Objective. Several studies have shown that cathep-sin K (CTK) is overexpressed in osteoarthritic (OA)cartilage and subchondral bone. However, it has notbeen well established whether CTK expression is harm-ful or beneficial. We undertook this study to investigatethe direct involvement of CTK in OA development usingCtsk-knockout (Ctsk�/�) mice in a joint instability–induced model of OA.
Methods. We analyzed the natural course of thephenotype of 25-week-old Ctsk�/� mice. OA develop-ment was evaluated with a modified Mankin histologicscore up to 8 weeks after surgery was performed todestabilize the knee in Ctsk�/� and Ctsk�/� mice. His-tologic analysis was used to evaluate expression of CTK,matrix metalloproteinase 13 (MMP-13), ADAMTS-5,and tartrate-resistant acid phosphatase (TRAP) pro-teins in chondrocytes, synovial cells, and osteoclasts.Bone architecture was analyzed by histomorphometry.
Results. Bone mineral content and bone volumewere higher in Ctsk�/� mice at 25 weeks, whereas OA did
not develop spontaneously in either Ctsk�/� or Ctsk�/�
mice. In a model of destabilization-induced OA, OAprogression was significantly delayed in Ctsk�/� mice.CTK was overexpressed in chondrocytes and synovialcells of knee joints developing OA in Ctsk�/� mice.MMP-13 and ADAMTS-5 were less strongly expressedin chondrocytes of Ctsk�/� mice, and MMP-13 was lessstrongly expressed in synovial cells. TRAP-positive os-teoclasts were overexpressed in Ctsk�/� mice.
Conclusion. These results indicate that CTKplays crucial direct roles in the early to intermediatestage of OA development. CTK-positive chondrocytesand synovial cells may be a possible target to preventdisease progression in OA.
Osteoarthritis (OA) has been considered a pri-mary disorder of articular cartilage as well as the cumu-lative result of mechanical and biologic events thatinduce an imbalance between the degradation and syn-thesis of cartilage within articular joint tissues. As theOA process progresses, there is evidence of increasedcatabolic activity related to enhanced expression ofdegradative proteinase genes associated with gradualloss of proteoglycans followed by type II collagen degra-dation (1,2). The key enzymes underlying cartilage de-struction have been identified as ADAMTS. Aggrecan isdegraded both by matrix metalloproteinases (MMPs)and by ADAMTS, whereas type II collagen is degradedprimarily by various MMPs (3–5).
Although the characteristic feature of OA isdegeneration of cartilage, it is now generally appreciatedthat all joint structures are affected, including calcifiedcartilage, subchondral cortical and trabecular bone, jointcapsular tissues, and synovium. Epidemiologic study hassuggested that increased subchondral bone density maycontribute to OA (6). However, a recent report has
Supported in part by the Ministry of Education, Culture,Sports, Science, and Technology of Japan (Grant-in-Aid for ScientificResearch [C] 20591751).
1Eiji Kozawa, MD, Yoshihiro Nishida, MD, PhD, Xian WuCheng, MD, PhD, Hiroshi Urakawa, MD, PhD, Eisuke Arai, MD,PhD, Naohisa Futamura, MD, Masafumi Kuzuya, MD, PhD, Lina Hu,BM, Naoki Ishiguro, MD, PhD: Nagoya University Graduate Schoolof Medicine, Nagoya, Japan; 2Guo-Ping Shi, DSc: Brigham andWomen’s Hospital and Harvard Medical School, Boston, Massachu-setts; 3Takeshi Sasaki, PhD: Hamamatsu University School of Medi-cine, Shizuoka, Japan.
Dr. Ishiguro has received consulting fees, speaking fees,and/or honoraria from Abbott, Eisai, Mitsubishi Tanabe, Chugai, andDaiichi-Sankyo (less than $10,000 each) and from Takeda and Astellas(more than $10,000 each).
Address correspondence to Yoshihiro Nishida, MD, PhD,Department of Orthopaedic Surgery, Nagoya University GraduateSchool of Medicine, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550,Japan. E-mail: [email protected].
Submitted for publication February 15, 2011; accepted inrevised form September 29, 2011.
454
suggested that decreased subchondral bone mineraldensity and strength may be associated with OA (7).
Cathepsin K (CTK), a cysteine protease pro-duced predominantly by osteoclasts, degrades triplehelix collagen (4) and is involved in bone metabolism(8). It is considered a pharmacologic target for thetreatment of osteoporosis, in which bone resorption isexcessive. This potentially very degradative enzyme isalso expressed by other cells, including chondrocytes inarticular cartilage (9).
CTK cleaves at distinct sites in the triple helixand N- and C-telopeptides (4,10–12) as well as in theaggrecan core protein (13). Given that CTK is synthe-sized by healthy and OA chondrocytes, it is now believedthat this enzyme could play an important role in thebreakdown of the cartilage collagen network in OA (14).
The involvement of CTK in the pathogenesis ofOA has been reported. Intense CTK staining is observedin areas of cartilage degeneration, particularly in chon-drocyte clusters, and in the synovium of the OA joint ina CTK overexpression mouse model (15) and transgenicDel1 mouse models (16). CTK staining is first observedin the cells in OA joints, but only to a slight extent innormal healthy joints, and with progression of thedisease, pericellular staining around chondrocytes be-comes evident with spread of the stain into the extracel-lular matrix (17). This clearly indicates that chondro-cytes are the source of CTK and, furthermore, that it isreleased from the cells with progression of the disease.Increased CTK expression has also been observed in thesynovial membrane of OA Del1 mice and patients witharthritis, suggesting a role for it in increasing the inva-siveness of synovial pannus and facilitating cartilage andbone erosion (16,18–20).
In contrast, a cartilage-protective role of CTKhas been reported. Down-regulation of CTK in syno-vium significantly accelerated cartilage degeneration,indicating that CTK may slow the progression of carti-lage degradation in the initial stage of OA (21). Thissuggests that CTK activity on type II collagen in OAcartilage could be beneficial by removing bioactive typeII collagen peptides/epitopes implicated in the patho-genesis of progressive cartilage breakdown in OA (22).To elucidate the true pathogenic role of CTK, particu-larly in the early stage of OA, other experimental modelsshould be applied.
