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Integrin expression in stem cells from bone marrow and

1
Department of Otolaryngology, Head and Neck Surgery, University Hospital Mannheim, University of Heidelberg,
Mannheim; 2Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg/
Hessen, Faculty of Clinical Medicine, Ruprecht Karls University of Heidelberg, Mannheim, Germany

Received October 3, 2007; Accepted November 29, 2007

Abstract. The use of adult mesenchymal stem cells (MSC) during chondrogenic differentiation. Particularly, the receptors
in cartilage tissue engineering offers new perspectives in the for fibronectin, vitronectin, osteopontin and the collagens
generation of transplants for reconstructive surgery. The may be involved in the generation of the ECM. Intra-
extracelular matrix (ECM) plays a key role in modulating the cellularly, their signals might be transduced by ILK and
function and phenotype of the embedded cells and contains CD47. To fully harness the potential of these cells, future
the integrins as adhesion receptors mediating cell-cell and studies should be directed to ascertain their cellular and
cell-matrix interactions. In our study, characteristic changes molecular characteristics for optimal identification, isolation,
in integrin expression during the course of chondrogenic and expansion.
differentiation of MSC from bone marrow and adipose tissue
were compared. MSC were isolated from bone marrow Introduction
biopsies and adipose tissue. During cell culture, chondrogenic
differentiation was performed. The expression of integrins and Each year millions suffer from organ failure or tissue loss
their signaling components were analysed with microarray due to injury, disease, or congenital malformation (1,2). With
and immunohistochemistry in freshly isolated MSC and after a progressively aging population, there is an increasing demand
chondrogenic differentiation. The fibronectin receptor for therapies to regenerate or replace musculoskeletal tissues.
(integrin α5ß1) was expressed by undifferentiated MSC, and More than three million musculoskeletal procedures are
expression rose during chondrogenic differentiation in both performed annually. The existing shortage of donor tissue and
types of MSC. The components of the vitronectin/osteopontin organs available for transplantation has driven a multi-
receptors ( α vß5) were not expressed by freshly isolated disciplinary effort to develop therapeutic solutions.
MSC, and expression rose with ongoing differentiation. The emerging field of tissue engineering promises to
Receptors for the collagens (α1ß1, α2ß1, α3ß1) were weakly deliver improvements in the technologies and therapies for
expressed by undifferentiated MSC and were activated during musculoskeletal disorders through the development of
differentiation. Intracellular signaling components integrin- biological substitutes for tissue replacement. The creation of
linked kinase (ILK) and CD47 showed increased expression functional tissue addresses very complex biological problems,
with ongoing differentiation. For all integrins, no significant comprising a wide range of engineering, science and clinical
differences were be found in the 2 types of MSC. Integrin- disciplines. The next generation of engineered musculo-
mediated signaling appeared to play an important role in the skeletal tissues will be more complex and structurally
generation and maintenance of the chondrocytic phenotype organized to better mimic normal tissue structure and function.
Tissue engineering as an interdisciplinary approach utilizes
specific combinations of cells, scaffolds and bioactive factors
_________________________________________ to create, influence and maintain cellular phenotype and
function (3).
Cartilage is a unique avascular, aneural and alymphatic
Correspondence to: Dr Ulrich Goessler, Department of
Otolaryngology, Head and Neck Surgery, University Hospital
loadbearing living tissue. It is unique in that the extracellular
Mannheim, D-68135 Mannheim, Germany matrix is composed of a complex combination of type II
E-mail: ulrich.goessler@hno.ma.uni-heidelberg.de collagen fibrils which are specifically arranged and are
bonded to very large water-retaining molecules called
Key words: integrin, cartilage, tissue engineering, differentiation, aggrecan molecules (4). The promise of tissue engineering is
extracellular matrix, mesenchymal stem cells, chondrogenic perhaps most relevant to chondrogenic defects because
differentiation cartilage has little self-healing potential.
