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The OARSI histopathology initiative – recommendations for histological assessments of osteoarthritis in the dog

      Summary

      The dog is a common model for study of osteoarthritis (OA). Subjective histologic scoring systems have often served as the reference standard for presence and severity of OA. However, these scoring systems have perceived shortcomings. The system developed for this report attempts to address these shortcomings by providing a standardized methodology for global assessment of the joint, versatility and the potential for relative weighting of pathology, allowing for comparison among time points, studies, and centers, and critical analysis of the system’s reliability. The proposed system for assessment of canine tissues appears to provide an effective method for global assessment of articular pathology in OA. The system is versatile, comprehensive, and reliable and appears to have advantages over conventional scoring systems.

      Keywords

      Introduction

      Histologic assessment of osteoarthritis (OA) is currently considered the gold standard for determining presence, extent and severity of OA. The dog is the most studied species with respect to models of OA (see Table I for an overview) and, importantly, spontaneously occurring OA is common in dogs in multiple joints from various etiologies. The vast majority of laboratory and clinical studies have used the Mankin or modified Mankin scoring systems to determine the presence or absence of OA and assess the extent and severity of OA based on subjective identification of histologic criteria of articular cartilage pathology (Table II). While this methodology has produced a vast amount of useful and relatively repeatable data, a comprehensive literature search (PubMed and Medline,
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      • et al.
      Experimental hyaline cartilage lesions: two-dimensional spin-echo versus three-dimensional gradient-echo MR imaging.
      • Altman R.D.
      • Dean D.D.
      • Muniz O.E.
      • Howell D.S.
      Therapeutic treatment of canine osteoarthritis with glycosaminoglycan polysulfuric acid ester.
      • Altman R.D.
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      Preliminary observations of chondral abrasion in a canine model.
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      • et al.
      Chondroscopy: a new method for scoring chondropathy.
      • Bendele A.M.
      Animal models of osteoarthritis.
      • Boileau C.
      • Martel-Pelletier J.
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      • Tardif G.
      • Schrier D.
      • Flory C.
      • et al.
      Oral treatment with PD-0200347, an a2d ligand, reduces the development of experimental osteoarthritis by inhibiting metalloproteinases and inducible nitric oxide synthase gene expression and synthesis in cartilage chondrocytes.
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      • Martel-Pelletier J.
      • Abram F.
      • Raynauld J.P.
      • Troncy E.
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      • et al.
      Magnetic Resonance Imaging can accurately assess the long-term progression of knee structural changes in experimental dog OA.
      • Bove S.E.
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      • et al.
      Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis.
      • Breinan H.A.
      • Hsu H.P.
      • Spector M.
      Chondral defects in animal models: effects of selected repair procedures in canines.
      • Burkhardt J.E.
      • Hill M.A.
      • Carlton W.W.
      • Kesterson J.W.
      Histologic and histochemical changes in articular cartilages of immature beagle dogs dosed with difloxacin, a fluoroquinolone.
      • Canapp Jr., S.O.
      • McLaughlin Jr., R.M.
      • Hoskinson J.J.
      • Roush J.K.
      • Butine M.D.
      Scintigraphic evaluation of dogs with acute synovitis after treatment with glucosamine hydrochloride and chondroitin sulfate.
      • Caplan A.I.
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      • Mochizuki Y.
      • Wakitani S.
      • Goldberg V.M.
      Principles of cartilage repair and regeneration.
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      • Gardin J.F.
      • Kraus V.B.
      Synovial fluid biomarker levels predict articular cartilage damage following complete medial meniscectomy in the canine knee.
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      • Martel-Pelletier J.
      • Tardif G.
      • Mineau F.
      • Geng C.
      • et al.
      Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expression.
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      • Garmong A.
      • Schultz D.R.
      • Howell D.S.
      • Altman R.D.
      • et al.
      Inhibition of insulin-like growth factor binding protein 5 proteolysis in articular cartilage and joint fluid results in enhanced concentrations of insulin-like growth factor 1 and is associated with improved osteoarthritis.
      • Cook J.L.
      • Marberry K.M.
      • Kuroki K.
      • Kenter K.
