Differential Matrix Degradation and Turnover in Early Cartilage Lesions of Human Knee and Ankle Joints

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1 ARTHRITIS & RHEUMATISM Vol. 52, No. 1, January 2005, pp DOI /art , American College of Rheumatology Differential Matrix Degradation and Turnover in Early Cartilage Lesions of Human Knee and Ankle Joints Matthias Aurich, 1 Ginette R. Squires, 2 Agnes Reiner, 2 Juergen A. Mollenhauer, 3 Klaus E. Kuettner, 4 A. Robin Poole, 2 and Ada A. Cole 4 Objective. To determine whether there are differences in matrix turnover within early cartilage lesions of the ankle (talocrural) joint compared with the knee (tibiofemoral) joint that may help explain differences in the prevalence of osteoarthritis in these 2 joints. Methods. Cartilage removed from lesions of the tali and femoral condyles was analyzed for type IIB collagen messenger RNA, C-terminal type II procollagen propeptide (CPII), the collagenase cleavage neoepitope (Col2-3/4C short ), and the denaturation epitope (Col2-3/4m). The content of collagen, glycosaminoglycan, and epitope 846 of aggrecan was quantitated. Results. In ankle lesions, there was an upregulation of markers of synthesis (CPII [P 0.07]; epitope 846 [P < ]), but these were downregulated in the knee (CPII [P 0.1]; epitope 846 [P 0.004]). In lesions of the knee, but not the ankle, there was an up-regulation of collagen degradation markers (P 0.008). On a molar basis, there was 24 times more cleavage epitope than denaturation epitope in knee lesions compared with ankle lesions. Dr. Aurich s work was supported by grants from the NIH (P50-AR-39239), the Max Kade Foundation, and the Deutsche Forschungsgemeinschaft (156/6-1). Dr. Kuettner s work was supported by NIH grant P50-AR Dr. Poole s work was supported by grants from the NIH (AG-13857), the Shriners Hospitals for Children, and the Canadian Institutes for Health Research. Dr. Cole s work was supported by NIH grant P50-AR Matthias Aurich, MD: Rush Medical College at Rush University Medical Center, Chicago, Illinois, and Friedrich-Schiller- Universität Jena, Eisenberg, Germany; 2 Ginette R. Squires, PhD, Agnes Reiner, MSc, A. Robin Poole, PhD, DSc: Shriners Hospitals for Children, and McGill University, Montreal, Quebec, Canada; 3 Juergen A. Mollenhauer, PhD, DSc: Friedrich-Schiller-Universität Jena, Eisenberg, Germany; 4 Klaus E. Kuettner, PhD, Ada A. Cole, PhD: Rush Medical College at Rush University Medical Center, Chicago, Illinois. Address correspondence and reprint requests to Ada A. Cole, PhD, Department of Biochemistry, Rush Medical College at Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL Ada_Cole@rush.edu. Submitted for publication February 19, 2004; accepted in revised form September 27, Conclusion. The up-regulation of matrix turnover that is seen in early cartilage lesions of the ankle would appear to represent an attempt to repair the damaged matrix. The increase in collagen synthesis and aggrecan turnover seen in ankle lesions is absent from knee lesions. Instead, there is an increase in type II collagen cleavage. Together with the differences in collagen denaturation, these changes point to an emphasis on matrix assembly during early lesion development in the ankle and to degradation in the knee, resulting in fundamental differences in matrix turnover in these lesions. Approximately 10% of individuals 65 years of age are affected by symptomatic knee osteoarthritis (OA) (1); however, 1% will develop OA in the ankle, even at an advanced age (2). Although differences in biomechanics between the joints undoubtedly play a role in the degenerative changes that precede the development of OA, we have proposed that there also may be biochemical and biomechanical property differences in the joint tissues themselves, including the articular cartilage. It has been suggested that the differences in susceptibility to OA might be due, in part, to an imbalance between degenerative and repair processes (3). Our studies have compared knees (tibiofemoral joints) and ankles (talocrural joints) obtained from human donors. These have provided evidence to support our hypothesis that the cartilage from these 2 joints has inherent metabolic, biochemical, and biomechanical tissue properties that might help explain the imbalance between higher degradation rates (knee) and a greater ability for repair (ankle). The extracellular matrix of ankle cartilage is denser and more highly charged, providing an increased resistance to loading and decreased sensitivity to mechanical damage (4,5). Ankle chondrocytes are less responsive to catabolic mediators, either fibronectin fragments (6,7) or interleukin-1 (8). 112

