ARTHRITIS & RHEUMATISM
Vol. 44, No. 7, July 2001, pp 1608–1619
Molecular Markers Predictive of the Capacity of
Expanded Human Articular Chondrocytes to
Form Stable Cartilage In Vivo
Francesco Dell’Accio, Cosimo De Bari, and Frank P. Luyten
Objective. To establish a model and associated
molecular markers for monitoring the capacity of in
vitro–expanded chondrocytes to generate stable carti-
lage in vivo.
Methods. Adult human articular chondrocytes
(AHAC) were prepared by collagenase digestion of
samples obtained postmortem and were expanded in
monolayer. Upon passaging, aliquots of chondrocyte
suspensions were either injected intramuscularly into
nude mice, cultured in agarose, or used for gene expres-
sion analysis. Cartilage formation in vivo was docu-
mented by histology, histochemistry, immunofluores-
cence for type II collagen, and proteoglycan analysis by
35
S-sulfate incorporation and molecular sieve chroma-
tography of the radiolabeled macromolecules. In situ
hybridization for species-specific genomic repeats was
used to discriminate human-derived from mouse-
derived cells. Gene expression dynamics were analyzed
by semiquantitative reverse transcription–polymerase
chain reaction.
Results. Intramuscular injection of freshly iso-
lated AHAC into nude mice resulted in stable cartilage
implants that were resistant to mineralization, vascular
invasion, and replacement by bone. In vitro expansion of
AHAC resulted in the loss of in vivo cartilage formation.
This capacity was positively associated with the expres-
sion of fibroblast growth factor receptor 3, bone mor-
phogenetic protein 2, and ␣1(II) collagen (COL2A1),
and its loss was marked by the up-regulation of activin
Supported by FWO grant G.0192.99.
Francesco Dell’Accio, MD, Cosimo De Bari, MD, Frank P.
Luyten, MD, PhD: University Hospitals Katholieke Universiteit Leu-
ven, Leuven, Belgium.
Address correspondence and reprint requests to Frank P.
Luyten, MD, PhD, Laboratory for Skeletal Development and Joint
Disorders, Onderwijs & Navorsing, Herestraat 49, 3000 Leuven,
Belgium.
Submitted for publication November 3, 2000; accepted in
revised form March 14, 2001.
1608
© 2001, American College of Rheumatology
Published by Wiley-Liss, Inc.
receptor–like kinase 1 messenger RNA. Anchorage-
independent growth and the reexpression of COL2A1 in
agarose culture were insufficient to predict cartilage
formation in vivo.
Conclusion. AHAC have a finite capacity to form
stable cartilage in vivo; this capacity is lost throughout
passaging and can be monitored using a nude mouse
model and associated molecular markers. This cartilage-
forming ability in vivo may be pivotal for successful
cell-based joint surface defect repair protocols.
Joint destruction represents the outcome of most
inflammatory and degenerative rheumatic diseases and
leads to severe disability. Damage of the joint surface
often results in end-stage osteoarthritis, requiring surgi-
cal intervention and total joint replacement. Its high
prevalence, heavy impact on working capacity, and the
high costs and morbidity associated with current thera-
peutic approaches make joint disease a major social
issue.
Recent developments in basic science and clinical
rheumatology have achieved a substantial understanding
of the mechanisms leading to cartilage breakdown and
considerable control of the progression of the damage to
the joint surface. In contrast, the range of interventions
for the repair of joint surface defects (JSD) is often
limited to prosthetic replacement of end-stage joints.
Modern tissue engineering strategies offer an opportu-
nity for biologic repair of JSD (1,2). Autologous chon-
drocyte transplantation (ACT) represents a paradigm in
biologic repair for JSD (3,4). In the original description,
autologous chondrocytes were obtained arthroscopically
via cartilage biopsy from a minor weight-bearing area of
the joint and were expanded in vitro in the presence of
autologous serum. In a subsequent open knee surgery
procedure, expanded chondrocytes were injected into
the JSD under a periosteal flap sutured to the cartilage
to seal the JSD (3).
MOLECULAR MARKERS OF STABLE CHONDROCYTES
Conceptual problems inherent to the technique
are the origin of the cells and the expansion in vitro. The
cartilage biopsy, although taken from a non–weight-
bearing area of the joint, is itself an additional injury to
the joint surface. Moreover, in vitro expansion of chon-
drocytes is known to result in a phenotypic derange-
ment, often referred to as dedifferentiation (5). There is
a possibility that the loss of phenotypic traits in vitro
could reflect a progressive loss of potential to form in
vivo stable cartilage that is resistant to vascular invasion,
mineralization, and bone formation, thereby jeopardiz-
ing the long-term outcome of ACT. The use of progen-
itor cells from, for example, bone marrow (6) or perios-
teum (7) may potentially circumvent these problems.
Progenitor cells can be obtained without inducing
irreversible damage, and they are extensively expandable
and phenotypically stable throughout expansion (8,9).
The use of mesenchymal cells is restricted by the as-yet-
insufficient knowledge of the long-term stability of the
repair tissue and by their tendency to differentiate
toward other cell lineages. Their multilineage potential
may present a risk of heterotopic tissue formation
(10,11).
