Group Title: Arthritis Research & Therapy
Title: Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer
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Title: Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer
Physical Description: Book
Language: English
Creator: Steinert, Andre
Proffen, Benedikt
Kunz, Manuela
Hendrich, Christian
Ghivizzani, Steven
Nöth, Ulrich
Rethwilm, Axel
Eulert, Jochen
Evans, Christopher
Publisher: Arthritis Research and Therapy
Publication Date: 2009
 Notes
Abstract: INTRODUCTION:The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.METHODS:Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.RESULTS:Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.CONCLUSIONS:BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.
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General Note: M3: 10.1186/ar2822
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Research article


Hypertrophy is induced during the in vitro chondrogenic
differentiation of human mesenchymal stem cells by bone

morphogenetic protein-2 and bone morphogenetic protein-4 gene

transfer
Andre F Steinert1,2, Benedikt Proffen1, Manuela Kunz1, Christian Hendrich1,
Steven C Ghivizzani2,3, Ulrich NSth1, Axel Rethwilm4, Jochen Eulert1 and Christopher H Evans2


1Orthopaedic Center for Musculoskeletal Research, Orthopaedic Clinic, Konig-Ludwig-Haus, Julius-Maximilians-University, Brettreichstrasse 11,
97074 Warzburg, Germany
2Center for Molecular Orthopaedics, Harvard Medical School, 221 Longwood Avenue, BLI 152, Boston, MA 02115, USA
3Department of Orthopaedics and Rehabilitation, University of Florida, 3450 Hull Road, Gainesville, FL 32607, USA
41nstitut for Virologie und Immunbiologie, Julius-Maximilians-University, Versbacherstrasse 7, 97078 Warzburg, Germany

Corresponding author: Christopher H Evans, cevans@bidmc.harvard.edu

Received: 1 May 2009 Revisions requested: 10 Jun 2009 Revisions received: 15 Sep 2009 Accepted: 2 Oct 2009 Published: 2 Oct 2009

Arthritis Research & Therapy 2009, 11 :R148 (doi:10.1186/ar2822)
This article is online at: http://arthritis-research.com/content/11/5/R148
C 2009 Steinert et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract


Introduction The present study compares bone morphogenetic
protein (BMP)-4 and BMP-2 gene transfer as agents of
chondrogenesis and hypertrophy in human primary
mesenchymal stem cells (MSCs) maintained as pellet cultures.

Methods Adenoviral vectors carrying cDNA encoding human
BMP-4 (Ad.BMP-4) were constructed by cre-lox combination
and compared to previously generated adenoviral vectors for
BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or
firefly luciferase (Ad.Luc). Cultures of human bone-marrow
derived MSCs were infected with 5 x 102 viral particles/cell of
Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured
for three weeks in a defined, serum-free medium. Untransduced
cells or cultures transduced with marker genes served as
controls. Expression of BMP-2 and BMP-4 was determined by
ELISA, and aggregates were analyzed histologically,
immunohistochemically, biochemically and by RT-PCR for
chondrogenesis and hypertrophy.


Results Levels of BMP-2 and BMP-4 in the media were initially
30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2
genes were equipotent inducers of chondrogenesis in primary
MSCs as judged by lacuna formation, strong staining for
proteoglycans and collagen type II, increased levels of GAG
synthesis, and expression of mRNAs associated with the
chondrocyte phenotype. However, BMP-4 modified aggregates
showed a lower tendency to progress towards hypertrophy, as
judged by expression of alkaline phosphatase, annexin 5,
immunohistochemical staining for type X collagen protein, and
lacunar size.
Conclusions BMP-2 and BMP-4 were equally effective in
provoking chondrogenesis by primary human MSCs in pellet
culture. However, chondrogenesis triggered by BMP-2 and
BMP-4 gene transfer showed considerable evidence of
hypertrophic differentiation, with, the cells resembling growth
plate chondrocytes both morphologically and functionally. This
suggests caution when using these candidate genes in cartilage
repair.


AGC: aggrecan core protein; ALP: alkaline phosphatase; Ann: Annexin; ATP: adenosine 5 triphosphate; Ad: adenoviral vector; BMP: bone morpho-
genetic protein; BSA: bovine serum albumin; CFDA: carboxyfluorescein diacetate; COL: collagen; CS: chondroitin sulphate; COMP: cartilage oligo-
meric matrix protein; DMEM: Dulbecco's modified eagle media; EF1 : elongation factor 1 ; ELISA: enzyme linked immunosorbent assay; FBS: fetal
bovine serum; FGF: fibroblast growth factor; FMD: fibromodulin; GAG: glycosaminoglycan; GFP: green fluorescent protein; H&E: hematoxylin and
eosin; Ig: immunoglobulin; IHH: indian hedgehog; Luc: luciferase; MSC: mesenchymal stem cell; OP: osteopontin; PBS: phosphate-buffered saline;
PCR: polymerase chain reaction; RUNX2: runt-related transcription factor 2; SD: standard deviation; SOX9: SRY (sex determining region Y) box9;
TBS: Tris-buffered saline; TGF: transforming growth factor.


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Arthritis Research & Therapy Vol 11 No 5 Steinert et al.



Introduction
Mesenchymal progenitor cells, also referred to as mesenchy-
mal stem cells (MSCs), provide an attractive alternative to
chondrocytes with regard to cell-based approaches to carti-
lage repair [1]. With the use of the proper three-dimensional
serum-free culture conditions, expanded MSCs can be stimu-
lated to differentiate along the chondrogenic pathway when
the appropriate factors, such as certain members of the trans-
forming growth factor (TGF)-P superfamily, are present [2-4].
This research has led to the first clinical application of autolo-
gous bone marrow stromal cells for the repair of full-thickness
articular cartilage defects in humans [5,6]. However, to date,
the delivery of MSCs into cartilaginous lesions has neither clin-
ically nor experimentally resulted in sustained regeneration of
hyaline cartilage in vivo [7]. Inadequate delivery of the soluble
factors necessary to drive the chondrogenic differentiation of
the transplanted cells in vivo is a major impediment to effective
chondrogenesis in situ [7]. To overcome this limitation, gene
transfer approaches are being explored clinically [8] and
experimentally [9-12] to enable the sustained delivery of chon-
drogenic and anti-inflammatory factors to cartilage defects.

Another obstacle was identified from studies of in vitro chon-
drogenesis using MSCs or chondrocytes treated with bone
morphogenetic proteins (BMPs), members of the TGF-jP
superfamily. BMPs are a group of secreted polypeptides with
pleiotropic roles in many different cell types and were originally
identified by their ability to induce endochondral bone forma-
tion in ectopic extraskeletal sites in vivo [1,7-10]. Among other
BMPs, BMP-2 and BMP-7 are known to induce differentiation
of mesenchymal progenitor cells and preosteoblasts into
mature osteoblasts, and to enhance the differentiated function
of osteoblasts, which have led to the clinical application of
these proteins for bone regeneration [1,7-10]. We and others
have tested several BMPs for their potential use in cartilage
regeneration including BMP-2, BMP-4, BMP-6 and BMP-7,
which were shown to induce chondrogenic differentiation of
mesenchymal progenitor cells and to up regulate the levels of
type II collagen and aggrecan in chondrocytes and chondro-
progenitor cells [1,7-11]. During development of the limbs,
however, BMPs along with other regulators also mediate the
replacement of chondrogenesis by endochondral ossification
comprising chondrocyte maturation, hypertrophy, transition
from type II to type X collagen with subsequent chondrocyte
apoptosis, while osteoprogenitor cells differentiate into oste-
oblasts and replace the cartilage with mineralized bone tissue.
Equivalently, chondrogenic cultures induced by BMPs
showed high expression of genes associated with chondro-
cyte hypertrophy, including collagen type (COL) X and indian
hedgehog (IHH), among others [1,7-11,13]. This suggests
that the chondrogenic differentiation of the MSCs advanced to
the end stage, hypertrophic state that is typical of endochon-
dral ossification during skeletal development. This conclusion
correlates well with existing in vivo data. For example, delivery
of BMP-2 expressing MSCs resulted in tissue hypertrophy and


the formation of osteophytes, when transplanted orthotopically
to osteochondral defects [14] or ectopically [15,16] in small
animal models. Moreover, such hypertrophy-associated
changes are not exclusively found in terminal differentiated
growth plate chondrocytes, but are also present in pathologi-
cal conditions such as osteoarthritis [17,18].

