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Research article

Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model Anke Lohan a,∗ , Ulrike Marzahn b , Karym El Sayed a , Andreas Haisch b , Riccarda Dolores Müller a , Benjamin Kohl a , Katharina Stölzel b , Wolfgang Ertel a , Thilo John a,1 , Gundula Schulze-Tanzil a,1 a b

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

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Article history: Received 26 September 2013 Received in revised form 7 March 2014 Accepted 8 March 2014 Available online xxx Keywords: Chondrocytes Cartilage repair Articular Auricular Rabbit model Polyglycolic acid

s u m m a r y Hypothesizing that the implantation of non-articular (heterotopic) chondrocytes might be an alternative approach to support articular cartilage repair, we analyzed joint cartilage defect healing in the rabbit model after implantation of autologous auricle-derived (auricular) chondrocytes. Autologous lapine articular and auricular chondrocytes were cultured for 3 weeks in polyglycolic acid (PGA) scaffolds before being implanted into critical sized osteochondral defects of the rabbit knee femoropatellar groove. Cell-free PGA scaffolds and empty defects served as controls. Construct quality was determined before implantation and defect healing was monitored after 6 and 12 weeks using vitality assays, macroscopical and histological score systems. Neo-cartilage was formed in the PGA constructs seeded with both articular and auricular chondrocytes in vitro and in vivo. At the histological level, cartilage repair was slightly improved when using autologous articular chondrocyte seeded constructs compared to empty defects and was significantly superior compared to defects treated with auricular chondrocytes 6 weeks after implantation. Although only the immunohistological differences were significant, auricular chondrocyte implantation induced an inferior healing response compared with the empty defects. Elastic auricular chondrocytes might maintain some tissue-specific characteristics when implanted into joint cartilage defects which limit its repair capacity. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction Injured cartilage possesses only a poor intrinsic healing capacity. Matrix-assisted autologous chondrocytes transplantation (MACT) is a strategy to cover larger cartilage defects. The limited availability of autologous articular chondrocytes restricts the application of this tissue engineering (TE) based technique. However, the implantation of mesenchymal stem cells (MSCs) as alternative cell source also presents several limits such as, low cell numbers of MSCs in the bone marrow (Beane and Darling, 2012), the necessity of their careful characterization, the presence of several cell subpopulations

∗ Corresponding author at: Department of Trauma and Reconstructive Surgery, Charité-University of Medicine, Campus Benjamin Franklin, FEM, Garystraße 5, 14195 Berlin, Germany. Tel.: +49 30 450 552385; fax: +49 30 450 552985. E-mail address: [email protected] (A. Lohan). 1 Joined senior authorship.

(Mafi et al., 2011), the content of already committed cells, the time consuming chondrogenic differentiation procedure, the instability of chondrogenic phenotype and their uncontrolled differentiation in other lineages (Pelttari et al., 2008) particularly in view of the influence of inflammatory mediators in injured cartilage (Wehling et al., 2009). Non-articular “heterotopic” chondrocytes such as nasoseptal chondrocytes or auricle-derived “auricular” chondrocytes might serve as an alternative cartilage source to cover joint cartilage defects as discussed previously (El Sayed et al., 2010, 2013; Van Osch et al., 2004). Heterotopic cartilage is easier to harvest, associated with lower donor site morbidity and heterotopic chondrocytes possess a higher proliferation rate (El Sayed et al., 2010, 2013; Van Osch et al., 2004). The compatibility of auricular chondrocytes with articular chondrocytes has been indicated by several in vitro co-culturing approaches (El Sayed et al., 2013; Kuhne et al., 2010). Previously, nasoseptal chondrocytes which represent another promising cartilage source were implanted into joint cartilage defects in rabbits resulting in a hyaline-like repair

http://dx.doi.org/10.1016/j.aanat.2014.03.002 0940-9602/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Lohan, A., et al., Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.03.002