Studies in humans have focused on late stages ofthe disease. However, studies in animal models allow usto evaluate time-dependent histomorphometric changesin articular cartilage in comparison to those in thesubchondral bone at disease onset. In the present study,
we established OA models with transection of the ante-rior cruciate ligament (ACL), medial meniscus, andmedial collateral ligament (MCL) in the Ctsk-knockoutmouse, analyzing the direct roles of this enzyme in thepathogenesis of OA onset and progression.
MATERIALS AND METHODS
Mice. Ctsk-heteroknockout (Ctsk�/�) mice on a 129/Sv � C57BL/6 background, originally generated by Li et al(23), were provided by the Cardiovascular Laboratory ofHarvard University. All mice (Ctsk�/� and Ctsk�/�) used forthe animal experiments were produced by repeated cross-breeding of Ctsk heterogeneous (Ctsk�/�) mice. The litterswere genotyped by Southern blotting using an external probeas previously described (23). All animal experiments wereperformed in accordance with the Institute for LaboratoryAnimal Research, National Research Council of the NationalAcademies Guide for the Care and Use of Laboratory Animalsand under approval of the Institutional Animal Ethics Com-mittee.
Initially, to investigate the natural history of kneejoints in Ctsk�/� mice, we removed knee joints of 25-week-oldmale Ctsk�/� and Ctsk�/� mice. In addition, their macroscopicphenotype was analyzed, including body weight and length,and a soft radiograph of the skeleton was obtained using aSoftex apparatus (Type EMB). Ten-week-old male Ctsk�/�
and Ctsk�/� mice were subjected to an experimental OAmodel of knee joints.
Experimental OA model. The mice were anesthetizedwith a 0.1-ml intraperitoneal injection of pentobarbital sodium(10 mg/ml). Under sterile conditions, the right knees of 8Ctsk�/� and 8 Ctsk�/� male mice were destabilized by partialresection of the medial meniscus and transection of the MCLand ACL. The skin and joint capsule were incised in the leftknees (sham side) of each mouse. After irrigation with saline,the skin was closed (24). At 8 weeks postoperatively, mice werekilled by cervical dislocation, and the knee joints were excisedand subjected to histologic analysis. To investigate the timecourse of OA development, 2 additional Ctsk�/� and Ctsk�/�
mice each were subjected to histologic analysis at 4 and 6weeks postoperatively.
Histologic evaluation of OA. OA change was analyzedin sagittal sections of knee joints and graded according to amodified Mankin histologic score for the tibia and femurarticular side (25,26). Each sample was stained with Safranin Oor hematoxylin and eosin. Serial sagittal sections were exam-ined, and the maximally affected section was evaluated andscored. Typically, the midline of the medial condyle wasscored. The grade of OA was determined by 2 observers (HUand EA) who were blinded with regard to the experimentalgroup to avoid observer bias. The grading scores were deter-mined by a combined score based on the following 7 para-meters: articular cartilage structure, grade 0–11; tidemarkduplication, grade 0–3; Safranin O staining, grade 0–8; fibro-cartilage, grade 0–2; chondrocyte clones in uncalcified carti-lage, grade 0–2; hypertrophic chondrocytes in calcified carti-lage, grade 0–2; subchondral bone, grade 0–2. In addition to
OA CHANGE IN Ctsk-KNOCKOUT MICE 455
the modified Mankin score, we applied the modified Cham-bers scoring system to evaluate OA progression (27,28).
Immunohistochemistry. Mice were transcardially per-fused with cold 4% paraformaldehyde (PFA), and the kneejoint was postfixed in 4% PFA at 4°C for 12 hours. Afterwashing with phosphate buffered saline (PBS), the sampleswere decalcified in 10% EDTA solution at 4°C for 3 weeks andthen embedded in paraffin. Sagittal sections (5 �m) were cutand mounted onto silane-coated slides. Tissue sections, whichwere selected to be adjacent to those evaluated for modified
Mankin score, were deparaffinized in xylenes and descendingethanols. The sections were unmasked with 8 �g/ml proteinaseK (Dako). Endogenous peroxidase activity was blocked byimmersion in 3% H2O2 in methanol for 5 minutes. Thesections were incubated in a blocking solution (3% goat serumwith PBS with 0.01% Triton X-100) at room temperature for15 minutes, followed by incubation with a diluted primaryantibody for 2 hours at room temperature. Primary antibodiesused were rabbit anti-CTK polyclonal antibody (1:100 dilu-tion; Proteintech Group), mouse anti–MMP-13 monoclonal
Figure 1. A–J, Histology of articular cartilage of knee joints from cathepsin K–homozygous positive (Ctsk�/�) mice (A, C, E, G, and I) and cathepsinK–knockout (Ctsk�/�) mice (B, D, F, H, and J). A and B, Safranin O–fast green staining showed no difference in stainable features of cartilageproteoglycan between 25-week-old Ctsk�/� (A) and Ctsk�/� (B) mice. C and D, The sham-operated side of both Ctsk�/� (C) and Ctsk�/� (D) miceat 8 weeks showed no obvious osteoarthritic (OA) change. E–H, The OA process was observed at 4 weeks (E) and 8 weeks (G) on the operated (Ope)side of Ctsk�/� mice, whereas the change was delayed in Ctsk�/� mice (F and H). I and J, More osteophyte formation (arrows) was observed onthe operated side of Ctsk�/� mice (I) than on that of Ctsk�/� mice (J). Original magnification � 100. K–M, A representative section was selectedbased on the average score from each experimental group. K, OA change at 4, 6, and 8 weeks was evaluated according to the modified Mankin scoreand graphed. L, This OA model was validated with the difference between scores of the sham-operated side and those of the operated side in Ctsk�/�
mice (P � 0.001). L and M, OA change was significantly delayed on the operated side of Ctsk�/� mice compared with that of Ctsk�/� mice at 8 weeks,according to the modified Mankin score (L) and the modified Chambers score (M). Values are the mean � SD.