Inspired by these observations, we aim to further explore the
effects of chondrogenic-induction by BMPs on hypertrophy,
maturation and apoptosis. We have previously shown that
adenoviral delivery of individual cDNAs encoding BMP-2 or
TGF-pl into primary MSCs is capable of driving chondrogen-
esis in culture [19,20]. In the present study, using adenoviral-
mediated gene transfer our aim was to compare the effects of
BMP-4 and BMP-2 expression on chondrogenesis of primary
MSCs and to investigate whether levels and extent of hyper-
trophy in vitro is influenced by the choice of transgene.

Materials and methods
Construction and preparation of recombinant adenoviral
vectors
The complete coding sequence of the human BMP-4 gene
[GenBank:M22490] cloned into X gt10 bacteriophage vec-
tors (ATCC No. 40342; Manassas, VA, USA) was isolated
and purified according to standard protocols [21]. The iso-
lated X gtl 0 DNA was then digested with EcoRI to release the
1.7 kB sized BMP-4 cDNA insert, which was then cloned into
the EcoRI site of the pAdlox shuttle vector, and first-genera-
tion, El, E3-deleted, serotype 5 adenoviral vectors carrying
the cDNAs for human BMP-4 were constructed by cre-lox
recombination as previously described [22]. The vectors
encoding BMP-2, firefly luciferase (Luc) or green fluorescent
protein (GFP) from jellyfish were generated previously [22].
The resulting vectors were designated Ad.BMP-2, Ad.BMP-4,
Ad.Luc and Ad.GFP, and suspensions of recombinant adeno-
virus were prepared by amplification in 293 cells followed by
purification using three consecutive CsCI gradients [22]. Viral
titers were estimated to be between 1012 and 1013 particles/
mL by optical density at 260 nm and standard plaque assay.

Culture of human bone marrow-derived MSCs and
adenoviral transduction
Bone marrow was harvested from the surgical waste of femurs
of six patients, aged 48 to 63 years (mean age 55 years),
undergoing total hip arthroplasty, after informed consent was
given and as approved by the institutional review board of the
University of Wuerzburg as described earlier [23]. The col-
lected cells were spun at 1 x 103 rpm for five minutes, resus-
pended in complete DMEM (containing 10% fetal bovine
serum (FBS) and 1% penicillin/streptomycin), and plated at 4
to 6 x 107 nucleated cells per 75 cm2 flask (Falcon, Beckton
Dickinson Labware, Franklin Lakes, NJ, USA). Unattached
cells were removed after three days, and adherent colonies
were cultured at 370C, 5% C02 in DMEM with 10% FBS sup-
plemented with 1 ng/mL fibroblast growth factor (FGF) -2 for


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expansion of chondroprogenitor cells. Medium changes were
performed every three to four days, and after 14 days adherent
colonies were trypsinized and related in several 75 cm2 tis-
sue culture flasks. At confluence (approximately 1.2 x 106
cells/T-75 flask), the cultures were infected in 750 U-L serum-
free DMEM for two hours at a dose of 5 x 103 vp/cell of
Ad.BMP-2, or Ad.BMP-4. Control cultures were similarly
infected with Ad.GFP or Ad.Luc at 5 x 103 vp/cell, or
remained uninfected. For comparison, an additional set of
untransduced recombinant human protein controls were main-
tained, which were cultured in the presence of 10 ng/mL TGF-
31 protein, or 25 ng/mL BMP-2, or 25 ng/mL BMP-4 (all R&D
Systems, Minneapolis, MN, USA). Following viral infection, the
supernatant was aspirated and replaced with 10 mL complete
DMEM.

Aggregate culture and transgene expression
Twenty-four hours post-infection, the MSC cultures were
trypsinized, washed and placed in aggregate culture as
described previously [24], and as modified by Penick and col-
leagues [25]. Briefly, MSCs were suspended to a concentra-
tion of 1 x 106 cell/mL in serum-free DMEM containing 1 mM
pyruvate, 1% ITS + Premix (insulin, transferring and selenous
acid containing culture supplement), 37.5 mg/mL ascorbate-
2-phosphate and 10-7 M dexamethasone (all Sigma, St. Louis,
MO, USA), and 200 U-L aliquots (2 x 105 cells) were distrib-
uted to a polypropylene, v-bottom 96-well plate (Corning,
Corning, NY, USA) to promote aggregate formation. As men-
tioned above, to particular control aggregates 25 ng/mL BMP-
2, 25 ng/mL BMP-4, or 10 ng/mL TGF-pl recombinant pro-
tein (all R&D Systems, Minneapolis, MN, USA) was added to
induce chondrogenesis. The cell pellets were cultured at
370C, 5% CO2 and formed spherical aggregates within 24
hours. Changes of media were performed every two to three
days, with the recombinant protein being also freshly added to
the respective controls. The aggregates were harvested at var-
ious time points for further analyses.

Media conditioned by the aggregates over a 24-hour period
were collected at day 3, 7, 14 and 21 of culture and assayed
for BMP-2 and BMP-4 expression using the appropriate com-
mercially available ELISA kits (R&D Systems, Minneapolis,
MN, USA).

Cell proliferation, glycosaminoglycan and alkaline
phosphatase assays
For analysis of cell proliferation in aggregates, the WST1 test
was performed at day 3, 7, 14 and 21 of culture according to
the directions of the supplier (Boehringer, Ingelheim, Ger-
many). Briefly, at time points indicated, pellets were washed
twice with PBS and incubated with the WST1 reagent for two
hours at 370C. After this incubation, the formazan dye pro-
duced by metabolically active cells was quantified by measur-
ing the absorbance at 450/690 nm in 96-well plates (Falcon).


Available online http://arthritis-research.com/content/1 1/5/R148



Cell proliferation in aggregates was further assessed by quan-
titative detection of adenosine 5'-triphosphate (ATP), which
correlates with the number of viable cells present using the
CellTiter-Glo Luminescent Cell Viability Assay (Promega,
Mannheim, Baden-Wurtemberg, Germany) according to the
manufacturer's instructions. Briefly, pellets were homogenized
mechanically using a pellet pestle and mixed with 100 U-L of
CellTiter-Glo reagent, which was generated by reconstitution
of CellTiter-Glo substrate with CellTiter-Glo buffer. After
incubation for 10 minutes at room temperature luminescence
was measured using a plate-reading luminometer.

For analysis of glycosaminoglycan (GAG) content, aggregates
were washed with PBS, digested with 200 U-L of papain digest
solution (1 ig/mL, Sigma, St. Louis, MO, USA), and incubated
for 16 hours at 650C. Total GAG content was measured by
reaction with 1,9-dimethylmethylene blue using the BlyscanTM
Sulfated Glycosaminoglycan Assay (Biocolor Ltd., New-
townabbey, Northern Ireland) as directed by the supplier. For
normalization, DNA content of aggregates was also deter-
mined fluorometrically using the Quant-iTTM PicoGreen kit as
directed by the supplier (Invitrogen GmbH, Karlsruhe, Ger-
many).

