Polyglycolic acid-hyaluronan scaffolds loaded with bone ...Jenel M. Patrascu,1* Jan Philipp...

Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenic differentiationin vitro and cartilage repair in the rabbit model

Jenel M. Patrascu,1* Jan Philipp Kr€uger,2* Hademar G. B€oss,1 Anna-Katharina Ketzmar,2

Undine Freymann,2 Michael Sittinger,3,4 Michael Notter,5 Michaela Endres,2,3 Christian Kaps2

1Department of Orthopaedic Surgery, V. Babes University of Medicine and Pharmacy, Timisoara, Romania2TransTissue Technologies GmbH, Charit�eplatz 1, 10117 Berlin, Germany3Department of Rheumatology, Tissue Engineering Laboratory, Charit�e Campus Mitte, Charit�e - Universit€atsmedizin Berlin,

Charit�eplatz 1, 10117, Berlin, Germany4Berlin-Brandenburg Center for Regenerative Therapies, Charit�e-Universit€atsmedizin Berlin, Augustenburger Platz 1, 13353

Berlin5Department of Hematology and Oncology, Charite-Universit€atsmedizin Berlin, Hindenburgdamm 30, 12200 Berlin, Germany

Received 5 November 2012; revised 28 January 2013; accepted 6 March 2013

Published online 10 May 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32944

Abstract: In cartilage repair, scaffold-assisted one-step

approaches are used to improve the microfracture (Mfx) tech-

nique. Since the number of progenitors in Mfx is low and may

further decrease with age, aim of our study was to analyze the

chondrogenic potential of freeze-dried polyglycolic acid-hyalur-

onan (PGA-HA) implants preloaded with mesenchymal

stem cells (MSCs) in vitro and in a rabbit articular cartilage

defect model. Human bone marrow-derived MSC from iliac

crest were cultured in freeze-dried PGA-HA implants for

chondrogenic differentiation. In a pilot study, implants were

loaded with autologous rabbit MSC and used to cover 5 mm 3

6 mm full-thickness femoral articular cartilage defects (n 5 4).

Untreated defects (n 5 3) served as controls. Gene expression

analysis and histology showed induction of typical

chondrogenic marker genes like type II collagen and formation

of hyaline-like cartilaginous tissue in MSC-laden PGA-HA

implants. Histological evaluation of rabbit repair tissue forma-

tion after 30 and 45 days showed formation of repair tissue,

rich in chondrocytic cells and of a hyaline-like appearance.

Controls showed no articular resurfacing, tissue repair in the

subchondral zone and fibrin formation. These results suggest

that MSC-laden PGA-HA scaffolds have chondrogenic potential

and are a promising option for stem cell-mediated cartilage

regeneration. VC 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part

B: Appl Biomater 101B: 1310–1320, 2013.

Key Words: cartilage repair, cartilage regeneration, cell-free

implant, microfracture, stem cells

How to cite this article: Patrascu JM, Kr€uger JP, B€oss HG, Ketzmar A-K, Freymann U, Sittinger M, Notter M, Endres M, Kaps C.2013. Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenicdifferentiation in vitro and cartilage repair in the rabbit model. J Biomed Mater Res Part B 2013:101B:1310–1320.

INTRODUCTION

In cartilage repair, a variety of techniques evolved that aimat cartilage resurfacing and=or regeneration. These techni-ques comprise debridement, bone marrow stimulation (e.g.,abrasion, drilling, and microfracturing), osteochondral graft-ing or mosaicplasty, and autologous chondrocyte implanta-tion with or without the use of scaffolds.1–5 Themicrofracture (Mfx) technique is a frequently used and costeffective first-line treatment option for the repair of focalcartilage defects.6,7 In Mfx, bleeding of the subchondralbone is induced by the introduction of multiple perforations

or Mfxs. In Mfx, repair tissue formation by mesenchymalstem cells (MSCs) may be stimulated by growth and differ-entiation factors from the subchondral bone and=or the sy-novial fluid.5,8,9 Although clinical studies demonstrated thatthe Mfx technique shows good results in the short and mid-term with up to 5 years follow-up,10 the repair tissueinduced by Mfxs has been shown to be of mostly fibrocarti-laginous appearance with limited short-term durability.11

Apparently, the Mfx treatment shows good short-termresults, but clinical results may be variable in the mid tolong terms. Apart from that, the technique may be limited

*These authors contributed equally to this work.

JPK, UF, ME, and CK are employees of TransTissue Technologies GmbH (TTT). TTT develops scaffold-based cartilage repair treatment strategies

and developed the PGA-HA scaffold. CK is consultant of BioTissue AG that distributes the PGA-HA based chondrotissue cartilage implant. MS is

consultant of BioTissue Technologies GmbH that produces the PGA-HA scaffold.Correspondence to: C. Kaps; e-mail: [email protected] grant sponsor: European Union, EU-FP7 program; contract grand number: TissueGEN: HEALTH-F4-2011-278955

1310 VC 2013 WILEY PERIODICALS, INC.

by its indication for relatively small focal defects with anintact defect shoulder surrounding the defect.

Recently, scaffold-assisted one-step approaches evolvedthat may improve cartilage repair by enhancing repair tissueformation in microfracturing and may extend the indicationeven to partly unshouldered defects by covering the micro-fractured defect with, for example, porcine collagen scaffoldswith fibrin12,13 or resorbable polyglycolic acid-hyaluronan(PGA-HA) scaffolds.14,15 PGA-HA scaffolds have been shownto recruit MSC into the scaffold and guide them toward car-tilage repair, with improvement of cartilage repair tissueformation compared to microfracturing alone.16 Clinically, ina group of 52 patients, implantation of PGA-HA scaffolds fortreatment of cartilage defects improved the patients’ situa-tion as shown by the Knee injury and Osteoarthritis Out-come Score (KOOS) at 1 year follow-up and formed hyaline-like cartilage tissue as assessed by second-look biopsies.17