After washing, sections were subjected to incubationwith either N-Histofine Simple Stain Rabbit MAX PO (R) orN-Histofine MOUSESTAIN (Nichirei Biosciences). The slideswere then reacted with streptavidin conjugated to peroxidase(Nichirei Biosciences) for 10 minutes, followed by incubationin a 3,3�-diaminobenzidine–H2O2 substrate medium (NichireiBiosciences) for 5 minutes. For negative controls, tissue sec-tions were incubated with a nonimmunized mouse IgG as asubstitute for the first antibody. Tissue sections were alsosubjected to tartrate-resistant acid phosphatase (TRAP) stain-ing using an acid phosphatase leukocyte kit (Sigma). The numberof TRAP-positive osteoclasts in metaphyseal areas in each mousewas counted and subjected to bone histomorphometric analysis.
The number of CTK- and MMP-13–positive stainablecells was counted in articular cartilage and synovium. Eachsection was observed under a light microscope at 400� mag-nification. The mean number of positive cells/50 cells wascalculated from 8 mice in each group. All slides were evaluatedindependently by 2 blinded observers (HU and EA).
Bone histomorphometry. To study the trabecular andcortical architecture of the proximal tibia (metaphyses andepiphyses) in the natural course of 25-week-old Ctsk�/� andCtsk�/� mice, a total of 160 slices of 5 �m thickness corre-sponding to a volume of from 0.3 mm to 1.1 mm below andabove the growth plate, respectively, were reconstructed andanalyzed using 3-dimensional reconstructions with micro–computed tomography (micro-CT) scanning (TRI/3D-Bon;Ratoc System Engineering). The indices examined includedbone mineral content (BMC), bone volume (BV), growth platelength, trabecular number (TbN), trabecular thickness (TbTh),and trabecular separation (TbSp).
For static bone histomorphometric analyses in theexperimental OA model, bone perimeter (BPm), TbTh, andcortical bone thickness (CtTh) were measured in proximaltibial metaphyses and epiphyses in the area between 1 mm and2 mm below and above the growth plate, respectively, usingScion Image. The number of TRAP-positive osteoclasts (OcN)per 1 mm BPm in each metaphysis was also calculated toevaluate the frequency of osteoclast expression. These para-meters were determined as described by Parfitt et al (29).
Statistical analysis. All data are expressed as themean � SD. Group means were compared using Student’st-test or Mann-Whitney U test. Analysis of variance followedby Bonferroni correction or Dunn’s post hoc test was used toassess differences among multiple groups. P values less than0.05 were considered significant. All analyses were performedusing SPSS software for Windows, version 17.0.
RESULTS
Natural course of skeletal formation in Ctsk�/�
and Ctsk�/� mice. Soft radiograph analyses of 25-week-old mice revealed no significant differences in skeletalformation (data not shown), which is consistent withfindings of previous studies (30–32). Safranin O stainingrevealed no significant differences in stainable features
of cartilage proteoglycans between Ctsk�/� and Ctsk�/�
mice (Figures 1A and B).Micro-CT analyses of the tibial metaphysis
showed that BMC, BV, TbTh, and TbN were higher inCtsk�/� mice than in Ctsk�/� mice. In contrast, TbSpwas lower in Ctsk�/� mice than in Ctsk�/� mice. Anal-yses of the tibial epiphysis (subchondral bone) showedthat there was no significant difference in any of thesevariables between Ctsk�/� and Ctsk�/� mice (Table 1).These results suggest that subchondral bone in thismodel may not affect OA development.
OA development. In the sham-operated (left)knee joints at 8 weeks, the articular cartilage was evenlystained with Safranin O in both Ctsk�/� and Ctsk�/�
mice (Figures 1C and D). Four weeks after the opera-tion, there was a slight difference in Safranin O stainingbetween the operated (right) knees of Ctsk�/� mice andthose of Ctsk�/� mice (Figures 1E and F). At 6 weeks,reduction of the staining was observed on the operatedside of Ctsk�/� mice compared with that of Ctsk�/� mice(results not shown). Moreover, the difference in stainingbetween Ctsk�/� and Ctsk�/� mice was more prominentat 8 weeks (Figures 1G and H). Interestingly, consider-ably more osteophyte formation was observed on theoperated side of Ctsk�/� mice than on that of Ctsk�/�
mice (Figures 1I and J); however, the difference was notanalyzed statistically due to the rare occurrence ofosteophytes. To quantify OA development in eachgroup, we used a modified Mankin score. The mean �SD score was 39.8 � 8.9 for the operated side in the
Table 1. Bone histomorphometry of 25-week-old mice*
* Values are the mean � SD. BV/TV � bone volume/tissue volume;BMC/TV � bone mineral content/tissue volume; TbTh � trabecularthickness; TbN � trabecular number; TbSp � trabecular separation;CtBV � cortical bone volume; CtTh � cortical bone thickness.† P � 0.01 versus Ctsk�/� mice.‡ P � 0.05 versus Ctsk�/� mice.
OA CHANGE IN Ctsk-KNOCKOUT MICE 457
Ctsk�/� group and 23.1 � 17.3 for the operated side inthe Ctsk�/� group, indicating that progression of OAchange was significantly inhibited in the Ctsk�/� groupcompared with that in the Ctsk�/� group (P � 0.028).The time course of the modified Mankin score on theoperated side of Ctsk�/� and Ctsk�/� mice is shown inFigure 1K. The mean � SD score was 3.2 � 1.8 for thesham-operated side in the Ctsk�/� group and 4.5 � 3.5for the sham-operated side in the Ctsk�/� group (P �1.0) (Figure 1L).
