Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model

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Annals of Anatomy

j ourna l h omepage: www.elsev ier .de /aanat

esearch article

steochondral articular defect repair using auricle-derivedutologous chondrocytes in a rabbit model

nke Lohana,∗, Ulrike Marzahnb, Karym El Sayeda, Andreas Haischb,iccarda Dolores Müllera, Benjamin Kohla, Katharina Stölzelb, Wolfgang Ertela,hilo Johna,1, Gundula Schulze-Tanzil a,1

Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-University of Medicine, Campus Benjamin Franklin, Berlin, GermanyDepartment of Otorhinolaryngology, Head and Neck Surgery, Charité-University of Medicine, Campus Benjamin Franklin, Berlin, Germany

r t i c l e i n f o

rticle history:eceived 26 September 2013eceived in revised form 7 March 2014ccepted 8 March 2014vailable online xxx

eywords:hondrocytesartilage repairrticularuricularabbit model

s u m m a r y

Hypothesizing that the implantation of non-articular (heterotopic) chondrocytes might be an alternativeapproach to support articular cartilage repair, we analyzed joint cartilage defect healing in the rabbitmodel after implantation of autologous auricle-derived (auricular) chondrocytes.

Autologous lapine articular and auricular chondrocytes were cultured for 3 weeks in polyglycolicacid (PGA) scaffolds before being implanted into critical sized osteochondral defects of the rabbit kneefemoropatellar groove. Cell-free PGA scaffolds and empty defects served as controls. Construct qualitywas determined before implantation and defect healing was monitored after 6 and 12 weeks using vitalityassays, macroscopical and histological score systems.

Neo-cartilage was formed in the PGA constructs seeded with both articular and auricular chondrocytesin vitro and in vivo. At the histological level, cartilage repair was slightly improved when using autolo-gous articular chondrocyte seeded constructs compared to empty defects and was significantly superior

olyglycolic acid compared to defects treated with auricular chondrocytes 6 weeks after implantation. Although only theimmunohistological differences were significant, auricular chondrocyte implantation induced an inferiorhealing response compared with the empty defects.

Elastic auricular chondrocytes might maintain some tissue-specific characteristics when implantedinto joint cartilage defects which limit its repair capacity.

© 2014 Elsevier GmbH. All rights reserved.

. Introduction

Injured cartilage possesses only a poor intrinsic healing capacity.atrix-assisted autologous chondrocytes transplantation (MACT)

s a strategy to cover larger cartilage defects. The limited availabil-ty of autologous articular chondrocytes restricts the application ofhis tissue engineering (TE) based technique. However, the implan-ation of mesenchymal stem cells (MSCs) as alternative cell source

Please cite this article in press as: Lohan, A., et al., Osteochondral articin a rabbit model. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.a

lso presents several limits such as, low cell numbers of MSCs in theone marrow (Beane and Darling, 2012), the necessity of their care-ul characterization, the presence of several cell subpopulations

∗ Corresponding author at: Department of Trauma and Reconstructive Surgery,harité-University of Medicine, Campus Benjamin Franklin, FEM, Garystraße 5,4195 Berlin, Germany. Tel.: +49 30 450 552385; fax: +49 30 450 552985.

E-mail address: [email protected] (A. Lohan).1 Joined senior authorship.

ttp://dx.doi.org/10.1016/j.aanat.2014.03.002940-9602/© 2014 Elsevier GmbH. All rights reserved.

(Mafi et al., 2011), the content of already committed cells, the timeconsuming chondrogenic differentiation procedure, the instabilityof chondrogenic phenotype and their uncontrolled differentiationin other lineages (Pelttari et al., 2008) particularly in view of theinfluence of inflammatory mediators in injured cartilage (Wehlinget al., 2009). Non-articular “heterotopic” chondrocytes such asnasoseptal chondrocytes or auricle-derived “auricular” chondro-cytes might serve as an alternative cartilage source to cover jointcartilage defects as discussed previously (El Sayed et al., 2010,2013; Van Osch et al., 2004). Heterotopic cartilage is easier to har-vest, associated with lower donor site morbidity and heterotopicchondrocytes possess a higher proliferation rate (El Sayed et al.,2010, 2013; Van Osch et al., 2004). The compatibility of auricu-lar chondrocytes with articular chondrocytes has been indicated

ular defect repair using auricle-derived autologous chondrocytesanat.2014.03.002

by several in vitro co-culturing approaches (El Sayed et al., 2013;Kuhne et al., 2010). Previously, nasoseptal chondrocytes whichrepresent another promising cartilage source were implanted intojoint cartilage defects in rabbits resulting in a hyaline-like repair

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Fig. 1. Implanted cartilage cylinder after in vitro culture. A 3.5 mm diameter disc is punchAn auricular chondrocytes containing (a2) scaffold cylinder is implanted into the osteochAB- (d1-d2) stainings after 3 weeks of culture. Native lapine articular cartilage (e1-f1) and

Table 1Macroscopical Score.

