 9% ± 3% of the cells
in the unloaded samples, whereas the loaded samples showed significantly
higher caspase-3 activation (31% ± 7% of the cells) (p < 0.01). When
the activated-caspase-3-positive cells were quantified as a function of depth
(Fig. 6, B), the
application of load was found to increase caspase-3 activation in the
uppermost 500 µm as well as the next 500-µm layer (p < 0.001) when
compared with the activation in the same region of the unloaded samples.
Osteochondral grafting has been used with varying degrees of success for
restoration of articular cartilage. Current clinical techniques utilizing
devices that impact the graft during insertion can lead to chondrocyte death,
which may diminish the performance of the graft. In the present study, we
sought to characterize the mechanical parameters and chondrocyte responses to
the current clinical technique of insertion. The impacts (average, ten
impacts) needed to insert 15-mm-diameter grafts resulted in an average force
of 2.4 kN and an area-averaged compressive stress of 13.3 MPa; the average
duration of the impacts was 0.57 ms, with an average impulse of 0.62
N·s. Cell death as a result of impact insertion was localized near the
impacted articular surface, extending from the top 500 µm at one hour after
impact to a depth of 1000 µm by forty-eight hours
(Fig. 4). An increase in
caspase-3 activation at eight hours and TUNEL-positive cells at forty-eight
hours, both of which are indicative of an apoptotic pathway, also demonstrated
a loss of viable cells that was accentuated near the articular surface (Figs.
5 and
6).
In this study, we employed techniques used in surgery. Previous in vitro
studies of impact loading have been performed on cartilage
explants13-16,18,26,45,
intact animal
knees10,46,47,
or osteochondral grafts from
animals12,48.
In the single other study of impact loading of human osteochondral grafts, of
which we are aware, the investigators used osteoarthritic
knees40 in contrast
to fresh normal human tissue (from donors just over the age limit of
forty-five years for osteochondral allografting). Also, the osteochondral
grafts were prepared to a size (15 mm in diameter) and shape (circular)
typical of those used in human osteochondral allografting, and they were
inserted into a recipient socket by a surgeon who was experienced with the
procedure.
The variability in the impact mechanical parameters (e.g., a 52%
coefficient of variation of impact force) could be due to a number of factors.
Surgeon control of the impact may be one factor. Other sources of variation
could be differences in the bone quality of the graft recipient, the tightness
of the fit of the grafts, and the relationship of the socket depth to the
graft length, which could affect the seating level. The peak impact occurred
near the completion of insertion (Fig.
2), suggesting that peak impact occurs when the base of the graft
is seated into the recipient socket. The standard manual preparation of the
graft base and recipient socket can result in variations of ~0.5 to 1 mm.
Thus, an important part of the surgical technique is to trim the bone to the
appropriate length, which can play a key role in the successful outcome of
allograft transplantation and long-term cell viability.
Several biomechanical features of impact loading may lead to cell death.
The peak stress determined in this study (13 ± 5 MPa) was in the range
(14 to 20 MPa) that has been reported to induce chondrocyte
death18
(Fig. 2). The high rate at
which the load (or stress) was applied may have contributed to the
distribution of cell death, as high stress rates produce chondrocyte death
localized to the superficial
layer45 and low
stress rates produce a more diffuse distribution of cell
death13,14,26.
The stress rates that were applied were comparable with the range of 35 to
1250 MPa/s reported in the
literature14,17,46.
The depth of cell death from the articular surface has been observed
previously to increase with peak stress and to decrease with an increasing
stress rate14.
Alternatively, it may be that strain and/or the strain rate are the key
components leading to
apoptosis21,49-51.
Probably just as important as the load magnitude and rate is the duration
over which the load is applied, or the impulse. In this study, the impulse was
generally constant, with amplitudes of ~0.6 N·s. Telemetry
measurements obtained from instrumented knee prostheses have shown axial
forces ranging from 1500 to 2400 N (~2.8 to 3.6 × body weight)
during typical levels of activity, such as walking, stair-climbing, and light
jogging52-54.
This finding suggests that high loads are probably present in the knee itself
but are transmitted and dissipated by various tissues in the knee, such as the
meniscus55-57,
to help create an environment that protects against cell injury during
activity.
