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ABSTRACT: The assessment of cartilage repair has largely been limited to macroscopic observation, magnetic ..... dylar lesion treatments demonstrated a high degree of variability ..... colleagues29 suggested that tissue-engineered car-.
Matrix-Induced Autologous Chondrocyte Implantation in Sheep: Objective Assessments Including Confocal Arthroscopy C.W. Jones,1,2 C. Willers,2 A. Keogh,2 D. Smolinski,1 D. Fick,2 P.J. Yates,2 T.B. Kirk,1 M.H. Zheng2 1

School of Mechanical Engineering, University of Western Australia, 35 Stirling Highway, Crawley WA, 6009, Australia

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School of Pathology and Surgery, Department of Orthopaedics, University of Western Australia, 2nd Floor M-block QEII Medical Centre, Nedlands, Perth WA, 6009, Australia Received 18 August 2006; accepted 10 July 2007 Published online 27 September 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20502 ABSTRACT: The assessment of cartilage repair has largely been limited to macroscopic observation, magnetic resonance imaging (MRI), or destructive biopsy. The aims of this study were to establish an ovine model of articular cartilage injury repair and to examine the efficacy of nondestructive techniques for assessing cartilage regeneration by matrix-induced autologous chondrocyte implantation (MACI). The development of nondestructive assessment techniques facilitates the monitoring of repair treatments in both experimental animal models and human clinical subjects. Defects (Ø 6 mm) were created on the trochlea and medial femoral condyle of 21 sheep randomized into untreated controls or one of two treatment arms: MACI or collagen-only membrane. Each group was divided into 8-, 10-, and 12-week time points. Repair outcomes were examined using laser scanning confocal arthroscopy (LSCA), MRI, histology, macroscopic ICRS grading, and biomechanical compression analysis. Interobserver analysis of the randomized blinded scoring of LSCA images validated our scoring protocol. Pearson correlation analysis demonstrated the correlation between LSCA, MRI, and ICRS grading. Testing of overall treatment effect independent of time point revealed significant differences between MACI and control groups for all sites and assessment modalities (Asym Sig < 0.05), except condyle histology. Biomechanical analysis suggests that while MACI tissue may resemble native tissue histologically in the early stages of remodeling, the biomechanical properties remain inferior at least in the short term. This study demonstrates the potential of a multisite sheep model of articular cartilage defect repair and its assessment via nondestructive methods. ß 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:292–303, 2008

Keywords: cartilage repair; matrix-induced autologous chondrocyte impantation (MACI); confocal microscopy

INTRODUCTION It has long been recognized that once damaged, articular cartilage may progress from difficult-totreat lesions to osteoarthritis. The problems associated in treating the initial lesions have prompted the development of a number of approaches, most recently matrix-induced autologous chondrocyte implantation (MACI).1,2 MACI utilizes a type I/III collagen bioscaffold to traffic cultured autologous chondrocytes into the defect, thereby obviating periosteal harvesting and assoThis article includes Supplementary Material available via the Internet at http://www.interscience.wiley.com/jpages/07360266/suppmat. Correspondence to: M.H. Zheng (Telephone: þ61 8 93464050; Fax: þ61 8 93463210; E-mail: [email protected]) ß 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

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ciated donor site morbidity on the tibia.2,3 While excellent clinical results have been achieved, significant controversy remains regarding the histological nature of the tissue formed.4,5 In conjunction with the International Cartilage Repair Society (ICRS), Brittberg and Winalski have standardized the clinical assessment, arthroscopic evaluation, and magnetic resonance imaging (MRI) of focal cartilage lesions and their repair.6 In combination with clinical measures, the ICRS system provides an overall assessment of cartilage repair, but provides no histological data. Histological assessment of cartilage repair tissue distinguishes between tissue types.4,7 Previous studies utilized mechanical biopsy to provide tissue samples for histological analysis.3,4,8,9 A limited amount of data exist regarding the cellular and microstructural nature of MACI repair tissue due to early demonstrations of clinical efficacy and

MACI IN SHEEP

reluctance to take biopsy samples in human patients. Indeed, Poole cautioned against mechanical biopsy of articular grafts due to the danger of sustaining further damage.5 Ergo, the development of the laser scanning confocal arthroscope (LSCA), an optical biopsy tool capable of nondestructively imaging articular cartilage repair tissue in situ and in vivo.10,11 The need for controlled trials of alternative repair techniques indicates the further development of reliable large animal models. In this study, our objectives were to establish a large animal model of articular cartilage injury repair and to analyze the effectiveness of MACI objectively using the nondestructive techniques of LSCA and MRI in comparison to the gold standard of histological assessment. Mechanical analysis of repair tissue was also conducted.

