Chondrogenesis of Human BoneMarrow-Derived Mesenchymal Stem

Cells in Agarose CultureC.-Y. CHARLES HUANG,1,2 PAUL M. REUBEN,1,2 GIANLUCA D’IPPOLITO,1

PAUL C. SCHILLER,1 AND HERMAN S. CHEUNG1,2*

1Research Service and Geriatrics Research, Education, and Clinical Center,Veterans Affairs Medical Center, Miami, Florida

2Department of Biomedical Engineering, University of Miami, Coral Gables, Florida

ABSTRACTMesenchymal stem cells derived from human bone marrow (hBM-MSCs) can differenti-

ate into chondrogenic cells for the potential treatment of injured articular cartilage. Toevaluate agarose gels as a supportive material for chondrogenesis of hBM-MSCs, this studyexamined chondrogenesis of hBM-MSCs in the agarose cultures. Pellet cultures were em-ployed to confirm the chondrogenic potential of the hBM-MSCs that were used in agarosecultures. The hBM-MSCs were seeded in 2% agarose constructs at the initial cell-seedingdensities of 3, 6, and 9 � 106 cells/ml while each of pellets was formed using 2.5 � 105 cells.Chondrogenesis of hBM-MSCs was induced by culturing cell-agarose constructs and pelletsfor 21 days in the presence of a defined medium containing transforming growth factor �3(TGF-�3). The analysis of reverse transcription-polymerase chain reaction showed thathBM-MSCs of agarose and pellet cultures expressed the chondrogenic markers of collagentype II and aggrecan in the presence of TGF-�3. The deposition of cartilage-specific macro-molecules was detected in both agarose and pellet cultures by histological and immunohis-tochemical assessments. Chondrogenesis of hBM-MSCs in agarose gels directly correlatedwith the initial cell-seeding density, with the cell-agarose constructs of higher initial cell-seeding density exhibiting more cartilage-specific gene expressions. This study establishes abasic model for future studies on chondrogenesis of hBM-MSCs using the agarose cultures.Anat Rec Part A 278A:428–436, 2004. © 2004 Wiley-Liss, Inc.

Key words: biomaterials; cartilage; adult stem cells; TGF-�3; agarose; tissueengineering

Due to the inaccessibility of reparative cells and lowcellularity, defects of articular cartilage, without the pen-etration of subchondral bone, result in insufficient healingresponses of adjacent chondrocytes. Such defects usuallyprogress to cartilage degeneration and finally advance toosteoarthritis.

Genzyme Biosurgery (Cambridge, MA) was the first tomarket autologous cultured chondrocytes (Carticel) forcell-based therapies such as cartilage repair. While theprocedure was used clinically, the protocol to harvestchondrocytes from adjacent normal sites in the joint wasinvasive and permanently damaged normal area of carti-lage. It has been suggested that this invasive procedurealone can initiate degenerative changes and eventuallylead to osteoarthritis in the joint (Erickson et al., 2002).

Mesenchymal stem cells derived from bone marrow(BM-MSCs) have demonstrated the multipotential to dif-ferentiate into several cell lineages, including chondro-cytes and osteocytes (Aydelotte and Kuettner, 1988;

Mackay et al., 1998; Yoo et al., 1998; Pittenger et al.,1999). In addition to their multipotency, BM-MSCs can beacquired by bone marrow aspiration without permanentlydamaging tissues, efficiently expanded in monolayers byserial passages without altering their differentiation po-tential, and capable of repairing joint defects involvingboth subchondral bone and articular cartilage. These

Grant sponsor: the National Institutes of Health; Grant num-ber: AR 38421; Grant sponsor: VA Merit Review Grant.

*Correspondence to: Dr. Herman S. Cheung, Research Service,Miami VA Medical Center, 1201 NW 16th Street, Miami, Fl33125. Fax: 305-575-3365. E-mail: hcheung@med.miami.edu

Received 14 July 2003; Accepted 4 December 2003DOI 10.1002/ar.a.20010

THE ANATOMICAL RECORD PART A 278A:428–436 (2004)

© 2004 WILEY-LISS, INC.

characteristics make BM-MSCs candidate cells for celltransplantation (Wakitani et al., 1994, 2002; Diduch et al.,2000) and the development of autologous cartilaginousimplants (Mackay et al., 1998; Caterson et al., 2001) forthe repair of injured articular cartilage that has verylimited capability for self-healing.

Biomaterials also play an important role in serving as adelivery vehicle in cell transplantation as well as provid-ing initial three-dimensional structures for developing tis-sues with required geometry. Generally, the suitability ofbiomaterials for cell transplantation or engineering carti-laginous tissues for the repair of articular cartilage de-pends on certain criteria: the biomaterial is biocompatible,can promote chondrogenic expressions of cells, can allowthe deposition of the extracellular matrix, and is mechan-ically stable. Recently, a number of biomaterials, such ascollagen gel (Wakitani et al., 1994, 2002), hyaluronic acid-based polymer (Angele et al., 1999; Solchaga et al., 1999),polylactide/aliginate amalgam (Caterson et al., 2001), gel-atin-based resorbable sponge (Ponticiello et al., 2000),photopolymerized hydrogels (Williams et al., 2003), andalginate (Diduch et al., 2000), have been proposed as cellcarriers of BM-MSCs for cell transplantation or cartilagetissue engineering. These biomaterials have been shownto support chondrogenesis of BM-MSCs under in vivo (Wa-kitani et al., 1994, 2002; Solchaga et al., 1999; Diduch etal., 2000) or in vitro (Angele et al., 1999; Caterson et al.,2001) conditions. However, other biomaterials such asagarose, which may potentially promote chondrogenesis ofBM-MSCs (Diduch et al., 2000), still have not been ex-plored.

Three-dimensional agarose cultures of chondrocyteshad been shown to maintain the stability of differentiatedphenotype (Benya and Shaffer, 1982; Aydelotte andKuettner, 1988) while promoting the chondrocyte biosyn-thesis of cartilage-specific aggrecan and collagen thatwere deposited within agarose gels to develop a cartilage-like tissue (Buschmann et al., 1992; Mauck et al., 2000).This suggests that the agarose culture of chondrocytesmay be able to synthesize a functional cartilaginous tissue(Buschmann et al., 1992; Mauck et al., 2000). Due to theirmechanical stability, agarose cultures have also beenwidely used as an in vitro model for studying the influenceof compressive loading on the biosynthetic activities andmechanotransduction events of chondrocytes, the deposi-tion of pericellular matrix (Buschmann et al., 1995; Leeand Bader, 1997; Knight et al., 1998; Mauck et al., 2000;Roberts et al., 2001), and chondrogenic differentiation ofchick limb-bud cells (Elder et al., 2000). Furthermore, arecent study has demonstrated promising results on therepair of full-thickness cartilage defects in rabbit kneejoints by transplanting chondrocyte-agarose allografts(Rahfoth et al., 1998). Therefore, agarose gels may serveas a reasonable model for studying chondrogenic re-sponses of BM-MSCs.

To evaluate agarose gels as a potential material forcell-based therapies or cartilage tissue engineering usingBM-MSCs, the fundamental step is to examine whetherthe three-dimensional agarose culture can support chon-drogenesis of BM-MSCs under in vitro control conditions.Since previous studies have shown that chondrogenic dif-ferentiation of human BM-MSCs (hBM-MSCs) can be in-duced in the suspension pellet culture (Yoo et al., 1998;Pittenger et al., 1999) and in the polymer culture (Angeleet al., 1999; Ponticiello et al., 2000; Caterson et al., 2001)

with the treatment of TGF-�, we hypothesize that thethree-dimensional agarose culture can provide a support-ive environment for chondrogenesis of hBM-MSCs in thepresence of such growth factors. The objective of this studywas to examine the chondrogenesis of hBM-MSCs cul-tured in agarose gels with the treatment of TGF-�3 byanalyzing the expressions of cartilage-specific macromol-ecules (collagen type II and aggrecan) as well as the dep-osition of the collagen type II proteins secreted by hBM-MSCs within agarose gels.