Alkaline phosphatase (ALP) activity was measured densito-
metrically using change in absorbance at 405 nm by the con-
version of p-nitrophenyl phosphate to p-nitrophenol and
inorganic phosphate, as described previously [26]. Briefly,
aggregates were homogenized mechanically and incubated
with 0.1 mL of alkaline lysis buffer (0.1 M glycin, 1% triton X-
100, 1 mM MgCI2, 1 mM ZnCI2) at room temperature for one
hour. Thereafter 100 U-L of lysis buffer was added which was
supplemented with p-nitrophenylphosphate (2 mg/mL; Sigma,
St. Louis, MO, USA), and stopped after 15 minutes with 50 U-L
50 mM NaOH before optical densities were determined at
405 nm in an ELISA reader. ALP activity was referred to a
standard curve made from p-nitrophenol (Sigma, St. Louis,
MO, USA), and normalized to the DNA content and given as
relative ALP activity in U/jLg.

Histological and immunohistochemical analyses
For histological analyses, aggregates were fixed in 4% para-
formaldehyde for one hour before tissue processing. After
dehydration in graded alcohols, the aggregates were paraffin
embedded, and sectioned to 5 jim. Representative sections
were stained using H&E for evaluation of cellularity and alcian
blue (Sigma, St. Louis, MO, USA) for the detection of matrix
proteoglycan. ALP activity was also detected by a histochem-
ical assay performed according to the manufacturer's protocol
(Sigma, St. Louis, MO, USA) and alternate sections were used
for immunohistochemistry.

For immunohistochemical analyses, sections were washed for
20 minutes in Tris-buffered saline (TBS), and incubated in 5%
BSA (Sigma, St. Louis, MO, USA). Following washing in TBS,


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Arthritis Research & Therapy Vol 11 No 5 Steinert et al.



sections were pre-digested with pepsin at 1 mg/mL in Tris-
HCI (pH 2.0) for 15 minutes at room temperature for COL II
detection, or with chondroitinase ABC (Sigma, St. Louis, MO,
USA) for 10 minutes for chondroitin-4-sulfate (CS4) detection
(5 U/mL in distilled water), or with 0.25% trypsin containing 1
mM EDTA for 15 minutes at 370C for COL X detection, before
sections were incubated overnight at 4C primary antibodies
diluted in 0.5% BSA. As primary antibodies monoclonal anti-
COL II (Acris Antibodies GmbH, Hiddenhausen, Germany),
anti-CS4 (Millipore GmbH, Schwalbach, Germany) or anti-
COL X antibodies (Calbiochem, Bad Soden, Germany) were
used. Immunostaining was visualized by treatment with perox-
idase-conjugated antibodies (Dako, Hamburg, Germany) fol-
lowed by diaminobenzidine staining (DAB kit; Sigma, St.
Louis, MO, USA). The slides were finally counterstained with
hemalaun (Merck, Darmstadt, Germany). For all immunohisto-
chemical analyses, controls with non-immune immunoglobulin
(Ig) G (Sigma, St. Louis, MO, USA) instead of the primary anti-
bodies were performed.

Although more sophisticated and accurate methods of lacu-
nae size determination have been described [27], we used a
simple random field histomorphometric cell surface area
measurement procedure to approximate cell sizes in aggre-
gates. For each aggregate analyzed, three individual mid-sec-
tions stained with H&E or alcian blue were taken, and the
surface areas of 10 randomly chosen lacunae by two inde-
pendent investigators (AFS and BP) in a blinded fashion were
measured from each of three representative microscope views
taken from the center or the periphery (outer 200 itm area)
section using the KS 400 computerized image analysis sys-
tem (Carl Zeiss GmbH, Jena, Germany). At least three differ-
ent aggregates per group and bone marrow preparations from
five different preparations were analyzed.

For comparison, we also analyzed the sizes of the lacunae
within different zones of growth plate cartilage obtained from
a four-year-old child, from whom a sixth toe was removed. Spe-
cifically, from the toe we obtained four physes (two joints) and
at least three sections per physis were analyzed by measuring
the surface areas of 10 randomly chosen lacunae from each of
three representative microscope views by two independent
investigators (AFS and BP). The lacunae were taken from the
reserve, proliferative, hypertrophic or calcifying zone.

Cell viability and apoptosis assay
As annexin 5 (Ann5) is expressed by hypertrophic chondro-
cytes and in osteoarthritic cartilage [17], we were next inter-
ested in the appearance of live and apoptotic cells within our
aggregate system after 10 and 21 days, which was visualized
using the Ann5-Cy3 apoptosis detection kit (APOAC; Sigma,
St. Louis, MO, USA) as directed by the supplier. The assay
uses the Cy3.18 dye as red fluorochome conjugated with
Ann5-Cy3 for apoptosis detection through binding to phos-
phatidylserine epitopes on the plasma membrane of early


apoptotic cells, and the hydrolysis of the non-fluorescent 6-
carboxyfluorescein diacetate (6-CFDA) to the green fluores-
cent compound 6-carboxyfluorescein by the esterases of living
cells to label viable cells. This combination allows the differen-
tiation among early apoptotic cells (Ann5 positive, 6-CFDA
positive), necrotic cells (Ann5 positive, 6-CFDA negative), and
viable cells (Ann5 negative, 6-CFDA positive). Aggregates
were incubated with 50 UiL of the double labelling staining
solution for 10 minutes at room temperature. After staining,
aggregates were washed five times with 100 UiL of binding
buffer, fixed overnight in PBS-buffered 4% paraformaldehyde,
dehydrated, infiltrated with isoamylacetate (Merck, Hohenb-
runn, Germany), embedded in paraffin, and sectioned to 4 uim.
Viable and non-viable cells were observed on the respective
mid-sections using a fluorescence microscope and the appro-
priate green and red filters.

Total RNA extraction, semi-quantitative and real-time
RT-PCR
RNA was extracted from MSC aggregates at the indicated
time-points. For this, 6 to 10 pellets per group and time point
for each donor were pooled and homogenized using a pellet
pestle and repeated titration in 1 mL of Trizol reagent (Invitro-
gen, Karlsruhe, Germany). Total RNA was subsequently
extracted using Trizol reagent with an additional purification
step using separation columns (NucleoSpin RNA II kit; Mach-
erey-Nagel GmbH, DUren, Germany) including a DNase treat-
ment step according to the manufacturer's instructions. RNA
from aggregates of each condition (2 uig each group) was
used for random hexamer primed cDNA synthesis using Bio-
Script reverse transcriptase (Bioline GmbH, Luckenwalde,
Germany).

