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Biomaterials 31 (2010) 6968e6980

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Biomaterials

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Cartilage repair using hyaluronan hydrogel-encapsulated human embryonicstem cell-derived chondrogenic cells

Wei Seong Toh a, Eng Hin Lee b, Xi-Min Guo b,c, Jerry K.Y. Chan d,e, Chen Hua Yeow f,Andre B. Choo f,g, Tong Cao a,*

a Stem Cell Laboratory, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083, SingaporebDepartment of Orthopaedic Surgery, Yong Loo Lin School of Medicine, NUS Tissue Engineering Program, National University of Singapore, 27 Medical Dr, Singapore 117510, SingaporecDepartment of Tissue Engineering & Regenerative Medicine, Beijing Institute of Basic Medical Sciences, 27 Tianping Road, Beijing 100850, PR Chinad Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore,5 Lower Kent Ridge Road, Singapore 119074, SingaporeeDepartment of Reproductive Medicine, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899, SingaporefDivision of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574, SingaporegBioprocessing Technology Institute, 20 Biopolis Way, Centros, #06-01, Singapore 138668, Singapore

a r t i c l e i n f o

Article history:Received 11 March 2010Accepted 25 May 2010Available online 17 June 2010

Keywords:Human embryonic stem cellsChondrogenicCartilageDifferentiationHyaluronanHydrogel

* Corresponding author. Tel.: þ65 65164630; fax: þE-mail address: omscaot@nus.edu.sg (T. Cao).

0142-9612/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.biomaterials.2010.05.064

a b s t r a c t

Human embryonic stem cells (hESCs) have the potential to offer a virtually unlimited source of chon-drogenic cells for use in cartilage repair and regeneration. We have recently shown that expandablechondrogenic cells can be derived from hESCs under selective growth factor-responsive conditions. Inthis study, we explore the potential of these hESC-derived chondrogenic cells to produce an extracellularmatrix (ECM)-enriched cartilaginous tissue construct when cultured in hyaluronic acid (HA)-basedhydrogel, and further investigated the long-term reparative ability of the resulting hESC-derived chon-drogenic cell-engineered cartilage (HCCEC) in an osteochondral defect model. We hypothesized thatHCCEC can provide a functional template capable of undergoing orderly remodeling during the repair ofcritical-sized osteochondral defects (1.5 mm in diameter, 1 mm depth into the subchondral bone) in a ratmodel. In the process of repair, we observed an orderly spatial-temporal remodeling of HCCEC over 12weeks into osteochondral tissue, with characteristic architectural features including a hyaline-likeneocartilage layer with good surface regularity and complete integration with the adjacent host cartilageand a regenerated subchondral bone. By 12 weeks, the HCCEC-regenerated osteochondral tissueresembled closely that of age-matched unoperated native control, while only fibrous tissue filled in thecontrol defects which left empty or treated with hydrogel alone. Here we demonstrate that transplantedhESC-derived chondrogenic cells maintain long-term viability with no evidence of tumorigenicity,providing a safe, highly-efficient and practical strategy of applying hESCs for cartilage tissue engineering.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The poor regenerative and reparative ability of articular carti-lage following injury or degenerative diseases makes this tissuea key target for cell-based therapy and tissue engineering [1,2].

Current strategies for articular cartilage repair have included theuse of expanded autologous chondrocytes injected to the lesionunderneath a periosteal flap, or attached to a scaffold support [1].Biocompatibility and functionality of these expanded chondrocyteshave been extensively studied and demonstrated on several natural

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and synthetic polymeric materials such as collagen, alginate, pol-yglycolic acid and polylactic acid [3e6]. Similar studies have alsobeen carried out on biomaterials such as hyaluronan-basedhydrogels (Glycosil�) andmeshes (Hyaff-11), which are derivativesof hyaluronan, a naturally occurring glycosaminoglycan andessential component of the cartilage extracellular matrix (ECM)[7e9].

Primary explanted chondrocytes de-differentiate in monolayerexpansion, compromising their utility for articular cartilage repairprocess [10]. Identification of a better cell source for cartilage repairis thus needed.

Human embryonic stem cells (hESCs) represent a promising cellsource for regenerative medicine because of their unlimited self-renewal capacity and ability to differentiate into various somatic

Table 1List of growth factor conditions.

Conditions Medium components and growth factors

Control Chondrogenic medium (CM) e DMEM-HG supplementedwith 1% ITSþ, 40 mg/ml L-proline, 1% sodium pyruvate, 1%non-essential amino acids, 50 mg/ml ascorbic acid 2-phosphate, 10�7 M dexamethasone and 100U/100 mgpenicillin/streptomycin

IGF1 CM þ IGF1 (100 ng/ml)BMP2 CM þ BMP2 (100 ng/ml)BMP7 CM þ BMP7 (100 ng/ml)GDF5 CM þ GDF5 (100 ng/ml)TGFb1 CM þ TGFb1 (10 ng/ml)IGF1/TGFb1 CM þ IGF1 (100 ng/ml) þ TGFb1 (10 ng/ml)BMP2/TGFb1 CM þ BMP2 (100 ng/ml) þ TGFb1 (10 ng/ml)BMP7/TGFb1 CM þ BMP7 (100 ng/ml) þ TGFb1 (10 ng/ml)GDF5/TGFb1 CM þ GDF5 (100 ng/ml) þ TGFb1 (10 ng/ml)

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cell lineages [11,12]. However, one of the challenges in this field is tounderstand, control and develop a stable and efficient culturemilieu for directing differentiation of hESCs into a specific lineage[13]. Several groups have reported chondrogenic differentiation ofhESCs by various means including three-dimensional (3D) culturewith growth factor induction using BMP2 and/or TGFb1 [14,15], co-culture with bovine chondrocytes [16], and biophysical inductioninvolving hypoxic and mechanical stimulations [17,18].

We have recently demonstrated that culturing hESCs in a high-density microenvironment, coupled with TGFb1 stimulation,results in its direct differentiation to a chondrogenic lineage [14,15].Further cultivation of these putative hESC-derived chondrogeniccells in defined selective growth factor combination (TGFb1, FGF2and PDGF-bb) enable hESC-derived chondrogenic cell lines to bereproducibly isolated and expanded up to 16 population doublingswhile retaining their chondrogenic potential for cartilaginoustissue formation [15]. However, the potential of hESC-derivedchondrogenic cells for cartilage tissue engineering and regenera-tion have yet to be demonstrated and examined functionally ina clinical-relevant critical-sized osteochondral defect, where theinfluence of mechanical loading, tissue injury and inflammatoryresponse play a part in the healing process. Here, we examined thepotential of hESC-derived chondrogenic cells to produce ECM-enriched cartilaginous construct when cultured in hyaluronic acid(HA)-based hydrogel (Glycosil�), and further investigated thelong-term reparative ability of the resulting hESC-derived chon-drogenic cell-engineered cartilage (HCCEC) in an osteochondraldefect model.