Color of defect areaBright white 2Dull white, translucent 1Yellow, rose, brown, red 0

Level of the defectAt the niveau of surrounding healthy tissue 3Slight above the level 2Prominent above the level 1No defect filling 0

Defect surfaceIntact, smooth 3Smooth and harsh area 2Uneven, harsh 1No defect filling 0

Defect marginDetectable 2Hardly detectable 1Not detectable 0

Integration to the surrounding boundingConnected 2Slightly connected 1Not connected 0

Inflammation/osteophytesNot existing 2Moderate (swelling, redness) 1Severe or osteophytes 0

ed out of a PGA scaffold seeded with articular chondrocytes (a1) for implantation.ondral defects. Vitality assays (green: vital, red: dead cells) (b1-b2), HE- (c1-c2) and

auricular cartilage surrounded by a perichondrium (e2-f2).

tissue (El Sayed et al., 2012; Vinatier et al., 2009a, 2009b). Addi-tionally, costal chondrocytes have been implanted in joint cartilagedefects of rabbits and pigs leading to defect filling (Gelse et al.,2009; Szeparowicz et al., 2006). Auricular chondrocytes culturedon PGA exhibited a high chondrogenic potential in the nude micexenograft model which was comparable with that of articular chon-drocytes (Lohan et al., 2011, 2013). Moreover, Zhang and Spectordetected lubricin expression in constructs seeded with auricularchondrocytes (Zhang and Spector, 2009). Lubricin is a typical jointcartilage glycoprotein produced by chondrocytes of the superficialzone which is important for joint lubrication (Jay et al., 2007). Incontrast to articular cartilage, auricular cartilage is covered by aperichondrium, contains elastic fibers and does not show a definedzonality. Growth parameters of auricular chondrocytes of the rab-bit have been thoroughly characterized previously (Frohlich et al.,2007; Van Osch et al., 2004). The rabbit is a very common model forthe study of cartilage repair (Ahern et al., 2009). PGA is a non-toxic,biodegradable biomaterial which has been characterized for carti-lage repair in large animal studies (Erggelet et al., 2009; Liu et al.,2002).


However, it remains unclear whether auricular chondrocytesseeded in a PGA scaffold produce a hyaline-like repair tissue dur-ing joint cartilage defect healing. Hence, in the present studywe analyzed the healing of osteochondral articular defects after

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Table 2Histological Score.

Nature of the predominant repair tissueCellular morphology

(After 6 Weeks) Hyaline articular cartilage 4Incomplete differentiated mesenchymal-like tissue 2Fibrous tissue or bone 0(After 12 weeks): completed healing of cartilage and bone 4heterogeneous healing of cartilage or bone 3no healing of cartilage and bone 2bone formation at the surface 0

Alcian blue staining of the matrixNormal or nearly normal 3Moderate 2Slight 1None 0

Structural characteristicsSurface regularity

Smooth and intact 3Superficial horizontal lamination 2Fissures–25 to 100 per cent of the thickness 1Severe disruption, including fibrillation 0

Structural integrityNormal 2Slight disruption, including cysts 1Severe disintegration 0

Thickness100 per cent of normal adjacent cartilage 250–<100 per cent of normal cartilage 10–50 per cent of normal cartilage 0

Bonding to the adjacent cartilageBonded at both ends of graft 2Bonded at one end, or partially at both ends 1Not bonded 0

Absence of cellular changes or degenerationHypo/hypercellularity

Normal cellularity 2Moderate hypo/hypercellularity 1Severe hypo/hypercellularity 0

Chondrocytes and matrix stainingNormal cell morphology, normal staining 3Cell cluster, normal staining 2Cell degeneration, reduced staining 1Severe cell degeneration, poor or no staining 0

Absence of degenerative changes in adjacent cartilageNormal cellularity, no clusters, normal staining 3Normal cellularity, mild clusters, moderate staining 2

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Table 3aImmunohistological Score after 6 weeks in vivo.

Type II collagenType II collagen content

Normal, physiological 3Slightly reduced 2Moderate reduced 1Not detectable 0

Type II collagen distributionHomogeneous, physiological 1.5Heterogeneous 1Predominantly heterogeneous 0.5

Zonation of the surface compared to profound defectIncreased staining 1.5Equal staining 1Decreased staining 0.5

Quality of the ECM>90% smooth, homogeneous 350–90% smooth, homogeneous 250–90% fibrous 1> 90% fibrous 0

Type I collagenType I collagen content

Normal, physiological (not detectable) 2Slightly increased 1Severely increased 0

Type I collagen distributionHomogeneous, physical 1.5

Mild to moderate hypocellularity, slight staining 1Severe hypocellularity, poor or no staining 0

mplantation of autologous auricular chondrocytes in the rabbitodel and compared it to the outcome in response to autologous

orthotopic) articular chondrocytes implantation.

. Materials and methods

.1. Isolation and culturing of rabbit chondrocytes

Autologous rabbit cartilage was harvested from the auricle orhe knee joints of female New Zealand White (NZW) rabbits (age:2 months, n = 27, Charles River, Sulzfeld, Germany). The study waspproved by the National Animal Care and Use Committee (LAGeSoerlin, Germany). The connective tissue and perichondrium ofuricular cartilage were carefully removed. One mm slices of rab-it cartilage samples were enzymatically digested with 0.4% (w/v)ronase with gentle shaking (7 U/mg; Roche Diagnostics, Basel,witzerland) diluted in Ham’s F-12/Dulbecco’s modified Eagle’sDMEM) medium 1:1 [Biochrom AG, Berlin, Germany] for 20 mint 37 ◦C and subsequently digested with 0.2% (w/v) collagenase


≥0.1 U/mg; SERVA Electrophoresis, Heidelberg, Germany) dilutedn growth medium for 1–2 h at 37 ◦C. Isolated chondrocytes wereesuspended at 28.000 cells/cm2 in T25 flasks in growth mediumHam’s F-12/DMEM 1:1 containing 10% fetal calf serum (FCS)

Heterogeneous 1Predominantly heterogeneous 0.5

[Biochrom AG], 25 �g/mL ascorbic acid [Sigma–Aldrich], 50 IU/mLstreptomycin, 50 IU/mL penicillin, 0.5 �g/mL partricin, 1 mg/mLessential amino acids, 2 mM l-glutamine [all: Biochrom AG]). Cellswere expanded for less than four monolayer passages.