MATERIALS AND METHODSCulture of hBM-MSCs

The hBM-MSCs were provided by the Diabetes Re-search Institute at University of Miami School of Medicineand the Geriatrics Research, Education, and Clinical Cen-ter at Miami Veterans Affairs Medical Center. The proce-dure for isolating hBM-MSCs from bone marrow of post-mortem thoracolumbar (T1–T5) vertical bodies had beenpreviously described (D’Iippolito et al., 1999). Briefly, thevertical columns were harvested from eight donors (agerange, 21–45) immediately after death from traumaticinjuries. The vertical bodies were separated along thesagittal midline craniocaudal axis and cut into bone chips(� 5 mm3). The bone chips were placed at room tempera-ture in 1,000 ml of processing medium (X-Vivo 10; Bio-whittaker, Walkersville, MD) containing 2.5% human se-rum albumin (Calbiochem, La Jolla, CA), bacitracin (50U/ml), heparin (10 U/ml), polymyxin B (500 U/ml), and 2mM HEPES buffer (pH 7.2). The cell suspension wasseparated from the bone chips by filtering through twoconsecutive stainless steel screens (pore diameters 450and 180 �m) and then centrifuged at 300 g for 10 min at4°C. The cell pellet was suspended in �-modified essentialmedium (�-MEM; Gibco-BRL, Grand Island, NY) with10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL). The remaining bone marrow cells trapped withinbone trabeculae were released at room temperature bytwo 30-min gentle rocking agitations (� 40 cycles/min) ofbone chips in 1,000 ml of RPMI-1640 (Gibco-BRL) contain-ing 2.5% human serum albumin, heparin (10 U/ml), gen-tamicin (0.5 mg/ml), and 2 mM HEPES buffer (pH 7.2).The suspension of bone marrow cells was filtered througha Y-Type Blood Set (McGaw, Irvine, CA) to remove bonefragments and clumps of cells. An aliquot of cells wastreated with 4% acetic acid to lyse erythrocyte and thencounted with a hemacytometer for trypan blue exclusion.Cells were plated in culture flasks with low-glucose Dul-becco’s modified Eagle’s medium (DMEM; Gibco-BRL)containing 10% FBS serum and 1% antibiotics and incu-bated at 37°C with 5% CO2. After 4 days of primaryculture, nonadherent cells were removed by changing me-dium. After 14 days of primary culture, hBM-MSCs wereharvested with trypsin/EDTA and frozen in 1 ml aliquotsin liquid nitrogen.

The hBM-MSCs were thawed and expanded in 10 cmculture dishes with low-glucose DMEM containing 10%FBS serum and 1% antibiotics (passage 1). The cells ofeach 10 cm culture disk were harvested and replatedequally into two 10 cm culture disks after 14 days (pas-sage 2), 21 days (passage 3), and 28 days (passage 4).Passage 4 cells were used in both agarose cultures andpellet cultures.

429CHONDROGENESIS OF hBM-MSC

Pellet Culture of hBM-MSCsChondrogenic potential of the hBM-MSCs used in this

study was initially confirmed by pellet cultures that havebeen shown to promote chondrogenesis of hBM-MSCs inother studies (Yoo et al., 1998; Pittenger et al., 1999).Approximately 2.5 � 105 cells were trypsinized and cen-trifuged in 15 ml polypropylene tube to form a pellet. Thepellets of the control group (n � 30) were cultured inserum-free medium consisting of high-glucose DMEM, 1%insulin-transferrin-selenium supplements (final concen-trations, 10 �g/ml bovine insulin, 5.5 �g/ml transferrin,6.7 ng/ml sodium selenite; Gibco-BRL), 1.25 mg/ml bovinealbumin, 5.33 �g/ml linoleic acid, 40 �g/ml proline, 50 �g/ml ascorbic acid, and 10-7 M dexamethasone (Sigma, St.Louis, MO), while the serum-free medium supplementedwith 10 ng/ml of recombinant human TGF-�3 (R&D Sys-tems, Minneapolis, MN) was used for the treated group(n � 30). Three pellets in each group were used for histo-logical and immunohistochemical evaluations while theexpression of the cartilage-specific markers were exam-ined on the remaining pellets. All pellet cultures wereperformed in humidified incubator maintained at 37°C in5% CO2 for 21 days. The culture medium was changedevery 2–3 days.