2. Materials and methods

2.1. Cell culture

The NIH-registered H9 cell line, isolated and established at the University ofWisconsin, was used in this study (Wicell Agreement No. 04-W094) [11]. HumanESC-derived chondrogenic cells were isolated from hESCs as previously described[15]. Briefly, hESCs were cultured as embryoid bodies for 5 days prior to dissociationinto single cells and cultured as high-density micromass cultures in the presence ofTGFb1 to induce chondrogenic lineage differentiation for up to 21 days. Followingdifferentiation, hESC-derived chondrogenic cells were reproducibly isolated bymeans of collagenase P (Roche Diagnostics GmbH, Mannheim, Germany) treatmentand selective plating on Type II collagen-coated plates, followed by expansion indefined growth factor conditions that comprised of Dulbecco’s modified Eagle’smedium high glucose (DMEM-HG, Invitrogen, USA) containing 10% fetal bovineserum (FBS, HyClone, USA), 0.1 mM non-essential acids (Invitrogen), 1 mM sodiumpyruvate (Sigma), 100U/100 mg penicillin/streptomycin (Invitrogen) with the addi-tion of TFP growth factors [1 ng/ml TGFb1 (R&D Systems), 5 ng/ml FGF2 (Invitrogen)and 10 ng/ml PDGF-bb (Peprotech, Rocky Hill, NJ, USA). These growth factors wereselected based on their reported capacity to enhance chondrogenic potential andpost-expansion differentiation ability of human chondrocytes as well as hESC-derived chondrogenic cells [15,19].

2.2. Differentiation assays

Human ESC-derived chondrogenic cells were expanded as monolayer culturesup to 5 passages (z9.4 population doublings) before subjected to two differentmodels (I & II) for assessment of chondrocytic differentiation.

2.2.1. Model I: 3D pellet culturesIn order to optimize the growth factor conditions for cartilage tissue engi-

neering, the conventional pellet system was set up to screen the effects of growthfactors on chondrogenic differentiation of hESC-derived chondrogenic cells [20].Aliquots of 2�105 cells/0.5 mlwere spun down at 1100 rpm for 5min to form pelletsand cultured in the serum-free chondrogenic medium without (Control) or withgrowth factor supplementation for 2weeks. Basic serum-free chondrogenicmediumconsisted of DMEM-HG (Invitrogen) supplemented with 1% ITSþ (BD Bioscience),40 mg/ml L-proline (Sigma), 1% sodium pyruvate (Sigma), 1% non-essential aminoacids (Invitrogen), 50 mg/ml ascorbic acid 2-phosphate (Sigma), 10�7 M dexameth-asone (Sigma) and 100U/100 mg penicillin/streptomycin (Invitrogen)[21]. Thegrowth factors included IGF1, BMP2, BMP7 and GDF5 applied at 100 ng/ml and/orTGFb1 applied at 10 ng/ml in a singly or combined fashion as described in Table 1.

2.2.2. Model II: 3D HA-based hydrogel culturesFor HCCEC construction, HA hydrogel was used for cell encapsulation and

prepared as per manufacturer’s protocol (Glycosan Biosystems, Salt Lake City, UT,USA). Briefly, hESC-derived chondrogenic cells were mixed gently with 2:2:1 ratio ofthiol-modified hyaluronic acid (Glycosil), thiol-modified gelatin (Gelin-S) andPEGDA, polyethylene glycol diacrylate (Extralink) to form constructs of cell density2 � 106 cells/100 ml hydrogel in 24-well transwell cultures. The cell-hydrogelconstructs were cultured for up to 4 weeks in both control and BMP7/TGFb1 sup-plemented chondrogenic medium, with medium changes thrice a week. Thecombination of BMP7 and TGFb1 was chosen, based on our testing results fromModel I. The constructs were harvested at 2 and 4 weeks for histology, immuno-histochemistry and biochemical analyses.

2.3. Animal models

Human ESC-derived chondrogenic cells encapsulated in HA hydrogel were pre-differentiated in the presence of BMP7/TGFb1 for 4 weeks to produce ECM-enrichedcartilaginous construct. Long-term reparative ability and tumorigenicity of theresulting hESC-derived chondrogenic cell-engineered cartilage (HCCEC) wereinvestigated in both orthotopic and ectopic animal models. Animal experimentswere performed in accordance with Institutional Animal Care and Use Committee(IACUC) of National University of Singapore.

2.3.1. Model I: osteochondral defect modelForty hind limbs of skeletally mature male Sprague Dawley rats weighing

250e300 g were used. Osteochondral defects (1.5 mm diameter and 1 mm depth)were created on the trochlear grooves of the distal femurs of these rats [15]. Usinga biopsy punch, HCCEC constructs (z5 � 105 cells) were cut out and press-fit intothe osteochondral defects. Defects left empty or implanted with HA hydrogel servedas controls. The rats were given daily subcutaneous injection of Cyclosporine A,Sandimmune (Novartis, Basel, Switzerland) at 14 mg/kg body mass to preventimmune rejection of the xenogenic human cells implanted. At designated time-points, animals were euthanized and distal femora were resected en bloc and pro-cessed for histological analysis. The histologic grading scale as described byWakitani[22,23] was used to evaluate the quality of the repaired tissue (Table 2) by threeblinded independent researchers.

2.3.2. Model II: subcutaneous ectopic modelHCCEC constructs were implanted subcutaneously in 5 week-old male Severely

Combined Immunodeficiency (SCID) mice to assess tumorigenicity. After 12 weeks,mice were euthanized and explants processed for histological analysis (n ¼ 6).

2.4. Histology and immunohistochemistry

For histological analysis, samples were fixed in 10% neutral buffered formalin(Sigma) overnight and embedded into paraffin. Sections were cut at 5 mm, depar-affinized and stained with Haematoxylin and Eosin (HE), Masson’s Trichrome (MT),Safranin-O (Saf-O), Alcian blue (AB) following the standard procedures as previouslydescribed [21]. Immunohistochemistry to detect Type I, II and X collagens wascarried out following the standard protocol [21]. Rat-specific ED1 and CD8 anti-bodies (Serotec, Oxford, UK) were used to investigate the infiltration of rat macro-phages (ED1) and T cells (CD8) in rat tissue, as previously described [24].

2.5. Human cell chimerism

Human-specific vimentin (hVIM) immunostaining to detect human cellchimerism was carried out following the procedures as previously described [24].Cell nuclei with cytoplasmic staining of hVIM were counted as human cells against

Table 2Histological grading scale for cartilage repair.

Category Points

Cell morphologyHyaline cartilage 0Mostly hyaline cartilage 1Mostly fibrocartilage 2Mostly non-cartilage 3Non-cartilage only 4

Matrix-staining (metachromasia)Normal (compared with host adjacent cartilage) 0Slightly reduced 1Markedly reduced 2No metachromatic stain 3

Surface regularitya

Smooth (>3/4) 0Moderate (>1/2e3/4) 1Irregular (1/4e1/2) 2Severely irregular (<1/4) 3

Thickness of cartilageb

>2/3 01/3e1/2 1<1/3 2

Integration of donor with host adjacent cartilageBoth edges integrated 0One edge integrated 1Neither edge integrated 2

Total maximum 14

a Total smooth area of the reparative cartilage compared with the entire area ofthe cartilage defect.

b Average thickness of the reparative cartilage compared with that of thesurrounding cartilage.

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cell nuclei without cytoplasmic stain as the rat cells. The number of human and ratcells within the regenerated cartilage layer was divided into centre and peripheralzones, and enumerated manually at 20� magnification to estimate the level ofchimerism of human cells. At least 2 sections from each sample were analyzed(n ¼ 16). A median of 481 cells was counted for each sample (range 234e741).