      Assessment of cellular, biochemical, and histologic effects of bipolar radiofrequency treatment of canine articular cartilage.
      • Roller B.L.
      • Stoker A.M.
      • Fox D.B.
      • Bal B.S.
      • Cook J.L.
      Characterization of pathology of knee menisci: correlation of radiographic, gross, histologic, biochemical, and molecular measures of disease.
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      • Van Roermund P.M.
      • et al.
      Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis.
      • Dew T.L.
      • Martin R.A.
      Functional, radiographic, and histologic assessment of healing of autogenous osteochondral grafts and full-thickness cartilage defects in the talus of dogs.
      • Fernandes J.C.
      • Jovanovic D.
      • Dehnade F.
      • Martel-Pelletier J.
      • Pelletier J.P.
      [Resection of the anterior cruciate ligament of the knee using arthroscopy induces arthrosis in dogs. Validity of the Pond-Nuki model].
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      • Jakob R.P.
      • Mainil-Varlet P.
      In situ compressive stiffness, biomechanical composition and structural integrity of articular cartilage in the human knee joint.
      • Garcia-Seco E.
      • Wilson D.A.
      • Cook J.L.
      • Kuroki K.
      • Kreeger J.M.
      • Keegan K.G.
      Measurement of articular cartilage stiffness of the femoropatellar, tarsocrural, and metatarsophalangeal joints in horses and comparison with biochemical data.
      • Goldenberg D.L.
      • Cohen A.S.
      Synovial membrane histopathology in the differential diagnosis of rheumatoid arthritis, gout, pseudo-gout, systemic lupus erythematosus, infectious arthritis, and degenerative joint disease.
      • Hunt N.
      • Sanchez-Ballester J.
      • Pandit R.
      • Thomas R.
      • Stachan R.
      Chondral lesions of the knee: A new localization method and correlation with associated pathology.
      • Hunt S.A.
      • Sherman O.
      Arthroscopic treatment of osteochondral lesions of the talus with correlation of outcome scoring systems.
      • Inerot S.
      • Heinegard D.
      • Olsson S.E.
      • Telhag H.
      • Audell L.
      Proteoglycan alterations during developing experimental osteoarthritis in a novel hip joint model.
      • Johnson K.A.
      • Francis D.J.
      • Manley P.A.
      • Chu Q.
      • Caterson B.
      Comparison of the effects of caudal pole hemi-meniscectomy and complete medial meniscectomy in the canine stifle joint.
      • Johnson K.A.
      • Hay C.W.
      • Chu Q.
      • Roe S.C.
      • Caterson B.
      Cartilage-derived biomarkers of osteoarthritis in synovial fluid of dogs with naturally acquired rupture of the cranial curciate ligament.
      • Johnson R.G.
      • Poole A.R.
      Degenerative changes in dog articular cartilage induced by a unilateral tibial valgus osteotomy.
      • Lafeber F.P.
      • van der Kraan P.M.
      • van Roy H.L.
      • Vitters E.L.
      • Huber-Bruning O.
      • van den Berg W.B.
      • et al.
      Local changes in proteoglycan synthesis during culture are different for normal and osteoarthritic cartilage.
      • Lahm A.
      • Uhl M.
      • Erggelet C.
      • Haberstroh J.
      • Mrosek E.
      Articular cartilage degeneration after acute subchondral bone damage: an experimental study in dogs with histopathological grading.
      • Leipold H.R.
      • Goldberg R.L.
      • Lust G.
      Canine serum keratan sulfate and hyaluronate concentrations. Relationship to age and osteoarthritis.
      • Lindhorst E.
      • Vail T.P.
      • Guilak F.
      • Wang H.
      • Setton L.A.
      • Vilim V.
      • et al.
      Longitudinal characterization of synovial fluid biomarkers in the canine meniscectomy model of osteoarthritis.
      • Lindhorst E.
      • Wachsmuth L.
      • Kimmig N.
      • Raiss R.
      • Aigner T.
      • Atley L.
      • et al.
      Increase in degraded collagen type II in synovial fluid early in the meniscectomy model of osteoarthritis.
      • Lorenz H.
      • Wenz W.
      • Ivancic M.
      • Steck E.
      • Richter W.