2.2. Dynamic 3D PGA cultures

For sterilization, biodegradable woven PGA meshes (Biofelt65 mg/cc, Concordia Medical, USA) were incubated for 5 min in100% isopropanol, dried for 30 min at 30 ◦C and washed three timeswith aqua dest. PGA meshes (5 mm × 5 mm × 3.5 mm) were soakedin 5 mL rabbit chondrocyte suspension in growth medium (8 × 106

chondrocytes of the second or third monolayer passage) preparedfrom each of the two chondrocyte sources. Dynamic cultures wereperformed by rotating (36 rpm) the cell suspension together withthe floating PGA felt in a bioreactor filter tube (TPP, Switzerland)on a rotatory shaker (digital tube roller Stuart SRT9D, Bibby Sci-entific, USA) at 37 ◦C and 5% CO2. Constructs were cultured for 21days before implantation whereby growth medium was changedthree-times a week.

2.3. Assessment of cell vitality in PGA cultures

After washing with PBS, the 21-day-old scaffolds were incu-bated in fluorescein diacetate (FDA, Sigma-Aldrich) (3 �g/mLdissolved in acetone [stock solution] and further diluted 1: 1000in PBS [working solution]) for 15 min at 37 ◦C, rinsed three timeswith PBS before being counterstained with propidium iodide (PI,Sigma–Aldrich) solution (1 mg/mL dissolved in PBS [stock solution]and further diluted 1:100 in PBS [working solution]) for 1 min inthe dark at room temperature (RT). The green or red fluorescencewas visualized using fluorescence microscopy (Axioskop 40, CarlZeiss, Jena, Germany) and a XC30 camera system (Olympus, EuropaHolding, Hamburg, Germany).


2.4. Rabbit osteochondral defect model of the knee joint

Chondrocyte-seeded PGA constructs were implanted (Fig. 1a1-a2) into osteochondral defects of adult NZW rabbits. The animals

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Table 3bImmunohistological Score after 12 weeks in vivo.

Type II collagen in the cartilage defect zoneType II collagen content

Normal, physiological 1.5Slightly reduced 1Moderate reduced 0.5Not detectable 0

Zonation of the surface compared profound defectIncreased staining 1.5Equal staining 1>50% decreased staining 0.5Not detectable 0

Type II collagen distributionHomogeneous, physiological 1.5Heterogeneous 1Predominantly heterogeneous 0.5

Quality of the ECM>90% smooth, homogeneous 1.550–90% smooth, homogeneous 150–90% fibrous 0.5>90% fibrous 0

Type II collagen in the bone defect zone:Type II collagen content

Physiological (not detectable) 1.5Slightly increased 1Moderate increased 0.5Severe increased 0

Type II collagen distributionNot detectable 1.5Slightly increased 1Moderate increased 0.5

Severe increased 0Type I collagen in the bone defect zone

Type I collagen contentNormal, physiological 1Slightly reduced 0.5Not detectable 0

Type I collagen distribution>50% positive staining 1.5<50% positive staining 1Not detectable 0

Type I collagen distributionHomogeneous, physical 1.5Heterogeneous 1Not detectable 0.5

Type I collagen in the cartilage defect zoneType I collagen content

Physiological (not detectable) 1Slightly increased 0.5Severe increased 0

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performed to determine E modulus and failure load. The osteochon-

ere divided randomly into four different groups consisting ofhree animals each, depending on the source of chondrocytes (scaf-olds seeded with articular or auricular chondrocytes) and usingon-seeded scaffolds and empty defects as controls. Two points inime (6 and 12 weeks) were monitored (experimental set up: sup-lemental Table 1). For macroscopical analysis, three additionalnimals receiving implants seeded with articular chondrocytesrovided for biomechanical analysis could be included (in total

= 27). These additional samples could not be used for histologyue to tissue damage. To get the auricular biopsy, the rabbits werenesthetized using 12 mg/kg ketanest (100 mg/ml ketamine, WDT,arbsen, Germany) and 120 �g/kg domitor (1 mg/ml medeto-idine, Janssen Animal Health, Neuss, Germany). Cylinders of

uricular cartilage tissue, 2 × 4 mm in diameter, were punchedut under sterile conditions. For the articular cartilage biopsy, thenimals were anesthetized as described above. In the right rab-it knees, the autologous cartilage biopsies were taken whereby


wo 2 mm diameter full thickness cartilage fragments were ablatedfter parapatellar arthrotomy from the ultimate cranial end ofhe trochlea groove. The isolation of autologous chondrocytes was

PRESSomy xxx (2014) xxx–xxx

performed as described above. The rabbits received carprofen(4 mg/kg body weight, rimadyl, Pfizer, Karlsruhe, Germany) and5 mg/kg gentamycin (genta 5%, WDT) subcutaneously for 3 daysas pre- and postmedication. The construct implantation was per-formed in the left knees. Therefore, a parapatellar skin and fascialincision of 1 cm was made. The patellar ligament was split longitu-dinally within its medial third. The patella was luxated in a lateraldirection. Using a diamond dusted hollow drill a reproducible3.5 mm diameter and 2.7 mm deep defect was created in the midof the trochlear groove. The constructs were punched to a size of3.5 mm diameter immediately before implantation (Fig. 1a1). Sub-sequently, they were rinsed with autologous rabbit serum beforebeing implanted “press fit” into the defects (Fig. 1a2). The cell-freescaffolds were soaked 24 h with growth medium before implan-tation and then treated in a similar manner. Finally, the woundwas closed and the rabbits received 4 mg/kg carprofen and 5 mg/kggentamycin subcutaneously for three days as pre- and post- med-ication. Animals were sacrificed at 6 or 12 weeks after constructimplantation using intracardial T61 injection (2 ml/kg, Intervet,Unterschleißheim, Germany). The defect site was macroscopicallyscored using a macroscopical scoring system which was devel-oped to assess the cartilage quality in vivo in the rabbit (Table 1).Then samples were documented photographically using a CanonEOS500D camera with a Canon EF-S60 mm macro objective (Canon,Krefeld, Germany). Subsequently, explanted osteochondral articu-lar blocks were fixed in 4% paraformaldehyde solution for 3 days,decalcified in 100 mM/L EDTA solution (pH 8.0, Merck, Darmstadt,Germany) before being embedded in paraffin.

