ARTHRITIS & RHEUMATISM
Vol. 56, No. 1, January 2007, pp 177–187
© 2007, American College of Rheumatology
Three-Dimensional Cartilage Tissue Engineering Using
Adult Stem Cells From Osteoarthritis Patients
Wael Kafienah, Sanjay Mistry, Sally C. Dickinson, Trevor J. Sims, Ian Learmonth,
and Anthony P. Hollander
Objective. To determine whether it is possible to
engineer 3-dimensional hyaline cartilage using mesen-
chymal stem cells derived from the bone marrow
(BMSCs) of patients with osteoarthritis (OA).
Methods. Expanded BMSCs derived from pa-
tients with hip OA were seeded onto polyglycolic acid
scaffolds and differentiated using transforming growth
factor ?3 in the presence or absence of parathyroid
hormone–related protein (PTHrP) to regulate hypertro-
phy. Micromass pellet cultures were established using
the same cells for comparison. At the end of culture, the
constructs or pellets were processed for messenger RNA
(mRNA) analysis by quantitative real-time reverse
transcription–polymerase chain reaction. Matrix pro-
teins were analyzed using specific assays.
Results. Cartilage constructs engineered from
BMSCs were at least 5 times the weight of equivalent
pellet cultures. Histologic, mRNA, and biochemical ana-
lyses of the constructs showed extensive synthesis of
proteoglycan and type II collagen but only low levels of
type I collagen. The protein content was almost identical
to that of cartilage engineered from bovine nasal chon-
drocytes. Analysis of type X collagen mRNA revealed a
high level of mRNA in chondrogenic constructs com-
pared with that in undifferentiated BMSCs, indicating
an increased risk of hypertrophy in the tissue-
engineered cells. However, the inclusion of PTHrP at a
dose of 1 ?M or 10 ?M during the culture period
resulted in significant suppression of type X collagen
mRNA expression and a significant decrease in alkaline
phosphatase activity, without any loss of the cartilage-
specific matrix proteins.
Conclusion. Three-dimensional hyaline cartilage
can be engineered using BMSCs from patients with OA.
This method could thus be used for the repair of
An intact articular cartilage surface is essential
for normal joint function (1). Loss of this tissue through
degradation of type II collagen and proteoglycan com-
ponents in the extracellular matrix is a well-described
feature of osteoarthritis (OA) (1–4). In adults there is
little or no capacity for self-repair of eroded articular
cartilage, presumably because this tissue is avascular
(5,6). Despite intensive research into the use of protein-
ase inhibitors to prevent cartilage loss in OA (7), no
effective pharmaceutical therapies have emerged (8,9).
In recent years, various methods for the repair of
articular cartilage lesions have been developed (5,10).
These include osteochondral transplantation (11), mi-
crofracture (6), and autologous chondrocyte implanta-
tion (12,13) with or without the assistance of a scaffold
matrix to deliver the cells (14). A feature of all of these
techniques is that their use is limited to the repair of
focal lesions. In patients with OA, cartilage lesions are
generally large and unconfined (15) and thus do not
provide an appropriate environment for the retention of
chondrocytes or stem cells over a sufficient length of
time to elaborate an extracellular matrix. Thus, the
majority of patients with OA are excluded from these
types of treatment. Successful repair of cartilage lesions
in OA is therefore likely to be achieved only when
3-dimensional cartilage implants that have enough ex-
Supported by a grant from the UK Biotechnology and Bio-
logical Sciences Research Council. Dr. Hollander’s work was funded in
part by an endowed chair from the UK Arthritis Research Campaign.
Wael Kafienah, PhD, Sanjay Mistry, BSc, Sally C. Dickinson,
PhD, Trevor J. Sims, Ian Learmonth, MD, Anthony P. Hollander,
PhD: Southmead Hospital, and University of Bristol, Bristol, UK.
Dr. Kafienah, Mr. Mistry, Dr. Dickinson, Mr. Sims, and Drs.
Learmonth and Hollander have a patent pending for a method of
engineering cartilage tissue implants using adult stem cells.
Address correspondence and reprint requests to Anthony P.
Hollander, PhD, Department of Clinical Science at North Bristol,
AMBI Research Laboratories, Avon Orthopaedic Centre, Southmead
Hospital, Bristol BS10 5NB, UK. E-mail: A.Hollander@Bristol.ac.uk.
