1393

INTRODUCTION

ARTICULAR CARTILAGE has limited capacity for self-re-newal, probably due to its avascularity and low cel-

lularity.1 The repair of joint surface defects by trans-plantation of autologous chondrocytes is well establishedin orthopedics.2,3 Major drawbacks of this method are thesource of cells and the change in biological propertiesseen in expanding chondrocytes in vitro. Articular carti-lage is harvested from a nonload-bearing area of the in-jured joint in a first operation. It has been suggested thatthis harvesting procedure leads to cartilage injury, whichis a predisposition for osteoarthritis.4 Harvested chon-

drocytes expanded in vitro change their phenotype to afibroblastic lineage5 and are not able to generate an ap-propriate matrix, as is possible in normal articular carti-lage.6

Attempts were made to circumvent this problem withthe use of mesenchymal stem cells (MSCs), which canbe easily expanded in vitro in an undifferentiated state.In the presence of the transforming growth factor �(TGF-�), mesenchymal stem cells from bone marrow areable to differentiate into a chondrogenic cell line pro-ducing cartilage-specific matrix proteins such as colla-gen type II, cartilage oligomeric matrix protein (COMP),and aggrecan.7,8 Initial studies in which MSCs were used

TISSUE ENGINEERINGVolume 12, Number 6, 2006© Mary Ann Liebert, Inc.

Mesenchymal Stem Cells Maintain TGF-�-MediatedChondrogenic Phenotype in Alginate Bead Culture

A.T. MEHLHORN,1 H. SCHMAL,1 S. KAISER,3 G. LEPSKI,4 G. FINKENZELLER,2G.B. STARK,2 and N.P. SÜDKAMP1

ABSTRACT

This article addresses the stability of chondrogenic phenotype and the transdifferentiation potentialof bone marrow–derived mesenchymal stem cells (MSCs) at distinct stages of differentiation. Differ-entiated MSCs were expected to maintain cartilage-like gene expression pattern in the absence of anychondrogenic growth factor or in the presence of osteogenic signals. MSCs encapsulated in alginatebeads were treated with transforming growth factor (TGF)-�3 for 3, 6, or 14 days and then culturedin absence of TGF-� for the remainder of the 2-week culture period. Additionally, cells were culturedin osteogenic medium after TGF-�-mediated chondroinduction. Gene expression of col2a1, aggrecan,COMP, alkaline phosphatase (AP), and correlating protein synthesis was analyzed. After short-termstimulation with TGF-�, MSCs maintained a chondrogenic phenotype. Chondrogenic gene expressionand protein synthesis directly correlated with the extent of stimulation time and the concentration ofTGF-�. Pretreatment with TGF-� could prevent AP mRNA expression of encapsulated MSCs. TGF-� stimulation within the first 3 days of culture seems to be crucial for the expression of a chondro-genic phenotype. Fully differentiated and encapsulated MSCs are not able to transdifferentiate intoosteoblasts. These findings give rise to a better understanding of the behavior of cartilage grafts af-fected by local factors of osteochondral transplantation sites in vivo.

1Department of Orthopaedic and Trauma Surgery, Albert-Ludwigs University Freiburg, Freiburg, Germany.2Department of Plastic and Hand Surgery, Albert-Ludwigs University Freiburg, Freiburg, Germany3Department of Haematology and Oncology, Albert-Ludwigs University Freiburg, Freiburg, Germany.4Department of Stereotactic Neurosurgery, Albert-Ludwigs University Freiburg, Freiburg, Germany.

as autologous tissue transplants showed promising resultsafter transplantation in vivo.9

TGF-�-induced commitment and differentiation ofmesenchymal progenitor cells are modulated by the typeof three-dimensional cell culture matrix and the nature ofexposure (e.g., concentration of protein and the timecourse of exposure).10,11 Alginate has been shown to bean appropriate matrix to study chondrogenesis of MSCs12

and is used as a suitable cell carrier for stem cell–basedcartilage repair in vivo.13,14 In this context, it is still un-clear whether TGF-�-mediated chondrogenic differenti-ation of MSCs in vitro enhances the tissue-specific properties of the graft after implantation or whether un-differentiated MSCs can be transplanted into osteochon-dral defects and chondrogenesis is solely driven by localfactors.

Recent data suggest that MSCs committed to a givenmesenchymal cell lineage can switch into another celltype of a different lineage through genetic reprogram-ming if the local environment is changed to osteogenic,chondrogenic, or adipogenic cues.15 This phenomenon isof importance at transplantation sites where two mes-enchymal tissues merge into each other and only one tis-sue is replaced by MSC-based engineered grafts as, forexample, cartilage grafts at the osteochondral bone plate.Recently, it has been suggested that MSCs of superficiallayers of the transplanted graft are influenced by chon-drogenic factors in the synovial fluid, whereas MSCsfrom the deep layers respond to osteogenic signals of thesubchondral bone plate.16

The aim of this study was to examine the biologicalresponse of cartilage grafts to osteogenic and chondro-genic signals. The stability of the chondrogenic pheno-type of MSCs was analyzed after withdrawal of TGF-�under in vitro conditions. We investigated the effect ofosteogenic signals on the chondro- and osteospecific geneexpression pattern of MSCs committed to the chondro-genic lineage. Our hypothesis was that fully differenti-ated MSCs maintain their chondrogenic phenotype in theabsence of TGF-�. It was expected that genetic repro-gramming to an osteogenic lineage does not take placeif cells have fully committed to a chondrogenic lineageand if culture was continued in a three-dimensional ma-trix. The hypothesis was tested comparing gene expres-sion and protein synthesis levels of MSCs, which wereexposed to TGF-� and later to control medium (CM) orosteogenic medium (OM).