However, from the technical and cell biology point of view,variability in the mid- to long-term outcome may be, forexample, due to different depths of the Mfxs,18 use of thedrilling or the Mfx procedure19 and maybe more impor-tantly due to a reduced availability of MSC with chondro-genic capacity in the subchondral bone marrow. Inparticular, with increasing age, the number of MSC in thebone marrow is reduced dramatically20 and in early osteo-arthritis, for instance, there may be a reduced potential ofcells to induce hyaline-like cartilage repair.21 To overcomethese hurdles, the enrichment of scaffolds with autologousbone marrow concentrate or cells are currently investigatedto improve and=or extend the Mfx technique and=or carti-lage repair.22,23 A recent report about bone marrow concen-trate has shown that cells within the concentrate grow onthe HA-based scaffold and are able to undergo chondrogenicdifferentiation.24 In addition, expanded bone marrow and=oradipose derived stem cells have been shown to differentiatealong the chondrogenic lineage in, for example, platelet-richplasma (PRP)-derived scaffolds, porous poly(ethyleneglycol) diglycidyl ether-cross-linked HA, poly-L-lactide-co-epsilon-caprolactone (PLCL)/chitosan scaffolds, or RGD-polyhydroxyalkanoate scaffolds.25–28 We hypothesize thatbone marrow-derived MSC show chondrogenic differentia-tion in clinically applicable PGA-HA scaffolds in vitro andthat implantation of these cells in PGA-HA scaffolds intofull-thickness articular cartilage defects leads to hyaline-likecartilage repair in the rabbit model.

MATERIALS AND METHODS

Isolation and culture of human MSCHuman adult MSC were isolated from iliac crest bone mar-row aspirates of healthy donors (n 5 11, 3 female, 8 male,age 43–72). Bone marrow samples were derived fromdonors who were examined to exclude hematopoietic neo-plasmas and were histologically diagnosed as normal. Thestudy was approved by the ethical committee of theCharit�e-Universit€atsmedizin Berlin. In brief, aspirates (1 mL)were suspended in 21 mL Dulbecco’s modified Eagle’s(DME) medium (Biochrom, Berlin, Germany) containing 2ng=mL basic fibroblast growth factor (Tebu-bio, Germany),

100 U=mL penicillin, 100 mg=mL streptomycin, and 10%human serum and transferred to cell culture flasks. Mediumwas exchanged after 72 h and then every 2 days thereafter.Cells were allowed to grow to 90% confluence, detached by0.05% trypsin–ethylenediaminetetraacetic acid (Biochrom)in phosphate-buffered saline (PBS; Biochrom) and useddirectly for further analysis.

Fluorescence-activated cell sortingFor characterization of MSC, typical MSC-related cell surfaceantigens were analyzed. MSC (2.5 3 105 cells, passage 0; n5 3 donors) were washed in PBS=0.5% bovine serum albu-min and incubated with monoclonal mouse anti-human anti-bodies CD34-phycoerythrin (PE), CD45-fluorescein-isothiocyanate (FITC), CD73-PE, CD90-FITC, CD105-FITC,and CD166-PE, conjugated with FITC or PE for 15 min onice. Staining of cell surface antigens was analyzed using thefluorescence-activated cell sorting Calibur equipped withCELLQUEST software (Becton Dickinson). Apoptotic cellswere excluded from analysis using propidium iodide. CD34stained cells served as isotypic control.

Three-dimensional tissue culture of human MSCin polymer scaffoldsThe chondrotissue matrix was prepared as described previ-ously.29 In brief, resorbable PGA scaffolds (BioTissue AG,Zurich, Switzerland) of 8 mm 3 8 mm 3 0.5 mm wereimmersed with 32 mL hyaluronic acid (HA, OstenilV

R

; TRBChemedica AG, Germany) and subsequently freeze-dried for16 h using a lyophilisator (Leybold-Heraeus, Germany). MSC(passage 0, n 5 3 donors) were seeded into the PGA-HAscaffolds at a density of 2 3 107 viable cells=mL by resus-pending 0.64 3 106 cells in 21 mL cell culture medium and11 mL fibrinogen (Tissucol, Baxter International). Polymer-ization of the fibrinogen was achieved by adding thrombin(10%, v=v in PBS; Tissucol) and incubation at 37 C for 15min.

Differentiation of human MSC in polymer scaffoldsFor chondrogenic differentiation, MSC-PGA-HA scaffolds (n5 4 per donor; n 5 3 donors) were cultured up to 14 daysin DME medium containing 1% Insulin–Transferrin–Sele-nium 1 1, 1 mM sodium pyruvate, 0.35 mM L-proline, 0.17mM L-ascorbic acid-2-phosphate, and 0.1 mM dexamethasone(all Sigma-Aldrich, St. Louis, MO) and 10 ng=mL transform-ing growth factor-b3 (TGFB3; Peprotech). MSC-PGA-HA scaf-folds in DME medium without TGFB3 served as controls.Medium was exchanged every 2–3 days.

Real-time polymerase chain reactionTotal RNA was isolated from MSC monolayer cultures (pas-sage 0, n 5 5 donors) at 90% confluence and from MSC cul-tured in PGA-HA-scaffolds (n 5 6) as described.30

Subsequently, total RNA (3 mg) was reversely transcribedwith the iScript cDNA Synthesis Kit according to the manu-facturer’s instructions (BioRad, M€unchen, Germany). The rel-ative expression level of the housekeeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | OCT 2013 VOL 101B, ISSUE 7 1311

used to normalize samples. Real-time reverse-transcriptasepolymerase chain reaction (PCR) using i-Cycler PCR System(BioRad) was performed with 1 mL of each cDNA sample intriplicate using the SYBR green PCR Core Kit (Applied Bio-systems, Foster City, CA). Relative quantification of markergenes (Table I) expression was performed and is given aspercentage of the GAPDH product.

Histology and immune-histochemistryMSC in PGA-HA scaffolds were embedded in optimal cuttingtemperature compound, frozen and cryosectioned (6 mm).Sections (n 5 9) were digested for 30 min. with 50 U=mLhyaluronidase (Sigma-Aldrich) at room temperature, followedby staining with rabbit anti-human type II collagen antibodies(Acris, Hiddenhausen, Germany) for 40 min. Colorimetricaldetection was done with 3-amino-9-ethylcarbazole (EnVi-sion

TM

; Dako, Glostrup, Denmark) and counterstaining withhematoxylin (Merk, Darmstadt, Germany). Proteoglycansand=or collagens were visualized by staining with Alcian blue8GS (Roth, Karlsruhe, Germany; n 5 9) and safranin O (n 5

9). Osteogenic differentiation was assessed by staining ofmineralized matrix components according to von Kossa (n 5

3). Adipogenic differentiation and intracellular lipid dropletswere visualized using Oil Red O (n 5 3).

