Development of Partial Thickness Articular Cartilage Injury in an Ovine Model

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1 Development of Partial Thickness Articular Cartilage Injury in an Ovine Model Yan Lu, 1 Mark D. Markel, 1 Carol Swain, 2 Lee D. Kaplan 2 1 Comparative Orthopaedic Research Laboratory, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin Division of Sports Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, Wisconsin Received 15 February 2006; accepted 12 May 2006 Published online 7 August 2006 in Wiley InterScience ( DOI /jor ABSTRACT: The purpose of this study was to create a controlled partial thickness cartilage lesion in a sheep model, and to provide a foundation to study the natural history of the progression of this lesion. Twenty-eight sheep divided into four groups (1, 12, 24, and 52 weeks, n ¼ 7/group) were used in this study. In one stifle, a mechanical tool was used to create a 200 mm partial thickness lesion ( cm 2 ) on the medial femoral condyle via arthroscopy. Joint fluid was drawn presurgery and after euthanasia for analysis of collage II 3/4 C long (C2C). After euthanasia, the condyle was analyzed by gross appearance, confocal laser microscopy (CLM) for cell viability, scanning electronic microscopy (SEM) for surface roughness, Artscan for cartilage stiffness, and histology for cartilage morphology. The gross appearance of the treated area appeared rough, soft, and swollen compared to untreated control over time. CLM demonstrated that the depth of cell death increased to 590 mm at 52 weeks after surgery. SEM demonstrated that the treated area became more irregular over time. Stiffness of the treated area was significantly less than control by 12 weeks after surgery. Histologic analysis demonstrated that the 12, 24, and 52 week groups had significantly poorer histologic scores than the 1 week group. Joint fluid analysis demonstrated that the treatment group at 1 week had significant higher levels of C2C than the pretreatment baseline data. The results of this study demonstrated that partial thickness injury of cartilage continued to propagate and degenerate over time in this sheep model. Options for the prevention or treatment of this lesion may be tested using this model in the future. ß 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24: , 2006 Keywords: articular cartilage; chondrocyte; partial thickness; sheep; confocal laser microscopy INTRODUCTION Partial thickness articular cartilage (PARC) lesions are commonly encountered in orthopedic surgery. 1 These lesions do not have the ability to heal themselves, due to lack of vascular supply and the reliance on synovial nutrition. 2 However, Hunziker and Rosenberg 3 demonstrated that the failure of PARC lesions to heal is not entirely due to the lack of access to mesenchymal cells in perivascular tissue, although their experiments did not result in repaired cartilage. Yoshioka et al. 4 also reported that the repair mechanisms of partial cartilage injury differed according to injury direction using a rat model. Better repair Correspondence to: Lee D. Kaplan (Telephone: ; Fax: ; kaplan@orthorehab.wisc.edu) ß 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. was obtained when the injury was parallel to the direction of joint motion rather than perpendicular to joint motion. The clinical quandary presented by PARC lesions is one with no clear answer. 5 Surgical techniques and options have focused on stabilization of PARC lesions to halt further degeneration. These techniques include mechanical and thermal chondroplasty The use of thermal energy whether laser or radio-frequency generated has been controversial. The primary issue is the viability and denaturation of the remaining articular cartilage after thermal treatment. 7,11 16 Recently, there has been increased focus on the various techniques used to evaluate the cartilage after treatment This has included histological techniques such as hematoxylin and eosin staining and confocal laser microscopy. The utilization of metabolic techniques may add significant information regarding the cellular chondrocyte activity 1974 JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2006

