Veterinary Pathology OnlineFirst, published on May 27, 2015 as doi:10.1177/0300985815586221
Original Article
Generalized Degenerative Joint Disease in Osteoprotegerin (Opg) Null Mutant Mice
Veterinary Pathology 1-10 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985815586221 vet.sagepub.com
B. Bolon1, M. Grisanti2, K. Villasenor2, S. Morony2, U. Feige3, and W. S. Simonet2
Abstract Bone structure is modulated by the interaction between receptor activator of nuclear factor–kB (RANK) and RANK ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor for RANKL, modifies osteoclast-mediated bone resorption directly and spares articular cartilage indirectly in rodents with immune-mediated arthritis by preventing subchondral bone destruction. The OPG/RANKL balance also seems to be critical in maintaining joint integrity in osteoarthritis, a condition featuring articular bone and cartilage damage in the absence of profound inflammation. The current study explored the role of OPG in sparing articular cartilage by evaluating joint lesions in adult C57BL/6J mice lacking osteoprotegerin (Opg/). At 3, 5, 7, 9, and 12 months of age, both sexes of Opg/ mice developed severe degenerative joint disease (DJD) characterized by progressive loss of cartilage matrix and eventually articular cartilage. Lesions developed earlier and more severely in Opg/ mice relative to age-matched, wild-type (Opgþ/þ), or heterozygous (Opgþ/) littermates (P .05). The femorotibial joint was affected bilaterally at 3 months, while other key weight-bearing diarthrodial joints (eg, coxofemoral, scapulohumeral, humeroradioulnar) were affected later and unilaterally. Cortical bone in subchondral plates and long bone diaphyses of Opg/ mice but not Opgþ/þ or Opgþ/ animals was osteoporotic by 3 months of age (P .05); the extent of porosity was less than the degree of DJD. Closure of the physes in long bones (P .05) and cartilage retention in the femoral primary spongiosa (P .05) affected chiefly Opg/ mice. These data suggest that OPG plays an essential direct role in maintaining cartilage integrity in the articular surfaces and physes. Keywords cartilage, degenerative joint disease, knockout, mouse, OPG, osteoarthritis, osteoprotegerin, RANKL
Osteoarthritis (OA) or degenerative joint disease (DJD) results from extensive remodeling of articular cartilage and subchondral bone in response to longstanding biochemical/metabolic abnormalities and/or imbalances in biomechanical stress. This disease is the most common cause of joint deterioration, affecting people in the United States22 with a prevalence of clinical disease estimated at between 2.4% and 8.5% of the population.16 The disease can affect any diarthrodial joint but tends to affect weight-bearing articulations such as the coxofemoral (hip) and femorotibial (knee or stifle) joints. Debilitation associated with chronic pain from OA is a major economic burden, in the United States accounting for approximately 21 million visits annually to a primary care physician16 and nearly a million hospitalizations yearly15 (usually ending in knee or hip arthroplasty [total joint replacement]) at a cumulative annual cost of $185.5 billion.13 Joint structure in health and disease depends on tightly coordinated, reciprocal, cell-to-cell communication between osteoblasts and osteoclasts, with osteoblasts acting to form bone and also to control the activity of bone-eroding osteoclasts.10,20 A key signaling pathway facilitating this communication involves the interactions among receptor activator of nuclear factor (NF)–kB (RANK), a membrane-bound receptor on osteoclasts;
its binding partner RANK ligand (RANKL); and osteoprotegerin (OPG, more formally designated as tumor necrosis factor receptor superfamily member 11b [Tnfrsf11b]), an osteoblastderived osteoclast inhibitor that functions as a soluble decoy receptor for RANKL. Abnormalities in cartilage architecture during OA have been linked to altered expression of RANK, RANKL, and OPG in cartilage;2,5,20 indeed, chondrocytes express and produce OPG and RANKL, and those taken from OA joints exhibit a shift toward overproduction of RANKL.19 Administration of OPG has been shown to protect cartilage in joints of mice with OA.17 Given the crosstalk between bone and cartilage in joints,6 whether or not such changes represent
1
The Ohio State University, Columbus, OH, USA Amgen, Inc., Thousand Oaks, CA, USA 3 EUROCBI GmbH, Benglen, Zurich, Switzerland 2
Supplemental material for this article is available on the Veterinary Pathology website at http://vet.sagepub.com/supplemental. Corresponding Author: B. Bolon, GEMpath, Inc, 307 Coffman Rd, Suite 4D, Longmont, CO 80501, USA. Email: [email protected]
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
2
Veterinary Pathology
Figure 1. Femorotibial joint (para-axial orientation), 3-month-old wild-type mouse (Opgþ/þ [control]). The femur (F) and tibia (T) have intact articular and physeal cartilages as well as dense subchondral bone plates. Hematoxylin and eosin (HE). Figure 2. Femorotibial joint, 3-month-old mouse lacking osteoprotegerin (Opg/). The femur and tibia exhibit slight indentations of the articular cartilage surface and increased porosity of the subchondral bone, and the physeal cartilage in the femur is partly closed (long arrow). HE.
a primary impact on cartilage, a secondary consequence of subchondral osteoblast dysfunction, or some combination of the two remains to be determined. The current study had 2 purposes. The first aim was to define the lesion progression in multiple weight-bearing joints of Opg/ mice over time. This goal was undertaken because the original description of the Opg/ mutation3 portrayed in detail the early-onset, generalized, severe osteoporosis that represents the fundamental skeletal phenotype in Opg/ mice while underemphasizing the potential for concurrent joint abnormalities. The second objective was to assess the
hypothesis that an Opg deficit (ie, an altered tissue OPG/ RANKL ratio) will lead to substantial anomalies in cartilage biochemistry (as assessed by altered cartilage matrix staining) and/or structure (indicated by cartilage fragmentation or loss) in cartilaginous organs, especially weight-bearing diarthrodial joints. Our results have achieved these 2 purposes by providing (1) a thorough description of the distribution and severity of joint lesions in adult Opg/ mice over time and (2) histopathologic evidence that Opg-deficient joints and physes (growth plates) experience significant alterations in cartilage structure and biochemistry.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
3
Figure 3. Femorotibial joint, 9-month-old wild-type (Opgþ/þ [control]) mouse. The femur and tibia retain their intact articular and physeal cartilages and dense subchondral bone. Hematoxylin and eosin (HE). Figure 4. Femorotibial joint, 9-month-old Opg/ mouse. The femur and tibia lack articular cartilage and exhibit markedly thinned subchondral and cortical bone, while the femoral physis is closed except for a small remnant (short arrow). The bone and cartilage of the meniscus (arrowhead) display similar disruption of the bone and cartilage. HE.
Materials and Methods Ethical Treatment of Animals The work was conducted in 2002 in accordance with federal (Guide for the Care and Use of Laboratory Animals) and California state guidelines following a protocol approved in advance by the Amgen Institutional Animal Care and Use Committee.
appropriately engineered ES cells derived from a 129/SvJ mouse embryo were microinjected into C57BL/6J blastocysts, after which chimeric offspring were mated to Tac:N:NIH(S)-BC (Black Swiss) mice at 6 to 8 weeks of age. All animals were supplied with pelleted rodent chow and filter-purified tap water ad libitum and were housed in a room with constant temperature (21 + 2 C) and humidity (45% + 10%). Subsequently, heterozygous (HET) breeding pairs were used to produce null mutant (knockout [KO]), HET, and wild-type (WT) mice.
Animals and Husbandry Mice with engineered mutations in Opg (Tnfrsf11btm1Wss mice; http://www.informatics.jax.org/allele/MGI:2183229) were created in which the Opg gene was ablated in RW-4 embryonic stem (ES) cells from the 129X1/SvJ mouse strain and established on a C57BL6 genetic background as described previously.3 Briefly,
Clinical Observations Multiple mice of each genotype were assessed weekly throughout their lives for any ambulatory difficulties by routine evaluation of the quantity (amount) and quality (character) of their gait and motility. The examination involved viewing each
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
4
Veterinary Pathology
Table 1. Summary Histopathology Data: Biologically Significant Structural Lesions in the Femorotibial Joint in Age-Matched OPG Null (KO), OPG HET, and WT Mice. Degenerative Lesions Age, mo Genotype No. Males No. Females Total No.
JD Score
Osteophyte Score
Cartilage Score
Changes Reflecting Opg Null Mutation Cortical Bone Porosity
Physeal Closure
Cartilage Retention
3
WT HET KO
2 5 4
3 5 5
5 10 9
0 0 *1.1 + 0.4
0 0.2 + 0.2 0 0.2 + 0.1 0.3 + 0.2 *1.8 + 0.6
0 0 *2.3 + 0.5
0 0 0 0 *1.2 + 0.3 *0.4 + 0.2
5
WT HET KO
5 5 5
5 4 5
10 9 10
0.5 + 0.2 0.2 + 0.2 0.9 + 0.3 *2.0 + 0.6 0.7 + 0.3 *2.3 + 0.5 *2.2 + 0.2 *0.8 + 0.2 *3.1 + 0.5
0 *1.6 + 0.5 *2.6 + 0.3
0 *0.8 + 0.3 *1.9 + 0.1
7
WT HET KO
5 5 6
5 4 5
10 9 11
0.3 + 0.2 0 0.2 + 0.2 0 0 0.2 + 0.1 *2.5 + 0.2 *1.0 + 0.3 *3.3 + 0.2
0 0 *2.7 + 0.2
0 0 0 0 *1.7 + 0.1 *0.4 + 0.2
9
WT HET KO
5 5 3
5 4 4
10 9 7
2.7 + 0.5 2.2 + 0.5 3.4 + 0.3
1.3 + 0.3 0.7 + 0.3 1.4 + 0.3
2.6 + 0.5 2.4 + 0.5 4.0 + 0
0 0.1 + 0.1 *3.3 + 0.3
0 0.1 + 0.1 *2.0 + 0
12
WT HET KO
5 5 3
5 5 4
10 10 7
2.3 + 0.7 *1.1 + 0.6 *4.6 + 0.2
1.0 + 0.3 0.6 + 0.3 1.7 + 0.3
2.0 + 0.6 1.0 + 0.5 4.0 + 0
0 0 *3.1 + 0.3
0.2 + 0.2 0 0 0 *1.7 + 0.3 *0.6 + 0.2
15
WT
5
4
9
2.4 + 0.6
0.7 + 0.3
2.4 + 0.5
0
0
0.2 + 0.1 0.1 + 0.1 0.5 + 0.2
0.1 + 0.1 0 0.4 + 0.2
0
Values represent mean + standard deviation (SD) of the histopathology scores. Bolded values indicate a substantial difference, and asterisks (*) denote a significant divergence from age-matched wild type animals, P .05, by the Wilcoxon rank-sum test. HET, heterozygous; JD, joint degeneration; KO, knockout; OPG, osteoprotegerin; WT, wild type.
mouse’s activity level from behind and from one or both sides during routine bedding changes. The observation period was 10 to 30 seconds per animal, with longer times spent confirming the presence and evaluating the degree of any abnormalities in locomotion. A specific notation of an animal’s performance was made only if the clinical examination was considered ‘‘abnormal.’’
Skeletal Processing Mice of each genotype were deeply anesthetized with carbon dioxide, euthanized by exsanguination and thoracotomy, and autopsied at 3, 5, 7, 9, 12, or 15 months of age. Viscera were removed and the carcasses were skinned so that the outlines of major appendicular and axial (chiefly vertebral) bones could be assessed for gross distortions. Multiple joints from each mouse were harvested and fixed by immersion in neutral buffered 10% formalin for 48 hours; knees were examined at all 6 time points, while other sites were assessed only at 3 or 9 months. Fixed specimens were decalcified with a 1:1 mixture of 8 N formic acid and 1 N sodium formate until bones could be cut easily with a razor blade (typically 36–48 hours). After decalcification, specimens were processed into paraffin, cut serially at 4 mm, and stained with hematoxylin and eosin (HE) to evaluate general joint structure or with toluidine blue to assess matrix integrity in articular cartilage.
Histopathologic Scoring Three main indices of cartilage damage in diarthrodial articulations were emphasized in the joint evaluation: the joint
degeneration (JD) score (a global index of joint destruction), the cartilage score (an index of matrix integrity in the articular cartilage), and the osteophyte score (an estimate of skeletal proliferation as a periarticular repair process to stabilize deformed joints). An inflammation score was obtained to evaluate the degree to which leukocyte infiltration associated with the joint cavity, periarticular tissues, and subchondral bone might have contributed to the induction of these cartilaginous changes. The extent of the expected osteoporotic phenotype associated with Opg ablation3 was determined by collecting a score for cortical porosity (a direct gauge of osteoporosis) for subchondral bone and the diaphyses of long bones. Finally, scores were acquired for physeal closure (an indication of premature growth plate attenuation that is another expected phenotype of Opg deficiency21) in the femur and tibia as well as cartilage retention (a potential index of altered endochondral ossification) in the femoral primary spongiosa; these 2 scores were collected to explore the consequences of Opg deficiency to induce generalized alterations in cartilage biology. These 6 semiquantitative histopathologic scores were devised specifically to focus on major anatomic attributes of articular cartilage and subchondral bone damage observed in Opg/ mice; the features chosen for analysis and the criteria used to assign scores were equivalent to those of many other scoring schemes used to grade joint lesions in mouse models of OA, which were established in the decade after this study was performed.1,4,8,9,11 For all scores, lesion severity was graded using tiered, semiquantitative scales (Suppl. Tables S1, S2), which were assigned during a coded (‘‘blinded’’) analysis.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
5
Figure 5. Femorotibial joint (para-axial orientation), 3-month-old Opgþ/þ (control) mouse. The femur (F) and tibia (T) have smooth articular surfaces and very few marrow-filled spaces extending from the epiphyseal marrow cavity into the dense plates of subchondral bone. Matrix (dark purple) in the articular cartilage extends across the entire length of both joint surfaces. Toluidine blue. Figure 6. Femorotibial joint, 3-monthold Opg/ mouse. The femur and tibia have thin articular cartilages and exhibit loss of cartilage matrix at multiple sites, especially near the joint margins (indicated by regions of pallor). Toluidine blue. Figure 7. Femorotibial joint, 9-month-old Opgþ/þ (control) mouse. Focal loss (between the arrows, femur only) or narrowing (femur and tibia) of the nonmineralized (deep purple) articular cartilages. Toluidine blue. Figure 8. Femorotibial joint, 9-month-old Opg/ mouse. The femur and tibia have rough articular surfaces due to the loss of nonmineralized cartilage and sometimes also the mineralized cartilage (between the arrows, femur only). The tibial physis is discontinuous (arrowheads), indicating partial closure. Large osteophytes (*) are evident near one meniscus (M). Toluidine blue.
Statistical Analysis
Results
Histopathologic (ordinal) data were compared using the nonparametric Wilcoxon rank-sum test. Because the spontaneous incidences of the lesions associated with Opg ablation are negligible in wild-type mice (C57BL/6 background), the significance and power were set at .05 and 90%, respectively, by selecting group sizes (n) of 5 or more mice per genotype per time point. Statistical calculations were done using JMP statistical software (v. 5.0; SAS Institute, Cary, NC).
Clinical Observations A few KO mice but no HET or WT animals were seen to be limping slightly on one hind limb just prior to autopsy. Animals with gait abnormalities were at least 9 months old.
Skeletal Pathology Gross joint abnormalities or bone distortions were not seen in mice of any genotype. However, the constellation of
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
6
Veterinary Pathology
Table 2. Incidence of Biologically Significant Structural Lesions in Other Joints of OPG Null (KO), OPG HET, and WT Mice at 9 Months of Age. Genotype
Sex
WT
Male Female Total affected Missing Male Female Total affected Missing Male Female Total affected Missing
HET
KO
No. of Mice
Scapulohumeral
Humeroradioulnar
Intercarpal
Coxofemoral
Intertarsal
5 5 10
1 0 1 (10) 0 1 0 1 (11) 0 2 4 *6 (100) 0
0 2 2 (20) 0 0 3 3 (33) 0 2 3 *5 (83) 0
0 0 0 (0) 0 0 0 0 (0) 0 0 3 *3 (50) 0
0 1 1 (13) 2 0 1 1 (11) 1 3 2 *5 (83) 0
0 0 0 (0) 0 0 0 0 (0) 0 0 2 2 (33) 0
5 4 9 2 4 6
Intervertebral facets of all animals exhibited multifocal cell loss and/or superficial fibrillation, regardless of genotype. Values represent the number of animals with affected joints; the values in parentheses represent the percentage of affected mice. Bolded values indicate a substantial difference, and asterisks (*) denote a significant difference from age-matched wild type animals, P .05, by the Wilcoxon rank-sum test. HET, heterozygous; KO, knockout; OPG, osteoprotegerin; WT, wild type.
histopathologic lesions in the KO mice was indicative of generalized DJD (based on articular cartilage damage) in tandem with osteoporosis. The interface between the affected articular cartilage and subjacent subchondral bone was intact in affected joints. Relative to age-matched WT controls, the knees of KO animals exhibited a significant (P .05) incidence of DJD at 3 months of age, which progressed in severity over time (Figs. 1–4). The changes were often but not always bilateral, and both sexes were affected equally. The mean JD score of KO mice was minimal at 3 months of age, mild at 5 months, moderate at 9 months, and severe at 12 months (Table 1). In like manner, the cartilage score of KO animals was mild at 3 months, moderate at 5 to 7 months, and marked at 9 to 12 months (Table 1). Both the JD score and the cartilage score were increased significantly (P .05) in KO mice relative to WT littermates at 3, 5, 7, and 12 months of age and exhibited a trend toward a substantial but nonsignificant increase at 9 months (Figs. 5–8). Osteophyte scores in knees of KO animals were significantly higher (P .05) by 1 grade than those of WT mice at 5 and 7 months (Table 1). At the other time points, the various joint degeneration scores in knees of KO animals exceeded those of WT mice, but the extent of joint degeneration in the WT animals was so substantial that statistical comparison could not distinguish the groups. Multiple other joints of KO animals also exhibited significantly more DJD (P .05) than did WT mice (Table 2). The principal cartilage abnormalities in affected joints were cartilage and chondrocyte loss and sometimes osteophyte production. Chondrocyte loss occurred to an equal extent in both sexes, while osteophyte production was more common in KO females. Sites with more DJD in KO mice included the scapulohumeral (shoulder; Figs. 9 vs 10), humeroradioulnar (elbow; Figs. 11 vs 12), coxofemoral (Figs. 13 vs 14), tibiotarsal (hock or ankle; Figs. 15 vs 16), intercarpal, and intertarsal joints. Neither the intervertebral facets (spine) nor the
temporomandibular (jaw) joints of KO mice were affected to a greater degree than in WT animals. Lesions occurred in 9-month-old KO mice but not in 3-month-olds and typically were unilateral. The absence of Opg was not associated with joint inflammation. Leukocytes were not visible in the synovium or periarticular tissues or within the joint cavity. Similarly, leukocytes were not observed within the bone marrow adjacent to the subchondral bone plate. Ablation of Opg in KO mice also was associated with alterations to bone and nonarticular cartilage. Relative to age-matched WT controls, the long bones of KO animals had a significantly increased (P .05) incidence of bony changes comprising hallmark features of Opg deficiency3 at 3 months of age (Table 1). Affected KO mice had more porous cortical bone (ie, osteoporosis), which was evident in both the diaphysis and subchondral bone as many narrow channels extending from the marrow cavity toward the joint; these spaces rarely reached the deep surface of the mineralized articular cartilage (Figs. 5 vs 6). The extent of osteoporosis was similar in both the diaphysis and subchondral bone. Furthermore, affected KO animals exhibited one or multiple regions of growth plate closure in the femur (Figs. 2, 4) and occasionally in the tibia along with small foci of retained cartilage in the trabeculae of the femoral primary spongiosa. The growth plate in the femur was partially closed in all KO mice at 3 months, while both femoral and tibial growth plates were partially and occasionally completely closed at 5 months or later. In contrast, bones of WT mice had solid cortices, intact growth plates, and trabeculae formed only of bone in the primary and secondary spongiosae (Figs. 1, 3). The HET and WT mice also had DJD, but both the severity and prevalence were lower compared with KO animals. Significant (P .05) DJD was seen in HET mice at 5 and 9 months of age but not at other time points (Table 1); some of these 5-month-old animals, but not the 9-month-old mice, had increased cortical porosity,
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
7
Figure 9. Scapulohumeral joint, 9-month-old Opgþ/þ (control) mouse. The scapula (S) and humeral head (H) have smooth articular surfaces, and the proximal physis of the humerus is intact. Matrix (dark purple) in the articular cartilage extends across the full length of both joint surfaces. Toluidine blue. Figure 10. Scapulohumeral joint, 9-month-old Opg/ mouse. The scapula and humeral head exhibit attenuation or loss of articular cartilage, and the humeral physis is partly absent. A massive osteophyte (asterisk) expands the peri-articular soft tissue. Toluidine blue. Figure 11. Humeroradioulnar joint, 9-month-old Opgþ/þ (control) mouse. The distal humerus (H), radius (R), and ulna (U) have smooth articular surfaces with extension of the intact matrix (dark purple) across the full length of the joint. Toluidine blue. Figure 12. Humeroradioulnar joint, 9-month-old Opg/ mouse. The humerus, radius, and ulna exhibit multifocal loss (arrows) or thinning of their articular cartilage. Toluidine blue. Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
8
Veterinary Pathology
Figure 13. Coxofemoral joint, 9-month-old Opgþ/þ (control) mouse. The pelvic acetabulum (A) and femoral head (F) exhibit smooth articular surfaces with uniform staining of the cartilage matrix (dark purple) across the full length of the joint. Toluidine blue. Note: The apparent increased thickness of the articular cartilage results from a plane of section that is slightly tangential to the main axis of the femoral head. Figure 14. Coxofemoral joint, 9-month-old Opg/ mouse. The articular surfaces of the acetabulum and femoral head are substantially attenuated across the entire extent of the joint. Toluidine blue. Figure 15. Tibiotarsal joint, 9-month-old Opgþ/þ (control) mouse. The distal tibia (Ti) and talus (Ta) have smooth articular surfaces subtended by dense plates of subchondral bone. Hematoxylin and eosin (HE). Figure 16. Tibiotarsal joint, 9-month-old Opg/ mouse. The articular surfaces of the tibia (Ti) and talus (Ta) exhibit multifocal loss (between the arrows) or roughening, and the subchondral bone is extremely porous. HE.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
9
partial physeal closure, and cartilage retention in the trabeculae of the femoral primary spongiosa (Table 1). The extent of the joint degeneration and the KO-like bone changes was lower in HET animals than in age-matched KO mice. In WT mice, both the JD score and the cartilage score were essentially absent until 9 months of age, at which time mild lesions were first observed (Table 1). Findings in WT mice did not progress beyond mild severity through 15 months of age.