For semi-quantitative PCR analyses equal amounts (100 ng)
of each cDNA were used as templates for amplification in a 30
UiL reaction volume using MangoTaq DNA Polymerase Taq
(Bioline GmbH, Luckenwalde, Germany) and 5 pmol of gene-
specific primers, which were used to detect mRNA transcripts
characteristic of chondrogenic, hypertrophic or osteogenic
differentiation states. The sequences, annealing temperatures
and product sizes of forward and reverse primers used for
COL II, aggrecan core protein (AGC), cartilage oligomeric
matrix protein (COMP), fibromodulin (FMD), SRY (sex deter-
mining region Y) box9 (SOX9), COL I, COL X, osteopontin
(OP), IHH, runt-related transcription factor 2 (RUNX 2) are
listed in Table 1, with elongation factor la (EF1a) serving as
housekeeping gene and internal control. The RT-PCR prod-
ucts were electrophoretically separated on 1.5% agarose gels
containing 0.1 mg/mL ethidium bromide and visualized using
the Bio Profile software (LTF, Wasserburg, Germany), allow-
ing correlation between EFla signals and cycle number for
each sample. The densities of the PCR bands were analyzed
with the Bio 1 D/Capt MW software (LTF, Wasserburg, Ger-
many) and the mean ratio (fold change), normalized to expres-


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Available online http://arthritis-research.com/content/1 1/5/R148


Table 1

Primer sequences and product sizes, for semi-quantitative and real-time RT-PCR


RT-PCR primer sequences (5'-3')


Annealing temp. (C)


Product size (bp) Cycles


Chondrogenic markers
COL II Sense: TTTCCCAGGTCAAGATGGTC
Antisense: CTTCAGCACCTGTC CACCA
AGC Sense: TGAGGAGGGCTGGAACAAGTACC
Antisense: GGAGGTGGTAATTGCAGGGAACA
COMP Sense: CAGGACGACTTTGATGCAGA
Antisense: AAGCTGGAGCTGTCTGGTA
FMD Sense: CTTACCCCTATGGGGTGGAT
Antisense: GTACATGGCCGTGAGGAAGT
SOX9 Sense: ATCTGAAGAAGGAGAGCGAG
Antisense: TCAGAAGTCTCCAGAGCTTG
SOX9 (rt) Sense: GGA GTGGAAGTTACTGACTGATG
Antisense: AGGCGTTTTGCTTCGTCAATG
Hypertrophy and osteogenic markers
COL I Sense: GGACACAATGGATTGCAAGG
Antisense: TAACCACTGCTCCACTCTGG
COL X Sense: CCCTTTTTGCTGCTAGTATCC
Antisense: CTGTTGTCCAGGTTTTCCTGGCAC
OP Sense: ACGCCGACCAAGGAAAACTC
Antisense: GTCCATAAACCACACTATCACCTCG
ALP (rt) Sense: TGGAGCTTCAGAAGCTCAACACCA
Antisense: ATCTCGTTGTCTGAGTACCAGTCC
IHH Sense: GAGGAGTCCCTGCATTATGA
Antisense: CAGGAAAATGAGCACATCGC
RUNX2 Sense: ACAGATGATGACACTGCCACC
Antisense: CATAGTAGAGATATGGAGTGCTGC
Internal control


EF1


Sense: AGGTGATTATCCTGAACCATCC
Antisense: AAAGGTGGATAGTCTGAGAAGC


rt: primer pairs, that have been used for real-time PCR only.
AGC = aggrecan core protein; ALP = alkaline phosphatase; COL = collar
factor 1 ; FMD = fibromodulin; IHH= indian hedgehog; OP = osteopontil
determining region Y) box9.

sion of the EFla housekeeping gene, was calculated from
three bands (one per patient).

For a more detailed mRNA expression profile of chondrogenic
and hypertrophy associated genes, genetically-modified MSC
aggregates were subjected to real-time quantitative PCR anal-
yses. One microliter of each cDNA was used as template for
amplification in a 50 1iL reaction volume using BioTaq DNA
Polymerase Taq (Bioline GmbH, Luckenwalde, Germany) and
50 pmol of gene-specific primers was used for COL II, SOX9,
ALP and COL X as listed in Table 1. Real-time PCR conditions
were as follows: 30 seconds at 94C, 30 seconds at anneal-
ing temperature, 60 seconds at 720C (see Table 1 for PCR
conditions). Real-time PCR was performed with the DNA
Engine Opticon system (MJ Research, Waltham, MA, USA)
using SYBR Green (Biozym Scientific GmbH, Hessisch Old-
endorf, Germany) as fluorescent dye allowing determination of


= cartilage oligomeric matrix protein; EF1 = elongation
runt-related transcription factor 2; SOX9 = SRY (sex


the threshold cycle at which exponential amplification of PCR
products begins. Specificities of amplicons were confirmed by
melting curve analyses by gel electrophoresis of test PCR
reactions. For quantification mRNA expression was normal-
ized to the expression levels of the housekeeping gene EF1a
and relative expression levels compared with values from
undifferentiated monolayer MSCs are shown using the relative
expression software tool (REST) [28]. Each PCR was per-
formed in triplicate on three separate bone marrow prepara-
tions for each independent experiment.

Statistical analysis
The data from the ELISA, WST1, ATP, GAG, DNA, and ALP
content, cell surface area and RT-PCR analyses were
expressed as mean values standard deviation (SD). Each
experiment was performed in quadruplicate (n = 4) and
repeated on at least three and up to six individual marrow prep-


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Gene


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321








Arthritis Research & Therapy Vol 11 No 5 Steinert et al




arations from different patients (m = 3 to 6), as indicated in the
respective experiments. All numerical data were subjected to
variance analysis (one or two factor analysis of variance) and
statistical significance was determined by student's t-test, and
level of P< 0.05 was considered significant.


Figure 1
(a)
60


'E 40


20

0-
0


BMP-2 ELISA


= Ad.GFP
FMM Ad.BMP-2


Results
Transgene expression by aggregates of genetically
modified MSCs
Consistent with previous findings [21], cultures infected with
these doses of Ad.BMP-2 and Ad.BMP-4 generated approxi-
mately 30 to 60 ng/mL of gene product per 24 hours at day 3
post-infection (Figures la, b). The amount of each transgene


(b)
60

0
'E 40

0.
oi 20


0


BMP-4 ELISA


=I Ad.GFP
M Ad.BMP-4


WST1 test ATP test *
0.6 16.
0 [ Ad.GFP 1 = Ad.GFP *
Ad.BMP-2 o 7 Ad.BMP-2
M Ad.BMP-4 12 Ad.BMP-4 *
Ad.BMP4 ~ 12
S0.4 *.
8.

M* 4
0
S0.2I
0 C 4
0C 0


Day 3


Day 7


Day 14 Day 21


GAG content


= Ad.GFP
F Ad.BMP-2
M Ad.BMP-4


Day 3 Day 7 Day 14 Day 21


(f)
0.8

0.6


I 0.4
a.
-J
0.2


0


Day 3 Day 7 Day 14 Day 21

ALP assay
Ad.GFP
- Ad.BMP-2
- Ad.BMP-4


Day 3


Day 7


Day 14


Day 21


Transgene expression and biochemical composition of MSCs during 21 days of aggregate culture following BMP-2 and BMP-4 gene transfer. Pri-
mary MSCs were infected with Ad.BMP-2, Ad.BMP-4 or Ad.GFP at 5 x 102 vp/cell, seeded into aggregates and analyzed biochemically during a
three-week time course. (a, b) Values represent levels of (a) BMP-2 and (b) BMP-4 transgene product expressed in ng/mL in the conditioned media
over a 24-hour period at days 3, 7, 14 and 21. At the same time-points cell proliferation was quantified using the (c) WST1 and (d) ATP cell prolif-
eration assay, (e) GAG content and (f) relative ALP activity normalized to DNA is shown. The data represent mean values + standard deviation from
four aggregates per condition and marrow preparation and was performed on five marrow preparations from different patients. Asterisks indicate val-
ues that are statistically different (P < 0.05) from marker gene vector-transduced control cultures or between samples. ALP = alkaline phosphatase;
ATP = adenosine 5 triphosphate; Ad = adenoviral vector; BMP = bone morphogenetic protein; GAG = glycosaminoglycan; MSC = mesenchymal
stem cell.


Page 6 of 15
(page number not for citation purposes)


(e)
80


S60

4 40

0
( 20
0

0











product steadily decreased thereafter, and reached levels of
about 3 to 6 ng/mL at day 21 (Figures la, b). Levels of BMP-
2 and BMP-4 in media conditioned by Ad.GFP or Ad.Luc
infected cultures were below 200 pg/mL (Figures la, b),
equivalent to the levels observed in the naiVe controls (data not
shown).