      Early and stable upregulation of collagen type II, collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading.
      • Lust G.
      • Pronsky W.
      • Sherman D.M.
      Biochemical studies on developing canine hip joints.
      • Mankin H.J.
      • Dorfman H.
      • Lippiello L.
      • Zarins A.
      Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. II. Correlation of morphology with biochemical and metabolic data.
      • Mankin H.J.
      The reaction of articular cartilage to injury and osteoarthritis (first of two parts).
      • Mankin H.J.
      • Thrasher A.Z.
      • Weinberg E.H.
      • Harris W.H.
      Dissociation between the effect of bovine growth hormone in articular cartilage and in bone of the adult dog.
      • Marijnissen A.C.
      • van Roermund P.M.
      • TeKoppele J.M.
      • Bijlsma J.W.
      • Lafeber F.P.
      The canine ‘groove’ model, compared with the ACLT model of osteoarthritis.
      • Marijnissen A.C.
      • van Roermund P.M.
      • Verzijl N.
      • Tekoppele J.M.
      • Bijlsma J.W.
      • Lafeber F.P.
      Steady progression of osteoarthritic features in the canine groove model.
      • Mastbergen S.C.
      • Marijnissen A.C.
      • Vianen M.E.
      • van Roermund P.M.
      • Bijlsma J.W.
      • Lafeber F.P.
      The canine ‘groove’ model of osteoarthritis is more than simply the expression of surgically applied damage.
      • Mastbergen S.C.
      • Marijnissen A.C.
      • Vianen M.E.
      • Zoer B.
      • van Roermund P.M.
      • Bijlsma J.W.
      • et al.
      Inhibition of COX-2 by celecoxib in the canine groove model of osteoarthritis.
      • McCarty Jr., D.J.
      • Phelps P.
      • Pyenson J.
      Crystal-induced inflammation in canine joints. I. An experimental model with quantification of the host response.
      • Myers S.L.
      • Brandt K.D.
      • Eilam O.
      Even low-grade synovitis significantly accelerates the clearance of protein from the canine knee. Implications for measurement of synovial fluid “markers” of osteoarthritis.
      • Myers S.L.
      • Brandt K.D.
      • O'Connor B.L.
      • Visco D.M.
      • Albrecht M.E.
      Synovitis and osteoarthritic changes in canine articular cartilage after anterior cruciate ligament transection. Effect of surgical hemostasis.
      • O’Driscoll S.W.
      • Marx R.G.
      • Beaton D.E.
      • Miura Y.
      • Gallay S.H.
      • Fitzsimmons J.S.
      Validation of a simple histological-histochemical cartilage scoring system.
      • Ogata K.
      • Whiteside L.A.
      • Andersen D.A.
      The intra-articular effect of various postoperative managements following knee ligament repair: an experimental study in dogs.
      • Outerbridge R.E.
      The etiology of chondromalacia patellae.
      • Panula H.E.
      • Helminen H.J.
      • Kiviranta I.
      Slowly progressive osteoarthritis after tibial valgus osteotomy in young beagle dogs.
      • Panula H.E.
      • Nieminen J.
      • Parkkinen J.J.
      • Arnala I.
      • Kroger H.
      • Alhava E.
      Subchondral bone remodeling increases in early experimental osteoarthrosis in young beagle dogs.
      • Pastoureau P.
      • Leduc S.
      • Chomel A.
      • De Ceuninck F.
      Quantitative assessment of articular cartilage and subchondral bone histology in the meniscectomize guinea pig model of osteoarthritis.
      • Pelletier J.-P.
      • Boileau C.
      • Brunet J.
      • Boily M.
      • Lajeunesse D.
      • Reboul P.
      • et al.
      The inhibition of subchondral bone resorption in the early phase of experimental dog osteoarthritis by licofelone is associated with a reduction in the synthesis of MMP-13 and cathepsin K.
      • Pelletier J.-P.
      • Martel-Pelletier J.
      • Ghandur-Mnaymneh L.
      • Howell D.S.
      • Woessner J.F.
      Role of synovial membrane inflammation in cartilage matrix breakdown in the Pond-Nuki dog model of osteoarthritis.
      • Pickvance E.A.
      • Oegema Jr., T.R.
      • Thompson Jr., R.C.
      Immunolocalization of selected cytokines and proteases in canine articular cartilage after transarticular loading.
      • Pond M.J.
      • Nuki G.
      Experimentally-induced osteoarthritis in the dog.
      • Rainsford K.D.
      • Skerry T.M.
      • Chindemi P.
      • Delaney K.
      Effects of the NSAIDs meloxicam and indomethacin on cartilage proteoglycan synthesis and joint responses to calcium pyrophosphate crystals in dogs.
      • Rogachefsky R.A.
      • Altman R.D.
      • Markov M.S.
      • Cheung H.S.
      Use of a permanent magnetic field to inhibit the development of canine osteoarthritis.
      • Rogachefsky R.A.
      • Dean D.D.
      • Howell D.S.
      • Altman R.D.
      Treatment of canine osteoarthritis with insulin-like growth factor-1 (IGF-1) and sodium pentosan polysulfate.
      • Rudert M.
      Histological evaluation of osteochondral defects: consideration of animal models with emphasis on the rabbit, experimental setup, follow-up and applied methods.
      • Sakakibara Y.
      • Miura T.
      • Iwata H.
      • Kikuchi T.
      • Yamaguchi T.
      • Yoshimi T.
      • et al.
      Effect of high molecular weight sodium hyaluronate on immobilized rabbit knee.
      • Scheck M.
      • Sakovich L.
      Degenerative joint disease of the canine hip: experimental production by multiple papain and prednisone injections.
      • Smith G.N.
      • Mickler E.A.
      • Albrecht M.E.
      • Myers S.L.
      • Brandt K.D.
      Severity of medial meniscus damage in the canine knee after anterior cruciate ligament transection.
      • Thompson Jr., R.C.
      • Vener M.J.
      • Griffiths H.J.
      • Lewis J.L.
      • Oegema Jr., T.R.
      • Wallace L.
      Scanning electron-microscopic and magnetic resonance-imaging studies of injuries to the patellofemoral joint after acute transarticular loading.
      • Verbruggen G.
      • Veys E.M.
      Numerical scoring systems for the progression of osteoarthritis of the finger joints.
      • Vener M.J.
      • Thompson Jr., R.C.
      • Lewis J.L.
      • Oegema Jr., T.R.
      Subchondral damage after acute transarticular loading: an in vitro model of joint injury.
      • Visco D.M.
      • Hill M.A.
      • Widmer W.R.
      • Johnstone B.
      • O’Connor B.L.
      • Myers S.L.
      Experimental Osteoarthritis in Dogs: a Comparison of the Pond-Nuki and Medial Arthrotomy Methods.
      • Wang Q.
      • Breinan H.A.
      • Hsu H.P.
      • Spector M.
      Healing of defects in canine articular cartilage: distribution of nonvascular alpha-smooth muscle actin-containing cells.
      • Wenz W.
      • Breusch S.J.
      • Graf J.
      • Stratmann U.
      Ultrastructural findings after intraarticular application of hyaluronan in a canine model of arthropathy.
      • Wind A.P.
      Elbow incongruity and developmental elbow diseases in the dog.
      • Wyland D.J.
      • Guilak F.
      • Elliott D.M.
      • Setton L.A.
      • Vail T.P.
      Chondropathy after meniscal tear or partial meniscectomy in a canine model.
      • Yabe K.
      • Satoh H.
      • Ishii Y.
      • Jindo T.
      • Sugawara T.
      • Furuhama K.
      • et al.
      Early pathophysiologic feature of arthropathy in juvenile dogs induced by ofloxacin, a quinolone antimicrobial agent.
      • Sniekers Y.H.
      • Intema F.
      • Lafeber F.P.
      • van Osch G.J.
      • van Leeuwen J.P.
      • Weinans H.
      • et al.
      A role for subchondral bone changes in the process of osteoarthritis; a micro-CT study of two canine models.
      • Boyd S.K.
      • Muller R.
      • Leonard T.
      • Herzog W.
      Long-term periarticular bone adaptation in a feline knee injury model for post-traumatic experimental osteoarthritis.
      • Intema F.