2.5. Histological analysis and type II and I collagenimmunolabelling

Deparaffinized paraffin sections (5 �m) of the tissue engi-neered constructs were stained with Hematoxylin Eosin (HE) andalcian blue (AB) using a protocol as already described (Lohanet al., 2013). For immunolabelling, they were rinsed in Trisbuffered saline (TBS: 0.05 M Tris, 0.015 M NaCl, pH 7.6) beforebeing incubated with 5 mg/mL pronase (7 U/mg, Roche, Basel,Switzerland) diluted in TBS for 5 min at 37 ◦C. Sections weresubsequently rinsed with TBS, blocked with protease-free don-key serum (5% diluted in TBS) for 30 min at RT and incubatedwith the polyclonal rabbit anti-type II or type I collagen anti-bodies (27.5 �g/mL, Acris Antibodies, Herford, Germany) in ahumidifier chamber overnight at 4 ◦C. Sections were subsequentlywashed with TBS before incubation with donkey-anti-rabbit-Alexa-Fluor®488 (10 mg/mL, Invitrogen) secondary antibody for30 min at RT. Negative controls included omitting the primaryantibody during the staining procedure. Cell nuclei were counter-stained using 4′,6- diamidino-2-phenylindole (DAPI) (0.1 �g/mL,Roche). Sections were rinsed several times with TBS and embeddedwith Fluoromount G (Southern Biotech, Biozol Diagnostica, Bir-mingham, USA). Histological or immunohistological samples wereanalyzed using a histological score based on O‘Driscoll (O’Driscollet al., 1988) and an immunohistological scoring system (Tables 2and 3) and using an Axioskop 40 microscope (Zeiss). Photos weretaken using an Olympus camera XC30 (Olympus Soft ImagingSolutions).

2.6. Biomechanical analysis

Biomechanical analysis of articular chondrocytes constructs 12weeks after implantation into joint cartilage defects of rabbits were


dral cylinders were obtained with a diamonded hollow drill fromthe defect area at the knee joint (n = 3). As controls, osteochondralcylinders from healthy cartilage and bone of the contralateral knee

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ig. 2. Results of macroscopical scoring after 6 and 12 weeks of implantation. A sum = 6). Macroscopical pictures of the healing response after 6 and 12 weeks of implb1-b2) and auricular (c1-c2) chondrocytes seeded and non-seeded PGA (d1-d2) scaffo

oint were prepared (n = 5). Immediately after explanation, biome-hanical testing was performed. The samples were placed betweenwo indentors (diameter: 4 mm) allowing free lateral deformation.

echanical indentation of the osteochondral cylinders were testedn 5 N steps starting with a compression of 5 N until failure loadcontrol: maximum load 100 N). The elastic (E-) modulus was cal-ulated according to the standard formula.

.7. Statistical analysis

All values were expressed as mean with standard deviation.ata was analyzed using unpaired Student’s t test (GraphPad


rism 5, GraphPad software inc, San Diego, USA). Statisticalignificance was set at a p value of ≤0.05. Gaussian distribu-ion was determined performing the Kolmogorov–Smirnov-testDallal–Wilkinson–Lillifor). The histological data were analyzed

of the macroscopical scoring is depictured (a, n = 3, articular chondrocytes 12 weekson. Representative pictures of the defect repair in response to autologous articularplanted into rabbit osteochondral defects. Empty defects served as controls (e1-e2).

using a non-parametric Kruskal–Wallis test followed by a post hocDunns test.

3. Results

3.1. Dynamic 3D PGA cultures: cell vitality and ECM formation

Most of the autologous articular and auricular chondrocytes sur-vived within the scaffolds for the three weeks of culturing, wherebyonly few dead cells could be demonstrated (Fig. 1b1-b2). Typicallacunae surrounded the chondrocytes within their abundant ECM


(Fig. 1c1-c2). The ECM was AB positive indicating its content of sul-phated proteoglycans (Fig. 1d1-d2), very similar to native lapinearticular (Fig. 1e1-f1) and auricular cartilage (Fig. 1e2-f2). PGA fiberremnants could still be detected during the 3 weeks of culture. The

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ig. 3. Results of histological scoring after 6 and 12 weeks of implantation, respectiistological pictures of the defect after 6 and 12 weeks of implantation. Representat

c1-c2) chondrocytes seeded and non-seeded PGA (d1-d2). Empty defects served as

ells were mostly homogenously distributed within the ECM of theonstructs.

.2. Macroscopical scoring: neo-cartilage quality

The differences observed macroscopically between the groupsere not significant (Fig. 2a and b1-e2). The empty defects revealed

ome self-healing. At 6 weeks post implantation, the rabbit grouphich received the cell-free scaffolds achieved the highest sco-

ing numbers, followed by the group which received the scaffoldseeded with articular chondrocytes when compared with thempty defect group. Auricular chondrocyte implantation resultedn inferior macroscopical scores compared with that of the emptyefects after both time points (6 or 12 weeks) post surgery. At 12eeks post implantation, the non-seeded scaffolds lead to slightly

nferior results when compared with the empty defect group.

.3. Histological assessment of defect repair


Different histological features such as height of repair tissue,ounding to the surrounding cartilage, integrity and qual-

ty of the neo-tissue within the defect were summarizedsing a histological scoring system (O’Driscoll et al., 1988)

he diagram summarizes the results of the histological scoring (a, p = 0.0284, n = 3).tures of slices of the defect in response to autologous articular (b1-b2) and auricularls (e1-e2). Asterisk: defect areas.