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Fig. 1. Implanted cartilage cylinder after in vitro culture. A 3.5 mm diameter disc is punched out of a PGA scaffold seeded with articular chondrocytes (a1 ) for implantation. An auricular chondrocytes containing (a2 ) scaffold cylinder is implanted into the osteochondral defects. Vitality assays (green: vital, red: dead cells) (b1 -b2 ), HE- (c1 -c2 ) and AB- (d1 -d2 ) stainings after 3 weeks of culture. Native lapine articular cartilage (e1 -f1 ) and auricular cartilage surrounded by a perichondrium (e2 -f2 ). Table 1 Macroscopical Score. Color of defect area Bright white Dull white, translucent Yellow, rose, brown, red Level of the defect At the niveau of surrounding healthy tissue Slight above the level Prominent above the level No defect filling Defect surface Intact, smooth Smooth and harsh area Uneven, harsh No defect filling Defect margin Detectable Hardly detectable Not detectable Integration to the surrounding bounding Connected Slightly connected Not connected Inflammation/osteophytes Not existing Moderate (swelling, redness) Severe or osteophytes

2 1 0 3 2 1 0 3 2 1 0 2 1 0 2 1 0 2 1 0

tissue (El Sayed et al., 2012; Vinatier et al., 2009a, 2009b). Additionally, costal chondrocytes have been implanted in joint cartilage defects of rabbits and pigs leading to defect filling (Gelse et al., 2009; Szeparowicz et al., 2006). Auricular chondrocytes cultured on PGA exhibited a high chondrogenic potential in the nude mice xenograft model which was comparable with that of articular chondrocytes (Lohan et al., 2011, 2013). Moreover, Zhang and Spector detected lubricin expression in constructs seeded with auricular chondrocytes (Zhang and Spector, 2009). Lubricin is a typical joint cartilage glycoprotein produced by chondrocytes of the superficial zone which is important for joint lubrication (Jay et al., 2007). In contrast to articular cartilage, auricular cartilage is covered by a perichondrium, contains elastic fibers and does not show a defined zonality. Growth parameters of auricular chondrocytes of the rabbit have been thoroughly characterized previously (Frohlich et al., 2007; Van Osch et al., 2004). The rabbit is a very common model for the study of cartilage repair (Ahern et al., 2009). PGA is a non-toxic, biodegradable biomaterial which has been characterized for cartilage repair in large animal studies (Erggelet et al., 2009; Liu et al., 2002). However, it remains unclear whether auricular chondrocytes seeded in a PGA scaffold produce a hyaline-like repair tissue during joint cartilage defect healing. Hence, in the present study we analyzed the healing of osteochondral articular defects after

Please cite this article in press as: Lohan, A., et al., Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.03.002

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Table 2 Histological Score. Nature of the predominant repair tissue Cellular morphology (After 6 Weeks) Hyaline articular cartilage Incomplete differentiated mesenchymal-like tissue Fibrous tissue or bone (After 12 weeks): completed healing of cartilage and bone heterogeneous healing of cartilage or bone no healing of cartilage and bone bone formation at the surface Alcian blue staining of the matrix Normal or nearly normal Moderate Slight None Structural characteristics Surface regularity Smooth and intact Superficial horizontal lamination Fissures–25 to 100 per cent of the thickness Severe disruption, including fibrillation Structural integrity Normal Slight disruption, including cysts Severe disintegration Thickness 100 per cent of normal adjacent cartilage 50–90% smooth, homogeneous 50–90% smooth, homogeneous 50–90% fibrous > 90% fibrous Type I collagen Type I collagen content Normal, physiological (not detectable) Slightly increased Severely increased Type I collagen distribution Homogeneous, physical Heterogeneous Predominantly heterogeneous

