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The American Journal of Sports


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

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60 Albrecht et al The American Journal of Sports Medicine

propagated in vitro, seeded onto a 3-dimensional scaffold, MATERIALS AND METHODS

and subsequently cultured for various periods (depending
on the standard operating procedures of the manufac- Study Design and Participants
turers) before implantation into the defect. The safety
and clinical effectiveness of this procedure for the treat- In this study, 27 patients with symptomatic traumatic
ment of moderate to large symptomatic full-thickness defects of the articular cartilage of the knee (tibiofemoral
articular cartilage defects have been demonstrated in sev- joint area) were included, who had been treated between Jan-
eral studies, showing significant improvement in pain, uary 2001 and July 2005 with MACT at a single academic
function, and activity up to at least 5 years.4,6,15,19,20,25,30,45 clinical center. During this time period, a total of 71 patients
Several parameters influencing clinical outcomes after with cartilage defects in the knee were treated with MACT
MACT have been identified including localization of the using Hyalograft C (Fidia Advanced Biomaterials, Abano
defect, duration of symptoms before surgery, and the Terme, Italy). Because differences in clinical outcomes have
patients age.21,28,45 However, the influence of cell differen- been described between cartilage transplantations in the
tiation and other specific cell characteristics of chondro- tibiofemoral and patellofemoral joint area,21 only patients
cytes used for transplantation on clinical results after with defects restricted to the tibiofemoral joint area (n =
autologous chondrocyte transplantation is still poorly 32) were selected for the study. From 3 patients, no trans-
investigated. In a recent study, we demonstrated that plant residual could be obtained, or the isolated RNA did
gene expression (collagen type II:type I ratio, aggrecan, not fulfill the quality standards. Two patients were lost to fol-
and interleukin-1 b) varied highly within cartilage trans- low-up. Ethical approval for the study was obtained from the
plants of the same scaffold type in different patients.1 institutional review board of the hospital.
For further optimization of transplants, it is important to Study participants were men and women between 19 and
know which parameters positively influence clinical out- 50 years of age with a defect size of .2 cm2 and no knee
comes after MACT. instability or misalignment (axis deviation .5). There
Collagen type II and aggrecan are 2 of the major compo- were no restrictions on the upper limit of the defect size
nents of the extracellular matrix in articular cartilage and or the number of defects. Patients were excluded from the
are responsible for its unique mechanical properties. Dur- study if they had a body mass index (BMI) of .30 kg/m2,
ing in vitro cultivation of chondrocytes, however, the totally or subtotally resected menisci, severe neurological
expression of these proteins is diminished.31,46 In place of disorders, metabolic arthritis, joint infections, tumors, psy-
collagen type II, the expression of collagen type I, which chiatric diseases, arthrofibrosis, or autoimmune diseases
is normally found only in fibrocartilage and the superficial or were pregnant. All patients provided written informed
zone of articular cartilage,31,50 increases. To quantify this consent before study enrollment.
process, called dedifferentiation, a differentiation index At the time of initial arthroscopic surgery and grading
defined as the ratio of COL2A1 to COL1A1 expression is of the defect, 200 to 300 mg of normal full-thickness hya-
used.2,13,24,29 This index, first introduced by Martin line cartilage was biopsied from a minimally weightbear-
et al,34 attains a higher value with a more chondrocytic ing area of the intercondylar notch, placed in a transport
phenotype and shows less variability than a single gene. medium, and then sent to Fidia Advanced Biomaterials.
Besides chondrocytes cultivated in vitro, osteoarthritic The graft, Hyalograft C, was produced by the manufac-
chondrocytes also exhibit a lower differentiation index.34 turer as follows: The patients chondrocytes were isolated
Interleukin-1 b (IL-1b) is a proinflammatory cytokine from biopsy specimens and multiplied in a monolayer cul-
that plays a central role in connective tissue destruction ture. According to the manufacturer, the maximum num-
and inflammation.22 In cartilage, IL-1b is considered a pri- ber of passages for 2-dimensional cultivation was defined
mary instigator of osteoarthritis, disrupting the metabolic in the production process, which was not further specified.
balance of chondrocytes and reducing COL2A1 expres- The cells were thereafter seeded onto a hyaluronan web at
sion.8,42 Different sites of action have been identified for a density of 1 3 106/cm2 and cultivated for at least 2 weeks.
IL-1b in chondrocytes. Bauge et al3 have demonstrated The cells were cultivated in a culture medium supple-
that IL-1b antagonizes the transforming growth factor b mented with fetal calf serum.
(TGFb) pathway through down-regulation of its TbRII
receptor. Others have shown that IL-1b promotes prolifer- Surgical Technique
ation and dedifferentiation by down-regulation of fibro-
blast growth factor receptor 3 (FGFR3) or up-regulation Through a mini-arthrotomy, the cartilage defect was pre-
of sirtuin 1 (SIRT1).22,48 Inhibition of IL-1b via RNA inter- pared by curettage to remove any fissured and undermined
ference, on the other hand, has been demonstrated to cartilage. Debridement of the subchondral bone plate was
increase transcript levels of TGFb.43 performed with care to prevent perforation or subchondral
There have not been any reports on the influence of IL- bone bleeding. The dimensions of the defect were trans-
1b expression or cell differentiation on the success rate ferred to a template; the hyaluronan fleece containing the
after MACT. The aim of this study, therefore, was to exam- seeded chondrocytes was trimmed to exactly fit into the
ine the effect of these parameters on clinical outcomes up defect and then implanted. Fibrin glue (Tissucol, Baxter,
to 5 years after MACT. Vienna, Austria) was applied on the edges to fix the graft;