MATERIALS AND METHODS Experimental Design Twenty-one sheep were randomly divided into three groups. Defect-only control animals were compared to animals surgically treated with MACI or type I/III collagen membrane alone. After surgical intervention, animals were divided into three time periods for final assessment at 8 (2  MACI, 2  collagen, 2  control), 10 (3  MACI, 3  collagen, 3  control), and 12 weeks (2  MACI, 2  collagen, 2  control; see also Supplementary Material). Surgical Procedure All operations were conducted under strict guidelines of the National Health and Medical Research Council (Canberra, Australia). All surgery was performed by the orthopedic surgeons named as coauthors. Animals were sedated and anaesthetized, administered with antibiotic prophylaxis, and provided with pre- and postoperative analgesia (ketamine 11 mg/kg, xylaxine 0.22 mg/kg, keflin 1 mg, carprofin 2–4 mg/kg, buprenorphine/temgesic 1 ml IM). Unilateral stifle joints were surgically accessed via a medial parapatellar approach, and standardized partial thickness (1.5 mm) trochlea and medial femoral condyle defects were created using a custom-designed 6 mm chondral punch. Nonweightbearing cartilage from the lateral supracondyle was harvested for cell culture. Defect debris was removed and the base of the defect leveled via surgical curette. Following defect creation the wound was closed via interrupted capsular (1 vicryl) and subcuticular sutures (3-0 monocryl). Animals were not immobilized, but movement was confined for 24 h postoperatively. Using a previously described protocol for cultivation of rabbit and human chondrocytes,3,12 autologous chondrocytes were isolated under high sterility conditions, expanded to >5  106 cells, and seeded onto collagen membrane (porcinederived type I/III collagen membrane; Matricel, Herzo-

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genrath, Germany). Six weeks after surgery, implantation was undertaken via the previous parapatellar approach with repunching and debridement performed to maintain a well-circumscribed defect and remove any fibrous repair tissue. The chondrocyte-seeded collagen membrane was shaped to match the defect geometry, and press-fit onto the floor of the defect with Tisseel1 fibrin sealant (Baxter, Vienna, Austria). The second treatment arm of the study involved implantation of defects with acellular type I/III collagen membrane in an identical manner to MACI surgical protocol. Defect-only controls were established as untreated partial-thickness lesions in comparable sites. Euthanasia of sheep was performed at each specified time point and imaging carried out immediately. ICRS Cartilage Repair Assessment ICRS grading was conducted during open joint examination (prior to LSCA imaging) by a single orthopedic surgeon. Grading was corroborated by the consultant histopathologists on en bloc specimens. Cartilage repair tissue was evaluated according to the ICRS Cartilage Injury Evaluation Package (Protocol A) considering macroscopic appearance, volume of defect filled, and integration with adjacent cartilage.6 Laser Scanning Confocal Arthroscopy LSCA immediately followed ICRS assessment and enabled imaging in situ without mechanical biopsy or tissue processing. The LSCA uses a proprietary optical fiber scanner both performing the laser delivery (488 nm argon-ion laser) and acting as the confocal pinhole and filtration mechanism.10,11,13 The LSCA is a 4.4-mm diameter arthroscopically mounted miniaturized confocal microscope providing an xy spatial resolution of 2 mm across a field of view of approximately 500  500 mm (512  512 pixels) and focal plane penetration of 200 mm at 2 Hz (see Supplementary Material). The fluorophores were acridine orange (0.5 g/L, 30 mL, 30–40 min) and fluorescein (5 g/L, 30mL, 40–50 min; Molecular Probes Inc., Eugene, OR). Stains were prepared immediately prior to imaging in 0.9% phosphate-buffered saline (PBS) at physiological pH and temperature (378C) and stored away from light. Optimal staining concentration, volume, and times were established during a previous study.10 Lavage with 0.9% PBS (300 mL) was performed prior to imaging until all excess stain was removed. More than 2,500 individual confocal images were edited to three representative images for each site, blinded, computer randomized, and distributed to two histopathologists and three orthopedic surgeons for modality scoring (Table 1). Blinding of the treatment group and time point was maintained by code known only to the chief investigator for the duration of the analysis. Histology and Immunohistochemistry Distal femora were fixed then decalcified with 10% formic acid. Specimen blocks (8  8 mm) were prepared JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008

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Table 1. Modality Scoring and Overall Repair Assessment Grading Modality Scoring MRI Cartilage Repair Assessment Criteria 1. Infill of the repair site Excellent: complete Good: >50% of the defect Fair: 75% homogeneous Good: >50% homogeneous Fair: heterogeneous with no clefts Poor: heterogeneous with clefts 5. Border integration Excellent: complete integration Good: minor gap Fair: incomplete, gap visible Poor: incomplete, visible defect 6. Subchondral lamina Excellent: fully reconstituted Good: >50% intact Fair: