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Influence of Cell Differentiation and IL-1 Expression on Clinical Outcomes After Matrix-Associated
Chondrocyte Transplantation
Christian Albrecht, Brigitte Tichy, Lukas Zak, Silke Aldrian, Sylvia Nrnberger and Stefan Marlovits
Am J Sports Med 2014 42: 59 originally published online November 6, 2013
DOI: 10.1177/0363546513507543

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Influence of Cell Differentiation
and IL-1b Expression on Clinical Outcomes
After Matrix-Associated Chondrocyte
Transplantation
Christian Albrecht,*yz MD, PhD, Brigitte Tichy,yz Lukas Zak,yz MD, Silke Aldrian,yz MD,
Sylvia Nurnberger,yz PhD, and Stefan Marlovits,yz MD
Investigation performed at the Department of Trauma-Surgery,
Medical University of Vienna, Vienna, Austria

Background: Several patient- and defect-specific factors influencing clinical outcomes after matrix-associated chondrocyte
transplantation (MACT) have been identified, including the patients age, location of the defect, or duration of symptoms before
surgery. Little is known, however, about the influence of cell-specific characteristics on clinical results after transplantation.
Purpose: The aim of the present study was to investigate the influence of cell differentiation and interleukin-1 b (IL-1b) expression
on clinical outcomes up to 5 years after MACT.
Study Design: Case series; Level of evidence, 4.
Methods: Twenty-seven patients who underwent MACT of the tibiofemoral joint area of the knee were included in this study. Clin-
ical assessments were performed preoperatively as well as 6, 12, 24, and 60 months after transplantation by using the following
scores: the Knee injury and Osteoarthritis Outcome Score (KOOS), the International Knee Documentation Committee (IKDC) Sub-
jective Knee Form, the Noyes sports activity rating scale, the Brittberg clinical score, and a visual analog scale (VAS) for pain. The
quality of repair tissue was assessed by magnetic resonance imaging using the magnetic resonance observation of cartilage
repair tissue (MOCART) score at 1 and 5 years. Cell differentiation (defined as collagen type II:type I expression ratio), aggrecan,
and IL-1b expression were determined by real-time polymerase chain reaction in transplant residuals and were correlated with
clinical outcomes.
Results: The largest improvements in clinical scores were found during the first year. Two years postoperatively, a stable
improvement was reached until 5 years after transplantation, with a mean IKDC score of 34.4 6 8.6 preoperatively to 77.9 6
16 after 24 months (P \ .001). Cell differentiation showed a significant positive correlation with nearly all clinical scores at different
time points, especially after 12 months (P \ .05). IL-1b expression negatively influenced clinical outcomes at 24 months (Brittberg
score) and 60 months (Brittberg and VAS scores) after surgery (P \ .05). No correlation was found between the MOCART score
and clinical outcomes or gene expression.
Conclusion: Our data demonstrate that cell differentiation and IL-1b expression influence clinical outcomes up to 5 years after
MACT.
Keywords: chondrocytes; MACT; transplantation; clinical outcome; differentiation; IL-1b

*Address correspondence to Christian Albrecht, MD, PhD, Depart-

ment of Trauma-Surgery, Medical University of Vienna, Waehringer Guer-
tel 18-20, 1090 Vienna, Austria (e-mail: christian.albrecht@meduni
wien.ac.at).
y
Department of Trauma-Surgery, Center for Joint and Cartilage, Med-
ical University of Vienna, Vienna, Austria.
z articular cartilage has a very limited capability to self-
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
One or more of the authors has declared the following potential con-
flict of interest or source of funding: The study was supported by the
research budget of the Department of Trauma-Surgery, Medical Univer-
sity of Vienna.
The American Journal of Sports Medicine, Vol. 42, No. 1
DOI: 10.1177/0363546513507543
2013 The Author(s)
Articular cartilage defects caused by trauma, knee insta-
bility, or improper mechanical loading often lead to ongo-
ing pain, negatively affect the quality of life, and
predispose to osteoarthritis in the long term.9,18 Because
repair, cartilage injuries are a prime target for regenera-
tive approaches such as tissue engineering. Matrix-associ-
ated chondrocyte transplantation (MACT) is a tissue-
engineering approach using the chondrogenic capacity of
autologous transplanted cells.33 In a 2-step procedure,
chondrocytes are isolated from a small biopsy specimen
taken from a nonweightbearing area of the joint,