Discussion Our current descriptive data demonstrate that mice that lack both Opg alleles (KO) exhibit more severe DJD, affecting many more weight-bearing joints than do age-matched animals with only 1 Opg allele (HET) or having the normal complement of 2 Opg alleles (WT). Specific changes in KO joints included matrix degeneration, superficial fibrillation, and chondrocyte loss affecting articular cartilage. These cartilage changes were accompanied by progressive osteoporosis of the subchondral bone and sometimes by osteophyte production at the joint margins as a preliminary repair process to stabilize the deforming joints. The femorotibial joint (ie, the major weight-bearing articulation in quadrupeds) had the most severe lesions, and the changes were obvious and generally bilateral at a young age (3 months vs 9 months in WT animals) (Table 1). Multiple other diarthrodial joints of KO mice also developed DJD (Table 2), but these tended to occur later in life and typically were unilateral. Importantly, the interfaces between the deep margins of the damaged articular cartilage and the osteoporotic subchondral bone associated with these margins were intact. Taken together, our data confirm our hypothesis that longstanding Opg deficiency is associated not only with a severe osteoporotic phenotype in bone but also with generalized, progressive, early-onset injury to articular cartilage within major weight-bearing joints. This outcome supports prior reports indicating that Opg modulates cartilage catabolism7 and fracture callus remodeling.14 Our data also provide indirect evidence that Opg plays a vital role in regulating the biology of cartilaginous tissues in general, not simply the health of articular cartilage. This inference is supported by the finding that significantly more Opg/ mice exhibited foci in the femur of physeal closure and, to a lesser extent, cartilage retention within the trabeculae of the primary spongiosa. These changes were not associated with prominent shifts in the structure of bony features in the immediate vicinity, suggesting that the cartilaginous alterations might occur independent of the osteoporosis phenotype affecting cortical bone of Opg/ animals. The possibility that OPG helps to maintain cartilage integrity, especially in joints, gains further credibility because OA in humans is characterized by altered expression of RANK, RANKL, and OPG in articular cartilage. These molecules are found in the superficial zone of normal cartilage, while their localization is shifted to the middle zone during OA.12 Alternatively, another causal factor in cartilage destruction during OA may be dysfunction of subchondral osteoblasts.18 Increased porosity of subchondral bone
in tandem with thinning of the articular cartilage in femorotibial joints of 3-month-old Opg/ mice supports this supposition, although the degree of bone damage in these animals was modest compared with the degree of matrix degeneration and cartilage fibrillation in the superficial articular cartilage. Further work will be needed to ascertain whether cartilage damage in Opg/ mice results chiefly from direct insults to chondrocytes, indirectly by anomalies of subchondral bone that secondarily lead to chondrocyte damage, or some combination of the two.
Conclusions Our current data provide a detailed description of the generalized joint lesions affecting articular cartilage and bone in weight-bearing diarthrodial joints of mice engineered to lack Opg. The most prominent changes were DJD characterized by early-onset, progressive loss of cartilage staining by toluidine blue (indicative of matrix degeneration) and subsequently fibrillation, chondrocyte loss, and ultimately cartilage loss affecting the femorotibial joint bilaterally and later the scapulohumeral, humeroradioulnar, and coxofemoral articulations unilaterally. The co-occurrence of severe, progressive osteoporosis (the expected skeletal phenotype in Opg/ mice), including increased formation of marrow-filled spaces in the subchondral bone, represents one explanation for the lesions in the articular cartilage. However, in the femur, premature physeal closure and cartilage retention in the cores of primary spongiosa trabeculae also occurred almost exclusively in Opg/ mice, suggesting that an Opg deficiency also might affect cartilage biology differently from the usual regulation of RANKL-induced osteoclast activity. Further work will be necessary to differentiate which of these two explanations is correct. However, regardless of the mechanism(s), the existence of progressive, severe DJD but not joint inflammation in mice lacking Opg implies that supplementation of human patients with arthritis to boost OPG levels may represent an appropriate strategy for sustaining cartilage integrity in arthritic joints. Acknowledgments The authors thank Reviewers 1 and 2 for their extensive comments, which have greatly improved the final paper, and Jill Findlay and Deborah Gillette for technical assistance with formatting the figures.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
References 1. Bomsta BD, Bridgewater LC, Seegmiller RE. Premature osteoarthritis in the Disproportionate micromelia (Dmm) mouse. Osteoarthritis Cartilage. 2006;14(5):477–485.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
10
Veterinary Pathology
2. Botter SM, van Osch GJ, Waarsing JH, et al. Quantification of subchondral bone changes in a murine osteoarthritis model using micro-CT. Biorheology. 2006;43(3–4):379–388. 3. Bucay N, Sarosi I, Dunstan CR, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998;12(9):1260–1268. 4. Coles JM, Zhang L, Blum JJ, et al. Loss of cartilage structure, stiffness, and frictional properties in mice lacking PRG4. Arthritis Rheum. 2010;62(6):1666–1674. 5. Cohen-Solal M, Funck-Brentano T, Hay E. Animal models of osteoarthritis for the understanding of the bone contribution. BoneKEy Rep. 2013;2:422. 6. Funck-Brentano T, Cohen-Solal M. Crosstalk between cartilage and bone: when bone cytokines matter. Cytokine Growth Factor Rev. 2011;22(2):91–97. 7. Funck-Brentano T, Lin H, Hay E, et al. Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism. PLoS One. 2012:7(3):e33543. 8. Glasson SS, Chambers MG, Van Den Berg WB, et al. The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage. 2010;18(suppl 3):S17–S23. 9. Kamekura S, Hoshi K, Shimoaka T, et al. Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthritis Cartilage. 2005;13(7):632–641. 10. Kikuta J, Ishii M. Osteoclast migration, differentiation and function: novel therapeutic targets for rheumatic diseases. Rheumatology. 2013;52(2):226–234. 11. Klopfleisch R. Multiparametric and semiquantitative scoring systems for the evaluation of mouse model histopathology—a systematic review. BMC Vet Res. 2013;9:123. 12. Komuro H, Olee T, Kuhn K, et al. The osteoprotegerin/receptor activator of nuclear factor kB/receptor activator of nuclear factor kB ligand system in cartilage. Arthritis Rheum. 2001;44(12):2768–2776.