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Vol. 42, No. 1, 2014 Role of Cell Differentiation in MACT 61

no periosteal cover or sutures were used. The joint was (2) integration with the border zone, (3) structure of the
manipulated intraoperatively to ensure adherence and sta- repair tissue, (4 and 5) signal intensity on 2 different
bility of the implant. At the conclusion of the procedure, the sequences (proton densityweighted turbo spin echo and
joint was wrapped in a compressive elastic bandage. dual fast spin echo; classified as normal, nearly normal,
and abnormal, relating to signal alteration in comparison
Postoperative Rehabilitation with the adjacent cartilage), integrity of the (6) subchondral
lamina and (7) bone, and presence of (8) adhesions and (9)
Immediately after surgery, immobilization of the joint was effusion. The total score ranges from 0 to 100. The MRI
recommended for 12 to 24 hours, followed by a standardized scans were viewed by 2 independent examiners experienced
rehabilitation program. Early rehabilitation began on the in musculoskeletal imaging and cartilage repair.
second postoperative day with continuous passive motion
(range in sagittal plane, 0-0-40) for 6 to 8 hours per Gene Expression Analysis
day and was continued for 6 weeks. Range of motion was
increased on a daily basis. In addition, patients were asked Residuals of 27 Hyalograft C autografts were collected dur-
to perform isometric muscle contractions and circulation ing surgery and stored in a transport medium at room tem-
exercises for their lower limb. perature. Immediately after surgery, the samples were
Patients were instructed to use crutches immediately brought to the laboratory for further processing. Trans-
after surgery to avoid full weightbearing. Toe-touch plant samples were immersed in TRI reagent (Sigma-
weightbearing was permitted for 4 weeks. After this, Aldrich, St Louis, Missouri) for lysis of the cells and stored
patients began partial weightbearing of 20% of their body at 80C until RNA extraction, which was performed
weight (up to week 6) and 50% of their body weight (up according to the standard protocol.
to week 8) and then progressed to full weightbearing by
8 weeks (up to week 10). Full weightbearing was defined cDNA Synthesis
as walking without crutches to an extent that allowed
patients to perform activities of daily living. A standard Total RNA (0.1-1 mg) was diluted with nuclease-free water
bathroom scale was used to instruct patients about relative to a volume of 15 mL. Thereafter, 4 mL of iScript Reaction
amounts of weightbearing. Mix as well as 1 mL of iScript reverse transcriptase were
For strengthening, patients were assigned isometric, added (Bio-Rad Laboratories, Hercules, California). The
concentric, and eccentric exercises (open and closed kinetic reaction mixture was incubated for 5 minutes at 25C,
chain) in the first week after surgery. Neuromuscular exer- for 30 minutes at 40C, and for 5 minutes at 85C.
cises were also implemented to improve the dynamic sta-
bility of the knee. Primers and Probes for Quantitative Analyses