Submitted for publication April 27, 2006; accepted in revised
form September 15, 2006.
tracellular matrix for fixation within the joint can be
Cartilage tissue engineering provides a potential
method for the production of 3-dimensional implants
(16,17). Effective engineering protocols have already
been developed in which chondrocytes, usually from
young animals, are seeded onto biodegradable scaffolds
and cultured in a bioreactor (18,19). Generating
3-dimensional cartilage using adult human chondrocytes
is far more challenging and, in the case of older OA
patients, is probably impossible in the clinic setting,
because of the lack of autologous donor tissue. One
study demonstrated that OA chondrocytes can generate
a cartilage-like matrix when grown on hyaluronic acid
scaffolds (20), indicating the potential utility of these
cells in OA. However, the cells synthesized significantly
less collagen than was generated by chondrocytes from
individuals without arthritis, suggesting that there may
be a need for additional up-regulation of chondrogenesis
if OA chondrocytes are ever to be used in a clinical
This has led several groups of investigators to
explore the use of mesenchymal stem cells (MSCs) for
the generation of autologous chondrocytes (21). MSCs
are multipotent cells with a self-renewing capacity
(22,23). Many studies have utilized adherent bone mar-
row MSCs (BMSCs) cultured as small micromass pellets
and stimulated with transforming growth factor ?
(TGF?) to drive chondrogenesis (24,25). From these
studies there is strong histologic evidence that under
these conditions, the BMSCs become chondrocytes and
synthesize both type II collagen and proteoglycan. How-
ever, micromass pellets were designed for use in exper-
imental models, and the amount of extracellular matrix
produced by the pellets is too small to be of practical
value for implantation (26). Furthermore, there is clear
evidence that BMSCs stimulated with TGF? express
type X collagen, an early marker of hypertrophy that is
normally absent from hyaline cartilage (27).
It is not yet known whether BMSCs derived from
OA patients have the capacity to become chondrocytes
and generate hyaline cartilage. Most studies have uti-
lized BMSCs from animals or healthy human donors
(23–25,28). Nevertheless, one study investigated OA
BMSCs cultured as pellets, and the results demonstrated
a reduced chondrogenic capacity of these cells (29). The
purpose of the present study was therefore to determine
whether OA BMSCs can be used for tissue engineering
of 3-dimensional hyaline cartilage.
PATIENTS AND METHODS
Patients. Bone marrow plugs were collected as trabec-
ular bone biopsy specimens from the femoral heads of 23
patients with OA who were undergoing hip arthroplasty at
Southmead Hospital (Bristol, UK), of whom 52% were men
and 48% were women. The mean age of the 23 patients was
65.8 years (range 42–90 years). The study was carried out in full
accordance with local ethics guidelines, and all of the patients
gave their informed consent.
Isolation and characterization of BMSCs. Cells were
isolated from the bone marrow plugs by washing in expansion
medium consisting of low-glucose Dulbecco’s modified Eagle’s
medium (DMEM) (Sigma, St. Louis, MO) supplemented with
10% fetal bovine serum (FBS), 1% (volume/volume) Glu-
tamax (1?; Invitrogen, Carlsbad, CA), and 1% (v/v) penicillin
(100 units/ml)–streptomycin (100 ?g/ml) (Invitrogen). The
serum batch was selected to promote the growth and differen-
tiation of MSCs (30).
The cell suspension was separated from any bone in
the sample using a 19-gauge needle. The cells were centrifuged
at 1,500 revolutions per minute for 5 minutes and the
supernatant/fat was removed. The resulting cell pellet was
resuspended in medium and then plated at a seeding density of
1.5–2.0 ? 105nucleated cells per cm2. The expansion medium
was supplemented with 1 ng/ml fibroblast growth factor 2
(FGF-2) (PeproTech, London, UK) to enhance BMSC prolif-
eration and differentiation (31,32). These flasks were incu-
bated at 37°C in a humidified atmosphere of 5% CO2and 95%
air. The first medium change was after 4 days, and the medium
was then changed every other day until adherent cells reached
90% confluence and were ready for passaging. The cells were
characterized for stem cell surface markers and multilineage
potential, as described previously (30).
Micromass pellet cultures using OA BMSCs. Ex-
panded BMSCs were trypsinized and cultured in micromass
pellets as described previously (24), with slight modification.
Briefly, 500,000 cells were placed in a 15-ml conical polypro-
pylene tube and resuspended in 1.0 ml chondrogenic differen-
tiation medium consisting of DMEM containing 4.5 gm/liter
glucose supplemented with 10 ng/ml of TGF?3 (R&D Systems,
Abingdon, UK), 1 mM sodium pyruvate (Sigma), 50 ?g/ml
ascorbic acid-2-phosphate (Sigma), 1 ? 10?7M dexamethasone
(Sigma), 1% insulin–transferrin–selenium (Invitrogen), and
1% (v/v) penicillin (100 units/ml)–streptomycin (100 ?g/ml)
(Invitrogen). Cells were centrifuged at 1,500 rpm for 5 minutes
at 20°C. The pellets were maintained in culture with 1 pellet/
tube and 1.0 ml chondrogenic medium/tube. Medium was
changed every 2–3 days. After the first week the medium was
further supplemented with 50 ?g/ml insulin (Sigma) until the
end of culture. Chondrogenic pellets were harvested at 21 days
for messenger RNA (mRNA), matrix proteins, and histologic
Tissue engineering using OA BMSCs. Polyglycolic acid
(PGA) scaffolds (a kind gift from Dr. James Huckle, Smith &
Nephew, York, UK) were produced as 5-mm (diameter) ? 2-
mm (thick) discs according to an established method (33). The
scaffolds were presoaked in 100 ?g/ml human fibronectin
(Sigma) in phosphate buffered saline (PBS) to support BMSC
adherence to PGA fibers. BMSCs from passage 2 or passage 3
were trypsinized and suspended in 30 ?l of expansion medium.