13. Kotlarz H, Gunnarsson CL, Fang H, et al. Insurer and out-ofpocket costs of osteoarthritis in the US: evidence from national survey data. Arthritis Rheum. 2009;60(12):3546–3553. 14. Ota N, Takaishi H, Kosaki N, et al. Accelerated cartilage resorption by chondroclasts during bone fracture healing in osteoprotegerindeficient mice. Endocrinology. 2009;150(11):4823–4834. 15. Pfuntner A, Weir LM, Stocks C. Most Frequent Conditions in U.S. Hospitals, 2011: Statistical Brief. Healthcare Cost and Utilization Project (HCUP) Statistical Brief 162. Rockville, MD: Agency for Healthcare Research and Quality; 2013. 16. Sacks JJ, Luo YH, Helmick CG. Prevalence of specific types of arthritis and other rheumatic conditions in the ambulatory health care system in the United States, 2001–2005. Arthritis Care Res. 2010;62(4):460–464. 17. Shimizu S, Asou Y, Itoh S, et al. Prevention of cartilage destruction with intraarticular osteoclastogenesis inhibitory factor/osteoprotegerin in a murine model of osteoarthritis. Arthritis Rheum. 2007;56(10):3358–3365. 18. Tat SK, Pelletier JP, Lajeunesse D, et al. Differential modulation of RANKL isoforms by human osteoarthritic subchondral bone osteoblasts: influence of osteotropic factors. Bone. 2008;43(2): 284–291. 19. Tat SK, Amiable N, Pelletier JP, et al. Modulation of OPG, RANK and RANKL by human chondrocytes and their implication during osteoarthritis. Rheumatology. 2009;48(12):1482–1490. 20. Tat SK, Pelletier JP, Velasco CR, et al. New perspective in osteoarthritis: the OPG and RANKL system as a potential therapeutic target? Keio J Med. 2009;58(1):29–40. 21. Yamazaki H, Sasaki T. Effects of osteoprotegerin administration on osteoclast differentiation and trabecular bone structure in osteoprotegerin-deficient mice. J Electron Microsc (Tokyo). 2005;54(5):467–477. 22. Zhang Y, Jordan JM. Epidemiology of osteoarthritis. Clin Geriatric Med. 2010;26(3):355–369.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Original Article
Generalized Degenerative Joint Disease in Osteoprotegerin (Opg) Null Mutant Mice
Veterinary Pathology 1-10 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985815586221 vet.sagepub.com
B. Bolon1, M. Grisanti2, K. Villasenor2, S. Morony2, U. Feige3, and W. S. Simonet2
Abstract Bone structure is modulated by the interaction between receptor activator of nuclear factor–kB (RANK) and RANK ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor for RANKL, modifies osteoclast-mediated bone resorption directly and spares articular cartilage indirectly in rodents with immune-mediated arthritis by preventing subchondral bone destruction. The OPG/RANKL balance also seems to be critical in maintaining joint integrity in osteoarthritis, a condition featuring articular bone and cartilage damage in the absence of profound inflammation. The current study explored the role of OPG in sparing articular cartilage by evaluating joint lesions in adult C57BL/6J mice lacking osteoprotegerin (Opg/). At 3, 5, 7, 9, and 12 months of age, both sexes of Opg/ mice developed severe degenerative joint disease (DJD) characterized by progressive loss of cartilage matrix and eventually articular cartilage. Lesions developed earlier and more severely in Opg/ mice relative to age-matched, wild-type (Opgþ/þ), or heterozygous (Opgþ/) littermates (P .05). The femorotibial joint was affected bilaterally at 3 months, while other key weight-bearing diarthrodial joints (eg, coxofemoral, scapulohumeral, humeroradioulnar) were affected later and unilaterally. Cortical bone in subchondral plates and long bone diaphyses of Opg/ mice but not Opgþ/þ or Opgþ/ animals was osteoporotic by 3 months of age (P .05); the extent of porosity was less than the degree of DJD. Closure of the physes in long bones (P .05) and cartilage retention in the femoral primary spongiosa (P .05) affected chiefly Opg/ mice. These data suggest that OPG plays an essential direct role in maintaining cartilage integrity in the articular surfaces and physes. Keywords cartilage, degenerative joint disease, knockout, mouse, OPG, osteoarthritis, osteoprotegerin, RANKL
Osteoarthritis (OA) or degenerative joint disease (DJD) results from extensive remodeling of articular cartilage and subchondral bone in response to longstanding biochemical/metabolic abnormalities and/or imbalances in biomechanical stress. This disease is the most common cause of joint deterioration, affecting people in the United States22 with a prevalence of clinical disease estimated at between 2.4% and 8.5% of the population.16 The disease can affect any diarthrodial joint but tends to affect weight-bearing articulations such as the coxofemoral (hip) and femorotibial (knee or stifle) joints. Debilitation associated with chronic pain from OA is a major economic burden, in the United States accounting for approximately 21 million visits annually to a primary care physician16 and nearly a million hospitalizations yearly15 (usually ending in knee or hip arthroplasty [total joint replacement]) at a cumulative annual cost of $185.5 billion.13 Joint structure in health and disease depends on tightly coordinated, reciprocal, cell-to-cell communication between osteoblasts and osteoclasts, with osteoblasts acting to form bone and also to control the activity of bone-eroding osteoclasts.10,20 A key signaling pathway facilitating this communication involves the interactions among receptor activator of nuclear factor (NF)–kB (RANK), a membrane-bound receptor on osteoclasts;
its binding partner RANK ligand (RANKL); and osteoprotegerin (OPG, more formally designated as tumor necrosis factor receptor superfamily member 11b [Tnfrsf11b]), an osteoblastderived osteoclast inhibitor that functions as a soluble decoy receptor for RANKL. Abnormalities in cartilage architecture during OA have been linked to altered expression of RANK, RANKL, and OPG in cartilage;2,5,20 indeed, chondrocytes express and produce OPG and RANKL, and those taken from OA joints exhibit a shift toward overproduction of RANKL.19 Administration of OPG has been shown to protect cartilage in joints of mice with OA.17 Given the crosstalk between bone and cartilage in joints,6 whether or not such changes represent
1
The Ohio State University, Columbus, OH, USA Amgen, Inc., Thousand Oaks, CA, USA 3 EUROCBI GmbH, Benglen, Zurich, Switzerland 2
Supplemental material for this article is available on the Veterinary Pathology website at http://vet.sagepub.com/supplemental. Corresponding Author: B. Bolon, GEMpath, Inc, 307 Coffman Rd, Suite 4D, Longmont, CO 80501, USA. Email: [email protected]
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
2
Veterinary Pathology
Figure 1. Femorotibial joint (para-axial orientation), 3-month-old wild-type mouse (Opgþ/þ [control]). The femur (F) and tibia (T) have intact articular and physeal cartilages as well as dense subchondral bone plates. Hematoxylin and eosin (HE). Figure 2. Femorotibial joint, 3-month-old mouse lacking osteoprotegerin (Opg/). The femur and tibia exhibit slight indentations of the articular cartilage surface and increased porosity of the subchondral bone, and the physeal cartilage in the femur is partly closed (long arrow). HE.
a primary impact on cartilage, a secondary consequence of subchondral osteoblast dysfunction, or some combination of the two remains to be determined. The current study had 2 purposes. The first aim was to define the lesion progression in multiple weight-bearing joints of Opg/ mice over time. This goal was undertaken because the original description of the Opg/ mutation3 portrayed in detail the early-onset, generalized, severe osteoporosis that represents the fundamental skeletal phenotype in Opg/ mice while underemphasizing the potential for concurrent joint abnormalities. The second objective was to assess the
hypothesis that an Opg deficit (ie, an altered tissue OPG/ RANKL ratio) will lead to substantial anomalies in cartilage biochemistry (as assessed by altered cartilage matrix staining) and/or structure (indicated by cartilage fragmentation or loss) in cartilaginous organs, especially weight-bearing diarthrodial joints. Our results have achieved these 2 purposes by providing (1) a thorough description of the distribution and severity of joint lesions in adult Opg/ mice over time and (2) histopathologic evidence that Opg-deficient joints and physes (growth plates) experience significant alterations in cartilage structure and biochemistry.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
3
Figure 3. Femorotibial joint, 9-month-old wild-type (Opgþ/þ [control]) mouse. The femur and tibia retain their intact articular and physeal cartilages and dense subchondral bone. Hematoxylin and eosin (HE). Figure 4. Femorotibial joint, 9-month-old Opg/ mouse. The femur and tibia lack articular cartilage and exhibit markedly thinned subchondral and cortical bone, while the femoral physis is closed except for a small remnant (short arrow). The bone and cartilage of the meniscus (arrowhead) display similar disruption of the bone and cartilage. HE.
Materials and Methods Ethical Treatment of Animals The work was conducted in 2002 in accordance with federal (Guide for the Care and Use of Laboratory Animals) and California state guidelines following a protocol approved in advance by the Amgen Institutional Animal Care and Use Committee.
appropriately engineered ES cells derived from a 129/SvJ mouse embryo were microinjected into C57BL/6J blastocysts, after which chimeric offspring were mated to Tac:N:NIH(S)-BC (Black Swiss) mice at 6 to 8 weeks of age. All animals were supplied with pelleted rodent chow and filter-purified tap water ad libitum and were housed in a room with constant temperature (21 + 2 C) and humidity (45% + 10%). Subsequently, heterozygous (HET) breeding pairs were used to produce null mutant (knockout [KO]), HET, and wild-type (WT) mice.
Animals and Husbandry Mice with engineered mutations in Opg (Tnfrsf11btm1Wss mice; http://www.informatics.jax.org/allele/MGI:2183229) were created in which the Opg gene was ablated in RW-4 embryonic stem (ES) cells from the 129X1/SvJ mouse strain and established on a C57BL6 genetic background as described previously.3 Briefly,
Clinical Observations Multiple mice of each genotype were assessed weekly throughout their lives for any ambulatory difficulties by routine evaluation of the quantity (amount) and quality (character) of their gait and motility. The examination involved viewing each
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
4
Veterinary Pathology
Table 1. Summary Histopathology Data: Biologically Significant Structural Lesions in the Femorotibial Joint in Age-Matched OPG Null (KO), OPG HET, and WT Mice. Degenerative Lesions Age, mo Genotype No. Males No. Females Total No.
JD Score
Osteophyte Score
Cartilage Score
Changes Reflecting Opg Null Mutation Cortical Bone Porosity
Physeal Closure
Cartilage Retention
3
WT HET KO
2 5 4
3 5 5
5 10 9
0 0 *1.1 + 0.4
0 0.2 + 0.2 0 0.2 + 0.1 0.3 + 0.2 *1.8 + 0.6
0 0 *2.3 + 0.5
0 0 0 0 *1.2 + 0.3 *0.4 + 0.2
5
WT HET KO
5 5 5
5 4 5
10 9 10
0.5 + 0.2 0.2 + 0.2 0.9 + 0.3 *2.0 + 0.6 0.7 + 0.3 *2.3 + 0.5 *2.2 + 0.2 *0.8 + 0.2 *3.1 + 0.5
0 *1.6 + 0.5 *2.6 + 0.3
0 *0.8 + 0.3 *1.9 + 0.1
7
WT HET KO
5 5 6
5 4 5
10 9 11
0.3 + 0.2 0 0.2 + 0.2 0 0 0.2 + 0.1 *2.5 + 0.2 *1.0 + 0.3 *3.3 + 0.2
0 0 *2.7 + 0.2
0 0 0 0 *1.7 + 0.1 *0.4 + 0.2
9
WT HET KO
5 5 3
5 4 4
10 9 7
2.7 + 0.5 2.2 + 0.5 3.4 + 0.3
1.3 + 0.3 0.7 + 0.3 1.4 + 0.3
2.6 + 0.5 2.4 + 0.5 4.0 + 0
0 0.1 + 0.1 *3.3 + 0.3
0 0.1 + 0.1 *2.0 + 0
12
WT HET KO
5 5 3
5 5 4
10 10 7
2.3 + 0.7 *1.1 + 0.6 *4.6 + 0.2
1.0 + 0.3 0.6 + 0.3 1.7 + 0.3
2.0 + 0.6 1.0 + 0.5 4.0 + 0
0 0 *3.1 + 0.3
0.2 + 0.2 0 0 0 *1.7 + 0.3 *0.6 + 0.2
15
WT
5
4
9
2.4 + 0.6
0.7 + 0.3
2.4 + 0.5
0
0
0.2 + 0.1 0.1 + 0.1 0.5 + 0.2
0.1 + 0.1 0 0.4 + 0.2
0
Values represent mean + standard deviation (SD) of the histopathology scores. Bolded values indicate a substantial difference, and asterisks (*) denote a significant divergence from age-matched wild type animals, P .05, by the Wilcoxon rank-sum test. HET, heterozygous; JD, joint degeneration; KO, knockout; OPG, osteoprotegerin; WT, wild type.
mouse’s activity level from behind and from one or both sides during routine bedding changes. The observation period was 10 to 30 seconds per animal, with longer times spent confirming the presence and evaluating the degree of any abnormalities in locomotion. A specific notation of an animal’s performance was made only if the clinical examination was considered ‘‘abnormal.’’
Skeletal Processing Mice of each genotype were deeply anesthetized with carbon dioxide, euthanized by exsanguination and thoracotomy, and autopsied at 3, 5, 7, 9, 12, or 15 months of age. Viscera were removed and the carcasses were skinned so that the outlines of major appendicular and axial (chiefly vertebral) bones could be assessed for gross distortions. Multiple joints from each mouse were harvested and fixed by immersion in neutral buffered 10% formalin for 48 hours; knees were examined at all 6 time points, while other sites were assessed only at 3 or 9 months. Fixed specimens were decalcified with a 1:1 mixture of 8 N formic acid and 1 N sodium formate until bones could be cut easily with a razor blade (typically 36–48 hours). After decalcification, specimens were processed into paraffin, cut serially at 4 mm, and stained with hematoxylin and eosin (HE) to evaluate general joint structure or with toluidine blue to assess matrix integrity in articular cartilage.
Histopathologic Scoring Three main indices of cartilage damage in diarthrodial articulations were emphasized in the joint evaluation: the joint
degeneration (JD) score (a global index of joint destruction), the cartilage score (an index of matrix integrity in the articular cartilage), and the osteophyte score (an estimate of skeletal proliferation as a periarticular repair process to stabilize deformed joints). An inflammation score was obtained to evaluate the degree to which leukocyte infiltration associated with the joint cavity, periarticular tissues, and subchondral bone might have contributed to the induction of these cartilaginous changes. The extent of the expected osteoporotic phenotype associated with Opg ablation3 was determined by collecting a score for cortical porosity (a direct gauge of osteoporosis) for subchondral bone and the diaphyses of long bones. Finally, scores were acquired for physeal closure (an indication of premature growth plate attenuation that is another expected phenotype of Opg deficiency21) in the femur and tibia as well as cartilage retention (a potential index of altered endochondral ossification) in the femoral primary spongiosa; these 2 scores were collected to explore the consequences of Opg deficiency to induce generalized alterations in cartilage biology. These 6 semiquantitative histopathologic scores were devised specifically to focus on major anatomic attributes of articular cartilage and subchondral bone damage observed in Opg/ mice; the features chosen for analysis and the criteria used to assign scores were equivalent to those of many other scoring schemes used to grade joint lesions in mouse models of OA, which were established in the decade after this study was performed.1,4,8,9,11 For all scores, lesion severity was graded using tiered, semiquantitative scales (Suppl. Tables S1, S2), which were assigned during a coded (‘‘blinded’’) analysis.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
5
Figure 5. Femorotibial joint (para-axial orientation), 3-month-old Opgþ/þ (control) mouse. The femur (F) and tibia (T) have smooth articular surfaces and very few marrow-filled spaces extending from the epiphyseal marrow cavity into the dense plates of subchondral bone. Matrix (dark purple) in the articular cartilage extends across the entire length of both joint surfaces. Toluidine blue. Figure 6. Femorotibial joint, 3-monthold Opg/ mouse. The femur and tibia have thin articular cartilages and exhibit loss of cartilage matrix at multiple sites, especially near the joint margins (indicated by regions of pallor). Toluidine blue. Figure 7. Femorotibial joint, 9-month-old Opgþ/þ (control) mouse. Focal loss (between the arrows, femur only) or narrowing (femur and tibia) of the nonmineralized (deep purple) articular cartilages. Toluidine blue. Figure 8. Femorotibial joint, 9-month-old Opg/ mouse. The femur and tibia have rough articular surfaces due to the loss of nonmineralized cartilage and sometimes also the mineralized cartilage (between the arrows, femur only). The tibial physis is discontinuous (arrowheads), indicating partial closure. Large osteophytes (*) are evident near one meniscus (M). Toluidine blue.
Statistical Analysis
Results
Histopathologic (ordinal) data were compared using the nonparametric Wilcoxon rank-sum test. Because the spontaneous incidences of the lesions associated with Opg ablation are negligible in wild-type mice (C57BL/6 background), the significance and power were set at .05 and 90%, respectively, by selecting group sizes (n) of 5 or more mice per genotype per time point. Statistical calculations were done using JMP statistical software (v. 5.0; SAS Institute, Cary, NC).
Clinical Observations A few KO mice but no HET or WT animals were seen to be limping slightly on one hind limb just prior to autopsy. Animals with gait abnormalities were at least 9 months old.