Statistical analysisFor statistical analysis of gene expression data (n 5 9 perpoint in time; n 5 3 independent donors with gene expres-sion analysis in triplicates), the Kolmogorov–Smirnov methodwas applied for testing normal distribution of the data. Theparametric t-test and the nonparametric Mann–Whitney ranksum test were applied. Differences were considered signifi-cant at p < 0.05 and a fold change of >3 or <23.

Implantation of MSC-PGA-HA scaffolds in rabbitcartilage defects and histologySeven New Zealand White rabbits (3.5–4.0 kg) were used inthis study. For anesthesia, rabbits were premedicated with

an intramuscular injection of 3 mg=kg xylazine and 10.0mg=kg ketamine. Anesthesia was maintained by inhalationof 1.5–2.0% isoflurane delivered in oxygen and air (FiO2

0.4). All surgical procedures were performed under asepticconditions. For bone marrow harvest, a 3 cm anterolateralincision was made at the hip to prepare the diaphysis of thefemoral bone. Bone marrow of 5 mL was aspirated from thediaphysis using an 18-gauge needle and a syringe. Fourweeks after bone marrow harvest and MSC preparation asdescribed above, the PGA-HA implant (chondrotissueVR , Bio-Tissue AG, Zurich, Switzerland) was cut into 6 mm 3 7 mm3 1.1 mm and loaded with 2 3 107=mL MSC in cell culturemedium. To prepare the cartilage defects, the joint wasopened by an anteromedial approach and the femoral con-dyles were exposed. Degenerative joint disease and skeletalabnormalities were excluded by visual inspection and full-thickness cartilage trochlear defects of 5 mm 3 6 mm werecreated in the weight bearing cartilage using a scalpel and asharp spoon. For covering of the defect, cell-laden PGA-HAimplants (n 5 4) were introduced using the press-fit tech-nique. Cartilage defects without covering with cell-ladenimplants served as controls (n 5 3). Wound closure wasperformed in layers and protected by a plaster. Postopera-tively, prophylactic maintenance of analgesia was achievedby daily application of 20 mg=kg amoxyclin and calvulanatefor 5 days. At 30 days and 45 days, rabbits were anesthe-tized and sacrificed by administration of 100 mg=kg thio-pentone and 2 mmol=kg potassium chloride intravenously.Joints were fixed in formalin, decalcified and embedded inparaffin. Sections (6 mm) were stained with eosin and sec-tions through the center of the defect (n 5 3 per defect)were evaluated histologically.

RESULTS

Cell surface antigen pattern of human bonemarrow-derived MSCDirectly seeded, low expanded (passage 0) iliac crest bonemarrow-derived MSC showed typical cell surface antigens

TABLE I. Oligonucleotide Sequences

Gene Name Accession no. Oligonucleotide (50 ! 30) (Up=Down)BasePairs

Collagen type I NM_000088 CGA TGG CTG CAC GAG TCA CAC=CAG GTT GGG ATG GAGGGA GTT TAC

180

Collagen type II NM_001844 CCG GGC AGA GGG CAA TAG CAG GTT=CAA TGA TGG GGAGGC GTG AG

128

Collagen type IX NM_001853 AAT CAG GCT CTC GAA GCT CAT AAA A=CCT GCC ACA CCC CCGCTC CTT CAT

100

Cartilage oligomericmatrix protein

NM_000095 CCG GAG GGT GAC GCG CAG ATT GA=TGC CCT CGA AGT CCACGC CAT TGA A

133

Aggrecan NM_001135 GGC TGC TGT CCC CGT AGA AGA=GGG AGG CCA AGT AGG AAG GAT 163Link-protein NM_001884 GCG TCC GCT ACC CCA TCT CTA=GCG CTC TAA GGG CAC ATT CAG TT 145Osteocalcin NM_199173 GAG CCC CAG TTC CCC TAC CC=GCC TCC TGA AAG CCG ATG TG 103Fatty acid-binding

protein 4NM_001442 CCT TAG ATG GGG GTG TCC TGG TA=AAC GTC CCT TGG CTT

ATG CTC TC156

Glyceraldehyde-3-phospatedehydrogenase

NM_002046 GGC GAT GCT GGC GCT GAG TAC=TGG TCC ACA CCC ATG ACG A 149

1312 PATRASCU ET AL. PROGENITOR-LOADED POLYGLYCOLIC ACID-HYALURONAN SCAFFOLDS FOR CARTILAGE REPAIR

known from MSC and progenitor cell. MSC showed ahomogenous population and were negative for CD34 andCD45, but presented the antigens CD73, CD90, CD105, andCD166 (Fig. 1).

Chondrogenic differentiation of human bonemarrow-derived MSC in PGA-HA scaffoldsTo test whether human progenitor cells are capable ofundergoing chondrogenic differentiation in PGA-HA scaf-folds, MSC were cultured for up to 14 days in PGA-HA scaf-folds using standard chondrogenic conditions andstimulated with TGFB3. Histological analysis of MSC-PGA-HAcultures (Fig. 2) revealed that control cultures not treatedwith TGFB3 showed virtually no deposition of proteoglycan[Fig. 2(A,C)] or type II collagen [Fig. 2(E)]. MSC [Fig. 2(A),arrowhead] were embedded in a HA-fibrin matrix [Fig. 2(A),asterisk], distributed evenly within the PGA-HA scaffold andscaffold fibers were partly fragmented, showing first signsof degradation [Fig. 2(A), double arrowhead]. Scaffold fibersappeared blue when stained with alcian blue [Fig. 2(A)],and stained red upon staining with safranin O [Fig. 2(B)].MSC-PGA-HA cultures that were treated with TGFB3 for 14

days showed formation of a cartilaginous extracellular ma-trix with deposition of proteoglycan [Fig. 2(B,D), arrow-heads] and type II collagen [Fig. 2(F), arrowhead].

