ARTHRITIS & RHEUMATISM
Vol. 48, No. 12, December 2003, pp 3382–3394
Indentation Testing of Human Cartilage
Sensitivity to Articular Surface Degeneration
Won C. Bae,1 Michele M. Temple,1 David Amiel,1 Richard D. Coutts,1
Gabriele G. Niederauer,2 and Robert L. Sah1
Objective. To determine, for clinical indentation
testing of human articular cartilage, the effects of aging
and degeneration on indentation stiffness and tradi-
tional indices of cartilage degeneration; the relationship
between indentation stiffness and indices of degenera-
tion; and the sensitivity and specificity of indentation
stiffness to cartilage degeneration.
Methods. Osteochondral cores from femoral con-
dyles of cadaveric human donors were harvested. Sam-
ples were distributed into experimental groups based on
donor age (young [20–39 years], middle [40–59 years],
and old [>60 years]), and a macroscopic articular
surface appearance that was either normal or mildly
degenerate, without deep erosion. Samples were ana-
lyzed for indentation stiffness, cartilage thickness, India
ink staining (quantitated as the reflected light score),
and Mankin-Shapiro histopathology score.
Results. Indentation stiffness, India ink staining,
and the histopathology score each varied markedly
between normal-sample and degenerate-sample groups
but varied relatively little between normal samples
obtained from different age groups. A decrease in
indentation stiffness (softening) correlated with a de-
crease in the reflectance score and an increase in the
overall histopathology score, especially the surface ir-
regularity component of the histopathology score. Re-
ceiver operating characteristic analysis suggested that
the indentation testing could accurately detect cartilage
1
Won C. Bae, MS, Michele M. Temple, MS, David Amiel,
PhD, Richard D. Coutts, MD, Robert L. Sah, MD, ScD: University of
California–San Diego, La Jolla; 2Gabriele G. Niederauer, PhD: Os-
teoBiologics, Inc., San Antonio, Texas.
Address correspondence and reprint requests to Robert L.
Sah, MD, ScD, Department of Bioengineering, 9500 Gilman Drive,
Mail Code 0412, University of California–San Diego, La Jolla, CA
92093-0412. E-mail: rsah@ucsd.edu.
Submitted for publication October 8, 2002; accepted in re-
vised form August 8, 2003.
3382
DOI 10.1002/art.11347
© 2003, American College of Rheumatology
degeneration as indicated by macroscopic appearance,
India ink staining, and histopathology score.
Conclusion. The indentation stiffness of the nor-
mal to mildly degenerate samples tested in this study
was sensitive to mild degeneration at the articular
surface and was insensitive to changes associated with
normal aging or to slight variations in cartilage thick-
ness. This suggests that indentation testing may be a
useful clinical tool for the evaluation of early-stage
degenerative changes in articular cartilage.
Short-duration indentation testing is being con-
sidered as a diagnostic tool for assessing the biomechani-
cal properties of human cartilage (1–6). In the labora-
tory setting, indentation tests of long duration have been
performed routinely on human or animal tissue as an
end point (post mortem) method of evaluation (7–13).
In the clinical setting, however, such traditional labora-
tory tests are not practical because of the long duration
of the test and the large size of the test apparatus.
Short-duration indentation testing, performed using an
arthroscopic probe, may be useful and appropriate in the
clinical setting.
A short-duration indentation test of articular
cartilage in adult humans may be affected by both
degenerative changes and normal age-associated
changes in the cartilage. Although several biomechanics
studies have analyzed human articular cartilage using a
rapid indentation test (2,4–6,12,14–17), and others have
examined the relationships between short-duration in-
dentation stiffness and traditional measures of cartilage
degeneration (5,14,17,18), the effects of normal aging
and mild cartilage degeneration remain to be estab-
lished. The establishment of baseline indentation mea-
surements for articular cartilage in the setting of normal
aging would be useful to help interpret an indentation
test result in the clinical setting. Determination of the
SENSITIVITY OF INDENTATION TESTING TO CARTILAGE DEGENERATION
sensitivity to features of mild degeneration would help
define the efficacy of indentation testing as a method of
cartilage evaluation and would also help determine the
basis for variations in indentation stiffness.
The extent of articular cartilage degeneration has
traditionally been analyzed by macroscopic viewing and
histopathologic grading. Macroscopically, the grading
system described by Collins (19) distinguishes early
degeneration by defining grade 0 as cartilage with a
smooth surface and grade 1 as cartilage with fibrillation
limited to the superficial zone; grades 2–4 describe
cartilage with increasing levels of erosive degeneration.
The application of India ink highlights surface abnor-
malities and characterizes the increase in cartilage de-
generation with increasing age (20). India ink contains
carbon black particles and clusters with a diameter of
⬃40–100 nm (21,22). Such India ink particles are pre-
vented from entering into normal articular cartilage, the
average pore size of which is ⬃6 nm (23), but are able to
stain cartilage that has articular surface fibrillation (24).
India ink particles are entrapped in surface irregular-
ities and also adhere to fibrillated cartilage (21). Be-
cause India ink particles absorb and scatter incident
light (25), the reflection of light from cartilage depends
on the degree of India ink staining. Application of
digital video imaging and analysis to the surface of
ink-stained cartilage samples allow for quantitation and
localization of degeneration over broad surface areas of
joints (26).
Histopathologic analysis has provided more de-
tailed structural information about cartilage degenera-
tion, from superficial fraying and splitting of the articu-
lar surface in mild cases, to deep fissures and extensive
erosion extending beyond the superficial layer of the
cartilage in advanced cases (27). Histopathologic fea-
tures of articular cartilage structure, cartilage cellularity,
and tidemark integrity, as well as histochemical staining
of glycosaminoglycan with Safranin O, are often tallied
using the Mankin scoring system (28), or the modified
Mankin scoring system described by Shapiro and Glim-
cher (29), to grade the severity of osteoarthritic cartilage
degeneration.
The relationships between the indentation stiff-
ness and the material and structural properties of carti-
lage have been analyzed theoretically with simplifying
model assumptions and have been examined experimen-
tally in a number of studies. Theoretically, the indenta-
tion stiffness of articular cartilage has been related to the
biomechanical material properties (i.e., Young’s modu-
lus and Poisson’s ratio) of cartilage tissue as well as the
thickness of the cartilage layer (30,31). As cartilage
3383
thickness increases to a sufficient degree, the indenta-
tion stiffness is predicted to become essentially indepen-
dent of thickness. However, the indentation stiffness of
human articular cartilage is likely to depend also on
complex features, such as depth variation in tissue
biomechanical material properties (32), especially the
tensile modulus of the superficial region (33), which
decreases with advancing age (34). Such variations, as
well as structural features (e.g., fissures) associated with
degeneration (27), are not easily incorporated into the-
oretical models.