MATERIALS AND METHODS

Cell samples

Bone marrow samples were obtained from consentinghealthy adult donors (n � 5, 25 yrs � 5 (mean � SDM),m:f � 1:4) by an iliac crest biopsy. The donor program

MEHLHORN ET AL.

was approved by the ethical committee of the Universityof Freiburg. Mononuclear cells (MNCs) were purified bydensity gradient centrifugation with Biocoll Separating So-lution17 (Biochrom AG, Berlin, Germany) and then cellswere filtered through 100-�m cell strainers (BD Labware,Franklin Lakes, NJ). MNCs were seeded in 175-cm2 cul-ture flasks at a density of 0.2–1.0 � 106/cm2 in 25-mLStem Cell Basal Medium supplemented with MSCGMSinglequots, penicillin/streptomycin, and L-glutamine(MSCGM; Cambrex, Walkersville, MD). The mediumwas changed completely twice a week, which washed out all nonadherent cells. Once adherent cells had grownto confluence, they were detached with trypsin-EDTA(Sigma, Steinheim, Germany), re-seeded at a density ofapproximately 2000–5000 cells/cm2 and cultured for twofurther passages. After MSCs have reached passage, twocell samples from each donor were separately used for theexperiments and each experiment was done in triplicates.

Flow cytometry

For flow cytometric analysis of in vitro expandedMSCs, cells were detached with trypsin-EDTA for en-dothelial cells, washed with phosphate-buffered saline(PBS) Dulbecco’s and stained with fluorescent antibod-ies against human CD34, CD44, CD45, CD73 (BectonDickinson, San Jose, CA), CD90, and CD105 (Pharmin-gen, San Diego, CA) and corresponding isotype controls.

Chondrogenesis in alginate bead culture

Cells were harvested with trypsin-EDTA, counted, andthen suspended in 1.2% alginate (Sigma, Taufkirchen,Germany) at a concentration of 4 � 106 cells/mL. Spher-ical beads were created by dispensing droplets of the al-ginate cell suspension from the tip of a 14G needle intoa bath of 102 mM CaCl2 or BaCl2. The bead volume wascontrolled by measuring bead diameters with Scion Im-age 4.02 software (Scion Cooperation, Frederick, MD).Alginate beads from each donor were cultured either inCM containing high-glucose Dulbecco’s Modified EagleMedium with sodium pyruvate (100 mg/L), L-glutamine(100 mg/L), pyridoxine hydrochloride (100 mg/L), 1%penicillin/streptomycin, L-ascorbate 2-phosphate (37.5�g/mL), ITS� Premix (6.25 mg/mL; each Gibco) and10–7 M dexamethasone or differentiation medium. Dif-ferentiation medium differed in that it contained 10ng/mL, 20 ng/mL, or 60 ng/mL TGF-�3 (R&D Systems,Minneapolis, MN). TGF-�3 was used for chondrogenicdifferentiation assays instead of TGF-�1 because it wasshown that TGF-�3 has a higher chondroinductive po-tential than TGF-�1.7 All samples were cultured for 14days in total, and medium was changed every third day.The beads were exposed for 3, 6, or 14 days to differen-tiation medium and for the remainder to CM. Controlsamples were cultured in CM for 14 days.

1394

For the osteogenic transdifferentiation assay, encapsu-lated or monolayer-cultured (seeding density: 2 � 105

cells/cm2) MSCs were exposed for 3, 6, or 14 days tochondrogenic medium containing 10 ng/mL TGF-�3(R&D Systems, Minneapolis, MN) or to CM, and after-wards the medium was replaced with OM containing �-MEM (Invitrogen, Paisley, UK), 10% fetal calf serum,100 nM dexamethasone, 200 �M L-ascorbic acid 2-phos-phate, and 10 mM �-glycerophosphate (all Sigma,Taufkirchen, Germany) for another 9 days of culture.

DNA content

Beads were digested in digestion buffer containingPBS, 50 mM NaCl, 55 mM sodium citrate, 2 mM L-cys-teine, and 2 mM EDTA (pH 6.5) (all Sigma). Aliquotswere collected for aggrecan protein analysis, glycosami-noglycan, and DNA quantification. For measurement ofDNA, aliquots of digested beads were diluted 1:5 in Tris-EDTA buffer (10 mM Tris-HCl, 2 mM EDTA, pH 7.5)and analyzed with fluorescent picoGreen dsDNA quan-tification assay (Molecular Probes, Eugene, OR). Fluo-rescence was measured with TECAN SpectrafluorPlusmicroplate reader at 485-nm excitation and 535-nm emis-sion wavelengths.

Quantification of aggrecan

Alginate beads were dissolved in digestion buffer, andthe concentration of aggrecan was detected in the digestof alginate beads using a commercial ELISA kit accord-ing to the manufacturer’s instructions (MD Biosource,Zurich, Switzerland). The amount of aggrecan was nor-malized to corresponding DNA content.

Quantification of glycosaminoglycans

Alginate beads were dissolved in digestion buffer andaliquots of solubilized beads were diluted 1:2.5, enzy-matically digested with chondroitinase ABC (5 units/mL)or hyaluronidase (0.5 units/mL; both Seikagaku Cooper-ation, Tokyo, Japan) for 2 h at 37°C, and ultrafiltered us-ing an Ultrafree C3GC system (Millipore). The HPLCanalysis of the unsaturated disaccharides �di-6-S, �di-4S, and �di-0S derived form hyaluronic acid and chon-droitin sulfate was performed as previously described.18

The amount of glycosaminoglycans was normalized tocorresponding DNA content.

Alkaline phosphatase detection

Detection of alkaline phosphatase (AP) activity in cel-lular extracts was performed as previously described.19

Two hundred fifty microliter aliquots of a single digestedbead were mixed with 250 �L assay buffer containing25 mM Tris-HCl (pH 8.5) and 0.5% Triton X-100.Twenty microliters of each sample was mixed with

MSCs MAINTAIN TGF-�-MEDIATED CHONDROGENIC PHENOTYPE

100 �L CSPD substrate (Applied Biosystems, ForsterCity, CA) and incubated at 37°C for 30 min. Light out-put was measured by a plate luminometer (Tecan Spec-trafluor Plus, MTX Lab Systems, Vienna, VA) in rela-tive luminescence units. Cellular AP activities werenormalized to DNA content analyzed with fluorescentpicoGreen dsDNA quantification assay (MolecularProbes, Eugene, OR).