178KAFIENAH ET AL
The suspension was loaded drop-wise onto the scaffold in
tissue-culture wells precoated with 1% (weight/volume) aga-
rose (Sigma) to prevent cell adherence to plastic. After incu-
bation for 4 hours, the scaffolds were turned over and incu-
bated for a further 4 hours to allow for even distribution of
cells across the scaffold.
The constructs were maintained in chondrogenic dif-
ferentiation medium, as described above for micromass pellet
cultures. After the first week, the medium was further supple-
mented with 50 ?g/ml insulin (PeproTech) until the end of
culture. Human recombinant parathyroid hormone–related
protein (PTHrP) (Sigma) was included in the differentiation
medium at 1 ?M or 10 ?M, as appropriate. The medium was
changed 3 times a week. The constructs were incubated at
37°C, in 5% CO2on a rotating platform at 50 rpm for 35 days.
Harvested samples were digested with collagenase to release
the cells, which were stored at ?70°C for subsequent RNA
extraction or alkaline phosphatase activity assay. Other sam-
ples were stored at ?20°C prior to quantitative biochemical
analysis (see below). In some experiments, cartilage was engi-
neered using bovine nasal chondrocytes (BNCs) that were
isolated as described previously (34).
Histologic and immunohistochemical analyses of mi-
cromass pellets and engineered cartilage. Micromass pellets
and mature cartilage engineered from stem cells were frozen in
OCT embedding matrix (BDH Chemicals, Poole, UK). Full-
depth sections (thickness 7 ?m) were cut with a cryostat and
fixed in 4% (w/v) paraformaldehyde (Sigma) in PBS, pH 7.6.
Some sections were stained with hematoxylin and eosin or
0.1% (w/v) Safranin O (both from Sigma) to evaluate the
distribution of matrix and proteoglycan, respectively. Other
sections were immunostained with monoclonal antibodies
against types I and II collagen (Southern Biotechnology,
Birmingham, AL) and type X collagen (a kind gift from Dr.
Alvin Kwan, Cardiff University, UK), as previously described
(34,35). Biotinylated secondary antibodies were detected with
a peroxidase-labeled biotin–streptavidin complex (Vectastain
Elite kit; Vector Laboratories, Peterborough, UK) with diami-
nobenzidine substrate (Vector Laboratories). Natural cartilage
and tendon were used as positive controls for detection of type
II collagen and type I collagen, respectively. Normal goat
serum was used as a negative control, and all sections were
counterstained with hematoxylin (Vector Laboratories).
Quantitative biochemical analyses of engineered car-
tilage. Dry weights of the constructs were determined after
freeze-drying. The samples were then solubilized with trypsin
and processed for complete biochemical analysis as recently
described (36). Briefly, samples were digested with 2 mg/ml
TPCK-treated bovine pancreatic trypsin containing 1 mM iodo-
acetamide, 1 mM EDTA, and 10 ?g/ml pepstatin A (all from
Sigma). An initial incubation for 15 hours at 37°C with 250 ?l
trypsin was followed by a further 2-hour incubation at 65°C
after the addition of a further 250 ?l of the freshly prepared
proteinase. All samples were boiled for 15 minutes at the end
of incubation to destroy any remaining enzyme activity. The
extracts were assayed by inhibition enzyme-linked immunosor-
bent assay (ELISA) using a mouse IgG monoclonal antibody
to denatured type II collagen, COL2-3/4m, as previously de-
scribed (2). Peptide CB11B (CGKVGPSGAP[OH]GEDGRP-
[OH]GPP[OH]GPQY) was synthesized using 9-fluorenyl-
methoxycarbonyl chemistry (a gift from Dr. A. Moir, Kreb’s
Institute, Sheffield University, UK) and was used as a standard
in all of the immunoassays. The extracts were also assayed by
inhibition ELISA using a rabbit antipeptide antibody to type I
collagen, as previously described (36). Peptide SFLPQPPQ
was synthesized using 9-fluorenyl-methoxycarbonyl chemistry
(also from Dr. A. Moir) and was used as a standard in all of the
immunoassays. Proteoglycan in the digests was measured by
the detection of sulfated glycosaminoglycan using colorimetric
assay with dimethylmethylene blue (Aldrich, Gillingham, UK)
as previously described (37).