Detailed parameters of modified Mankin gradingscores are shown in Table 2. Safranin O staining wassignificantly more marked on the operated side ofCtsk�/� mice than on that of Ctsk�/� mice (P � 0.01).There were significant differences in all parameters ofmodified Mankin scores between the operated andsham-operated sides of Ctsk�/� mice, indicating that theexperimental OA model used in this study was properlyvalidated. According to the modified Chambers scoringsystem, there was a significant difference in scoresbetween the operated side of Ctsk�/� mice and that ofCtsk�/� mice (P � 0.042) (Figure 1M).
Immunolocalization. CTK localization was deter-mined in joints of Ctsk�/� mice. As expected in acontrol, negative staining for CTK in joints of Ctsk�/�
mice was confirmed (Figures 2C, F, I, and L). Minimalimmunostaining was observed in chondrocyte lacunaefrom the sham-operated side of Ctsk�/� mice at 8 weeks(Figure 2B). In contrast, staining was greater in chon-drocyte lacunae from the operated side of Ctsk�/� mice(Figure 2A). Similarly, more synovial cells staining pos-itive for CTK were observed on the operated side ofCtsk�/� mice (Figure 2D) than on the sham-operatedside of Ctsk�/� mice (Figure 2E). The mean � SD
number of positively stained chondrocytes and synovio-cytes was significantly higher on the operated side thanon the sham-operated side of Ctsk�/� mice at 8 weeks (P� 0.002 and P � 0.009, respectively) (Figures 2M andN). Interestingly, slightly more CTK-positive osteoclastswere observed in metaphyseal trabeculae on the oper-ated side (Figure 2G) than on the sham-operated side(Figure 2H) of Ctsk�/� mice; however, the differencewas not significant. CTK expression in subchondral boneis shown in Figures 2J and K. There was no significantdifference in CTK expression between the operated andsham-operated sides of Ctsk�/� mice.
Since MMP-13 is thought to be most importantfor degradation of collagen within the cartilage due to itspreferential digestion of type II collagen over type I andtype III collagens (33,34), MMP-13 expression in carti-lage and synovial tissues was evaluated in this model.Eight weeks after operation, MMP-13–positive chondro-cytes were located in the area adjacent to that ofdecreased Safranin O staining on the operated side ofCtsk�/� mice, particularly in the superficial layer of thecartilage (Figure 3A). In contrast, fewer MMP-13–positive chondrocytes were observed on the sham-operated side of Ctsk�/� mice (Figure 3B) or on theoperated or sham-operated sides of Ctsk�/� mice (Fig-ures 3C and D). Statistical analysis revealed that thenumber of MMP-13–positive chondrocytes and synovialcells was higher on the operated side of Ctsk�/� micethan on the operated side of Ctsk�/� mice (P � 0.024and P � 0.057, respectively) (Figures 3M and N). Therewas no significant difference between the sham-operatedside of Ctsk�/� mice and that of Ctsk�/� mice. InCtsk�/� mice, the numbers of MMP-13–positive chon-drocytes and synovial cells were significantly higher on
Table 2. Comparison in parameters of modified Mankin scores (tibia plus femur)*
* Values are the mean � SD.† P � 0.001 versus operated Ctsk�/� mice.‡ P � 0.01 versus operated Ctsk�/� mice.§ P � 0.05 versus operated Ctsk�/� mice.¶ P � 0.05 versus operated Ctsk�/� mice.
458 KOZAWA ET AL
Figure 2. Immunostaining for cathepsin K (CTK) in Ctsk�/� mice (on the operated side, n � 8; on the sham-operated side, n � 7) and Ctsk�/�
mice (on the operated side, n � 6). A–L, Shown is staining for CTK in chondrocytes (A–C), synovial cells (D–F), metaphyseal trabeculae (G–I), andsubchondral bone (J–L). As expected, the operated side of Ctsk�/� mice showed no positive staining in chondrocytes, synovial cells, metaphysealtrabeculae, or subchondral bone. A and B, More chondrocytes in the superficial layer were positively stained on the operated side than on thesham-operated side in Ctsk�/� mice. D, E, G, and H, More cells were positive in synovium and metaphyseal trabeculae on the operated side ofCtsk�/� mice (D and G, respectively) than on the sham-operated side of Ctsk�/� mice (E and H, respectively). J and K, There was no increase ofCTK-positive osteoclasts in subchondral bone on the operated side of Ctsk�/� mice (J) compared with the sham-operated side of Ctsk�/� mice (K).Arrows in A and B indicate CTK-positive chondrocytes; arrows in D and E indicate CTK-positive synovial cells; arrows in G, H, J, and K indicateCTK-positive osteoclasts. Boxed areas in A, D, G, and J depict the location of the staining and are shown at higher magnification in A, D, G, andJ, respectively. Original magnification � 100. M and N, The number of CTK-positive chondrocytes (M) and synovial cells (N) was significantly higheron the operated side of Ctsk�/� mice than on the sham-operated side of Ctsk�/� mice at 6 and 8 weeks. � � P � 0.05; �� � P � 0.01 versussham-operated side of Ctsk�/� mice. Values are the mean � SD. See Figure 1 for other definitions.
OA CHANGE IN Ctsk-KNOCKOUT MICE 459
the operated side than on the sham-operated side at 8weeks (P � 0.002 and P � 0.005, respectively) (Figures3M and N).
The expression of CTK and MMP-13 at earlytime points was determined. The mean � SD numbersof CTK-positive chondrocytes at 4 weeks (4.0 � 1.0) and6 weeks (10 � 2.0) on the operated side of Ctsk�/� micewere higher than those at 4 weeks (2.3 � 0.6) and 6weeks (2.3 � 0.6) on the sham-operated side of Ctsk�/�
mice (Figure 2M). Similarly, the mean � SD numbers ofCTK-positive synovial cells at 4 weeks (5.0 � 1.7) and 6weeks (8.3 � 1.5) on the operated side of Ctsk�/� mice
were higher than those at 4 weeks (2.7 � 0.6) and 6weeks (2.7 � 0.6) on the sham-operated side of Ctsk�/�
mice (Figure 2N). The differences in the numbers ofCTK-positive chondrocytes and synovial cells betweenthe operated and sham-operated sides were statisticallysignificant at 6 weeks (P � 0.05) (Figures 2M and N).