2.6. Biochemical analysis

Samples were digested as previously described [25]. Sulfated Glycosamino-glycan (s-GAG) content was measured using Biocolor Blyscan GlycosaminoglycanAssay Kit (Biocolor Ltd, Newtownabbey, Ireland). Type II collagen was measuredusing the Type II Collagen Detection Kit (Chondrex Inc., Redmond, WA, USA). Fornormalization, DNA content was measured spectrophotometrically using theHoechst 33 258 method [26]. Standard curves of s-GAG and Type II collagen wereconstructed using different concentrations of bovine trachea chondroitin sulfate andType II collagen respectively. Calf thymus DNA was used for construction of thestandard curve for DNA quantification.

2.7. Micro-computational tomography (MicroCT)

The samples were analyzed using the MicroCT scanner (SMX-100CT X-ray CTSys, Shimadzu, Japan). The scan settings were: X-ray voltage ¼ 56 kV, X-raycurrent ¼ 61 mA, detector size ¼ 9”, scaling coefficient ¼ 50 and voxelresolution ¼ 0.012 mm. The scans were then reconstructed to create the 3Dgeometry using VGStudioMax (Version 1.2, Volume Graphics, Germany). To estimatethe bone and cartilage content of the repair site, we set a 1.9 mm � 0.8 mm rect-angular region-of-interest (ROI) encompassing the desired region in the micro-CTslices for each specimen using CTAnalyser (v1.9, Skyscan, Belgium). The bone andcartilage portions were then segmented using the thresholding function, wherebywe noted that the threshold ranges of 167e255 and 154e166 were appropriate fordemarcating the bone and cartilage regions respectively in themicro-CT images, andfulfilled the requirement of bordering the individual histogram signal intensitypeaks exhibited by the cartilage and bone [27]. Thereafter, their respective 3Dvolumes were computed from the segmented ROI in each micro-CT slice. At least 2samples were analyzed at each time-point of harvest.

2.8. Statistical analysis

All quantitative data reported here was analyzed using Student’s T-test oranalysis of variance (one-way ANOVA) followed by Tukey’s post-hoc test as appli-cable. A p-value of less than 0.05 was considered statistically significant. For in vitro

experiments, eachmeasurement reportedwas based on duplicate analysis of at leastthree independent experiments.

3. Results

3.1. Derivation and differentiation of hESC-derivedchondrogenic cells

Human ESC-derived chondrogenic cells demonstrated chon-drogenic differentiation and cartilaginous tissue formation,a normal karyotype and normal somatic cell cycle kinetics, as wellas absence of teratoma formationwhen injected intramuscularly inthighs of a SCID mouse model, as previous reported [15].

We tested the effects of growth factors such as TGFs, BMPs andIGFs on chondrogenic differentiation of hESC-derived chondrogeniccells in the pellet system either in a singly or combinatorial fashion.From our screening, the combination of BMP7 and TGFb1 hassynergistic effects in stimulating maximum chondrogenesis, asobserved by the highest level of s-GAG synthesis by day 14 ofdifferentiation over other growth factors (Fig. 1A).

To create a cartilaginous tissue of clinical-relevant size, a tissueengineering approach of hydrogel-encapsulation of hESC-derivedchondrogenic cells, coupled with the optimized growth factorinduction (BMP7/TGFb1), was further investigated. Whencompared to the conventional pellet system under parallel BMP/TGFb1 treatment, the hydrogel system demonstrated superiorchondrogenesis, with a remarkable 4.4-fold increase in s-GAG/DNA(p< 0.05) and a 5.9-fold increase in Type II collagen/DNA (p< 0.05)by day 14 of differentiation (Fig. 1B).

To further evaluate BMP7/TGFb1-induced chondrogenic differ-entiation of hESC-derived chondrogenic cells in the 3D HA hydrogelsystem, quantitative assessments of matrix synthesis (s-GAG andType II collagen) were carried out at day 14 and 28 of differentia-tion. The resultant hESC-derived chondrogenic cell-engineeredcartilage (HCCEC) constructs at the end of 28-day differentiationunder BMP7/TGFb1 treatment were also evaluated by the end-point histological assessment.

Quantitative assessment of matrix synthesis indicated signifi-cant increase in s-GAG and Type II collagen in the presence ofBMP7/TGFb1 treatment, as compared to the untreated control. Byday 28 of differentiation, BMP7/TGFb1 induction resulted ina striking 33.5-fold increase in s-GAG/DNA (p < 0.001) and a 5.8-fold increase in Type II collagen/DNA (p < 0.001) over the control(Fig. 1C and D).

During the course of BMP7/TGFb1 treatment, HCCEC demon-strated characteristic time-dependent patterns of matrix synthesis.There was robust increase in s-GAG content between day 0 and 14(2.2 � 0.1 to 41.7 � 10.9 mg/mg s-GAG/DNA, p < 0.01), before pla-teauing between day 14 and 28 (Fig. 1C). On the other hand, therewas a relatively slower onset in Type II collagen synthesis, with lowlevels at day 14, but showed a significant increase between day 14and 28 (30.3 � 16.5 to 78.9 � 25.7 ng/mg Type II collagen/DNA,p < 0.05, Fig. 1D).

Consistent with the quantitative assessment of matrix synthesis,HCCEC exhibited morphological similarities to neocartilage withround cells embedded in lacunae, and appeared hyaline-like, withhigh amounts of s-GAG and Type II collagen, low amount of Type Icollagen and almost no Type X collagen by the end of 4-weekinduction (Fig. 1E).

3.2. Cartilage regeneration in osteochondral defect

We assessed the cartilage regenerative capability of HCCEC afterorthotopic transplantation into a critical-sized osteochondraldefect bymeans of gross morphology examination, histological and

Fig. 1. Growth factor induction and tissue engineering of hESC-derived chondrogenic cells (A) Determination of optimal growth factor combination for in vitro chondrogenicdifferentiation of hESC-derived chondrogenic cells. Combination of BMP7/TGFb1 demonstrated synergistic effects of stimulating maximum chondrogenesis, indicated by highestlevel of s-GAG synthesis by day 14 of differentiation. (B) Comparison of 3D HA hydrogel systemwith the pellet system. Superior chondrogenesis indicated by significant higher levelsof s-GAG and Type II collagen was observed in the hydrogel system. (*p < 0.05, n ¼ 3) Quantitative analysis of (C) s-GAG and (D) Type II collagen indicated significant increase incartilage matrix protein synthesis in the presence of BMP7/TGFb1 treatment (*p < 0.05; ***p < 0.001 compared to control, n ¼ 3) (E) Histological assessment of hESC-derivedchondrogenic cell-engineered cartilage (HCCEC) using Haematoxylin and Eosin (HE) and Alcian Blue (AB), and immunohistochemical staining specific for Type II (COL II), Type I (COLI) and Type X (COL X) collagens. Human origin of HCCEC was further verified by immunohistochemical staining of human-specific vimentin (hVIM).

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immunohistochemical evaluation, semi-quantitative histologicalscoring analysis, as well as quantitative micro-CT evaluation ofcartilage and bone volume.