(Fig. 3a, 3b1-e2, 4a1-d4 and 5a1-d4). At 6 and 12 weeks post implan-tation, the defect repair was superior in the groups which receivedarticular chondrocytes containing constructs compared with theempty defects (not significant). The non-seeded scaffold, althoughnot statistically significant, lead to slightly better results at 6 and12 weeks post implantation compared with the empty defects.At 6 weeks, the results with auricular chondrocytes were sig-nificantly inferior compared to the articular chondrocyte treatedgroup. At 12 weeks, the difference was no longer significant. Occa-sional singular fiber remnants could be found after 6 or 12 weeksin the repair tissue (not shown). Only faint inflammatory fea-tures could be detected such as some immigrated inflammatorycells surrounding the repair tissue, whereby this observation wasmore sporadic when articular chondrocytes were implanted com-pared with auricular chondrocyte implantation (not shown). Insome cases, a perichondrium-like tissue became evident after 6 and12 weeks when auricular chondrocyte constructs were implanted(Fig. 4b1, black double-head arrow).


3.4. Immunohistological results of defect repair

Type II and type I collagen expression was evaluated in the repairtissue (Fig. 6a and supplementary Fig. 1a1-d4). The implants seeded

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Fig. 4. Histological pictures of healing response after 6 weeks of implantation. Representative pictures of the HE and AB staining of articular (a1-a4) and auricular (b1-b4)chondrocyte seeded and non-seeded PGA scaffolds (c -c ) implanted into rabbit cartilage defects (knee joint). Empty defects served as controls (d -d ). The border betweenu , aste

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noffended and regenerated area in two magnifications is shown. Scale bars 100 �m

ith articular chondrocytes at 6 weeks lead to superior resultsompared with the other rabbit groups. Implantation of the earhondrocytes into the artificial joint cartilage defect resulted in aignificantly inferior repair tissue compared with the empty defectroup after 12 weeks. Although the same difference was detectablet 6 weeks, it was not significant. The implants containing articu-ar or auricular chondrocytes or cell-free scaffolds lead to inferioresults compared to the empty defect group at 12 weeks (not sig-ificant). Cysts could also be detected e.g. in non-seeded implantsFig. 3d1).

.5. Biomechanical

Macroscopically, the explanted osteochondral cylinder of therticular chondrocytes showed no difference to the native con-rol cylinders. However, the E-moduli were significantly lowerhan the moduli of the healthy osteochondral cylinder (Fig. 6b1).ealthy native osteochondral cylinders could be loaded with aaximum force of 100 N with no apparent loss of structure

Fig. 6b2). In contrast, the osteochondral defects which received


onstructs seeded with articular chondrocytes showed a failuref an average of 60 N. Here, the osteochondral remodeling zonef the osteochondral cylinder breaks down resulting in a loss oftructure.

1 4

risk: defect; black double-head arrow: perichondrium-like tissue.

4. Discussion

Heterotopic non-articular cartilage might be utilized as arecruiting source for additional autologous chondrocytes for artic-ular cartilage repair. In 2009, Vinatier et al. reported that theimplantation of nasoseptal chondrocytes embedded in fibrin ora cellulose-based hydrogel into joint cartilage defects of the rab-bit knee lead to a hyaline-like repair tissue (Vinatier et al., 2009a,2009b). In the present study, we decided to test auricular chon-drocytes as a promising heterotopic cartilage source of articularcartilage healing based on the results of our previous experiment(Lohan et al., 2011, 2013) which indicated their high chondrogenicpotential. Additionally, biopsies of auricular cartilage are associatedwith low donor morbidity (El Sayed et al., 2010, 2013; Van Oschet al., 2004). Since the articular cartilage layer in the rabbit kneejoint is rather small (∼0.3 mm) (Ahern et al., 2009) an osteochon-dral defect model was performed in this study. In this setting, theimmigration of MSCs from the opened bone marrow cavity into theosteochondral defects contributes considerably to the defect repair(Chu et al., 2010). In fact, the ingrowth of bone marrow-derivedcells into the defects was clearly detectable in some of the histo-


logical samples. In the present study the implanted chondrocyteswere not traced to pursue and distinguish them from immigratinghost cells since cell tracking could influence chondrocyte biology,viability and therefore, could also affect cartilage repair. Paracrine

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ig. 5. Histological pictures of healing response after 12 weeks of implantation. Rehondrocyte seeded and non-seeded PGA scaffolds (c1-c4) implanted into rabbit carnoffended and regenerated area is shown in a low and higher magnifications. Scal

ffects of the implanted chondrocytes e.g. on the immigrating MSCsight play an essential role in osteochondral defect healing (Gelse

t al., 2009). Various co-culture models combining chondrocytesith MSCs revealed a chondro-inductive effect of chondrocytes onSCs and vice versa (El Sayed et al., 2012). The implanted chondro-

ytes were pre-cultured for 21 days in the PGA scaffolds beforen vivo implantation was performed. This is a rather long time,

hich was chosen to gain a stable preformed ECM in the constructased on previous investigations (Lohan et al., 2011). For futurepplications the pre-incubation period could be shortened – a facthich might influence the integration of the construct and bound-

ng to the margin of the defects. PGA was chosen as a scaffold sincet is a well established reference biomaterial for cartilage repairErggelet et al., 2009; Liu et al., 2002; Zhou et al., 2006) which allowsartilage formation in vitro (El Sayed et al., 2013; Lohan et al., 2011).

and 12 weeks are rather short, but commonly used investigationoints of time in view of cartilage defect healing (Funayama et al.,008; Lubiatowski et al., 2006; Nagura et al., 2007; Shangkai et al.,007; Szeparowicz et al., 2006). Nonetheless they are suitable torovide a first impression of the repair response. Cartilage and boneealing has to be distinguished in the osteochondral defect modelt both observation points of time. At 12 weeks, bone healing haddvanced substantially compared with 6 weeks, but it remainedncomplete, however.