Alkaline phosphatase activity. Cells in engineered
cartilage constructs were assayed for alkaline phosphatase
activity after collagenase digestion of the extracellular matrix,
as described previously (38). Although it is theoretically pos-
sible that the collagenase digestion could itself influence
alkaline phosphatase activity, this is unlikely to affect the
interpretation of our results, since all samples were extracted
under identical conditions and assayed as soon as possible after
completion of the extraction process. Briefly, the cells were
lysed with 0.1 ml of 25 mM sodium carbonate (pH 10.3), 0.1%
(v/v) Triton X-100. After 2 minutes, each sample was treated
with 0.2 ml of 15 mM p-nitrophenyl phosphate (di-tri salt;
Sigma) in 250 mM sodium carbonate (pH 10.3), 1.5 mM
MgCl2. Lysates were then incubated at 37°C for 2 hours. After
the incubation period, 0.1-ml aliquots were transferred to a
96-well microtiter plate and the absorbance was read at 405
nm. An ascending series of p-nitrophenol (25–500 ?M) pre-
pared in the incubation buffer enabled quantification of prod-
RNA isolation and reverse transcription. RNA was
extracted from cell cultures using the GenElute Mammalian
Total RNA kit (Sigma), according to the manufacturer’s
instructions. Reverse transcription (RT) was carried out using
the Superscript II system (Invitrogen). Total RNA (2 ?g) was
reverse transcribed in a 20-?l reaction volume containing
Superscript II (200 units), random primers (25 ?M), and dNTP
(0.5 mM each) at 42°C for 50 minutes.
Primer design. The coding sequences for human type
X, type II, and type I collagens (accession no. NM_000493,
NM_001844, and NM_000088, respectively) were used to
design primers using the online software, Primer3 (Whitehead
Institute for Biomedical Research, Cambridge, MA). The
primers span intronic junctions to avoid the amplification of
genomic sequences. They were also checked for the amplifica-
tion of potential pseudogenes. A BLAST search against all
known sequences confirmed specificity. Published primers for
the housekeeping gene ?-actin (39) were used as a reference
for normalization in all RT–polymerase chain reactions (RT-
PCRs). In preliminary studies (results not shown) we found
that this gene was expressed more stably than GAPDH or
ribosomal RNA for a range of differentiated progeny of stem
cells. All of our primers were specifically designed not to
coamplify processed pseudogenes in contaminating genomic
DNA and they all generated the correct sizes of the PCR
fragments with no nonspecific products, thus confirming the
specificity of the real-time RT-PCR (results not shown).
Details of the primers used in the study are as follows: for type
X collagen ?, forward GACACAGTTCTTCATTCCCTACAC
and reverse GCAACCCTGGCTCTCCTT; for type II collagen
?1 (A?B), forward CAACACTGCCAACGTCCAGAT and
reverse CTGCTTCGTCCAGATAGGCAAT; for type I colla-
CARTILAGE TISSUE ENGINEERING USING STEM CELLS FROM OA PATIENTS179
Figure 1. Chondrogenesis in pellet cultures of bone marrow mesenchymal stem cells (BMSCs) from
patients with hip osteoarthritis (OA). Expanded OA BMSCs from passages 2 or 3 were cultured as
3-dimensional pellets. Representative results are shown for 1 of 5 different patients. A, Macroscopic
appearance of pellets. Bar ? 3 mm. B, Histologic appearance of pellets at the end of culture. Sections were
stained with hematoxylin and eosin (H&E) or Safranin O (Saf O) for sulfated proteoglycans. C,
Immunohistochemical detection of type I collagen (Col I) and type II collagen (Col II) at the end of
culture, using specific antibodies for immunostaining of sections. D, Controls for immunostaining,
comprising the negative control (tissue-engineered cartilage with normal goat serum) (left) in which
staining of the remaining polyglycolic acid scaffold but not the extracellular matrix is evident, and positive
controls for Col II in hyaline cartilage (middle) and Col I in tendon (right). Bar ? 100 ?m. (Original
magnification ? 40 in B and C; ? 10 in D.)