59
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60 Albrecht et al
propagated in vitro, seeded onto a 3-dimensional scaffold,
and subsequently cultured for various periods (depending
on the standard operating procedures of the manufac-
turers) before implantation into the defect. The safety
and clinical effectiveness of this procedure for the treat-
ment of moderate to large symptomatic full-thickness
articular cartilage defects have been demonstrated in sev-
eral studies, showing significant improvement in pain,
function, and activity up to at least 5 years.4,6,15,19,20,25,30,45
Several parameters influencing clinical outcomes after
MACT have been identified including localization of the
defect, duration of symptoms before surgery, and the
patients age.21,28,45 However, the influence of cell differen-
tiation and other specific cell characteristics of chondro-
cytes used for transplantation on clinical results after
autologous chondrocyte transplantation is still poorly
investigated. In a recent study, we demonstrated that
gene expression (collagen type II:type I ratio, aggrecan,
and interleukin-1 b) varied highly within cartilage trans-
plants of the same scaffold type in different patients.1
For further optimization of transplants, it is important to
know which parameters positively influence clinical out-
comes after MACT.
Collagen type II and aggrecan are 2 of the major compo-
nents of the extracellular matrix in articular cartilage and
are responsible for its unique mechanical properties. Dur-
ing in vitro cultivation of chondrocytes, however, the
expression of these proteins is diminished.31,46 In place of
collagen type II, the expression of collagen type I, which
is normally found only in fibrocartilage and the superficial
zone of articular cartilage,31,50 increases. To quantify this
process, called dedifferentiation, a differentiation index
defined as the ratio of COL2A1 to COL1A1 expression is
used.2,13,24,29 This index, first introduced by Martin
et al,34 attains a higher value with a more chondrocytic
phenotype and shows less variability than a single gene.
Besides chondrocytes cultivated in vitro, osteoarthritic
chondrocytes also exhibit a lower differentiation index.34
Interleukin-1 b (IL-1b) is a proinflammatory cytokine
that plays a central role in connective tissue destruction
and inflammation.22 In cartilage, IL-1b is considered a pri-
mary instigator of osteoarthritis, disrupting the metabolic
balance of chondrocytes and reducing COL2A1 expres-
sion.8,42 Different sites of action have been identified for
IL-1b in chondrocytes. Bauge et al3 have demonstrated
that IL-1b antagonizes the transforming growth factor b
(TGFb) pathway through down-regulation of its TbRII
receptor. Others have shown that IL-1b promotes prolifer-
ation and dedifferentiation by down-regulation of fibro-
blast growth factor receptor 3 (FGFR3) or up-regulation
of sirtuin 1 (SIRT1).22,48 Inhibition of IL-1b via RNA inter-
ference, on the other hand, has been demonstrated to
increase transcript levels of TGFb.43
There have not been any reports on the influence of IL-
1b expression or cell differentiation on the success rate
after MACT. The aim of this study, therefore, was to exam-
ine the effect of these parameters on clinical outcomes up
to 5 years after MACT.
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The American Journal of Sports Medicine
MATERIALS AND METHODS
Study Design and Participants
In this study, 27 patients with symptomatic traumatic
defects of the articular cartilage of the knee (tibiofemoral
joint area) were included, who had been treated between Jan-
uary 2001 and July 2005 with MACT at a single academic
clinical center. During this time period, a total of 71 patients
with cartilage defects in the knee were treated with MACT
using Hyalograft C (Fidia Advanced Biomaterials, Abano
Terme, Italy). Because differences in clinical outcomes have
been described between cartilage transplantations in the
tibiofemoral and patellofemoral joint area,21 only patients
with defects restricted to the tibiofemoral joint area (n =
32) were selected for the study. From 3 patients, no trans-
plant residual could be obtained, or the isolated RNA did
not fulfill the quality standards. Two patients were lost to fol-
low-up. Ethical approval for the study was obtained from the
institutional review board of the hospital.
Study participants were men and women between 19 and
50 years of age with a defect size of .2 cm2 and no knee
instability or misalignment (axis deviation .5). There
were no restrictions on the upper limit of the defect size
or the number of defects. Patients were excluded from the
study if they had a body mass index (BMI) of .30 kg/m2,
totally or subtotally resected menisci, severe neurological
disorders, metabolic arthritis, joint infections, tumors, psy-
chiatric diseases, arthrofibrosis, or autoimmune diseases
or were pregnant. All patients provided written informed
consent before study enrollment.
At the time of initial arthroscopic surgery and grading
of the defect, 200 to 300 mg of normal full-thickness hya-
line cartilage was biopsied from a minimally weightbear-
ing area of the intercondylar notch, placed in a transport
medium, and then sent to Fidia Advanced Biomaterials.
The graft, Hyalograft C, was produced by the manufac-
turer as follows: The patients chondrocytes were isolated
from biopsy specimens and multiplied in a monolayer cul-
ture. According to the manufacturer, the maximum num-
ber of passages for 2-dimensional cultivation was defined
in the production process, which was not further specified.
The cells were thereafter seeded onto a hyaluronan web at
a density of 1 3 106/cm2 and cultivated for at least 2 weeks.
The cells were cultivated in a culture medium supple-
mented with fetal calf serum.
Surgical Technique
Through a mini-arthrotomy, the cartilage defect was pre-
pared by curettage to remove any fissured and undermined
cartilage. Debridement of the subchondral bone plate was
performed with care to prevent perforation or subchondral