MMP-13 staining at 4 and 6 weeks revealed thatthere were significantly more MMP-13–positive chon-drocytes on the operated side of Ctsk�/� mice (13 � 1.7)than on the operated side of Ctsk�/� mice (8.3 � 0.6) at6 weeks (P � 0.01), whereas there was no difference at4 weeks. There were significantly more MMP-13–
Figure 3. Immunostaining for matrix metalloproteinase 13 (MMP-13) and ADAMTS-5 in Ctsk�/� mice (on the operated side, n � 8; on thesham-operated side, n � 7) and Ctsk�/� mice (on the operated side, n � 6; on the sham-operated side, n � 6). A–L, Shown is staining for MMP-13in chondrocytes (A–D) and synovial cells (E–H) as well as staining for ADAMTS-5 in chondrocytes (I–L). MMP-13 was highly expressed inchondrocytes of the superficial layer on the operated side of Ctsk�/� mice compared with the sham-operated side of Ctsk�/� mice or the operatedside of Ctsk�/� mice. Synovial cells in the lining layer were stained more on the operated side of Ctsk�/� mice than on the sham-operated side ofCtsk�/� mice or the operated side of Ctsk�/� mice. More ADAMTS-5 was expressed in chondrocytes of the superficial layer on the operated sideof Ctsk�/� mice than on the sham-operated side of Ctsk�/� mice or on the operated side of Ctsk�/� mice. Arrows in A, B, and C indicateMMP-13–positive chondrocytes; arrows in E, F, and G indicate MMP-13–positive synovial cells; arrows in I, J, and K indicate ADAMTS-5–positivechondrocytes. M and N, Shown are numbers of MMP-13–positive chondrocytes (M) and synovial cells (N) in operated and sham-operated Ctsk�/�
and Ctsk�/� mice. There were significant differences in the numbers of stained chondrocytes between the operated side of Ctsk�/� mice and thatof Ctsk�/� mice at 6 weeks (## � P � 0.01) and 8 weeks (# � P � 0.05), and between the operated and sham-operated sides of Ctsk�/� mice at6 weeks (��� � P � 0.001) and 8 weeks (�� � P � 0.01). There were significant differences in the numbers of stained synovial cells between theoperated and sham-operated sides of Ctsk�/� mice at 6 and 8 weeks (�� � P � 0.01 for both). Values are the mean � SD. O–V, Shown istartrate-resistant acid phosphatase (TRAP) staining for metaphyseal trabeculae (O–R) and subchondral bone (S–V). TRAP-positive cells includingosteoclasts were up-regulated on the operated and sham-operated sides of Ctsk�/� mice. Boxed areas in A, E, I, O, and S depict the location of thestaining and are shown at higher magnification in A, E, I, O, and S, respectively. Original magnification � 100. See Figure 1 for other definitions.
460 KOZAWA ET AL
positive chondrocytes at 6 weeks on the operated side ofCtsk�/� mice (13 � 1.7) than on the sham-operated sideof Ctsk�/� mice (3.3 � 0.6) (P � 0.001) (Figure 3M).Similar results were obtained for MMP-13 staining insynovial cells (Figure 3N). These results suggest thatexpression of both enzymes causes the decrease inSafranin O staining. Expression of another aggrecan-degrading enzyme, ADAMTS-5, was also decreased inchondrocytes on the operated side of Ctsk�/� micecompared with that on the operated side of Ctsk�/� mice(3 sections each counted) (P � 0.069) (Figures 3I and K).
Bone histomorphometry findings. The tibia ofOA model mice was subjected to histomorphometricanalysis (Table 3). As described for the results of thenatural course of Ctsk�/� and Ctsk�/� mice, analyses oftibial metaphysis revealed that TbTh was significantlyhigher on the sham-operated side of Ctsk�/� mice thanon that of Ctsk�/� mice (P � 0.05). Similarly, TbTh wassignificantly higher on the operated side of Ctsk�/� micethan on that of Ctsk�/� mice (P � 0.05). In contrast, theresults for tibial epiphysis (subchondral bone) showedno significant difference in BV/bone marrow space,TbTh, TbN, or TbSp, either between Ctsk�/� andCtsk�/� mice or between the operated and sham-operated sides. The OcN/BPm in sham-operatedCtsk�/� mice was higher than that in sham-operatedCtsk�/� mice. Analyses of tibial diaphysis indicated thatCtTh was greater in operated Ctsk�/� mice than inoperated Ctsk�/� mice and greater in sham-operated
Ctsk�/� mice than in sham-operated Ctsk�/� mice, butthese differences did not reach statistical significance.
There was higher expression of TRAP-positiveosteoclasts in the metaphysis on the operated side ofCtsk�/� mice (Figure 3O) than on the sham-operatedside (Figure 3P), suggesting stimulation of osteoclasto-genesis in the course of OA development. Interestingly,in Ctsk�/� mice, TRAP-positive osteoclasts were highlyexpressed in the metaphysis on both the operated andsham-operated sides (Figures 3Q and R). This may beexplained by positive feedback of the absence of CTKactivity in Ctsk�/� mice. Densities of TRAP-positiveosteoclasts were expressed as OcN/BPm of Ctsk�/�
mice, and results are shown in Table 3. In subchondralbone, TRAP-positive osteoclasts were also highly ex-pressed in Ctsk�/� mice (Figures 3U and V) comparedwith Ctsk�/� mice (Figures 3S and T).
DISCUSSION
The most interesting findings of the current studywere that the absence of CTK delayed the developmentof OA in a knee instability mouse model evaluated by amodified Mankin score, in parallel with a decrease inMMP-13 expression in both chondrocytes and synovialcells and a decrease in ADAMTS-5 expression in chon-drocytes. CTK is highly expressed within osteoclasts,predominantly degrades type I collagen in bone, andhelps to maintain bone metabolism (35). However,
Table 3. Bone histomorphometry of mice with induced osteoarthritis*
* Values are the mean � SD. BV/BMS � bone volume/bone marrow space; OcN/BPm � osteoclast number/bone perimeter (see Table 1 for otherdefinitions).† P � 0.05 versus operated Ctsk�/� mice.‡ P � 0.01 versus sham-operated Ctsk�/� mice.§ P � 0.05 versus sham-operated Ctsk�/� mice.