180 KAFIENAH ET AL
gen ?1, forward AGGGCCAAGACGAAGACATC and re-
verse CAACACTGCCAACGTCCAGAT; and for ?-actin,
forward GACAGGATGCAGAAGGAGATTACT and re-
Quantitative real-time RT-PCR. Quantitative real-
time RT-PCR was performed in a 25-?l reaction volume
containing 12.5 ?l of the SYBR Green PCR master mix
(Sigma), 5 ?l of the RT reaction mixture, and 300 nM each
primer using the Smart Cycler II System (Cepheid, Sunnyvale,
CA). For the ?-actin gene, the RT reaction mixture was diluted
100 times. The amplification program consisted of initial
denaturation at 95°C for 2 minutes followed by 40 cycles of
95°C for 15 seconds, annealing at 58°C for 30 seconds, and
extension at 72°C for 15 seconds. After amplification, melt
analysis was performed by heating the reaction mixture from
60°C to 95°C at a rate of 0.2°C/second. The threshold cycle (Ct)
value for each gene of interest was measured for each RT
sample. The Ctvalue for ?-actin was used as an endogenous
reference for normalization. Real-time RT-PCR assays were
done in duplicate or triplicate, with each set of assays repeated
Statistical analysis. Comparison of differences be-
tween individual groups was performed using the 2-tailed
Mann-Whitney U test. For multiple comparisons, the groups
were compared by analysis of variance using the nonpara-
metric Kruskal-Wallis test. When significant variance was
demonstrated, differences between individual groups were
then determined using the 2-tailed Mann-Whitney U test with
Dunn’s post hoc correction. In all analyses, P values less than
0.05 were considered significant.
Phenotype of isolated OA BMSCs. A well-
characterized population of stem cells was used for the
3-dimensional engineering of cartilage tissue. We have
previously described this cell population as being posi-
tive for CD105, CD106, CD49a, CD117, STRO-1, and
bone morphogenetic protein receptor type 1A and neg-
ative for CD34 (30). We have also shown the population
to be multipotential, in that it consistently is able to
differentiate into adipogenic, chondrogenic, and osteo-
genic lineages (30). In the present study there was no
significant variation in the extent of stem cell differen-
tiation among the patient samples (n ? 23), between
male and female patients, or between different age
groups (results not shown). Therefore, we were able to
confirm that the BMSC population used was consistently
multipotent, as expected for stem cells that have been
expanded with the use of FGF-2 (30–32).
Chondrocyte formation from OA BMSCs. In our
initial experiments, we cultured OA BMSCs with
TGF?3 in high-density pellets. Under these conditions,
the pellets grew into cartilage-like nodules that were
visible to the naked eye (Figure 1A). At the histologic
level, these nodules contained a large number of cells as
well as an extracellular matrix that stained consistently
for proteoglycan, although this was largely in a pericel-
lular location (Figure 1B). Similarly, there was staining
for types I and II collagen throughout the pellets and
this was most intense around the cells (Figure 1C). The
extent of immunostaining for type I collagen was rela-
tively similar to that for type II collagen, suggesting that
the pellet tissue was likely to be predominantly fibrocar-
tilage rather than hyaline cartilage. Negative and posi-
tive controls for the immunostaining are shown in Figure
Cartilage tissue engineering using OA BMSCs.
We were able to successfully engineer 3-dimensional
cartilage using a carefully ordered sequence of signals, as
described above. First, the OA BMSCs were expanded
in 10% FBS and 1 ng/ml FGF-2. Second, the expanded
cells were seeded onto PGA scaffolds that had been
precoated with fibronectin. Third, the cells were cul-
tured on a gently rotating platform for 1 week with 10
ng/ml TGF?3 in differentiation medium. Fourth, the
cells were cultured for a further 4 weeks on the rotating
platform in differentiation medium with 50 ?g/ml insulin
as well as 10 ng/ml TGF?3.
Under these carefully defined conditions, we
were able to generate a white, shiny tissue that resem-
bled hyaline cartilage at a macroscopic level (Figure
2A). On histologic analysis, these cartilage constructs
were found to contain an extracellular matrix that
stained extensively for proteoglycan, moderately for type
II collagen, and weakly for type I collagen (Figure 2B).
At higher magnification, it was possible to observe
rounded cells, some pericellular staining for type X
collagen, and staining for type II collagen (strongly) and
type I collagen (weakly) in the interterritorial matrix.
Biochemical findings in engineered cartilage. We
undertook an extensive quantitative analysis of the en-
gineered cartilage using a series of well-validated and
specific assays (36). For comparison, micromass pellet
cultures were also analyzed. Despite the sensitive nature
of our assays (36), we had to use a minimum of 500,000
cells per micromass pellet, and at the end of culture we
had to combine 3 of these pellets in order to generate
enough extracellular matrix for quantitative analysis. For
tissue engineering, we were able to use as few as 300,000
cells per scaffold and to analyze one sample at a time.
Cultures were maintained in TGF?3 for up to 35 days
prior to analysis. However, in cultures with the micro-
mass pellets, there was evidence of some loss of matrix
beyond 21 days, and therefore the pellet cultures were
CARTILAGE TISSUE ENGINEERING USING STEM CELLS FROM OA PATIENTS 181
stopped at this optimal time point. After 35 days in
culture, the mean dry weight was 0.08 mg, the type II
collagen content was 0.4%, and proteoglycan content
was 0.75%, whereas the type I collagen content was
undetectable; the equivalent findings after 21 days in
pellet cultures were a mean 0.23 mg dry weight, 0.57%
type II collagen, 1.14% proteoglycan, and 0.26% type I
collagen. The dry weights of pellets and engineered
tissue were calculated after freeze-drying. In the case of
engineered tissue, the weight of any remaining PGA
scaffold was determined after enzymatic digestion of the
extracellular matrix, and this was subtracted from the
total dry weight.