      • Sniekers Y.H.
      • Weinans H.
      • Vianen M.E.
      • Yocum S.A.
      • Zuurmond A.M.
      • et al.
      Similarities and discrepancies in subchondral bone structure in two differently induced canine models of osteoarthritis.
      ) in conjunction with review by recognized experts in the field (authors) suggest that important limitations exist including:
      • This methodology only assesses articular cartilage pathology and does not consider other tissues including synovium, subchondral bone, or menisci, which are known to be important components of initiation and progression of OA (i.e., the joint is an organ comprised of multiple cell and tissue types).
      • This methodology does not provide a global assessment of the joint, only those areas evaluated histologically.
      • The scoring systems arbitrarily assign values to different degrees of pathology and overall values to various categories evaluated (e.g., structural changes, changes in matrix composition, and cellular changes) without making attempts to “weight” the scores for each category based on relative importance in OA.
      • There is no standardized methodology for number of sections scored, location of sections for scoring, or ensuring normalization of staining for comparison between batches, studies, or institutions.
      • This methodology has not been extensively analyzed for statistical validity nor has it been truly validated to clinical or functional outcome measures.
      Table ITable listing the most important models available in dog for studying joint degeneration
      Type of modelsPublished modelsReferences
      Spontaneous OA-modelsCanine Hip Dysplasia
      • Lust G.
      • Pronsky W.
      • Sherman D.M.
      Biochemical studies on developing canine hip joints.
      (and >200 others)
      Canine Elbow Dysplasia
      • Wind A.P.
      Elbow incongruity and developmental elbow diseases in the dog.
      (and >100 others)
      Anterior Cruciate Deficiency
      • Johnson K.A.
      • Hay C.W.
      • Chu Q.
      • Roe S.C.
      • Caterson B.
      Cartilage-derived biomarkers of osteoarthritis in synovial fluid of dogs with naturally acquired rupture of the cranial curciate ligament.
      (and >100 others)
      Surgically induced OA modelsAnterior Cruciate Transection
      • Pond M.J.
      • Nuki G.
      Experimentally-induced osteoarthritis in the dog.
      (and >400 others)
      Meniscectomy
      • Bendele A.M.
      Animal models of osteoarthritis.
      ,
      • Carlson C.S.
      • Guilak F.
      • Vail T.P.
      • Gardin J.F.
      • Kraus V.B.
      Synovial fluid biomarker levels predict articular cartilage damage following complete medial meniscectomy in the canine knee.
      ,
      • Johnson K.A.
      • Francis D.J.
      • Manley P.A.
      • Chu Q.
      • Caterson B.
      Comparison of the effects of caudal pole hemi-meniscectomy and complete medial meniscectomy in the canine stifle joint.
      ,
      • Lindhorst E.
      • Vail T.P.
      • Guilak F.
      • Wang H.
      • Setton L.A.
      • Vilim V.
      • et al.
      Longitudinal characterization of synovial fluid biomarkers in the canine meniscectomy model of osteoarthritis.
      ,
      • Lindhorst E.
      • Wachsmuth L.
      • Kimmig N.
      • Raiss R.
      • Aigner T.
      • Atley L.
      • et al.
      Increase in degraded collagen type II in synovial fluid early in the meniscectomy model of osteoarthritis.
      ,
      • Wyland D.J.
      • Guilak F.
      • Elliott D.M.
      • Setton L.A.
      • Vail T.P.
      Chondropathy after meniscal tear or partial meniscectomy in a canine model.
      and others
      Abrasion
      • Adam G.
      • Nolte-Ernsting C.
      • Prescher A.
      • Buhne M.
      • Bruchmuller K.
      • Kupper W.
      • et al.
      Experimental hyaline cartilage lesions: two-dimensional spin-echo versus three-dimensional gradient-echo MR imaging.
      ,
      • Altman R.D.
      • Kates J.
      • Chun L.E.
      • Dean D.D.
      • Eyre D.
      Preliminary observations of chondral abrasion in a canine model.
      Groove
      • Marijnissen A.C.
      • van Roermund P.M.
      • TeKoppele J.M.
      • Bijlsma J.W.
      • Lafeber F.P.