The successful isolation of human stem cells from bone
marrow, periosteum and fat tissue was established by
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272 GOESSLER et al: INTEGRIN EXPRESSION DURING CHONDROGENIC DIFFERENTIATION

different groups (5-8). These cells are highly proliferative investigation of the molecular basis of distinct changes during
and are capable of differentiating into different types of developmental processes, for the generation of cartilage
tissue such as bone, cartilage, tendon, muscle or fat. Human transplants especially the process of chondrogenic differ-
mesenchymal stem cells are characterized by a specific pattern entiation, might broaden the understanding of impediments
of cell surface markers, growth factors, cytokine receptors, in the field of tissue engineering. As BM-MSC are best
integrins and other adhesion molecules (9,10). characterized, we asked whether MSC derived from other
Although bone marrow (BM) has been the main source sources share the characteristic expression patterns of BM-
for the isolation of multipotent MSC and BM-MSC are well MSC. The aim of our study was to analyse MSC isolated
characterized and safe for handling, the harvesting of BM is a from BM and AT under identical in vitro conditions and
highly invasive procedure and the number, differentiation during chondrogenic differentiation with respect to integrin
potential, and maximal life span of MSC from BM decline expression.
with increasing age (11-13). Therefore, alternative sources
from which to isolate MSC are subject to intensive investi- Materials and methods
gation. Adipose tissue (AT) is an alternative source that can
be obtained by a less invasive method and in larger quantities Collection and isolation of MSC from bone marrow (BM).
than BM. It has been demonstrated that AT contains stem Bone marrow was obtained from the femoral shaft of patients
cells similar to BM-MSC, which are termed processed lipo- undergoing total hip replacement at the orthopedic department
aspirate (PLA) cells, and these cells can be isolated from of the University Hospital Mannheim. Cells were aspirated
cosmetic liposuction in large numbers and grown easily into a 5-ml syringe containing CPD anticoagulant. In total,
under standard tissue culture conditions (14,15). The multi- six specimens from female patients were obtained, with the
lineage differentiation capacity of PLA cells has been proven donor age ranging from 68 to 84 years.
in previous studies (14,15). To isolate mononuclear cells (MNC), the bone marrow
As cellular function and phenotype are influenced by aspirates were diluted 1:5 with PBS/2 mM EDTA (Nexell,
intrinsic and extrinsic stimuli, the cell-cell and cell-matrix Baxter, Unterschleissheim, Germany, and Merck, Darmstadt,
interactions are of special interest in understanding factors Germany) and carefully loaded into Ficoll-Hypaque solution
crucial to generation of a distinct cellular phenotype. The (Amersham, Freiburg, Germany). After density gradient
integrin family of cell surface receptors appears to play a centrifugation at 435 x g for 30 min at room temperature,
major role in the mediation of the cell-ECM interactions MNC were removed from the interphase and washed two to
associated with structural and functional changes in three times with PBS/EDTA. Cell counts were performed
surrounding tissues (16-19). The integrins are heterodimeric using an automated cell analyzer (Cell-Dyn 3200, Abbott,
glycoproteins that are composed of an α- and a ß-subunit, Wiesbaden, Germany).
each of which has extracellular and cytoplasmic domains. The BM-derived MNC were set in culture at a density of
extracellular domains bind to a number of ECM proteins, 1x105/cm2 into 75 cm2 tissue culture flasks (Nunc, Wiesbaden,
including collagen types II and VI, fibronectin and matrix Germany) in MSCGM medium (MSCGM BulletKit™,
Gla protein. Several previous studies have provided evidence Cambrex, St. Katharinen, Germany).