ACT offers the advantage of using cells that are
stably committed to the appropriate phenotype and natu-
rally resistant to vascular invasion, mineralization, and
ossification. Therefore, it is crucial to ensure that this
phenotypic stability is retained throughout expansion. To
date, unfortunately, the choice of the appropriate assay to
measure this property is rather unsatisfactory, being lim-
ited to the expression of some extracellular matrix (ECM)
molecules such as type II collagen and to anchorage-
independent growth in agarose culture (5).
Tissue regeneration is thought to recapitulate the
molecular events that take place during embryonic de-
velopment (12–18). It requires not only cell proliferation
and deposition of the proper ECM, but also reproduc-
tion of the tissue architecture and patterning (1,19). This
appears to be particularly relevant for articular cartilage,
since the expression of cartilage-specific ECM mole-
cules, such as type II collagen, and anchorage-
independent growth in agarose culture (5) have been
used to evaluate the phenotypic stability of expanded
chondrocytes for ACT (3). Type II collagen expression
reports the differentiation state of the cells at the
moment of the test. The agarose assay consists of
evaluating the capacity to grow in anchorage-
independent conditions and to reexpress cartilage-
specific markers in low temperature melting (LTM)
agarose in in vitro culture (5). Anchorage-independent
growth and capacity to differentiate are likely to be
1609
necessary, but are not necessarily sufficient for the
formation of cartilage tissue in vivo, starting from a
suspension of isolated chondrocytes. These assays, in-
deed, have never been shown to predict the capacity to
form stable cartilage in vivo. An assay that directly
measures cartilage formation in vivo is therefore
needed.
We have standardized an in vivo assay in nude
mice to measure the potential of adult human articular
chondrocytes (AHAC) to form stable cartilage in vivo
that is resistant to vascular invasion, dystrophic calcifi-
cation, and bone formation. We have identified a set of
molecular markers that reproducibly predicts the out-
come of our in vivo assay, independently of donor age.
This set of molecular markers proved to be more
accurate than the agarose assay in predicting the in vivo
cartilage-forming ability, and may therefore be useful in
assessing the suitability of chondrocyte suspensions for
cell-based joint surface repair protocols.
PATIENTS AND METHODS
Cartilage harvest and chondrocyte isolation. Normal
articular cartilage was obtained from the femoral condyles of
human donors within 12 hours postmortem. Cartilage was
sliced full thickness, excluding the mineralized cartilage and
the subchondral bone, and placed in Hanks’ balanced salt
solution (HBSS; Life Technologies, Merelbeke, Belgium) sup-
plemented with 2⫻ antibiotic–antimycotic solution (final con-
centrations 200 units/ml of penicillin, 200 ␮g/ml of streptomy-
cin, and 0.5 ␮g/ml of amphotericin B; Life Technologies).
Cartilage slices were minced and washed twice in HBSS
supplemented with 2⫻ antibiotic–antimycotic solution for 5
minutes at 37°C.
Chondrocytes were released by overnight digestion at
37°C in 0.2% crude type IV collagenase (Life Technologies) in
high-glucose Dulbecco’s modified Eagle medium (DMEM;
Life Technologies) containing 10% fetal bovine serum (FBS;
BioWhittaker, Verviers, Belgium), and 2⫻ antibiotic–
antimycotic solution. Cells were washed twice in DMEM
supplemented with 10% FBS and 2⫻ antibiotic–antimycotic
solution and then counted. Trypan blue exclusion was used to
adjust for the number of viable cells. Routinely, ⬎95% of the
cells were viable.
Chondrocyte expansion and agarose culture. Chon-
drocytes were plated at a density of 5 ⫻ 104 cells/cm2 in growth
medium (DMEM supplemented with 10% FBS and 1⫻
antibiotic–antimycotic solution at final concentrations of 100
units/ml of penicillin, 100 ␮g/ml of streptomycin, and 0.25
␮g/ml of amphotericin B) and cultured at 37°C in a humidified
atmosphere containing 5% CO2. Cells were released 10 days
after confluence by trypsin–EDTA (0.25% trypsin, 1 mM
disodium EDTA; Life Technologies) treatment. Cultures in
LTM agarose were performed as described elsewhere (5).
Nude mouse assay. Five million viable cells were
resuspended in 50 ␮l of phosphate buffered saline (PBS) and
injected intramuscularly into the adductor muscles of the
1610
Table 1. Sequences of the primers used for polymerase chain reaction and size of the products*
Genes Sense primer
COL2A1 5⬘-CTGCTCGTCGCCGCTGTCCTT-3⬘
FGFR-3 5⬘-GCTGAAAGACGATGCCACTG-3⬘
BMP-2 5⬘-CAGAGACCCACCCCCAGCA-3⬘
ALK-1 5⬘-CGACGGAGGCAGGAGAAGCAG-3⬘
␤-actin 5⬘-TGACGGGGTCACCCACACTGTGCCCATCTA-3⬘
Aggrecan 5⬘-GTTGTCATCAGCACCAGCATC-3⬘
Versican 5⬘-AACATTTTTCAGGTGGTGAG-3⬘
Biglycan 5⬘-TCCCAGACCTCAAGCTCCTC-3⬘
Decorin 5⬘-ATTGTCATAGAACTGGGCAC-3⬘
Fibromodulin 5⬘-CTGGACGGGAACGAGATCAA-3⬘
Lumican 5⬘-ACCTTGAAAACTATTACCTGGAG-3⬘
␤2m 5⬘-GTGTCTGGGTTTCATCAATC-3⬘
* COL2A1 ⫽ ␣1(II) collagen; FGFR-3 ⫽ fibroblast growth factor receptor 3; BMP-2 ⫽ bone morphogenetic protein 2; ALK-1 ⫽ activin
receptor–like kinase receptor 1; ␤2m ⫽ ␤2-microglobulin.
thighs of 4–5-week-old female NMRI nu/nu mice. Animals
were maintained in isolator cages on an unrestricted diet. All
the procedures were approved by the local ethics committee.
Mice were killed by cervical dislocation after 3 weeks unless
otherwise specified.
Histochemistry. Formalin-fixed paraffin-embedded
sections were stained with Alcian blue, pH 2.5, toluidine blue,
Masson’s trichrome, and Safranin O according to standard
protocols.
Indirect immunofluorescence. After deparaffinization,
sections were digested with 0.25 mg/ml of pepsin (Sigma,
Bornem, Belgium) in 0.2N HCl for 1 hour at 37°C, postfixed in
buffered 4% paraformaldehyde for 10 minutes at room tem-
perature, and washed extensively with PBS. Slides were incu-
bated twice in 50 mM ammonium chloride in PBS for 10
minutes, washed in PBS, and blocked with Blocking Reagent
(Roche Molecular Biochemicals, Brussels, Belgium) supple-
mented with 0.2% (volume/volume) Triton X-100 (Bio-Rad,
Nazareth Eke, Belgium).
Slides were incubated overnight at 4°C with a mouse
anti-human type II collagen antibody (Chemicon, Hofheim,
Germany) diluted 1:5, extensively washed with PBS containing
0.2% Triton X-100, and incubated for 1 hour at room temper-
ature with Cy3-conjugated goat anti-mouse IgG antibody
(Jackson ImmunoResearch, De Pinte, Belgium) diluted 1:200.
After 3 washes in PBS–0.2% Triton X-100, slides were coun-
terstained with 4⬘,6-diamidino-2-phenylindole (DAPI; ICN,
Asse-Relegem, Belgium) and mounted with Mowiol
(Calbiochem-Merck Belgolabo, Overijse, Belgium). Red and
blue digital images were acquired by Spot camera and super-
imposed using Spot software version 3.0.4 (Diagnostic Instru-
ments, Sterling Heights, MI).
Chondrocyte devitalization and irradiation. To obtain
nonviable chondrocytes, cell suspensions were snap-frozen and
thawed at 37°C for 3 minutes for 3 consecutive times as
described in detail elsewhere (20). To arrest cell division while
preserving cell viability, chondrocytes were x-irradiated with a
single radiation dose of 50 Gy (21,22).
35
S-sulfate incorporation and hydrodynamic profile of
proteoglycans. The analysis of 35S-sulfate incorporation into
macromolecules was performed as described elsewhere (23).
DELL’ACCIO ET AL
Product
Reverse primer size (bp)
5⬘-AAGGGTCCCAGGTTCTCCATC-3⬘ 511
5⬘-AGGACCCCAAAGGACCAGAC-3⬘ 522
5⬘-CTGTTTGTGTTTGGCTTGAC-3⬘ 672
5⬘-TGAAGTCGCGGTGGGCAATGG-3⬘ 565
5⬘-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3⬘ 661
5⬘-ACCACACAGTCCTCTCCAGC-3⬘ 509
5⬘-GATGCAGAACTTGGAACTAT-3⬘ 304
5⬘-TGGAGACTTGGACACTTGGG-3⬘ 544
5⬘-AAGACTCACACCCGAATAAG-3⬘ 459
5⬘-TGTGGAGGAAAGTGGAGTGG-3⬘ 577
5⬘-ATTGAGTTTGATGTCTATTCGTG-3⬘ 407
5⬘-GGCAGGCATACTCATCTTTT-3⬘ 163
Briefly, explants from adult human knee articular cartilage and
3 implants from injected AHAC were minced and radiolabeled
for 6 hours at 37°C in DMEM supplemented with 35S-sulfate at
50 ␮Ci/ml. Radiolabeled tissues were extracted for 48 hours at
room temperature in 4M guanidine HCl, 0.2% (v/v) Triton
X-100, and 50 mM sodium acetate, pH 5.8, containing protease
inhibitors (5 mM benzamidine HCl, 10 mM EDTA, 10 mM
N-ethylmaleimide, and 1 mM phenylmethylsulfonyl fluoride).
The residual material was then extracted with 0.5M sodium
hydroxide to determine the efficiency of the extraction procedure.
Unincorporated radiolabel was removed by Sephadex
G-25 gel chromatography (PD-10 columns; Amersham Pharma-
cia Biotech, Roosendaal, The Netherlands) equilibrated in 7M
urea, 0.2M NaCl, 0.2% (v/v) Triton X-100, and 50 mM sodium
acetate, pH 6.0. An aliquot of the radiolabeled macromolecular
fraction was analyzed on a Sephacryl S-500 HR column (1 ⫻ 30
cm; Amersham Pharmacia Biotech) equilibrated with 4M guani-
dine HCl, 0.5% (v/v) Triton-X 100 in 50 mM sodium acetate, pH
6. The flow rate was 0.4 ml/minute. Fractions were collected at
1-minute intervals and counted for radioactivity.