It is well known that the rabbit shows some self-healing capac-ty of cartilage (Ahern et al., 2009; Chu et al., 2010). Since thentrinsic cartilage repair capacity is greater in immature animals,nly adult animals (at least more than 12 month old) were includedn the present study (Wei et al., 1997). The epiphyses are closeds a common reference point for adolescence after 12 month inhe rabbit (Woo et al., 1990). Maturity in rabbits is achieved at


n age between 16 and 39 weeks or 9 month (Ahern et al., 2009;einholz et al., 2004). The critical defect size is reported to be

mm in diameter (Ahern et al., 2009; Chu et al., 2010) and hence,efects of a size larger than 3.5 mm were created in the present

ntative pictures of the HE and AB staining of articular (a1-a4) and auricular (b1-b4) defects (knee joint). Empty defects served as controls (d1-d4). The border between

100 �m, asterisk: defect; arrow: immigrating bone marrow-derived cells.

study. The defect depth of 2.7 mm used here was also proposedby Chu et al. for cartilage repair in the rabbit model (Chu et al.,2010). Auricular chondrocytes are known to produce elastic fibers(Moskalewski, 1981) which were not analyzed in the present study,since we did not find them in a previous study after 12 weeksin the nude mice xenograft model (Gelse et al., 2009). Moreover,the zonal architecture seems to be less distinct and is mostlyunknown for cartilage of the auricle (Fig. 1e2-f2). The repair tis-sue did not display any zonality, either in response to articular orto auricular chondrocytes, or cell-free PGA implantation as well asin the empty defects. Madry et al. could show an enhanced car-tilage repair following overexpression of IGF-I tissue-engineeredconstructs in an osteochondral defect, but in accordance with ourresults, they could not see a zonality of the chondrocytes (Madryet al., 2013). However, the formation of distinct cartilage zonesmight appear at a later investigation point of time after 12 weeks.Also bone healing remained mostly incomplete in all treatmentgroups at 12 weeks post implantation. This result is confirmed byKim et al. who analyzed osteochondral tissue regeneration usinga bilayered composite hydrogel with modulating dual growth fac-tor release kinetics in an osteochondral rabbit model. At 12 weekspost-implantation, the quality of tissue repair in subchondral lay-ers was analyzed based on quantitative histological scoring (Kimet al., 2013). The formation of a complete subchondral bone platein the defect area should be monitored using later investigationpoints of time. In addition to the greater cell content in auricularcompared with articular cartilage, cultured auricular chondrocytesare known to proliferate four times faster than articular chondro-cytes (Henderson et al., 2007; Panossian et al., 2001; Van Oschet al., 2004). However, the cell content in the repair tissues didnot show major differences. Perichondrium-like structures could


be observed during cartilage repair in some animals in the presentstudy when auricular chondrocytes were implanted. This cell-richconnective tissue seemed to interfere with the integration of therepair tissue into the joint cartilage. The cell-free PGA scaffolds

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Fig. 6. Results are shown of immunohistological scoring after 6 and 12 weeks ofimplantation and biomechanical analyses of articular chondrocytes 12 weeks afterimplantation. A summary of the immunohistological scoring is shown (a, p = 0.002,n = 3). Biomechanical analyses of articular chondrocytes seeded on PGA scaffolds(ua

ledstdamLctluqhTldca

aatsd

6tTg

n = 3) 12 weeks after implantation into an osteochondral defect and a native,ntreated osteochondral cylinder (n = 5) was performed. E-modulus (b1, p = 0.0019)nd failure load (b2, 100 N is the end of the measurement) was analyzed.

ed to good results when compared to the empty defects. Emanst al. analyzed the effect of scaffold architecture on osteochon-ral defect repair and the development of (OA) using the Mankincore. They found less OA in the animal group which receivedhe three-dimensional fiber scaffold group compared to emptyefects (Emans et al., 2013). These cell-free scaffolds might provide

matrix for cell adhesion and allow a more rapid ingrowth ofesenchymal stromal cells from the open bone marrow cavities.

ooking at different aspects of cartilage defect healing (macroscopi-al appearance, histological organization, distribution of type II andype I collagen), the constructs based on auricular chondrocytesead to a slightly inferior repair tissue quality compared with artic-lar chondrocyte implantation. In contrast to this observation, theuality of in vitro cultured auricular chondrocyte constructs wasighly comparable with that containing articular chondrocytes.he biomechanical analysis of the constructs seeded with articu-ar chondrocytes and implanted for 12 weeks in the osteochondralefects revealed an inferior repair tissue elasticity and stabilityompared to native osteochondral cylinders, probably mainly as

result of incomplete subchondral bone healing.Taken together, the results of this study indicate that elastic

uricular chondrocytes might maintain some tissue-specific char-cteristics during the in vitro culture and in vivo implantation, e.g.he formation of a perichondrium like tissue and, therefore, ourtudy suggests that auricular chondrocytes are not suitable for theefect repair of osteochondral defects.

Further, monitoring of the advanced healing processes, e.g. after


months, would provide additional information in future. The syn-hetic program of auricular chondrocytes might be adapted usingE strategies supported by supplementation of particular anabolicrowth factors or cytokines.

PRESSomy xxx (2014) xxx–xxx 9

Acknowledgement

The authors are grateful for technical assistance of Carola Meierand Claudia Conrad. This study was supported by grants from BayerInnovation GmbH, Duesseldorf, Germany and the Sonnenfeld Foun-dation, Berlin, Germany.