2 PARC INJURY IN AN OVINE MODEL 1975 that occurs following cartilage injury. 14 A combination of all available techniques for cartilage evaluation should provide the most accurate assessment of treatment modalities. The basic science of the natural history of PARC lesions is necessary to fully evaluate current and future treatment techniques. Therefore, an in vivo animal model would provide important information with regard to the clinical applicability of these techniques. To date, the literature on articular cartilage lesions has focused primarily on fulldepth lesions, with only a few studies methodically addressing partial-thickness lesions. 3,20,21 These studies have examined the ability of the lesion to repair itself, or to improve repair through supplemental growth factors in rabbits and miniature swine. 3,20,21 In 2002, Marijnissen et al. 22,23 reported using a Kirschner-wire to make grooves on the weight-bearing area of the femoral condyles in order to induce osteoarthritis (OA) in a canine model. The results of these studies demonstrated that the degenerative joint damage in the canine groove model was slowly progressive over time and showed characteristics identical to those seen in the anterior cruciate ligament (ACL) transaction model. In 2006, Mastbergen et al. 24 further demonstrated that changes observed in the canine groove model were not just the expression of the surgically applied damage but were the result of progressive features of experimental OA. However, the Kirschner-wire used to create damage of the articular cartilage in these studies was not well controlled. The purpose of this study was to create a more precisely controlled PARC lesion that was confined to the cartilage and did not lead to global OA, which would be evaluated by morphology, histology, cell viability, and collagen II destruction over time. This study should serve the orthopedic community by establishing a natural history of PARC lesions and providing a novel, well-characterized model for testing current and future treatment options for these lesions. MATERIALS AND METHODS Twenty-eight mature female sheep, ranging in age from 2 to 5 years and weighing between 60 and 100 kg ( kg: mean SD) were utilized in the study. All experimental protocols were approved by the Institutional Animal Use and Care Committee. Joint fluid was collected from both treatment and contralateral control knee joints in each sheep just before surgery as baseline data for the analysis of collagen II 3/4 C long (C2C) using a technique as previously described. 25 Surgery was carried out under general anesthesia with halothane and oxygen inhalation via endotracheal intubation. Using aseptic techniques, arthroscopy was performed in one randomly selected ovine stifle. Each joint was approached via a stab incision lateral to the distal aspect of the patellar ligament to allow insertion of the arthroscope for the visualization of the medial condyle. A mechanical shaver was used to remove the fat pad for a clearer view if necessary. A second stab incision was made medial to the distal aspect of the patellar ligament to allow insertion of a custom-designed Dremel tool. The Dremel tool with spring-coil controlled pressure tip (depth of tool teeth: 200 mm) was applied to the central surface of the medial condyle to create an approximately cm 2 roughened area (lesion depth of approximately 200 mm) under arthroscopic guidance (Fig. 1A, B). The spring-coil on the tip Figure 1. Image demonstrating the Dremel tool with spring-coil controlled pressure tip (A) roughening the ovine medial condyle (B). After treatment, the condylar surface appeared rough and irregular (C). DOI /jor JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2006

3 1976 LU ET AL. maintained a pressure force of approximately 250 g. If the pressure force applied on the tip was greater than 250 g, the spring-coil would be over-bent to prevent deeper damage on the cartilage surface. The roughened cartilaginous surface approximated grade 2 chondromalacia with protruding fine fronds based on the Outerbridge system (Fig. 1C). The joints were irrigated by lactated Ringer s solution at room temperature during the operation. The contralateral stifle served as an untreated control. After surgery, the operated sheep were allowed to move freely. Sheep were sacrificed at 1, 12, 24, and 52 weeks after surgery (n ¼ 7/time group). After euthanasia, joint fluid was drawn from both treatment and control joints for the analysis of C2C as previously described, 25 then both treatment and control medial condyles were harvested and digitally photographed for evaluation of gross appearance of the cartilaginous surfaces. Following photography, articular cartilage stiffness measurements using the Artscan 2000 TM probe (Artscan Oy., Helsinki, Finland) were taken of the treated area in the treated stifle and the relevant identical area of the control stifle as described by a previous study. 