Agarose Culture of hBM-MSCsThis study examined chondrogenesis of hBM-MSCs cul-

tured in 2% agarose gels that have been shown to main-tain chondrocyte phenotype, promote the cartilage-specificmacromolecule biosynthesis of chondrocyte, and develop acartilage-like tissue (Buschmann et al., 1992; Mauck etal., 2000). After trypsinizing and cell counting, hBM-MSCs were suspended in the high-glucose DMEM solu-tion and then mixed with an equal volume of 4% (wt/vol)agarose solution at 37°C to produce mixtures of 3 � 106,6 � 106, and 9 � 106 cells/ml. The cell-agarose constructs(8 mm in diameter and 1.5 mm thick) were formed bycasting the cell-agarose mixture in a custom-designedmold and gelling for 15 min at room temperature. Thecell-agarose mixtures of 3 � 106, 6 � 106, and 9 � 106

cells/ml produced 20, 20, and 12 cell-agarose constructs,respectively. For each cell-seeding density, the cell-agar-ose constructs were equally separated into two groups (thecontrol group and the TGF-�3-treated group). Two cell-

agarose constructs in each group were analyzed histolog-ically and immunohistochemically while the remainingconstructs were used for the analyses of cartilage-specificgene expression. All cell-agarose constructs were culturedin three 24-well culture plates in a humidified 5% CO2atmosphere at 37°C for 21 days with the culture mediaused in the pellet culture study. The culture medium waschanged every 2–3 days.

Reverse Transcription-Polymerase ChainReaction (RT-PCR) Analysis

The expressions of cartilage-specific markers (collagentype II and aggrecan) and collagen type I gene were ex-amined to determine whether the chondrogenic differen-tiation of hBM-MSCs occurred or not. Because of the lowcell number in specimens and the low recovery rate ofRNA from agarose gels, the total RNA of hBM-MSCs wasextracted and combined from all pellets or all cell-agaroseconstructs in the same group using the TRIzol reagent(Gibco-BRL) according to the manufacturer’s instructions.The RT-PCR was performed in GeneAmp PCR system(9600, Perkin Elmer Ceyus, Norwalk, CT) using the Ther-moScript RT-PCR system (Gibco-BRL). The cDNA synthe-sis was performed by a 60-min incubation at 50°C with anavian RNase H-minus reverse transcriptase and Oli-go(dT)20 primer, followed by enzyme inactivation at 85°Cfor 5 min. PCR amplifications for the resulting cDNAsamples were carried out for 30 cycles (for pellets) or 40cycles (for cell-agarose constructs) by denaturing at 95°Cfor 30 sec, annealing at 58°C for 45 sec, and extending at72°C for 45 sec, with final extension at 72°C for 10 min.The primers used in the PCR amplifications were listed inTable 1. As an internal control, 353 bp of the constitutivelyexpressed housekeeping gene, �-actin, was also synthe-sized and used to normalize the amount of mRNA in eachRT-PCR reaction. The PCR products were analyzed byelectrophoresis on 2% agarose gel containing ethidiumbromide and subsequently photographed using a low-lightimage system (ChemiImager 4000, Alpha Innotech, SanLeandro, CA). Integrated density value (IDV) of each PCRproduct on the image of electrophoresis was measured bythe AlphaEase software (Alpha Innotech) and normalizedby the IDV of the PCR product of �-actin gene for compar-

TABLE 1. PCR primer sequences used in this study

Gene Sequence Size Reference

Collagen type ISense 5�-CGTGGTGACAAGGGTGAGAC-3� 827 bp GenbankAntisense 5�-TAGGTGATGTTCTGGGAGGC-3� Z74615

Collagen type IISense 5�-AACCAGATTGAGAGCATCCGC-3� 517 bp (Su et al., 1989)Antisense 5�-CCTTCAGGGCAGTGTACGTGA-3�

Collagen XSense 5�-GCCCAAGAGGCGTGATGGCTTATTTGT-3� 703 bp Genbank NM000493Antisense 5�-CCTGAGAAAGAGGAGTGGACATAC-3�

AggrecanSense 5�-ACCGTTGCAGACATTGACGAGTG-3� 300 bp (Freije et al., 1994)Antisense 5�-ACTCCTGCTCC-TCGGGGGTGACG-3�

�-actinSense 5�-GCTCGTCGTCGACAACGGCTC-3� 353 bp GibcoBRLAntisense 5�-CAAACATGATCTGGGTCATCTTCTC-3�

430 HUANG ET AL.

ison between the groups of different initial cell-seedingdensities.