In situ hybridization for species-specific genomic re-
peats. In situ hybridization for human-specific ALU repeats
was performed as described elsewhere (24), with a few modi-
fications. Briefly, sections were deparaffinized and rehydrated.
Matrix digestion was obtained by enzymatic treatment with 1
mg/ml of pepsin in 0.2N HCl for 10 minutes at 37°C. Sections
were immediately postfixed in 3% formaldehyde for 10 min-
utes and equilibrated in PBS. Sections were acetylated with
0.25% acetic acid containing 0.1M triethanolamine, pH 8, for
10 minutes, and prehybridized with 50% deionized formamide
containing 4⫻ saline–sodium citrate (SSC) at 37°C for 15
minutes. Sections were covered with hybridization buffer (1⫻
Denhardt’s solution, 5% Dextran sulfate, 0.2 mg/ml denatured
sheared salmon sperm DNA, 4⫻ SSC, and 50% deionized
formamide) containing 1 ng/␮l of a digoxigenin (DIG)–labeled
double-stranded DNA probe specific for human ALU genomic
repeats (a kind gift from K. Satomura, First Department of
Oral and Maxillofacial Surgery, University of Tokushima,
Tokushima, Japan) and covered with a glass coverslip.
Denaturation of both the probe and the genomic DNA
template was achieved by heating the slides at 95°C for 45
MOLECULAR MARKERS OF STABLE CHONDROCYTES 1611

seconds. Hybridization was performed at 42°C for 3 hours.

Sections were washed twice for 30 minutes at room tempera-
ture with 2⫻ SSC and 0.1⫻ SSC. Digoxigenin was detected
using a commercially available kit (DIG Nucleic Acid Detec-
tion kit; Roche Molecular Biochemicals) according to the
manufacturer’s protocol.
DIG-labeled mouse-specific L1 probe was obtained as
described previously (25). In situ hybridization for m-specific
L1 repeats was carried out as described above for human ALU
repeats, with a few modifications. Hybridization was allowed
overnight at 42°C, and stringency washes were twice for 30
minutes at room temperature in 1⫻ SSC and once for 30
minutes at 50°C in 0.5⫻ SSC.
Reverse transcription–polymerase chain reaction (RT-
PCR) analysis. Total RNA was isolated using TRIzol reagent
(Life Technologies) according to the manufacturer’s instruc-
tions. Total RNA from agarose cultures was obtained as
described elsewhere (26). Complementary DNA (cDNA) were
obtained by reverse transcription of 1 ␮g of total RNA
(Thermoscript; Life Technologies) using oligo(dT)20 as a
primer. PCR was performed in a 10-␮l volume. The cDNA
were added to the following PCR mixture: 0.5 units of Taq
polymerase (Eurogentec, Seraing, Belgium), 0.2 mM dNTPs,
0.5 ␮M specific primers, and 1.5 mM MgCl2. Negative controls
were either RT without enzyme or PCR without cDNA
template. PCR reactions were carried out in a Perkin Elmer
Thermal Cycler model 9600 (Applied Biosystems, Lennik,
Belgium). The sequences of the primers and the expected sizes
of the amplification products are listed in Table 1.
Marker analysis by PCR was preceded by equalization
of cDNA for the housekeeping gene ␤-actin. Figure 1. Generation of cartilage tissue by freshly isolated adult
human articular chondrocytes after intramuscular injection into nude
mice. a and b, Safranin O staining of adult human articular cartilage
RESULTS harvested from the femoral condyle. c and d, Safranin O staining of a
cartilage implant. b and d, Higher-magnification view of the boxed
Generation of a stable hyaline-like cartilage im- areas shown in a and c, respectively. Compared with adult human
plant in nude mice. The intramuscular injection of 5 ⫻ articular cartilage, the implant is hypercellular. e, Masson’s trichrome
106 freshly isolated AHAC into the posterior compart- staining displays the absence of neoangiogenesis or endochondral bone
ment of the thigh of NMRI nu/nu mice resulted in the formation. f, Immunofluorescence for type II collagen is positive in the
extracellular matrix of the implant. Asterisk indicates adjacent muscle
reproducible (15 of 15) retrieval of a distinct cartilage
tissue. Nuclei are counterstained with 4⬘,6-diamidino-2-phenylindole.
implant as early as 7 days after injection. The implant Bars ⫽ 200 ␮m.
was stable for at least 12 weeks, without signs of vascular
invasion, dystrophic calcification, or bone formation.
