Special Focus Section:

ENGINEERED IN VITRO MODELS OF TUMOR ANGIOGENESIS

Tissue Engineering& Regenerative MedicineInternational Society

The Official Journal of

ISSN 19373341

VOLUME 16 NUMBER 7JULY 2010

TissueTissueEngineeringEngineeringPart A

43986_TEA_cover.indd 143986_TEA_cover.indd 1 6/21/10 8:56 PM6/21/10 8:56 PM

Parameters in Three-Dimensional Osteospheroidsof Telomerized Human Mesenchymal (Stromal)Stem Cells Grown on Osteoconductive Scaffolds

That Predict In Vivo Bone-Forming Potential

Jorge S. Burns, Ph.D.,1,2,* Pernille L. Rasmussen, B.Sc., M.Sc.,1,* Kenneth H. Larsen, B.Sc., M.Sc.,1

Henrik Daa Schrøder, M.D., D.M.Sc.,3 and Moustapha Kassem, M.D., Ph.D., D.Sci.1,4

Osteoblastic differentiation of human mesenchymal stem cells (hMSC) in monolayer culture is artefactual,lacking an organized bone-like matrix. We present a highly reproducible microwell protocol generating three-dimensional ex vivo multicellular aggregates of telomerized hMSC (hMSC-telomerase reverse transcriptase(TERT)) with improved mimicry of in vivo tissue-engineered bone. In osteogenic induction medium the hMSCwere transitioned with time-dependent specification toward the osteoblastic lineage characterized by productionof alkaline phosphatase, type I collagen, osteonectin, and osteocalcin. Introducing a 1–2mm3 crystallinehydroxyapatite/beta-tricalcium phosphate scaffold generated osteospheroids with upregulated gene expressionof transcription factors RUNX2/CBFA1, Msx-2, and Dlx-5. An organized lamellar bone-like collagen matrix,evident by birefringence of polarized light, was deposited in the scaffold concavities. Here, mature osteoblastsstained positively for differentiated osteoblast markers TAZ, biglycan, osteocalcin, and phospho-AKT.Quantification of collagen birefringence and relatively high expression of genes for matrix proteins, in-cluding type I collagen, biglycan, decorin, lumican, elastin, microfibrillar-associated proteins (MFAP2 andMFAP5), periostin, and tetranectin, in vitro correlated predictively with in vivo bone formation. The three-dimensional hMSC-TERT/hydroxyapatite-tricalcium phosphate osteospheroid cultures in osteogenic inductionmedium recapitulated many characteristics of in vivo bone formation, providing a highly reproducible andresourceful platform for improved in vitro modeling of osteogenesis and refinement of bone tissue engineering.

Introduction

Osteoblasts, derived from mesenchymal stem cells(MSC; also known as, skeletal stem cells or bone

marrow stromal cells), reside in the nonhematopoietic com-partment of bone marrow stroma. Since isolation of colony-forming MSC from the hematopoietic component of bonemarrow cultures has principally relied on preferential plasticadherence,1 it is not surprising that most studies of MSCdifferentiation and function cultured the cells in a two-dimensional (2D) monolayer environment. The ex vivo oste-oblastic phenotype has been traditionally described in terms

of expression of genes known to play a role in bone forma-tion, for example, alkaline phosphatase (ALP), collagen type 1(COL1), and osteocalcin (OCN). However, 2D primary cul-tures have several limitations. The cells lose the endogenousthree-dimensional (3D) microenvironment architecture in-cluding cell–cell and cell–matrix interactions resulting inheterogeneous cell populations at different stages of differ-entiation.2 Also, the functional capacity of the osteoblasticcells is usually demonstrated by their ability to form min-eralized matrix in presence of organic phosphate donor(b-glycerophosphate) and ascorbic acid. However, ex vivo ma-trix mineralization assays lack specificity, may be confounded

1Laboratory for Molecular Endocrinology (KMEB), Department of Endocrinology and Metabolism, Odense University Hospital andMedical Biotechnology Center, Odense, Denmark.

2Laboratory of Cell Biology and Advanced Cancer Therapies, Department of Oncology, Hematology and Respiratory Disease, UniversityHospital of Modena and Reggio Emilia, Modena, Italy.

3Institue of Pathology, Odense University Hospital and Medical Biotechnology Center, University of Southern Denmark, Odense,Denmark.

4Stem Cell Unit, Department of Anatomy, King Saud University, Riyadh, Saudi Arabia.*These two authors contributed equally to the work.

TISSUE ENGINEERING: Part AVolume 16, Number 7, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089/ten.tea.2009.0735

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by simple precipitation of calcium phosphate, and exhibit asurprisingly poor concordance with the cellular capacity toform bone in vivo.2–4 To overcome such problems, severalinvestigators have established 3D culture system for osteo-blastic cells5–9 and reported induction of better osteoblastdifferentiation compared to 2D culture.10,11 However, mostof the described 3D culture studies, certainly those usingosteogenic cells derived from osteosarcoma cell lines, didnot use bone formation as their final outcome and employedinstead a modified form of ex vivo mineralization assay.5