To confirm chondrogenic differentiation of MSC in PGA-HA scaffolds, semiquantitative gene expression analysis ofgenes coding for typical cartilage matrix molecules was per-formed (Fig. 3). Treatment of MSC cultured in PGA-HA scaf-folds with TGFB3 significantly (p < 0.05) induced theexpression of the chondrogenic marker genes type II colla-gen (from mean 62% of the GAPDH expression level foundin untreated controls to mean 8201% of the GAPDH expres-sion level found in TGFB3 treated MSC), type IX collagen(from 2% to 191%), cartilage oligomeric matrix protein(COMP, from 8.9% to 180%), aggrecan (from 0.3% to 7.8%),and cartilage link protein (from 14% to 531%), comparedto untreated MSC in PGA-HA scaffolds. Compared to mono-layer cultures of MSC, the three-dimensional assembly andtissue culture of MSC in PGA-HA scaffolds significantly (p <0.05) induced the chondrogenic marker genes type II colla-gen (from 2.3% found in monolayer to 62% in PGA-HA scaf-folds) and COMP (from 1.6% to 8.9%), even in the absenceof the chondrogenic inducer TGFB3.

Adipogenic or osteogenic differentiation of human bonemarrow-derived MSC in PGA-HA scaffoldsSince MSC show a multipotential differentiation capacity to-ward cartilage as well as—unwanted in cartilage repair—bone and fat, the osteogenic and adipogenic differentiationpotential of MSC in PGA-HA scaffolds was evaluated by his-tological staining and gene expression analysis of typicaladipogenic and osteogenic markers (Fig. 4). At day 14, MSCcultured in PGA-HA scaffolds with or without TGFB3showed no mineralization of the extracellular matrix and alow expression of the osteogenic marker gene osteocalcin[Fig. 4(A)]. MSC-PGA-HA cultures with or without TGFB3treatment showed no lipid droplet formation. Compared toMSC grown in monolayer cultures, MSC in PGA-HA scaffoldsshowed significantly increased levels of the adipogenicmarker gene fatty acid-binding protein 4 (FABP4, from172% in monolayer to 5568% in MSC-PGA-HA withoutTGFB3). Treatment of MSC-PGA-HA cultures with TGFB3 sig-nificantly decreased (from 5568% in untreated controls to115% in MSC-PGA-Ha treated with TGFB3) FABP4 expres-sion levels [Fig. 4(B)].

Implantation of MSC-laden PGA-HA scaffolds in rabbitarticular defectsRectangular full-thickness trochlear cartilage defects of 5mm 3 6 mm were left untreated [Fig. 5(A)] or filled pressfit with MSC-laden PGA-HA scaffolds [Fig. 5(B)]. At 45 days,rabbits showed no lameness or abnormal behavior. Therewere no clinical signs of inflammation, infection or allergicreaction. The exposed joints showed no signs of synovialinflammation or irritation. The synovial fluid was clear andappeared normal. In contrast to untreated controls, theMSC-PGA-HA treated cartilage defects showed macroscopi-cally cartilaginous repair tissue formation with a smooth

FIGURE 1. Cell surface antigen profile of adult human bone marrow-

derived MSC. Flow cytometric analysis shows that low expanded

MSC (passage 0) are positive for the antigens CD73, CD90, CD105,

and CD166, while cells were negative for CD34 and CD45. [Color fig-

ure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]



surface and almost complete filling of the defect (data notshown).

At days 30 and 45, untreated controls showed some car-tilaginous repair tissue formation adjacent to the subchon-dral bone [Fig. 5(C,D), black arrows]. The chondral area waspredominantly filled with fibrin that showed few cells [Fig.5(C,D), asterisk]. At day 30, the subchondral bone showedsigns of bone remodeling [Fig. 5(C), double arrowhead],while the subchondral bone was clearly delineated at day45 [Fig. 5(D), double arrowhead].

Defects treated with MSC-laden PGA-HA scaffoldsshowed formation of cell-rich cartilaginous repair tissue atday 30 that partially filled the defect [Fig. 5(E,F)]. The tissueshowed good bonding to the remodeling subchondral boneand appeared unstructured with round-shaped chondrocytic

cells that were scattered through the repair tissue [Fig. 5(F),arrows]. At day 45, the cartilage repair tissue covered thedefect and showed chondrocytic cells, with in part columnardistribution [Fig. 5(G,H), arrows]. The repair tissue was of ahyaline-like to hyaline appearance and the subchondralbone showed tidemark formation [Fig. 5(G), double arrow].

DISCUSSION

In the present study, we demonstrated that human bonemarrow-derived MSCs have the capacity to undergo chon-drogenic lineage development in vitro when embedded inresorbable PGA-HA scaffolds. Transplantation of autologousMSC-laden PGA-HA scaffolds into full-thickness articular car-tilage defects resulted in hyaline-like cartilage repair in therabbit model.

FIGURE 2. Chondrogenic differentiation of MSC-laden PGA-HA scaffolds in vitro. Untreated controls showed no deposition of proteoglycan as

assessed by Alcian blue (A; *HA–fibrin matrix; arrowhead MSC; double arrowheads PGA fiber) and Safranin O staining (C). Immunostaining for

type II collagen was negative (E). MSC-laden PGA-scaffolds treated with TGFB3 for 14 days showed deposition of proteoglycan according to

Alcian blue (B, arrowheads) and Safranin O staining (C, arrowheads). The newly formed extracellular matrix was rich in type II collagen (F,

arrowheads).