Experimentally, short-duration indentation stiff-
ness has been related to structural measures of cartilage
degeneration. The indentation stiffness of human fem-
oral head samples was found to be markedly decreased
in samples with a gross structural grade of osteoarthritis
but variably decreased in samples with a gross grade of
mild degeneration (17,18). The relationship between
indentation stiffness and cartilage degeneration has
been clarified somewhat by more detailed histopatho-
logic analysis. Indentation stiffness was decreased in
human patellar samples with surface fibrillation (14) and
in relatively old (mean age 65 years) human femoral
condyle samples with increasing Mankin scores (28).
More detailed studies would be useful to assess further
whether indentation stiffness is affected by age or
surface-specific structural measures of cartilage degen-
eration, especially in early stages.
The receiver operating characteristic (ROC)
curve can be used to define the performance of a
diagnostic test (35,36). A ROC plot illustrates the rela-
tionship between the proportion of true-positive and
false-positive cases for a population of test samples and
either specific test results or result thresholds that
attempt to distinguish disease from nondisease cases. In
addition, the shape of the ROC plot and the area under
the ROC plot are features that indicate the diagnostic
accuracy of a test (36,37). Such ROC analysis has not
been applied previously to indentation stiffness to test
for various indicators of cartilage degeneration.
The objectives of this study on human articular
cartilage were to determine 1) how indentation cartilage
stiffness, measured using a handheld arthroscopic probe
(3), varies within a sample as well as between samples, as
a function of donor age and tissue degeneration, 2) how
these indentation stiffness values correlate with struc-
tural indices of cartilage degeneration, including India
ink staining, histopathology, and cartilage thickness, and
3) the sensitivity and specificity of indentation stiffness
measurements to the indices of degeneration by ROC
analysis.
3384
MATERIALS AND METHODS
Sample preparation. Using a surgical coring device
(Osteochondral Autograft Transfer System; Arthrex, Naples,
FL), 10-mm–diameter osteochondral cores were harvested
from the anterior region of the lateral and medial femoral
condyles of fresh (not previously frozen) cadaveric human
knee joints, obtained from tissue banks. Each core was notched
at the posterior edge to maintain orientation throughout
testing, immersed in a solution of Dulbecco’s phosphate
buffered saline (PBS; 2.667 mM KCl, 1.471 mM KH2PO4, 138
mM NaCl, 8.1 mM Na2HPO4–7H2O [pH 7.2]), and proteinase
inhibitor (PI; 1 mM phenylmethanesulfonyl fluoride, 2 mM
disodium ethylenediamine tetraacetate, 5 mM benzamidine–
HCl, and 10 mM N-ethylmaleimide) (38) at 4°C for 1 hour, and
then stored at ⫺70°C until the time of testing. Previous studies
demonstrated that indentation stiffness measurements are not
affected by a single freeze–thaw cycle (18,39–41).
Indentation testing. Each sample was thawed by im-
mersion in PBS plus PI at ⬃22°C and clamped at the bony
region into a custom jig to stabilize the sample. Then, inden-
tation testing was performed at a site near the center, 3.5 mm
away from the edge opposite the notch, using a handheld
ACTAEON Probe (OsteoBiologics, San Antonio, TX). With
each manual trigger, the ACTAEON Probe advanced its
nonporous, hemispheric 1-mm–diameter tip to a depth of up
to 100 ␮m for a duration of up to 0.25 second, and provided a
stiffness measurement on a scale of 0–99. The probe was
calibrated to American Society for Testing and Material type
A durometer standards (ASTM D2240). On this scale, a
relatively low value indicates a relatively soft sample. For each
cartilage specimen, a total of 9 stiffness measurements were
obtained at the indentation site. To achieve this, the probe tip
was applied 3 times to the test site location, triggered consec-
utively 3 times during each application, and removed from the
test site. The stiffness of the sample was calculated as the
average of these 9 measurements. This measurement proce-
dure typically required ⬍15 seconds for an individual sample.
India ink staining and image analysis. Samples were
analyzed by India ink staining and video image analysis to
obtain a light reflectance score, as described previously (42).
Briefly, the articular surface was swabbed with a solution of
India ink in PBS plus PI (1:5), wiped to remove excess ink, and
imaged (8-bit, gray scale, 640 ⫻ 480 pixels, 0.1 mm ⫻ 0.1 mm
per pixel) using a video acquisition card (LG3; Scion, Freder-
ick, MD) and NIH Image 1.59 software (National Institutes of
Health, Bethesda, MD) on a Power Macintosh 7100/80 (Apple
Computer, Cupertino, CA). Light reflectance scores from the
cartilage surface in the images were normalized to gray-scale
calibration targets (Q13; Eastman Kodak, Rochester, NY) that
were chosen to approximate the reflectance of light from
normal, nonstaining cartilage (gray no. 3 normalized value ⫽
1) and fibrillated, maximally ink-stained cartilage (gray no. 19
normalized value ⫽ 0). A relatively low reflectance score
corresponds to relatively high India ink staining and cartilage
degeneration.
Cartilage thickness measurement. The thickness of
articular cartilage of each core was measured by obtaining and
analyzing cartilage images. Each core was positioned to enable
imaging at 6 equally spaced circumferential locations. The
thickness of cartilage was calculated as the average of the 6
measurements for each sample. Control studies confirmed that
BAE ET AL
this method of cartilage thickness determination provided
values similar to those measured on histologic sections ob-
tained from near the center of the core (Pearson’s correlation,
slope ⫽ 0.99, R2 ⫽ 0.42).
Histopathologic analysis. An osteochondral fragment
containing the indentation site was obtained for histopatho-
logic analysis. The fragment was isolated using a 3.5-mm–
diameter biopsy punch (part no. 33-33; Miltex Instrument
Company, Bethpage, NY), fixed by immersion in 10% neutral-
buffered formalin with 1% cetylpyridinium chloride, decalci-
fied, embedded in paraffin, sectioned to 6 ␮m, and stained with
Safranin O–fast green (43). Images of the full-thickness carti-
lage were obtained on an inverted microscope (Nikon Eclipse
TE300; Nikon, Melville, NY) using transmitted light micros-
copy at magnifications of 4⫻ and 10⫻ (field of view [FOV]
2.5 ⫻ 3.9 mm2 and 1.0 ⫻ 1.5 mm2, respectively) and a digital
camera (Kodak Microscopy Documentation System 290; East-
man Kodak, New Haven, CT). The 4⫻ images were used to
observe overall structural characteristics of the section, while
the 10⫻ images were used in the analysis of cellularity.