Cell proliferation, GAG content and ALP activity
As primary MSCs were shown to be capable of expressing the
BMP-2 or the BMP-4 transgene in aggregate culture, we
examined the effects of BMP-2 and BMP-4 gene delivery on
cell proliferation using the WST1 cell proliferation assay. At
day 3 and 7 of culture the cell proliferation rate in MSC aggre-
gates was approximately equal in all groups tested (Figure 1 c).
BMP-2 and BMP-4 transduced MSC aggregates maintained
their proliferation rate over 21 days while Ad.GFP cells (Figure
1c) and unmodified control cultures (not shown) decreased
rate of proliferation (Figure 1c). The same pattern was
observed using the ATP test, where sustained high cell prolif-
eration rates were observed at day 14 and 21 in BMP-2- and
BMP-4-modified aggregates compared with the controls,
while at the same time points, levels in the BMP-2-modified
aggregates were significantly elevated compared with the
BMP-4 cultures (Figure 1 d). To quantitatively compare extra-
cellular matrix synthesis among treatment groups, GAG levels


Available online http://arthritis-research.com/content/1 1/5/R148



in the aggregates after 21 days in culture were determined
(Figure le). All aggregates infected with Ad.BMP-2 or
Ad.BMP-4 showed significantly increased GAG production
relative to those receiving Ad.GFP (Figure le), Ad.Luc or
untransduced aggregates (not shown), which showed no evi-
dence of chondrogenesis. At days 14 and 21, significantly ele-
vated levels of GAG synthesis in the BMP-2 compared with
the BMP-4 transduced cultures became apparent (Figure 1 e).
Indicative of hypertrophic chondrocytes we analyzed ALP
activity, which was found to be significantly elevated at all time
points in the BMP-2-modified aggregates compared with the
GFP controls and BMP-4 transduced cultures, whereas signif-
icantly higher values in the BMP-4 modified cultures compared
with the GFP controls could only be resolved at day 14 and 21
(Figure 1f).

Histological and immunohistochemical analyses of
chondrogenesis
Transduction of MSCs with adenoviral vectors encoding
BMP-2 (Figure 2b) or BMP-4 (Figure 2c) using viral doses suf-
ficient to generate 30 to 60 ngs transgene product at day 3
induced a significant chondrogenic response in the respective
aggregate cultures compared with the controls (Figure 2a),
which were not chondrogenic. This was demonstrated by
increased aggregate size and strong production of proteogly-


Figure 2


H & E Alcian blue
Day 10 Day 21 Day 10 Day 21
50 X 200X 50 X 200 X 50 X 200 X 50 X .200X
"; r "'" "_
n ~ -.. .__...,,.__._.,..... ., .



N
(b) 0..



(c) C .
IM O'I


Control
U)

> ~
0 *~
Z


rhTGF-P~ rhBMP-2 rhBMP-4
F 'fP "

; f ,- =


Histological appearance of MSC pellets after chondrogenic induction with BMP-2 or BMP-4 gene transfer. Monolayer cultures of MSCs were
infected with (a) Ad.GFP, (b) Ad.BMP-2 or (c) Ad.BMP-4 at 5 x 102 vp/cell as indicated, seeded into aggregates 24 hours after infection and cul-
tured in serum-free medium for 21 days. Representative sections after 10 and 21 days are shown. (Left panels) H&E staining for evaluation of cellu-
larity and cell morphology. (Right panels) Alcian blue staining for detection of matrix proteoglycan. (a to c) Panels are reproduced at low (50x; bar=
200 um) or high (200x; bar = 50 or 100 um) magnification as indicated. (d) Comparative uninfected aggregate cultures after 21 days, that were
maintained in the absence (control) or presence of recombinant human TGF-p 1 (10 ng/mL), or BMP-2 (25 ng/mL), or BMP-4 (25 ng/mL) protein as
indicated. Panels are reproduced at low (50x; bar = 100 gm) magnification. Ad = adenoviral vector; BMP = bone morphogenetic protein; GFP =
green fluorescent protein; H&E = hematoxylin and eosin; MSC = mesenchymal stem cell; TGF = transforming growth factor.


Page 7 of 15
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Arthritis Research & Therapy Vol 11 No 5 Steinert et al.



cans as indicated by metachromatic staining with alcian blue
in the Ad.BMP-2 or Ad.BMP-4 transduced cultures (Figures
2b, c) compared with the Ad.GFP controls (Figure 2a). Inter-
estingly, the phenotype of the Ad.BMP-4 (Figure 2c) infected
aggregates appeared chondrogenic but less hypertrophic at
day 10 and 21 compared with the Ad.BMP-2 cultures in that
the BMP-2-modified cells were more rounded with greater
cytoplasmic volume (Figure 2b).

Correspondingly, immunohistochemical analyses for COL II,
the predominant collagen type in cartilage, and CS4, one of
the monomers of the polysaccharide portion of proteoglycan,
showed significantly enhanced production of these cartilage
matrix proteins at days 10 and 21 of culture in the aggregates
receiving Ad.BMP-2 (Figure 3b) or Ad.BMP-4 (Figure 3c) rel-
ative to the Ad.GFP (Figure 3a) controls.

Uninfected aggregates maintained in the presence of recom-
binant BMP-2, BMP-4, or TGF-pl protein were also chondro-
genic as evidenced by lacunae formation, positive staining for
alcian blue (Figure 2d), COL II and CS4 (not shown), although
the stage of chondrogenesis seemed less progressed com-
pared with that in the aggregates genetically modified with
BMP-2 or BMP-4 (Figures 2b, c) after 21 days, while control
cultures where growth factor supplementation was absent
were non-chondrogenic.

Hypertrophic differentiation and apoptosis
We used staining for ALP and immunohistochemistry for COL
X as markers for chondrocyte hypertrophy (Figure 4). No
detectable ALP and only weak COL X immunostaining was
seen in the control aggregates transduced with Ad.GFP (Fig-


ure 4a). ALP staining was primarily pericellular in the aggre-
gates infected with Ad.BMP-4 (Figure 4c). In contrast,
aggregates transduced with Ad.BMP-2 showed more abun-
dant staining for ALP throughout the extracellular matrix at day
10 and was most extensive at day 21 of culture (Figure 4b).
Correspondingly, immunohistochemical analyses of the
Ad.BMP-2 infected aggregates revealed strong abundant
staining for COL X in the aggregate matrix at day 10 and 21 of
culture (Figure 4b). In the Ad.BMP-4-modified cultures COL X
immunostaining of the matrix was strongly observed at day 21
in the aggregate matrix, while staining tended to be pericellular
at day 10 of culture (Figure 4c); no significant differences
were noted among the aggregates. Notably, the distribution
pattern of the hypertrophy markers was somewhat heteroge-
neous in the aggregates, which we attribute to the rather inho-
mogeneous aggregate morphologies obtained during culture
in v-bottom plates as opposed to more homogeneous aggre-
gate morphologies seen after centrifugation and culture in 15
mL conical tubes [20].

Double fluorescence staining with Ann5-Cy3/6-CFDA
allowed visualisation of Ann5 expressions. The high levels of
green fluorescence found in BMP-modified (Figures 5b, c) and
control groups (Figure 5a) revealed high viability of adenoviral
infected MSCs in aggregate cultures after 10 and 21 days. At
day 10, only very few cells in the Luc (Figure 5a) and BMP-2
(Figure 5b) and BMP-4 (Figure 5c) modified aggregates
appeared to be annexin 5 positive. At day 21, the BMP-2 (Fig.
5B) and the BMP-4 (Fig. 5C) modified groups showed many
Ann5-positive cells, as evidenced by red fluorescence, com-
pared with the Ad.Luc transduced (Figure 5a) and untrans-


Figure 3


Collagen type II
Day 10 Day
50 X 200 X 50 X

(a) D. :Y.