that chondrocytes express integrins (20-25). Salter et al used After overnight incubation at 37˚C in a humidified
immunohistochemical staining in normal adult articular atmosphere containing 5% CO2, non-adherent cells were
cartilage, and noted that integrin α 5ß1 was the most removed and fresh medium was added to the flasks. Cultures
prominently expressed chondrocyte integrin (24). A subsequent were maintained and remaining non-adherent cells were
study demonstrated that the chondrocyte expression of α1ß1, removed by complete exchange of culture medium every
α5ß1 and αvß5 were accompanied by weak expression of three to four days. The flasks were screened continuously to
integrin α3ß1 and αvß3 (23). Other integrins are known to access developing colonies of adherent cells. Fibroblastoid
have distinct functions in binding components of the ECM cells were recovered between day 7 and 10 after initial
(Table I). plating by using 0.04% trypsin/0.03% EDTA (PromoCell,
Integrin-mediated signaling is involved in a variety of Heidelberg, Germany). Recovered cells were replated at a
cellular processes such as differentiation, adhesion and density of 4000-5000 cells/cm2 as passage 1 (P1) cells and
migration. Hannigan et al found that integrin-linked kinase thereafter.
(ILK) co-immunoprecipitated with ß-1 integrin from cell
lysates, and that overexpression of ILK disrupted cell Collection of adipose tissue (AT). AT was obtained from 18
architecture and inhibited adhesion to integrin substrates, donors ranging in age from 26 to 57 years undergoing lipo-
suggesting that ILK regulates integrin-mediated signal suction procedures. Lipoaspirates were obtained in accordance
transduction (26). In addition to ILK, integrin cytoplasmic with the standard of ethics of the local ethics committee.
domain-associated protein 1 (ICAP-1) interacts with the
cytoplasmic domain of ß-1 integrin (27). CD47 or integrin- Isolation and culture of cells from adipose tissue (AT). The
associated protein (IAP) is a membrane protein that is involved raw lipoaspirate (50-100 ml) was processed as described
in the increase in intracellular calcium concentration that previously by Goessler et al (17). To isolate the stromal
occurs upon cell adhesion to the extracellular matrix (28). vascular fraction (SVF), lipoaspirates were washed vigorously
As the stem cell is responsible for modulating its with PBS. Thereafter the lipoaspirates were digested with an
environment and the chondrocyte phenotype is influenced by equal volume of 0.075% collagenase type I (Sigma-Aldrich,
the diverse components of the extracellular matrix, the St. Louis, MO, USA) for 30-60 min at 37˚C with gentle
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agitation. The activity of the collagenase was neutralized Table I. Different integrins and their functions.
with DMEM-lg containing 10% FCS. To obtain the high- –––––––––––––––––––––––––––––––––––––––––––––––––
density SVF pellet, the digested lipoaspirate was centrifuged Integrin Receptor for
at 1,200 x g for 10 min. The pellet was re-suspended in –––––––––––––––––––––––––––––––––––––––––––––––––
MSCGM or DMEM-lg containing 10% MSCGS and filtered Integrin α5ß1 Fibronectin
through a 100-fim nylon cell strainer (Falcon). The filtered Integrin α4ß1 VCAM, fibronectin
cells were centrifuged at 1,200 x g for 10 min. The re- Integrin α6ß1 and α7ß1 Laminin
suspended SVF cells were plated at a density of 1x106/cm2
Integrin α1ß1 Collagen, laminin, tenascin
into T75 or T175 culture flasks. Nonadherent cells were
removed 12-18 h after the initial plating by vigorously Integrin α2ß1 Laminin, collagen
washing the plates. The resulting fibroblastoid adherent cells Integrin αvß5 Osteopontin
–––––––––––––––––––––––––––––––––––––––––––––––––
were termed AT-derived fibroblastoid adherent cells (AT-
FACs). AT-FACs were cultivated under the same conditions
as described for BM-FACs. AT-FACs were harvested at
subconfluence using trypsin. Cells at the second passage and hybridization chamber for 16 h at 42˚C in a water bath. After
thereafter were replated at a mean density of 1.8±3.1x103/cm2. stringent washing of the glass slides according to the
The generation of single separated fibroblast colony manufacturer's specifications the hybridization signals of the
forming units (CFU-F) was achieved by initially seeding the Cy3 and the Cy5 dyes were measured using a microarray
SVF cells at a low density (1x102 to 1x103 cells per cm2). laser scanner (GMS418; Affymetrix, MWG-Biotech).