In cartilage repair, mesenchymal progenitor cells play akey role in bone marrow stimulating treatment strategieslike drilling or microfracturing. The Mfx technique is a fre-quently used first line cartilage repair option and inducesthe formation of cartilage repair tissue by perforating thesubchondral bone. These Mfxs allow mesenchymal progeni-tor cells, which have a considerable chondrogenic potential,to populate the defect and form cartilaginous tissue.5,31 InMfx-mediated cartilage repair, unstructured repair tissueformation is often apparent that is predominantly of a fibro-cartilaginous type. This may be underlined by a recent

clinical study in young athletes with a 3-year follow-up thatshowed fibrocartilage and surface fibrillation in 8 out of 14biopsies.11 Therefore, a variety of one-step cartilage repairtechniques evolved that aim at the improvement of the Mfx-mediated cartilage repair tissue formation. These techniquesutilize different scaffolds and blood derivatives to cover themicrofractured defect. The autologous matrix-induced chon-drogenesis technique uses a porcine collagen type I=IIImembrane to cover the microfractured defect, followed bythe injection of a serum–fibrin mixture and=or PRP gels.32–34 The use of the PGA-HA scaffold, also used in the current

FIGURE 3. Semiquantitative real-time gene expression analysis of MSC-laden PGA-HA scaffolds treated with TGFB3. The expression level of typ-

ical chondrogenic marker genes such as types I, II and IX collagens, aggrecan, cartilage oligomeric matrix protein (COMP), and cartilage link-pro-

tein was calculated as percentage of the expression level of the housekeeping gene GAPDH. The bars show the mean (n 5 3) and the SD.

*Significantly different (p < 0.05) compared to MSC-PGA-HA without TGFB3 treatment. #Significantly different (p < 0.05) compared to mono-

layer MSC.



study in vitro, in the treatment of chondral cartilage defectshave been shown to be safe and effective, and is accompa-nied by complete defect filling and hyaline-like cartilagerepair tissue formation.12,14,15 In the ovine model, coveringof microfractured defects with PGA-HA scaffolds immersedin autologous serum significantly improved the formation ofcartilage repair tissue with type II collagen compared toMfx treatment alone.16 Recently, in a case series with 52patients suffering from tibial and femoral cartilage defects,the PGA-HA scaffold was combined with PRP and showedhyaline-like cartilage formation as well as significant andclinically meaningful patients’ improvement at 12 monthsfollow-up as assessed by the KOOS.17 The textile PGA-HAscaffold seems to have good chondrogenesis supporting oreven inducing properties by potentially helping to enrichmesenchymal progenitor cells in the defect and guidingthem toward cartilage formation.16,29 This is in concordancewith our findings that show chondrogenic differentiation ofhuman MSCs in PGA-HA scaffolds in vitro. Although thechondrogenic differentiation of MSC in PGA-HA scaffolds

was more pronounced in the presence of the chondrogenicinducer TGFB3, the PGA-HA scaffold alone induced key car-tilage marker genes in MSC. This is in line with our previ-ous studies that showed that the particular hyaluronicacid—that is a component of the PGA-HA scaffold—inducesor initiates the chondrogenic differentiation sequence inequine and human MSCs.29,35 In addition, the PGA-HA scaf-fold alone, without TGFB3, showed cartilage repair tissueformation in the rabbit full thickness cartilage defect modelwhen augmented with autologous bone marrow-derivedcells. Therefore, it is suggested that the PGA-HA scaffoldalone has a chondrogenesis inducing effect on MSC, in vitroand in vivo.

Bone marrow-derived stem and=or progenitor cells havea multilineage differentiation capacity and have been shownto undergo chondrogenic differentiation upon stimulationwith, for example, all TGFB isoforms in pellet cultures.36,37

There seems to be no difference in the chondrogenic poten-tial regarding collagen deposition induced by different TGFBsubtypes in human MSC.38 Recently, human bone marrow-

FIGURE 4. Absence of osteogenic and adipogenic differentiation in chondrogenic MSC-laden PGA-HA scaffolds. MSC-laden PGA-HA scaffolds

treated with TGFB3 showed no deposition of mineralized matrix as assessed by von Kossa staining, expression level of the osteogenic marker

gene osteocalcin were low in MSC cultured in monolayer and in MSC-laden PGA-HA scaffolds with and without TGFB3 treatment (A). Oil red O

staining showed no lipid droplets or vacuoles filled with lipids in MSC-laden PGA-HA scaffolds. The adipogenic marker gene fatty acid-binding

protein 4 (FABP4) was elevated in MSC-laden PGA-HA scaffolds compared to MSC in monolayer (#p < 0.05), while TGFB3 repressed FABP4 (*p

< 0.05) compared to the expression level found MSC without TGFB3 treatment (B). [Color figure can be viewed in the online issue, which is

available at wileyonlinelibrary.com.]


FIGURE 5. Articular cartilage repair with MSC-laden PGA-HA scaffolds in the rabbit cartilage defect model. Large full-thickness cartilage trochlear

defects of 5 mm 3 6 mm were prepared (A) and covered with MSC-laden PGA-HA scaffolds using the press-fit technique (B). Untreated control

defects showed some cartilaginous repair tissue (arrowhead) adjacent to the remodeling subchondral bone (double arrowhead). The chondral

defect area was filled with fibrin (asterisks), at day 30 (C) and day 45 (D). At day 30, defects treated with MSC-laden PGA-HA scaffolds (E and F)

showed unstructured cartilaginous repair tissue that partially filled the defect and that was rich in chondrocytic cells (F, arrowheads). At day 45

after MSC-laden PGA-HA implantation (G and H), the defects were covered with hyaline-like cartilage repair tissue showing chondrocytic cells

partially in a columnar distribution (G and H, arrowheads). The subchondral bone showed the formation of a clearly delineated tidemark (G,

double arrowheads). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



derived MSC have been shown to undergo chondrogenic dif-ferentiation in chitosan-based (chitosan-poly-butylene ter-ephtalate adipate) scaffolds upon TGFB3 treatment.39 RatMSC have been shown to undergo chondrogenic differentia-tion when assembled three-dimensionally in silk fibroin orsilk fibroin–chitosan scaffolds, as assessed by PCR analysisof cartilage marker genes.40 These studies are in line withour current study that showed cartilaginous tissue forma-tion in vitro with extracellular cartilage matrix formationand induction of typical cartilage marker genes, after stimu-lation of less expanded human bone marrow-derived pro-genitors with TGFB3 in PGA-HA scaffolds. Interestingly, arecent report showed that human MSC in poly-lactic acid(PLLA) scaffolds formed extracellular matrix that containedaggrecan and collagens type I and X,41 which are knownfrom fibrous and=or hypertrophic cartilage tissue. Obviously,the type of scaffold and chondrogenic stimulus is importantfor the induction of a hyaline-like cartilage tissue, as shownhere for the PGA-HA scaffold.