(D , ... :t ; ''


X 50 X


Chondroitin-4-Sulfate
Day 10 Day 21
200 X 50 X "

"'' 5 .,'
i L t .t L


Immunohistochemical analyses for cartilage matrix proteins of MSC pellets after chondrogenic induction with BMP-2 or BMP-4 gene transfer. Mon-
olayer cultures of MSCs were infected with (a) Ad.GFP, (b) Ad.BMP-2 or (c) Ad.BMP-4 at 5 x 102 vp/cell as indicated and placed into aggregate
cultures. Immunohistochemical staining was performed on culture days 10 and 21 for collagen type II (left panels) and chondroitin-4-sulfate (right
panels). Regions of positive immunostaining appear brown. Panels are reproduced at low (50x; bar = 200 p.m) or high (200x; bar = 50 or 100 p.m)
magnification as indicated. Ad = adenoviral vector; BMP = bone morphogenetic protein; GFP = green fluorescent protein; MSC = mesenchymal
stem cell.


Page 8 of 15
(page number not for citation purposes)







Available online http://arthritis-research.com/content/1 1/5/R148


Figure 4


Alkaline
Day 10
50 X 200 X


phosphatase
Day 21
50 X 200 X


Day 10


50 X


Collagen type X


200 X


50 X


- r


.~*JL~ ,~i

I.


Histological and immunohistochemical analyses for hypertrophy of MSC pellets after chondrogenic induction with BMP-2 or BMP-4 gene transfer.
Following genetic modification with (a) Ad.GFP, (b) Ad.BMP-2 or (c) Ad.BMP-4 aggregates after 10 and 21 days of culture stained for ALP (left
panels) and collagen type X (right panels) are shown. Regions of positive immunostaining appear brown. Panels are reproduced at low (50x; bar =
200 uim) or high (200x; bar = 50 or 100 uim) magnification as indicated. Ad = adenoviral vector; ALP = alkaline phosphatase; BMP = bone morpho-
genetic protein; GFP = green fluorescent protein; MSC = mesenchymal stem cell.


duced (not shown) cultures where only very few such cells
were seen.

A similar pattern of hypertrophy and apoptosis was observed
in the untransduced control aggregates that were maintained
in the presence or absence of recombinant BMP-2, BMP-4 or
TGF-pl protein (not shown).


Figure 5


6-CFDA
Day 10 Day 21


Comparison of BMP-2 and BMP-4 modified MSC
aggregates with immature growth plate chondrocytes
In the different types of aggregates examined in Figures 2 to
5, different cell morphologies were apparent, especially with
respect to incidence and extent of lacunae formation. Thus we
were next interested to know if it was possible to distinguish
the types of aggregates produced by measuring the sizes of
the respective lacunae, approximated by simple histomorpho-
metric cell surface area measurement on aggregate sections.
For comparison, we first analyzed the sizes of the lacunae



Annexin 5
Day 10 Day 21
X 0 X 200 X 5.0 X 200 X


(b)







Analyses for cell viability and apoptosis within MSC pellets after chondrogenic induction with BMP-2 or BMP-4 gene transfer. Following genetic
modification with (a) Ad.GFP, (b) Ad.BMP-2 or (c) Ad.BMP-4 aggregates were double-stained with 6-CFDA (left panels) and annexin 5-Cy3 (right
panels) at day 10 and 21 of culture. Representative fluorescence microscopy images are shown. Note that living cells are stained green with 6-
CFDA, late apoptotic cells red with annexin 5-Cy3, while early apoptotic cells stained for both 6-CFDA and annexin 5-Cy3. Panels are reproduced at
low (50x; bar = 200 gim) or high (200x; bar = 50 gim) magnification as indicated. Ad = adenoviral vector; BMP = bone morphogenetic protein;
CFDA = carboxyfluorescein diacetate; GFP = green fluorescent protein; MSC = mesenchymal stem cell.


Page 9 of 15
(page number not for citation purposes)


(a) LL
0
1w ,,.,'-

(b) .


Ct) -
(c) =E
OQf -.


Day 21


200 X


,144







Arthritis Research & Therapy Vol 11 No 5 Steinert et al.


Figure 6
(a)
Juvenile human cartilage


4)



C
0
N
10_



a.

0
N
.2
0.
0
"1-
t
0
C.



0
N4

U)


R.-r~ P.~Ifer.ti- Hyp~rt,ophl. C.ki0&9~


(c)


Ad.GFP


Aggregate center
Aggregate center


(d)

Adult human MSC aggregates
Ad.BMP-2


EZAd.GFP
EJAd.BMP-2
M Ad.BMP-4 *


(e)


Ad.BMP-4


CL
4.
CL


Analysis of hypertrophic cell morphology in MSC pellets after chondrogenic induction with BMP-2 or BMP-4 gene transfer in comparison to growth
plate chondrocytes. Lacunar sizes were measured in the different zones of growth plate cartilage obtained from a four-year-old child, from which a
sixth toe was removed. (a) The different cell morphologies and lacunar sizes in the reserve, proliferative, hypertrophic and calcifying zone of growth
plate cartilage can be observed. (b) Measurements of the lacunar sizes in the respective zones are shown. Note that the mean values +/- SD repre-
sent analyses of size-measurements of 10 lacunae per zone, which were performed on three representative mid-sections per growth plate, and a
total of four physes (one digit, two joints) were examined. (c) The GFP-modified aggregates showed no lacunae formation. (d, e) In contrast the
BMP-2 and BMP-2 modified aggregates displayed a strong chondrogenic phenotype with formation of large lacunae in the (d) BMP-2 and (e) BMP-
4 modified aggregates at day 21 of culture. (f) Analyses of lacunae surface areas in the center and periphery (outer 200 um area) of the different
aggregate types at day 21 of culture. The data represent mean values + SD from four aggregates per condition and marrow preparation and was
performed on six marrow preparations from different patients. Asterisks indicate values that are statistically different (P < 0.05) from marker gene
vector-transduced control cultures. Thus both, the BMP-2 and the BMP-4 were significantly larger compared with the non-chondrogenic controls
and displayed lacunar sizes comparable with those of the hypertrophic and calcifying zones of growth plate cartilage. Original magnification: 200x;
scale bar = 50 uim. BMP = bone morphogenetic protein; GFP = green fluorescent protein; MSC = mesenchymal stem cell; SD = standard devia-
tion.


within different zones of growth plate cartilage obtained from
a four-year-old child, from whom a sixth toe was removed.
These measurements were compared with those of the lacu-
nae found in the center and periphery of the different treatment
groups of genetically modified aggregates.

As shown in Figure 6a, the reserve, proliferative, hypertrophic
and calcifying zone of cartilage could be clearly separated by
the proximity of the cells to the joint space and the bone


respectively, alignment of the chondrocytes along the arcades
of Benninghoff [29] and by the appearance of hypertrophic
cells. Analyses of lacunae surface areas in the different growth
plate zones revealed mean lacunae surface areas SD of
100.8 25.8 upm2 in the reserve zone, 113.3 25.5 upm2 in the
proliferative zone, 288.5 111.0 upm2 in the hypertrophic zone
and 421.8 131.9 upm2 in the calcifying zone of growth plate
cartilage (Figure 6b). The mean values SD represent meas-
urements of 10 lacunae per zone, which were performed on


Page 10 of 15
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three representative mid-sections per growth plate. A total of
four physes (1 digit, 2 joints) was examined.