26 The Artscan 2000 TM probe is an electromechanical indentation probe that can be used to measure mechanical stiffness of tissue. It consists of a measurement rod (5 mm in diameter and 150 mm in length), an indenter (300 mm in diameter and 1.3 mm in length) located at the end of the rod and in the center of a 208 inclined reference plate, a bending beam attached to the indenter via two force transducers that measure force applied to the tissue surface, and another pair of transducers that measure force applied on the reference plate against the cartilage surface. Consistent measurements were taken by applying a constant 10 N force to the cartilage surface for 1 s intervals over 60 s, and recording the mean indenter force, which is a measure of stiffness. An average of three mean stiffness values was taken and calculated. After Artscan mechanical testing, harvested medial condyles were cut by band saw with phosphate-buffered saline (PBS) irrigation to prevent frictional heat into small osteochondral blocks including the treatment area with 0.5 cm of its associated subchondral bone. A lowspeed saw equipped with PBS solution flushing (Buehler, Isomet 2000, Lake Bluff, IL) was used to cut the osteochondral block into two slices with a thickness of 1.5 and 2.5 mm, respectively. The 1.5 mm thick slices were analyzed for chondrocyte viability by confocal laser microscopy (CLM) on the same day of the harvest. After taking the CLM image, the same specimen was used for general histologic analysis. The 2.5 mm thick slice was processed for scanning electron microscopy (SEM). The 1.5 mm thick cartilage slices were stained by incubation in a 1.0 ml PBS containing 0.4 ml calcein (acetoxymethylester)/10 ml ethidium homodimer (Molecular Probes, Eugene, OR) for 30 min at room temperature. The method of determining the location of surviving cells was based on the knowledge that viable and nonviable cells differ in their ability to exclude fluorescent dyes. The cell membranes of dead, damaged, or dying cells are penetrated by ethidium homodimer to stain their nuclei red. Living cells with intact plasma membranes and active esterases metabolize calcein (acetoxymethyl ester) and show cytoplasmic green fluorescence. The method of analysis utilized a 1.5 mm thick cartilage slice that was placed on a glass slide and moistened by several drops of PBS. A confocal laser microscope (MRC-1024; Bio-Rad, Hemel Hempsted/ Cambridge, England) equipped with a krypton/argon laser and the necessary filter systems (fluorecein: 522DF32 and rhodamine: 585EFLP) was employed using the triple-labeling technique as previously described. 15,27 All cartilage samples were examined blinded to group assignment. The confocal laser microscope was calibrated using a micrometer measured through the objective lens (2X) used for this project. The pixel length measured on images was converted to micrometers. The mean depth of chondrocyte death (mean depth of three deepest areas of chondrocyte death) was determined in each CLM image of the osteochondral section with Adobe PhotoShop TM (Version 5.0.2; San Jose, CA). After CLM analysis, the same 1.5 mm thick cartilage slice was fixed in 10% neutral buffered formalin, then decalcified and embedded in paraffin. Sections 5 mm thick were cut and stained with hematoxylin-eosin to show general morphology and with Safranin-O to stain proteoglycan in the extracellular matrix. The microscope was calibrated using a micrometer measured through the objective lens (4) used for this project. The pixel length measured on images was converted to micrometers. The mean depth of clefts (mean depth of three deepest clefts on cartilage surface) was determined in each histologic image of the osteochondral section with Adobe Photo- Shop TM The section from each specimen was examined blindly by three senior researchers (Y.L., M.D.M., L.K.), and then the cartilage structure, Safranin-O staining, cell morphology, chondrocyte viability, and tidemark integrity were assessed. Cell viability was evaluated based on the data from CLM. Each of these characteristics was given a score according to a modified Mankin s scoring system described in a previous study. 28 Lower scores indicate better histologic appearance. The 2.5 mm thick cartilage slice cut for SEM was rinsed thoroughly in three changes of normal saline to remove synovial fluid. Then, the cartilage slice was fixed in modified Karnovsky s solution (2% glutaraldehyde in 0.1 mol/l sodium cacodylate buffer, ph 7.4) for 2 h, and then washed in 0.1 mol/l sodium cacodylate buffer twice at room temperature. The samples were stored in 0.1 mol/l sodium phosphate buffer for 8 h at 48C. The samples were dehydrated in a graded series of ethanol (50, 70, 80, 95, 100%), air dried, coated with gold in a Bio-Rad E5000M gold coater, and examined with a Hitachi S570 SEM (Hitachi High Technologies Corp., Tokyo, Japan). Analysis of variance (ANOVA) was used to evaluate the data of mean depth of chondrocyte death, mean depth of clefts on cartilage surface, cartilage stiffness, and C2C levels. When ANOVA revealed significant differences over time, a Duncan s Multiple Range Test was performed to separate these differences. Comparison of JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2006 DOI /jor