Skeletal Pathology Gross joint abnormalities or bone distortions were not seen in mice of any genotype. However, the constellation of
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
6
Veterinary Pathology
Table 2. Incidence of Biologically Significant Structural Lesions in Other Joints of OPG Null (KO), OPG HET, and WT Mice at 9 Months of Age. Genotype
Sex
WT
Male Female Total affected Missing Male Female Total affected Missing Male Female Total affected Missing
HET
KO
No. of Mice
Scapulohumeral
Humeroradioulnar
Intercarpal
Coxofemoral
Intertarsal
5 5 10
1 0 1 (10) 0 1 0 1 (11) 0 2 4 *6 (100) 0
0 2 2 (20) 0 0 3 3 (33) 0 2 3 *5 (83) 0
0 0 0 (0) 0 0 0 0 (0) 0 0 3 *3 (50) 0
0 1 1 (13) 2 0 1 1 (11) 1 3 2 *5 (83) 0
0 0 0 (0) 0 0 0 0 (0) 0 0 2 2 (33) 0
5 4 9 2 4 6
Intervertebral facets of all animals exhibited multifocal cell loss and/or superficial fibrillation, regardless of genotype. Values represent the number of animals with affected joints; the values in parentheses represent the percentage of affected mice. Bolded values indicate a substantial difference, and asterisks (*) denote a significant difference from age-matched wild type animals, P .05, by the Wilcoxon rank-sum test. HET, heterozygous; KO, knockout; OPG, osteoprotegerin; WT, wild type.
histopathologic lesions in the KO mice was indicative of generalized DJD (based on articular cartilage damage) in tandem with osteoporosis. The interface between the affected articular cartilage and subjacent subchondral bone was intact in affected joints. Relative to age-matched WT controls, the knees of KO animals exhibited a significant (P .05) incidence of DJD at 3 months of age, which progressed in severity over time (Figs. 1–4). The changes were often but not always bilateral, and both sexes were affected equally. The mean JD score of KO mice was minimal at 3 months of age, mild at 5 months, moderate at 9 months, and severe at 12 months (Table 1). In like manner, the cartilage score of KO animals was mild at 3 months, moderate at 5 to 7 months, and marked at 9 to 12 months (Table 1). Both the JD score and the cartilage score were increased significantly (P .05) in KO mice relative to WT littermates at 3, 5, 7, and 12 months of age and exhibited a trend toward a substantial but nonsignificant increase at 9 months (Figs. 5–8). Osteophyte scores in knees of KO animals were significantly higher (P .05) by 1 grade than those of WT mice at 5 and 7 months (Table 1). At the other time points, the various joint degeneration scores in knees of KO animals exceeded those of WT mice, but the extent of joint degeneration in the WT animals was so substantial that statistical comparison could not distinguish the groups. Multiple other joints of KO animals also exhibited significantly more DJD (P .05) than did WT mice (Table 2). The principal cartilage abnormalities in affected joints were cartilage and chondrocyte loss and sometimes osteophyte production. Chondrocyte loss occurred to an equal extent in both sexes, while osteophyte production was more common in KO females. Sites with more DJD in KO mice included the scapulohumeral (shoulder; Figs. 9 vs 10), humeroradioulnar (elbow; Figs. 11 vs 12), coxofemoral (Figs. 13 vs 14), tibiotarsal (hock or ankle; Figs. 15 vs 16), intercarpal, and intertarsal joints. Neither the intervertebral facets (spine) nor the
temporomandibular (jaw) joints of KO mice were affected to a greater degree than in WT animals. Lesions occurred in 9-month-old KO mice but not in 3-month-olds and typically were unilateral. The absence of Opg was not associated with joint inflammation. Leukocytes were not visible in the synovium or periarticular tissues or within the joint cavity. Similarly, leukocytes were not observed within the bone marrow adjacent to the subchondral bone plate. Ablation of Opg in KO mice also was associated with alterations to bone and nonarticular cartilage. Relative to age-matched WT controls, the long bones of KO animals had a significantly increased (P .05) incidence of bony changes comprising hallmark features of Opg deficiency3 at 3 months of age (Table 1). Affected KO mice had more porous cortical bone (ie, osteoporosis), which was evident in both the diaphysis and subchondral bone as many narrow channels extending from the marrow cavity toward the joint; these spaces rarely reached the deep surface of the mineralized articular cartilage (Figs. 5 vs 6). The extent of osteoporosis was similar in both the diaphysis and subchondral bone. Furthermore, affected KO animals exhibited one or multiple regions of growth plate closure in the femur (Figs. 2, 4) and occasionally in the tibia along with small foci of retained cartilage in the trabeculae of the femoral primary spongiosa. The growth plate in the femur was partially closed in all KO mice at 3 months, while both femoral and tibial growth plates were partially and occasionally completely closed at 5 months or later. In contrast, bones of WT mice had solid cortices, intact growth plates, and trabeculae formed only of bone in the primary and secondary spongiosae (Figs. 1, 3). The HET and WT mice also had DJD, but both the severity and prevalence were lower compared with KO animals. Significant (P .05) DJD was seen in HET mice at 5 and 9 months of age but not at other time points (Table 1); some of these 5-month-old animals, but not the 9-month-old mice, had increased cortical porosity,
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
7
Figure 9. Scapulohumeral joint, 9-month-old Opgþ/þ (control) mouse. The scapula (S) and humeral head (H) have smooth articular surfaces, and the proximal physis of the humerus is intact. Matrix (dark purple) in the articular cartilage extends across the full length of both joint surfaces. Toluidine blue. Figure 10. Scapulohumeral joint, 9-month-old Opg/ mouse. The scapula and humeral head exhibit attenuation or loss of articular cartilage, and the humeral physis is partly absent. A massive osteophyte (asterisk) expands the peri-articular soft tissue. Toluidine blue. Figure 11. Humeroradioulnar joint, 9-month-old Opgþ/þ (control) mouse. The distal humerus (H), radius (R), and ulna (U) have smooth articular surfaces with extension of the intact matrix (dark purple) across the full length of the joint. Toluidine blue. Figure 12. Humeroradioulnar joint, 9-month-old Opg/ mouse. The humerus, radius, and ulna exhibit multifocal loss (arrows) or thinning of their articular cartilage. Toluidine blue. Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
8
Veterinary Pathology
Figure 13. Coxofemoral joint, 9-month-old Opgþ/þ (control) mouse. The pelvic acetabulum (A) and femoral head (F) exhibit smooth articular surfaces with uniform staining of the cartilage matrix (dark purple) across the full length of the joint. Toluidine blue. Note: The apparent increased thickness of the articular cartilage results from a plane of section that is slightly tangential to the main axis of the femoral head. Figure 14. Coxofemoral joint, 9-month-old Opg/ mouse. The articular surfaces of the acetabulum and femoral head are substantially attenuated across the entire extent of the joint. Toluidine blue. Figure 15. Tibiotarsal joint, 9-month-old Opgþ/þ (control) mouse. The distal tibia (Ti) and talus (Ta) have smooth articular surfaces subtended by dense plates of subchondral bone. Hematoxylin and eosin (HE). Figure 16. Tibiotarsal joint, 9-month-old Opg/ mouse. The articular surfaces of the tibia (Ti) and talus (Ta) exhibit multifocal loss (between the arrows) or roughening, and the subchondral bone is extremely porous. HE.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
Bolon et al
9
partial physeal closure, and cartilage retention in the trabeculae of the femoral primary spongiosa (Table 1). The extent of the joint degeneration and the KO-like bone changes was lower in HET animals than in age-matched KO mice. In WT mice, both the JD score and the cartilage score were essentially absent until 9 months of age, at which time mild lesions were first observed (Table 1). Findings in WT mice did not progress beyond mild severity through 15 months of age.