In recent years, the differentiation potential of MSC toosteoblastic cells has been tested by demonstrating theirability to form heterotopic bone in vivo when mixed with anosteoconductive material: hydroxyapatite/beta tricalciumphosphate (HA/b-TCP) and implanted subcutaneously inimmune deficient mice.12,13 Here, we introduced, stepwise,two main aspects of the in vivo microenvironment: three-dimensionality and growth on HA/b-TCP scaffold, to gen-erate a highly reproducible ex vivo model for osteoblastdifferentiation of human mesenchymal stem cells (hMSC)overespressing the telomerase reverse transcriptase (TERT)gene, referred to here as hMSC-TERT. The hMSC-TERTstrain is known to have a full potential for bone forma-tion.14,15 We found that in osteogenic induction medium(OIM) 3D cell aggregates of hMSC (spheroids) and suchaggregates grown on HA/b-TCP scaffolds (osteospheroids)developed into mature osteoblastic cells and deposited or-ganized lamellar collagen bone–like matrix that could beobserved and quantified by a characteristic birefringencewhen examined by polarized light. When comparing hMSC-TERT clones with different bone-forming potential, ourex vivo culture method with measurement of birefringenceand expression of associated matrix proteins was now able topredict the in vivo extent of stem cell osteogenesis in tissue-engineered bone.

Materials and Methods

Cell culture

The hMSC strain developed from hMSC cultured from ahealthy male donor (age 33) and transduced at popula-tion doubling level (PDL) 12 with a retroviral vector over-expressing the human telomerase reverse transcriptasegene (hMSC-TERT cells)14,15 was used at PDL 95. Cells wereexpanded inmaintenancemedium (MM), consisting ofMEMwithout phenol red (Gibco, Invitrogen), with 10% fetal bovineserum (PAA, Biotechline AS) and 1% penicillin/streptomycin(Gibco, Invitrogen). Cells seeded at *1000 cells/cm2 werepassaged 1:4 into Nunclon T175 flasks (Nunc) kept at 378C in ahumidified incubator (Hereus) containing 5% CO2. Cultureswere routinely passaged when 80%–90% confluent. Two indi-vidual clonal cell lines were derived from the hMSC-TERTpopulation (PDL 77) by limited dilution as described.4

3D multicellular aggregates and osteogenic induction

Cells were seeded at 10,000 cells per microwell in poly-styrene nonadhesive round-bottomed 96-well microplates(Greiner, Invitrogen) or Ultra Low Adhesion round-bottomed 96-well microplates (Corning). To improve spheroiduniformity, the medium with 0.25% carboxymethylcellulose(CMC) was filtered via prefilter pads into 0.45 mm filter

bottles (Corning). Once stable multicellular spheroids hadformed (72 h), the osteogenic differentiation time-course wasstarted (day 0). Parallel cell populations were fed either MMwith CMC, or OIM with added 50 mg/mL L-ascorbic acid(Wako Chemicals GmbH), 10 nM b-glycerol phosphate(Calbiochem), 10 nM dexamethasone (Sigma), and 10 nMCalcitrol (1,25 dihydroxy vitamin D3; Leo Pharma). Re-feeding was every third day before harvesting the multicel-lular spheroids at days 0, 7, 15, and 21. We examined theinfluence of a bioceramic scaffold, composed of HA Ca10(PO4)6(OH)2 and b-TCP, Ca3(PO4)2 (HA/b-TCP, Triosite;MBPC Biomatlant; Zimmer Scandinavia!). The HA/b-TCPweight ratio was 60/40% with 30% of microporosity lessthan 1mm and a specific crystal surface area of 4! 0.02m2/g.Macroporosity was 50% macropore size ranging between 150and 700 (mean 400mm). One 0.5–1-mm-diameter granulewas placed in each well before seeding with cells. A 96-wellplate required about 40mg of HA/b-TCP granules, equiva-lent to the amount for each ectopic murine implantationstudy.

RNA extraction and reverse transcriptase real-time poly-merase chain reaction

Briefly, as described previously,15 total cellular RNA wasisolated using a TRIzol (Invitrogen) single-step RNA ex-traction method, according to manufacturer’s instructions.First-strand complementary cDNA was synthesized from2 mg of total RNA using a Revertaid" H minus first-strandcDNA synthesis kit (Fermentas), according to manufacturer’sinstructions. Real-time polymerase chain reaction (RT-PCR)utilized the iCycler IQ detection system (Bio-Rad) withdouble-strand DNA-specific SYBR! Green I luminescentdye. In a total reaction volume of 20mL, 20 pmol/mL of eachprimer (DNA Technology A/S) and 10pmol/mL for eachreference gene was used together with 2"iQ" SYBR GreenSupermix (Bio-Rad). The cycle conditions consisted of aninitial denaturation step at 908C for 1min and 50 cycles of958C for 30 s, 608C for 30 s, and 728C for 1min. The meanvalue from glucoronidase, glyceraldehyde-3-phosphate de-hydrogenase, and b2-microglobulin housekeeping genes wasused for reference (see Table 1 for primer sequences).

Immunohistochemistry

For each sample, pooled 3D cellular aggregates from threeplates (n¼ 288) were washed with phosphate-buffered salinewithout Ca2þ Mg2þ and fixed for 24 h in 4% formaldehyde.Then, samples were washed three times in isotonic salineand embedded using the Shandon Cytoblock! Cell Pre-paration System (Thermo Electron Coorporation). Afterparaffin embedding, 4-mm-thick sections were mounted onslides, deparaffinized with xylene, and rehydrated by step-wise washing in ethanol with increasing quantities of water.To reverse formaldehyde-induced protein cross-linking,slides were demasked by boiling in a microwave oven.Staining followed the EnVisionþprotocol (Techmate; Dako).The target proteins, primary antibodies, dilutions used, andsuppliers were as follows: Ki67 (MIB-1, 1:100; DAKOCyto-mation); Biglycan (LF-112, 1:500, NIDCR NIH); CD146(N1238, 1:100; Novocastra); Col 1a (LF-67, 1:1000, NIDCRNIH); OCN (Rabbit polyclonal BT-593, 1:400; BiomedicalTechnologies); Osteonectin (ON) (15G12, 1:100; Novocastra);

2332 BURNS ET AL.

Osteopontin (OP3N, 1:100; Novocastra); Phospho-AKT(736E11, 1:100; Cell Signaling Technology); RUNX2 (2302,1:100; R&D Systems); TAZ (Rabbit polyclonal, 1:100; NovusBiologicals). Slides were incubated with primary antibodyfor 1 h, washed with TNT-buffer (0.1M Tris HCl, 0.15MNaCl, and 0.05% Tween-20, pH 7.5), and incubated withsecondary antibody, peroxidase-labeled EnVisionþpolymer,for 30min. After washing with TNT-buffer, slides were in-cubated with DABþ for 10min to produce the stain. Afterrinsing with water, slides were counterstained with Mayer’shematoxylin and rinsed again, and a cover slip was mountedwith Harleco Aquatex mounting medium.

Osteoblastic differentiation

Parallel 3D cultures with or without OIM were assayed at0, 7, 15, and 21 days (n¼ 288 spheroids per data point). Inaddition, a set of osteosphere samples was prepared byseeding 10,000 cells on a singe 1–2mm3 granule (diameter*1500 mm) of HA/b-TCP (*25,000 cells/mg scaffold), cul-tured in parallel conditions to the 3D cultures until analysison day 15. Immunohistochemical analysis was com-plemented with quantitative RT-PCR analysis of lineagebiomarkers pertaining to osteoblasts (COL1a and ALP),chondrocytes (collagen type II and collagen X), and adipo-cytes (LPL and PPARª).

Collagen matrix birefringence measurement

The picrosirius polarization staining method16 is a sensi-tive selective stain for collagen, which, after acquiring a ma-ture tertiary structure, becomes birefringent under polarizedlight.17 Sirius-red-stained 4-mm sections from ex vivo day 15HA/TCP osteosphere cultures (n¼ 5 sections per data point)or from in vivo xenograft bone formation assays (n¼ 9 wholesections for each data point for each hMSC-TERT) were ob-served under linear or, for quantitation, circular polarizedlight. Images were obtained at 200" magnification using aLeica microscope and DFC480 camera. Survey 5.5.4.3 auto-mated specimen scanning software (Objective Imaging, Ltd.)was used to obtain mosaic images of whole sections. Visio-Morph VIS Histoinformatics image segmentation software(Visiopharm A/S) quantified the percentage of birefringencewithin the cell matrix areas. Segmentation of background,scaffold, matrix, and birefringent areas used pixel classifierswith Bayesian decision rules derived from the trainingset obtained by highlighting relevant areas of the image.Postprocessing correction involved redefinition of areas ofscaffold that resembled matrix. The relative extent ofbirefringence was quantified from five randomly selectedfields of view by the formula %Birefringence¼Birefringentarea (pixels)/total area of extracellular matrix (pixels)"100.

In vivo bone formation assay in immuno-deficient mice

Cells (5"105) mixed with 40mg of 0.5–1-mm-diametergranules of HA/b-TCP scaffold were transplanted subcu-taneously into the dorsal surface of 8-week-old femaleNOD/SCID mice (NOD/LtSz-Prkdcscid) as described.18

The implants were recovered after 8 weeks and transferredto 4% neutral buffered formalin for about 45min, andthen formic acid was added for 2 days. Using standard

Table 1. Primer Sequences for Polymerase ChainReactions

Gene Primer sequence

ALP 50-ACGTGGCTAAGAATGTCATC-30 (sense)50-ACGTGGCTAAGAATGTCATC-30 (antisense)

B2M 50-GAGTATGCTGCCGTGTG-30 (sense)50-AATCCAAATGCGGCATCT-30 (antisense)

COL1A2 50-GGACACAATGGATTGCAAGG-30 (sense)50-TAACCACTGCTCCACTCTGG-30 (antisense)

COL2A1 50-TTTCCCAGGTCAAGATGGTC-30 (sense)50-CTTCAGCACCTGTCTCACCA-30 (antisense)

COL10A1 50-CACCAGGCATTCCAGGATTCC-30 (sense)50-AGGTTTGTTCTGATAGCTC-30 (antisense)

COL12A1 50-ACCCAAGCTGGCATGAGACC-30 (Sense)50-TGACGTTTCCCTCCAGAAACTG-30

(antisense)DLX5 50-TGGCAAACCAAAGAAAGTTC-30 (sense)

50-AATAGAGTGTCCCGGAGG-30 (antisense)GAPDH 50-ACCACAGTCCATGCCATCAC-30 (sense)

50-TTCACCACCCTGTTGCTGTA-30 (antisense)GUS-B 50-GAAAATATGTGGTTGGAGAGCTCATT-30

(sense)50-CCGAGTGAAGATCCCCTTTTTA-30 (antisense)

LPL 50-CTTGGAGATGTGGACCAGC-30 (sense)50-GTGCCATACAGAGAAATCTC-30 (antisense)

MSX2 50-CGGTCAAGTCGGAAAATTCA-30 (sense)50-GCATAGGTTTTGCAGCCATT-30 (antisense)

OCN 50-CATGAGAGCCCTCACA-30 (sense)50-AGAGCGACACCCTAGAC-30 (antisense)

ON 50-CGAAGAGGAGGTGGTGGCGGAAA-30 (sense)50-GGTTGTTGTCCTCATCCCTCTCATAC-30

(antisense)OP 50-CCAAGTAAGTCAACGAAAG-30 (sense)

50-GGTGATGTCCTCGTCTGTA-30 (antisense)PPARg-2 50-TTCTCCTATTGACCCAGAAAGC-30 (sense)

50-CTCCACTTTGATTGCACTTTGG-30 (antisense)RUNX2 50-CACCATGTCAGCAAAACTTCTT-30 (sense)

50-TCACGTCGCTCATTTTGC-30 (antisense)LUM 50CGTGACTGGGCTGGGTCTC-30 (sense)

50CCACCAATCAATGCCAGGAA-30 (antisense)MFAP2 50-CAGTTCCAGTCCCAGCAGCA-30 (sense)

50-ACACGGCGGAGGCTGTAGAA-30 (antisense)MFAP5 50-GCCAAAGGACTCGGTGGAAA-30 (sense)

50-GGGCAGACCAGCCATCTGAC-30 (antisense)ELN 50-TGTCCATCCTCCACCCCTCT-30 (sense)

50-CTCCTGGGACACCACCAAGC-30 (antisense)DCN 50-CGCCTCATCTGAGGGAGCTT-30 (sense)

50-GGACCGGGTTGCTGAAAAGA-30 (antisense)BGN 50-CCTCCCCTCTCCAGGTCCAT-30 (sense)

50-CTTCTGCAGCTTCCGCAGTG-30 (antisense)POSTN 50-ATTGAAGGCAGTCTTCAGCCTA-30 (sense)

50-TGAAGTCAACTTGGCTCTCACA-30

(antisense)TNA 50-GGGGGCCTACCTCCTCCTCT-30 (sense)

50-ACGCTCTGGCGCAGGTACTC-30 (antisense)

ALP, alkaline phosphatase; B2M, beta-2-microglobulin; COL1A2,collagen type I alpha2; COL2A1, collagen type II alpha1; COL10A1,collagen type X alpha1; COL12A1, collagen type XII alpha1; DLX5,distal-less homeo box 5; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; GUS-B, beta-glucoronidase; LPL, lipoprotein lipase;MSX2, Msh homeobox 2; OCN, osteocalcin; ON, osteonectin; OP,osteopontin; PPARg-2, peroxisome proliferator-activated receptorgamma; RUNX2, Runt-related transcription factor 2; LUM, lumican;MFAP2, microfibrillar-associated protein 2; MFAP5, microfibrillar-associated protein 5; ELN, elastin; DCN, decorin; BGN, biglycan;POSTN, periostin; TNA, tetranectin.

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histopathologic methods, the HA/b-TCP implants wereembedded in paraffin and 4-mm tissue sections werestained with hematoxylin and eosin Y (Bie & BerntsensReagenslaboratorium). The bone volume per total volumewas quantified in a manner similar to that described18 usingVisioMorph Histoinformatics image segmentation software(Visiopharm A/S).

Statistical analysis

A two-tailed t-test was applied to analyze the induction ofosteogenic transcription factors or birefringence between thegroups and for analysis of predictive osteogenic biomarkers,and the data were log transformed for a two-tailed unpairedt-test. A p-value of <0.05 was used as a threshold for sta-tistical significance.

Results

Highly reproducible, uniform synchronized3D multicellular aggregate cultures

Use of the stated concentration of CMC, filtration of themedium, and round-bottomed rather than flat-bottomed orV-shaped low adhesion wells in 96-well plates was critical forhighly uniform 3D multicellular spheroids. Overnight ag-gregation of hMSC-TERT cells formed a spheroid*450mm indiameter (Fig. 1B). Further self-aggregation led to spheroidcontraction to a stable diameter of *300 mm within 72 h(Fig. 1C). Self-aggregation and spheroid formation was veryuniform and reproducible. In OIM, the spheroid diameterremained unchanged, whereas in MM the 3D multicellularaggregate grew to twice the original diameter within 21 days(Fig. 1D). Larger spheroid size correlated with more cells

showing cell cycle activity, evidenced by Ki67 staining in MM(Fig. 1E) versus OIM (Fig. 1F).

Concurrent osteoblastic differentiationof the 3D multicellular aggregates

The time-course study of 3D hMSC spheroids revealedmultipotent differentiation. Expression analysis of biomarkergenes for osteogenic (COL1, ON, and OCN), chondrogenic(collagen II and collagen X), and adipogenic (PPARg2 andLPL) lineages revealed a three-phase kinetic pattern (Fig. 2).First, coinduction of osteogenic, chondrogenic, and (at a lowyet detectable level) adipogenic biomarkers was seen within7 days. The next week there was reduced expression ofadipogenic biomarkers and a continued rise in chondrogenicand osteogenic biomarkers. After 15 days osteoblastic com-mitment involved no expression of adipogenic biomarkers,plateaued expression of chondrogenic markers, and contin-ued increase of only the osteogenic lineage markers.