CFU-F were selected and isolated as described for BM-CFU-F.
Subcultivation of the cells was performed as described for Microarray data analysis and statistics. The ArrayVision
the AT-FACs. (Imaging Research, Inc., St. Catharines, ON, Canada) software
was used for evaluation and calculation of signal intensities
Chondrogenic differentiation. To promote chondrogenic from the raw data images in 16-bit tagged-image-file (TIF)
differentiation, 2.5x105 cells were gently centrifuged (150 x g, format as previously described by Bugert et al (30). In brief,
5 min) in a 15-ml polypropylene tube (Greiner) to form a for evaluation of hybridization results, we defined a negative
pellet according to the protocol of Mackay et al (29). (<3,000), a grey area (3,000-4,999) and a positive range
Without disturbing the pellet, the cells were cultured for four (≥5,000) of hybridization signal intensities. Signal-to-
weeks in complete chondrogenic differentiation medium background (S/B) values were calculated by dividing the
(Cambrex) including 10 ng/ml TGFß3 (Strathmann Biotec signal intensity for each spot with the background signal
AG, Hamburg, Germany) by feeding twice a week. After the intensities of the hybridized glass slide. Computer-assisted
culture period, cryosections were analyzed by Safranin O evaluation of the raw data provided the mean signal intensity
staining. The sections were fixed with ice-cold acetone and the signal-to-background ratio for each individual gene
(Sigma) and stained with 0.1% aqueous Safranin O solution spot. For statistical evaluation the mean signal intensity and
(Sigma). Cell nuclei were counterstained with Weigert's iron standard deviation (SD) were calculated for each spot from the
hematoxylin (Sigma). values obtained in the 10 individual experiments. Functional
For the RNA analysis we harvested and lysed the grouping of genes was performed on the basis of the database
aggregates in RLT buffer (Qiagen). The lysis was aggravated supplied by the array manufacturer.
by freezing the pellet repeatedly in liquid nitrogen.
Immunohistochemistry. Immunohistochemistry for integrin
RNA extraction and microarray hybridization. Extraction of integrin αv, integrin ß1, integrin ß5, CD47 and the integrin-
RNA was performed using the RNA Mini Kit (Qiagen, linked kinase (ILK) was performed by using a streptavidin-
Hilden, Germany) according to the manufacturer's protocol biotin complex procedure. Endogenous peroxidase was
and as previously published (30). The RNA concentration blocked with 0.3% hydrogen peroxide for 30 min. Sections
was estimated from the absorbance at 260 nm. were washed with phosphate-buffered saline (PBS) and
Approximately 1 μg total RNA was used in each micro- incubated with normal rabbit serum in PBS for 30 min at
array experiment, and for amplification and labeling of mRNA room temperature to block non-specific antibody reaction.
the Smart technique (Smart Fluorescent Probe Amplification The sections were then incubated overnight at 4˚C with the
Kit; BD Clontech, Heidelberg, Germany) was applied primary antibody (all from Santa Cruz Biotechnologies,
according to the manufacturer's protocol. RNA samples from Heidelberg, Germany). The slides were washed by several
day 1 were labeled with Cy3, and day 6 or day 21 samples changes of PBS. The sections were then incubated with a
were labeled with Cy5 (Cy™3- and Cy™5-monoreactive peroxidase-conjugated secondary antibody (Dako, Hamburg,
dye; Amersham Pharmacia Biotech). Corresponding Cy3- Germany). After being washed twice in PBS, sections were
and Cy5-labeled samples were mixed, vacuum dried and re- then treated with a streptavidin-biotin-peroxidase complex
suspended in 25 μl microarray hybridization buffer (MWG- and peroxidase reaction was performed using diamino-
Biotech; Ebersberg, Germany). Prior to hybridization, the benzidine (DAB) (Dako) as chromogen. The various
samples were heat denaturated at 95˚C for 5 min. The human antibodies were diluted to the desired concentrations in PBS.