These images were then analyzed for degeneration by
determining a histopathology score (29). This histopathology
score combined the gross characteristics of articular cartilage
with the histologic appearances of surface irregularity, vertical
clefts into the transitional zone, vertical clefts into the radial
zone, transverse clefts, cloning, hypocellularity, and Safranin O
staining. Each characteristic was given a score of 0–2 or 0–3,
with 0 representing normal and 2 or 3 representing severe
degeneration. The sum of these scores provided the overall
histopathology score (scale 0–15), with a relatively high score
corresponding to a relatively degenerate cartilage sample. In
addition, the surface irregularity component of the histopa-
thology score was added separately to assess surface-specific
structural degeneration in each sample.
Experiment 1: sources of variation in indentation
stiffness. The sources of variability in indentation stiffness
measurements were assessed in twenty 10-mm–diameter hu-
man osteochondral cores. These samples were chosen at
random and thus included a wide range of donor ages and
stages of cartilage degeneration. Three observers indepen-
dently tested the same cores, and both intraobserver and
interobserver sources of variability were assessed. Because
variability was likely to depend on the extent of degeneration,
the samples were categorized into 4 experimental groups,
based on their average indentation stiffness score (⬍30, 30–50,
50–70, and ⬎70).
The data were analyzed to determine the extent of
intraobserver variability, particularly that attributable to “trig-
gering” of the probe (3 consecutive triggers without moving the
probe) and “manual repositioning” of the probe (removal of
the probe tip from the cartilage surface, reapplication of the
probe tip, and measurement). The measures of variability were
the standard deviation (SD), the root mean squared coefficient
of variation (CV) of repeated measurements (44), and the
standardized CV (sCV; CV normalized to population variabil-
ity) (45,46). Interobserver variability was calculated as the SD,
the CV, and the sCV of the sample indentation stiffness (i.e.,
the average of all 9 measurements on a sample as determined
by an individual observer) between the 3 observers. In addi-
tion, to check for any systematic changes in indentation
stiffness (e.g., those attributable to cumulative creep) during
triggering, each of the 3 consecutive measurements (3 trigger-
SENSITIVITY OF INDENTATION TESTING TO CARTILAGE DEGENERATION 3385

Figure 1. Representative photographs (A and B) and corresponding histologic micrographs

(C and D; Safranin O–fast green stained) of young-age normal and old-age degenerate human
cartilage specimens, respectively. The articular surface is intact in the young-age normal sample
and is roughened in the old-age degenerate sample.

ings) were linearly regressed to values of 1, 2, and 3, respec-
tively, and the resulting slopes were averaged. A slope of 0
indicated zero variability during triggering. Similarly, system-
atic changes during repositioning were assessed for 3 reposi-
tionings in each sample. No cumulative creep was observed
during either triggering or repositioning, because the slopes
(mean ⫾ SD) of linear fits were 0.3 ⫾ 2.5 and ⫺0.6 ⫾ 3.5,
respectively.
Experiment 2: indentation stiffness and indices of
cartilage degeneration. The relationship between indentation
stiffness and indices of cartilage degeneration was analyzed
using seventy 10-mm–diameter osteochondral cores. These
samples were selected based on a macroscopic articular surface
appearance that was either smooth (normal, n ⫽ 58) (Figure
1A) or roughened (degenerate, n ⫽ 12) (Figure 1B). In
addition, samples were categorized into 3 adult age groups:
young (20–39 years, n ⫽ 18), middle (40–59 years, n ⫽ 24), and
old (ⱖ60 years, n ⫽ 28). This provided sample groups of
young-age normal (YN), middle-age normal (MN), middle-age
degenerate (MD), old-age normal (ON), and old-age degen-
erate (OD). Insufficient numbers of samples were obtained for
a young-age degenerate group. Together, these samples were
obtained from 38 human donors (17 women and 21 men), with
36 from the lateral femoral condyle (LFC) and 34 from the
medial femoral condyle (MFC). Samples were tested by inden-
tation testing, India ink staining and image analysis, cartilage
thickness measurements, and histopathologic analysis, as de-
scribed above. The resultant data were analyzed statistically (as
follows), using SYSTAT 9 software (Systat, Richmond, CA).
Effects of location, aging, and degeneration. Several
statistical methods were used to determine the effects of
anatomic location (LFC and MFC) and experimental group
(YN, MN, MD, ON, and OD) on indentation stiffness, the
reflectance score, cartilage thickness, and histopathology
scores (overall score as well as scores for individual character-
istics). First, to investigate the effect of LFC and MFC
3386
locations, t-tests were performed to compare LFC and MFC
samples for each of the 5 experimental groups, with ␣ ⬍ 0.01
(Bonferroni adjustment) used as the significance level to
reflect multiple planned comparisons.
The effect of each experimental group (YN, MN, MD,
ON, and OD) within each of the LFC and MFC locations was
analyzed separately by one-way analysis of variance
(ANOVA). Using Tukey’s test, the effect of aging was deter-
mined by comparing the normal-sample groups (YN, MN, and
ON), while the effect of degeneration was assessed by com-
paring the YN (young adult control tissue) against all
degenerate-sample groups (MD and OD) as well as by com-
paring normal and degenerate samples in age-matched groups
(i.e., MN versus MD, and ON versus OD). (Because many of
the LFC and MFC samples were obtained from different
donors, the alternative statistical approach of repeated-
measures ANOVA and nonparametric Wilcoxon’s tests was
not used, because such an approach would have disregarded a
large portion of the data.) Analogously, for the nonparametric
measures (i.e., the histopathology score), the effects of LFC
and MFC locations were analyzed using the Mann-Whitney U
test (with adjusted significance level), and the effect of the
experimental group within each of the LFC and MFC locations
was analyzed with the Kruskal-Wallis test and Dunn’s test.
Data are presented as the mean ⫾ SEM, unless indicated
otherwise.
Relationships between indentation stiffness and indices of
degeneration. To determine which of the many parameters
were closely related to each other, a factor analysis using the
method of principal components was performed. Indentation
stiffness, reflectance score, cartilage thickness, overall histopa-
thology score, and donor age were the parameters included for
this analysis. The principal components matrix was computed,
and the loading coefficients (describing how each parameter
contributed to each factor) were obtained. For each factor, the
dominant contributors were identified as those whose coeffi-
cients (ranging from ⫺1 to ⫹1) had an absolute value of ⬎0.5.