Semi-quantitative reversetranscription–polymerase chain reaction

RNA samples were taken at days 3, 6, and 14, tran-scribed into cDNA and reverse transcription–polymerasechain reaction (RT-PCR) analysis for gene expression ofcartilage oligomeric matrix protein (COMP), aggrecan,�1-collagen type II (col2a1), and AP was carried out asdescribed.20 Total mRNA was prepared using TRIzolreagent according to the manufacturer’s instructions (Invitrogen, Life Technologies). Total RNA (1 �g) wastreated with 1 U of deoxyribonuclease I (DNase I; Invit-rogen, Life Technologies) to digest genomic DNA cont-amination. Random-primed cDNA synthesis was per-formed using 1 �g of DNase I-treated total RNA and 50U of StrataScript reverse transcriptase according to themanufacturer’s instructions (Stratagene, La Jolla, CA).TaqMan PCR assays were performed in 96-well opticalplates on an ABI Prism 7700 Sequence Detection system(Applied Biosystems, Forster City, CA) using AbsoluteQPCR ROX Mix (Abgene, Hamburg, Germany) accord-ing to the manufacturer’s instructions. Oligonucleotideprimers and TaqMan probes (Table 1) were designed us-ing Primer Express (Applied Biosystems, Forster City,CA) according to company guidelines. The thermal cy-cling conditions were 95°C for 15 min followed by 40cycles at 95°C for 15 s and at 60°C for 1 min. Data wereanalyzed using the relative standard curve method, witheach sample being normalized to GAPDH to correct fordifferences in mRNA quality and quantity. Only COMPgene expression of treated samples was reported relativeto the corresponding gene expression of control samplesat day 14. Control samples did not express col2a1 andaggrecan genes at day 14. Data are expressed as arbitraryunits. The negative control for chondrogenic gene ex-pression was mRNA samples of human fibroblast.

Histology

For paraffin sections, alginate beads complexed withBaCl2 were fixed in 4% phosphate buffered formalin, de-hydrated, and embedded in paraffin. Sections were cutdry (2.5 �m) on a Leica RM 2165 microtome. For col-lagen type II immunohistochemistry, sections were incu-bated for 30 min with 5% normal goat serum, followedby incubation with 1:50 monoclonal mouse anti-collagentype II antibody (Clone 6B3, Chemicon, Temecula) for

1395

12 h, washed three times with PBS, and incubated witha fluorescein isothiocyanate–labeled goat anti-mouse im-munoglobulin for 45 min (Sigma, Taufkirchen, Ger-many). Sections were mounted with Vectashield mount-ing medium containing DAPI (Vectashield, Vector Labs,Burlingame, CA).

Statistical analysis

Numerical data were analyzed by computer softwarepackage for statistical analysis (SPSS statistical program,version 11.5, SPSS Inc., Chicago, IL). All values are re-ported as mean � SE of the mean. Statistical significancewas determined using Student’s t-test using a confidencelevel of 95% (p � 0.05). Power analysis was performedusing StatMate Software 2.00 (Graph Pad Software, Inc.).

RESULTS

Flow cytometry

MSCs were characterized with respect to the expres-sion of surface antigens. The expression of all surfaceantigens studied was similar for the entire expansion pe-riod and for all samples used in the experiment: MSCswere strongly positive for CD90 (Thy-1), CD44(hyaluronate receptor), CD105 (endoglin), CD73 (ecto-5-nucleoditase), and negative for CD34 (sialomucin/he-matopoietic progenitors) and CD45 (leukocyte commonantigen/haematopoietic progenitors) surface antigens.

Quantification of aggrecan

Aggrecan was analyzed in all samples using ELISA.All samples were cultured for 14 days in total. They weretreated with TGF-�3 for either 14 days (G1), for 6 days

MEHLHORN ET AL.

(G2), or 3 days (G3). One sample was cultured for 14days in CM only (G4). For the remainder of the total cul-ture time, alginate beads (G2, G3) were cultured in theabsence of TGF-�3 in CM (Fig. 1). In group G1, aggre-can content slightly increased between day 0 and 3 from0 to 365 � 110 �g aggrecan/�g DNA (mean � SE of themean) followed by an enhanced synthesis between days3 and day 12 from 365 � 110 up to 5742 � 520 �g ag-grecan/�g DNA. Aggrecan content finally reached6389 � 955 �g aggrecan/�g DNA at day 14. In groupsG2 and G3, cells were exposed to TGF-�3-containing

1396

TABLE 1. NUCLEOTIDE SEQUENCES OF TAQMAN PRIMERS AND PROBES FOR TARGET GENES

Gene Primer/probe Sequence Gene Bank acc. no.

GAPDH Forward 5�-TGGGCTACACTGAGCACCAG-3� BC029618Reverse 5�-CAGCGTCAAAGGTGGAGGAG-3�Probe 5�-FAM-TCTCCTCTGACTTCAACAGCGACACCC-TAMRA-3�

col2a1 Forward 5�-GAGACAGCATGACGCCGAG-3� BC007252Reverse 5�-GCGGATGCTCTCAATCTGGT-3�Probe 5�-FAM-TGGATGCCACACTCAAGTCCCTCAAC-TAMRA-3�

Aggrecan Forward 5�-AGCCTGCGCTCCAATGACT-3� NM_013227Reverse 5�-TGGAACACGATGCCTTTCAC-3�Probe 5�-FAM-CGCTGCGAGGTGATGCATGGC-TAMRA-3�

COMP Forward 5�-CAATGAACAGCGACCCAGG-3� NM_000095Reverse 5�-TCACATGGAACGTGCCCTC-3�Probe 5�-FAM-TTACACTGCCTTCAATGGCGTGGACTT-TAMRA-3�

AP Forward 5�-ATGCCCTGGAGCTTCAGAAG-3� NM_000478Reverse 5�-TGGTGGAGCTGACCCTTGAG-3�Probe 5�-CGTGGCTAAGAATGTCATCATGTTCCTGG-TAMRA-3�

FIG. 1. MSCs were cultured for 14 days in total. They weretreated with TGF-�3 for either 14 days (G1), for 6 days (G2),or 3 days (G3). One sample was cultured for 14 days in con-trol medium only (G4). For the remainder of the total culturetime, alginate beads (G2, G3) were cultured in the absence ofTGF-�3 in control medium. All samples were analyzed for ag-grecan with ELISA. Values were normalized to the DNA con-tent of the corresponding sample.

medium for 3 days or 6 days, respectively. After themedium was switched to CM, cells continued to producelower amounts of aggrecan than under permanent treat-ment for the rest of the culture period shown by an in-crease from 1123 � 44 to 2291 � 815 �g aggrecan/�gDNA in group G2 and 294 � 114 to 880 � 534 �g ag-grecan/�g DNA in group G3 between day 6 (G2) or day3 (G3) and day 14. Aggrecan was not detected in cellstreated with CM for 14 days.

Quantification of glycosaminoglycans (GAG)

All alginate beads were cultured for 14 days in totaland exposed to TGF-�3 as described above. On day 14,beads were dissolved in digestion buffer and glycosami-noglycan analysis revealed 9.61 � 2.23 �g glycosami-noglycans (GAG)/�g DNA (mean � SE of the mean) ofhyaluronic acid, 3.71 � 1.94 �g GAG/�g DNA of chon-droitin-4-sulfate, and 1.18 � 0.74 �g GAG/�g DNA ofchondroitin-6-sulfate in group G1, 8.99 � 0.98 �gGAG/�g DNA of hyaluronic acid, 3.54 � 0.88 �gGAG/�g DNA of chondroitin-4-sulfate, and 1.19 �0.68 �g GAG/�g DNA of chondroitin-6-sulfate in groupG2 and 5.22 � 1.54 �g GAG/�g DNA of hyaluronicacid, 1.19 � 0.28 �g GAG/�g DNA of chondroitin-4-sulfate and 0.85 � 0.2 �g GAG/�g DNA of chondroitin-6-sulfate in group G3 (Fig. 2). In group G4, 3.19 � 0.76�g GAG/�g DNA of hyaluronic acid, 1.01 � 0.12 �gGAG/�g DNA of chondroitin-4-sulfate, and 0.76 �

MSCs MAINTAIN TGF-�-MEDIATED CHONDROGENIC PHENOTYPE

0.12 �g GAG/�g DNA of chondroitin-6-sulfate was ob-tained.

Quantitative RT-PCR–stability of chondrogenic phenotype

MSCs were cultured in the presence or absence ofTGF-�3 as described above. RNA samples were taken atdays 3, 6, and 14, and RT-PCR analysis was performedfor mRNA expression of COMP, aggrecan, �1-collagentype II (col2a1), and AP.

COMP mRNA was elevated 1.3-fold � 0.4 (mean �SE of the mean) at day 3 (10 ng/mL TGF-�3) and afterwithdrawal of TGF-�3 2.8-fold � 0.9 (10 ng/mL TGF-�3), 2.8-fold � 0.8 (20 ng/mL TGF-�3), and 3.1-fold �0.7 (60 ng/mL TGF-�3) at day 14 (p � 0.05). After 6days of continuous exposure to TGF-�3, gene expressionof COMP was elevated 1.8-fold � 0.2 at day 6 (10 ng/mLTGF-�3) and after withdrawal of TGF-�3 1.9-fold � 0.3(10 ng/mL TGF-�3), 2.3-fold � 0.4 (20 ng/mL TGF-�3)and 2.7-fold � 0.6 (60 ng/mL TGF-�3) at day 14. Con-tinuous stimulation with 10 ng/mL TGF-�3 over 14 daysled to a 4.0-fold � 1.0 upregulation of COMP mRNA ex-pression in MSCs (Fig. 3A).

Aggrecan mRNA was elevated 0.07-fold � 0.03 at day3 (10 ng/mL TGF-�3) and after withdrawal of TGF-�30.12-fold � 0.03 (10 ng/mL TGF-�3), 0.14-fold � 0.05(20 ng/mL TGF-�3), and 0.16-fold � 0.05 (60 ng/mLTGF-�3) at day 14 (p � 0.05). After 6 days of continu-ous exposure to TGF-�3, gene expression of aggrecanwas elevated 0.09-fold � 0.02 at day 6 (10 ng/mL TGF-�3) and after withdrawal of TGF-�3 0.13-fold � 0.2 (10ng/mL TGF-�3), 0.16-fold � 0.01 (20 ng/mL TGF-�3)and 0.17-fold � 0.03 (60 ng/mL TGF-�3) at day 14 (p �0.05). Continuous stimulation with 10 ng/mL TGF-�3over 14 days led to a 0.21-fold � 0.04 upregulation ofaggrecan mRNA expression in MSCs (Fig. 3B).

Col2a1 mRNA was not expressed at day 3 (10 ng/mLTGF-�3) and after withdrawal of TGF-�3 0.17-fold �0.03 (10 ng/mL TGF-�3), 0.21-fold � 0.06 (20 ng/mLTGF-�3), and 0.52-fold � 0.08 (60 ng/mL TGF-�3) el-evated at day 14 (Fig. 4). After 6 days of continuous ex-posure to TGF-�3, gene expression of col2a1 was ele-vated 0.11-fold � 0.04 on day 6 (10 ng/mL TGF-�3) andafter withdrawal of TGF-�3 0.10-fold � 0.01 (10 ng/mLTGF-�3), 0.34-fold � 0.08 (20 ng/mL TGF-�3), and0.44-fold � 0.1 (60 ng/mL TGF-�3) at day 14 (Fig. 4).Continuous stimulation with 10 ng/mL TGF-�3 over 14days led to a 0.52-fold � 0.11 upregulation of col2a1mRNA expression in MSCs (Fig. 3C).