      The canine ‘groove’ model, compared with the ACLT model of osteoarthritis.
      ,
      • Marijnissen A.C.
      • van Roermund P.M.
      • Verzijl N.
      • Tekoppele J.M.
      • Bijlsma J.W.
      • Lafeber F.P.
      Steady progression of osteoarthritic features in the canine groove model.
      ,
      • Mastbergen S.C.
      • Marijnissen A.C.
      • Vianen M.E.
      • van Roermund P.M.
      • Bijlsma J.W.
      • Lafeber F.P.
      The canine ‘groove’ model of osteoarthritis is more than simply the expression of surgically applied damage.
      ,
      • Mastbergen S.C.
      • Marijnissen A.C.
      • Vianen M.E.
      • Zoer B.
      • van Roermund P.M.
      • Bijlsma J.W.
      • et al.
      Inhibition of COX-2 by celecoxib in the canine groove model of osteoarthritis.
      ALD/Valgus Osteotomy
      • Johnson R.G.
      • Poole A.R.
      Degenerative changes in dog articular cartilage induced by a unilateral tibial valgus osteotomy.
      ,
      • Panula H.E.
      • Helminen H.J.
      • Kiviranta I.
      Slowly progressive osteoarthritis after tibial valgus osteotomy in young beagle dogs.
      ,
      • Panula H.E.
      • Nieminen J.
      • Parkkinen J.J.
      • Arnala I.
      • Kroger H.
      • Alhava E.
      Subchondral bone remodeling increases in early experimental osteoarthrosis in young beagle dogs.
      Cartilage Defects
      • Boileau C.
      • Martel-Pelletier J.
      • Brunet J.
      • Tardif G.
      • Schrier D.
      • Flory C.
      • et al.
      Oral treatment with PD-0200347, an a2d ligand, reduces the development of experimental osteoarthritis by inhibiting metalloproteinases and inducible nitric oxide synthase gene expression and synthesis in cartilage chondrocytes.
      ,
      • Boileau C.
      • Martel-Pelletier J.
      • Abram F.
      • Raynauld J.P.
      • Troncy E.
      • D’Anjou M.A.
      • et al.
      Magnetic Resonance Imaging can accurately assess the long-term progression of knee structural changes in experimental dog OA.
      ,
      • Breinan H.A.
      • Hsu H.P.
      • Spector M.
      Chondral defects in animal models: effects of selected repair procedures in canines.
      ,
      • Wang Q.
      • Breinan H.A.
      • Hsu H.P.
      • Spector M.
      Healing of defects in canine articular cartilage: distribution of nonvascular alpha-smooth muscle actin-containing cells.
      , and others
      Reverse Triple Pelvic Osteotomy
      • Inerot S.
      • Heinegard D.
      • Olsson S.E.
      • Telhag H.
      • Audell L.
      Proteoglycan alterations during developing experimental osteoarthritis in a novel hip joint model.
      Enzymatic/Chemically induced OAIodoacetate
      • Bendele A.M.
      Animal models of osteoarthritis.
      Papain and Prednisone
      • Scheck M.
      • Sakovich L.
      Degenerative joint disease of the canine hip: experimental production by multiple papain and prednisone injections.
      Papain/Chymopapain
      • Canapp Jr., S.O.
      • McLaughlin Jr., R.M.
      • Hoskinson J.J.
      • Roush J.K.
      • Butine M.D.
      Scintigraphic evaluation of dogs with acute synovitis after treatment with glucosamine hydrochloride and chondroitin sulfate.
      Calcium Pyrophosphate Crystals
      • McCarty Jr., D.J.
      • Phelps P.
      • Pyenson J.
      Crystal-induced inflammation in canine joints. I. An experimental model with quantification of the host response.
      ,
      • Myers S.L.
      • Brandt K.D.
      • Eilam O.
      Even low-grade synovitis significantly accelerates the clearance of protein from the canine knee. Implications for measurement of synovial fluid “markers” of osteoarthritis.
      ,
      • Rainsford K.D.
      • Skerry T.M.
      • Chindemi P.
      • Delaney K.
      Effects of the NSAIDs meloxicam and indomethacin on cartilage proteoglycan synthesis and joint responses to calcium pyrophosphate crystals in dogs.