As many factors as were necessary were calculated, in order to
account for each parameter as a dominant contributor.
In addition, analyses were conducted to determine
whether indentation stiffness was related to cartilage thickness
or surface damage. Indentation stiffness was correlated to the
reflectance score and cartilage thickness by parametric univar-
iate linear regression analysis (47). Indentation stiffness was
correlated to the histopathology scores by the nonparametric
Spearman’s rank method (47) (because the subjective histopa-
thology score was an ordinal variable rather than a continuous
variable).
Sensitivity and specificity of indentation stiffness to indi-
ces of degeneration. To determine the accuracy of the indenta-
tion stiffness measurements in distinguishing various tradi-
tional indices of cartilage degeneration, sensitivity and
specificity values were determined. The sensitivity of indenta-
tion stiffness to macroscopic degeneration was determined as
the proportion of the number of data points from macroscop-
ically degenerate samples (by indentation stiffness), of the total
number of data points from macroscopically degenerate sam-
ples (true positives/total positives), at a particular indentation
stiffness cutoff value. For example, at an indentation stiffness
cutoff value of 50, there were 12 data points from macroscop-
ically degenerate samples (total positive), of which 11 had
indentation stiffness values of ⬍50 (and therefore were true
BAE ET AL
positives for macroscopic degeneration). Thus, the sensitivity
at the indentation stiffness cutoff value of 50 was 0.92 (11 of
12). For specificity, at the indentation stiffness cutoff value of
50, there were 58 data points from macroscopically normal
samples (total negative), of which 55 had indentation stiffness
values of ⬎50 (true negative). Thus, the specificity was 0.95 (55
of 58). Using these methods, sensitivity and specificity values
were calculated for a range of indentation stiffness cutoff
values.
For the calculation of the sensitivity and specificity of
indentation stiffness to other indices of degeneration (i.e., the
reflectance score and the overall histopathology score), thresh-
old values for each index were needed in order to classify the
sample as apparently normal or degenerate, based on the value
of the index. This allowed segregating the scatter plot of data
(the indentation stiffness score versus the index of degenera-
tion) into 4 quadrants of true positive, true negative, false
positive, and false negative. The status of being true or false
would be determined by the value of the index, and the status
of being positive or negative would be determined by the value
of the indentation stiffness score. Examples chosen to illustrate
the effects of varying threshold values were determined as a)
the average value for all macroscopically normal samples, b)
the average for all macroscopically degenerate samples, and c)
the midpoint value between the two, to segregate samples that
are roughly normal from those that are roughly degenerate.
For reflectance scores, values below the threshold value indi-
cated degenerate samples. For histopathology scores, values
above the threshold value indicated degenerate samples. Once
the data were segregated in this way, sensitivity and specificity
were computed for a range of indentation stiffness cutoff
values.
To describe the tradeoff between sensitivity to and
specificity for each of the above indices of degeneration, ROC
plots were generated. For an ideal test, all of the normal and
diseased samples would be segregated perfectly (e.g., by using
a single cutoff value). In such a case, the ROC plot would rise
vertically and reach a plateau after achieving maximum sensi-
tivity, with a resulting area under the curve of 1. Conversely,
for the worst possible test, the ROC plot would be a straight
line with a slope of 1, resulting in an area under the curve of
0.5. Thus, the ability of indentation testing to distinguish
between normal and degenerate samples, as indicated by
various indices of degeneration, was determined as the area
under the ROC curve.
RESULTS
Experiment 1: sources of variation in indentation
stiffness. The variation in measurements of indentation
stiffness was attributable to both intrasample and inter-
sample factors. For an individual sample, the variation
due to triggering was generally less than that due to re-
positioning, in all groups (Table 1). The variation in the
indentation stiffness score due to triggering (SDtrigger)
ranged from 2.4 to 3.4 for the different experimental
groups, with the corresponding CVtrigger ranging from
3.5% to 13.1%. The variation due to repositioning
(SDreposition) ranged from 2.9 to 5.6 (CVreposition 6.7–
SENSITIVITY OF INDENTATION TESTING TO CARTILAGE DEGENERATION 3387

Table 1. Intersample and intrasample indentation stiffness variability in experiment 1*

Intrasample variability

Intraobserver
Intersample variability
Group, Interobserver Repositioning Triggering
indentation No. of
score† Avg SD SD/Avg samples SD CV sCV SD CV sCV SD CV sCV
⬍30 24 2.2 9.4 4 4.4 21.4 13.7 3.3 16.4 10.4 2.6 13.1 8.4
30–50 37 4.5 12.0 4 8.3 24.3 15.5 5.6 18.2 11.6 3.4 10.8 6.9
50–70 63 4.1 6.6 9 5.3 9.6 6.1 3.4 7.6 4.9 2.9 5.6 3.6
⬎70 77 5.5 7.2 3 8.1 12.5 8.0 2.9 6.7 4.3 2.4 3.5 2.3
All 52 19.4 37.4 20 6.2 16.6 10.6 3.7 11.9 7.6 2.0 9.0 5.7

* Values for SD/Avg (standard deviation/average), CV (coefficient of variation), and sCV (standardized CV) are expressed as percentages. For each
group, there were 3 observers, 3 repositionings, and 3 triggerings.
† Samples were analyzed as 4 experimental groups, based on their average indentation stiffness score.

18.2%). In comparison, the variation between observers
was slightly greater, with the SDobserver ranging from 4.4
to 8.3 (CVobserver 9.6–24.3%). However, the interob-
server variation in stiffness measurement for a particular
sample was smaller than the overall variation between
cartilage samples (SD 19, CV 37.4%) (Table 1).
Experiment 2: indentation stiffness and indices
of cartilage degeneration. Effects of aging and degenera-
tion. Indentation stiffness. The major source of variation
in experiment 2, like that in experiment 1, was between
samples. The intrasample variations for repositioning
(SDreposition 2.5–4.1, CVreposition 5.8–16.0%) (Table 2)
and triggering (SDtrigger 2.4–3.2, CVtrigger 4.9–12.0%)
(Table 2) in experiment 2 were generally similar to those
in experiment 1 (Table 1), especially considering groups
with similar (average) stiffness values. The interobserver
variations in experiment 1 (Table 1) were also generally
smaller than the intersample variations within the
groups of experiment 2 (SD 4.2–13.9, CV 4.2–37%)
(Table 2). Thus, the indentation test protocol provided
measurements that were reasonably precise for an indi-
vidual sample, relative to the variation between samples
of the same experimental group.
Indentation stiffness was slightly different be-
tween the MFC and LFC locations for certain experi-
mental groups, and was markedly different among the
experimental groups within the LFC and MFC locations
(Figures 2A and B). YN samples were stiffer at the LFC
location (P ⬍ 0.05) than at the MFC location (difference
in indentation stiffness score ⫽ 9). Other experimental
groups (MN, MD, ON, and OD) did not show statisti-
cally significant differences between the LFC and MFC
locations. In contrast, at both the LFC and MFC loca-
tions, indentation stiffness varied markedly (P ⬍ 0.001)
among the sample groups, particularly between the
normal-sample and degenerate-sample groups. For both
the LFC and MFC locations, the normal-sample age
groups (YN, MN, and ON) had stiffness values that were
indistinguishable (P ⬎ 0.8). In contrast, the degenerate-
sample groups (OD and MD) had stiffness values that
were significantly lower than those of the YN group
(P ⬍ 0.001 in each case) as well as the age-matched

Table 2. Indentation stiffness variability in experiment 2*

Intrasample variability
Intersample variability
Repositioning Triggering
No. of
Group Avg SD SD/Avg samples SD CV sCV SD CV sCV
YN 68 7.1 10.4 18 3.2 5.8 5.6 2.6 4.9 4.7
MN 65 10.2 15.7 20 2.5 6.3 6.0 2.8 5.6 5.4
MD 30 4.2 14.0 4 2.9 9.9 9.5 2.4 10.5 10.1
ON 65 10.0 15.5 20 4.1 9.6 9.2 2.8 5.7 5.5
OD 37 13.9 37.3 8 4.0 16.0 15.4 3.2 12.0 11.5
All 60 15.2 25.2 70 3.4 9.0 8.6 2.6 5.5 5.2

* Values for SD/Avg (standard deviation/average), CV (coefficient of variation), and sCV (standardized CV) are expressed as percentages. For each
group, there were 3 repositionings and 3 triggerings. YN ⫽ young-age normal; MN ⫽ middle-age normal; MD ⫽ middle-age degenerate; ON ⫽
old-age normal; OD ⫽ old-age degenerate.
3388
Figure 2. Indentation stiffness score (A and B), reflectance score
(C and D), cartilage thickness (E and F), and histopathology scores of
overall (G and H) and surface irregularity characteristics (I and J),
grouped according to adult age and macroscopic grade, as well as
anatomic location. Surf. irreg. ⫽ surface irregularity; YN ⫽ young-age
normal, MN ⫽ middle-age normal, MD ⫽ middle-age degenerate,
ON ⫽ old-age normal, OD ⫽ old-age degenerate; LFC ⫽ lateral
femoral condyle; MFC ⫽ medial femoral condyle. Values are the
mean and SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001.
normal-sample groups (ON and MN, respectively; P ⬍
0.05 in each case). The difference in indentation stiffness
between the normal-sample and degenerate-sample
groups was ⬃30, which was much greater than the
difference between the LFC and MFC locations for each
experimental group.
Reflectance score. The reflectance score was
markedly different between the MFC and LFC locations
for certain experimental groups, and among the experi-
BAE ET AL
mental groups within the LFC and MFC locations
(Figures 2C and D). Both the MN and ON groups had
significantly (P ⬍ 0.05) greater reflectance scores (by
⬃0.2) at the LFC location than at the MFC location,
while the other groups (YN, MD, and OD) had similar
(P ⫽ 1.0) reflectance scores at both locations. Similar to
the pattern for indentation stiffness, normal and degen-
erate samples showed large differences in the reflec-
tance score at the LFC location. Although normal-
sample groups (YN, ON, and MN) had reflectance
scores that were not statistically different (P ⬎ 0.08 in
each case), degenerate-sample groups (MD and OD)
had reflectance scores that were generally lower than
those of the YN group (by ⬃0.3–0.5) as well as the
corresponding age-matched normal-sample groups (MN
and ON, respectively, by ⬃0.3). At the MFC, the reflec-
tance score was also higher in the YN group than in all
other groups. However, there was no difference (P ⬎
0.90 in each case) between normal-sample and
degenerate-sample groups of either middle or old age
(i.e., MN versus MD, and ON versus OD). These results
indicate that after India ink staining, the reflectance
score varied with normal aging and degeneration in a
location-dependent manner.
Cartilage thickness. Cartilage thickness was sim-
ilar for the LFC and MFC locations and between all
experimental groups. Planned comparisons between the
MFC and LFC for each experimental group did not
reveal any effect of anatomic location (P ⫽ 1.0 for each
group). In addition, the experimental groups showed
negligible effects of normal aging and degeneration at
the LFC (P ⫽ 0.6) (Figure 2E) and MFC (P ⫽ 1.0)
(Figure 2F) locations. Cartilage thickness averaged
2.19 ⫾ 0.07 mm for LFC samples and 2.13 ⫾ 0.07 mm
for MFC samples.
Histopathology score. The overall histopathology
scores were similar between LFC and MFC locations but
varied markedly among experimental groups within the
LFC and MFC locations (Figures 2G and H). The
histopathology scores were similar for LFC and MFC
locations in each experimental group (P ⬎ 0.4). For the
LFC location (Figure 2G), the normal-sample groups
(YN, MN, ON) had histopathology scores (average
2.1 ⫾ 1.3) that were indistinguishable (P ⫽ 1.0). Here,
the degenerate-sample groups had histopathology scores
that either were significantly higher than those of the
YN group as well as the age-matched normal-sample
groups or that exhibited strong trends for such differ-
ences. For the MFC location (Figure 2H), the normal-
sample groups showed an age-related change, with a
significant (P ⬍ 0.01) increase in histopathology score
from YN (mean ⫾ SD 1.6 ⫾ 0.2) to ON (3.9 ⫾ 0.3). In
SENSITIVITY OF INDENTATION TESTING TO CARTILAGE DEGENERATION 3389

Table 3. Principal components analysis* (␳2 ⫽ 0.44) (Figure 3C). A decrease in indentation
Parameter Factor 1 Factor 2 stiffness was also related to an increase in the surface
irregularity characteristic of the histopathology score
Reflectance score ⴚ0.858 ⫺0.048
Histopathology overall score 0.843 0.012 (␳2 ⫽ 0.34) (Figure 3D). However, indentation stiffness
Indentation stiffness score ⴚ0.804 0.079 showed no correlation with cartilage thickness (R2 ⫽
Age 0.726 0.292 0.01) (Figure 3B).