Discussion Our current descriptive data demonstrate that mice that lack both Opg alleles (KO) exhibit more severe DJD, affecting many more weight-bearing joints than do age-matched animals with only 1 Opg allele (HET) or having the normal complement of 2 Opg alleles (WT). Specific changes in KO joints included matrix degeneration, superficial fibrillation, and chondrocyte loss affecting articular cartilage. These cartilage changes were accompanied by progressive osteoporosis of the subchondral bone and sometimes by osteophyte production at the joint margins as a preliminary repair process to stabilize the deforming joints. The femorotibial joint (ie, the major weight-bearing articulation in quadrupeds) had the most severe lesions, and the changes were obvious and generally bilateral at a young age (3 months vs 9 months in WT animals) (Table 1). Multiple other diarthrodial joints of KO mice also developed DJD (Table 2), but these tended to occur later in life and typically were unilateral. Importantly, the interfaces between the deep margins of the damaged articular cartilage and the osteoporotic subchondral bone associated with these margins were intact. Taken together, our data confirm our hypothesis that longstanding Opg deficiency is associated not only with a severe osteoporotic phenotype in bone but also with generalized, progressive, early-onset injury to articular cartilage within major weight-bearing joints. This outcome supports prior reports indicating that Opg modulates cartilage catabolism7 and fracture callus remodeling.14 Our data also provide indirect evidence that Opg plays a vital role in regulating the biology of cartilaginous tissues in general, not simply the health of articular cartilage. This inference is supported by the finding that significantly more Opg/ mice exhibited foci in the femur of physeal closure and, to a lesser extent, cartilage retention within the trabeculae of the primary spongiosa. These changes were not associated with prominent shifts in the structure of bony features in the immediate vicinity, suggesting that the cartilaginous alterations might occur independent of the osteoporosis phenotype affecting cortical bone of Opg/ animals. The possibility that OPG helps to maintain cartilage integrity, especially in joints, gains further credibility because OA in humans is characterized by altered expression of RANK, RANKL, and OPG in articular cartilage. These molecules are found in the superficial zone of normal cartilage, while their localization is shifted to the middle zone during OA.12 Alternatively, another causal factor in cartilage destruction during OA may be dysfunction of subchondral osteoblasts.18 Increased porosity of subchondral bone
in tandem with thinning of the articular cartilage in femorotibial joints of 3-month-old Opg/ mice supports this supposition, although the degree of bone damage in these animals was modest compared with the degree of matrix degeneration and cartilage fibrillation in the superficial articular cartilage. Further work will be needed to ascertain whether cartilage damage in Opg/ mice results chiefly from direct insults to chondrocytes, indirectly by anomalies of subchondral bone that secondarily lead to chondrocyte damage, or some combination of the two.
Conclusions Our current data provide a detailed description of the generalized joint lesions affecting articular cartilage and bone in weight-bearing diarthrodial joints of mice engineered to lack Opg. The most prominent changes were DJD characterized by early-onset, progressive loss of cartilage staining by toluidine blue (indicative of matrix degeneration) and subsequently fibrillation, chondrocyte loss, and ultimately cartilage loss affecting the femorotibial joint bilaterally and later the scapulohumeral, humeroradioulnar, and coxofemoral articulations unilaterally. The co-occurrence of severe, progressive osteoporosis (the expected skeletal phenotype in Opg/ mice), including increased formation of marrow-filled spaces in the subchondral bone, represents one explanation for the lesions in the articular cartilage. However, in the femur, premature physeal closure and cartilage retention in the cores of primary spongiosa trabeculae also occurred almost exclusively in Opg/ mice, suggesting that an Opg deficiency also might affect cartilage biology differently from the usual regulation of RANKL-induced osteoclast activity. Further work will be necessary to differentiate which of these two explanations is correct. However, regardless of the mechanism(s), the existence of progressive, severe DJD but not joint inflammation in mice lacking Opg implies that supplementation of human patients with arthritis to boost OPG levels may represent an appropriate strategy for sustaining cartilage integrity in arthritic joints. Acknowledgments The authors thank Reviewers 1 and 2 for their extensive comments, which have greatly improved the final paper, and Jill Findlay and Deborah Gillette for technical assistance with formatting the figures.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
References 1. Bomsta BD, Bridgewater LC, Seegmiller RE. Premature osteoarthritis in the Disproportionate micromelia (Dmm) mouse. Osteoarthritis Cartilage. 2006;14(5):477–485.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
10
Veterinary Pathology
2. Botter SM, van Osch GJ, Waarsing JH, et al. Quantification of subchondral bone changes in a murine osteoarthritis model using micro-CT. Biorheology. 2006;43(3–4):379–388. 3. Bucay N, Sarosi I, Dunstan CR, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998;12(9):1260–1268. 4. Coles JM, Zhang L, Blum JJ, et al. Loss of cartilage structure, stiffness, and frictional properties in mice lacking PRG4. Arthritis Rheum. 2010;62(6):1666–1674. 5. Cohen-Solal M, Funck-Brentano T, Hay E. Animal models of osteoarthritis for the understanding of the bone contribution. BoneKEy Rep. 2013;2:422. 6. Funck-Brentano T, Cohen-Solal M. Crosstalk between cartilage and bone: when bone cytokines matter. Cytokine Growth Factor Rev. 2011;22(2):91–97. 7. Funck-Brentano T, Lin H, Hay E, et al. Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism. PLoS One. 2012:7(3):e33543. 8. Glasson SS, Chambers MG, Van Den Berg WB, et al. The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage. 2010;18(suppl 3):S17–S23. 9. Kamekura S, Hoshi K, Shimoaka T, et al. Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthritis Cartilage. 2005;13(7):632–641. 10. Kikuta J, Ishii M. Osteoclast migration, differentiation and function: novel therapeutic targets for rheumatic diseases. Rheumatology. 2013;52(2):226–234. 11. Klopfleisch R. Multiparametric and semiquantitative scoring systems for the evaluation of mouse model histopathology—a systematic review. BMC Vet Res. 2013;9:123. 12. Komuro H, Olee T, Kuhn K, et al. The osteoprotegerin/receptor activator of nuclear factor kB/receptor activator of nuclear factor kB ligand system in cartilage. Arthritis Rheum. 2001;44(12):2768–2776.
13. Kotlarz H, Gunnarsson CL, Fang H, et al. Insurer and out-ofpocket costs of osteoarthritis in the US: evidence from national survey data. Arthritis Rheum. 2009;60(12):3546–3553. 14. Ota N, Takaishi H, Kosaki N, et al. Accelerated cartilage resorption by chondroclasts during bone fracture healing in osteoprotegerindeficient mice. Endocrinology. 2009;150(11):4823–4834. 15. Pfuntner A, Weir LM, Stocks C. Most Frequent Conditions in U.S. Hospitals, 2011: Statistical Brief. Healthcare Cost and Utilization Project (HCUP) Statistical Brief 162. Rockville, MD: Agency for Healthcare Research and Quality; 2013. 16. Sacks JJ, Luo YH, Helmick CG. Prevalence of specific types of arthritis and other rheumatic conditions in the ambulatory health care system in the United States, 2001–2005. Arthritis Care Res. 2010;62(4):460–464. 17. Shimizu S, Asou Y, Itoh S, et al. Prevention of cartilage destruction with intraarticular osteoclastogenesis inhibitory factor/osteoprotegerin in a murine model of osteoarthritis. Arthritis Rheum. 2007;56(10):3358–3365. 18. Tat SK, Pelletier JP, Lajeunesse D, et al. Differential modulation of RANKL isoforms by human osteoarthritic subchondral bone osteoblasts: influence of osteotropic factors. Bone. 2008;43(2): 284–291. 19. Tat SK, Amiable N, Pelletier JP, et al. Modulation of OPG, RANK and RANKL by human chondrocytes and their implication during osteoarthritis. Rheumatology. 2009;48(12):1482–1490. 20. Tat SK, Pelletier JP, Velasco CR, et al. New perspective in osteoarthritis: the OPG and RANKL system as a potential therapeutic target? Keio J Med. 2009;58(1):29–40. 21. Yamazaki H, Sasaki T. Effects of osteoprotegerin administration on osteoclast differentiation and trabecular bone structure in osteoprotegerin-deficient mice. J Electron Microsc (Tokyo). 2005;54(5):467–477. 22. Zhang Y, Jordan JM. Epidemiology of osteoarthritis. Clin Geriatric Med. 2010;26(3):355–369.
Downloaded from vet.sagepub.com at CMU Libraries - library.cmich.edu on September 14, 2015