Clinical Outcomes The design of the primers and probes has previously been
described.1 To avoid the amplification of genomic DNA, the
Five clinical scores with documented reliability and valid- probes were placed at the junction of 2 exons. For IL-1b,
ity were used to assess outcomes. This included the Inter- the predeveloped TaqMan assay (Applied Biosystems, Fos-
national Knee Documentation Committee (IKDC) ter City, California) was used.
Subjective Knee Form, which measures subjective symp-
toms, joint function, and sports activities,23,27,47 and the Real-Time PCR Amplification and Analysis
Knee injury and Osteoarthritis Outcome Score (KOOS)
with the following subscores: pain, symptoms, activities Real-time polymerase chain reaction (PCR) amplification
of daily living, function in sport and recreation, and was performed and monitored using an ABI Prism 7500
knee-related quality of life.16,30,41 Furthermore, the Noyes Fast Real-time PCR System (Applied Biosystems). The
sports activity rating scale26,30,38 grading the level and master mix was based on the SensiMix Probe Kit (Quan-
intensity of athletic activity, the Brittberg clinical tace, London, United Kingdom). The thermal cycling condi-
score,7,36,51 and a visual analog scale (VAS) grading pain tions comprised the initial steps at 50C for 2 minutes and
only5,14,47 were evaluated. Clinical assessments were per- at 95C for 10 minutes. Amplification of the cDNA products
formed preoperatively and at 6, 12, 24, and 60 months after was performed with 40 PCR cycles, consisting of a denatur-
transplantation. Patients were asked to keep diaries of ation step at 95C for 15 seconds and an extension step at
their activities and exercise regimen. 60C for 1 minute. b-2 microglobulin (B2M) was chosen as
the housekeeping gene, using the predeveloped TaqMan
Magnetic Resonance Imaging assay.17 For a given amount of cDNA, the range of the
housekeeping gene was within 61.5 Ct of the median
Characteristics of the articular cartilage repair tissue were value; samples outside of this range were excluded from
evaluated by magnetic resonance imaging (MRI) performed analysis. All cDNA samples (2.4 mL of cDNA in a total vol-
at 12 and 60 months after MACT in accordance with the ume of 20 mL) were analyzed in triplicate. Relative quanti-
magnetic resonance observation of cartilage repair tissue fication of gene expression was performed using the
(MOCART) score.32 Scoring was recorded for 9 separate comparative DDCT method. Native cartilage was used as
parameters of graft outcome: (1) degree of the defect fill, a calibrator.

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62 Albrecht et al The American Journal of Sports Medicine