OA CHANGE IN Ctsk-KNOCKOUT MICE 461
recent studies have demonstrated that CTK degradesnot only type I collagen but also type II collagen incartilage (11,12). Hou et al demonstrated that CTK candegrade aggrecan complexes at specific cleavage sites,and CTK activity alone is sufficient to provide glycos-aminoglycan fragments (13). Previous observations raisethe question of whether CTK expression in the course ofOA is harmful or beneficial for joint homeostasis, whilethe demonstrated role of CTK in OA raises the questionof “which comes first?” No previous studies have ana-lyzed the effects of CTK absence on the development ofOA, and since Ctsk-knockout mice are available at ourinstitution, we undertook an investigation of CTK in-volvement in OA development using an ACL and MCLtransection model.
CTK was overexpressed in chondrocytes andsynovial cells on the operated side compared with thesham-operated side of Ctsk�/� mice, concurrently withOA progression as determined by the modified Mankinscore, particularly regarding the decrease of Safranin Ostaining. CTK has catabolic activity for proteoglycan(13), and MMP-13 activation possibly mediated by CTKactivation might abrogate the positivity of Safranin Ostaining. These results indicate that CTK in chondro-cytes and synovial cells plays pivotal roles in the promo-tion of OA, which was also confirmed by the finding thatOA was delayed on the operated side of Ctsk�/� mice.
Inconsistent with the results of the current study,Takahashi et al reported beneficial roles of CTK in OApathogenesis (21). However, in their study, CTK expres-sion in synovium was up-regulated 7 days after ACLtransection, whereas at 5 and 15 days, the expressionlevel was not significantly different from that in syno-vium on the sham-operated side. The time course ofCTK expression was completely different from that inour study, in which CTK was overexpressed 8 weeksafter the operation compared with its expression on thesham-operated side.
Expression of CTK in OA cartilage (16,36) andinflamed synovial tissues (18,19) has been reported inearlier studies. A previous study on the cleavage site ofCTK revealed that the neoepitope by CTK (C2K) isobserved in the very superficial zone, and the pattern ofstaining increases with the degree of disease, progressingdeeper into the tissues, similar to the observations ofothers (37). The current study examined the period up to8 weeks after operation, representing an early to inter-mediate stage of OA development, resulting in positivestaining of CTK mainly in the superficial layer ofarticular cartilage. This finding was consistent with thefinding that Safranin O staining was the only variable
among modified Mankin scores that differed signifi-cantly between the operated sides of Ctsk�/� andCtsk�/� mice. Excluding the factors of tidemark dupli-cation and subchondral bone from the scoring system,summed scores differed significantly between the oper-ated sides of Ctsk�/� and Ctsk�/� mice (P � 0.01).
Hou et al demonstrated that CTK expression inrheumatoid arthritis (RA)–derived synovial specimenswas 2–5 times higher than that in OA-derived synovialspecimens. On the other hand, interleukin-1� and tumornecrosis factor � stimulated expression of CTK in pri-mary synovial fibroblast cultures, without any differ-ences in expression between RA- and OA-derived syno-vial fibroblasts (20). Our finding that CTK expression inthe synovium may have been implicated in the develop-ment of OA in an experimental mouse model indicatesthat synovial CTK could be an important factor in OAdevelopment in humans.
Investigators in previous studies have reportedthe involvement of subchondral bone in OA develop-ment. Studies using isotope-labeled bone-seeking agentsand new radiographic techniques indicate that altera-tions in subchondral bone occur early in the course ofOA and confirm that changes in bone turnover precedethe evidence of bony changes detected with standardradiographic techniques (38,39). Those observationswere only partly consistent with those in the currentstudy, in which slightly more CTK-positive osteoclastswere observed in metaphyseal trabeculae on the oper-ated side of Ctsk�/� mice, while there was no increase ofCTK-positive osteoclasts in subchondral bone on theoperated side of Ctsk�/� mice. The current study re-vealed higher BV and TbTh in the metaphysis, but not inthe epiphysis, of Ctsk�/� mice compared with Ctsk�/�
mice. Consistent with the previous report that bonehistomorphometric change in Ctsk�/� mice was due notonly to decreased bone resorption but also to increasedbone formation (40), the effects of CTK deficiency onbone metabolism differ between the epiphysis and me-taphysis. Although the current study could not show theinvolvement of subchondral bone in OA development,alterations of bone remodeling will depend on the stageof OA progression.
One of the limitations of this study was thepossibility of alterations of musculoskeletal phenotypein Ctsk�/� mice. CTK deficiency in humans leads topyknodysostosis due to bone resorption disorder, whichis characterized by short-limbed dwarfism, skeletal de-formities, bone brittleness, multiple pathologic fracture,and joint deformity mainly in the phalangeal joint. Incontrast, despite the strain-dependent differences, those
462 KOZAWA ET AL
morphologic characteristics were less marked in Ctsk�/�
mice than in CTSK�/� humans due to a weak diseasephenotype. Because of compensatory mechanisms formaintenance of bone metabolism, CTK-deficient miceshow significantly milder osteopetrosis and grow nor-mally (40). In previous reports, an alternative bone-resorptive function of osteoclasts was noted to exist inCtsk�/� mice (32,41). The number of osteoclasts tendedto increase because the RANK/RANKL pathway wasup-regulated in Ctsk�/� mice (30,40). Those reportedfindings are consistent with those of the current study inwhich osteoclasts were overexpressed in Ctsk�/� mice.Pennypacker et al reported that the absence of CTK inyoung adult mice was associated with the presence ofbone tissue of normal quality (40). Although BMC andBV were higher in Ctsk�/� mice than in Ctsk�/� mice,analyses of the natural course in these mice indicatedthere were no signs of OA development in either strain,suggesting that the effects of bone quality on OAdevelopment in Ctsk�/� and Ctsk�/� mice are minimalin the current experimental design.