Calcium deposits were undetectable by alizarin red performed. Masson’s trichrome as well as hematoxylin
staining at all time points examined, up to 12 weeks and eosin preparations did not reveal the presence of
(data not shown). dense collagen bundles as in fibrocartilage. These over-
Safranin O staining was approximately as strong all characteristics allowed us to consider the cartilage
in the implants (Figures 1c and d) as in normal adult implants to be of hyaline-like nature.
articular cartilage (Figures 1a and b), indicating the To study the specificity of the assay, we injected
presence of sulfated proteoglycans, but the implants human primary skin fibroblasts and primary cells and
were hypercellular and lacked typical columnar organi- cell lines known to have chondrogenic potential. The
zation. Chondrocytes were embedded in typical lacunae. injection of primary human skin fibroblasts (kindly
Masson’s trichrome staining (Figure 1e) revealed no provided by S. Tejpar, Centrum Menselijke Erfelijkheid,
vascular invasion or bone formation. The detection of Katholieke Universiteit Leuven, Leuven, Belgium)
type II collagen protein by immunostaining further failed to produce any detectable implant. The injection
supported the cartilaginous nature of the implant (Fig- of primary adult human periosteal cells resulted in
ure 1f). The ECM was homogeneous with all the stains fibrous tissue formation (data not shown). The injection
1612 DELL’ACCIO ET AL

Figure 2. Hydrodynamic profiles of sulfated proteoglycans and proteoglycan expression profile.

35
S-sulfate incorporation and size fractionation of macromolecules in 3 implants (a) shows the
presence of high molecular weight proteoglycans with the same hydrodynamic size as those in adult
human articular cartilage explants (b). V0 ⫽ void volume; Vt ⫽ total volume. Proteoglycan
expression profile generated by semiquantitative reverse transcription–polymerase chain reaction
analysis with RNA obtained from a cartilage implant (IMP) (c). Confluent primary adult human
articular chondrocytes (P0) were used as positive control. Uninjected mouse muscle (MM) was
used as a negative control. The template cDNA from MM and IMP were equalized for the
expression of ␤-actin using primers that amplify both the mouse and human (mh) ␤-actin cDNA.
Human cDNA from IMP and P0 were normalized for the number of human cells using
human-specific primers (h) for ␤2-microglobulin. All the primers used to detect proteoglycan
cDNA were human-specific.

of cell lines that have in vitro chondrogenic potential,
such as the mouse teratocarcinoma cell line ATDC5 (27)
or the rat calvaria cell lines RCJ (28), RCJ 3.1 C5.18
(29), and CFK2 (30), did not produce any retrievable
implants under the conditions we used.
It is known that cartilage formation can be in-
duced in similar systems by nonviable materials contain-
ing chondrogenic factors, such as transforming growth
factor ␤ (TGF␤) and bone morphogenetic proteins
(BMPs) (31–33) or nonchondrogenic cells producing
those factors (34–36). To address whether, in our sys-
tem, living chondrocytes were required, we implanted, in
parallel and in duplicate, living chondrocytes and chon-
drocytes that had been lysed by repeated freezing and
thawing (20). Only the injection of living chondrocytes
resulted in cartilage implants.
To investigate whether cell proliferation was re-
quired, we x-irradiated chondrocytes with a single 50-Gy
dose, which blocks proliferation but does not kill the
cells (21,22). Under our conditions, chondrocytes that
had been made unable to proliferate by x-irradiation
could still generate a cartilage implant in vivo (data not
shown).
Proteoglycan synthesis and characterization.
High molecular weight sulfated proteoglycans constitute an
important component of the cartilage matrix (23) and are
critical for the biomechanical properties of articular carti-
lage, such as elasticity and resistance to load and impact
(37). Incorporation of 35S-sulfate and size fractionation of
macromolecules (Figure 2) showed the presence in the
implants of high molecular weight sulfated proteoglycans
comparable in hydrodynamic size to human articular car-
tilage from an age-matched donor (73 years old). The
lower ratio of high:low hydrodynamic–size sulfated proteo-
MOLECULAR MARKERS OF STABLE CHONDROCYTES 1613

Figure 3. Origin of the implant. To determine the relative contribution of the donor (human) and
host (mouse) tissue to the formation of the cartilage implant, consecutive sections were hybridized
with a, human-specific or b, mouse-specific probes recognizing genomic repeats (ALU and
mouse-specific L1, respectively). c, Superimposition of a and b using artificial colors. The
intermingling of human and mouse cells at the left edge of the implant was due to infiltration of
chondrocytes between the muscle fibers, as shown by the toluidine blue staining in d. Bar ⫽ 200 ␮m.

glycans in the extracts from articular cartilage explants may
be related to the lower extraction efficiency (data not
shown).
To better characterize the proteoglycan expres-
sion profile of the implants, we performed RT-PCR
analysis using human-specific primers for aggrecan, ver-
sican, biglycan, decorin, and lumican (Figure 2c). Unin-
jected mouse muscle was used as the negative control.
Primers for both human and mouse ␤-actin were used to
normalize cDNA from the implant and from the unin-
jected mouse muscle. Confluent primary AHAC were
the positive control. Since the implant may have con-
tained a considerable number of mouse cells, cDNA
from the implant and from primary human chondrocytes
were normalized for the expression of the housekeeping
gene ␤2-microglobulin using human-specific primers.
This set of data confirms the expression of cartilage
proteoglycans at the messenger RNA (mRNA) level.
The absence of versican transcript suggests that most of
the high molecular weight peak represents sulfate incor-
poration into aggrecan.
Contribution of the injected cells to the forma-
tion of the implant. In our in vivo system, the injected
human cells and the surrounding muscle are free to
interact. Chondrogenesis in the mouse muscle could
potentially be induced by the injected chondrocytes, for
example, by sustained secretion of growth factors and
morphogens (33). We explored the relative contribution
of donor-derived (human) and host-derived (mouse)
cells to the formation of the implant by the detection of
human-specific and mouse-specific genomic repeats by
in situ hybridization.