2 MATRIX METABOLISM IN CARTILAGE LESIONS 113 The rebound response of ankle chondrocytes to anabolic factors, such as osteogenic protein 1 (bone morphogenetic protein 7), is higher than that of knee chondrocytes (8), suggesting that ankle chondrocytes are better able to repair their matrix following damage. Differences between knee and ankle chondrocytes are maintained when the cells are released from their native matrix and placed in culture (9), suggesting that these differences are not based on biomechanical differences alone, but are programmed into the chondrocytes themselves. Our population-based studies comparing human donor knees with ankles have revealed that degenerative morphologic changes develop in knees and ankles (10 13). Recently, we reported the degenerative changes in the tali and distal femoral condyles of 2,142 donors between the ages of 13 and 96 years (13). The data were obtained from 4,005 ankles and 409 knees that were graded based on the appearance of articular cartilage and osteophytes. In both the ankles and the knees, there was an increase in degenerative changes from the second to the eighth decade of life. By the seventh decade, only 15% of the knees and 34% of the ankles were considered macroscopically normal; of the ankles, 59% had surface fissures and fibrillations, and 0.07% had full-thickness defects. By comparison, 56% of the knees had surface fissures and fibrillations, and 29% had full-thickness defects. These data suggest that ankle cartilage may possess mechanisms to reduce the rate of progression of degenerative changes that occur with aging. The focus of the current study was to determine whether there are differences in the cartilage from the 2 joints with respect to matrix synthesis or degradation that might indicate how the progression of degenerative changes may be enhanced in the knee and/or retarded in the ankle cartilage. In a previous study of aging of normal ankle cartilage (14), we reported that the content of type II collagen (CII) and proteoglycan did not change throughout life and that both synthesis and degradation are maintained at a steady state in cartilage from donors who were years of age. The matrix content of the C-terminal type II procollagen propeptide (CPII) of CII directly reflects synthesis of this molecule (15), whereas epitope 846 is a marker of aggrecan turnover (16), which is increased in OA cartilage and present on the largest, most intact, molecules (17). Degeneration of CII involves cleavage of the triple helix by collagenases, which produces a three-quarter length fragment (TC A ) and a smaller, one-quarter length fragment (TC B ). This cleavage creates a neoepitope (Col2-3/4C short ; hereafter referred to as Col2-3/4C), which is located on the carboxy-terminus of the TC A fragment (18). Denaturation of the triple helix following its cleavage exposes a hidden (intrachain) epitope (Col2-3/4m) that reflects the denaturation of CII (19). One aim of our studies was to determine whether there were differences in CII degradation from the initial specific cleavage of the triple helix by collagenases (hereafter referred to as cleavage) to the denaturation of the -chains (hereafter referred to as denaturation) in early cartilage lesions from the knee and ankle. The second aim was to investigate whether there were changes in CII and aggrecan synthesis and turnover and to monitor changes in the matrix content of CII and proteoglycan (mainly aggrecan). We hypothesized that there may be differences in matrix turnover that may influence the development of pathology in these cartilages, which determines lesion formation. MATERIALS AND METHODS Tissue acquisition. The tali of the ankles and the femoral condyles of the knees of cadaver donors were obtained with institutional approval within 24 hours of death through the Gift of Hope Organ and Tissue Donor Network. All donors tested negative for hepatitis and human immunodeficiency virus; none had a diagnosed skeletal pathology. The joints were opened and graded according to a modified Collins scale (20), as previously reported (5). This is a 5-point scale with grades as follows: 0 no signs of cartilage degeneration, 1 limited minor articular surface roughening or fibrillation, 2 fibrillations and fissuring (the surface irregularities of 1 and 2 reflect early degenerative lesions), 3 and 4 fullthickness defects covering less than or more than 30% of the articular surface, respectively. Joints with grades 3 and 4 changes were excluded from the study. Figure 1 shows a knee and an ankle with a histologic grade of 2. Antibodies. The specificities of polyclonal rabbit antisera to the Col2-3/4C neoepitope and CPII and of the mouse monoclonal antibody for the Col2-3/4m intrachain epitope and epitope 846 of aggrecan have been previously described (15 19,21). Tissue processing. Full-thickness, uncalcified, articular cartilage plugs ( 25-mm 2 surface area) with no signs of degeneration (grade 0) were harvested from the medial aspect of the talar dome (n 10; ages years) and from the medial femoral condyle of the knee (n 10; ages years) for histology, in situ hybridization, immunohistochemistry, enzyme-linked immunosorbent assay, and biochemical analyses. Plugs were also removed from the early degenerative lesions (grade 1 or 2) of 10 ankles (talar dome) and 7 knees (femoral condyles). Histology. The cartilage plugs were frozen in embedding compound (TFM; Triangle Biomedical Sciences, Durham, NC) and cryosectioned at 8 m thickness. The sections were stained with Safranin O fast green and graded as previously described (22).

3 114 AURICH ET AL Figure 1. Tibiofemoral joint of the knee and talus from the ankle of a 56-year-old man. Both A, the knee and B, the ankle were assigned a Collins grade of 2. Arrows indicate superficial fibrillations or lesions. C, Photomicrograph of talar cartilage from a grade 2 lesion that was cryosectioned and stained with Safranin O fast green (original magnification 25). In situ hybridization. Cartilage was processed under RNase-free conditions following the procedure developed at our laboratory (22), except that the tissues were not embedded in paraffin. Cryosections were incubated with a 35 S-labeled probe for human type IIB collagen as previously described (14). Immunohistochemistry. The immunohistochemistry protocol described by Hollander et al (19) was used, with slight modifications. Frozen sections (8 m thickness) were fixed in 4% paraformaldehyde for 5 minutes and digested for 1 hour at room temperature with 0.1 units/ml chondroitin ABC lysate (ICN Biochemicals, Cleveland, OH) in 0.1M Tris, ph 8. The sections were incubated with 1% bovine serum albumin and 1% nonimmune serum (goat for Col2-3/4C and CPII; horse for Col2-3/4m) to block nonspecific binding sites. The primary antibodies were used at a 1:500 dilution in the 1% nonimmune serum described above and incubated overnight at 4 C. A biotinylated secondary antibody (anti-goat for Col2-3/4C and CPII; anti-horse for Col2-3/4m) was applied at a 1:333 dilution according to the manufacturer s protocol (Pierce, Rockford, IL) and detected with an alkaline phosphatase complex using the 1-Step NBT/BCIP kit (plus suppressor; Pierce). Controls included omitting the first antibody, using normal rabbit serum instead of the first antibody (Col2-3/4C, CPII), or using a nonimmune serum (goat or horse) or IgG instead of the first antibody (Col2-3/4m). Controls also included preincubation of the primary antibody with peptide (Col2-3/4C or Col2-3/4m epitopes) or anti-cpii with purified CPII, as previously described (15,21). Cartilage analysis. Full-thickness cartilage plugs from the talus of normal (grade 0) ankles and from the femoral condyle of normal (grade 0) knees, as well as from areas of superficial fibrillation (grades 1 and 2) of the tali and the femoral condyle of the knees were harvested and stored frozen ( 80 C) until assayed for glycosaminoglycans (GAGs), CPII, Col2-3/4C, Col2-3/4m, and epitope 846 of aggrecan. The cartilage samples were incubated for 48 hours at 4 C with 4M guanidinium hydrochloride, 1 mm EDTA, 1 mm iodoacetamide, 1 mm phenylmethylsulfonyl fluoride, and 5 g/ml pepstatin A, in 50 mm sodium acetate, ph 5.8, and 1% CHAPS. The extract was dialyzed against 50 mm sodium acetate, ph 6.3, and assayed for CPII (15) and epitope 846 (17). Separate cartilage samples were treated sequentially with -chymotrypsin and proteinase K (19). The collagenasegenerated cleavage neoepitope (Col2-3/4C) and the denatured epitope (Col2-3/4m) were measured in the -chymotrypsin digest. Total CII content was determined from the total amount of Col2-3/4m epitope in both the -chymotrypsin digest and the proteinase K digest by immunoassay, as previously described (19). GAG content was measured in combined -chymotrypsin and proteinase K digests by dimethylmethylene blue assay (23). Statistical analysis. Analysis was performed using SigmaPlot 5.0 and SigmaStat 2.0 (SPSS, Chicago, IL). Student s t-test was used to assess significant differences between the groups. Data are presented as the mean SD. P values less than or equal to 0.05 were considered significant. RESULTS Histologic findings. Histologic assessment of the normal and lesional cartilage samples were performed on the sections stained with Safranin O fast green (Figure 1). For the normal knee and ankle cartilage, the grades ranged from 0 to 3, with a mean SD of for the knee and for the ankle. There was no statistical difference between the histologic grades of the knee and ankle cartilage. The early lesions from the knee and ankle were graded 2 5, with a mean SD of for the knee and for the ankle. The grades for the lesions in both the knee and the ankle were significantly different from those of normal cartilage (P ). In situ hybridization and immunohistochemistry findings. Messenger RNA for type IIB collagen was detectable in chondrocytes by in situ hybridization in normal ankle cartilage (Figure 2A) and in lesions (Figure 2B), where cell clusters were evident in the upper layers and there was a clear up-regulation of message. The CPII epitope was evident by immunohistochemistry in normal ankle cartilage (Figure 2C), and was seen in both the chondrocyte and its pericellular matrix. In the lesion, however, there was a strong increase in the staining pattern that was territorial or even interterritorial (Figure 2D). This up-regulation of collagen synthesis in the lesion site compared with normal cartilage oc-

4 MATRIX METABOLISM IN CARTILAGE LESIONS 115 curred in correlation with matrix damage in the ankle cartilage. Both Col2-3/4C (Figures 3A and B) and Col2-3/4m (Figures 3C and D) were detectable immunohistochemically, as shown in sections from the ankle. Staining in the lesion was interterritorial, while in normal cartilage, only the pericellular matrix of chondrocytes was positive for Col2-3/4C. Col2-3/4m was hardly detectable in normal cartilage. The immunohistochemistry of the knee was similar to that shown for the ankle. Quantitative analysis. There was no difference (P 0.67) in collagen cleavage within the triple helix, as measured by immunoassay with antibodies to the neoepitope Col2-3/4C, between the normal and lesional cartilage of the ankle (Figure 4A). However, there was a 5-fold increase in knee cartilage lesions as compared with normal cartilage (P 0.008). By contrast, denaturation, as measured by the Col2-3/4m immunoassay (Figure 4B), was increased 2.6-fold in knee lesions (mean SD %) compared with normal cartilage ( %; P 0.15), whereas in the ankle, there was an 11.6-fold increase in lesions ( %) compared with normal cartilage ( %; P ). Interestingly, when the molar ratio of cleaved collagen to denatured collagen was calculated (Figure 4C), similarities were seen in normal cartilage; however, there was a marked increase in the ratio in knee lesions (mean SD molar ratio) compared with Figure 2. Type IIB procollagen expression and type II collagen (CII) synthesis in normal ankle cartilage compared with cartilage from a lesion. A, Type IIB collagen mrna detected by in situ hybridization is present throughout the thickness of normal cartilage, from the superficial to the deep zone. B, Cluster formation and increased expression are seen in the lesion site. C, As a marker of CII synthesis, C-terminal type II procollagen propeptide (CPII) is shown immunohistochemically in the pericellular matrix in normal cartilage. Inset, Enlargement of a chondrocyte showing pericellular staining (arrow). D, At the lesion site, there is a clear up-regulation of CPII, with more pronounced territorial and interterritorial staining. (Original magnification 25.) Figure 3. Type II collagen degradation in normal ankle cartilage and cartilage from a lesion, as detected by immunohistochemistry. The collagenase-generated cleavage neoepitope (Col2-3/4C) is detectable in the pericellular matrix in normal cartilage (A). A clear up-regulation with interterritorial staining is seen in the fissure site (B). Compared with normal cartilage (C), the denaturation intrachain, or hidden, epitope (Col2-3/4m) is also significantly up-regulated in the lesion site (D). (Original magnification 25.)