Histological and Immunohistochemical Analysis

Deposition of extracellular matrix proteins was exam-ined by histological and immunohistochemical evalua-tions. Pellets and cell-agarose constructs were fixed in10% buffered formalin for 2 hr at 4°C and 4% paraformal-dehyde for 15 min at room temperature, respectively. Af-ter washing in PBS, the specimens were dehydrated in agraded series of increasing concentrations of ethanol,cleaned with xylene, embedded in paraffin, and cut into 5�m sections. Proteoglycans were detected by staining sec-tions with alcian blue solution. The deposition of collagentype I and type II proteins was identified by immunohis-tochemical analysis. After deparaffinizing, sections wereincubated in 0.3% hydrogen peroxide in distilled water for30 min to remove endogenous peroxidase. After rinsingwith distilled water and PBS, 10% normal horse serumwas placed on the sections to block nonspecific back-ground, then incubated with mouse monoclonal antihu-man collagen antibody (type I: I-8H5, 500 �g/ml; type II:II-4CII, 500 �g/ml; ICN Biomaterials, Aurora, OH) at 4°Covernight. Following extensive washing with PBS to re-move the primary antibody, immunoactivity was detectedby incubating the sections with biotinylated horse anti-mouse antibody, followed by incubation with avidin-bi-otin-peroxidase complex (ImmunoPure ABC peroxidasestaining kits; Pierce, Rockford, IL). Peroxidase activitywas visualized using 3-3�-diaminobenzidine (DAB) as sub-strate. The sections were incubated with 0.06% DAB in 0.1M Tris-HCL, pH 7.5, containing 0.03% H2O2, followed bycounterstaining with hematoxyline. All incubations wereperformed in a humidified chamber.

RESULTSChondrogenic Differentiation in Pellet Culture

After a 21-day culture, chondrogenesis of hBM-MSCs inthe TGF-�3-treated pellet culture was detected by immu-nohistochemical and histological assessments, as illus-trated in Figures 1 and 2. Only the specimens of theTGF-�3-treated group exhibited immunopositive reactionfor collagen type II, as visualized by strong brown prod-ucts in Figure 1. The specimens of the TGF-�3-treatedgroup exhibited stronger alcian blue staining as comparedwith the specimens of the control group (Fig. 2), indicatingthat more proteoglycans were deposited in the TGF-�3-treated specimens. The deposition of collagen type I pro-teins was also present in pellets of both groups (data notshown). Chondrogenic differentiation of hBM-MSCs inpellet cultures was further confirmed by the RT-PCR anal-ysis. Figure 3 shows the results of the RT-PCR analysis forboth groups, demonstrating that the pellet culture ofhBM-MSCs in the presence of TGF-�3 expressed the car-tilage-specific genes (collagen type II and aggrecan),whereas only weak expression of the aggrecan gene wasexhibited in pellets of the control group. However, pelletsof both groups still maintained a small expression of col-lagen type I gene after a 21-day culture (data not shown),whereas collagen type X gene expression was not detectedin pellets of either groups.

Chondrogenic Differentiation in AgaroseCulture

Figure 4a and b show the typical phase-contrast lightmicrographs of hBM-MSCs cultured in agarose gel at theinitial seeding density of 9 � 106 cells/ml with and withoutthe treatment of TGF-�3 for 21 days, demonstrating thatthe agarose gel maintained the spherical morphology ofthe hBM-MSCs; they were able to proliferate and depositextracellular matrix surrounding cells to form larger cel-lular aggregates within the agarose gel in the presence ofTGF-�3. The cartilage-specific extracellular matrix mac-romolecues, collagen type II and proteoglycans, were de-tected in the cellular aggregate formed by hBM-MSCscultured in the agarose gel with the treatment of TGF-�3(Fig. 4d and f), whereas the deposition of collagen type IIand proteoglycans were not detected in the cell-agaroseconstructs of the control group (Fig. 4c and e). The resultsof the RT-PCR analysis showed that the hBM-MSCs of theTGF-�3-treated group with the initial seeding density of9 � 106 cells/ml expressed both chondrogenic markers ofcollagen type II and aggrecan (Fig. 5). However, the TGF-�3-treated specimens with the initial cell-seeding densityof 3 � 106 and 6 � 106 cells/ml only exhibited the expres-sion of collagen type II gene (Fig. 6) but not aggrecan. Bydoubling cell-seeding density from 9 � 106 to 1.8 � 107

cells/ml, the difference is less than 20% increase in bothcartilage-specific gene expressions (data not shown), sug-gesting that cell seeding density of 9 � 106 cells/ml is nearthe optimum condition. The specimens of the controlgroup for the three initial cell-seeding densities expressedneither collagen type II gene nor aggrecan gene. A dimin-ished expression of collagen type I gene was still found inthe cell-agarose constructs of the TGF-�3-treated groupand the control group after a 21-day culture (data notshown).

Figure 6 shows a typical comparison on the normalizedIDVs of PCR products for the collagen type II gene be-tween the TGF-�3-treated groups of different initial cell-seeding densities, demonstrating that the cell-agaroseconstructs of higher cell-seeding density exhibited moreexpression of collagen type II gene.

DISCUSSIONDue to the inaccessibility of reparative cells and low

cellularity, defects of articular cartilage, without the pen-etration of subchondral bone, result in insufficient healingresponses of adjacent chondrocytes. Such defects usuallyprogress to cartilage degeneration and finally advance toosteoarthritis. When injury penetrates the subchondralbone, mesenchymal stem cells can be recruited from bonemarrow to regenerate reparative tissues (Coletti et al.,1972; Cheung et al., 1978, 1980; Furukawa et al., 1980;Johnson, 1986). However, these reparative tissues wereusually identified as fibrocartilage that is histologicallysimilar yet mechanically inferior to hyaline cartilage (Co-letti et al., 1972; Cheung et al., 1980; Furukawa et al.,1980; Johnson, 1986). Since adult BM-MSCs exhibit apromising potential for cartilage regeneration, a possiblestrategy for the restoration of injured articular cartilage tothe normal status is the transplantation of BM-MSCs orBM-MSC-generated cartilaginous implants using the bio-materials that potentially support the regeneration of newfunctional cartilage.

431CHONDROGENESIS OF hBM-MSC

Figure 1.

Figure 2.

432 HUANG ET AL.

Chondrogenic differentiation of hBM-MSCs used in thisstudy was initially confirmed by the findings of immuno-histochemical assessments and the RT-PCR analysis inthe pellet culture study, which is consistent with previousstudies (Yoo et al., 1998). This study provides supportingevidence for the hypothesis that hBM-MSCs are able toundergo chondrogenic differentiation in the three-dimen-sional agarose culture with the treatment of TGF-�3. Theresults of RT-PCR analysis showed that the TGF-�3-treated specimens expressed chondrogenic markers of col-lagen type II and aggrecan, wherein the deposition ofcollagen type II proteins was detected by immunohisto-chemical assessments. These findings suggest that theagarose gel is able to provide a supportive environment forchondrogenesis of hBM-MSCs. To the best of our knowl-edge, this study is the first to demonstrate chondrogenesisof hBM-MSCs in agarose cultures.

The study of Yoo et al. (1998) demonstrated that themonolayer cultures of hBM-MSCs with the treatment ofTGF-�1 did not exhibit the deposition of proteoglycans,whereas the collagen type II proteins and proteoglycanswere accumulated in the TGF-�1-treated pellet cultures ofhBM-MSCs. Other previous studies also demonstrated the

induction of chondrogenic differentiation of hBM-MSCs inthe polylactide/aliginate amalgam with the treatment ofTGF-�1 (Caterson et al., 2001) and the gelatin-basedresorbable sponge with the treatment of TGF-�3 (Pontici-ello et al., 2000). These studies indicate that suspensionculture may play an important role in promoting chondro-genesis of hBM-MSCs. Indeed, suspension culture hasbeen shown to maintain the phenotypic expression ofchondrocytes. Benya and Shaffer (1982) found that rabbitarticular chondrocytes lost the differentiated phenotypeand expressed the fibroblastic phenotype during serialmonolayer culture, whereas dedifferentiated chondrocytesreexpressed the differentiated phenotype during suspen-sion cultures in agarose gels.