Quantitative RT-PCR–osteogenictransdifferentiation

After 3, 6, and 14 days of exposure to TGF-�-con-taining or TGF-�-free medium (CM), encapsulated or

1397

FIG. 2. MSCs were cultured for 14 days in total. They weretreated with TGF-�3 for either 14 days (G1), for 6 days (G2),or 3 days (G3). One sample was cultured for 14 days in con-trol medium only (G4). For the remainder of the total culturetime, alginate beads (G2, G3) were cultured in the absence ofTGF-�3 in control medium. All samples were analyzed for gly-cosaminoglycan content with high performance liquid chro-matography. Values were normalized to the DNA content ofthe corresponding sample.

monolayer-seeded MSCs were cultured in OM for an-other 9-day period (Fig. 5).

AP mRNA of encapsulated MSCs was elevated 3.3-fold � 0.92 in MSCs pretreated for 14 days with CM and1.26-fold � 0.27 in samples pretreated for 3 days withTGF-�3-containing medium. In all other groups, APmRNA expression was not detected.

AP mRNA of monolayer-cultured MSCs was ele-vated 11.34-fold � 1.94 in samples pretreated for 14days with TGF-�3-containing medium and then treated9 days with OM and 14.39-fold � 1.57 in samples ex-posed to CM for 14 days and then to OM for 9 days(Fig. 5).

Encapsulated MSCs exposed for 3 days or 6 days toTGF-�3 did not express col2a1 after 9-day culture in OM.After 14 days of treatment with TGF-�3 and 9 days ofOM, col2a1 mRNA expression was elevated 1.3-fold �0.4 in MSCs. Col2a1 mRNA was not detected in anysamples cultured in monolayer (Fig. 5).

MEHLHORN ET AL.

AP detection

All samples were pretreated for 3, 6, 14 days withTGF-�3-containing medium or 14 days with CM andthen exposed to OM for 9 days of culture (Fig. 6).

AP enzyme activity of encapsulated MSCs was 0.55 �0.01 Radioluminescence Units (RLU)/�g DNA (mean �SE of the mean) in samples pretreated with TGF-�3-con-taining medium for 3 days, 0.10 � 0.01 RLU/�g DNAin samples pretreated with TGF-�3-containing mediumfor 6 days, 0.08 � 0.01 RLU/�g DNA in samples pre-treated with TGF-�3-containing medium for 14 days, and0.62 � 0.12 RLU/�g DNA after pretreatment with TGF-�3-free CM for 14 days. Comparing the group pretreatedfor 14 days (6 days) with TGF-� and then for 9 days withOM and the group pretreated for 14 days with CM andthen for 9 days with OM, a power of 80% was calculatedto detect a difference between means of 0.157 (0.158)with a significance level of � � 0.05.

1398

FIG. 3. mRNA was isolated from MSC cultured in serum-free medium containing 10 ng/mL TGF-�3 for 3, 6, or 14 days.For the remainder of the 2 weeks total culture period, MSCswere cultured in the absence of TGF-�3 (CM). Gene expres-sion analysis was performed for cartilage oligomeric matrix pro-tein (COMP) (A), aggrecan (B), and col2a1 (C) using quanti-tative RT-PCR. Gene expression levels were reported relativeto gene expression of the housekeeping gene GAPDH forcol2a1 and aggrecan and relative to GAPDH and gene expres-sion of the control samples for COMP. Asterisks indicate sta-tistically significant differences (*p � 0.05).

AP enzyme activity of monolayer-cultured MSCs was246.07 � 22.24 RDU/�g DNA in samples pretreated withTGF-�3-containing medium for 14 days and afterwardstreated with OM for 9 days and 370.16 � 45.1 RDU/�gDNA in samples exposed to CM for 14 days and then toOM for 9 days. Comparing monolayer-cultured samplespretreated with TGF for 14 days before exposure to OMfor 9 days and monolayer-cultured samples pretreated withCM for 14 days before exposure to OM, a power of 80%was calculated to detect a difference between means of104.40 with a significance level of � � 0.05.

Histology

Samples from MSCs treated with 10 ng/mL, 20 ng/mL,and 60 ng/mL TGF-�3 for 3 days and an additional 11days with CM were embedded in paraffin, cut, andstained for collagen type II and counterstained for nucleiwith 4,6-Diamidino-2-phenylindole (DAPI) (Fig. 7). Allbeads showed a homogeneous distribution of cells andconsistent size of beads. The control group, and samplestreated with 10 ng/mL or 20 ng/mL TGF-�3 for 3 daysand an additional 11 days with CM were negative for col-lagen type II staining. Collagen type II was detected insamples treated with 60 ng/mL TGF-�3 for 3 days andan additional 11 days with CM. Additionally, positivestaining for collagen type II was observed in samplestreated continuously with 10 ng/mL TGF-�3 for 14 days.

MSCs MAINTAIN TGF-�-MEDIATED CHONDROGENIC PHENOTYPE

DISCUSSION

By exposing differentiated pluripotent mesenchymalcell cultures to CM or OM, the ability of committed MSCto maintain chondrogenic gene expression was demon-strated. Furthermore, it was shown that commitment anddifferentiation to the chondrogenic lineage of MSCs weredependent on the concentration and duration of exposureto TGF-�.

After transient exposure to TGF-�, MSCs showed astable chondrogenic phenotype. MSCs stimulated for 3days expressed chondrospecific genes or proteins such asCOMP, aggrecan, and �1-collagen type II at the end ofthe culture period. A significant increase in mRNA ex-pression of COMP and aggrecan at day 14 compared today 3 was observed. Col2a1 gene was not expressed inMSCs during the first 3 days of TGF-� stimulation, butwas finally upregulated at day 14 if culture was contin-ued in CM for another 11 days. The upregulation ofcol2a1 gene at day 14 was dependent on the concentra-tion of TGF-� used for transient stimulation.