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

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 1.3 cm2, and the mean BMI was 24.8 6 3.6 kg/m2. There
were 22 male and 5 female participants.
The statistical analysis included a tabular description of the
demographic data, the subjective clinical scores, and the gene
Clinical Assessment
expression data. Statistical evaluation was performed using
SPSS software version 20.0 (SPSS Inc, Chicago, Illinois). Overall, all patients showed a significant improvement in
For statistical analysis, DDCT values were used.52 Values all clinical results after 5 years (Figure 1). Patients experi-
are presented as the mean 6 standard deviation. A Fried- enced the greatest improvements during the first year and
man test with post hoc analysis (Wilcoxon signed-rank test) reached a stable improvement 2 years postoperatively. The
conducted with a Bonferroni correction was applied to com- mean IKDC score increased from 34.4 6 8.6 preoperatively
pare the respective scores at different time points. The Wil- to 77.9 6 16.0 after 24 months (P \ .001). This status was
coxon test was used to compare differences between the maintained through year 5 after transplantation (74.7 6
MOCART score after 12 and 60 months. For correlation anal- 22.8; P \ .753 compared with 24 months). A similar curve
yses, the Spearman rank correlation coefficient was calcu- was observed for the Noyes score, which increased from
lated. Mann-Whitney U tests were applied to compare the a mean of 23.1 6 19.8 preoperatively to 73.0 6 15.7 after
group of samples with the highest COL2A1:COL1A1 ratio 24 months (P \ .001) and was maintained at a high level
to the group with the lowest ratio and to compare the group (75.8 6 21.5; P \ .084 compared with 24 months) through
of samples with the highest IL-1b expression to the group year 5. The mean Brittberg score declined from 3.33 6 0.70
with the lowest expression. Statistical tests were considered before transplantation to 2.15 6 0.85 after 24 months (P \
statistically significant when P values were \.05. .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
Twenty-seven patients who underwent MACT (Hyalograft
C) between January 2001 and July 2005 were included in The IKDC score was used as a representative clinical score
this study (Table 1). Because differences in clinical out- to correlate the improvement in clinical outcomes with
comes have been described between cartilage transplanta- patients age, duration of symptoms, defect size, and BMI
tions in the tibiofemoral and patellofemoral joint area,21 (Table 2). The improvement was calculated by subtracting
only patients with defects in the tibiofemoral joint area preoperative values from follow-up values. The patients
were included. Twenty of the defects were located in the age showed a significantly negative correlation with the
medial femoral condyle, 4 in the lateral femoral condyle, IKDC score at all follow-up time points (6 months: r =
and 1 in the tibial plateau, and 2 had combined localiza- 0.602, P \ .005; 12 months: r = 0.406, P \ .049; 24
tions. The mean age of the patients at the time of implanta- months: r = 0.541, P \ .014; 60 months: r = 0.636, P \
tion was 36.2 6 9.5 years, the mean duration of symptoms .003). The duration of symptoms correlated negatively
was 60.9 6 89.5 months, the mean defect size was 4.2 6 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,
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
P \ .01. expression ranged from 5.41 to 12.48 DDCT (mean, 9.08
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-
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).
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

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

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.
P \ .05.
P \ .01.

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

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.
P \ .05.
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 scaffold material used, only little effort has been made to
identified that influence clinical results. They include the analyze the influence of cell-specific characteristics such as
patients age, duration of symptoms before surgery, loca- cell differentiation on clinical outcomes after transplantation.
tion of the defect, and even insurance status such as work- The differentiation status of chondrocytes, however, greatly
ers compensation.28,35,37,44 In our study, we also found changes the physiological activity of the cells, as is the case
a negative influence of age, duration of symptoms, and in the course of in vitro expansion. During cultivation, chon-
BMI on clinical outcomes. However, considering the fact drocytes undergo a dedifferentiation process characterized by
that cartilage transplants are tissue-engineering products a loss of COL2A1 and aggrecan production and an increase in
whose properties mainly arise from the cells and the 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- points, the inverse Brittberg score and VAS pain score
plantation. However, there is still no scientific proof to showed a negative one. The best correlation was found at
what extent cell differentiation positively influences clinical 12 months, where the COL2A1:COL1A1 ratio significantly
results. The main aim of this study was therefore to evaluate correlated with most of the clinical scores (IKDC, Noyes,
if cell differentiation determined by the COL2A1:COL1A1 VAS pain, and KOOS pain). Two of the 5 KOOS subscores
expression ratio affects clinical outcomes, which may then (KOOS pain and KOOS symptoms) were additionally cor-
be used as quality criteria for MACT. related and are illustrated in Table 4. The KOOS symp-
We did find a correlation between the differentiation toms subscore significantly correlated with 3 of 4 follow-
index and clinical scores at various follow-up time points. up time points. It seems that high or low cell differentia-
Whereas the IKDC, KOOS, and Noyes scores showed a pos- tion has a particular effect on the parameters of this sub-
itive Spearman rank correlation coefficient at all time 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 Evidence that cell quality may affect clinical outcome
of the KOOS symptoms subscore are restricted to basic after MACT was already presented by Pietschmann
knee function such as knee motion or knee swelling, so et al,40 who correlated a high number of morphological
the answers of this subscore may therefore be more inde- abnormal cells found in histological stainings of transplant
pendent of other external factors. The hypothesis that samples with poor clinical results. In another recent study,
high cell differentiation results in good clinical outcomes Saris et al44 referred to a patented gene scoring system
was further supported by comparing the clinical curve of that claims to predict the capacity of chondrocytes to
patients with the highest and lowest 10 COL2A1:COL1A1 form hyaline cartilage in vivo. However, this gene scoring
ratios. In all scores, the 2 groups began to drift apart at system was not further specified. A positive correlation
about 6 months after surgery, showing a better curve for between CD44 and collagen type II expression with clinical
the group with the high differentiation index. outcomes was demonstrated very recently by Niemeyer