10K (MWG-Biotech) oligo microarray systems on glass Controls were carried out by omitting the primary antibody.
slides were used for mRNA profiling. Hybridization of Light microscopy was performed using a Zeiss Axiophot
Cy3/Cy5-cDNA was performed using cover slips and a microscope.
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274 GOESSLER et al: INTEGRIN EXPRESSION DURING CHONDROGENIC DIFFERENTIATION

Table II. Signal intensities of hybridization signals as measured using the microarray laser scanner and calculated by the
ArrayVision software in MSC from bone marrow.a
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Receptor for Day 0 Day 20 Day 0 SD Day 20 SD Ratio day 20/day 0
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Fibronectin
Integrin α5 11,790 9,144 4,269 4,093 0.78
Integrin ß1 32,134 15,557 3,040 3,698 0.48
VCAM, fibronectin
Integrin α4 811 2,584 307 821 3.19
Integrin ß1 32,134 15,557 3,040 3,698 0.48
Laminin
Integrin α6 1,206 773 241 132 0.64
Integrin α7 2,872 4,826 1,412 2,412 1.68
Integrin ß1 32,134 15,557 3,040 3,698 0.48
Collagen, laminin, tenascin
Integrin α1 7,345 4,639 6,079 4,269 0.63
Integrin ß1 32,134 15,557 3,040 3,698 0.48
Laminin, collagen
Integrin α2 23,892 30,795 9,674 3,988 1.29
Integrin ß1 32,134 15,557 3,040 3,698 0.48
Osteopontin
Integrin αv 4,044 3,306 1,112 1,036 0.82
Integrin ß5 2,958 15,601 804 8,541 5.27
Signaling cascade
CD47 1,931 2,625 1,224 1,921 1.36
ILK 22,758 23,360 11,452 11,496 1.03
ICAP-1 1,041 2,147 117 594 2.06
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
aSignals were measured on day 1 and after chondrogenic differentiation on day 20. Signals are shown with standard deviation (SD) and the

ratio day 20/day 1.

Results (day 0, 32,134; day 20, 15,557; ratio day 20/day 0, 0.48). For
the receptor for osteopontin (integrin αv/ß5), an inactivation
Microarray analysis. In MSC from bone marrow (BM-MSC; of integrin αv (day 0, 4,044; day 20, 3,306; ratio day 20/day
Table II, Fig. 1), for the components of the fibronectin receptor 0, 0.82) and an activation of integrin ß5 (day 0, 2,958; day
(integrin α5/ß1) a constant expression for integrin α5 (day 0, 20, 15,601; ratio day 20/day 0, 5.27) was found. The
11,790; day 20, 9,144; ratio day 20/day 0, 0.78) was found, components of the intracellular signaling cascade revealed a
and integrin ß1 was inactivated (day 0, 32,134; day 20, constant expression of ILK (day 0, 1,931; day 20, 2,625; ratio
15,557; ratio day 20/day 0, 0.48). The components of the day 20/day 0, 1.36) and of CD47 (day 0, 22,758; day 20,
receptor for VCAM and fibronectin (integrin α4/ß1) were not 23,360; ratio day 20/day 0, 1.03). ICAP-1 was not expressed
expressed (integrin α4: day 0, 811; day 20, 2,548; ratio day 20/ (day 0, 1,041; day 20, 2,147; ratio day 20/day 0, 2.06).