Another limitation of this study was the timecourse of the analyses. Eight weeks after operationseemed to represent a relatively early stage of OA.Long-term analyses are needed in the future to clarifythe roles of CTK in more advanced stages of OA.Evaluation of OA according to the modified Mankinscore is not sufficient for the mouse OA model. How-ever, OA progression was also evaluated with the mod-ified Chambers score, which focuses more on the struc-tures of cartilage tissues.
In conclusion, the current study demonstratedthat the absence of CTK slowed OA development injoint instability–induced OA in a mouse model. Further-more, CTK was overexpressed in chondrocytes, synovialcells, and osteoclasts on the operated side of Ctsk�/�
mice, and MMP-13 was more highly induced in Ctsk�/�
mice than in Ctsk�/� mice. These findings indicate thatCTK plays a significant direct role in the early stage ofOA development and that CTK in chondrocytes, syno-vial cells, and osteoclasts can be a potential target fortreatment of OA.
ACKNOWLEDGMENT
We thank Ms Eri Ishihara for secretarial assistance.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Nishida had full access to all of
the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Study conception and design. Kozawa, Nishida, Kuzuya, Ishiguro.Acquisition of data. Kozawa, Urakawa, Arai, Futamura, Hu, Sasaki.Analysis and interpretation of data. Kozawa, Nishida, Cheng,Urakawa, Arai, Futamura, Shi, Hu, Sasaki.
REFERENCES
1. Ishiguro N, Kojima T, Poole AR. Mechanism of cartilage destruc-tion in osteoarthritis. Nagoya J Med Sci 2002;65:73–84.
2. Poole AR. Cartilage in health and disease. In: Koopman WJ,editor. Arthritis and allied conditions: a textbook of rheumatology.14th ed. Baltimore: Williams & Wilkins; 2000. p. 226–84.
3. Glasson SS, Askew R, Sheppard B, Carito B, Blanchet T, Ma HL,et al. Deletion of active ADAMTS5 prevents cartilage degradationin a murine model of osteoarthritis. Nature 2005;434:644–8.
4. Dahlberg L, Billinghurst RC, Manner P, Nelson F, Webb G,Ionescu M, et al. Selective enhancement of collagenase-mediatedcleavage of resident type II collagen in cultured osteoarthriticcartilage and arrest with a synthetic inhibitor that spares collagen-ase 1 (matrix metalloproteinase 1). Arthritis Rheum 2000;43:673–82.
5. Wu W, Billinghurst RC, Pidoux I, Antoniou J, Zukor D, TanzerM, et al. Sites of collagenase cleavage and denaturation of type IIcollagen in aging and osteoarthritic articular cartilage and theirrelationship to the distribution of matrix metalloproteinase 1 andmatrix metalloproteinase 13. Arthritis Rheum 2002;46:2087–94.
6. Antoniades L, MacGregor AJ, Matson M, Spector TD. A cotwincontrol study of the relationship between hip osteoarthritis andbone mineral density. Arthritis Rheum 2000;43:1450–5.
7. Hunter DJ, Spector TD. The role of bone metabolism in osteoar-thritis. Curr Rheumatol Rep 2003;5:15–9.
8. Konttinen YT, Ma J, Ruuttila P, Hukkanen A, Santavirta S.Chondrocyte-mediated collagenolysis correlates with cartilage de-struction grades in osteoarthritis. Clin Exp Rheumatol 2005;23:19–26.
9. Charni-Ben Tabassi N, Desmarais S, Bay-Jensen AC, Delaisse JM,Percival MD, Garnero P. The type II collagen fragments Helix-IIand CTX-II reveal different enzymatic pathways of human carti-lage collagen degradation. Osteoarthritis Cartilage 2008;16:1183–91.
10. Lohmander LS, Atley LM, Pietka TA, Eyre DR. The release ofcrosslinked peptides from type II collagen into human synovialfluid is increased soon after joint injury and in osteoarthritis.Arthritis Rheum 2003;48:3130–9.
11. Kafienah W, Bromme D, Buttle DJ, Croucher LJ, Hollander AP.Human cathepsin K cleaves native type I and II collagens at theN-terminal end of the triple helix. Biochem J 1998;331:727–32.
12. Hou WS, Li ZQ, Gordon RE, Chan K, Klein MJ, Levy R, et al.Cathepsin K is a critical protease in synovial fibroblast-mediatedcollagen degradation. Am J Pathol 2001;159:2167–77.
13. Hou WS, Li Z, Buttner FH, Bartnik E, Bromme D. Cleavage sitespecificity of cathepsin K toward cartilage proteoglycans andprotease complex formation. Biol Chem 2003;384:891–7.
14. Dejica VM, Mort JS, Laverty S, Percival MD, Antoniou J, ZukorDJ, et al. Cleavage of type II collagen by cathepsin K in humanosteoarthritic cartilage. Am J Pathol 2008;173:161–9.
15. Morko J, Kiviranta R, Joronen K, Saamanen AM, Vuorio E,Salminen-Mankonen H. Spontaneous development of synovitisand cartilage degeneration in transgenic mice overexpressingcathepsin K. Arthritis Rheum 2005;52:3713–7.
16. Morko JP, Soderstrom M, Saamanen AM, Salminen HJ, VuorioEI. Up regulation of cathepsin K expression in articular chondro-cytes in a transgenic mouse model for osteoarthritis. Ann RheumDis 2004;63:649–55.
OA CHANGE IN Ctsk-KNOCKOUT MICE 463
17. Vinardell T, Dejica V, Poole AR, Mort JS, Richard H, Laverty S.Evidence to suggest that cathepsin K degrades articular cartilagein naturally occurring equine osteoarthritis. Osteoarthritis Carti-lage 2009;17:375–83.