Using these calculations, we determined that the
extracellular matrix of engineered tissue was at least 5
times that of pellet cultures, and this difference was
significant (Table 1). Furthermore, the engineered car-
tilage contained significantly more proteoglycan and
type II collagen than was observed in micromass pellet
cultures. There was also a slightly higher type I collagen
content in engineered cartilage, although this was still
?10% of the type II collagen content in engineered
We previously demonstrated that the best results
from cartilage tissue engineering could be achieved
using BNCs (34). We therefore compared the results of
cartilage engineering using human OA BMSCs with
those using BNCs. There was no significant difference in
the content of type II collagen, type I collagen, or
proteoglycan between the 2 cell types (Figure 3), indi-
cating that chondrocytes derived from OA BMSCs are
as effective as BNCs.
Inhibition of hypertrophy by PTHrP. In prelimi-
nary experiments, we observed that OA BMSCs cultured
with TGF?3 in micromass pellets displayed increased
expression of type X collagen, indicating that these cells
Figure 2. Cartilage tissue engineering from BMSCs. Expanded OA
BMSCs from passages 2 or 3 were used to engineer cartilage on
polyglycolic acid scaffolds. Representative results are shown for 1 of 8
different patients. A, Macroscopic appearance of engineered cartilage.
Bar ? 3 mm. B, Histologic appearance of engineered cartilage at the
end of culture, in sections stained with H&E or Saf O for sulfated
proteoglycans and immunostained for Col II and Col I using specific
antibodies. Bar ? 100 ?m. C, Histologic appearance of engineered
cartilage at the end of culture, in sections stained with H&E and
immunostained for types X, II, and I collagens using specific antibod-
ies; arrows indicate staining for Col X limited to the pericellular
region. See Figure 1 for other definitions. (Original magnification ? 10
in B; ? 40 in C.)
Biochemical analysis of cartilage extracellular matrix
(n ? 10)
cartilage (n ? 15)
Number of cells seeded*
Dry weight, mean ? SEM mg
Type II collagen, mean ?
SEM % of dry weight‡
Proteoglycan, mean ? SEM %
of dry weight§
Type I collagen, mean ? SEM
% of dry weight‡
0.23 ? 0.03
0.57 ? 0.07
1.25 ? 0.77†
17.21 ? 2.88†
1.14 ? 0.1329.96 ? 3.45†
0.26 ? 0.11 1.76 ? 0.25†
* Minimum number of cells required for accurate quantification using
† P ? 0.0001 versus pellet culture, by Mann-Whitney U test.
‡ Determined by specific immunoassay after selective extraction of
§ Determined by dimethylmethylene blue colorimetric assay of glycos-
182 KAFIENAH ET AL
were likely to generate hypertrophic cartilage rather
than hyaline cartilage. This was confirmed by immuno-
staining of sections from tissue-engineered cartilage (see
Figure 2C). We therefore investigated the potential of
inhibiting this hypertrophy using PTHrP, which has been
shown to prevent maturation of prehypertrophic chon-
drocytes in the growth plate.
BMSCs cultured in monolayer without TGF?3
expressed very little type X collagen; however, expres-
sion of type X collagen was significantly up-regulated
when the same cells were used to engineer cartilage in a
TGF?3-driven system (Figure 4A). PTHrP suppressed
this up-regulation of type X collagen mRNA in a
significant and dose-dependent manner (Figure 4A).
Similarly, PTHrP at 10 ?M significantly reduced the
alkaline phosphatase content of the cells from our
engineered cartilage (Figure 4B).
Having demonstrated that PTHrP can suppress
early markers of hypertrophy, we considered it impor-
tant to establish that there was no reduction in the
quality of engineered cartilage in the PTHrP cultures.
PTHrP had no effect on cartilage-specific type II colla-
gen mRNA or protein (Figure 5A). Type I collagen,
which is normally absent from hyaline cartilage, was
further reduced from its already low level in these
cartilage constructs, at both the mRNA and protein
levels (Figure 5B). This led to a 4-fold, significant
improvement in the ratio of type II collagen to type I
Figure 4. Inhibition of hypertrophy by parathyroid hormone–related
protein (PTHrP). Expanded osteoarthritis bone marrow mesenchy-
mal stem cells from passages 2 or 3 were cultured in monolayer
(stippled bar) or used to engineer cartilage on polyglycolic acid
scaffolds with or without PTHrP (shaded bars). A, Analysis of type
X collagen mRNA by quantitative real-time polymerase chain reac-
tion at the end of culture. Results were normalized to values in
the control culture with transforming growth factor ? alone, and are
shown as the mean and SEM in 7 patients. B, Alkaline phosphatase
content determined by reaction with p-nitrophenyl phosphate; the
enzyme activity was normalized to the values in the control culture
without PTHrP. Results are the mean and SEM in 6 patients. ? ? P ?