4 PARC INJURY IN AN OVINE MODEL 1977 the histologic subjective scores among treatment groups based on the medians was performed by the Kruskal- Wallis test. If the Kruskal-Wallis test demonstrated differences among the groups, the Mann-Whitney U test was used to evaluate these differences. Interobserver precision error was also determined among researchers for histological analysis. Differences were considered to be significant at a probability level of 95% ( p < 0.05). All statistical analyses were performed with a commercially available software program (SAS Version 8e; SAS Institute Inc., Cary, NC). RESULTS Gross appearance of the treated area appeared rough and swollen compared to control over time. Probing of the cartilage surface indicated that the treated area was softer than control beginning at 12 weeks after surgery (Fig. 2). Stiffness of the treated area measured by Artscan was significantly less than control in the 12, 24, and 52 week groups ( p < 0.05). There was no significant difference in the stiffness between treatment and control in the 1 week group (Table 1). CLM demonstrated that the depth of cell death significantly increased over time in the treatment groups, whereas all chondrocytes were viable in the control group for each time interval (Figs. 3 and 4). The depths of chondrocyte death in the 12 (mean SD: mm), 24 ( mm), and 52 week treatment groups ( mm) were significantly greater than that in the 1 week treatment group ( mm) ( p < 0.05). The depths of chondrocyte death in the 24 and 52 week treatment groups were also significantly greater compared to that in the 12 week treatment group ( p < 0.05), whereas there was no significant difference in the depth of chondrocyte death between the 24 and 52 week treatment groups ( p > 0.05). Interobserver precision error for histologic scores was 3.6%. The control in all time groups demonstrated a smooth cartilaginous surface with normal Safranin-O staining (Fig. 5). Histologic images showed that the depth of clefts on the cartilage surface in the 1 week treatment group was significantly less than those in 12, 24, and 52 week treatment groups (Fig. 6) ( p < 0.05). In addition, cloned cells were clearly present in the treated area beginning at 12 weeks. Histologic analysis demonstrated that the 1 week treatment group had a significantly better histologic appearance than the 12, 24, and 52 week treatment groups ( p < 0.05). There were no significant differences in the depths of clefts or histologic scores among the 12, 24, and 52 week treatment groups ( p > 0.05). SEM revealed that the cartilaginous surfaces in all control groups appeared smooth (Fig. 7). SEM demonstrated that the treated area became more irregular over time. The cartilaginous surfaces in the 1 and 12 week treatment groups had fibrillation without clearly detected clefts and fissures. Wider Figure 2. Gross appearance of the medial condylar surface of control (A) and treated specimens at 24 weeks after surgery (arrows) (B). DOI /jor JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2006

5 1978 LU ET AL. Table 1. Artscan Data for Control and Treatment Groups 1 Week 12 Weeks 24 Weeks 52 Weeks Control a a a Treatment b b b Values represent mean standard deviation. Different letters within a column represent differences between control and treatment groups ( p < 0.05). and deeper clefts were easily detected in the 24 and 52 week treatment groups (Fig. 7). For the control knee joints, joint fluid analysis demonstrated that there were no significant differences in the C2C level between any time point and the control baseline data ( p > 0.05). There were also no significant differences in the C2C levels over time ( p > 0.05). For the treatment knee joints, the C2C level in the 1 week treatment group was significantly higher than the pretreatment baseline data ( p < 0.05). There were no significant differences in the C2C level between the treatment and the baseline data at 12 and 52 weeks ( p > 0.05). The C2C level in the 24 week group significantly decreased compared to the baseline data ( p < 0.05). In addition, the C2C levels in the 24 and 52 week treatment groups were significantly less compared to that in the 1 week treatment group ( p < 0.05), whereas there were no significant differences in the C2C levels among the 12, 24, and 52 week treatment groups ( p < 0.05). Figure 3. CLM demonstrating the cartilage surface