Histology and gene expression analysisof 3D multicellular aggregates

In OIM spheroids evolved an organized 2–3-cell deepouter cortical layer of elongated fusiform cells surrounded aninner core of more cuboidal cells that stained strongly for ON(Fig. 3H), OP (Fig. 3K), and collagen type Ia (COL1a) (Fig.3N). RT-PCR analysis showed a maximal induction of ALP(Fig. 3C), ON (Fig. 3I), OP (Fig. 3L), and near-maximal in-duction of COL1a (Fig. 3O) preceding that of OCN, whichwas half-maximal after 15 days (Fig. 3F). OIM induced atime-dependent increase in gene expression that was notablyhigh for genes with very low basal levels of expression, forexample, ALP (*50-fold) and OC (200-fold) and in the range

FIG. 1. Highly reproducible, uniform, osteospheroid culture. Spheroids were produced allowing defined number of cells toaggregate in 96-well plates. (A) Phase-contrast photomicrograph of hMSC-TERT cells in monolayer expansion culture. (B)Seeding and aggregation of spheroids. (C) 72 h after seeding, the spheroid had condensed to a stable size. (D) Spheroiddiameter over time. Mean diameter of n¼ 5 spheroids, in medium without differentiation factors (white diamonds) and ionmedium with differentiation factors (black circles). Spheroids were harvested at days 0, 7, 14, and 21. (E) 5-mm tissue sectionof multicellular spheroids in MM stained for Ki67 and (F) equivalent cultures after growth in OIM for 7 days. Scalebar¼ 100 mm. hMSC, human mesenchymal stem cell; MM, maintenance medium; OIM, osteogenic induction medium; TERT,telomerase reverse transcriptase.

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2–3-fold for ON, OP, and Col Ia (Fig. 3C, F, I, L, O). Osteogenicdifferentiation in the 3D multicellular aggregates was also as-sociated with a loss of CD146þ cells (Fig. 3P, Q). Notably,compared to the central core region, the cell matrix in the outerlayer stained more intensely with picrosirius red (Fig. 3R), in-dicating a cortical concentration of collagen fibers. Yellow/orange birefringence with a lamellar pattern first emerged inthis outer cortical region, whereas in the spheroid’s core regionthe birefringence was green, indicating thinner collagen fibersforming a more woven lattice pattern (Fig. 3S).

HA/b-TCP scaffold induced lamellar bone-like structuresin ex vivo osteospheroid cultures

We first examined the bone formed in the heterotopicbone xenografts using the picrosirius-polarization method.As seen in Figure 4D, a characteristic feature of regions ofin vivo early bone formation was the lamellar organizationand periodic structure of mature collagen. The birefringentpattern and hue was proportional to the orientation and

thickness of the collagen fibrils. In vivo, scaffold concavitieswere regions of more extensive bone deposition with abrighter more orange birefringence. In contrast, thin collagenfibers with green birefringence bordered convex surfaces(Fig. 4G). Osteospheroids cultured ex vivo on HA/TCP inOIM exhibited a similar lamellar pattern of collagenbirefringence (Fig. 4E) that was totally absent when the cellswere only grown in MM (Fig. 4F). Confirming consistency,the denser picrosirius red staining and associated birefrin-gence of sections derived from the 8-week in vivo bone for-mation assay (Fig. 4H, J) was more closely mimicked whenosteospheroid culture was extended for an equivalent8-week period (Fig. 4I, K).

Focal areas of high birefringence correlatedwith bone formation

The hMSC-TERT cells grew closely associated with thesurface of the scaffold, cells migrated deep into any creviceson the scaffold surface, yet did not transpass the scaffold

FIG. 2. Gene expression kinetics for tissue-specific lineage biomarkers in cell spheroids grown under osteogenic inductionconditions. (A) Collagen type Ia2, (B) osteonectin, (C) osteocalcin; constituted (H) osteogenic biomarkers. (D) Collagen II and(E) collagen X; constituted (I) chondrogenic biomarkers. (F) LPL and (G) PPARg2; constituted (J) adipogenic biomarkers.Graphs (A–G) represent the formula 2%(CT,X%CT,R), where CT,X is the mean cycle threshold for the gene in question, and CT,R

is the mean cycle threshold for the reference housekeeping genes. For Graphs (I–J) relative QRT-PCR expression wasnormalized to the lowest CT,X values (maximum expression) for the osteogenic biomarkers on day 21. QRT-PCR, quantitativereal-time polymerase chain reaction.

3D HUMAN MESENCHYMAL STEM OSTEOSPHEROIDS 2335

material. To demonstrate that niches of high birefringencecorresponded to sites of bone formation, we examined thetopographical distribution of osteoblastic marker expressionin heterotopoic bone implants and in ex vivo cultured os-teospheroids. We observed that in vivo, concavities were ni-ches for cells positive for Biglycan, ON, RUNX2/CBFA1,TAZ, and Phospho-AKT (Fig. 5A, D, G, J, P). With regard toCD146, cells aligned immediately adjacent to the HA/b-TCPsurface were negative, whereas more distal cells were CD146positive (Fig. 5M). This staining pattern was retained whenosteospheroids were cultured in OIM (Fig. 5B, E, H, K, N, Q)but absent when cells were cultured in MM (Fig. 5C, F, I, L,O, R). Although cells not associated with the scaffold inscaffold spheroids grown in MM did express ON andRUNX2/CBFA1, only a few cells immediately adjacent to thescaffold were positive (Fig. 5F, I). Moreover, markers asso-ciated with mature osteoblasts, such as ON and TAZ, werenot associated with cells near the scaffold in scaffold spher-oids grown in MM (Fig. 5F, L) but were associated with cellsnear the scaffold in osteospheroids grown in osteogenicmedium (Fig. 5E, K). These observations suggested that areasof high birefringence were sites of more mature osteoblaticcells. Compared to multicellular aggregates without scaffold,RT-PCR analysis revealed that the HA/TCP scaffoldenhanced expression of the osteogenic transcription factors

RUNX2/CBFA1, MSX2, and DLX5 (Fig. 5S), but this was notaccompanied by a significantly enhanced production of ALP,Col I, ON, or OCN (data not shown).

Quantitation of areas of birefringence as a surrogatemarker for in vivo bone formation

Having established that highly birefringent areas corre-sponded to more mature osteoblastic cells with a pattern ofcollagen deposition similar to that of in vivo bone formation,we explored whether quantitation of birefringent areas couldserve as a surrogate marker for in vivo bone formation. Forsuch purposes, circular polarized light presented advantagesover linear polarized light.19 We identified two clonal cellpopulations, with a relatively high (hMSC-TERTþBone, cloneDD8) or low (hMSC-TERT%Bone, clone CF1) bone-formingphenotype.4 Quantification of birefringent areas in the osteo-spheroid cultures (Fig. 6A–M) showed that after 2 weeksin OIM, hMSC-TERTþBone showed a significant sixfoldhigher birefringence (52.1%) than when the cells were grownin MM (8.8%), which was still over twice that of the hMSC-TERT-Bone in OIM (20%) (Fig. 6M). The amount of birefrin-gence when the two clones were implanted in vivo was thencorrelated with the amount of ectopic bone. The hMSC-TERTþBone clone produced more ectopic bone compared

FIG. 3. Osteogenic biomarker expression. Immunocytochemistry of spheroid sections and QRT-PCR expression data ofthree-dimensional spheroids grown in selective media for 21 days. Cells were grown in (A, D, G, J, M) MM or (B, E, H, K, N)OIM. (C, F, I, L, O) QRT-PCR expression data for the corresponding genes. (P) Immunocytochemical staining for CD146(brown) in spheroids grown in MM versus (Q) spheroids grown in OIM. (R) Picrosirius red staining of a sectioned spheroidcultured in OIM for 15 days. (S) Polarized light birefringence of a picrosirius-red-stained spheroid. Scale bar¼ 100 mm.

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with hMSC-TERT%Bone showing more birefringence whencomparing whole sections from the bone implantation model(Fig. 6N–U). Notably, the extent of birefringence formed bythe cells in ex vivo osteospheroid culture was positivelycorrelated with the amount of bone formation (Fig. 6V).Thus, the extent of collagen birefringence in culture couldserve as a surrogate predictive marker for the amount ofbone formed when the cells were implanted with HA/b-TCPscaffold subcutaneously in immune-deficient mice.

Bone formation correlated with expressionof extracellular matrix genes

Previous studies using monolayer cultures of the hMSC-TERTþBone and hMSC-TERT%Bone clones showed that theydiffered in their basal expression level for a number ofextracellular matrix genes.4 Here, RT-PCR analysis confirmedthat these differences persisted in 3D osteospheroid culture.

Notably, when comparing the clones after 14 days in eitherMM or OIM, the hMSC-TERTþBone clone showed constitu-tively higher levels of COL1a, collagen 12A1, biglycan, lumi-can, elastin, microfibrillar-associated proteins (MFAP2 andMFAP5) and Periostin (Fig. 7A–C, E–I). Further, although basalexpression levels of decorin and tetranectin inMMwere similarfor the two clones, induction of their gene expression by OIMwas only evident in the hMSC-TERTþBone clone (Fig. 7D, J).

Discussion

In the present study, we sought to improve upon tradi-tional ex vivo assays for osteogenic differentiation, placingemphasis on improved correlation between ex vivo mea-surements and in vivo bone formation. It was logical todevelop a 3D culture platform given that many studiesbroadly agree that a 3D microenvironment enhances osteo-genic differentiation5–9,11 with more extracellular mineralized

FIG. 4. Collagen birefringence in hMSC-TERT osteospheroids. Bright field (A–C, H, I) and polarized light (D–F, J, K)illumination of picrosirius-red-stained 4-mm sections of cells grown on HA/b-TCP scaffolds (‘‘ha’’). (A, D, H) In vivo, 8 weeks.(B, E) Ex vivo, 15 days in OIM. (C, F) Ex vivo, 15 days in MM. (G) In vivo, 8 weeks. ‘‘C’’ indicates concavities where denseyellow/orange birefringence contrasted with ‘‘W,’’ regions of woven green birefringence overlying convex scaffold surfaces.(H, J) Picrosirius-red-stained section depicting a scaffold concavity from a bone formation assay after 8 weeks in vivo withnewly formed bone ‘‘b’’ deposited on the scaffold ‘‘ha.’’ (I, K) Picrosirius-red-stained section depicting a scaffold concavityfrom a bone formation assay after 8 weeks ex vivo. Scale bar¼ 100 mm. HA, hydroxyapatite; TCP, tricalcium phosphate.

3D HUMAN MESENCHYMAL STEM OSTEOSPHEROIDS 2337

matrix and improved in vivo mimicry relative to 2D sys-tems.20 3D spheroid culture also downregulated genesmaintaining hMSC self-renewal.21 Corroborating these find-ings our 3D culture platform presents further advantages.Our careful protocol with uniform immortalized hMSCstrains robustly generated highly reproducible 3D hMSCmulticellular aggregates, suitable for a screening platform.While the use of cell lines for any screening purposes is usefulfor their high level of reproducibility, one must take intoaccount that the biological effects of the immortalizing agentmay modulate the responses of the cells to differentiationagents as well as the tendency of cell lines for phenotypicdrift. In our hands, stringently maintained cultures of hMSC-TERT cells expressed all surface markers of primary hMSC,15

and even extensively expanded single-cell-derived cloneshave shown excellent bone-forming potential in vivo.4 How-ever, the reproducibility and consistency of multicellularaggregates need to be confirmed in primary hMSC obtainedfrom different donors.

The uniform kinetics of lineage biomarkers expression,often difficult to detect in 2D cultures, showed coordinatedtime-dependent commitment from multipotentiality to oste-ogenic differentiation. Since histology did not show foci ofdifferent lineage within the osteospheroid and multilineagegene expression observed in single stromal cells,22 our datasupported the hierarchical model whereby hMSC transiently

express trilineage biomarkers upon initiation of the hMSCdifferentiation program.23 Subsequent prompt loss of adipo-genic lineage markers would be expected given the inverserelationship between osteoblast and adipocyte differentiaton24

and conflict between PPAR-g and RUNX2 expression.25

The 3D osteospheroid culture model exhibited featuresthat mimic intramembranous ossificiation, an essential pro-cess during the healing of bone fractures. This involves bonesynthesis without the mediation of a cartilage phase, withmesenchymal cells differentiating along a preosteoblast toosteoblast pathway. Despite transient expression of chon-drocyte biomarker genes, we did not observe the formationof cartilage tissue or alcian-blue-positive chondrocytic cells(data not shown). The woven collagen fiber organization inthe core of 3D spheroids in OIM surrounded by a corticallayer of cells with more mature collagen fibers resembled thein vivo patterning of bone matrix collagen from an initialwoven to an eventual lamellar orientation, characteristics ofintramembraneous bone formation, and required for bone todevelop its maximal strength.26

Osteogenic differentiation of 3D culture of hMSC wasenhanced by HA/b-TCP scaffold. HA is the predomi-nant calcium phosphate analogue in bone tissue, providingmechanical strength. Scaffolds made of this material arebioactive in vivo, since an ion exchange reaction with sur-rounding body fluids forms a biologically active carbonate

FIG. 5. Expression of osteogenic and stem-cell-specific genes in con-cavities of MSCs seeded on HA/TCP. Immunostaining was performedon cells from 8-week-old in vivo implants (A, D, G, J, M, and P); cellsdifferentiated ex vivowith osteogenic induction factors (OIM) for 3 weeks(B, E, H, K, N, andQ); and cells grown ex vivo in MM for 3 weeks (C, F, I,L, O, and R). Samples were stained for biglycan (A–C), osteonectin (D–F), RUNX2 (G–I), TAZ (J–L), CD146 (M–O), and phospho-AKT (P–R).(A–R) Scale bar¼ 100 mm. (S) QRT-PCR fold induction of transcriptionfactors RUNX2, MSX-2, and DLX-5 for cells grown for 21 days in OIMversus MM without HA/b-TCP (unshaded columns) compared to cellsgrown on HA/b-TCP scaffold (shaded columns). *p< 0.05.

2338 BURNS ET AL.

apatite surface layer, equivalent to the mineral phase in bone.In addition, b-TCP serves as a key component for advancingcollagen and mineral deposition in cells grown on scaffoldsex vivo.13 We observed modest yet significant increased ex-pression of Runx2, Dlx5, andMsx2 in osteospheres with HA/

b-TCP versus 3D spheroids without scaffold. Notably, up-regulation of the osteogenic transcription factors was de-pendent on osteoinductive factors in the medium; thus, theHA/b-TCP scaffold was osteoconductive rather than inher-ently osteoinductive. Failure to find a significantly enhanced

FIG. 6. Collagen birefringence correlated with bone formation in CF1, low bone-forming, and DD8, high bone-forming,hMSC-TERT clones. (A–L) Sections of osteospheroids stained with picrosirius red and viewed (A, D, G, J) under bright fieldillumination or (B, E, H, K) under circular polarized light. (C, F, I, L) Polarized light images after software processing ofsegmentation for quantitative assessment of birefringence (red) versus tissue matrix (green). (M) Histogram of % birefrin-gence for hMSC-TERT4 clone osteospheroids grown in MM (unshaded bars) or OIM (shaded bars) for 14 days. (N–S) Mosaicimage of a whole section of HA/b-TCP scaffold and hMSC cells after implantation for 8 weeks in an immunocompromisedmouse, stained for (N, Q) hematoxylin and eosin, viewed under bright field illumination, and (O, R) picrosirius red, viewedunder circular polarized light. (P, S) Sections corresponding to (O) and (R), respectively, after software processing ofsegmentation for quantitative assessment of birefringence (red) versus tissue matrix (green). (T) Histogram of % bone matrixcalculated from segmentation of bone matrix versus tissue matrix areas. (U) Histogram of % birefringence calculated fromsegmentation of birefringent areas versus tissue matrix. (V) Linear regression curve of % bone formation versus % bire-fringence for both high and low bone-forming clones. Scale bar¼ 100 mm. *p< 0.05.

3D HUMAN MESENCHYMAL STEM OSTEOSPHEROIDS 2339

production of ALP, COL1a2, ON, or OCNmay reflect that 3Dculture per se may already elevate their expression.

Osteospheroids in OIM recapitulated a broad range offeatures important for bone formation. The principal osteo-genic transcription factor RUNX2 has roles in cell prolifera-tion and differentiation. Thus, it was expressed in scaffoldspheroids fed MM as well as osteospheroids induced to dif-ferentiate by OIM conditions. A more stringently specificbiomarker for osteogenic differentiation was the Runx2binding-transcription coactivator TAZ.27 In a pivotal manner,TAZ activates Runx2 but represses PPARg favoring commit-ment to an osteogenic versus adipogenic cell fate. TAZmRNA

and protein levels increase substantially asMSCs are stimulatedto undergo osteoblast differentiation.28 TAZ expression in theconcavities of the scaffold in vivo and ex vivo highlighted thatscaffold topology greatly influences hMSC differentiation po-tential,29 through induction of specific signaling pathways.30

The importance of concavities as niches for osteogenesis washighlighted by Ser473 phospho-AKT. This phosphorylationcan be evoked by at least three anabolic stimuli: mechanicalloading and sheer stress,31 activation of the parathyroid hor-mone (PTH) receptor,32 and response to the hormonally activemetabolite of vitamin D3.

33 Given the topologically restrictedexpression of Ser473 phospho-AKT-positive cells to a zone

FIG. 7. Gene expression of matrix proteins presented as expression relative to b-Actin, in low versus high bone-forming hMSC-TERT osteospheroids grown in MM(unshaded bars) or OIM (shaded bars) for 14 days. (A) Collagen type Ia1, (B)collagen 12A1, (C) biglycan, (D) decorin, (E) lumican, (F) elastin, (G) MFAP2, (H)MFAP5, (I) periostin, and (J) tetranectin. *p< 0.05, **p< 0.01, ***p< 0.001. MFAP,microfibrillar-associated proteins.

2340 BURNS ET AL.

adjacent to the HA/b-TCP scaffold, mechanical signals pre-sumably integrate with hormonal signals and extent of oste-oblast differentiation to determine AKT activation. Notably,osteoblasts of Akt1%/% mice showed increased susceptibilityto apoptosis plus suppressed differentiation and function.34

Collagen birefringence, enhanced in osteospheroids versus3D spheroid culture without scaffold, was not observed inmonolayer cultures of hMSC-TERT cells (data not shown),substantiating reports that 3D cultures of murine osteoblastsformed a well-organized bone matrix that was not obtainedwith traditional 2D cultures.35 The anionic dye picrosiriusred reacts avidly and stoichiometrically with the many basicamino acids in collagen, thereby enhancing its normal bire-fringence.36 Different interstitial collagens have distinct pat-terns of physical aggregation, influencing the color hueviewed with the polarized light, which changes from greento yellow to orange to red as the fiber thickness increases.37

Thus, thinner fibers, for example, newly formed fibers orthose belonging to collagen type III, show a weaker greenbirefringence.38 Advantageously, analysis of birefringenceunder polarized light provided a robust and cost-effectivemethod for assessing osteoblast differentiation that did notdepend on the performance of biological reagents such asantibodies or nucleotide sequences.

Consistent with observations concerning collagen birefrin-gence, close correlation for gene expression of select matrixproteins and bone formation4 was clearly reproduced in 3Dcultures—in particular, osteospheroid ex vivo cultures incorpo-rating the same HA/b-TCP scaffold as used in vivo. Decorin,biglycan, and lumican directly interact with collagen fibrils andHA39 maintaining collagen fibril structure and geometry40 toensure that bone formation is restricted to appropriate sites. Inaddition, microfibrils and their associated proteins, MAGP1and MAGP2, collectively provide regulatory functions that in-fluence type I procollagen protein stability, posttranscription-ally increasing the amount of COL1 in the matrix.41

A paucity of standardized and reproducible realisticex vivo models for systematically comparing bioactive ma-terials reflects the challenge of mimicking the natural 3Dmicroenvironment. This includes the combined interactionsbetween cell types and concurrently active biochemical fac-tors. We show that despite some ultimate differences be-tween in vivo and ex vivo situations, in particular the absenceof a fully mineralized true bone matrix in culture, ex vivohMSC can independently differentiate and show appropriateresponses to the osteoconductive scaffold without requir-ing a contribution from other cell types, for example, non-resorbing osteoclasts.42 Quantitative measurement of ex vivobirefringence after just 2 weeks together with RT-PCR anal-ysis of a subset of matrix proteins was predictive for thebone-forming potential in heterotopic xenograft bone for-mation assays after 8 weeks. Thus, we identified 3D culture-specific birefringence and expression of matrix proteinsgenes as physiologically relevant parameters, measurablewithin the context of a multiwell platform useful for ex vivoscreens that aim to improve tissue-engineered bone.

Acknowledgments

Lone Christiansen kindly provided excellent technical as-sistance in the preparation of histological sections. This workwas support by grants from EU FP6 Osteocord, the Danish

Medical Research Council, Danish Centre for Stem Cell Re-search (DASC), the Velux Foundation, and the InnovationConsortia program, Biomimetic 3D structures for tissue re-generation ‘‘3D Scaffolds,’’ Danish Ministry of Science,Technology and Innovation.

Disclosure Statement

All authors declare that no competing financial interests exist.

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Address correspondence to:Moustapha Kassem, M.D., Ph.D., D.Sci.

Laboratory for Molecular Endocrinology (KMEB)Department of Endocrinology and Metabolism

Odense University Hospital and Medical Biotechnology CenterWinsløwparken 25.1DK-5000 Odense C

Denmark

E-mail: mkassem@health.sdu.dk

Received: November 14, 2009Accepted: March 2, 2010

Online Publication Date: April 28, 2010

2342 BURNS ET AL.