      Oral Quinolones
      • Burkhardt J.E.
      • Hill M.A.
      • Carlton W.W.
      • Kesterson J.W.
      Histologic and histochemical changes in articular cartilages of immature beagle dogs dosed with difloxacin, a fluoroquinolone.
      Impact modelsAcute patellofemoral loading
      • Pickvance E.A.
      • Oegema Jr., T.R.
      • Thompson Jr., R.C.
      Immunolocalization of selected cytokines and proteases in canine articular cartilage after transarticular loading.
      ,
      • Thompson Jr., R.C.
      • Vener M.J.
      • Griffiths H.J.
      • Lewis J.L.
      • Oegema Jr., T.R.
      • Wallace L.
      Scanning electron-microscopic and magnetic resonance-imaging studies of injuries to the patellofemoral joint after acute transarticular loading.
      ,
      • Vener M.J.
      • Thompson Jr., R.C.
      • Lewis J.L.
      • Oegema Jr., T.R.
      Subchondral damage after acute transarticular loading: an in vitro model of joint injury.
      Genetically induced Models – Transgenic animalsNone for dogs
      Table IITable listing previously proposed grading and staging systems for canine cartilage degeneration
      ReferencesComment
      Grading systems
      • Mankin H.J.
      • Dorfman H.
      • Lippiello L.
      • Zarins A.
      Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. II. Correlation of morphology with biochemical and metabolic data.
      Cartilage grading system (Mankin grade)
      • Burkhardt J.E.
      • Hill M.A.
      • Carlton W.W.
      • Kesterson J.W.
      Histologic and histochemical changes in articular cartilages of immature beagle dogs dosed with difloxacin, a fluoroquinolone.
      ,
      • Lafeber F.P.
      • van der Kraan P.M.
      • van Roy H.L.
      • Vitters E.L.
      • Huber-Bruning O.
      • van den Berg W.B.
      • et al.
      Local changes in proteoglycan synthesis during culture are different for normal and osteoarthritic cartilage.
      ,
      • Sakakibara Y.
      • Miura T.
      • Iwata H.
      • Kikuchi T.
      • Yamaguchi T.
      • Yoshimi T.
      • et al.
      Effect of high molecular weight sodium hyaluronate on immobilized rabbit knee.
      ,
      • Wenz W.
      • Breusch S.J.
      • Graf J.
      • Stratmann U.
      Ultrastructural findings after intraarticular application of hyaluronan in a canine model of arthropathy.
      Cartilage grading system (modified Mankin grade)
      • Caplan A.I.
      • Elyaderani M.
      • Mochizuki Y.
      • Wakitani S.
      • Goldberg V.M.
      Principles of cartilage repair and regeneration.
      For treatment of cartilage defects, rather than for OA grading
      • Goldenberg D.L.
      • Cohen A.S.
      Synovial membrane histopathology in the differential diagnosis of rheumatoid arthritis, gout, pseudo-gout, systemic lupus erythematosus, infectious arthritis, and degenerative joint disease.
      ,
      • Myers S.L.
      • Brandt K.D.
      • O'Connor B.L.
      • Visco D.M.
      • Albrecht M.E.
      Synovitis and osteoarthritic changes in canine articular cartilage after anterior cruciate ligament transection. Effect of surgical hemostasis.
      ,
      • Pelletier J.-P.
      • Martel-Pelletier J.
      • Ghandur-Mnaymneh L.
      • Howell D.S.
      • Woessner J.F.
      Role of synovial membrane inflammation in cartilage matrix breakdown in the Pond-Nuki dog model of osteoarthritis.
      Synovium grading system
      • Roller B.L.
      • Stoker A.M.
      • Fox D.B.
      • Bal B.S.
      • Cook J.L.
      Characterization of pathology of knee menisci: correlation of radiographic, gross, histologic, biochemical, and molecular measures of disease.
      Meniscus
      Staging systems
      • Outerbridge R.E.
      The etiology of chondromalacia patellae.
      Primarily developed for humans
      • Hunt N.
      • Sanchez-Ballester J.
      • Pandit R.
      • Thomas R.
      • Stachan R.