Appendix A. Supplementary data

Supplementary material related to this article can befound, in the online version, at http://dx.doi.org/10.1016/j.aanat.2014.03.002.

References

Ahern, B.J., Parvizi, J., Boston, R., Schaer, T.P., 2009. Preclinical animal models insingle site cartilage defect testing: a systematic review. Osteoarthritis Cartilage17, 705–713.

Beane, O.S., Darling, E.M., 2012. Isolation, characterization, and differentiation ofstem cells for cartilage regeneration. Ann. Biomed. Eng. 40, 2079–2097.

Chu, C.R., Szczodry, M., Bruno, S., 2010. Animal models for cartilage regenerationand repair. Tissue Eng. Part B Rev. 16, 105–115.

El Sayed, K., Haisch, A., John, T., Marzahn, U., Lohan, A., Muller, R.D., Kohl, B., Ertel,W., Stoelzel, K., Schulze-Tanzil, G., 2010. Heterotopic autologous chondrocytetransplantation—a realistic approach to support articular cartilage repair? TissueEng. Part B Rev. 16, 603–616.

El Sayed, K., Zreiqat, H., Ertel, W., Schulze-Tanzil, G., 2012. Stimulated chondro-genesis via chondrocytes co-culturing. J. Biochip Tissue Chip S2:003, 2–11,http://dx.doi.org/10.4172/2153-0777.S2-003.

El Sayed, K., Marzahn, U., John, T., Hoyer, M., Zreiqat, H., Witthuhn, A., Kohl, B.,Haisch, A., Schulze-Tanzil, G., 2013. PGA-associated heterotopic chondrocytecocultures: implications of nasoseptal and auricular chondrocytes in articularcartilage repair. J. Tissue Eng. Regen. Med. 7, 61–72.

Emans, P.J., Jansen, E.J., van Iersel, D., Welting, T.J., Woodfield, T.B., Bulstra, S.K.,Riesle, J., van Rhijn, L.W., Kuijer, R., 2013. Tissue-engineered constructs: theeffect of scaffold architecture in osteochondral repair. J. Tissue Eng. Regen. Med.7, 751–756.

Erggelet, C., Endres, M., Neumann, K., Morawietz, L., Ringe, J., Haberstroh, K., Sit-tinger, M., Kaps, C., 2009. Formation of cartilage repair tissue in articular cartilagedefects pretreated with microfracture and covered with cell-free polymer-basedimplants. J. Orthop. Res. 27, 1353–1360.

Frohlich, M., Malicev, E., Gorensek, M., Knezevic, M., Kregar Velikonja, N., 2007. Eval-uation of rabbit auricular chondrocyte isolation and growth parameters in cellculture. Cell Biol. Int. 31, 620–625.

Funayama, A., Niki, Y., Matsumoto, H., Maeno, S., Yatabe, T., Morioka, H., Yanagimoto,S., Taguchi, T., Tanaka, J., Toyama, Y., 2008. Repair of full-thickness articularcartilage defects using injectable type II collagen gel embedded with culturedchondrocytes in a rabbit model. J. Orthop. Sci. 13, 225–232.

Gelse, K., Brem, M., Klinger, P., Hess, A., Swoboda, B., Hennig, F., Olk, A., 2009.Paracrine effect of transplanted rib chondrocyte spheroids supports formationof secondary cartilage repair tissue. J. Orthop. Res. 27, 1216–1225.

Henderson, J.H., Welter, J.F., Mansour, J.M., Niyibizi, C., Caplan, A.I., Dennis, J.E.,2007. Cartilage tissue engineering for laryngotracheal reconstruction: compari-son of chondrocytes from three anatomic locations in the rabbit. Tissue Eng. 13,843–853.

Jay, G.D., Torres, J.R., Warman, M.L., Laderer, M.C., Breuer, K.S., 2007. The role oflubricin in the mechanical behavior of synovial fluid. Proc. Natl. Acad. Sci. U. S.A. 104, 6194–6199.

Kim, K., Lam, J., Lu, S., Spicer, P.P., Lueckgen, A., Tabata, Y., Wong, M.E., Jansen, J.A.,Mikos, A.G., Kasper, F.K., 2013. Osteochondral tissue regeneration using a bila-yered composite hydrogel with modulating dual growth factor release kineticsin a rabbit model. J. Control. Rel. 168, 166–178.

Kuhne, M., John, T., El-Sayed, K., Marzahn, U., Aue, A., Kohl, B., Stoelzel, K., Ertel,W., Blottner, D., Haisch, A., Schulze-Tanzil, G., 2010. Characterization of auricu-lar chondrocytes and auricular/articular chondrocyte co-cultures in terms of anapplication in articular cartilage repair. Int. J. Mol. Med. 25, 701–708.

Liu, Y., Chen, F., Liu, W., Cui, L., Shang, Q., Xia, W., Wang, J., Cui, Y., Yang, G., Liu, D., Wu,J., Xu, R., Buonocore, S.D., Cao, Y., 2002. Repairing large porcine full-thicknessdefects of articular cartilage using autologous chondrocyte-engineered carti-lage. Tissue Eng. 8, 709–721.

Lohan, A., Marzahn, U., El Sayed, K., Bock, C., Haisch, A., Kohl, B., Stoelzel, K., John,T., Ertel, W., Schulze-Tanzil, G., 2013. Heterotopic and orthotopic autologouschondrocyte implantation using a minipig chondral defect model. Ann. Anat..

Lohan, A., Marzahn, U., El Sayed, K., Haisch, A., Kohl, B., Muller, R.D., Ertel, W.,Schulze-Tanzil, G., John, T., 2011. In vitro and in vivo neo-cartilage formation


by heterotopic chondrocytes seeded on PGA scaffolds. Histochem. Cell Biol. 136,57–69.