day 0, 3.19) and the gene for integrin ß1 (day 0, 32,134; day In MSC from adipose tissue (Table III, Fig. 2), the
20, 15,557; ratio day 20/day 0, 0.48) was inactivated. The components of the fibronectin receptor (integrin α 5/ß1)
components of the receptor for laminin (integrin α6/ß1 and showed an inactivation for integrin α5 (day 0, 29,474; day 20,
integrin α7/ß1) showed a constant expression of integrin α7 15,615; ratio day 20/day 0, 0.353), and for integrin ß1 (day 0,
(day 0, 2,872; day 20, 4,826; ratio day 20/day 0, 1.68) and an 7,345; day 20, 4,639; ratio day 20/day 0, 0.63). The
inactivation of integrin ß1 (day 0, 32,134; day 20, 15,557; components of the receptor for VCAM and fibronectin
ratio day 20/day 0, 0.64). The components of the receptor for (integrin α4/ß1) were not expressed (integrin α4: day 0, 1,038;
collagen, laminin and tenascin (integrin α1/ß1) were inactivated day 20, 1,186; ratio day 20/day 0, 1.14) and inactivated
(integrin α1: day 0, 7,345; day 20, 4,639; ratio day 20/day 0, (integrin ß1: day 0, 7,345; day 20, 4,639; ratio day 20/day 0,
0.63; and integrin ß1: day 0, 32,134; day 20, 15,557; ratio 0.63). The components of the receptor for laminin (integrin
day 20/day 0, 0.48). The components of the receptor for α6/ß1 and integrin α7/ß1) revealed no expression of integrin
laminin and collagen (integrin α2/ß1) revealed a constant α7 (day 0, 1,162; day 20, 848; ratio day 20/day 0, 0.73) and
expression of integrin α2 (day 0, 23,892; day 20, 30,795; an inactivation of integrin ß1 (day 0, 7,345; day 20, 4,639;
ratio day 20/day 0, 1.29) and an inactivation of integrin ß1 ratio day 20/day 0, 0.63). Integrin α6 (day 0, 1,169; day 20,
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Table III. Signal intensities of hybridization signals as measured using the microarray laser scanner and calculated by the
ArrayVision software in MSC from adipose tissue.a
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Receptor for Day 0 Day 20 Day 0 SD Day 20 SD Ratio day 20/day 0
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Fibronectin
Integrin α5 29,474 15,615 5,283 3,389 0.53
Integrin ß1 7,345 4,639 6,079 4,269 0.63
VCAM, fibronectin
Integrin α4 1,038 1,186 322 821 1.14
Integrin ß1 7,345 4,639 6,079 4,269 0.63
Laminin
Integrin α6 1,169 775 373 54 0.66
Integrin α7 1,162 848 399 177 0.73
Integrin ß1 7,345 4,639 6,079 4,269 0.63
Collagen, laminin, tenascin
Integrin α1 824 7,498 442 2,854 9.10
Integrin ß1 7,345 4,639 6,079 4,269 0.63
Laminin, collagen
Integrin α2 5,060 11,110 2,954 4,956 2.20
Integrin ß1 7,345 4,639 6,079 4,269 0.63
Osteopontin
Integrin αv 4,592 5,147 1,112 1,036 1.12
Integrin ß5 8,409 15,706 4,467 2,542 1.87
Signaling cascade
CD47 4,745 7,618 2,586 6,470 1.61
ILK 6,468 5,532 734 266 0.86
ICAP-1 1,041 2,147 117 594 2.06
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
aSignals were measured on day 1 and after chondrogenic differentiation on day 20. Signals are shown with standard deviation (SD) and the

ratio day 20/day 1.

Figure 1. Expression levels of genes for different integrins and integrin-associated proteins in MSC by microarray hybridization analysis. Results from
mesenchymal stem cells from bone marrow (BM-MSC) during chondrogenic differentiation for the given genes in undifferentiated MSC (white bars) and
chondrocytes differentiated from the MSC (black bars).
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276 GOESSLER et al: INTEGRIN EXPRESSION DURING CHONDROGENIC DIFFERENTIATION

Figure 2. Expression levels of genes for different integrins and integrin-associated proteins in MSC by microarray hybridization analysis. Results from
mesenchymal stem cells from adipose tissue (PLA-MSC) during chondrogenic differentiation for the given genes in undifferentiated MSC (white bars) and
chondrocytes differentiated from the MSC (black bars).