Consecutive sections were hybridized separately
with DNA probes specific for human-specific ALU and
mouse-specific L1 genomic repeats. Images were
aligned, converted to artificial colors, and superimposed.
As seen in Figure 3, although some chondrocytes infil-
trated the muscle tissue, there was no significant contri-
bution of mouse-derived cells to the implant.
Loss of in vivo cartilage-forming capacity on
serial passaging. It is well known that expansion of
chondrocytes in monolayer culture results in the loss of
1614 DELL’ACCIO ET AL

Figure 4. Serial expansion of human adult articular chondrocytes, resulting in the loss of their
capacity to form cartilage in vivo. Samples from 2 independent donors (S1 and S2; ages 86 and 28
years, respectively) were expanded. Upon passaging, aliquots of the cell suspension were injected
into nude mice or were used for gene expression analysis. After 2 population doublings,
chondrocytes could still form mature cartilage tissue, as shown by Alcian blue (a and aⴕ) and
Safranin O (c and cⴕ) staining. After 4 population doublings (solid arrow), the loss of cartilage-
forming ability was heralded by the formation of more immature implants, as shown by Alcian blue
(b and bⴕ) and Safranin O (d and dⴕ) staining. Chondrocytes from further passages (open arrow)
did not form any retrievable implant. The loss of cartilage-forming potential was marked by a
down-regulation of ␣1(II) collagen (COL2A1), fibroblast growth factor receptor 3 (FGFR3), and
bone morphogenetic protein 2 (BMP2) mRNA, while the expression of activin receptor–like kinase
1 (ALK-1) mRNA was up-regulated. FI is freshly isolated chondrocytes. Bar ⫽ 200 ␮m.

their phenotype, as measured by type II collagen expres-
sion and proteoglycan synthesis (5). To investigate
whether this phenotypic alteration was associated with a
loss of the capacity to generate stable cartilage in vivo,
we tested serially passaged human articular chondro-
cytes in our in vivo assay. Primary AHAC lost their in
vivo cartilage-forming potential after 2–3 passages in
vitro (about 4–6 population doublings). In the samples
shown in Figure 4, after 4 population duplications,
chondrocytes generated an implant that stained poorly
with Alcian blue and Safranin O. Chondrocytes from
later passages failed to generate any retrievable implant.
Molecular markers that predict the in vivo car-
tilage forming ability of articular chondrocytes indepen-
dently of donor age. To identify molecular markers that
are predictive of cartilage-forming capacity in our assay,
we monitored throughout in vitro expansion the expres-
sion of molecules that are known to play a role in the
formation and maintenance of the cartilage phenotype.
The molecular analysis included the expression of matrix
molecules and ligands, receptors, and intracellular com-
ponents of signaling pathways (TGF␤ superfamily, fibro-
blast growth factor [FGF], parathyroid hormone/
parathyroid hormone–related peptide, and hedgehog
proteins) (19). This analysis resulted in the selection of a
restricted number of molecular markers, the expression
of which tightly and reproducibly correlated with the
capacity of expanded chondrocytes to form stable carti-
lage in vivo. The results of the molecular analysis are
summarized in Figure 4.
The expression of ␣1(II) collagen (COL2A1) and
FGF receptor 3 (FGFR-3) correlated positively with our
in vivo assay. These markers were consistently down-
regulated when the cartilage-forming potential of ex-
MOLECULAR MARKERS OF STABLE CHONDROCYTES 1615

Figure 5. Markers predict the capacity of adult human articular chondrocytes to form stable
cartilage in vivo, independently of the donor age. Freshly isolated (FI) and serially passaged
chondrocytes from donors of different ages (range 28–86 years) were challenged in our in vivo
assay throughout expansion. Solid arrows mark the passage when a decline of the maturity of the
implant was first detected. Open arrows mark the first passage from which no implant could be
retrieved. COL2A1 ⫽ ␣1(II) collagen; FGFR3 ⫽ fibroblast growth factor receptor 3; BMP2 ⫽
bone morphogenetic protein 2; ALK-1 ⫽ activin receptor–like kinase 1.

panded chondrocytes was reduced and immature carti-
lage implants were retrieved. The expression of these
markers became undetectable when chondrocytes were
unable to organize a retrievable implant.
In contrast to the steep decrease of FGFR-3 and
COL2A1 mRNA levels, BMP-2 was strongly expressed
in freshly isolated chondrocytes, decreased throughout
passaging, and never completely disappeared within the
passages examined. The expression of activin receptor–
like kinase 1 (ALK-1) mRNA, as yet reported to be
predominantly expressed by endothelial cells (38), ap-
peared to be a negative marker of chondrocyte stability,
being steadily up-regulated during expansion.
Under our conditions, this set of molecular mark-
ers appeared to be predictive of the in vivo capacity to
generate cartilage tissue reproducibly, independently of
age. Figure 5 shows the molecular profiles of 3 chondro-
cyte populations from donors of different age (24, 28,
and 86 years old) throughout in vitro expansion.