bone bleeding. The dimensions of the defect were trans-
ferred to a template; the hyaluronan fleece containing the
seeded chondrocytes was trimmed to exactly fit into the
defect and then implanted. Fibrin glue (Tissucol, Baxter,
Vienna, Austria) was applied on the edges to fix the graft;
Vol. 42, No. 1, 2014
no periosteal cover or sutures were used. The joint was
manipulated intraoperatively to ensure adherence and sta-
bility of the implant. At the conclusion of the procedure, the
joint was wrapped in a compressive elastic bandage.
Postoperative Rehabilitation
Immediately after surgery, immobilization of the joint was
recommended for 12 to 24 hours, followed by a standardized
rehabilitation program. Early rehabilitation began on the
second postoperative day with continuous passive motion
(range in sagittal plane, 0-0-40) for 6 to 8 hours per
day and was continued for 6 weeks. Range of motion was
increased on a daily basis. In addition, patients were asked
to perform isometric muscle contractions and circulation
exercises for their lower limb.
Patients were instructed to use crutches immediately
after surgery to avoid full weightbearing. Toe-touch
weightbearing was permitted for 4 weeks. After this,
patients began partial weightbearing of 20% of their body
weight (up to week 6) and 50% of their body weight (up
to week 8) and then progressed to full weightbearing by
8 weeks (up to week 10). Full weightbearing was defined
as walking without crutches to an extent that allowed
patients to perform activities of daily living. A standard
bathroom scale was used to instruct patients about relative
amounts of weightbearing.
For strengthening, patients were assigned isometric,
concentric, and eccentric exercises (open and closed kinetic
chain) in the first week after surgery. Neuromuscular exer-
cises were also implemented to improve the dynamic sta-
bility of the knee.
Clinical Outcomes
Five clinical scores with documented reliability and valid-
ity were used to assess outcomes. This included the Inter-
national Knee Documentation Committee (IKDC)
Subjective Knee Form, which measures subjective symp-
toms, joint function, and sports activities,23,27,47 and the
Knee injury and Osteoarthritis Outcome Score (KOOS)
with the following subscores: pain, symptoms, activities
of daily living, function in sport and recreation, and
knee-related quality of life.16,30,41 Furthermore, the Noyes
sports activity rating scale26,30,38 grading the level and
intensity of athletic activity, the Brittberg clinical
score,7,36,51 and a visual analog scale (VAS) grading pain
only5,14,47 were evaluated. Clinical assessments were per-
formed preoperatively and at 6, 12, 24, and 60 months after
transplantation. Patients were asked to keep diaries of
their activities and exercise regimen.
Magnetic Resonance Imaging
Characteristics of the articular cartilage repair tissue were
evaluated by magnetic resonance imaging (MRI) performed
at 12 and 60 months after MACT in accordance with the
magnetic resonance observation of cartilage repair tissue
(MOCART) score.32 Scoring was recorded for 9 separate
parameters of graft outcome: (1) degree of the defect fill,
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Role of Cell Differentiation in MACT 61
(2) integration with the border zone, (3) structure of the
repair tissue, (4 and 5) signal intensity on 2 different
sequences (proton densityweighted turbo spin echo and
dual fast spin echo; classified as normal, nearly normal,
and abnormal, relating to signal alteration in comparison
with the adjacent cartilage), integrity of the (6) subchondral
lamina and (7) bone, and presence of (8) adhesions and (9)
effusion. The total score ranges from 0 to 100. The MRI
scans were viewed by 2 independent examiners experienced
in musculoskeletal imaging and cartilage repair.
Gene Expression Analysis
Residuals of 27 Hyalograft C autografts were collected dur-
ing surgery and stored in a transport medium at room tem-
perature. Immediately after surgery, the samples were
brought to the laboratory for further processing. Trans-
plant samples were immersed in TRI reagent (Sigma-
Aldrich, St Louis, Missouri) for lysis of the cells and stored
at 80C until RNA extraction, which was performed
according to the standard protocol.
cDNA Synthesis
Total RNA (0.1-1 mg) was diluted with nuclease-free water
to a volume of 15 mL. Thereafter, 4 mL of iScript Reaction
Mix as well as 1 mL of iScript reverse transcriptase were
added (Bio-Rad Laboratories, Hercules, California). The
reaction mixture was incubated for 5 minutes at 25C,
for 30 minutes at 40C, and for 5 minutes at 85C.
Primers and Probes for Quantitative Analyses
The design of the primers and probes has previously been
described.1 To avoid the amplification of genomic DNA, the
probes were placed at the junction of 2 exons. For IL-1b,
the predeveloped TaqMan assay (Applied Biosystems, Fos-
ter City, California) was used.
Real-Time PCR Amplification and Analysis
Real-time polymerase chain reaction (PCR) amplification
was performed and monitored using an ABI Prism 7500
Fast Real-time PCR System (Applied Biosystems). The
master mix was based on the SensiMix Probe Kit (Quan-
tace, London, United Kingdom). The thermal cycling condi-
tions comprised the initial steps at 50C for 2 minutes and
at 95C for 10 minutes. Amplification of the cDNA products
was performed with 40 PCR cycles, consisting of a denatur-
ation step at 95C for 15 seconds and an extension step at
60C for 1 minute. b-2 microglobulin (B2M) was chosen as
the housekeeping gene, using the predeveloped TaqMan
assay.17 For a given amount of cDNA, the range of the
housekeeping gene was within 61.5 Ct of the median
value; samples outside of this range were excluded from
analysis. All cDNA samples (2.4 mL of cDNA in a total vol-
ume of 20 mL) were analyzed in triplicate. Relative quanti-
fication of gene expression was performed using the
comparative DDCT method. Native cartilage was used as
a calibrator.
62 Albrecht et al The American Journal of Sports Medicine