These findings suggest that within the first 3 days ofexposure to TGF-�, MSCs underwent a genetic pro-gramming of differentiation that was maintained by a

1399

FIG. 4. mRNA was isolated from MSC cultured in serum-free medium containing TGF-�3 (10, 20, 60 ng/mL) for 3, 6,or 14 days. For the remainder of the 2 weeks total culture pe-riod, MSCs were cultured in the absence of TGF-�3 (CM).Gene expression analysis was performed for col2a1 using quan-titative RT-PCR. Gene expression levels were reported relativeto the gene expression of the housekeeping gene GAPDH. As-terisks indicate statistically significant differences (*p � 0.05).

FIG. 5. After 3, 6, and 14 days of exposure to TGF-�3, en-capsulated (3D) MSCs were cultured in osteogenic medium foranother 9-day period. Additionally, encapsulated (3D) cellswere cultured for 14 days in control medium (CM) and after-wards exposed to osteogenic medium (OM). Alternatively,MSCs were cultured in monolayer (ML) in the presence of CMor TGF-�-containing medium for 14 days and then in OM for9 days. Alkaline phosphatase (AP) and col2a1 gene expressionwere measured with quantitative RT-PCR and normalized tothe housekeeping gene GAPDH.

growth factor–free medium. After withdrawal of TGF-�,the chondrogenic maturation from a collagen type II neg-ative MSC to a chondrocyte-like cell being able to ex-press chondrospecific �1-collagen type II was continuedin CM. Our results differ from previous studies where theeffects of various exposure times of cytokines weretested, e.g., those found in a myoblastic cell line stimu-lated with bone morphogenetic protein (BMP)-2, anothermember of the TGF-�-superfamily. For this cell line, thephenotypic changes to an osteoblastic cell line were notheritable, and BMP-2 was required to maintain the dif-ferentiation pathway.21 Furthermore, Shea and colleaguescould show that the stem cell–like fibroblastic cell lineC3H10T1/2 needs to be continuously exposed to BMP-7 for more than 8 days in order to maintain a differen-tiated osteochondroblastic phenotype in monolayer culture.22 In contrast, MSCs encapsulated in polylac-tide/alginate amalgam constructs showed similar colla-gen type II and glycosaminoglycan production in histo-logic sections after continuous and transient exposure toTGF-�1.23 Possible reasons for these different findingscould be differences in the cell types used, the culturematrix, or the type of growth factor. A three-dimensionalculture environment as chosen by Caterson seems to becrucial for MSCs to maintain a chondrogenic phenotypeafter withdrawal of growth factors. However, during cul-ture in monolayer, even mature articular chondrocytesloose their cell-specific properties and develop to a fi-broblast-like phenotype.6,24 In the work carried out byShea, culture medium was supplemented by fetal bovineserum containing various growth factors that might favortransdifferentiation to other cell lineages. Medium con-taining insulin, transferrin, selenium, ascorbic acid, and

MEHLHORN ET AL.

dexamethasone instead of bovine serum establishes achondro-conductive culture environment that is able topreserve chondrogenic phenotype in the absence of anystimulating growth factor.23

Our results indicate that the prolonged time of tran-sient exposure from 3 days to 6 days to TGF-� does notenhance the chondrogenic differentiation and the stabil-ity of the chondrogenic phenotype. We could not find adifference in quantitative gene expression of aggrecan,COMP, and collagen type II at day 14 between samplescultured for 3 days or 6 days in TGF-containing mediumand for the remainder of the culture period in CM. Thesefindings suggest that key events that are responsible forthe differentiation of mesenchymal cells to the chondro-genic lineage take place during the early initial period ofexposure to the growth factor rather than between day 3and day 6 of TGF-� stimulation. From our in vitro model,it can be concluded that TGF-� has the strongest effecton MSCs within the first 3 days and at the end of the cul-ture period as shown by enhanced chondrogenesis aftercontinuous treatment over 14 days. It is stated in the lit-erature that growth factors of the TGF-� superfamily playa pivotal role in early chondrogenesis enhancing mes-enchymal condensation, for example, by overexpressionof N-cadherin.25 Additionally, TGF-� has been reportedto stimulate terminal differentiation of already commit-ted chondrocytes shown by an enhanced production ofcartilage-specific matrix components such as proteogly-cans and type-II collagen.21,26

MSCs are a potential source of cells for joint repairbecause they are able to differentiate into chondrocytesand can be transplanted in suitable cell carriers into car-tilage defects.9 It was thought that cells have to undergo

1400

FIG. 6. (A) After 3, 6, and 14 days of exposure to TGF-�3, encapsulated (3D) MSCs were cultured in osteogenic medium foranother 9 days period. Alternatively, encapsulated (3D) cells were cultured for 14 days in control medium (CM) and afterwardsexposed to osteogenic medium (OM). (B) MSCs were cultured in monolayer (ML) in presence of CM or TGF-�-containingmedium for 14 days and then in OM for 9 days. Alkaline phosphatase enzyme activity was measured with a chemiluminescenceassay and enzyme activity was normalized to DNA amount of the corresponding sample.

MSCs MAINTAIN TGF-�-MEDIATED CHONDROGENIC PHENOTYPE 1401

FIG. 7. Alginate beads of MSCs were cultured in mediumcontaining 10, 20, and 60 ng/mL TGF-�3 for 3 days and an-other 11 days in control medium (A–C), in medium containing10 ng/mL TGF-�3 for 14 days (D) or in control medium for 14days (E). Samples were embedded in paraffin, cut, and stainedfor collagen type II and counterstained with DAPI (magnifica-tion: 40�).