of a 52 week control specimen (top of the image) and subchondral bone (bottom of the image) (original magnification 20). The green dots indicate viable cells, and the red dots indicate dead cells. In the 52 week control group, all chondrocytes were viable. DISCUSSION The results of this study demonstrated that the development of a PARC lesion in an ovine model was successfully established, and biomechanical, biochemical, and histological characteristics and surface ultrastructural appearance of this PARC lesion continued to progress over time. Although the goat has recently been recommended by ICRS (International Cartilage Research Society) as the animal choice for articular cartilage repair, the reason that we chose the sheep in this study was that the size of the joint in a sheep is larger than in a goat of the same weight, which made it easier to insert the arthroscope and Dremel tool in the joint to roughen the medial condylar cartilage. Importantly, sheep are much easier to obtain in our area than goats. Using arthroscopic techniques to create a PARC lesion may significantly reduce the damage to the surrounding tissue and prevent unnecessary edema and stress on the animal during the perioperative period. The PARC lesion created by the Dremel tool in this study was selected for the weight bearing area of the medial condyle in order to mimic the clinical situation. A recent study performed by Hjelle et al. 29 demonstrated that 58% of articular cartilage lesions in 1,000 knee arthroscopies occurred on the medial femoral condyle. The stiffness analysis by Artscan demonstrated that the treated area was softer compared to contralateral control beginning at 12 weeks after surgery. By 24 weeks after surgery, probing the treated area revealed even more swelling and softening as typically occurs in chondromalacia. We hypothesized that this was caused by the Dremel tool breaking down and rupturing the superficial layers of cartilage during the surgery. The clefts on the cartilaginous surface progressed to deeper levels and the proteoglycan concentration continued to decrease over time as seen in Figures 6 and 7. The indentation response of articular cartilage is dependent upon the high tensile stiffness of the superficial collagen layer, in addition to the JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2006 DOI /jor

6 PARC INJURY IN AN OVINE MODEL 1979 Figure 4. CLM of treated medial femoral condyles. (A) One week group. (B) Twelve week group. (C) Twenty-four week group. (D) Fifty-two week group. The depth of chondrocyte death increased over time (original magnification 20). Figure 5. Safranin-O (S/O) staining of a decalcified section of the medial femoral condyle in the control group at 52 weeks. The morphology of the cartilage appeared normal with smooth surface (S/O staining; original magnification 40). compressive stiffness provided by proteoglycans in the matrix. The superficial collagen layer in the treated area was damaged, which might be partially responsible for the reduced stiffness properties seen in the treated area as previously reported. 30,31 In response to traumatic injury, chondrocytes appear to undergo a response that includes apoptosis with cumulative changes for as long as 1 week after injury. This apoptotic response is thought to be important in the degenerative process. 32,33 This is the reason why we selected 1 week after surgery as the first time point to analyze. The CLM demonstrated that the mean depth of chondrocyte death at 1 week was approximately 250 mm and progressed to 590 mm at 52 weeks after surgery. This result indicated that the chondrocytes in the treated area continued to die over time. Whether this cell death is the result of simple necrosis or apoptosis remains unclear. We hypothesized that the cell death may result from the combination of necrosis and apoptosis. However, there are indications that the apoptosis accounts for the cell death DOI /jor JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2006

7 1980 LU ET AL. Figure 6. S/O staining of the decalcified sections of the medial femoral condyles in the treated groups. (A) Articular surface in the 1 week group appeared fibrillated with fine fronds protruding and clefts were around 250 mm deep. (B) The depth of clefts in the 12 week group increased with S/O staining decreased. Cloned chondrocytes appeared in the treated area (arrows). (C, D) In the 24 and 52 week groups, the clefts were deeper and the S/O staining decreased compared to the 12 week group (S/O staining; original magnification 40). Figure 7. (A D) SEM demonstrating the control at 1, 12, 24, and 52 weeks with smooth cartilage surface. (E, F) In 1 and 12 week groups, the cartilage surface appeared fibrillated with fine fronds. (G, H) In 24 and 52 week groups, the clefts and fissures were wider and deeper (original magnification 120). JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2006 DOI /jor