Figure 3. Immunohistochemical staining against different integrins in MSC during chondrogenic differentiation. Day 1 (left) and day 20 (right) of cell culture.
(A) Integrin ß1 (day 1) in PLA-MSC; (B) integrin ß1 (day 20) in PLA-MSC; (C) ILK (day 1) in PLA-MSC; and (D) ILK (day 20) in PLA- MSC.
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Table IV. Immunohistochemical detection of integrins and integrin-associated proteins in freshly isolated MSC (day 1) and
during chondrogenic differentiation (days 1, 10, 20 and 30).
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Antibody specific Staining patterna
for BM-MSC –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Day 0 Day 1 Day 10 Day 20 Day 30
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Integrin αv +++ + ++ +++ ++
Integrin ß5 ++ + +++ +++ ++
Integrin ß1 ++ + ++ ++ +
CD47 +++ ++ +++ +++ ++
ILK +++ +++ +++ ++ ++
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Antibody specific Staining patterna
for PLA-MSC –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Day 0 Day 1 Day 10 Day 20 Day 30
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Integrin αv +++ ++ ++ ++ ++
Integrin ß5 +++ +++ ++ ++ ++
Integrin ß1 ++ ++ ++ ++ ++
CD47 +++ ++ ++ +++ +++
ILK ++ ++ +++ +++ +++
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
aThe amount of cells stained by the monoclonal antibodies is symbolized by ++++ (80-100%), +++ (60-70%), ++ - +++ (50-60%), ++ (40-50%),
+ - ++ (30-40%), + (20-30%), ± (<20%), and - no staining.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

775; ratio day 20/day 0, 0.66) was not expressed. As for the regenerative medicine, follows the principles of cell trans-
receptor for collagen, laminin and tenascin (integrin α1/ß1), a plantation, materials science, and engineering towards the
strong activation of integrin α1 (day 0, 824; day 20, 7,498; development of biological substitutes that can restore and
ratio day 20/day 0, 9.1) and an inactivation of integrin ß1 maintain normal function.
(day 0, 7,345; day 20, 4,639; ratio day 20/day 0, 0.63) was Bone marrow-derived stem cells have been studied for
found. The receptor for laminin and collagen (integrin α2/ß1) decades, and are well characterized and safe even in clinical
revealed an activation of integrin α2 (day 0, 5,060; day 20, settings. For clinical applications of stem cell transplantation
11,110; ratio day 20/day 0, 2.20) and an inactivation of therapy, direct manipulation of cells and their interactions
integrin ß1 (day 0, 7,345; day 20, 4,639; ratio day 20/day 0, would be desirable. Hitherto, establishing distinct protocols for
0.63). For the receptor for osteopontin (integrin αv/ß5), a precisely inducing and maintaining cellular differentiation
constant expression of integrin αv (day 0, 4,592; day 20, with a defined phenotype and function has been extremely
5,147; ratio day 20/day 0, 1.12) and an activation of integrin challenging. In addition, the elucidation of cell-cell and cell-
ß5 (day 0, 8,409; day 20, 15,706; ratio day 20/day 0, 1.87) matrix interactions is necessary, and the integrins as a family
was found. The components of the intracellular signaling of adhesion receptors mediating these stimuli are a promising
cascade showed a constant expression of ILK (day 0, 6,488; target for research.
day 20, 5,532; ratio day 20/day 0, 0.86) and CD47 (day 0, As mentioned above the harvesting of BM is a highly
4,745; day 20, 7,618; ratio day 20/day 0, 1.61). ICAP-1 was invasive procedure and the number, differentiation potential,
not expressed (day 0, 1,041; day 20, 2,147; ratio day 20/day 0, and maximal life span of MSC from BM decline with
2.06). increasing age (11,12). Therefore, alternative sources from
which to isolate MSC are subject to intensive investigation.