3 2 1 0 1.5 1 0.5 1.5 1 0.5 3 2 1 0

2 1 0 1.5 1 0.5

[Biochrom AG], 25 ␮g/mL ascorbic acid [Sigma–Aldrich], 50 IU/mL streptomycin, 50 IU/mL penicillin, 0.5 ␮g/mL partricin, 1 mg/mL essential amino acids, 2 mM l-glutamine [all: Biochrom AG]). Cells were expanded for less than four monolayer passages. 2.2. Dynamic 3D PGA cultures For sterilization, biodegradable woven PGA meshes (Biofelt 65 mg/cc, Concordia Medical, USA) were incubated for 5 min in 100% isopropanol, dried for 30 min at 30 ◦ C and washed three times with aqua dest. PGA meshes (5 mm × 5 mm × 3.5 mm) were soaked in 5 mL rabbit chondrocyte suspension in growth medium (8 × 106 chondrocytes of the second or third monolayer passage) prepared from each of the two chondrocyte sources. Dynamic cultures were performed by rotating (36 rpm) the cell suspension together with the floating PGA felt in a bioreactor filter tube (TPP, Switzerland) on a rotatory shaker (digital tube roller Stuart SRT9D, Bibby Scientific, USA) at 37 ◦ C and 5% CO2 . Constructs were cultured for 21 days before implantation whereby growth medium was changed three-times a week.

2. Materials and methods 2.3. Assessment of cell vitality in PGA cultures 2.1. Isolation and culturing of rabbit chondrocytes Autologous rabbit cartilage was harvested from the auricle or the knee joints of female New Zealand White (NZW) rabbits (age: 12 months, n = 27, Charles River, Sulzfeld, Germany). The study was approved by the National Animal Care and Use Committee (LAGeSo Berlin, Germany). The connective tissue and perichondrium of auricular cartilage were carefully removed. One mm slices of rabbit cartilage samples were enzymatically digested with 0.4% (w/v) pronase with gentle shaking (7 U/mg; Roche Diagnostics, Basel, Switzerland) diluted in Ham’s F-12/Dulbecco’s modified Eagle’s (DMEM) medium 1:1 [Biochrom AG, Berlin, Germany] for 20 min at 37 ◦ C and subsequently digested with 0.2% (w/v) collagenase (≥0.1 U/mg; SERVA Electrophoresis, Heidelberg, Germany) diluted in growth medium for 1–2 h at 37 ◦ C. Isolated chondrocytes were resuspended at 28.000 cells/cm2 in T25 flasks in growth medium (Ham’s F-12/DMEM 1:1 containing 10% fetal calf serum (FCS)

After washing with PBS, the 21-day-old scaffolds were incubated in fluorescein diacetate (FDA, Sigma-Aldrich) (3 ␮g/mL dissolved in acetone [stock solution] and further diluted 1: 1000 in PBS [working solution]) for 15 min at 37 ◦ C, rinsed three times with 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 in the dark at room temperature (RT). The green or red fluorescence was visualized using fluorescence microscopy (Axioskop 40, Carl Zeiss, Jena, Germany) and a XC30 camera system (Olympus, Europa Holding, 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

Please cite this article in press as: Lohan, A., et al., Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.03.002

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4 Table 3b Immunohistological Score after 12 weeks in vivo. Type II collagen in the cartilage defect zone Type II collagen content Normal, physiological Slightly reduced Moderate reduced Not detectable Zonation of the surface compared profound defect Increased staining Equal staining >50% decreased staining Not detectable Type II collagen distribution Homogeneous, physiological Heterogeneous Predominantly heterogeneous Quality of the ECM >90% smooth, homogeneous 50–90% smooth, homogeneous 50–90% fibrous >90% fibrous Type II collagen in the bone defect zone: Type II collagen content Physiological (not detectable) Slightly increased Moderate increased Severe increased Type II collagen distribution Not detectable Slightly increased Moderate increased Severe increased Type I collagen in the bone defect zone Type I collagen content Normal, physiological Slightly reduced Not detectable Type I collagen distribution >50% positive staining