8 PARC INJURY IN AN OVINE MODEL 1981 after the creation of lesions in cultured fetal sternal cartilage. 34,35 Histologic analysis demonstrated that the fissures or clefts created by the Dremel tool in the treated area continued to progress to deeper levels over time as affirmed by SEM images shown in Figure 6. This finding implies that repetitive loading on the treated area after surgery degraded the articular cartilage matrix and initiated progressive cartilage degeneration and fibrillation as other investigators have reported. 36,37 Histologic scores for the 1 week group were much better than those groups from 12 weeks and greater. This further demonstrated that the degradation of articular cartilage occurred over time in this ovine model. Damage to type II collagen with increased collagenase activity is one fundamental feature in OA. 38,39 Determination of C2C level in the joint fluid has been used as one of the useful indicators to predict OA. 25,40 Compared to pretreatment baseline data, the C2C level of the treated joint in this study increased significantly at 1 week after surgery but then decreased to pretreatment levels over time. We hypothesized that the type II collagen degradation products may correlate with the early inflammatory process, when the collagenase levels are likely to be the highest in the joint fluid. However, the lack of a sham-operated control group in this study may influence the results of C2C analysis when compared to the baseline data in a normal knee. The CLM and histologic analyses demonstrated that the depths of chondrocyte death and clefts on the cartilage surface did not propagate from 24 to 52 weeks after surgery. SEM analysis also revealed that the wider and deeper fissures were present on cartilaginous surface at both 24 and 52 weeks after surgery. These findings may indicate that the process of the PARC lesion did not progress significantly between 24 and 52 weeks after surgery. This may suggest that this PARC lesion model could be produced at 24 weeks after the treatment by the technique used in this ovine model. Several issues of this study deserve discussion. First, an untreated contralateral stifle was used as a control, instead of a sham operated stifle (arthroscopy, irrigation, remove the fat pad if necessary). For this study, we felt it important to compare the lesion to a completely normal joint rather than one where the synovium and fat pad were disrupted. Unfortunately, based on this study design we can not differentiate the effects of the arthroscope approach from the cartilage lesion that was created. Second, no cartilage metabolic analysis such as MTT conversion and proteoglycan 35 S incorporation was performed in this study because the size of the PARC lesion was not large enough for these analyses. Third, this study was designed to evaluate a PARC lesion that did not lead to global OA in the joint; therefore it was different from studies utilizing ACL transaction or thermal treatment to induce joint instability as a trigger for development of OA. 41,42 Fourth, although there were no significant differences in cell viability, depth of clefts, and surface morphology for the PARC lesion between 24 and 52 weeks, the outcome for this PARC lesion at a longer time point than 52 weeks has not been determined. Future studies need to be performed. In summary, this study demonstrated that the PARC lesions created on the weight bearing area of the medial femoral condyle may progress up to and then stabilize at 24 weeks after treatment. Currently, surgical options for this lesion primarily focus on removing fronds and fibrillated cartilage with mechanical debridement and/or thermal modification. This study provided a simple and safe animal model for observing the natural history and propagation of PARC lesions. This study also enabled the study of different treatment options for PARC lesions and early articular lesions. ACKNOWLEDGMENTS The authors thank John Bogdanske, Todd Forsythe, Matt Wisniewski, and Julie Sauer for their assistance with this project. This project was funded by grant support from the Orthopaedic Research and Education Foundation (OREF) and the UW Sports Medicine Fund. REFERENCES 1. Curl WW, Krome J, Gordon ES, et al Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy 13: Buckwalter JA, Mankin HJ Articular cartilage Part II: degeneration and osteoarthrosis, repair, regeneration, and transplantation. 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