in vitro commitment to a chondrogenic cell lineage be-fore transplantation. Once committed cells are trans-planted, they are exposed either to chondro-conductivecues from the synovial fluid or osteogenic signals fromthe underlying subchondral bone plate.16 We have shownthat MSCs stimulated with TGF-� for 3, 6, or 14 daysexpress a stable chondrogenic phenotype in a chondro-conductive medium containing dexamethasone, ascorbicacid, insulin, transferrin, and selenium. To study the bi-ological response of committed MSCs abutting on thesubchondral bone plate, we cultured differentiated MSCfor 9 days in OM. Under these conditions, encapsulatedMSCs exposed to TGF-� for more than 6 days did notexpress the osteogenic marker AP. In MSCs cultured for14 days in TGF-�-free CM or for 3 days in TGF-�-con-taining medium, AP mRNA expression and enzyme ac-tivity was detected. Song and colleagues were able toshow that MSCs fully differentiated to a chondrogeniclineage are able to transdifferentiate into adipogenic andosteogenic lineages. For transdifferentiation, the cellswere released from the alginate matrix and were culturedin monolayer in the presence of osteogenic stimuli.15 Be-cause we could show that AP enzyme activity and geneexpression was obviously higher in monolayer-culturedsamples compared to encapsulated cell samples and wasdecreased by pre-exposure to TGF-� in monolayer andalginate bead culture, the combination of TGF-� pre-treatment and the maintenance of a three-dimensionalculture environment prevents transdifferentiation ofMSCs from a chondrogenic to an osteogenic lineage.

We examined whether committed MSCs lose theirability to express �1-collagen type II gene after os-teogenic induction. MSCs pretreated with TGF-� for theentire period of 14 days continued expressing col2a1 geneduring culture in osteogenic differentiation medium. Insamples pretreated for 3 or 6 days with TGF-�, expres-sion of col2a1 was not detected after exposure to OM.However, a transient treatment with TGF-� was suffi-cient enough to induce col2a1 expression if the cultureof cells was continued in chondro-conductive CM. Thus,short-term treatment can be effective for chondro-spe-cific gene induction, but the presence of a chondro-con-ductive and not osteogenic environment is needed for further maturation of the chondrogenic phenotype. There-fore, full commitment of MSCs to a chondrogenic lin-eage is necessary to express a stable chondrogenic phe-notype and to prevent osteogenic transdifferentiation ofmatrix-encapsulated MSCs.

It is controversially discussed whether MSCs have tobe differentiated in vitro before transplantation to carti-lage defects or if the local environment of the transplan-tation site alone can induce a chondrogenic phenotype inundifferentiated MSCs. From our study, we conclude thatafter short-term in vitro exposure to TGF-�, MSCs of thesuperficial layer of cartilage grafts could express a sta-ble chondrogenic phenotype due to the close contact to

MEHLHORN ET AL.

the chondro-conductive synovial fluid. Autologous syn-ovial fluid has been shown to enhance the chondrogenicphenotype of mesenchymal stem cells, but it was less ef-fective than medium containing 10 ng/mL TGF-�.27,28

MSCs of deeper graft layers might be more strongly in-fluenced by an osteogenic environment coming from sub-chondral bone plate and have to be fully committed to achondrogenic cell line in vitro in order to express a sta-ble phenotype after implantation.

In summary, we characterized the conditions that enableMSCs to maintain a chondrogenic phenotype in vitro. Cul-ture of MSCs has to be continued in a three-dimensionalmatrix, and MSCs have to be fully differentiated to chon-drogenic lineage before they can be exposed to an os-teogenic environment. For the development of chondro-genic tissue, an initial stimulus of TGF-� is essential andchondrogenic gene expression levels can be improved byan increased dose of transient TGF-�. Because only fullydifferentiated MSCs can be prevented from transdifferen-tiation into an osteogenic cell lineage and maintain chon-drogenic phenotype in the presence of osteogenic factors,our data encourage the necessity of an in vitro pretreat-ment with TGF-� before in vivo implantation of MSC-seeded grafts. However, in vivo studies have to be doneshowing superior tissue-specific properties of pretreatedcell-seeded grafts compared to untreated transplants. Theseresults support the application of MSCs for tissue engi-neering purposes and can give rise to further studies usingMSCs for cartilage repair in vivo.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Björn Hackanson(Department of Haematology and Oncology, Freiburg)for performing the iliac crest biopsies. The authors aregrateful to Dr. K. Naruse (Department of OrthopaedicSurgery, Kitasato University, Japan) for advice and dis-cussion. This study was supported by the Kompetenz Net-zwerk Biomaterialien Baden-Württemberg and by a grantof the Forschungs-kommission of the Freiburg Univer-sity Medical School.

REFERENCES

1. Caplan, A.I., Elyaderani, M., Mochizuki, Y., Wakitani, S.,and Goldberg, V.M. Principles of cartilage repair and re-generation. Clin. Orthop. 342, 254, 1997.

2. Jobanputra, P., Parry, D., Fry-Smith, A., and Burls, A. Ef-fectiveness of autologous chondrocyte transplantation forhyaline cartilage defects in knees: a rapid and systematicreview. Health Technol. Assess. 5, 1, 2001.

3. Gillogly, S.D., Voight, M., and Blackburn, T. Treatment ofarticular cartilage defects of the knee with autologous chon-drocyte implantation. J. Orthop. Sports Phys. Ther. 28, 241,1998.

1402

4. Lee, C.R., Grodzinsky, A.J., Hsu, H.P., Martin, S.D., andSpector, M. Effects of harvest and selected cartilage repair pro-cedures on the physical and biochemical properties of articu-lar cartilage in the canine knee. J. Orthop. Res. 18, 790, 2000.

5. Marlovits, S., Hombauer, M., Truppe, M., Vecsei, V., andSchlegel, W. Changes in the ratio of type-I and type-II col-lagen expression during monolayer culture of human chon-drocytes. J. Bone Joint Surg. Br. 86, 286, 2004.

6. Binette, F., McQuaid, D.P., Haudenschild, D.R., Yaeger,P.C., McPherson, J.M., and Tubo, R. Expression of a sta-ble articular cartilage phenotype without evidence of hy-pertrophy by adult human articular chondrocytes in vitro.J. Orthop. Res. 16, 207, 1998.

7. Barry, F., Boynton, R.E., Liu, B., and Murphy, J.M. Chon-drogenic differentiation of mesenchymal stem cells frombone marrow: differentiation-dependent gene expression ofmatrix components. Exp. Cell Res. 268, 189, 2001.

8. Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M.,and Yoo, J.U. In vitro chondrogenesis of bone marrow-de-rived mesenchymal progenitor cells. Exp. Cell Res. 238,265, 1998.

9. Wakitani, S., Imoto, K., Yamamoto, T., Saito, M., Murata,N., and Yoneda, M. Human autologous culture expandedbone marrow mesenchymal cell transplantation for repairof cartilage defects in osteoarthritic knees. OsteoarthritisCartilage 10, 199, 2002.

10. Leonard, C.M., Fuld, H.M., Frenz, D.A., Downie, S.A.,Massague, J., and Newman, S.A. Role of transforminggrowth factor-beta in chondrogenic pattern formation in theembryonic limb: stimulation of mesenchymal condensationand fibronectin gene expression by exogenous TGF-betaand evidence for endogenous TGF-beta-like activity. Dev.Biol. 145, 99, 1991.

11. Awad, H.A., Wickham, M.Q., Leddy, H.A., Gimble, J.M.,and Guilak, F. Chondrogenic differentiation of adipose-de-rived adult stem cells in agarose, alginate, and gelatin scaf-folds. Biomaterials 25, 3211, 2004.

12. Ma, H.L., Hung, S.C., Lin, S.Y., Chen, Y.L., and Lo, W.H.Chondrogenesis of human mesenchymal stem cells encapsu-lated in alginate beads. J. Biomed. Mater. Res. 64A, 273, 2003.

13. Fragonas, E., Valente, M., Pozzi-Mucelli, M., Toffanin, R.,Rizzo, R., Silvestri, F., and Vittur, F. Articular cartilage re-pair in rabbits by using suspensions of allogenic chondro-cytes in alginate. Biomaterials 21, 795, 2000.

14. Diduch, D.R., Jordan, L.C., Mierisch, C.M., and Balian, G.Marrow stromal cells embedded in alginate for repair ofosteochondral defects. Arthroscopy 16, 571, 2000.

15. Song, L., and Tuan, R.S. Transdifferentiation potential ofhuman mesenchymal stem cells derived from bone mar-row. FASEB J. 18, 980, 2004.

16. Wakitani, S., and Yamamoto, T. Response of the donor andrecipient cells in mesenchymal cell transplantation to car-tilage defect. Microsc. Res. Tech. 58, 14, 2002.

17. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K.,Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W.,Craig, S., and Marshak, D.R. Multilineage potential of adulthuman mesenchymal stem cells. Science 284, 143, 1999.

18. Shinmei, M., Miyauchi, S., Machida, A., and Miyazaki, K.Quantitation of chondroitin 4-sulfate and chondroitin 6-sul-

MSCs MAINTAIN TGF-�-MEDIATED CHONDROGENIC PHENOTYPE

fate in pathologic joint fluid. Arthritis Rheum. 35, 1304,1992.

19. Blum, J.S., Li, R.H., Mikos, A.G., and Barry, M.A. An op-timized method for the chemiluminescent detection of al-kaline phosphatase levels during osteodifferentiation bybone morphogenetic protein 2. J. Cell Biochem. 80, 532,2001.

20. Stahl, A., Wenger, A., Weber, H., Stark, G.B., Augustin,H.G., and Finkenzeller, G. Bi-directional cell contact-depen-dent regulation of gene expression between endothelial cellsand osteoblasts in a three-dimensional spheroidal coculturemodel. Biochem. Biophys. Res. Commun. 322, 684, 2004.

21. Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Taka-hashi, N., Ikeda, T., Rosen, V., Wozney, J.M., Fujisawa-Sehara, A., and Suda, T. Bone morphogenetic protein-2converts the differentiation pathway of C2C12 myoblastsinto the osteoblast lineage. J. Cell Biol. 127, 1755, 1994.

22. Shea, C.M., Edgar, C.M., Einhorn, T.A., and Gerstenfeld,L.C. BMP treatment of C3H10T1/2 mesenchymal stemcells induces both chondrogenesis and osteogenesis. J. CellBiochem. 90, 1112, 2003.

23. Caterson, E.J., Nesti, L.J., Li, W.J., Danielson, K.G., Al-bert, T.J., Vaccaro, A.R., and Tuan, R.S. Three-dimen-sional cartilage formation by bone marrow-derived cellsseeded in polylactide/alginate amalgam. J. Biomed. Mater.Res. 57, 394, 2001.

24. Grundmann, K., Zimmermann, B., Barrach, H.J., andMerker, H.J. Behaviour of epiphyseal mouse chondrocytepopulations in monolayer culture. Morphological and im-munohistochemical studies. Virchows Arch. A Pathol.Anat. Histol. 389, 167, 1980.

25. Haas, A.R., and Tuan, R.S. Chondrogenic differentiationof murine C3H10T1/2 multipotential mesenchymal cells:II. Stimulation by bone morphogenetic protein-2 requiresmodulation of N-cadherin expression and function. Differ-entiation 64, 77, 1999.

26. Iwamoto, M., Sato, K., Nakashima, K., Fuchihata, H.,Suzuki, F., and Kato, Y. Regulation of colony formationof differentiated chondrocytes in soft agar by transforminggrowth factor-beta. Biochem. Biophys. Res. Commun. 159,1006, 1989.

27. Rodrigo, J.J., Steadman, J.R., Syftestad, G., Benton, H.,and Silliman, J. Effects of human knee synovial fluid onchondrogenesis in vitro. Am. J. Knee Surg. 8, 124, 1995.

28. Hegewald, A.A., Ringe, J., Bartel, J., Kruger, I., Notter,M., Barnewitz, D., Kaps, C., and Sittinger, M. Hyaluronicacid and autologous synovial fluid induce chondrogenicdifferentiation of equine mesenchymal stem cells: a pre-liminary study. Tissue Cell 36, 431, 2004.

Address reprint requests to:Alexander Mehlhorn, M.D.

Department of Orthopaedic and Trauma SurgeryUniversity Medical Center Freiburg

Hugstetterstrasse 5579106 Freiburg, Germany

E-mail: mehlhorn@ch11.ukl.uni-freiburg.de

1403