The overall density of chondrocytes in cartilage is affected by the balance
between cell death and cell proliferation. Cell death induced by impact may be
due to both apoptosis and necrosis. Necrosis does not require activation of
specific intracellular signaling cascades, and, after the application of
mechanical impact to
cartilage15,16,58,
necrosis has been observed as an earlier event than apoptosis, occurring as
soon as twenty to 120 minutes after
impact15. In the
present study, apoptosis was evaluated eight and forty-eight hours after graft
insertion. The percentages of cells shown to be apoptotic by TUNEL (26%) and
caspase-3 activation (31%) assays (Figs.
3 and
4) were similar, but they were
lower than the percentage of dead cells determined by viability assay (47%).
This could have been due to compression causing cell death by necrosis and the
fact that the evaluation times were past the period when cell death by
necrosis occurred. In addition, insertional loading could also increase the
chondrocyte number by causing proliferation, which would counteract the
injurious effect of the impact. Furthermore, although the grafts were prepared
from en bloc knee specimens in a manner typical of the clinical procedure, it
is unknown whether a fresh osteochondral allograft inserted in vivo may be
protected by exposure of the injured chondrocytes to synovial fluid or the
blood supply in the subchondral bone.
The chondrocytes responded differently to load according to their depth
from the articular surface. In other studies, cell death was often localized
at the superficial surface as a result of the high compaction and loss of
fluid14,45.
In the present study, chondrocytes were similarly affected by load, as evident
in the vertical profiles. However, chondrocytes in the deep layer of cartilage
appeared to be protected from damage. Although the cartilage was not evaluated
below a depth of 1500 µm (the overall thickness of human cartilage in the
femoral condyle ranges from 1.5 to 3
mm59-62),
the deeper layers appear to be protected by the subchondral bone, possibly by
preventing the cartilage from radially expanding at the cartilage-bone
interface and thus constraining the tissue to allow the fluid to pressurize,
sustain load, and diminish impact-induced damage and
apoptosis20. In the
present study, application of load did not grossly affect cell viability
(i.e., increase death) in the deep profiles. The effect of the donor site on
cell death was not assessed. Nonetheless, in the control tissues, the majority
of the chondrocytes were viable, suggesting methods to protect the
chondrocytes and their functions at the surface of the tissue may be
beneficial.
Apoptosis is known to occur after an ordered sequence of cellular events.
Abnormal biomechanical loading can trigger apoptosis by activation of caspases
that lead to characteristic changes in DNA and other cellular constituents.
The triggers of apoptosis induced by mechanical stimulation are not known, but
several candidates can be proposed, including nitric oxide
donors63,
expression of the Fas
receptor64, and
stress-induced rearrangement of the
cytoskeleton65.
Mechanical loading could disrupt cell-matrix interactions or lead to
cytoskeletal reorganization by means of three major pathways (death receptors,
mitochondria, and endoplasmic reticulum), all of which are pathways in the
sequential activation of caspases that converge at
caspase-366.
Pharmacologic or other therapeutic interventions may assist in protecting
grafts during insertion and may improve cell viability. Caspases can be
sensitive to pharmacologic
intervention11,67,68.
Injection of the caspase inhibitor Z-VAD.fmk into a rabbit knee joint for more
than seven days after an acute injury reduced cell apoptosis by 20% to 30% in
one study68. This
suggests that pharmacologic treatments may be possible and that these agents
may be used to inhibit apoptosis at various points in the pathway. The profile
of cell death in our study is consistent with previous trends in the
literature36, in
which cell death due to impact loading has been reported to have been located
primarily at the superficial region of cartilage. We found that, with time,
the region of cell death extended into the middle zone, a finding consistent
with other reports of delayed cell
death36. In
addition, this profile quantified how cell death increased not only with time,
but also with the depth from the articular surface. A pharmacologic or
technical intervention to protect regions from cell death may be useful during
graft procedures.
Maintaining chondrocyte viability is a fundamental principle of
osteochondral grafting. The results of this study suggest that modification of
graft insertion techniques may be warranted to protect graft viability.
Indeed, this study has led to a change in our clinical practice in that we now
minimize impact loading of grafts during insertion. Furthermore, understanding
the results of cell death with impact loading may lead to therapeutic
interventions that protect the graft. This may have implications not only for
osteochondral grafting techniques, but also for therapies for chondral impact
injuries. It is unknown what effect impact loading and subsequent chondrocyte
death have on the function or longevity of the graft in vivo; thus additional
studies are warranted. ?
Note: The authors thank Dr. Sanna Rosengren for statistical
assistance, Dr. Darryl D. D'Lima for valuable discussion, and the Lifesharing
Program of the University of California-San Diego for providing human donor
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