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68 Albrecht et al The American Journal of Sports Medicine

et al.37 In this study, extracellular expression of cell-bound to 5 years after MACT. Efforts to increase cell differentia-
collagen type II, aggrecan, and CD44 was measured by tion before transplantation should therefore be forced to
flow cytometry and correlated with the IKDC score up to further improve clinical results. The use of osteoarthritic
24 months after MACT. In their study, howeverand the chondrocytes, however, seems not to be suitable for
authors stated this as a possible limitationcells of a sepa- MACT as long as techniques capable of reducing proin-
rate alginate culture, destined for quality assessment, flammatory cytokine expression such as IL-1b have not
were used. In our study, we used real-time PCR to analyze been developed. Based on our findings, the COL2A1:
residuals of the original transplant. Real-time PCR is COL1A1 ratio in combination with IL-1b could also serve
a very sensitive and reproducible method that allows for as a promising parameter for assessing the quality of car-
the processing of very small sample sizes. This makes it tilage transplants before implantation.
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 1. Albrecht C, Tichy B, Nurnberger S, et al. Gene expression and cell
known to cause matrix degradation in cartilage and was differentiation in matrix-associated chondrocyte transplantation
therefore chosen as a possible negative predictor of clinical grafts: a comparative study. Osteoarthritis Cartilage. 2011;19(10):
outcomes for this study. Although osteoarthritic chondro- 1219-1227.
2. Barlic A, Drobnic M, Malicev E, Kregar-Velikonja N. Quantitative anal-
cytes were shown to be capable of producing a cartilage-
ysis of gene expression in human articular chondrocytes assigned for
like tissue in vitro and in vivo,10,49 we found a negative cor- autologous implantation. J Orthop Res. 2008;26(6):847-853.
relation between IL-1b expression and clinical results at 24 3. Bauge C, Girard N, Leclercq S, Galera P, Boumediene K. Regulatory
and 60 months after transplantation, indicating that high mechanism of transforming growth factor beta receptor type II deg-
IL-1b expression possibly caused by the use of cells predis- radation by interleukin-1 in primary chondrocytes. Biochim Biophys
posed to osteoarthritis leads to worse clinical outcomes. In Acta. 2012;1823(5):983-986.
4. Behrens P, Bitter T, Kurz B, Russlies M. Matrix-associated autolo-
contrast to the COL2A1:COL1A1 ratio in which effects in
gous chondrocyte transplantation/implantation (MACT/MACI): 5-
clinical results can be seen as early as 6 months after trans- year follow-up. Knee. 2006;13(3):194-202.
plantation, the action of IL-1b on tissue formation in vivo 5. Beris AE, Lykissas MG, Kostas-Agnantis I, Manoudis GN. Treatment
seems to play a role much later. Patient age has already of full-thickness chondral defects of the knee with autologous chon-
been demonstrated to negatively affect clinical out- drocyte implantation: a functional evaluation with long-term follow-
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6. Brittberg M. Cell carriers as the next generation of cell therapy for car-
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therapies such as IL-1 receptor antagonist protein or RNA 7. Brittberg M, Faxen E, Peterson L. Carbon fiber scaffolds in the treat-
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with a recently published meta-analysis that concluded
accelerates the development of knee osteoarthritis in mice after sur-
strong evidence is lacking to support using morphological gical transection of the medial collateral ligament and partial medial
MRI for predicting clinical outcome.11 It seems that several meniscectomy. Arthritis Rheum. 2003;48(12):3452-3463.
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to the gene expression of the COL2A1:COL1A1 ratio and
differentiation potential of osteoarthritic chondrocytes and their pos-
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ation and IL-1b expression influence clinical outcomes up 2012;40(7):1527-1537.

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