0.05; ?? ? P ? 0.01; ??? ? P ? 0.0001, by 2-tailed Mann-Whitney U
test with Dunn’s post hoc correction.
Figure 3. Quantitative comparison of cartilage engineered from bo-
vine nasal chondrocytes (BNCs) (hatched bars; n ? 18 animals) and
cartilage engineered from human OA BMSCs (shaded bars; n ? 19
patients). Cartilage was engineered from BNCs or from expanded OA
BMSCs at passages 2 or 3 and then digested with trypsin. Digests were
assayed for types I and II collagen using specific immunoassays.
Proteoglycan was measured as sulfated glycosaminoglycans using the
dimethylmethylene blue colorimetric assay. Bars show the mean and
SEM content of each protein expressed as a percentage of dry weight.
NS ? P not significant, by 2-tailed Mann-Whitney U test. See Figure
1 for other definitions.
CARTILAGE TISSUE ENGINEERING USING STEM CELLS FROM OA PATIENTS 183
Figure 5. Effect of parathyroid hormone–related protein (PTHrP) on the extracellular matrix of
engineered cartilage. A and B, Expanded osteoarthritis bone marrow mesenchymal stem cells from
passages 2 or 3 were cultured in monolayer (stippled bar) or used to engineer cartilage on
polyglycolic acid scaffolds with or without PTHrP (shaded bars). Type II collagen (A) and type I
collagen (B) were analyzed by quantitative real-time polymerase chain reaction for mRNA
expression (left) (n ? 6 patients in each) and by specific immunoassay of trypsin digests for protein
expression (right) (n ? 7 patients in each). The mRNA results were normalized to the values in
control culture with transforming growth factor ? alone. C, Ratio of type II collagen to type I
collagen measured as protein (n ? 7 patients). D, Proteoglycan content measured as sulfated
glycosaminoglycans using the dimethylmethylene blue colorimetric assay (n ? 7 patients). All
results are the mean and SEM. ? ? P ? 0.05; ?? ? P ? 0.01, by 2-tailed Mann-Whitney U test with
Dunn’s post hoc correction. NS ? not significant.
184KAFIENAH ET AL
collagen (Figure 5C), whereas no effect of PTHrP on the
proteoglycan content was observed (Figure 5D).
The present study is the first to demonstrate the
feasibility of tissue engineering of hyaline cartilage from
OA BMSCs. Biochemically, the cartilage quality was
comparable with that achieved using the best available
cell source, namely BNCs (34). Furthermore, we were
able to show that the tendency of BMSCs to become
hypertrophic can be down-regulated using PTHrP.
These findings suggest that it will be feasible to develop
a method of cartilage repair in OA patients using their
own BMSCs to generate 3-dimensional cartilage im-
This study investigated the use of stem cells
derived from patients with hip OA who were undergoing
arthroplasty at a large orthopedic referral center in the
UK. These patients are likely to be typical of individuals
who might benefit from cartilage implantation. We
demonstrated that despite the poor capacity of the cells
to produce any extracellular matrix in micromass pellet
cultures, the cells could be directed to produce cartilage
through the use of a series of specific molecular signals,
applied in appropriate order. We cannot be certain that
this set of conditions is the best possible for cartilage
formation, but our results demonstrate that the se-
quence of signals described herein can be used success-
fully to generate hyaline cartilage.
First, the adherent mesenchymal cells must be
driven to proliferate so that their cell number can be
expanded within a reasonable time frame. Murphy et al
(29) found that the proliferation rate of OA BMSCs
cultured in 10% serum was reduced compared with that
of control cells. In the present study we used 1 ng/ml
FGF-2 in addition to serum. This growth factor has been
previously shown to enhance the proliferation of normal
BMSCs (31,32). More recently, we found that prolifer-
ation of OA BMSCs is enhanced by FGF-2 and that the
mechanism is dependent on the stem cell nucleolar
protein nucleostemin (30). This suggests that the re-
duced proliferative capacity identified by Murphy et al
can be overcome by using this growth factor.
The second molecular signal used was fibronec-
tin, coated onto the PGA scaffolds in order to enhance
adhesion of the OA BMSCs. Fibronectin has been
previously shown to promote the adhesion of normal
mesenchymal cells (40), and our experiments revealed
the same effect on BMSCs derived from OA patients.
The third molecular signal was TGF?3. There is exten-
sive evidence indicating that growth factors of the TGF
superfamily promote chondrogenesis in micromass pel-
let cultures of normal human or animal BMSCs (23–
25,28), and our results showed that TFG?3 is effective at
driving chondrogenesis in BMSCs from OA patients.