TABLE 1
Descriptive Statisticsa

COL2A1:COL1A1 Ratio IL-1b Expression

Total (N = 27) 10 Highest 10 Lowest 10 Highest 10 Lowest

Sex, n (%)
Male 22 (81.5) 9 (90) 7 (70) 8 (80) 8 (80)
Female 5 (18.5) 1 (10) 3 (30) 2 (20) 2 (20)
Age, y 36.2 6 9.5 37.3 6 11.8 37.2 6 7.0 36.4 6 12.1 33.9 6 9.2
Duration of symptoms, mo 60.9 6 89.5 45.3 6 91.3 72.4 6 91.2 70.6 6 93.0 41.1 6 68.8
Defect size, cm2 4.2 6 1.3 3.9 6 1.0 4.2 6 1.5 4.7 6 1.1 3.6 6 1.5
BMI, kg/m2 24.8 6 3.6 23.8 6 3.7 25.4 6 3.9 25.9 6 3.4 24.4 6 3.5
Defect location, n
MFC 20 7 8 8 9
LFC 4 2 0 1 0
Tibial plateau 1 0 1 0 0
Combined 2 1 1 1 1

a
Values are expressed as mean 6 standard deviation unless otherwise indicated. BMI, body mass index; COL2A1:COL1A1, collagen type
II:type I; IL-1b, interleukin-1 b; LFC, lateral femoral condyle; MFC, medial femoral condyle.

Statistical Analysis
The statistical analysis included a tabular description of the
demographic data, the subjective clinical scores, and the gene
expression data. Statistical evaluation was performed using
SPSS software version 20.0 (SPSS Inc, Chicago, Illinois).
For statistical analysis, DDCT values were used.52 Values
are presented as the mean 6 standard deviation. A Fried-
man test with post hoc analysis (Wilcoxon signed-rank test)
conducted with a Bonferroni correction was applied to com-
pare the respective scores at different time points. The Wil-
coxon test was used to compare differences between the
MOCART score after 12 and 60 months. For correlation anal-
yses, the Spearman rank correlation coefficient was calcu-
lated. Mann-Whitney U tests were applied to compare the
group of samples with the highest COL2A1:COL1A1 ratio
to the group with the lowest ratio and to compare the group
of samples with the highest IL-1b expression to the group
with the lowest expression. Statistical tests were considered
statistically significant when P values were \.05.
RESULTS
Demographics
Twenty-seven patients who underwent MACT (Hyalograft
C) between January 2001 and July 2005 were included in
this study (Table 1). Because differences in clinical out-
comes have been described between cartilage transplanta-
tions in the tibiofemoral and patellofemoral joint area,21
only patients with defects in the tibiofemoral joint area
were included. Twenty of the defects were located in the
medial femoral condyle, 4 in the lateral femoral condyle,
and 1 in the tibial plateau, and 2 had combined localiza-
tions. The mean age of the patients at the time of implanta-
tion was 36.2 6 9.5 years, the mean duration of symptoms
was 60.9 6 89.5 months, the mean defect size was 4.2 6
1.3 cm2, and the mean BMI was 24.8 6 3.6 kg/m2. There
were 22 male and 5 female participants.
Clinical Assessment
Overall, all patients showed a significant improvement in
all clinical results after 5 years (Figure 1). Patients experi-
enced the greatest improvements during the first year and
reached a stable improvement 2 years postoperatively. The
mean IKDC score increased from 34.4 6 8.6 preoperatively
to 77.9 6 16.0 after 24 months (P \ .001). This status was
maintained through year 5 after transplantation (74.7 6
22.8; P \ .753 compared with 24 months). A similar curve
was observed for the Noyes score, which increased from
a mean of 23.1 6 19.8 preoperatively to 73.0 6 15.7 after
24 months (P \ .001) and was maintained at a high level
(75.8 6 21.5; P \ .084 compared with 24 months) through
year 5. The mean Brittberg score declined from 3.33 6 0.70
before transplantation to 2.15 6 0.85 after 24 months (P \
.001) or 2.25 6 1.04 after 5 years (P \ .004). The mean VAS
pain score improved from 5.42 6 2.20 to 1.35 6 1.74 after
24 months (P \ .001). The difference between 24 months
(1.35 6 1.74) and 60 months (2.05 6 2.38) reached no sta-
tistical significance (P \ .221).
Correlation Analyses With Clinical Outcomes
The IKDC score was used as a representative clinical score
to correlate the improvement in clinical outcomes with
patients age, duration of symptoms, defect size, and BMI
(Table 2). The improvement was calculated by subtracting
preoperative values from follow-up values. The patients
age showed a significantly negative correlation with the
IKDC score at all follow-up time points (6 months: r =
0.602, P \ .005; 12 months: r = 0.406, P \ .049; 24
months: r = 0.541, P \ .014; 60 months: r = 0.636, P \
.003). The duration of symptoms correlated negatively
with clinical outcomes after 24 months (r = 0.486, P \

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Vol. 42, No. 1, 2014 Role of Cell Differentiation in MACT 63

Figure 1. Mean (A) International Knee Documentation Committee (IKDC) and Noyes scores and (B) Brittberg and visual analog
scale (VAS) for pain scores in patients treated with matrix-associated chondrocyte transplantation over time. Error bars = stan-
dard deviation. *P \ .05 for all scores at 6, 12, 24, and 60 months compared with preoperative values.

TABLE 2 0.419, P \ .047, respectively). At 60 months, no correla-

Correlations With Clinical Outcomesa tions were found (data not shown).

IKDC Score
6 mo 12 mo 24 mo 60 mo
Correlation Analyses of Gene Expression
With Clinical Outcomes
Age 0.602b 0.406c 0.541c 0.636b
Duration of symptoms 0.057 0.177 0.486c 0.527c Gene expression data of COL2A1:COL1A1 ratio, aggrecan,
Defect size 0.098 0.088 0.091 0.125 and IL-1b are shown in Figure 2B. The COL2A1:COL1A1
BMI 0.435 0.454c 0.436 0.175 expression ratio ranged from 3.95 to 13.96 DDCT (mean,
a
10.71 6 2.25), which corresponds to a 1031-fold difference
Values are Spearman rank correlations with an improvement in in mRNA expression. For IL-1b, a range from 4.48 to 18.42
IKDC scores from preoperative levels to levels at different follow-up
DDCT (mean, 13.64 6 2.64) was found, which corresponds
time points. Significant values are in bold. BMI, body mass index;
IKDC, International Knee Documentation Committee.
to a 15,716-fold difference in mRNA expression. Aggrecan
b
P \ .01. expression ranged from 5.41 to 12.48 DDCT (mean, 9.08
c
P \ .05. 6 1.53), which corresponds to a 134-fold difference in
mRNA expression.
None of the patient-specific factors such as age, dura-
.03) and 60 months (r = 0.527, P \ .017). Whereas BMI tion of symptoms, BMI, or defect size showed any signifi-
was found to correlate negatively with the IKDC score cant correlation with gene expression (P \ .898, P \
after 12 months (r = 0.454, P \ .029), defect size did not .621, P \ .735, and P \ .762, respectively, for COL2A1:-
show any correlation with clinical outcomes. COL1A1 expression ratio; P \ .476, P \ .072, P \ .579,
and P \ .159, respectively, for IL-1b expression). Further
correlation analyses were performed between the
Correlation Analyses With MRI COL2A1:COL1A1 expression ratio and the improvement
in clinical scores at different follow-up time points (Table
The mean MOCART score was 61.9 6 12.8 at 12 months 4). The improvement was calculated by subtracting preop-
and almost did not change at 60 months after surgery erative values from follow-up values. The best correlation
(59.8 6 17.7) (P \ .923) (Figure 2A). In contrast to clinical was found after 12 months, where the COL2A1:COL1A1
outcomes, patients age and duration of symptoms did not ratio correlated positively with the IKDC (r = 0.517, P \
correlate with the MOCART score after 12 or 60 months .02) and Noyes (r = 0.457, P \ .028) scores but correlated
(Table 3). Defect size showed a negative correlation with negatively with the inverse (ie, in contrast to the other
the MOCART score after 12 months (r = 0.406, P \ .04), scores, a high score corresponds to a bad clinical outcome)
and BMI showed a negative correlation with the MOCART VAS pain score (r = 0.458, P \ .032). When looking at the
score after 60 months (r = 0.550, P \ .008). Neither gene KOOS subscales, the KOOS pain subscore showed a signif-
expression (COL2A1:COL1A1 ratio, aggrecan, IL-1b) nor icantly positive correlation (r = 0.585, P \ .004) at 12
IKDC score exhibited any correlation with the MOCART months. The KOOS symptoms subscore revealed the best
score at 12 or 60 months. When looking at MOCART sub- correlations among all scores, being significant with nearly
scores, only signal intensity (proton densityweighted all follow-up times (6 months: r = 0.558, P \ .011; 24
turbo spin echo) and swelling showed a correlation with months: r = 0.571, P \ .013; and 60 months: r = 0.498, P
the IKDC score at 12 months (r = 0.414, P \ .049 and r = \ .035). At 6 months, a further negative correlation was

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64 Albrecht et al The American Journal of Sports Medicine

Figure 2. Gene expression data and magnetic resonance observation of cartilage repair tissue (MOCART) scores. (A) Mean
MOCART scores at 12 months (61.9 6 12.8) and 60 months (59.8 6 17.7) after surgery. Black line, median; top of gray box,
25th percentile; bottom of gray box, 75th percentile; whisker, 1.5-fold box height or minimum and maximum value, respectively.
(B) Relative gene expression data of COL2A1:COL1A1 ratio, IL-1b, and aggrecan (normalized to B2M expression and related to
COL2A1 expression in native cartilage). Dots indicate outliers.

representative clinical scores are illustrated by scatterplots

TABLE 3 in Figure 3. Because of the demonstrated correlations, the
Correlations With MRIa 10 samples with the highest and lowest COL2A1:COL1A1
ratios of all samples were selected, and the clinical score
MOCART Score data are illustrated in Figure 4 (for descriptive statistics,
12 mo 60 mo see Table 1). The 2 groups did not significantly differ in
age, defect size, duration of symptoms, or BMI (P \ .970,
Age 0.074 0.079 P \ .774, P \ .364, and P \ .384, respectively). The 10 high-
Duration of symptoms 0.014 0.065 est COL2A1:COL1A1 ratio samples showed a better curve for
Defect size 0.406b 0.276 all clinical scores when compared with the 10 samples with
BMI 0.183 0.550b the lowest COL2A1:COL1A1 ratios. The largest differences
COL2A1:COL1A1 ratio 0.014 0.025
were seen beginning at month 6 through month 60. Because
IL-1b expression 0.113 0.077
IKDC score at 12 mo 0.203
the preoperative values sometimes slightly differ in both
IKDC score at 60 mo 0.055 groups, clinical improvement was calculated by subtracting
the preoperative values from the values of different follow-
a
Values are Spearman rank correlations with the MOCART up time points to test for statistical significance between
score at 12 and 60 months. Significant values are in bold. BMI, the 2 groups. As in the correlation analyses, most significant
body mass index; COL2A1:COL1A1, collagen type II:type I; differences were found at 12 months (IKDC: P \ .011; KOOS
IKDC, International Knee Documentation Committee; IL-1b, pain: P \ .009; Noyes: P \ .017; and VAS pain: P \ .018). At
interleukin-1 b; MOCART, magnetic resonance observation of car- 6 months, there was a significant difference between the 2
tilage repair tissue; MRI, magnetic resonance imaging. groups in the Brittberg score (P \ .036).
b
P \ .05.
The same procedure was carried out for IL-1b (Figure
5). These 2 subgroups did not significantly differ in age,
found between the COL2A1:COL1A1 ratio and the inverse defect size, duration of symptoms, or BMI (P \ .381, P \
Brittberg score (r = 0.506, P \ .027). In general, all posi- .086, P \ .211, and P \ .414, respectively). In contrast to
tive scores showed a positive Spearman rank correlation the results of the COL2A1:COL1A1 ratio, a better clinical
coefficient at all time points, whereas the inverse Brittberg curve in all scores was obtained for the samples with the
and VAS pain scores showed a negative one. No correlation lowest IL-1b expression. Because of a high standard devi-
was found between aggrecan expression and clinical out- ation, only the difference in the Brittberg score after 60
comes (data not shown). months reached statistical significance (P \ .03).
In contrast to the COL2A1:COL1A1 ratio, IL-1b expres-
sion was found to positively correlate with the inverse Britt-
berg score at 24 months (r = 0.534, P \ .022) and 60 months DISCUSSION
(r = 0.583, P \ .009) (Table 5). Another positive correlation
with IL-1b was revealed for the inverse VAS pain score at The major finding of this study was that cell differentiation
60 months (r = 0.459, P \ .048). The correlations of the (COL2A1:COL1A1 ratio) and IL-1b expression signifi-
COL2A1:COL1A1 ratio and IL-1b expression with 2 cantly influenced clinical outcomes after MACT. Several

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Vol. 42, No. 1, 2014 Role of Cell Differentiation in MACT 65

TABLE 4
Correlations Between COL2A1:COL1A1 Expression Ratio and Clinical Scores at Different Time Pointsa

Brittberg IKDC KOOS Pain KOOS Symptoms Noyes VAS Pain

6 mo 0.506b 0.261 0.353 0.558b 0.245 0.381

12 mo 0.326 0.517b 0.585c 0.373 0.457b 0.458b
24 mo 0.24 0.209 0.273 0.571b 0.387 0.385
60 mo 0.147 0.275 0.387 0.498b 0.149 0.230

a
Values are Spearman rank correlations between the COL2A1:COL1A1 expression ratio and the improvement in clinical scores from pre-
operative levels to levels at different follow-up time points. Significant values are in bold. COL2A1:COL1A1, collagen type II:type I; IKDC,
International Knee Documentation Committee; KOOS, Knee injury and Osteoarthritis Outcome Score; VAS, visual analog scale.
b
P \ .05.
c
P \ .01.