5 116 AURICH ET AL Figure 4. Collagen degradation revealed by enzyme-linked immunosorbent assay in normal and lesional ankle cartilage compared with normal and lesional knee cartilage. A, Cleaved type II collagen (CII) was detected by the cleavage neoepitope Col2-3/4C and expressed as a percentage of the total collagen content. The difference between normal ankle cartilage and ankle lesion cartilage was not significant (P 0.67); however, there was a significant difference between normal knee cartilage and knee lesion cartilage (P 0.008). B, Denatured CII was detected by the intrachain epitope Col2-3/4m and expressed as a percentage of the total collagen content. The difference between normal ankle cartilage and ankle lesion cartilage was significant (P ), while that between normal knee cartilage and knee lesion cartilage was not (P 0.15). C, The molar ratio of cleaved to denatured collagen in knee lesions was increased 24.3-fold in lesions compared with normal cartilage, whereas the ratio in ankle lesion cartilage was 1.1. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and 90th percentiles. Solid circles represent values below the 10th percentile or above the 90th percentile. Lines inside the boxes represent the median. normal cartilage ( molar ratio). In the ankle, there was a significant (P ) decrease in normal cartilage (mean SD molar ratio) compared with lesions ( molar ratio). The molar ratio for knee lesions was 24.3:1, indicating that in knee lesions, there was 24 times more cleavage than denaturation product. In ankle lesions, however, this ratio was 1.1:1, which means that there was an equal amount of cleavage and denaturation. Thus, there is evidence that following cleavage, there is an accumulation of denatured collagen -chains in knee lesions compared with ankle lesions and normal cartilage. Markers of anabolism (CPII) (Figure 5A) revealed an almost 2-fold increase in CPII in ankle lesions, whereas in knee lesions there was a decrease of 75% compared with normal cartilage. In epitope 846 (Figure 5B), there was an even more pronounced increase in ankle lesions compared with normal ankle cartilage. As for CPII, there was no detectable increase in epitope 846 in knee lesions but a clear reduction to 10% of the

6 MATRIX METABOLISM IN CARTILAGE LESIONS 117 normal control. Values for epitope 846 are given in Table 1. Quantitative analysis (Table 1) also revealed a trend toward an increase in total CII and GAG content in ankle lesions compared with normal ankle cartilage, with the levels being statistically significant for total collagen (P 0.04) but not for GAG content (P 0.45). In the knee, however, there was clearly no increase in Table 1. Quantitative analysis of total collagen and proteoglycan components in cartilage removed from knee and ankle joints or from lesions in knee and ankle joints* Epitope 846, g/mg wet weight Total collagen, nmoles/mg wet weight GAG, g/mg wet weight Ankle Normal (n 10) Lesion (n 10) P Knee Normal (n 10) Lesion (n 7) P * Normal ankle cartilage was obtained from ankles with a histologic grade of 0, ankle lesion cartilage was obtained from the fibrillated area of an ankle with a histologic grade of 2, normal knee cartilage was obtained from knees with a histologic grade of 0, and knee lesion cartilage was obtained from the fibrillated area of a knee with a histologic grade of 2. Values are the mean SD. GAG glycosaminoglycan. CII and GAG in the lesion site compared with normal cartilage. Figure 5. Synthesis of CII and aggrecan. A, CPII content in normal and lesional ankle cartilage compared with normal and lesional knee cartilage. In ankle lesions, there was a 2-fold increase in CPII, whereas in knee lesion cartilage there was a decrease of 75%. B, Epitope 846 of aggrecan in normal and lesional ankle cartilage compared with normal and lesional knee cartilage. There was a pronounced increase of epitope 846 in ankle lesions (mean SD g/mg wet weight) compared with normal ankle cartilage ( g/mg wet weight) (P ). In the knee lesions, there was a significant decrease of epitope 846 (see Table 1). Data are shown as box plots. Each box represents the 25th to 75th percentiles. Solid circles represent values below the 10th percentile or above the 90th percentile. Lines outside the boxes represent the 10th and 90th percentiles. Lines inside the boxes represent the median. See Figure 2 for definitions. DISCUSSION The present investigation points to fundamental differences in cartilage matrix damage and turnover in early lesions found in the knee compared with the ankle. The up-regulation of matrix synthesis involving collagen and proteoglycan in more advanced degeneration of the hip and knee has been previously reported (24 26). Our data for very early lesions of ankle cartilage support this observation. Lesional cartilage of the ankle joint clearly showed up-regulation of CII synthesis and proteoglycan turnover at sites of early focal matrix degeneration. Previously, we had also shown that the type II procollagen synthesis reflected by CPII (15) and the aggrecan turnover reflected by epitope 846 (17) were up-regulated in advanced knee cartilage lesions at arthroplasty. In early lesions in the ankle, collagen denaturation was up-regulated, with no evidence of the increase in CII cleavage that was observed in early knee lesions, albeit in the absence of significant changes in denaturation, and no increase in synthetic and turnover markers that were observed in ankle lesions. In previous studies on more advanced lesions of the knee at arthroplasty, we found that CII denaturation (19) and cleavage (18) were also increased. In a related recent study of more advanced focal lesions in knee articular cartilage, we used

7 118 AURICH ET AL the same quantitative analysis to characterize the changes in the biomarkers examined in the present study (27). In that study, we examined knee lesions with more degeneration (median Mankin grade of 6.0); however, in the present study, the histologic assessment for the knee (and ankle) lesions had a median grade of only 2.0. Thus, in knee lesions that exhibited more degeneration, we observed increases in CII synthesis (CPII) and aggrecan turnover (epitope 846), as well as increased cleavage and denaturation of CII. Any differences in lesions of the knee would thus appear to relate to the lower severity of the knee lesions examined here and presumably reflect their earlier development. The principal difference that distinguishes these very early lesions in the ankle and knee is the pronounced increase in CII synthesis and aggrecan turnover in the ankle, which is not seen in the knee. Furthermore, the increase in CII cleavage in knee lesions is not seen in the ankle. The absence of denatured collagen in knee lesions may relate to increased secondary proteolysis of the TC A and TC B fragments in knee lesions, which is associated with the increased cleavage of CII. The early increase in CII cleavage seen in the knee but not in the ankle points to an early onset of pathology in the knee with lesion onset, with an emphasis not on repair (as in the ankle) but on cartilage collagen degradation. Together, the results point to a net anabolic response in early ankle lesions and a net catabolic response in early knee lesions. These differences may account in part for the more frequent presence of OA in the knee than in the ankle joint. They also point to differences in the responses of chondrocytes to early matrix damage in these different joints. An improved understanding of the underlying mechanisms may provide important insights into how cartilage degeneration may be regulated. The observations further suggest that early up-regulation of matrix synthesis and turnover may serve to control lesion progression, whereas an early increase in collagenase activity in the absence of increased assembly and turnover favors progression of the pathology. ACKNOWLEDGMENTS We wish to thank the Gift of Hope Organ and Tissue Donor Network and the donor families for providing access to human donor cartilage. REFERENCES 1. Felson DT, Naimark A, Anderson J, Kazis L, Castelli W, Meenan RF. The prevalence of knee osteoarthritis in the elderly: The Framingham Osteoarthritis Study. Arthritis Rheum 1987;30: Peyron JG. The epidemiology of osteoarthritis. In: Moskowitz RW, Howell DS, Goldberg VM, Mankin HJ, editors. Osteoarthritis: diagnosis and treatment. Philadelphia: W. B. Saunders; p Poole AR, Ionescu M, Swan A, Dieppe PA. 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