The fourth signal was 50 ?g/ml insulin, which was added
to the tissue-engineering cultures 1 week after the start
of differentiation by TGF?, to promote the formation of
extracellular matrix by the differentiated cells (41).
Finally, as a fifth molecular signal, we investi-
gated the use of PTHrP. Previous studies (23,27) have
shown that the TGF?s promote the formation of hyper-
trophic chondrocytes, as shown by the up-regulation of
type X collagen mRNA. It is also possible that dexa-
methasone, which was included in our culture medium,
can contribute to hypertrophy. PTHrP is known to
down-regulate the maturation of prehypertrophic chon-
drocytes in the growth plate (42), and we therefore
considered it logical to investigate the effects of PTHrP
in our tissue-engineering cultures. Not only did PTHrP
down-regulate the early hypertrophic markers, it also
enhanced the biochemical quality of our extracellular
matrix, as shown by the down-regulation of type I
collagen and the maintenance of both type II collagen
We were able to generate cartilage that was of
the highest quality, comparable with that of cartilage
generated using BNCs, which we have previously shown
to be an excellent source of chondrocytes for cartilage
engineering (34). However, this engineered cartilage
had a lower collagen content than that found in natural
tissue, a feature that is true of all cartilage engineered
in vitro (18,19,34,41,43). Although there is growing
evidence that even very immature cartilage constructs
can mature into natural hyaline cartilage once implanted
within the joint (44,45), it would nevertheless be prefer-
able to engineer fully matured tissue in vitro prior to
implantation. It is, at present, unclear whether such
maturation can be achieved in vitro.
Our findings support and build on the work of
other investigators who have shown that normal BMSCs
can be used to generate chondrocytes (21,23–26,28).
Murphy et al (29) described the poor capacity of OA
BMSCs to proliferate and to form chondrocytes in
micromass pellet cultures. We were able to overcome
this reduced potential of the OA-derived cells by testing
the range of molecular signals as described above and by
the use of a PGA scaffold. Li et al (26) described the
importance of using scaffolds to generate cartilage with
a sufficient volume and mass to be implanted. However,
in their studies, the histologic results suggested that the
CARTILAGE TISSUE ENGINEERING USING STEM CELLS FROM OA PATIENTS 185
cartilage quality was no better than that achieved using
micromass pellet cultures. The reason that we were able
to successfully generate constructs of enhanced quality is
presumably because of the use of the specific molecular
signals in conjunction with the use of a biomaterial
Our conclusion that OA BMSCs can be used to
generate relatively mature cartilage implants opens up
the possibility of developing a cartilage therapy utilizing
autologous stem cells. The use of autologous cells has
several advantages. It avoids the risk of immune system
rejection or the need for immunosuppression that would
be required for donor cells. It also avoids the risk of
disease transmission from donor to patient. There is
currently intensive research into the use of embryonic
stem cells (22,46) as well as other cells to generate
chondrocytes. Albeit this is of scientific importance, it is
currently unclear whether embryonic cell lines will ever
be used in the clinical setting. Apart from the concerns
of some patients regarding ethics, there is an inherent
risk of teratoma formation as well as the potential for
immune system rejection that must be managed (22).
Autologous stem cells provide an attractive op-
tion for patients and clinicians. However, it must also be
recognized that autologous therapies are expensive,
requiring growth of cells and tissue over several weeks in
specialized ultraclean rooms. Therefore, it will be im-
portant to develop our tissue-engineering protocol so
that it can be undertaken in the shortest possible time in
order to reduce costs. We also need to develop methods
of attaching the cartilage implants to the subchondral
bone and of promoting integration of the implant with
surrounding tissue. Despite these challenges, our find-
ings represent a step forward in the development of an
autologous cartilage replacement therapy.
We are grateful to Ms Maureen Lee (University of
Bristol) for her assistance in providing patient samples, and to
Dr. Alvin Kwan (Cardiff University) for kindly providing us
with the anti–type X collagen antibody.
Dr. Hollander had full access to all of the data in the
study and takes responsibility for the integrity of the data and
the accuracy of the data analysis.
Study design. Dr. Kafienah, Mr. Mistry, and Dr. Hollander.
Acquisition of data. Dr. Kafienah, Mr. Mistry, Dr. Dickinson,
Mr. Sims, and Drs. Learmonth and Hollander.
Analysis and interpretation of data. Dr. Kafienah, Mr. Mistry,
Dr. Dickinson, Mr. Sims, and Drs. Learmonth and Hollander.
Manuscript preparation. Dr. Kafienah, Mr. Mistry, Dr. Dick-
inson, Mr. Sims, and Drs. Learmonth and Hollander.
Statistical analysis. Mr. Mistry and Dr. Hollander.
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CARTILAGE TISSUE ENGINEERING USING STEM CELLS FROM OA PATIENTS 187