Macroscopically, all defects appeared depressed and irregularwith poor integration with the host cartilage at 2 weeks post-operatively (Fig. 2). By 6 weeks, the entire HCCEC-treated defect

appearedwith neo-tissue graft of moderately flat surface regularity.In contrast, the control group with empty defect displayedincomplete neo-tissue coverage with depressed and irregulararticular surface, while HA-treated group displayed moderate neo-tissue coverage and surface regularity (Fig. 3). At 12 weeks, theHCCEC-treated defect displayed good surface regularity and

Fig. 2. In vivo cartilage repair at 2 weeks post-implantation. Osteochondral defects (1.5 mm in diameter and 1 mm depth) were created at the trochlear grooves at both knees ofSprague Dawley rats. Experimental group (Human ESC-derived chondrogenic cell-engineered cartilage, HCCEC) was compared against the control groups either with defects leftempty or implanted with the scaffold alone (HA hydrogel). The rats were euthanized at 2 weeks post-implantation and observed by macroscopic examination of the grossappearance (GA), as well as histological analysis using HE and Saf-O, and immunohistochemical staining specific for COL II, COL I and COL X. At 2 weeks, experimental groupimplanted with HCCEC showed complete filling of the defect, with partial integration with the adjacent host cartilage. Control groups either implanted with HA hydrogel or leftempty were filled with fibrous tissue. Grafted sites are indicated by the concave open boxes. Scale bar: 500 mm.

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appeared close morphological similarities to the age-matchednative unoperated control. In contrast, the control groups leftempty or treated with HA displayed complete neo-tissue coverage,but with irregular articular surfaces (Fig. 4).

Histologically, 2 weeks after transplantation of HCCEC, thecompact engineered cartilage implant occupied the entire osteo-chondral defect, and expressed high amounts of s-GAG and colla-gens including high amounts of Type I and II collagens, but no Type

W.S. Toh et al. / Biomaterials 31 (2010) 6968e6980 6973

X collagen. 2 out of 6 implants demonstrated partial integration,while the remaining 4 implants demonstrated poor integration. Incontrast, defects left empty or transplanted with HA alone resultedin limited repair, with negligible amount of s-GAG and Type IIcollagen. At 2 weeks, the overall histological scores for group I

Fig. 3. In vivo cartilage repair at 6 weeks post-implantation. The rats were euthanized aappearance (GA), as well as histological analysis using HE and Saf-O, and immunohistochemrepair in the control groups. In the HCCEC-treated group, HCCEC implant regenerated a mintegration to the adjacent host cartilage, but no obvious tidemark was observed. There wasType X collagen indicative of hypertrophy was mainly observed at the lower half of the im

(Empty), II (HA alone) and III (HCCEC) were 13.6, 13.7 and 5.4respectively (p < 0.001 compared to group I (Empty) and II (HA),Figs. 2 and 5).

By 6 weeks, there was mostly fibrous repair in both controlgroups, with severe surface irregularity and noticeable bone in-

t 6 weeks post-implantation and observed by macroscopic examination of the grossical staining specific for COL II, COL I and COL X. At 6 weeks, there were mostly fibrousoderately flat cartilage surface with comparable matrix staining and good structurallimited amount of Type I collagen distributed at the periphery surrounding the implant.plant in the deep zone of the subchondral bone. Scale bar: 500 mm.

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Fig. 5. Histological scoring for reparative tissues. The reparative tissues were evaluatedfor cell morphology, matrix-staining, surface regularity, thickness of cartilage andintegration of donor with host adjacent cartilage. The repair is inversely correlatedwith the score. HCCEC demonstrated significant improvement in cartilage repair, whencompared to the control groups at all time-points. (ns: not significant; *p < 0.05;**p < 0.01; ***p < 0.001) There was also significant improvement in scores of HCCEC at6 weeks (#p < 0.05) and 12 weeks (##p < 0.01) when compared to 2 weeks post-transplantation.

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growth in the empty defects. Some chondrocytic cells could beobserved in the HA-treated defects as observed by Safranin-Ostaining. In the HCCEC-treated group, HCCEC implant regenerateda smooth cartilage surface with comparable matrix staining to theadjacent host cartilage. These HCCEC repaired tissues exhibiteduniform staining for s-GAG but with relatively lower intensity ofType II collagen, when compared to the results at 2 weeks. Therewas limited amount of Type I collagen present in chondrocytic cells,distributed primarily at the periphery surrounding the implant.Hypertrophic chondrocytes that exhibited pericellular staining forType X collagen were mainly observed at the lower half of theimplant in the deep zone of the subchondral bone. 2 out of 4implants demonstrated partial integration to one end of hostcartilage, while the remaining 2 implants demonstrated poorintegration. At 6 weeks, the overall histological scores for group I(Empty), II (HA alone) and III (HCCEC) were 13.0, 9.1 and 2.8respectively (p < 0.001; p < 0.01 compared to Group I (Empty) andII (HA alone) respectively, Figs. 3 and 5).

At 12 weeks post-operation, there were only fibrous tissuerepair found in the control groups, with little observable matrixstaining. The subchondral bone in both control groups wascompletely regenerated. In the HCCEC-treated group, a new carti-lage layer bridging both ends of the host cartilage could beobserved. The regenerated cartilage layer appeared hyaline-likewith comparable matrix staining to the native cartilage andexhibited good structural integration to the adjacent host cartilage.Under high magnification view, the cells in the reparative tissueappeared spherical and clustered within lacuna. In addition, therewas almost complete subchondral bone regeneration, marked bynew bone formation sequestered within the matrix network ofhypertrophic cartilage. At 12 weeks, the overall histological scoresfor group I (Empty), II (HA alone) and III (HCCEC) were 11.5, 8.0 and

Fig. 4. In vivo cartilage repair at 12 weeks post-implantation. The rats were euthanized aappearance (GA), as well as histological analysis using HE and Saf-O, and immunohistochemtissue repair in the control groups, with little observable matrix staining. In the HCCEC-trestructural integration, could be observed. The regenerated neocartilage appeared hyaline-likview (200�), cells in the regenerated neocartilage appeared spherical and clustered with lasequestered within the matrix network of hypertrophic cartilage. Dotted lines indicate the b(right) while the arrow indicates the tidemark.

2.6 respectively (p < 0.001 and p < 0.05 compared to Group I(Empty) and II (HA) respectively, Figs. 4 and 5). 4 out of 6 implantsin the HCCEC-treated group demonstrated complete integration toboth ends of the host cartilage, while the remaining 2 demon-strated partial integration. Furthermore, there was significantimprovement in overall scores of HCCEC at 6 weeks (p < 0.05) and12 weeks (p < 0.01) when compared to 2 weeks post-trans-plantation, indicating that the engineered cartilage underwenta dynamic remodeling process over 12 weeks, enhancing itsreparative effect (Fig. 5).

These findings were consistent with the quantitative micro-CTresults, where we observed characteristic remodeling processesmarked by changes in the ratio of cartilage to bone volume of therepaired tissue (Fig. 6B,C). The percentage of cartilage and bonevolume of the HCCEC-treated defect progressed from 86.2 � 5.7%cartilage and 13.8 � 5.7% bone at 6 weeks post-implantation to30.5 � 9.1% cartilage and 69.5 � 9.1% bone at 12 weeks post-implantation. The HCCEC-regenerated osteochondral tissue at 12weeks resembled closely to the age-matched unoperated nativecontrol of 40.2 � 6.6% cartilage and 59.8 � 6.6% bone (p ¼ 0.207).