Immunohistochemistry. The analysis of integrin expression at Adipose tissue (AT) is an alternative source that can be
the protein level was analysed with monoclonal antibodies obtained by a less invasive method and in larger quantities
against integrin αv, integrin ß1, integrin ß5, CD47 and the than BM.
integrin-linked kinase (ILK). During the entire process of Expression of integrins was analysed in MSC from bone
chondrogenic differentiation, a constant expression for all marrow and adipose tissue during chondrogenic differ-
markers was found (Fig. 3, Table IV). entiation. The components of the fibronectin receptor
(integrin α5/ß1) demonstrated a diminished expression with
Discussion ongoing differentiation in both types of MSC at the RNA and
protein level alike.
The field of regenerative medicine encompasses various areas With RT-PCR analysis it was previously shown that
of technology, such as tissue engineering, stem cells, and freshly isolated MSC express collagen 2 and 10 (31). These
cloning. Tissue engineering, one of the major components of previous results agree with the finding of high expression of
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278 GOESSLER et al: INTEGRIN EXPRESSION DURING CHONDROGENIC DIFFERENTIATION

integrin α5ß1 in undifferentiated MSC, as the interaction of However, we believe that due to the high quantities of AT-
MSC and components of the ECM (e.g. collagen 2) via this MSC, small fat reservoirs are also sufficient for MSC
receptor are known. Thus, integrin α 5ß1 may exert an isolation. For the past decades, BM has been utilized as the
influence on the cellular phenotype in undifferentiated MSC, main source of MSC for clinical applications, such as the
and with ongoing differentiation this receptor seems to become treatment of osteogenesis imperfecta, graft versus host
less important in both types of MSC at the same time. disease, or acute myocardial infarction (35-37). As BM-MSC
The receptor for VCAM and fibronectin (integrin α4/ß1) demonstrate an age-dependent decrease in number, frequency,
was not expressed by MSC from BM and AT in terms of and differentiation capacity, they may be clinically inefficient
integrin α4 and was inactivated during chondrogenic differ- when derived from elderly patients. Taking into account all
entiation for integrin ß1, respectively. This receptor does not of these factors, PLA-MSC may provide a reliable first source
seem to be involved in signaling during chondrogenic in reference to their abundance, easy harvest, and high MSC
differentiation. frequency.
The components of the receptor for laminin (integrin In the present study, we analysed the expression patterns
α6/ß1 and α7/ß1) showed no expression of integrin α6 and α7 of integrins and integrin-related signaling proteins. One of
in the two types of MSC as well as a diminished expression of the candidates for signal transmission is the fibronectin
integrin ß1 during chondrogenic differentiation. Thus, this receptor which may play a role in freshly isolated cells. Other
receptor does not appear to play an important role during receptors, e.g. for collagen, laminin and tenascin, do not
chondrogenic differentiation. appear to be involved in signal transduction. The receptor for
The receptor for collagen, laminin and tenascin (integrin osteopontin seems to play a role during chondrogenic differ-
α1/ß1) revealed a strong activation of integrin α1 in both entiation. In addition, the receptor for laminin and collagen
stem cell types, whereas integrin ß1 showed a diminished may assist the beginning of chondrogenic differentiation.
expression with ongoing differentiation. These results do not Intracellularly, ILK and CD47, but not ICAP-1, may be
allow a specific conclusion to be made concerning the role of involved in transduction of the integrin-dependent signals.
this receptor during chondrogenic differentiation. Integrin-mediated signaling seems to play an important
The osteopontin receptor (integrin α v/ß5) showed role in the generation and maintenance of the chondrocytic
constant expression for integrin αv in both types of MSC. phenotype during chondrogenic differentiation. To fully
Integrin ß5 was inactivated in BM-MSC and was activated in harness the potential of these cells, future studies should be
PLA-MSC. It has been established that integrin ß5 plays a directed to ascertain their cellular and molecular characteristics
role in binding vitronectin (32). In addition, it has been for optimal identification, isolation, and expansion.
suggested that the osteopontin receptor might be involved in
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