18. Dodds RA, Connor JR, Drake FH, Gowen M. Expression ofcathepsin K messenger RNA in giant cells and their precursors inhuman osteoarthritic synovial tissues. Arthritis Rheum 1999;42:1588–93.
19. Hummel KM, Petrow PK, Franz JK, Muller-Ladner U, AicherWK, Gay RE, et al. Cysteine proteinase cathepsin K mRNA isexpressed in synovium of patients with rheumatoid arthritis and isdetected at sites of synovial bone destruction. J Rheumatol1998;25:1887–94.
20. Hou WS, Li W, Keyszer G, Weber E, Levy R, Klein MJ, et al.Comparison of cathepsins K and S expression within the rheuma-toid and osteoarthritic synovium. Arthritis Rheum 2002;46:663–74.
21. Takahashi D, Iwasaki N, Kon S, Matsui Y, Majima T, Minami A,et al. Down-regulation of cathepsin K in synovium leads toprogression of osteoarthritis in rabbits. Arthritis Rheum 2009;60:2372–80.
22. Yasuda T, Tchetina E, Ohsawa K, Roughley PJ, Wu W, Mousa A,et al. Peptides of type II collagen can induce the cleavage of typeII collagen and aggrecan in articular cartilage. Matrix Biol 2006;25:419–29.
23. Li CY, Jepsen KJ, Majeska RJ, Zhang J, Ni RJ, Gelb BD, et al.Mice lacking cathepsin K maintain bone remodeling but developbone fragility despite high bone mass. J Bone Miner Res 2006;21:865–75.
24. Kamekura S, Hoshi K, Shimoaka T, Chung U, Chikuda H,Yamada T, et al. Osteoarthritis development in novel experimen-tal mouse models induced by knee joint instability. OsteoarthritisCartilage 2005;13:632–41.
25. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical andmetabolic abnormalities in articular cartilage from osteo-arthritichuman hips. II. Correlation of morphology with biochemical andmetabolic data. J Bone Joint Surg Am 1971;53:523–37.
26. Furman BD, Strand J, Hembree WC, Ward BD, Guilak F, OlsonSA. Joint degeneration following closed intraarticular fracture inthe mouse knee: a model of posttraumatic arthritis. J Orthop Res2007;25:578–92.
28. Glasson SS, Chambers MG, Van Den Berg WB, Little CB. TheOARSI histopathology initiative—recommendations for histolog-ical assessments of osteoarthritis in the mouse. OsteoarthritisCartilage 2010;18:S17–23.
29. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H,Meunier PJ, et al. Bone histomorphometry: standardization ofnomenclature, symbols, and units. Report of the ASBMR Histo-morphometry Nomenclature Committee. J Bone Miner Res 1987;2:595–610.
30. Kiviranta R, Morko J, Alatalo SL, NicAmhlaoibh R, Risteli J,Laitala-Leinonen T, et al. Impaired bone resorption in cathepsinK-deficient mice is partially compensated for by enhanced osteo-clastogenesis and increased expression of other proteases via anincreased RANKL/OPG ratio. Bone 2005;36:159–72.
31. Saftig P, Hunziker E, Wehmeyer O, Jones S, Boyde A, Rommer-skirch W, et al. Impaired osteoclastic bone resorption leads toosteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci US A 1998;95:13453–8.
32. Gowen M, Lazner F, Dodds R, Kapadia R, Feild J, Tavaria M, etal. Cathepsin K knockout mice develop osteopetrosis due to adeficit in matrix degradation but not demineralization. J BoneMiner Res 1999;14:1654–63.
33. Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL,Yocum SA, Rosner PJ, et al. Cloning, expression, and type IIcollagenolytic activity of matrix metalloproteinase-13 from humanosteoarthritic cartilage. J Clin Invest 1996;97:761–8.
34. Knauper V, Will H, Lopez-Otin C, Smith B, Atkinson SJ, StantonH, et al. Cellular mechanisms for human procollagenase-3 (MMP-13) activation: evidence that MT1-MMP (MMP-14) and gelatinaseA (MMP-2) are able to generate active enzyme. J Biol Chem1996;271:17124–31.
35. Bromme D, Okamoto K, Wang BB, Biroc S. Human cathepsin O2,a matrix protein-degrading cysteine protease expressed in osteo-clasts: functional expression of human cathepsin O2 in Spodopterafrugiperda and characterization of the enzyme. J Biol Chem1996;271:2126–32.
36. Konttinen YT, Mandelin J, Li TF, Salo J, Lassus J, Liljestrom M,et al. Acidic cysteine endoproteinase cathepsin K in the degener-ation of the superficial articular hyaline cartilage in osteoarthritis.Arthritis Rheum 2002;46:953–60.
37. Hollander AP, Pidoux I, Reiner A, Rorabeck C, Bourne R, PooleAR. Damage to type II collagen in aging and osteoarthritis startsat the articular surface, originates around chondrocytes, andextends into the cartilage with progressive degeneration. J ClinInvest 1995;96:2859–69.
38. Buckland-Wright JC, MacFarlane DG, Fogelman I, Emery P,Lynch JA. Techetium 99m methylene diphosphonate bone scan-ning in osteoarthritis hands. Eur J Nucl Med 1991;18:12–6.
39. Hutton CW, Higgs ER, Jackson PC, Watt I, Dieppe PA. 99m TcHMDP bone scanning in generalised nodal osteoarthritis. 2. The 4hour bone scan image predicts radiographic change. Ann RheumDis 1986;45:622–6.
40. Pennypacker B, Shea M, Liu Q, Masarachia P, Saftig R, Rodan S,et al. Bone density, strength, and formation in adult cathepsinK�/� mice. Bone 2009;44:199–207.
41. Saftig P, Hunziker E, Everts V, Jones S, Boyde A, Wehmeyer O,et al. Functions of cathepsin K in bone resorption: lessons fromcathepsin K deficient mice. In: Langner J, Ansorge S, editors.Cellular peptidases in immune functions and diseases 2. NewYork: Kluwer Academic/Plenum; 2000. p. 293–303.