3.3. Human cell chimerism

Immunostaining with human-specific vimentin (hVIM) anti-body in this xenogeneic model revealed a high level of human cellchimerism in the regenerated cartilage layer at 2 weeks (centre:74.3 � 14.4% vs. periphery: 72.3 � 13.3%, p > 0.05), but reducedsignificantly at 6 weeks (centre: 26.6 � 11.4% vs. periphery:37.2 � 6.2%, p< 0.05), and was only detectable at very low levels by12 weeks post-transplantation (centre: 1.3 � 0.9% vs. periphery:6.7 � 5.9%, p < 0.05) (Fig. 7AeC). Double immunostaining with rat-specific ED1 and hVIM antibodies demonstrated presence of ED1positive rat macrophages at the periphery of the HCCEC implant inthe deep zone of the subchondral bone at 2 weeks, which laterinfiltrated into the implant in close occurrence with hVIM positivehuman cells at 6 weeks post-surgery. At 12 weeks, only lownumbers of host macrophages could be observed in the tidemarkregion underneath the regenerated neocartilage layer. Similardegree of host macrophage infiltration was found in animalstransplanted with acellular HA controls across all time-points(Fig. 7D). Only very low numbers of CD8 positive T cells could bedetected in both control (HA) and HCCEC-implanted defects at alltime-points (data not shown).

4. Discussion

Direct chondrogenic differentiation of ESCs is currentlyhampered by low differentiation efficiency and cellular heteroge-neity of differentiated cell populations, which poses the risk oftumorigenicity in vivo, as previously reported by us and otherresearch groups [14,28e30]. Therefore, deriving lineage-committed chondrogenic cell lines from hESCs bereft of anyundifferentiated tumorigenic hESCs has been pursued [15]. Notably,no report to date has investigated the fate of hESC-derived cells incartilage regeneration. In this study, we aimed to engineer a carti-laginous tissue construct based on hESC-derived chondrogenic cells

t 12 weeks post-implantation and observed by macroscopic examination of the grossical staining specific for COL II, COL I and COL X. At 12 weeks, there were only fibrousated group, a new cartilage layer bridging both ends of the host cartilage, with goode with comparable matrix staining to the native cartilage and under high magnificationcunae. There was also subchondral bone regeneration, marked by new bone formationoundaries between the regenerated neocartilage (left) and the adjacent host cartilage

Fig. 6. MicroCT model of HCCEC remodeling process in cartilage regeneration (A) 3D geometry of the implant site. Cross-sectional view of the implant site and its surroundingnative tissue is obtained along the line AA. (B) Cartilage (yellow) and bone growth (grey) within the HCCEC implant were isolated out in micro-CT models, and compared to thenative unoperated control. The host cartilage and bone are denoted pink and blue respectively. (C) The cartilage and bone portions of the reparative tissues were segmented usingthe thresholding function, and their respective 3D volumes were computed from the segmented ROI. Quantitative CT results observed characteristic time-dependent remodelingprocess marked by significant changes in ratio of cartilage to bone volume of the repaired tissue (ns: not significant; *p < 0.05).

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and assess the long-term reparative ability in repairing large crit-ical-sized osteochondral defects in adult rat model.

Our in vitro findings revealed that the chondrogenic efficiencyof hESC-derived chondrogenic cells is largely influenced by theinducing growth factors as well as the culture systems used. In ouroptimization study, we identified combination of BMP7/TGFb1being the potent inducers of chondrogenic differentiation of hESC-derived chondrogenic cells. A recent report also documentedsynergistic effects of BMP7 and TGFb1 in inducing chondrogenicdifferentiation of hESC-derived mesenchymal cells in a pelletculture system [31]. There has also been use of this growth factorcombination (BMP7/TGFb1) for chondrogenic induction of adultmesenchymal stem cells (MSCs) [32]. Therefore, we applied thecombination of BMP7 and TGFb1 in our study for application incartilage tissue engineering using hESC-derived chondrogenic cells.

Athough several studies reported chondrogenic differentiationof hESCs, low efficiency and cellular heterogeneity of the resultingcell populations impair the generation of homogenous cartilagi-nous graft for further applications in cartilage tissue engineeringand regenerative medicine [28e30]. Herein, we report a high effi-ciency chondrogenic system employing BMP7/TGFb1-induceddifferentiation of hESC-derived chondrogenic cells in the HAhydrogel to yield a homogenous hyaline-like cartilaginous tissue.HA-based hydrogel is selected as the cell carrier in this study due tothe widespread application of HA-based biomaterials in cartilagetissue engineering and regeneration [7,33,34]. Our protocol ofutilizing lineage-restricted chondrogenic cells for cartilage tissueengineering enables up-scalability for producing clinically relevantamounts of cartilage for tissue engineering and regenerative

medicine applications. We further demonstrated in this study thatorthotopic implantation of hESC-derived chondrogenic cell-engi-neered cartilage (HCCEC) in a rat osteochondral defect regeneratedosteochondral tissue that resembled closely that of age-matchedunoperated native control, while only fibrous tissue filled in thecontrol defects which left empty or treated with hydrogel alone.

Chondrogenic pre-induction of tissue-engineered cartilageconstructs prior to implantation has been demonstrated ina number of recent studies to have beneficial effects in cartilagerepair [3,35,36]. More recently, Chen[35] demonstrated that theduration of in vitro pre-differentiation of cartilage constructsinfluences the in vivo cartilage repair. In that study, chondrocyte-mediated cartilage constructs cultured for 3 weeks in bioreactorsinduced better in vivo cartilage repair than those cultured forshorter durations. However, there have also been concerns thatprolonged periods (>4 weeks) of cultivation of the tissue-engi-neered cartilage constructs may result in poor integration betweenthe cartilage graft and the host cartilage [37,38]. In our study,chondrogenic differentiation of the HCCEC constructs in the pres-ence of BMP7/TGFb1 growth factor conditions resulted in forma-tion of ECM-enriched cartilaginous constructs that exhibitedmorphological similarities to an neocartilage with round cellsembedded in lacunae, and appeared hyaline-like, with highamounts of s-GAG and Type II collagen, low amount of Type Icollagen and almost noType X collagen by the end of 4weeks. UponHCCEC implantation, two-thirds of the cases demonstratedcomplete integration in cartilage repair at the end of 12 weeks.Consistent with other studies [35,36], chondrogenic pre-differen-tiation induced ECM synthesis, which in turn probably improved

Fig. 7. Chimerism analysis of hESC-derived chondrogenic cells. (A) The presence and survival of hESC-derived chondrogenic cells were observed by hVIM immunostaining.Arrowheads indicate the boundaries of the neocartilage tissue and the adjacent host cartilage. (B) Schematic model to illustrate the centre (C) and periphery (P) zones of theregenerated neocartilage layer. Cells in the dotted boxes were counted. (C) Human ESC-derived chondrogenic cells demonstrated high degree of human cell chimerism in theregenerated cartilage layer at 2 weeks, but reduced drastically at 6 weeks, and only detectable at very low levels at 12 weeks post-transplantation. (D) The decline in hVIM positivehuman cells (red) was accompanied with the infiltration of ED1 positive rat macrophages (green). Rat macrophages were observed in the subchondral bone (SB) at the periphery ofthe HCCEC implant at 2 weeks, which later infiltrated into the implant at 6 weeks, and only detected at low numbers in the tidemark region underlying the regenerated neocartilagelayer (CL) at 12 weeks post-transplantation. Similar degree of infiltration was observed in the HA-treated controls. White arrows indicate regions of close occurrence of hVIMpositive human cells (red) and ED1 positive rat macrophages (green).