In the rabbit model, autologous progenitors from bonemarrow embedded in a collagen gel were transplanted intolarge (3 mm 3 6 mm) full-thickness cartilage defects for upto 24 weeks. The cells developed into chondrocytesthroughout the defect and formed hyaline-like cartilagerepair tissue.42 For osteochondral repair, rabbit bone mar-row-derived MSC were seeded in sponge-like PLCL scaffoldsand implanted into full-thickness (diameter 4.5 mm, depth5 mm) osteochondral femoral condylar defects. After 3–6months, hyaline cartilage-like repair tissue was evident,while empty defects and defects treated with the PLCL scaf-folds alone showed limited repair.43 Another approachfavored the use of bone marrow-derived MSC and polylac-tic-co-glycolic acid scaffolds (PLGA) that were pretreatedwith TGB3 for osteochondral repair in the rabbit model.Twelve weeks after transplantation, MSC-PLGA grafts pre-treated with TGFB3 showed repair tissue that was not dif-ferent from normal, hyaline cartilage. In addition, the TGFB3treated grafts showed improved cartilage regeneration com-pared to MSC-PLGA grafts that were not pretreated with thegrowth factor.44 As shown here, autologous MSC in PGA-HAscaffolds, not treated with TGFB3, developed hyaline-likecartilage repair tissue that covered larger defects of 5 mm3 6 mm after 45 days. However, clinical studies have toshow that these particular scaffolds loaded with bone mar-row-derived cells or bone marrow do repair cartilagedefects. Further limitations of this study are the lack ofimmune-histochemical proof of hyaline cartilage formationwith type II collagen deposition and the relatively short fol-low-up.

Meanwhile, the use of autologous bone marrow-derivedprogenitors for cartilage repair was challenged clinically andhas been shown to be safe in 41 patients with a mean fol-low-up of 75 months,45 and some case reports suggest theeffectiveness of bone marrow-derived progenitor cells forcartilage repair in traumatic, osteoarthritic, and osteogenesisimperfecta defects.46–50 It has to be taken into account thatusing expanded bone marrow-derived MSC is a two-stepprocedure that is time and cost intensive. Therefore,

autologous bone marrow concentrate seems to be moreattractive as a source for bone marrow MSC in one-step car-tilage repair procedures.22,46 Recently, it has been shownthat cells present in bone marrow concentrate are able toundergo chondrogenic differentiation in vitro when seededonto a HA-based scaffold.24 Nevertheless, for clinical appli-cation, a potential drawback is that the number of stemcells in bone marrow concentrate is low (0.04%),22 suggest-ing that grafts with expanded bone marrow-derived MSCmay be more effective in cartilage repair. Comparative clini-cal trials have to show whether bone marrow concentrateand=or expanded MSC lead to a better outcome in cartilagerepair compared to Mfx alone or scaffold-assisted Mfxapproaches. Previously, we have shown in the ovine modelthat the PGA-HA scaffold alone leads to cartilage repair andimprovement of the Mfx techniques, when used to covermicrofractured defects.16,29 In the current study, we did notinclude an empty PGA-HA scaffold group. This is a clear li-mitation of the study and we cannot conclude from the datathat augmentation of the PGA-HA scaffold with MSCimproves cartilage repair compared to Mfx or to implanta-tion a cell-free PGA-HA scaffold. A further limitation may bethat we used human and not rabbit MSC to show that stemcells undergo proper chondrogenic differentiation in thePGA-HA scaffold. Consequently, the data obtained from thein vitro and in vivo studies are not comparable and maytherefore not prove the intrinsic chondrogenic potential ofhuman MSC in PGA-HA scaffolds, in vivo. In particular forclinical approaches, a limitation is that rabbit or animal jointdefect models do not resemble the human situation. There-fore, clinical pilot studies and=or clinical trials are needed,before recommending such an approach for clinical use.

In conclusion, we have shown that bone marrow-derivedMSC show chondrogenic differentiation in PGA-HA scaffoldsin vitro and that repair of large full-thickness cartilagedefects was feasible in the rabbit model by implanting MSC-laden PGA-HA scaffolds. These findings suggest that PGA-HAscaffolds are suited for regenerative medicine cartilagerepair approaches based on bone marrow-derived stemand=or progenitor cells.

ACKNOWLEDGMENT

The authors are very grateful to Samuel Vetterlein for theexcellent technical assistance.

REFERENCES

1. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peter-

son L. Treatment of deep cartilage defects in the knee with auto-

logous chondrocyte transplantation. N Engl J Med

1994;331(14):889–895.

2. Hubbard MJ. Articular debridement versus washout for degenera-

tion of the medial femoral condyle. A five-year study. J Bone

Joint Surg Br 1996;78(2):217–219.

3. Kreuz PC, Muller S, Freymann U, Erggelet C, Niemeyer P, Kaps C,

Hirschmuller A. Repair of focal cartilage defects with scaffold-

assisted autologous chondrocyte grafts: Clinical and biomechani-

cal results 48 months after transplantation. Am J Sports Med

2011;39(8):1697–1705.

4. Matsusue Y, Yamamuro T, Hama H. Arthroscopic multiple osteo-

chondral transplantation to the chondral defect in the knee


associated with anterior cruciate ligament disruption. Arthroscopy

1993;9(3):318–321.

5. Steadman JR, Rodkey WG, Briggs KK, Rodrigo JJ. The microfrac-

ture technic in the management of complete cartilage defects in

the knee joint. Orthopade 1999;28(1):26–32.

6. Steadman JR, Briggs KK, Rodrigo JJ, Kocher MS, Gill TJ, Rodkey

WG. Outcomes of microfracture for traumatic chondral defects of

the knee: Average 11-year follow-up. Arthroscopy 2003;19(5):477–

484.

7. Steadman JR, Rodkey WG, Rodrigo JJ. Microfracture: Surgical

technique and rehabilitation to treat chondral defects. Clin Orthop

Relat Res 2001(391 Suppl):S362–S369.

8. Endres M, Neumann K, Haupl T, Erggelet C, Ringe J, Sittinger M,

Kaps C. Synovial fluid recruits human mesenchymal progenitors

from subchondral spongious bone marrow. J Orthop Res

2007;25(10):1299–1307.