The agarose assay is not sufficient to predict the
potential of expanded chondrocytes to generate stable
cartilage in vivo. Anchorage-independent growth and
rescue of type II collagen expression in anchorage-
independent culture (5) have been considered to be
predictive of the phenotypic stability of chondrocytes
(3). To validate the agarose assay as a predictor of the
cartilage-forming potential in our assay, we challenged
AHAC at passage 5 (⬃10 population doublings) with
our in vivo assay and with the agarose assay.
Passage 5 AHAC were still able to form colonies
(Figure 6a) and to recover to the original levels of
COL2A1 and FGFR-3 mRNA (Figure 6b) when cul-
tured in LTM agarose. However, no cartilaginous im-
plant was retrieved when AHAC were injected into nude
mice in 2 independent experiments each performed in
duplicate. Interestingly the up-regulation of ALK-1 in
serially passaged chondrocytes could not be reversed to
normal by the agarose culture.
DISCUSSION
ACT is a conceptually attractive therapeutic ap-
proach to JSD repair (3). The capacity to organize stable
cartilage in vivo is an important requisite for the cell
population used for JSD repair. Many independent
factors can interfere with the in vivo cartilage-forming
capability of the chondrocyte preparation. These include
the age of the donor (39) and the variability of the
autologous serum, both of which affect the growth rate
and, potentially, the phenotypic stability of chondrocytes
in vitro. Indeed, it is well known that in vitro expansion
of articular chondrocytes results in the progressive loss
of phenotypic traits (5). Consequently, the risk that this
process of dedifferentiation may result in a reduced
capacity to form stable cartilage in vivo is clearly a
concern in cartilage engineering. The importance of this
issue has been underscored by the variability of the
repair tissue formed after ACT (4). Importantly, the
formation of either hyaline-like cartilage tissue or disor-
ganized fibrocartilage seems to affect the clinical out-
come (4) and is likely to depend on the phenotypic
stability of the implanted chondrocytes.
To pursue a more consistent and reproducible
outcome of ACT, it would be useful to establish para-
meters that would allow us to monitor the capacity of
expanded chondrocytes to form cartilage in vivo. Ideally,
1616
Figure 6. The agarose assay is not sufficient to predict the cartilage-
forming ability of expanded chondrocytes in our in vivo assay. Freshly
isolated (FI) and serially passaged adult human articular chondrocytes
were injected into nude mice and tested in the agarose assay. a,
Although the cells lost their in vivo cartilage tissue–forming ability
after the second passage, they could still grow under anchorage-
independent conditions in agarose after passage 5 (⬃10 population
doublings). b, The molecular profile of the same chondrocytes
throughout passaging and after agarose culture [A(10)]. Note that
while the expression of ␣1(II) collagen (COL2A1), fibroblast growth
factor receptor 3 (FGFR3), and bone morphogenetic protein 2
(BMP-2) was rescued in the agarose culture system, the up-regulation
of activin receptor–like kinase 1 (ALK-1) was not reversed. Arrow
indicates the first passage from which no implant could be retrieved.
these parameters should be independent of the stage of
differentiation of the cell population, the age of the
patient, and any variable in the culture system and cell
handling. So far, anchorage-independent growth and the
rescue of type II collagen expression in LTM agarose
culture (5) have been used to validate chondrocyte
expansion procedures for ACT (3). Their value for the
specific use in cartilage engineering may be limited,
however, because none of them necessarily predicts the
capacity of expanded chondrocytes to form stable carti-
lage tissue in vivo.
In the present study, we standardized and vali-
dated an assay in nude mice that assesses the capacity of
AHAC to organize stable cartilage in vivo. Freshly
isolated human articular chondrocytes reproducibly
formed cartilage implants when injected intramuscularly
DELL’ACCIO ET AL
into nude mice. The implants consisted of differentiated
hyaline-like cartilage, as evaluated by histology and
confirmed by polarized light microscopy (data not
shown), containing a significant amount of high molec-
ular weight proteoglycans and type II collagen. The
implant was stable for at least 12 weeks, without signs of
vascular invasion or endochondral bone formation, un-
like in fetal swine chondrocytes, where these phenomena
were evident as early as after 3 weeks (data not shown).
This cartilage-forming ability was progressively lost dur-
ing in vitro expansion. Subsequently, we identified a set
of molecular markers predictive of the outcome of our in
vivo assay. Importantly, we have shown that the
anchorage-independent growth and the rescue of
COL2A1 expression in agarose culture are not sufficient
to predict the cartilage-forming ability in our in vivo
model.
We are aware that the nude mouse assay does not
exactly reproduce the same environment of a JSD in a
human joint such as the knee. Experimental evidence is
still lacking that this, or any other assay, can directly
predict the outcome of ACT. Ideally, studies in large
animals would represent the optimum system for vali-
dating chondrocyte expansion procedures for ACT. Un-
fortunately, due to significant anatomic and biologic
differences between human cartilage and the articular
cartilage of other animals typically used (essentially,
rabbit, dog, sheep, and goat), none of the animal models
of ACT available today is close enough to the human to
be considered truly representative (40). In addition,
some spontaneous tissue repair takes place in these
animal models, and the actual contribution of the im-
planted cells to the repair tissue is not always clear. On
the other hand, studies in humans cannot be properly
controlled for ethical reasons.