In this study, the initial cell-seeding density of cell-agarose constructs was found to influence chondrogenicdifferentiation of hBM-MSCs, as demonstrated by stron-ger expression of collagen type II gene exhibited in thecell-agarose constructs of higher initial cell-seeding den-sity. The expression of aggrecan gene was only detected inthe cell constructs of 9 � 106 cells/ml. This finding isconsistent with the study of Ponticiello et al. (2000), whofound that hBM-MSCs cultured in the gelatin matrix witha higher initial cell-seeding density produced more proteo-glycans in the presence of TGF-�3. Since a previous studyhad noted that the interactions between cells were impor-tant for in vitro chondrogenic differentiation and mainte-nance of chondrocyte phenotype (Solursh, 1991), the highcell-seeding density may improve the cell interactions thatpromote chondrogenic differentiation of hBM-MSCs. Cellsare not uniformly dispersed when cast into the cell-agar-ose constructs. Microscopic examination revealed cell ag-gregates throughout the agarose gel. Not surprising, thenumber of cell aggregates increases with higher cell num-bers; perhaps this may account for higher efficiency ofinduction by high cell–seeding density. Moreover, extra-cellular matrix proteins were deposited around individualcells in agarose cultures, high cell-seeding density mayfacilitate the integration of extracellular matrices secretedby individual cells to form a cartilaginous tissue.

Collagen type I gene was found to be expressed in theTGF-�3-treated pellets and cell-agarose constructs, sug-gesting that hBM-MSCs have not been completely trans-formed into chondrocyte-like cells after the 21-day treat-ment of TGF-�3. The TGF-�3-treated pellets alsoexhibited the deposition of collagen type I proteins after a21-day culture, indicating that incomplete chondrogenictransformation of hBM-MSCs may produce an extracellu-lar matrix of different collagen fibers. Indeed, the abun-dant deposition of collagen type I proteins was found inthe fibrocartilaginous tissues that are usually regeneratedat injury sites of articular cartilage by bone marrow cellsrecruited by penetrating subchondral bone (Furukawa etal., 1980; Johnson, 1986). These reparative fibrocartilagi-nous tissues were found to be functionally inferior to ar-ticular cartilage and degenerate over time (Coletti et al.,1972; Furukawa et al., 1980). Therefore, the accelerationand efficiency of chondrogenic transformation of hBM-MSCs are important subjects for the clinical applicationsof hBM-MSCs on the repair of articular cartilage.

It has been demonstrated that compressive loading notonly stimulated chondrogenic differentiation of chick em-bryonic limb-bud cells (Elder et al., 2000), but also modu-lated the cartilage matrix biosynthesis of mature chondro-cytes (Sah et al., 1989; Buschmann et al., 1995; Lee and

Fig. 3. Typical RT-PCR analysis of cartilage-specific genes on pelletspecimens of hBM-MSCs after a 21-day culture.

Fig. 1. Typical immunohistochemical analysis of collagen type IIproteins on the hBM-MSC pellet sections of the control group (a, 40�;c, 400�) and the TGF-�3-treated group (b, 40�; d, 400�) after a 21-dayculture (a and b, scale bar � 150 �m; c and d, scale bar � 20 �m). TheTGF-�3-treated specimen exhibits the deposition of collagen type II thatis illustrated as dark brown staining.

Fig. 2. Typical histological analysis of proteoglycans on the hBM-MSC pellet sections of the control group (a, 40�; c, 400�) and theTGF-�3-treated group (b, 40�; d, 400�) after a 21-day culture (a and b,scale bar � 150 �m; c and d, scale bar � 20 �m). The TGF-�3-treatedspecimen deposits more proteoglycans, as manifested by dark bluestaining.