TABLE 5
Correlations Between IL-1b Expression and Clinical Scores at Different Time Pointsa

Brittberg IKDC KOOS Pain KOOS Symptoms Noyes VAS Pain

6 mo 0.330 0.04 0.271 0.280 0.222 0.338

12 mo 0.396 0.212 0.075 0.147 0.103 0.318
24 mo 0.534b 0.235 0.222 0.047 0.176 0.446
60 mo 0.583c 0.278 0.184 0.273 0.426 0.459b

a
Values are Spearman rank correlations between IL-1b expression and the improvement in clinical scores from preoperative levels to lev-
els at different follow-up time points. Significant values are in bold. IKDC, International Knee Documentation Committee; IL-1b, interleu-
kin-1 b; KOOS, Knee injury and Osteoarthritis Outcome Score; VAS, visual analog scale.
b
P \ .05.
c
P \ .01.

Figure 3. Correlations between (A) collagen type II:type I (COL2A1:COL1A1) expression ratio and improvement in the Interna-
tional Knee Documentation Committee (IKDC) score after 12 months and (B) between interleukin-1 b (IL-1b) expression and
improvement in the Brittberg score after 60 months. Gene expression data are shown in DDCT values (high DDCT values corre-
spond to low gene expression levels). The improvement in clinical scores was calculated by subtracting preoperative values from
follow-up values.

patient- or defect-specific parameters have already been
identified that influence clinical results. They include the
patients age, duration of symptoms before surgery, loca-
tion of the defect, and even insurance status such as work-
ers compensation.28,35,37,44 In our study, we also found
a negative influence of age, duration of symptoms, and
BMI on clinical outcomes. However, considering the fact
that cartilage transplants are tissue-engineering products
whose properties mainly arise from the cells and the
scaffold material used, only little effort has been made to
analyze the influence of cell-specific characteristics such as
cell differentiation on clinical outcomes after transplantation.
The differentiation status of chondrocytes, however, greatly
changes the physiological activity of the cells, as is the case
in the course of in vitro expansion. During cultivation, chon-
drocytes undergo a dedifferentiation process characterized by
a loss of COL2A1 and aggrecan production and an increase in
COL1A1 expression.31,46 Much effort is deployed to reverse

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66 Albrecht et al The American Journal of Sports Medicine

Figure 4. Comparison of clinical outcomes between transplants with the highest 10 (solid line) and lowest 10 (dashed line) col-
lagen type II:type I (COL2A1:COL1A1) expression ratios over time. (A) Brittberg, (B) International Knee Documentation Committee
(IKDC), (C) Knee injury and Osteoarthritis Outcome Score (KOOS) pain, (D) KOOS symptoms, (E) Noyes, and (F) visual analog
scale (VAS) for pain scores. Error bars = standard deviation. *P \ .05 for differences in clinical improvement (calculated by sub-
tracting preoperative values from follow-up values) between the 2 groups.

this process to obtain highly differentiated cells for trans-
plantation. However, there is still no scientific proof to
what extent cell differentiation positively influences clinical
results. The main aim of this study was therefore to evaluate
if cell differentiation determined by the COL2A1:COL1A1
expression ratio affects clinical outcomes, which may then
be used as quality criteria for MACT.
We did find a correlation between the differentiation
index and clinical scores at various follow-up time points.
Whereas the IKDC, KOOS, and Noyes scores showed a pos-
itive Spearman rank correlation coefficient at all time
points, the inverse Brittberg score and VAS pain score
showed a negative one. The best correlation was found at
12 months, where the COL2A1:COL1A1 ratio significantly
correlated with most of the clinical scores (IKDC, Noyes,
VAS pain, and KOOS pain). Two of the 5 KOOS subscores
(KOOS pain and KOOS symptoms) were additionally cor-
related and are illustrated in Table 4. The KOOS symp-
toms subscore significantly correlated with 3 of 4 follow-
up time points. It seems that high or low cell differentia-
tion has a particular effect on the parameters of this sub-
score. In contrast to the other subscores such as activities

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Vol. 42, No. 1, 2014 Role of Cell Differentiation in MACT 67