In contrast the GFP-modified aggregates showed no lacuna
formation, either in the center or in the periphery of the pellets
(Figure 6c). However the BMP-2- and BMP-4-modified aggre-
gates displayed a chondrogenic phenotype with lacunae for-
mation throughout the aggregates (Figures 6d, e). Analyses of
cell surface areas in the different aggregate types revealed a
mean value of 60.6 14.5 upm2 in the center and 57.3 12.4
1pm2 the periphery of the Ad.GFP transduced aggregates,
which showed no lacunae formation, of 541.3 166.3 upm2 in
the center and 386.1 108.7 upm2 the periphery of the
Ad.BMP-2 transduced aggregates, and of 307.8 75.6 upm2
in the center and 248.7 65.4 upm2 the periphery of the
Ad.BMP-4 transduced aggregates (Figure 6f). Thus lacunae
formed in both the BMP-2 and BMP-4 transduced pellets and
led to significantly larger cell surface areas compared with the
non-chondrogenic controls. Nevertheless, the lacunae formed
in the presence of BMP-2 were larger than those formed by
BMP-4 and approximated the size of lacunae noted in the cal-
cifying zone of the human growth plate. In contrast, the lacu-
nae that formed in the presence of BMP-4 were closer in size
to those of the hypertrophic zone (Figures 6e, f).

Time course of chondrocytic and hypertrophic marker
gene expression
To examine further the effects of BMP-2 and BMP-4 gene
delivery on hypertrophic differentiation, we analyzed the tem-
poral expression profiles of genes associated with chondro-
cyte maturation and osteogenic differentiation using semi-
quantitative and real-time RT-PCR (Figure 7). These genes
included AGC, COL II, COMP, FMD, SOX9, RUNX2, COL X,
COLI, ALP, OP and IHH. Consistent with preceding analyses
[30], the aggregate cultures transduced with BMP-2 showed
evidence of chondrogenic differentiation at the RNA level with
upregulation of the chondrogenic markers AGC, COL II, FMD,
COMP and SOX-9 over time, compared with the non-chon-
drogenic Ad.GFP controls where these markers were
expressed only at low levels (Figure 7). Expression of these
genes was upregulated to a similar degree in the BMP-4- and
BMP-2-modified aggregates and marked differences between
the BMP-2 and BMP-4 groups were not observed (Figure 7).

Evidence of chondrocyte hypertrophy at the mRNA level in the
BMP-2- and BMP-4-modified aggregates was reflected by a
subsequent upregulation of COL X and OP at day 3, IHH and
ALP at day 7 and RUNX2 at day 14 compared with Ad.GFP
controls (Figure 7). These results suggest that BMP-2 and
BMP-4 gene transfer induced a significant chondrogenic and
hypertrophic response in MSC aggregates on mRNA level
over time.


Available online http://arthritis-research.com/content/1 1/5/R148



Discussion
We and others have shown previously that primary MSCs
undergo chondrogenesis following genetic modification with
Ad.BMP-2 or Ad.TGF-pl in aggregate culture in vitro [30-32]
or when transplanted into chondral defects in vivo [14]. In the
present study we adapted the MSC aggregate culture system
to determine whether adenoviral delivery of BMP-4 can lead to
chondrogenesis of primary MSCs in vitro, and to evaluate the
extent of hypertrophy compared with BMP-2-modified cul-
tures.

Adenoviral delivery of BMP-4 led to reliable chondrogenesis in
human MSC aggregate cultures in a fashion comparable with
that noted when the same dose of the BMP-2 transgene was
administered as shown by staining with alcian blue, COL II and
CS4 and the quantitative GAG assay, indicating increased
GAG levels at days 14 and 21 in the BMP-2-modified aggre-
gates. Notably, chondrogenic differentiation induced by either
transgene increase levels of metabolic activity and cell prolif-
eration compared with controls as evidenced by the WST1
and ATP assays. Moreover, high levels of chondrocyte hyper-
trophy occurred in MSC pellet cultures modified with either
BMP transgene, as assessed by lacunar size, and expression
of ALP, COL X and Ann5, and was overall slightly more
advanced in the BMP-2-modified cultures compared with the
BMP-4 modified cultures reaching significance levels in the
ALP assay at all time points. Notably, exact the lacunar size
comparisons between growth plate tissues and in vitro cell
pellets might be inaccurate (Figure 6) due to artifacts that may
appear during fixation and processing of these different types
of tissues.

The RT-PCR data are in general agreement with the biochem-
ical and histological observations, showing high levels of
chondrogenic mRNAs in aggregates after BMP-stimulation,
such as AGC, COMP, COL II, SOX9 and FMD. Likewise, tran-
scripts encoding the hypertrophy associated genes COL X,
OP, ALP, RUNX2 and IHH were also strongly present in both
types of BMP-modified aggregates compared with controls.

These observations are in broad agreement with our previous
study using alginate cultures of the murine mesenchymal
C3H10 OT1/2 cell line, stably transfected with BMP-2 or BMP-
4 cDNAs, where similar differences in the pattern of chondro-
genesis and hypertrophy were observed [33]. Although in this
previous study the expression of osteogenic and hypertrophy
markers were partly attributed to the presence of p-glycero-
phosphate, similar increases in hypertrophy associated genes
were seen in the present study where p-glycerophosphate
was absent. Our results are consistent with those reported by
Mackay and colleagues [34] and Mueller and Tuan [35] who
likewise showed that the addition of p-glycerophosphate is not
necessary to obtain a hypertrophic chondrocyte phenotype.


Page 11 of 15
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Arthritis Research & Therapy Vol 11 No 5 Steinert et al.





Figure 7


4-5
CZAd.GFP Aggrecan 5
3 = ]Ad.BMP-2 4
: Ad.BMP-4 3

1t


Day 3 Day 7 Dayl14 Day 21


Collagen type II


^} t


0


Day 3 Day 7 Day 14 Day 21


2 COMP 4Fibromodulin



0,: 1




1 ,2 SOX9 1,6 6RNX2
.- Day 3 Day 7 Day 14 Day 21 Day 3 Day 7 Day 14 Day 21
) 1,2 1,6

> SO -* *0T
0 1.2I




Day 3 Day 7 Day 14 Day 21 Day 3 Day 7 Day 14 Day 21

4) Collagen type X Collagen type I







Day 3 Day 7 Day 14 Day 21 Day 3 Day 7 Day 14 Day 21


Osteopontin


8




Day 3 Day 7 Day 14 Day 21


Indian hedgehog






Day 3 Day 7 Day 14 Day 21


400 IAd.GFP
=Ad.BMP-2
O 300 -MAd.BMP-4

m 200

100


E Day 3
p


16

12


4


Collagen type II 40 SOX9
30

S 20 I



Day 7 Day 14 Day 21 Day 3 Day 7 Day 14 Day 21


Day 3


Alkaline phosphatase *






Day 7 Day 14 Day 21


Profiles of temporal gene expression determined by semi-quantitative and real-time RT-PCR in MSC pellet cultures genetically modified with BMP-2
and BMP-4. Genes analyzed include collagen type (COL) II, aggrecan core protein (AGC), cartilage oligomeric matrix protein (COMP), fibromodulin
(FMD), SRY (sex determining region Y) box9 (SOX9), COL I, COL X, osteopontin (OP), indian hedgehog (IHH), runt-related transcription factor 2
(RUNX2) and alkaline phosphatase (ALP). Primer sequences and product sizes are listed in Table 1, with elongation factor 1 a (EF1 a) serving as
housekeeping gene and internal control. For each marrow preparation/patient, treatment group and time point indicated RNA was extracted from 10
aggregates, and four patients were analyzed depending on group and time point. For the semi-quantitative RT-PCR analyses (upper panels), values
are mean +/- SD raw data of optical band intensities of RT-PCR products between groups and time points (one band per patient), which were nor-
malized using the EF1 a reaction products. Values of the real-time RT-PCR analyses (lower panels) represent mean expression ratios +/- SD normal-
ized to the expression levels of the housekeeping gene EF1 a and relative to values from undifferentiated monolayer MSCs. Asterisks indicate values
that are statistically different (P < 0.05) from marker gene vector-transduced control cultures or between samples. BMP = bone morphogenetic pro-
tein; MSC= mesenchymal stem cell; SD = standard deviation.