Cartilage thickness 0.090 0.985
Sensitivity and specificity of indentation stiffness to
% of total variance explained by the factor 53.3 20.0 the indices of degeneration. As expected, the sensitivity of
* Dominant parameters are shown in boldface. Except where indicated indentation stiffness to macroscopic degeneration in-
otherwise, values are the loading coefficient of variation. creased with an increasing cutoff value for the indenta-
tion stiffness score, while specificity exhibited the oppo-
addition, at the MFC location, the degenerate samples site trend. Sensitivity to macroscopic degeneration
had histopathology scores that were significantly higher (Figure 4A) increased with indentation stiffness cut-
than those of the YN group (P ⬍ 0.05 in each case) and off values ranging from 15 to 50, reaching 90% at the
showed trends toward being higher than those of their indentation stiffness cutoff value of 48. The specificity
respective age-matched normal-sample groups. for macroscopic degeneration (Figure 4A) was maxi-
Among histopathologic features, the surface ir- mum at relatively low indentation stiffness cutoff
regularity characteristic was the one that varied signifi- values, and decreased to 90% specificity at the in-
cantly between groups and contributed to the variation dentation stiffness cutoff of 52. Thus, the indentation
in the overall score (Figures 2I and J). The representa- stiffness cutoff range of 48–52 provided an area of
tive micrographs of YN (Figure 1C) and OD (Figure
1D) illustrate the typical surface irregularity and vertical
and horizontal clefts into the transitional zone in the
degenerate-sample groups. Both the overall histopathol-
ogy score and the surface irregularity characteristic of
the histopathology score showed a pattern (Figures
2G–J) that was fairly similar (inversely related) to that of
the indentation stiffness values (Figures 2A and B).
Relationship between indentation stiffness and in-
dices of degeneration. Principal components analysis. A
total of 2 principal components was needed to account
for the major variations in sample parameters (Table 3).
Factor 1, which accounted for 53% of the total variance,
was determined by 4 dominant (loading coefficient of
⬎0.5) parameters: indentation stiffness, the reflectance
score, the histopathology score, and donor age. Factor 2,
which accounted for 20% of the total variance, com-
prised only one dominant parameter, cartilage thickness.
The association of the dominant parameters (all para-
meters listed in Table 3 except cartilage thickness)
contributing to factor 1 indicates generally strong corre-
lations between any 2 individual dominant parameters.
In contrast, cartilage thickness was the only nondomi-
nant parameter contributing to factor 1 while being the
Figure 3. Relationship between indentation stiffness and the indices
only dominant parameter contributing to factor 2. This of degeneration. A, Reflectance score. B, Cartilage thickness.
indicates that cartilage thickness is weakly correlated C, Overall histopathology score. D, Surface irregularity histopathology
with or unrelated to any of the other parameters. score. Experimental groups include young-age normal (䊐), middle-age
Linear regression. The indentation stiffness val- normal (‚), middle-age degenerate (Œ), old-age normal (E), and
old-age degenerate (F). Lines represent the linear regression fits of
ues correlated with certain indices of degeneration. A
the data and are shown only to indicate trends. For A and B,
decrease in indentation stiffness was related to a de- parametric linear regression analysis was performed to determine
crease in the reflectance score (R2 ⫽ 0.35) (Figure 3A), P and R2 values. For C and D, Spearman’s rank correlation was used
as well as an increase in the overall histopathology score to determine P and ␳2 values.
3390
Figure 4. Sensitivity analysis. Left column, Sensitivity (– –, —, – – –)
and specificity (‚, }, and E) of indentation stiffness to macroscopic
degeneration (A), the reflectance score (C), and the overall histopa-
thology score (E). Right column, Receiver operating characteristic
plots (‚, }, and E) of indentation cutoff sensitivity and specificity to
macroscopic degeneration (B), the reflectance score (D), and the
overall histopathology score (F). Different symbols and lines (C–F)
represent the threshold values for the index of degeneration, which
were calculated as the average of all macroscopically normal samples
(E and – –), the average of all macroscopically degenerate samples
(‚ and – – –), and the average of the 2 values (} and —). DGN ⫽
degenerate; NL ⫽ normal.
high sensitivity and specificity, with each parameter
being ⬎90%. The corresponding ROC curve (Figure
4B) rose rapidly and reached maximum sensitivity at a
low 1 ⫺ sensitivity value. When sensitivity increased to
0.9, 1 ⫺ specificity had already decreased to 0.04. The
area under the ROC curve was 0.96, which was close to
the ideal test value of 1.0. This ROC curve analysis
indicates that indentation testing is highly accurate in
discriminating between macroscopically normal and de-
generate samples.
Indentation stiffness was also sensitive to degen-
eration, as judged by the reflectance and histopathology
scores. For the reflectance score, the threshold values,
calculated as a) the average of macroscopically normal
samples, b) the average of macroscopically degenerate
samples, and c) the midpoint between a and b, were
0.59, 0.35, and 0.47, respectively. The sensitivity and
specificity curves right-shifted as the reflectance score
threshold value increased (i.e., became increasingly
normal) (Figure 4C), without marked differences be-
tween the ROC plots (Figure 4D). For the reflectance
BAE ET AL
score threshold values of 0.59 (average of normal sam-
ples), 0.35 (average of degenerate samples), and 0.47
(midpoint), the area under the ROC curve was 0.82,
0.78, and 0.76, respectively. For the overall histopathol-
ogy score, the threshold values were 2.3 (average of
normal samples), 7.3 (average of degenerate samples),
and 4.8 (midpoint). The sensitivity and specificity curves
also right-shifted as the histopathology score threshold
value decreased (i.e., became increasingly normal)
(Figure 4E). However, the amount of shift was greater
for sensitivity than for specificity. Consequently, ROC
plots (Figure 4F) were distinct for the 3 threshold values.
For the histopathology threshold values of 2.3, 7.3, and
4.8, the area under the ROC plots was 0.79, 0.95, and
0.91, respectively. These results indicate that indentation
stiffness can distinguish between samples of distinct
histopathologic grades.
DISCUSSION
This study provides a detailed examination of the
short-duration indentation stiffness of human articular
cartilage, gross morphologic and structural indices of
early cartilage degeneration in the same samples, and
relationships between indentation stiffness and indices
of degeneration. The results indicate that indentation
stiffness generally decreases as articular cartilage degen-
eration progresses to a mild stage, as assessed by gross
morphology (Figures 2A and B), India ink staining
(Figures 2C and D), and histopathology (Figures 2G–J).