9. Shapiro F, Koide S, Glimcher MJ. Cell origin and differentiation in

the repair of full-thickness defects of articular cartilage. J Bone

Joint Surg Am 1993;75(4):532–553.

10. Kreuz PC, Erggelet C, Steinwachs MR, Krause SJ, Lahm A, Nie-

meyer P, Ghanem N, Uhl M, Sudkamp N. Is microfracture of

chondral defects in the knee associated with different results in

patients aged 40 years or younger? Arthroscopy

2006;22(11):1180–1186.

11. Gudas R, Kalesinskas RJ, Kimtys V, Stankevicius E, Toliusis V,

Bernotavicius G, Smailys A. A prospective randomized clinical

study of mosaic osteochondral autologous transplantation versus

microfracture for the treatment of osteochondral defects in the

knee joint in young athletes. Arthroscopy 2005;21(9):1066–1075.

12. Dhollander AA, Verdonk PC, Lambrecht S, Almqvist KF, Elewaut

D, Verbruggen G, Verdonk R. The combination of microfracture

and a cell-free polymer-based implant immersed with autologous

serum for cartilage defect coverage. Knee Surg Sports Traumatol

Arthrosc 2011.

13. Gille J, Schuseil E, Wimmer J, Gellissen J, Schulz AP, Behrens P.

Mid-term results of autologous matrix-induced chondrogenesis

for treatment of focal cartilage defects in the knee. Knee Surg

Sports Traumatol Arthrosc 2010;18(11):1456–1464.

14. Patrascu JM, Freymann U, Kaps C, Poenaru DV. Repair of a post-

traumatic cartilage defect with a cell-free polymer-based cartilage

implant: A follow-up at two years by MRI and histological review.

J Bone Joint Surg Br 2010;92(8):1160–1163.

15. Zantop T, Petersen W. Arthroscopic implantation of a matrix to

cover large chondral defect during microfracture. Arthroscopy

2009;25(11):1354–1360.

16. Erggelet C, Endres M, Neumann K, Morawietz L, Ringe J, Haber-

stroh K, Sittinger M, Kaps C. Formation of cartilage repair tissue

in articular cartilage defects pretreated with microfracture and

covered with cell-free polymer-based implants. J Orthop Res

2009;27(10):1353–1360.

17. Siclari A, Mascaro G, Gentili C, Cancedda R, Boux E. A cell-free

scaffold-based cartilage repair provides improved function hya-

line-like repair at one year. Clin Orthop Relat Res 2012;470(3):910–

919.

18. Chen H, Hoemann CD, Sun J, Chevrier A, McKee MD, Shive MS,

Hurtig M, Buschmann MD. Depth of subchondral perforation

influences the outcome of bone marrow stimulation cartilage

repair. J Orthop Res 2011;29(8):1178–1184.

19. Chen H, Sun J, Hoemann CD, Lascau-Coman V, Ouyang W,

McKee MD, Shive MS, Buschmann MD. Drilling and microfracture

lead to different bone structure and necrosis during bone-marrow

stimulation for cartilage repair. J Orthop Res 2009;27(11):1432–

1438.

20. Caplan AI. The mesengenic process. Clin Plast Surg 1994;21(3):

429–435.

21. Kaul G, Cucchiarini M, Remberger K, Kohn D, Madry H. Failed car-

tilage repair for early osteoarthritis defects: A biochemical, histo-

logical and immunohistochemical analysis of the repair tissue

after treatment with marrow-stimulation techniques. Knee Surg


22. de Girolamo L, Bertolini G, Cervellin M, Sozzi G, Volpi P. Treat-

ment of chondral defects of the knee with one step matrix-

assisted technique enhanced by autologous concentrated bone

marrow: In vitro characterisation of mesenchymal stem cells from

iliac crest and subchondral bone. Injury 2010;41(11):1172–1177.

23. Shi J, Zhang X, Zeng X, Zhu J, Pi Y, Zhou C, Ao Y. One-step artic-

ular cartilage repair: Combination of in situ bone marrow stem

cells with cell-free poly(L-lactic-co-glycolic acid) scaffold in a rab-

bit model. Orthopedics 2012;35(5):e665–e671.

24. Cavallo C, Desando G, Columbaro M, Ferrari A, Zini N, Facchini

A, Grigolo B. Chondrogenic differentiation of bone marrow con-

centrate grown onto a hylauronan scaffold: Rationale for its use

in the treatment of cartilage lesions J Biomed Mater Res A 2012

[Epub ahead of print].

25. Xie X, Wang Y, Zhao C, Guo S, Liu S, Jia W, Tuan RS, Zhang C.

Comparative evaluation of MSCs from bone marrow and adipose

tissue seeded in PRP-derived scaffold for cartilage regeneration.

Biomaterials 2012;33(29):7008–7018.

26. Yang Z, Wu Y, Li C, Zhang T, Zou Y, Hui JH, Ge Z, Lee EH.

Improved mesenchymal stem cells attachment and in vitro carti-

lage tissue formation on chitosan-modified poly(L-lactide-co-epsi-

lon-caprolactone) scaffold. Tissue Eng Part A 2012;18(3-4):242–

251.

27. Yoon IS, Chung CW, Sung JH, Cho HJ, Kim JS, Shim WS, Shim

CK, Chung SJ, Kim DD. Proliferation and chondrogenic differen-

tiation of human adipose-derived mesenchymal stem cells in po-

rous hyaluronic acid scaffold. J Biosci Bioeng 2011;112(4):402–

408.

28. You M, Peng G, Li J, Ma P, Wang Z, Shu W, Peng S, Chen GQ.

Chondrogenic differentiation of human bone marrow mesenchy-

mal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated

with PHA granule binding protein PhaP fused with RGD peptide.

Biomaterials 2011;32(9):2305–2313.

29. Erggelet C, Neumann K, Endres M, Haberstroh K, Sittinger M,

Kaps C. Regeneration of ovine articular cartilage defects by cell-

free polymer-based implants. Biomaterials 2007;28(36):5570–5580.

30. Chomczynski P. A reagent for the single-step simultaneous isola-

tion of RNA, DNA and proteins from cell and tissue samples. Bio-

techniques 1993;15(3):532–534, 536–537.