Figure 5. Comparison of clinical outcomes between transplants with the highest 10 (solid line) and lowest 10 (dashed line) inter-
leukin-1 b (IL-1b) expression over time. (A) Brittberg, (B) International Knee Documentation Committee (IKDC), (C) Knee injury and
Osteoarthritis Outcome Score (KOOS) pain, (D) KOOS symptoms, (E) Noyes, and (F) visual analog scale (VAS) for pain scores.
Error bars = standard deviation. *P \ .05 for differences in clinical improvement (calculated by subtracting preoperative values
from follow-up values) between the 2 groups.

of daily living or function in sport and recreation, questions
of the KOOS symptoms subscore are restricted to basic
knee function such as knee motion or knee swelling, so
the answers of this subscore may therefore be more inde-
pendent of other external factors. The hypothesis that
high cell differentiation results in good clinical outcomes
was further supported by comparing the clinical curve of
patients with the highest and lowest 10 COL2A1:COL1A1
ratios. In all scores, the 2 groups began to drift apart at
about 6 months after surgery, showing a better curve for
the group with the high differentiation index.
Evidence that cell quality may affect clinical outcome
after MACT was already presented by Pietschmann
et al,40 who correlated a high number of morphological
abnormal cells found in histological stainings of transplant
samples with poor clinical results. In another recent study,
Saris et al44 referred to a patented gene scoring system
that claims to predict the capacity of chondrocytes to
form hyaline cartilage in vivo. However, this gene scoring
system was not further specified. A positive correlation
between CD44 and collagen type II expression with clinical
outcomes was demonstrated very recently by Niemeyer

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68 Albrecht et al
et al.37 In this study, extracellular expression of cell-bound
collagen type II, aggrecan, and CD44 was measured by
flow cytometry and correlated with the IKDC score up to
24 months after MACT. In their study, howeverand the
authors stated this as a possible limitationcells of a sepa-
rate alginate culture, destined for quality assessment,
were used. In our study, we used real-time PCR to analyze
residuals of the original transplant. Real-time PCR is
a very sensitive and reproducible method that allows for
the processing of very small sample sizes. This makes it
an ideal method for quality assessment of cartilage trans-
plants before implantation.
Besides matrix components, it is hypothesized that
matrix-catabolic factors also influence the quality of tissue
formation by chondrocytes in vivo.39 In fact, IL-1b is
known to cause matrix degradation in cartilage and was
therefore chosen as a possible negative predictor of clinical
outcomes for this study. Although osteoarthritic chondro-
cytes were shown to be capable of producing a cartilage-
like tissue in vitro and in vivo,10,49 we found a negative cor-
relation between IL-1b expression and clinical results at 24
and 60 months after transplantation, indicating that high
IL-1b expression possibly caused by the use of cells predis-
posed to osteoarthritis leads to worse clinical outcomes. In
contrast to the COL2A1:COL1A1 ratio in which effects in
clinical results can be seen as early as 6 months after trans-
plantation, the action of IL-1b on tissue formation in vivo
seems to play a role much later. Patient age has already
been demonstrated to negatively affect clinical out-
comes.35,37 Perhaps higher IL-1b expression in patients of
a greater age is responsible for this effect. Whether this neg-
ative effect on results can be inhibited by anti-inflammatory
therapies such as IL-1 receptor antagonist protein or RNA
interference remains to be evaluated in further studies.
Neither clinical outcome (IKDC score at 12 and 60
months) nor gene expression showed any correlation with
the MOCART score. These findings are in concordance
with a recently published meta-analysis that concluded
strong evidence is lacking to support using morphological
MRI for predicting clinical outcome.11 It seems that several
parameters influence clinical outcomes that do not affect
morphological MRI, at least under currently established
MRI scoring systems. We hypothesize that this also applies
to the gene expression of the COL2A1:COL1A1 ratio and
IL-1b because with the MOCART score, for instance, the
exact matrix composition is not known. In the future,
more sophisticated MRI protocols such as T2 mapping
might allow a more differentiated evaluation of cartilage
repair tissue and a better correlation with cell-specific
factors.
A limitation of this study is the relatively low number of
patients due to its restriction to defects in the tibiofemoral
joint to obtain a statistically homogeneous group. This
makes a comprehensive confounding analysis including
all patient-specific parameters impossible, and the results
are therefore mostly descriptive. Nonetheless, similar
case numbers are also found in other studies that report
correlations with MACT.12,40
In conclusion, our data demonstrate that cell differenti-
ation and IL-1b expression influence clinical outcomes up
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The American Journal of Sports Medicine
to 5 years after MACT. Efforts to increase cell differentia-
tion before transplantation should therefore be forced to
further improve clinical results. The use of osteoarthritic
chondrocytes, however, seems not to be suitable for
MACT as long as techniques capable of reducing proin-
flammatory cytokine expression such as IL-1b have not
been developed. Based on our findings, the COL2A1:
COL1A1 ratio in combination with IL-1b could also serve
as a promising parameter for assessing the quality of car-
tilage transplants before implantation.
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