The variation in indentation stiffness correlated well
with parameters that are indicative of early changes in
the articular surface, including India ink staining (Figure
3A) and the surface irregularity characteristic (Figure
3D) of the histopathology score (Figure 3C). For these
samples that ranged from normal to an early stage of
degeneration (without demonstrating cartilage thin-
ning), indentation stiffness did not depend detectably on
cartilage thickness (Figure 3B). For macroscopically
normal samples, the indentation stiffness score did not
depend on age groups (Figures 2A and B). The inden-
tation stiffness values were diagnostically sensitive to
(and specific for) macroscopic degeneration (Figures 4A
and B), as well as for degeneration as described by
reflectance scores after India ink staining (Figures 4C
and D) and histopathology scores (Figures 4E and F).
Because variations in indentation stiffness will
depend markedly on the types of samples studied, the
samples were chosen in accordance with the specific
objective of determining whether early stages of degen-
eration could be discerned by indentation stiffness mea-
surement. Samples from donors ages 28–91 years were
SENSITIVITY OF INDENTATION TESTING TO CARTILAGE DEGENERATION
chosen to represent a wide range of adult (skeletally
mature) samples. In addition, only macroscopically nor-
mal and mildly degenerate samples were selected for all
age groups, because more severe erosion can be readily
identified by macroscopic inspection. It should be noted
that such samples are not representative of randomly
selected samples from the particular age group, because
the frequency and severity of degeneration increase with
age (48).
Both the LFC and the MFC were of interest to
examine, because both sites are prone to age-associated
degeneration, with the MFC showing such degeneration
at an earlier age (48). Other studies of indentation of
human articular cartilage samples have tested these
same sites (2,4,5,39) as well as sites on the patellofemo-
ral groove (2,4,5,39), patellae (2,4), tibial plateau
(2,4,39), and femoral head (7,17). However, those sam-
ples were not chosen according to both age and degen-
eration grade criteria.
The repeatability of measurements from any
instrument, when used by one or many individuals, is
important to characterize. This is especially true for a
handheld mechanical test instrument. The variability in
indentation stiffness measurements, expressed as the
standard deviation of intraobserver and interobserver
measurements (SD 2.4–5.6 and 4.4–8.3, respectively)
(Table 1), was small relative to the differences between
the normal-sample and degenerate-sample groups (dif-
ference in indentation stiffness scores of ⬃30) (Table 2
and Figures 2A and B). Therefore, the probe was able to
distinguish differences associated with degeneration.
The variability in repeated measurements has not been
reported often but would likely be affected by the exact
setting in which measurements are made and by the type
of samples that are analyzed.
Additional studies in clinical situations, or in
arthroscopic simulations on cadaveric knee joints, may
be useful to characterize the probe further. The intraob-
server variation of stiffness measurements obtained by
successively placing the probe tip at the “same” site was
generally the highest for moderately soft samples. This
suggests that spatial gradients in indentation stiffness
exist, and that the indentation stiffness measurement is
also sensitive to very local variations in cartilage struc-
ture. Nevertheless, the standard protocol used here, 9
measurements from 3 tip applications, yielded a precise
measure. It is possible, however, that fewer measure-
ments (i.e., only 1 triggering) can be made with more tip
applications (i.e., ⬎3 repositionings) to increase the
speed of measurement at a particular site.
The strong dependence of indentation stiffness
on certain indices of degeneration is generally consistent
3391
with the findings of previous studies, whereas the weak
variation among the different age groups indicates that
age is not a strong confounding variable. Previous
studies showed that cartilage indentation stiffness de-
creased with osteoarthritic degeneration (17,18). Negli-
gible effects of normal aging on indentation stiffness
have also been reported previously, although in those
studies only gross morphology was used to distinguish
between normal and degenerate specimens (18,39).
The variation in India ink staining among normal
samples at the MFC location (Figure 2D) is consistent
with the general increase in ink staining with increasing
age in randomly selected samples, at various cartilage
surfaces in the human knee joint (20), as well as in
osteoarthritis (28). The slight increase in histopathology
score, and in particular surface irregularity, also con-
firms the selection of relatively normal samples but also
of slight age- and site-related variations in the normal-
sample groups. In addition, the lack of significant vari-
ation in thickness of the samples in the 5 experimental
groups used in this study also indicates that the samples
used in this study were appropriately chosen to be in
early stages of degeneration without deep erosion, be-
cause cartilage is known to become thinner with the
more extensive degenerative changes of aging (49,50)
and osteoarthritis (49,51).
The difference in indentation stiffness scores,
determined by ACTAEON probe, between young-
normal LFC and young-normal MFC samples (mean ⫾
SD of 73 ⫾ 6 and 64 ⫾ 7, respectively) (Figures 2A and
B) is generally consistent with biomechanical measures
in similar human cartilage samples. Another short-
duration indentation study using a handheld instrument
showed mean ⫾ SD indenter forces of 5.6 ⫾ 1.2
Newtons and 4.9 ⫾ 1.2 Newtons (4) for the LFC and
MFC sites, respectively. A long-duration indentation
study showed similar results, with a mean ⫾ SD aggre-
gate modulus of 0.70 ⫾ 0.23 MPa for the LFC and
0.59 ⫾ 0.11 MPa for the MFC (13).
The tensile strength of the superficial layer of
cartilage is another measure that may be closely related
to indentation stiffness (33). In a recent study, the
mean ⫾ SD tensile strength for LFC and MFC cartilage
was found to be 19.6 ⫾ 4.8 MPa and 7.8 ⫾ 7.4 MPa,
respectively (52), which also is in agreement with the
site-associated difference seen in the present study.
However, it is not necessarily straightforward to com-
pare a rapid, clinically applicable indentation test result,
as determined here, with biomechanical measures that
are commonly derived from laboratory techniques. Fac-
tors such as inhomogeneity (32) and anisotropy (53) of
articular cartilage affect the manner in which a cartilage
3392
sample behaves under different loading configurations.
In a confined compression test, for example, induced
strains are mostly in the axial (loading) direction (32). In
comparison, short-term indentation loading induces
complex intra-tissue strain that consists of axial com-
pression, transverse tension, and shear (30). In addition,
comparisons among various indentation methods may
even be difficult, because slight differences in indenter
geometry and indentation loading protocol significantly
affect the extent and magnitude of strains in the human
cartilage samples (30).
Results of this study are consistent with and
extend results of a past study that examined how inden-
tation stiffness varies with cartilage degeneration (5), by
defining in more detail the relationship between stiffness
and specific features of cartilage degeneration. The
significant negative correlation between indentation
stiffness and histopathology score (Figure 3C) confirms
the findings of a recent study that used a different
indentation probe and the Mankin histopathology scor-
ing system (5,28). Because histopathologic grading
schemes involve many components of structural and
cellular organization of articular cartilage (28,29), it was
not clear whether specific components of the degenera-
tion process were responsible for the correlation with
indentation stiffness. The detailed histopathologic ana-
lyses in this study indicated a particularly strong corre-
lation of indentation stiffness with surface disruption, as
indicated by the reflectance score (Figure 3A) and the
surface irregularity characteristic of the histopathology
score (Figure 3D). This result seems quite reasonable,
because indentation causes extension and shear of car-
tilage near and underneath the indenter and would be
predicted to be sensitive to alteration of the properties
of the cartilage tissue near and at the articular surface.