31. Neumann K, Dehne T, Endres M, Erggelet C, Kaps C, Ringe J, Sit-

tinger M. Chondrogenic differentiation capacity of human mesen-

chymal progenitor cells derived from subchondral cortico-

spongious bone. J Orthop Res 2008;26(11):1449–1456.

32. Behrens P. Matrix-coupled microfracture—A new concept for car-

tilage defect repair. Arthroskopie 2005;18:193–197.

33. Benthien JP, Behrens P. The treatment of chondral and osteo-

chondral defects of the knee with autologous matrix-induced

chondrogenesis (AMIC): Method description and recent develop-

ments. Knee Surg Sports Traumatol Arthrosc 2011;19(8):1316–

1319.

34. Dhollander AA, De Neve F, Almqvist KF, Verdonk R, Lambrecht S,

Elewaut D, Verbruggen G, Verdonk PC. Autologous matrix-

induced chondrogenesis combined with platelet-rich plasma gel:

Technical description and a five pilot patients report. Knee Surg


35. Hegewald AA, Ringe J, Bartel J, Kruger I, Notter M, Barnewitz D,

Kaps C, Sittinger M. Hyaluronic acid and autologous synovial

fluid induce chondrogenic differentiation of equine mesenchymal

stem cells: A preliminary study. Tissue Cell 2004;36(6):431–438.

36. Barry F, Boynton RE, Liu B, Murphy JM. Chondrogenic differentia-

tion of mesenchymal stem cells from bone marrow: Differentia-

tion-dependent gene expression of matrix components. Exp Cell

Res 2001;268(2):189–200.

37. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In

vitro chondrogenesis of bone marrow-derived mesenchymal pro-

genitor cells. Exp Cell Res 1998;238(1):265–2672.

38. Cals FL, Hellingman CA, Koevoet W, Baatenburg de Jong RJ, van

Osch GJ. Effects of transforming growth factor-beta subtypes on

in vitro cartilage production and mineralization of human bone

marrow stromal-derived mesenchymal stem cells. J Tissue Eng

Regen Med 2012;6(1):68–76.

39. Alves da Silva ML, Martins A, Costa-Pinto AR, Correlo VM, Sol P,

Bhattacharya M, Faria S, Reis RL, Neves NM. Chondrogenic differ-

entiation of human bone marrow mesenchymal stem cells in chi-

tosan-based scaffolds using a flow-perfusion bioreactor. J Tissue

Eng Regen Med 2011;5(9):722–732.



40. Bhardwaj N, Kundu SC. Chondrogenic differentiation of rat MSCs

on porous scaffolds of silk fibroin=chitosan blends. Biomaterials

2012;33(10):2848–2857.

41. Izal I, Aranda P, Sanz-Ramos P, Ripalda P, Mora G, Granero-Molto

F, Deplaine H, Gomez-Ribelles JL, Ferrer GG, Acosta V, Ochoa I,

Garc�ıa-Aznar JM, Andreu EJ, Monle�on-Pradas M, Doblar�e M,

Pr�osper F. Culture of human bone marrow-derived mesenchymal

stem cells on of poly(L-lactic acid) scaffolds: Potential application

for the tissue engineering of cartilage. Knee Surg Sports Trauma-

tol Arthrosc 2012 [Epub ahead of print].

42. Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan

AI, Goldberg VM. Mesenchymal cell-based repair of large, full-

thickness defects of articular cartilage. J Bone Joint Surg Am

1994;76(4):579–592.

43. Xie J, Han Z, Naito M, Maeyama A, Kim SH, Kim YH, Matsuda T.

Articular cartilage tissue engineering based on a mechano-active

scaffold made of poly(L-lactide-co-epsilon-caprolactone): In vivo

performance in adult rabbits. J Biomed Mater Res B Appl Bio-

mater 2010;94(1):80–88.

44. Han SH, Kim YH, Park MS, Kim IA, Shin JW, Yang WI, Jee KS,

Park KD, Ryu GH, Lee JW. Histological and biomechanical proper-

ties of regenerated articular cartilage using chondrogenic bone

marrow stromal cells with a PLGA scaffold in vivo. J Biomed

Mater Res A 2008;87(4):850–861.

45. Wakitani S, Okabe T, Horibe S, Mitsuoka T, Saito M, Koyama T,

Nawata M, Tensho K, Kato H, Uematsu K, Kuroda R, Kurosaka M,

Yoshiya S, Hattori K, Ohgushi H. Safety of autologous bone mar-

row-derived mesenchymal stem cell transplantation for cartilage

repair in 41 patients with 45 joints followed for up to 11 years

and 5 months. J Tissue Eng Regen Med 2011;5(2):146–150.

46. Gigante A, Calcagno S, Cecconi S, Ramazzotti D, Manzotti S, Enea

D. Use of collagen scaffold and autologous bone marrow concen-

trate as a one-step cartilage repair in the knee: Histological results

of second-look biopsies at 1 year follow-up. Int J Immunopathol

Pharmacol 2011;24(1 Suppl 2):69–72.

47. Giordano A, Galderisi U, Marino IR. From the laboratory bench to

the patient’s bedside: An update on clinical trials with mesenchy-

mal stem cells. J Cell Physiol 2007;211(1):27–35.

48. Kuroda R, Ishida K, Matsumoto T, Akisue T, Fujioka H, Mizuno K,

Ohgushi H, Wakitani S, Kurosaka M. Treatment of a full-thickness

articular cartilage defect in the femoral condyle of an athlete with

autologous bone-marrow stromal cells. Osteoarthritis Cartilage

2007;15(2):226–231.

49. Wakitani S, Imoto K, Yamamoto T, Saito M, Murata N, Yoneda M.

Human autologous culture expanded bone marrow mesenchymal

cell transplantation for repair of cartilage defects in osteoarthritic

knees. Osteoarthritis Cartilage 2002;10(3):199–206.

50. Wakitani S, Mitsuoka T, Nakamura N, Toritsuka Y, Nakamura Y,

Horibe S. Autologous bone marrow stromal cell transplantation

for repair of full-thickness articular cartilage defects in human

patellae: Two case reports. Cell Transplant 2004;13(5):595–600.


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