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W.S. Toh et al. / Biomaterials 31 (2010) 6968e69806978

the ability of the cartilage implant to withstand load-bearing at thetime of implantation, and helped in subsequent integration withthe host cartilage for a better repair.

Secure fixation of tissue-engineered cartilage constructs indefects is another critical issue for success in cartilage regenerationas it ensured good integration of engineered cartilage with the hostcartilage and subchondral bone. Preliminary findings using hESC-derived chondrogenic cells in cartilage repair indicated the limi-tations of transplantation of cells alone in the form of cell pellet,which resulted in suboptimal repair with formation of fibroustissue and little cartilage [15]. To circumvent this problem, a press-fit biomaterial-assisted strategy of implanting hESC-derivedchondrogenic cell-hydrogel construct was employed. Followingpress-fit implantation into osteochondral defects in adult rats,HCCEC demonstrated remodeling over a 12-week period into anosteochondral tissue with characteristic features includinga hyaline-like neocartilage layer with complete integrationwith theadjacent host cartilage and underlying subchondral bone. It is likelythat press-fit implantation enabled secure fixation of HCCEC in theosteochondral defect, supported close interaction between theimplant and the host cartilage, and improved eventual cartilageregeneration. The enhanced cartilage regeneration observed in vivocorrelated well with the superior chondrogenic efficiency of theHCCEC in the hydrogel system in vitro, suggesting a pivotal role inwhich the HA-based biomaterial played in cartilage regeneration.HA-based biomaterials, besides being the essential component ofcartilage ECM, also have the advantage of suitable degradation ratewhich matches the kinetics of new cartilage formation in vivo. Inthis study, residual HA was evident in the HA-treated defects at 2weeks but was completely resorbed by 6 weeks, thus implying thatthe HA hydrogel that was used as a cell carrier for construction ofHCCEC is highly biocompatible and biodegradable [7].

Defects repaired with HCCEC demonstrated a progressivedevelopment from a prechondrogenic state with synthesis of Type Iand II collagens to a hypertrophic state with synthesis of Type II andX collagens. Our findings support the hypothesis that HCCEC canprovide a functional template capable of orchestrating orderlyremodeling during the repair of critical-sized osteochondral defectsin adult rats. Remodeling process which is important for thepreservation of the cartilage layer and the regeneration of thesubchondral bone can be attributed to the local microenvironmentand the chemical and physical factors at the defect site [39]. It isimportant to note that functional restoration and survival of thecartilage layer is also largely dependent on the underlying sub-chondral bone regeneration [40,41]. In this model, we observed anorderly remodeling process where HCCEC induced a hyaline-likecartilage layer at the articular surface, with good structural inte-grationwith the host cartilage at the lateral boundaries. Within thedeep zone of vascularized subchondral bone, HCCEC induceda progressive endochondral process of ossification repair. Thissupports an ordered spatial-temporal remodeling of the osteo-chondral repair being induced by the transplanted HCCEC.

Further examination revealed good survival of human chon-drogenic cells at 6 weeks, followed by gradual decline up to 12weeks post-transplantation. By 12 weeks when the remodelingprocess has largely been completed, human cells account only for4% of all cells in the regenerated cartilage layer, while none could bedetected in the regenerated subchondral bone. Remodeling processseemed to be most active in the centre of the defect with relativelylower human cell chimerism than in the periphery. This overalldecline in human cell chimerism and survivability is in linewith theendochondral ossification process, where cells in the vascularizedmicroenvironment of the subchondral bone underwent hyper-trophy maturation, apoptosis and eventual replacement by bonecells. Another explanation may have been graft cellular apoptosis

described in many cellular transplantation paradigms even afterimmune suppression [24,42,43]. This is often associated withinfiltration of host macrophages to the injury site during repair. Inthis instance, rapid decline in human cell chimerism might havebeen due to non-specific immune reactions between species aswell as the remodeling process during osteochondral regeneration,which host macrophages are likely to be implicated in theseprocesses, as reported in several other transplantation paradigms[42,43]. Our findings highlight a key aspect of cellular trans-plantation not fully addressed in many studies of cell trans-plantation for cartilage regeneration: the host inflammatory/immune response to the xenograft and its role in remodelingprocess during cartilage and bone repair, which can only beaddressed through clinical trials.

Importantly, we showed that hESC-derived chondrogenic cellsplayed a direct pivotal role in cartilage regeneration by forminga preliminary neocartilaginous tissue within 2 weeks of trans-plantation that undergoes continuous remodeling over time tobring about regeneration of a neocartilage layer. This regenerationprocess is likely to be mediated by the implanted human cells sinceboth control groups of scaffold alone and nil-transplant resultedonly in fibrous tissue repair. We do not however exclude thepossibility of hESC-derived chondrogenic cells inducing hostcartilage regeneration via paracrine activity, as have been reportedin a number of other cell transplantation paradigms [44e46].Indeed, hESC-derived cells have been reported to secret a broadspectrum of growth factors including BMPs, TGFs, FGFs that arelikely to play important roles in recruitment and differentiation ofhost progenitors, and overall tissue regeneration [45e47].

To our knowledge, there is only one other study that has appliedhESC-derived cells in cartilage repair in an orthotopic model [48]. Inthat study, hESC-derived MSCs were differentiated under co-culture with bovine chondrocytes before implanted as cell pelletsinto the osteochondral defects in nude rats. Cartilage tissueformation could be observed, which remodeled over 8 weeks torepair the osteochondral defects. Although there was no teratomaformation, it was not clear if the regenerated tissue was indeedhyaline cartilage. Furthermore, the fate of transplanted cells wasnot investigated. By contrast, our studies have described a stepwiseapproach of generating non-tumorigenic, lineage-restricted chon-drogenic cells from hESCs under defined conditions [15], applyingtissue engineering techniques of creating engineered cartilageconstructs and demonstrating the orderly remodeling process thatthe engineered cartilage based on hESC-derived chondrogenic cellsundergoes during the repair of critical-sized osteochondral defects,and resulted in complete osteochondral regeneration, witha hyaline-like cartilage composition. In addition, our study alsoprovided strong evidence of hESC-derived chondrogenic cells andtheir role in cartilage regeneration.

Major challenges impeding clinical application of hESCs includesafety issues of tumorigenicity and immunogenicity upon trans-plantation. In our study, no teratomas were found throughout thecourse of implantation of HCCEC up to 12 weeks in the immuno-suppressed rat osteochondral defect model. As the osteochondraldefect site has been reported to be chondroinductive and preventstumor formation by mouse ESCs [49,50], we further transplantedHCCEC constructs into a subcutaneous SCID mouse model over 12weeks without any evidence of teratoma formation (data notshown). Therefore, we can conclude that HCCEC is non-tumorigenicand this property is independent of the transplantation site. Ourstrategy of utilizing lineage-restricted chondrogenic cells fromhESCs is similar to several studies where lineage-restricted cells aredifferentiated and selected from hESCs before transplantation toavoid teratoma formation [46,51,52]. Transplantation of hESCs andtheir derivatives pose the risk of immune rejection [53,54]. Recent

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studies have suggested that tissue-engineered neocartilages couldregulate host immune response by expressing factors related toimmune privilege [55,56]. Further research is now directed atextending this technology for hESCs and induced pluripotent stemcells (iPS)-derived cells [57] for cartilage repair in immunocom-petent models.