Page 12 of 15
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0


S1,

0

0,


0 Collagen type X 16
0 12

0. _* 8



Day 3 Day 7 Day 14 Day 21


0












Our study is also in agreement with studies of in vitro chondro-
genesis with primary MSCs using recombinant proteins,
where BMP-4 was identified as a strong inducer of chondro-
genesis [36], which produced less hypertrophy compared
with BMP-2 [37]. Correspondingly, in vivo implantation of
BMP-4 into abdominal muscles of rats led to ectopic cartilage
and bone formation when delivered as recombinant protein
[38] or via genetically modified cells [39]. Notably, the latter
study revealed differential effects on chondrogenesis and
osteogenesis depending on the type of cell analyzed [39]. Our
study is limited to the use of bone marrow-derived MSCs and
other effects may be seen when different cells are employed.
Orthotopic BMP-4 gene delivery via retrovirus transduction of
muscle-derived stem cells was shown to improve cartilage
repair in rat osteochondral defects [40] and also when it was
administered via adenovirus to dedifferentiated chondrocytes
in osteochondral defects in rabbits [41]. In both studies
improved repair in the BMP-4-treated defects compared with
non-chondrogenic controls at 12 or 24 weeks respectively
was observed, but detailed analyses of hypertrophy and apop-
tosis have not been performed [40,41].

BMP-2 and BMP-4 have been implicated in embryogenesis
and morphogenesis of various tissues and organs, where they
regulate growth, differentiation, chemotaxis and apoptosis of a
variety of cell types, including mesenchymal, epithelial, hemat-
opoietic and neuronal cells [42]. Interestingly, in conditional
knock-out experiments it has been found that a threshold level
of BMP signaling is required for the onset of chondrogenesis,
and hence some chondrogenic condensations failed to form in
limbs deficient in both BMP-2 and BMP-4 [43]. However, in
the condensations that do form, subsequent chondrogenic dif-
ferentiation proceeds normally even in the absence of BMP-2
and BMP-4 [43]. In contrast, it was found that the loss of both
BMP-2 and BMP-4 results in a severe impairment of osteogen-
esis. Deletion of BMP-4 alone did not impair osteogenesis or
fracture repair, while deletion of BMP-2 alone did not impair
osteogenesis but strongly prevented fracture repair [43-45].
This indicates that the presence of BMP-2 or BMP-4 is a pre-
requisite for osteoblastogenesis and these morphogens can
apparently compensate for each other to a certain extent.
However, they are less important for chondrogenesis [43-45].

During limb development, cartilage is gradually replaced by
endochondral ossification, a process in which the chondro-
cytes mature, hypertrophy and express COL X with reduced
production of COL II. Subsequently the cartilage becomes
vascularized and infiltrated by osteoprogenitor cells, while the
chondrocytes undergo apoptosis. The osteoprogenitor cells
differentiate into osteoblasts, replacing the cartilage with min-
eralized bone; BMP-2 and BMP-4 are important regulators of
these processes [46-48]. By using chondroprogenitor cells in
high density, three-dimensional cultures these regulatory
mechanisms can be partially recapitulated. Thus it is not sur-
prising that studies on in vitro chondrogenesis using MSCs or


Available online http://arthritis-research.com/content/1 1/5/R148



chondrocytes incubated with members of the TGF-P super-
family reveal considerable hypertrophy and high levels of COL
X expression. Although the use of COL X as a marker of chon-
drogenic hypertrophy in MSC-based systems has been ques-
tioned [13], it correlates well thus far to the existing in vivo
data. For example, MSCs genetically modified to express
BMP-2 display a significant level of tissue hypertrophy and
osteophyte formation, when transplanted orthotopically to
osteochondral defects [14] or ectopically [15,49] in small ani-
mal models. TGF-pl has been shown to induce hypertrophic
and osteometaplastic changes in the synovium of rabbit joints,
when directly delivered by first-generation adenovirus [50].
Furthermore, implantation of chondrocytes genetically modi-
fied to express BMP-7 has been shown to generate good hya-
line cartilage repair tissue after six weeks in vivo, but after one
year the repair cartilage is no better than that of controls, with
only 0 to 28% of the transplanted cells being detectable at
that time point [51]. This is agreement with a recent large ani-
mal study in pigs, that showed good hyaline cartilage repair
after six weeks, when chondral defects were filled with perios-
teum cells genetically modified with BMP-2, while at six
months the hyaline repair tissue had almost completely van-
ished and was replaced by fibrocartilage [52]. These observa-
tions might be attributed to mechanisms of hypertrophic
differentiation and subsequent apoptosis, although clarifying
analyses in vivo have not been conducted thus far. However,
the presence of Ann5-positive cells in our hypertrophic aggre-
gates modified with BMP-2 or BMP-4 in vitro correspond with
these data.

Our data suggest that the degree of hypertrophic differentia-
tion can be modulated by the choice of morphogenetic stimu-
lus, while still maintaining efficient chondrogenesis. This
permits cautious optimism that it may prove possible ultimately
to achieve effective regeneration of articular cartilage in the
absence of hypertrophic differentiation. Hypertrophic differen-
tiation of neo-cartilage tissue with subsequent apoptosis
development is certainly an undesired effect in cartilage
defects in vivo, because this would lead to loss of the trans-
planted repair cells with subsequent matrix degradation. How-
ever, the relevance of chondrogenic hypertrophy and
apoptosis of human MSCs induced by TGF-P superfamily
members for cartilage repair in vivo has to be considered still
unclear to this end, because this study is limited by its in vitro
nature. Therefore, clarifying in vivo experiments are necessary
before such factors can be recommended for further clinical
use.

Conclusions
Adenoviral BMP-4 gene transfer efficiently induces the chon-
drogenic differentiation of human primary MSCs as effectively
as BMP-2 gene transfer. However, both transgenes induced
high levels of chondrocyte hypertrophy after three weeks of in
vitro culture. It remains to be seen, whether it may be possible
to develop methods for allowing robust chondrogenesis while


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Arthritis Research & Therapy Vol 11 No 5 Steinert et al.




preventing hypertrophic differentiation using different genes or
proteins, which would presumably improve the outcome of
cell-based approaches to cartilage repair in vivo.


Competing interests
The authors declare that they have no competing interests.


Authors' contributions
All authors have read and approved the manuscript and con-
tributed to the study design, data analysis, interpretation of
data and drafting and revision of the manuscript. The data have
been generated by AFS, BP, MK, SCG, and a data review
committee (AFS, CH, SCG, AR, UN, JE and CHE) analysed
the data.


Acknowledgements
We are grateful to Nadja Karl, Viola Monz and Christa Amrehn for their
excellent technical assistance. This work was supported in parts by
grants AR48566 and AR50249 from to National Institute of Arthritis and
Musculoskeletal and Skin Diseases to SCG and CHE, by grant STE
1051/2-1 from the Deutsche Forschungsgemeinschaft (DFG) to AFS
and UN, and by grant D-23 to AFS and AR from the Interdisciplinary
Center for Clinical Research (IZKF) Wairzburg.

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