433CHONDROGENESIS OF hBM-MSC

Bader, 1997; Knight et al., 1998; Mauck et al., 2000;Roberts et al., 2001). In addition, a recent study trans-planted BM-MSCs to large full-thickness cartilage defectsof rabbit knee joints and showed that 6 months after the

implantation, the defects were repaired by regeneratedhyaline-like cartilage, which exhibited different mechani-cal properties on posterior and anterior aspects of the joint(Wakitani et al., 1994). It suggested that mechanical dif-

Fig. 4. Typical phase-contrast light micrographs of hBM-MSCs cul-tured in the agarose gel at the initial seeding density of 9 � 106 cells/ml(a) without the treatment of TGF-�3 and (b) with the treatment of TGF-�3for 21 days (400�; scale bar � 20 �m). The hBM-MSCs are able toproliferate and deposit extracellular matrix to form larger cellular aggre-gates (arrowhead) within the agarose gel with the treatment of TGF-�3.Typical immunohistochemical analysis of collagen type II on the typicalcellular aggregate formed by hBM-MSCs in the cell-agarose constructsof (c) the control group and (d) the TGF-�3-treated group after a 21-dayculture (400�; scale bar � 20 �m). Immunopositive reaction for collagen

type II proteins (arrow) illustrated by dark brown staining is only seenwithin the cellular aggregate in the TGF-�3-treated specimen. Nuclei(arrowhead) were counterstained using hematoxyline. Typical alcian bluestaining of proteoglycans on the typical cellular aggregate formed byhBM-MSCs in the cell-agarose constructs of (e) the control group and (f)the TGF-�3-treated group after a 21-day culture (400�; scale bar � 20�m). The deposition of proteoglycans (arrow) illustrated by strong bluestaining was detected outside and inside the cellular aggregate in theTGF-�3-treated specimen. Nuclei (arrow head) were counterstained us-ing nuclear fast red.

434 HUANG ET AL.

ferences might result from substantially different me-chanical environments at two locations (Wakitani et al.,1994). The cartilage repair studies of autogenous perios-teal grafts in which MSCs reside have demonstrated thatcontinuous passive motion enhanced the repair of full-thickness defect of articular cartilage (O’Driscoll et al.,1986). Based on these previous studies, mechanical stim-uli may influence chondrogenesis of BM-MSCs as well asthe deposition of extracellular matrix secreted by trans-formed cells. Agarose cultures have been widely used tostudy the effects of mechanical loading on the responses ofchondrocytes due to their mechanical stability. Thus, thisstudy establishes a basic model of agarose culture of hBM-MSCs for future studies to investigate the effects of me-

chanical loading on chondrogenesis of hBM-MSCs usingagarose cultures.

In summary, this study has demonstrated that chondro-genesis of hBM-MSCs can be induced in agarose gels withthe treatment of TGF-�3, as attested by the results of geneexpression analyses and histological and immunohisto-chemical assessments. The agarose culture study of hBM-MSCs also demonstrated the effect of initial cell-seedingdensity on chondrogenesis of hBM-MSCs. Since agarosegels are mechanically stable, this study has established abasic agarose culture system for further investigations ofthe effects of mechanical loading on chondrogenesis ofhBM-MSCs. Furthermore, the pellet culture study con-firms the chondrogenic potential of hBM-MSCs and showsthe mixing deposition of collagen type I and II proteinsduring chondrogenic differentiation of hBM-MSCs. It ex-plains that collagen type I and II proteins are found inreparative fibrocartilaginous tissues that are usuallyformed by bone marrow cells when injury on articularcartilage penetrates the subchondral bone. Therefore,complete chondrogenic transformation of hBM-MSCscould be an important factor for regeneration of functionalarticular cartilage using hBM-MSCs.

ACKNOWLEDGMENTSWe thank Ms. Leonor Wenger and Mr. Felix Soto for

their technical assistance with cell cultures and histolog-ical and immunohistochemical analyses and Ms. KristenHagar, Lauren Frost, and Marella Kurikose for their crit-ical reading of the article.

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Fig. 5. Typical RT-PCR analysis of cartilage-specific genes and�-actin gene on cell-agarose constructs of hBM-MSCs with the initialseeding density of 9 � 106 cells/ml after a 21-day culture.

Fig. 6. Typical comparison on the normalized IDVs of PCR productsfor the collagen type II gene between the TGF-�3-treated groups ofdifferent initial cell-seeding densities. Cells were obtained from a 24-year-old female donor.

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