5. Conclusion

We have demonstrated a safe, highly-efficient and practicalstrategy of utilizing non-tumorigenic lineage-restricted chondro-genic cell lines derived from hESCs for cartilage tissue engineeringand regeneration. Further characterization of in vivo outcomes andrepair mechanisms in immunocompetent models may lead todevelopment of new therapeutic applications for cartilage repair.

Acknowledgement

The authors would like to thank Dr. Hossein Nejadnik ofDepartment of Orthopaedic Surgery for his help in histologicalscoring and Mark Chong and Eddy Lee for their valuable technicaladvice. This work was partially supported by grants from theMinistry of Education of Singapore (R223000014112 andR223000018112). WST is supported by National University ofSingapore (NUS) research scholarship and President graduatefellowship. JC received salary support from the Clinician ScientistAward, NMRC, Singapore.

Appendix. Supplementary data

The supplementary data associated with this article can befound in the on-line version at 10.1016/j.biomaterials.2010.05.064.

Appendix

Figures with essential colour discrimination Figs. 1e4,6,7. Mostof the figures in this article have parts that are difficult to interpretin black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.biomaterials.2010.05.064.

References

[1] Hardingham T, Tew S, Murdoch A. Tissue engineering: chondrocytes andcartilage. Arthritis Res 2002;4:S63e8.

[2] Chung C, Burdick JA. Engineering cartilage tissue. Adv Drug Deliv Rev2008;60:243e62.

[3] Lee CR, Grodzinsky AJ, Hsu HP, Spector M. Effects of a cultured autologouschondrocyte-seeded type II collagen scaffold on the healing of a chondraldefect in a canine model. J Orthop Res 2003;21:272e81.

[4] Lee CS, Gleghorn JP, Won Choi N, Cabodi M, Stroock AD, Bonassar LJ. Inte-gration of layered chondrocyte-seeded alginate hydrogel scaffolds. Biomate-rials 2007;28:2987e93.

[5] Cohen SB, Meirisch CM, Wilson HA, Diduch DR. The use of absorbable co-polymer pads with alginate and cells for articular cartilage repair in rabbits.Biomaterials 2003;24:2653e60.

[6] Zwingmann J, Mehlhorn AT, Südkamp N, Stark B, Dauner M, Schmal H.Chondrogenic differentiation of human articular chondrocytes differs inbiodegradable PGA/PLA scaffolds. Tissue Eng 2007;13:2335e43.

[7] Liu Y, Shu XZ, Prestwich GD. Osteochondral defect repair with autologousbone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix. Tissue Eng 2006;12:3405e16.

[8] Katopodi T, Tew SR, Clegg PD, Hardingham TE. The influence of donor andhypoxic conditions on the assembly of cartilage matrix by osteoarthritichuman articular chondrocytes on hyalograft matrices. Biomaterials2009;30:535e40.

[9] Candrian C, Vonwil D, Barbero A, Bonacina E, Miot S, Farhadi J, et al. Engi-neered cartilage generated by nasal chondrocytes is responsive to physicalforces resembling joint loading. Arthritis Rheum 2008;58:197e208.

[10] Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differen-tiated collagen phenotype when cultured in agarose gels. Cell1982;30:215e24.

[11] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ,Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts.Science 1998;282:1145e7.

[12] Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem celllines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol2000;18:399e404.

[13] Heng BC, Cao T, Lee EH. Directing stem cell differentiation into the chon-drogenic lineage in vitro. Stem Cells 2004;22:1152e67.

[14] Toh WS, Yang Z, Liu H, Heng BC, Lee EH, Cao T. Effects of culture conditionsand bone morphogenetic protein 2 on extent of chondrogenesis from humanembryonic stem cells. Stem Cells 2007;25:950e60.

[15]. Toh WS, Guo XM, Choo AB, Lu K, Lee EH, Cao T. Differentiation and enrich-ment of expandable chondrogenic cells from human embryonic stem cells invitro. J Cell Mol Med 2009;13:3570e90.

[16] Hwang NS, Varghese S, Elisseeff J. Derivation of chondrogenically-committedcells from human embryonic cells for cartilage tissue regeneration. PLoS One2008;3:e2498.

[17] Koay EJ, Athanasiou KA. Hypoxic chondrogenic differentiation of humanembryonic stem cells enhances cartilage protein synthesis and biomechanicalfunctionality. Osteoarthritis Cartilage 2008;16:1450e6.

[18] Terraciano V, Hwang N, Moroni L, Park HB, Zhang Z, Mizrahi J, et al. Differ-ential response of adult and embryonic mesenchymal progenitor cells tomechanical compression in hydrogels. Stem Cells 2007;25:2730e8.

[19] Francioli SE, Martin I, Sie CP, Hagg R, Tommasini R, Candrian C, et al. Growthfactors for clinical-scale expansion of human articular chondrocytes: rele-vance for automated bioreactor systems. Tissue Eng 2007;13:1227e34.

[20] Toh WS, Liu H, Heng BC, Rufaihah AJ, Ye CP, Cao T. Combined effects ofTGFbeta1 and BMP2 in serum-free chondrogenic differentiation of mesen-chymal stem cells induced hyaline-like cartilage formation. Growth Factor2005;23:313e21.

[21]. Toh WS, Lee EH, Richards M, Cao T. In vitro derivation of chondrogenic cellsfrom human embryonic stem cells. Methods Mol Biol 2010;584:317e31.

[22] Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI, et al.Mesenchymal cell-based repair of large, full-thickness defects of articularcartilage. J Bone Joint Surg Am 1994;76:579e92.

[23] Nawata M, Wakitani S, Nakaya H, Tanigami A, Seki T, Nakamura Y, et al. Use ofbone morphogenetic protein 2 and diffusion chambers to engineer cartilagetissue for the repair of defects in articular cartilage. Arthritis Rheum2005;52:155e63.

[24] Lee ES, Chan J, Shuter B, Tan LG, Chong MS, Ramachandra DL, et al. Microgeliron oxide nanoparticles for tracking human fetal mesenchymal stem cellsthrough magnetic resonance imaging. Stem Cells 2009;27:1921e31.

[25] Toh WS, Yang Z, Heng BC, Cao T. Differentiation of human embryonic stemcells toward the chondrogenic lineage. Methods Mol Biol 2007;407:333e49.

[26] Kim YJ, Sah RL, Doong JY, Grodzinsky AJ. Fluorometric assay of DNA incartilage explants using Hoechst 33258. Anal Biochem 1988;174:168e76.

[27] Jones AC, Milthorpe B, Averdunk H, Limaye A, Senden TJ, Sakellariou A, et al.Analysis of 3D bone ingrowth into polymer scaffolds via micro-computedtomography imaging. Biomaterials 2004;25:4947e54.

[28]. Koay EJ, Hoben GM, Athanasiou KA. Tissue engineering with chondrogeni-cally differentiated human embryonic stem cells. Stem Cells2007;25:2183e90.