The possibility of addressing these critical issues
relies on the development of an adequate animal model
of ACT where every experimental step is controlled.
Our nude mouse model represents a well-validated,
stringent, flexible, and tightly controlled screening assay
for addressing specific biologic questions in vivo. The
use of costly and time-consuming animal studies could
be restricted to a further, preclinical validation step for
cell-based JSD repair protocols.
The use of the nude mouse model in cartilage
biology was introduced by Lipman et al (41) and has
become popular over the last few years in tissue engi-
neering. In a wide array of technical variants and using
cells from disparate origins, the nude mouse model has
been used to test cell–polymer constructs for cartilage
engineering (42) and to optimize culture conditions (43).
MOLECULAR MARKERS OF STABLE CHONDROCYTES
The nude mouse system is a powerful assay and
provides in vivo data, but a careful validation is recom-
mended for specific experimental settings. The mouse
tissues are sensitive to signaling molecules that are
produced by the implant or present in the matrix.
Cartilage and/or bone formation derived from the host
(mouse) tissue is induced by implantation of a variety of
either mesenchyme- or epithelium-derived cells (32,34–
36,44–46). The type of tissue obtained may also depend
on the site of the injection (32). Importantly, species
specificity of the assay has been described. Injections
into nude mice of either rabbit or dog articular chon-
drocytes that have been expanded for 14 days resulted in
hyaline cartilage implants in the former case, and in
fibrous tissue in the latter (41).
Therefore, for the specific use as a quality control
and a tool for investigating JSD repair protocols, it was
necessary to standardize and validate the nude mouse
assay with the use of human adult articular chondro-
cytes. We have shown that, under our experimental
conditions, the implants were constituted by the human
injected cells with no significant contribution from the
mouse host. In addition, cell viability, but not cell
proliferation, was required.
Use of the nude mouse assay requires time and
skills that are not compatible with a routine, consistent,
and reproducible quality control analysis for individual
patients. To circumvent this problem, we have identified
positive and negative molecular markers that predict the
outcome of the in vivo assay independently of the age of
the donor. COL2A1, FGFR-3, and BMP-2 were posi-
tively associated with the stable phenotype, while the
up-regulation of ALK-1 marked the loss of the in vivo
cartilage-forming potential. This set of molecular mark-
ers has proven to be more stringent than the agarose
assay in predicting the capacity to form cartilage in vivo.
In addition, the up-regulation of ALK-1 in serially
passaged chondrocytes could not be reversed to normal
by the agarose culture.
We do not address in this study the molecular
mechanisms underlying the phenomena described, espe-
cially since, with the exception of type II collagen (47),
the transcriptional regulation of these genes is not
completely elucidated. In addition, while the role of
BMP-2 (23) and FGFR-3 (48) in cartilage development
and homeostasis has been extensively studied, ALK-1 is
known to predominantly play a role in angiogenesis in
early development (38). ALK-1 up-regulation, in our
model, was unlikely to be due to endothelial contamina-
tion, since it was not associated with detectable levels of
1617
the endothelial marker Flk-1 (49) in late-passage chon-
drocytes, as evaluated by RT-PCR (data not shown).
The identification of a molecular profile of the
phenotypically stable chondrocyte opens an array of
clinical applications, from quality control for cell expan-
sion for ACT to the optimization of culture conditions
for specific applications in cartilage tissue engineering.
The presence of a negative marker such as ALK-1
mRNA offers the opportunity of an internal control for
gene expression analysis. Possible quantitative applica-
tions of this principle can be real-time quantitative
RT-PCR and flow cytometry.
The rescuing/enhancement of the cartilage phe-
notype has been documented in a large number of
systems such as the culture in agarose or alginate (5,50),
culture on Matrigel (51), and treatment with dihydrocy-
tochalasin B (52), retinoic acid (53), and members of the
TGF␤ superfamily (14,54–56). The possibility that some
of these systems also rescue the potential of late-passage
chondrocytes to form cartilage in vivo is very relevant for
cartilage tissue engineering and is being tested in our
laboratory.
Our experimental settings do not address the
question of the homogeneity of the chondrocyte popu-
lation throughout in vitro expansion. Based on our data,
however, it is likely that the in vivo potential to generate
cartilage in our assay is related to the predominance of
cells expressing the positive markers over cells express-
ing the negative ones. If this assumption proves to be
true, the development of specific antibodies to extracel-
lular domains of FGFR-3 and ALK-1 would offer the
opportunity of enriching cell populations with stable
chondrocytes by cell sorting techniques. More consistent
and homogeneous cell populations for ACT would prob-
ably ensure more reproducible clinical outcomes.
ACKNOWLEDGMENTS
The DIG-labeled ALU probe was kindly provided by
Dr. K. Satomura (First Department of Oral and Maxillofacial
Surgery, University of Tokushima, Tokushima, Japan). Nor-
mal adult human skin fibroblasts were a gift of Dr. S. Tejpar
(Centrum Meiselijke Erfelijkheid, Katholieke Universiteit
Leuven, Leuven, Belgium). Thanks are due to people working
in the Mortuary of the University Hospitals of Leuven, Bel-
gium, for providing the cartilage samples. We thank Drs. M.
Moos, T. Thomas, and P. Tylzanowski for many suggestions
and for critically reviewing the manuscript.
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