One of the factors that affected the strength of
correlation may be the proximity of the portions of the
individual cartilage samples that were used for the
various analyses. Indentation testing would be expected
to be affected the most by the properties of tissue near
the indenter (54). The reflectance score was measured
for the whole articular surface of the sample, although
additional analyses of more localized regions near the
indentation site did not markedly alter the extent of
correlation between indentation stiffness and the reflec-
tance score (data not shown). In addition, the histopa-
thology grading examines cartilage that is only a thin
vertical tissue section, which represents only a small
fraction of the volume of cartilage that is likely to affect
the indentation measurements. Nevertheless, the corre-
lations observed suggest that such sample analysis and
BAE ET AL
comparison provide useful information on the relation-
ships among the different tissue properties.
The relationship between indentation stiffness
and cartilage thickness is an important factor to consider
for clinical application of indentation stiffness. In theory
(30,31,55,56), as the cartilage becomes thin, the appar-
ent stiffness measured by the indentation device will
increase as the device starts to be influenced by the
bone. If cartilage is modeled as a homogeneous, isotro-
pic, and incompressible layer bonded to a rigid base
(30), for a sphere-ended indentation depth of 100 ␮m,
there is an increase of ⬃20% in the apparent stiffness
(defined as load/displacement) as the thickness de-
creases from 3.5 to 1.5 mm, which encompasses the
thickness of cartilage sample in this study. This phenom-
enon becomes prominent when the cartilage thickness is
less than or similar to the indenter characteristic size
(i.e., radius). Because it is not always possible in vivo to
determine the thickness of the cartilage sample being
indented, it is preferred to have an indentation probe
that outputs similar stiffness measurements irrespective
of the cartilage thickness. In this regard, the lack of
correlation between indentation stiffness and cartilage
thickness (Figure 3B) indicates that for the normal and
mildly degenerate sample populations used in this study,
the variation in thickness has no marked effects on
indentation stiffness measured by the probe. As cartilage
becomes thinner, as it will in more advanced stages of
degeneration, the sample thickness may begin to influ-
ence the indentation stiffness measurements consider-
ably. However, at these later stages of degeneration, it is
likely that the diagnosis of severe degeneration can be
made without the aid of an indentation instrument.
The indentation stiffness scores can help a user
interpret the results of the indentation testing in a
number of ways. The values for YN cartilage samples
(73 ⫾ 6 and 64 ⫾ 7 for the LFC and MFC, respectively)
(Figures 2A and B) may be used as reference (normal)
values against which other values measured by the probe
on a patient can be compared. In addition, it may be
possible to monitor progression of cartilage disease or
repair by indentation testing. Based on the values of
standard deviation of repeated measurements for inden-
tation stiffness scores (⬃3.5 for repositioning over all of
the test populations) (Tables 1 and 2), and based on
performing 3 successive measurements as was done
here, the 95% confidence interval for a site measure-
ment is ⫾5 (47). This is much less than the difference
(⬃30) in indentation stiffness scores between normal-
sample and degenerate-sample groups. In contrast, vari-
ability of measurements between sites and between
SENSITIVITY OF INDENTATION TESTING TO CARTILAGE DEGENERATION
operators is a factor that would complicate use of
indentation stiffness probes, in general.
ROC analyses defined the sensitivity and speci-
ficity of the indentation stiffness measurement, as well
as the accuracy of indentation stiffness as a diagnostic
test. The observed area under the ROC plots of 0.76–
0.95 (Figures 4D and F) compares favorably with many
of the typical values (of the area under the ROC curve)
found for medical diagnostic tests, in particular imaging
methods. In a study examining the relationship between
radiographic osteoarthritis and patient-reported knee
pain, the area under the ROC plots was at best 0.79 (57).
In another study comparing the ability of various mag-
netic resonance imaging field strengths and pulse se-
quences to detect relatively large (diameter of 4–12 mm)
experimental cartilage lesions, the area under the ROC
plots ranged from 0.59 to 0.96 for 10 different protocols,
with a mean ⫾ SD of 0.76 ⫾ 0.13 (58). The sensitivity
and specificity, along with the ROC plots, can help a
user interpret the results of the indentation testing by
providing objective bases for the accuracy of indentation
stiffness measurements for detecting cartilage degener-
ation. This type of analysis is particularly useful in that
the user can adopt it for a wide range of diagnostic
situations. For example, for screening situations in which
one would be willing to sacrifice specificity to achieve
high sensitivity, the user would select an indentation
cutoff value that is relatively high (thus more samples
are diagnosed as degenerate), and know exactly what the
trade-off would be.
Although this study focuses on the use of inden-
tation testing as a clinical diagnostic device, there are
many other possible uses for indentation testing. In the
past, the selection criterion for distinguishing between
normal and degenerate cartilage has been gross macro-
scopic observation. It would be possible to use indenta-
tion stiffness alone as a categorization tool, or to use
indentation stiffness along with the relationships be-
tween indentation stiffness and other indices of degen-
eration, found in this study, to target sample groups with
certain values for these other degeneration indices.
In conclusion, the marked changes in indentation
stiffness, the reflectance score, and the histopathology
score were observed to be primarily associated with
cartilage degeneration and not with aging or cartilage
thickness. The indentation stiffness appeared to be
particularly sensitive to the articular surface disruption
evident in degenerate specimens, but insensitive to the
range of cartilage thickness seen in this study, which
makes it useful for assessing the early-stage degenera-
tion as well as a repaired or regenerated tissue after a
3393
therapeutic intervention. The ROC analyses gave a
guideline for diagnosis and a quantitative measure of
comparing the accuracy of this device with other means
of diagnosing the health of articular cartilage. In princi-
ple, indentation stiffness could be used to identify local
areas of cartilage degeneration and to instigate appro-
priate interventions designed to prevent more severe
deterioration. Although it is ultimately the responsibility
of the user to interpret and apply the indentation
stiffness results, these guidelines provide detailed and
objective bases for clinical applications.
ACKNOWLEDGMENTS
We thank Karen Bowden for preparation of the histo-
logic slides, and Arthrex Inc. for providing the Osteochondral
Autograft Transfer System.
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