[29] Jukes JM, Moroni L, van Blitterswijk CA, de Boer J. Critical steps towarda tissue-engineered cartilage implant using embryonic stem cells. Tissue EngPart A 2008;14:135e47.

[30] Vats A, Bielby RC, Tolley N, Dickinson SC, Boccaccini AR, Hollander AP, et al.Chondrogenic differentiation of human embryonic stem cells: the effect of themicro-environment. Tissue Eng 2006;12:1687e97.

[31] Nakagawa T, Lee SY, Reddi AH. Induction of chondrogenesis from humanembryonic stem cells without embryoid body formation by bone morpho-genetic protein 7 and transforming growth factor beta1. Arthritis Rheum2009;60:3686e92.

[32] Miyamoto C, Matsumoto T, Sakimura K, Shindo H. Osteogenic protein-1with transforming growth factor-beta1: potent inducer of chondrogenesisof synovial mesenchymal stem cells in vitro. J Orthop Sci 2007;12:555e61.

[33] Lee KB, Hui JH, Song IC, Ardany L, Lee EH. Injectable mesenchymal stem celltherapy for large cartilage defectsea porcine model. Stem Cells2007;25:2964e71.

[34] Chang CH, Kuo TF, Lin CC, Chou CH, Chen KH, Lin FH, et al. Tissue engineering-based cartilage repair with allogenous chondrocytes and gelatin-chondroitin-hyaluronan tri-copolymer scaffold: a porcine model assessed at 18, 24, and 36weeks. Biomaterials 2006;27:1876e88.

[35] Chen HC, Chang YH, Chuang CK, Lin CY, Sung LY, Wang YH, et al. The repair ofosteochondral defects using baculovirus-mediated gene transfer with de-differentiated chondrocytes in bioreactor culture. Biomaterials2009;30:674e81.

[36] Cui L, Wu Y, Cen L, Zhou H, Yin S, Liu G, et al. Repair of articular cartilagedefect in non-weight bearing areas using adipose derived stem cells loadedpolyglycolic acid mesh. Biomaterials 2009;30:2683e93.

[37] Schaefer D, Martin I, Jundt G, Seidel J, Heberer M, Grodzinsky A, et al. Tissue-engineered composites for the repair of large osteochondral defects. ArthritisRheum 2002;46:2524e34.

[38] Obradovic B, Martin I, Padera RF, Treppo S, Freed LE, Vunjak-Novakovic G.Integration of engineered cartilage. J Orthop Res 2001;19:1089e97.

W.S. Toh et al. / Biomaterials 31 (2010) 6968e69806980

[39] Caplan AI, Elyaderani M, Mochizuki Y, Wakitani S, Goldberg VM. Principlesof cartilage repair and regeneration. Clin Orthop Relat Res 1997;342:254e69.

[40] Ho ST, Hutmacher DW, Ekaputra AK, Hitendra D, James HH. The evaluation ofa biphasic osteochondral implant coupled with an electrospun membrane ina large animal model. Tissue Eng Part A 2010;16:1123e41.

[41] Amin AK, Huntley JS, Simpson AH, Hall AC. Chondrocyte survival in articularcartilage: the influence of subchondral bone in a bovine model. J Bone JointSurg Br 2009;91:691e9.

[42] Zhang ZY, Teoh SH, Chong MS, Lee ES, Tan LG, Mattar CN, et al. Neo-vascu-larization and bone formation mediated by fetal mesenchymal stem celltissue-engineered bone grafts in critical-size femoral defects. Biomaterials2010;31:608e20.

[43] Carr AJ, Vugler AA, Hikita ST, Lawrence JM, Gias C, Chen LL, et al. Protectiveeffects of human iPS-derived retinal pigment epithelium cell transplantationin the retinal dystrophic rat. PLoS One 2009;4:e8152.

[44]. Gelse K, Brem M, Klinger P, Hess A, Swoboda B, Hennig F, et al. Paracrineeffect of transplanted rib chondrocyte spheroids supports formation ofsecondary cartilage repair tissue. J Orthop Res 2009;27:1216e25.

[45] Laurila JP, Laatikainen L, Castellone MD, Trivedi P, Heikkila J, Hinkkanen A,et al. Human embryonic stem cell-derived mesenchymal stromal celltransplantation in a rat hind limb injury model. Cytotherapy 2009;11:726e37.

[46] Chen X, Song XH, Yin Z, Zou XH, Wang LL, Hu H, et al. Stepwise differen-tiation of human embryonic stem cells promotes tendon regeneration bysecreting fetal tendon matrix and differentiation factors. Stem Cells 2009;27:1276e87.

[47] Sze SK, de Kleijn DP, Lai RC, Khia Way Tan E, Zhao H, Yeo KS, et al. Elucidatingthe secretion proteome of human embryonic stem cell-derived mesenchymalstem cells. Mol Cell Proteomics 2007;6:1680e9.

[48] Hwang NS, Varghese S, Lee HJ, Zhang Z, Ye Z, Bae J, et al. In vivo commitmentand functional tissue regeneration using human embryonic stem cell-derivedmesenchymal cells. Proc Natl Acad Sci USA 2008;105:20641e6.

[49] Wakitani S, Aoki H, Harada Y, Sonobe M, Morita Y, Mu Y, et al. Embryonic stemcells form articular cartilage, not teratomas, in osteochondral defects of ratjoints. Cell Transpl 2004;13:331e6.

[50] Nakajima M, Wakitani S, Harada Y, Tanigami A, Tomita N. In vivo mechanicalcondition plays an important role for appearance of cartilage tissue in ES celltransplanted joint. J Orthop Res 2008;26:10e7.

[51] Lian Q, Lye E, Suan Yeo K, Khia Way Tan E, Salto-Tellez M, Liu TM, et al.Derivation of clinically compliant MSCs from CD105þ, CD24� differentiatedhuman ESCs. Stem Cells 2007;25:425e36.

[52] Daadi MM, Maag AL, Steinberg GK. Adherent self-renewable human embry-onic stem cell-derived neural stem cell line: functional engraftment inexperimental stroke model. PLoS One 2008;3:e1644.

[53] Swijnenburg RJ, Schrepfer S, Govaert JA, Cao F, Ransohoff K, Sheikh AY, et al.Immunosuppressive therapy mitigates immunological rejection of humanembryonic stem cell xenografts. Proc Natl Acad Sci USA 2008;105:12991e6.

[54] Dai W, Field LJ, Rubart M, Reuter S, Hale SL, Zweigerdt R, et al. Survival andmaturation of human embryonic stem cell-derived cardiomyocytes in rathearts. J Mol Cell Cardiol 2007;43:504e16.

[55]. Fujihara Y, Takato T, Hoshi K. Immunological response to tissue-engineeredcartilage derived from auricular chondrocytes and a PLLA scaffold in trans-genic mice. Biomaterials 2010;31:1227e34.

[56] Adkisson HD, Milliman C, Zhang X, Mauch K, Maziarz RT, Streeter PR. Immuneevasion by neocartilage-derived chondrocytes: implications for biologic repairof joint articular cartilage. Stem Cell Res 2010;4:57e68.

[57] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al.Induction of pluripotent stem cells from adult human fibroblasts by definedfactors. Cell 2007;131:861e72.