Lubiatowski, P., Kruczynski, J., Gradys, A., Trzeciak, T., Jaroszewski, J., 2006. Artic-ular cartilage repair by means of biodegradable scaffolds. Transplant. Proc. 38,320–322.

dx.doi.org/10.1016/j.aanat.2014.03.002

http://dx.doi.org/10.1016/j.aanat.2014.03.002

http://dx.doi.org/10.1016/j.aanat.2014.03.002

http://refhub.elsevier.com/S0940-9602(14)00031-4/sbref0005









































































dx.doi.org/10.4172/2153-0777.S2-003















































































































































































































































































































ING ModelA

1 f Anat

M

M

N

O

M

P

P

R

S

ARTICLEANAT-50858; No. of Pages 10

0 A. Lohan et al. / Annals o

adry, H., Kaul, G., Zurakowski, D., Vunjak-Novakovic, G., Cucchiarini, M., 2013. Car-tilage constructs engineered from chondrocytes overexpressing IGF-I improvethe repair of osteochondral defects in a rabbit model. Eur. Cell Mater. 25,229–247.

oskalewski, S., 1981. The elastogenetic process in transplants and cultures of iso-lated auricular chondrocytes. Connect. Tissue Res. 8, 171–174.

agura, I., Fujioka, H., Kokubu, T., Makino, T., Sumi, Y., Kurosaka, M., 2007. Repairof osteochondral defects with a new porous synthetic polymer scaffold. J. BoneJoint Surg. Br. 89, 258–264.

’Driscoll, S.W., Keeley, F.W., Salter, R.B., 1988. Durability of regenerated articularcartilage produced by free autogenous periosteal grafts in major full-thicknessdefects in joint surfaces under the influence of continuous passive motion. Afollow-up report at one year. J. Bone Joint Surg. Am. 70, 595–606.

afi, P., Hindocha, S., Mafi, R., Griffin, M., Khan, W.S., 2011. Adult mesenchymal stemcells and cell surface characterization - a systematic review of the literature.Open Orthop. J. 5, 253–260.

anossian, A., Ashiku, S., Kirchhoff, C.H., Randolph, M.A., Yaremchuk, M.J., 2001.Effects of cell concentration and growth period on articular and ear chondrocytetransplants for tissue engineering. Plast. Reconstr. Surg. 108, 392–402.

elttari, K., Steck, E., Richter, W., 2008. The use of mesenchymal stem cells for chon-drogenesis. Injury 39 (Suppl 1), S58–S65.

einholz, G.G., Lu, L., Saris, D.B., Yaszemski, M.J., O’Driscoll, S.W., 2004. Animal mod-


els for cartilage reconstruction. Biomaterials 25, 1511–1521.hangkai, C., Naohide, T., Koji, Y., Yasuji, H., Masaaki, N., Tomohiro, T., Yasushi, T.,

2007. Transplantation of allogeneic chondrocytes cultured in fibroin spongeand stirring chamber to promote cartilage regeneration. Tissue Eng. 13, 483–492.

PRESSomy xxx (2014) xxx–xxx

Szeparowicz, P., Popko, J., Sawicki, B., Wolczynski, S., 2006. Is the repair of articularcartilage lesion by costal chondrocyte transplantation donor age-dependent?An experimental study in rabbits. Folia Histochem. Cytobiol. 44, 201–206.

Van Osch, G.J., Mandl, E.W., Jahr, H., Koevoet, W., Nolst-Trenite, G., Verhaar, J.A.,2004. Considerations on the use of ear chondrocytes as donor chondrocytes forcartilage tissue engineering. Biorheology 41, 411–421.

Vinatier, C., Gauthier, O., Fatimi, A., Merceron, C., Masson, M., Moreau, A., Moreau, F.,Fellah, B., Weiss, P., Guicheux, J., 2009a. An injectable cellulose-based hydrogelfor the transfer of autologous nasal chondrocytes in articular cartilage defects.Biotechnol. Bioeng. 102, 1259–1267.

Vinatier, C., Gauthier, O., Masson, M., Malard, O., Moreau, A., Fellah, B.H., Bilban,M., Spaethe, R., Daculsi, G., Guicheux, J., 2009b. Nasal chondrocytes and fibrinsealant for cartilage tissue engineering. J. Biomed. Mater. Res. A 89, 176–185.

Wehling, N., Palmer, G.D., Pilapil, C., Liu, F., Wells, J.W., Muller, P.E., Evans, C.H., Porter,R.M., 2009. Interleukin-1beta and tumor necrosis factor alpha inhibit chon-drogenesis by human mesenchymal stem cells through NF-kappaB-dependentpathways. Arthritis Rheum. 60, 801–812.

Wei, X., Gao, J., Messner, K., 1997. Maturation-dependent repair of untreated osteo-chondral defects in the rabbit knee joint. J. Biomed. Mater. Res. 34, 63–72.

Woo, S.L., Ohland, K.J., Weiss, J.A., 1990. Aging and sex-related changes in the biome-chanical properties of the rabbit medial collateral ligament. Mech. Ageing Dev.56, 129–142.


Zhang, L., Spector, M., 2009. Comparison of three types of chondrocytes in collagenscaffolds for cartilage tissue engineering. Biomed. Mater. 4, 045012.

Zhou, G., Liu, W., Cui, L., Wang, X., Liu, T., Cao, Y., 2006. Repair of porcine artic-ular osteochondral defects in non-weightbearing areas with autologous bonemarrow stromal cells. Tissue Eng. 12, 3209–3221.

dx.doi.org/10.1016/j.aanat.2014.03.002

































































































































































































































































































































































































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Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model