      Chondral lesions of the knee: A new localization method and correlation with associated pathology.
      Primarily developed for humans
      • Mastbergen S.C.
      • Marijnissen A.C.
      • Vianen M.E.
      • Zoer B.
      • van Roermund P.M.
      • Bijlsma J.W.
      • et al.
      Inhibition of COX-2 by celecoxib in the canine groove model of osteoarthritis.
      Macroscopic score based on high-resolution photographs
      Therefore, the purpose of this work is to attempt to address the majority of these limitations by developing, and then subsequently validating, a comprehensive histologic assessment system that is standardized, repeatable, and comparable among studies and institutions. For the purpose of this work, we use the term “grading” for microscopic/macroscopic scoring of tissues and the term “staging” for the overall assessment of the disease status.

      Anatomy and joint pathology of the dog knee joint

      The focus of the majority of previous research, and of the present work, is the knee (stifle) joint of the dog. However, other joints have also been extensively studied in the dog and have importance especially for spontaneously occurring disease, particularly dysplasias which cause secondary OA in the hip and elbow. However, the present work will focus only on the knee as this joint is the most frequently used as a model of OA.
      The anatomy of the canine knee is closely matched to that of the human (Fig. 1). Both macroscopic and microscopic anatomies are very similar apart from size. The canine knee has medial and lateral femorotibial compartments and a patellofemoral compartment as does man. The anterior (cranial) and posterior (caudal) cruciate ligaments, menisci, meniscal ligaments, fat pad, and patellar ligaments match the human in form and function very closely. The only major gross anatomical differences are that the dog has an intra-articular long digital extensor tendon, which crosses the joint in the anterior–lateral compartment, and that the dog has lateral and medial fabella (sesamoids) in the heads of the gastrocnemius muscle, as well as a popliteal sesamoid. Biomechanically, there are differences with respect to amount of load transmission, relative joint congruency and laxity, range of motion, weight bearing angle, tibial slope, and tibial thrust. Histologically and biochemically, articular cartilage, subchondral bone, synovium, joint capsule, and menisci are very well conserved between these two species. Importantly, spontaneously occurring pathology in terms of anterior cruciate ligament deficiency, meniscal pathology, osteochondrosis, and trauma is comparable in all facets between man and dog. This is one major advantage when using dogs for translational research in OA compared to smaller species where macro and microscopic anatomy, cartilage composition, and matrix turnover may significantly differ from that of humans.
      Figure thumbnail gr1
      Fig. 1Comparative radiographic (A) and gross (B, C) anatomy of the canine stifle (top row) and human knee (bottom row). The radiographic images show osteoarthritic joints while the gross images show normal patellofemoral joints (B) and normal tibial plateaus (C). The canine stifle joint is approximately 3.5–5 cm from medial to lateral edge for medium to large breed dogs (upper left panel A) compared to approximately 7–10 cm for the human knee joint (lower left panel A).
      There are many variables such as age, gender, reproductive status, and breed, which may significantly influence joint physiology and even more so affect outcomes after experimentally induced OA. To the authors’ knowledge, none of these variables have been studied to a degree that allows definitive recommendations to be made with respect to choice or management of research animals. However, it is recommended to use animals that have no clinical or radiographic signs of spontaneous joint pathology in any weight bearing joint. It is also advisable to use dogs that are skeletally mature (9–18 months old depending on breed and gender), as defined by radiographically closed growth plates. If possible, use of non-chondrodystrophic breeds (i.e., not Beagles, Dachshunds, etc. which are chondrodystrophic by definition) may be optimal for avoiding any potential confounding variables associated with chondrodystrophy. Use of a batch of dogs that are genetically closely related, of the same gender, and within narrow age and weight ranges may limit variability in outcome measures. However, use of dogs of both genders that are genetically diverse may provide a more valid representation of spontaneously occurring OA for translation to the human patient population. Pre-study power analyses are always recommended; based on presently available data in the literature, a minimum of eight animals per group is suggested.

      Scoring of Alterations in Joint structures

      Macroscopic scoring of cartilage alterations

      Macroscopic scoring is critical to include, in order to provide some measure or indication of global joint pathology. Ideally, this should be objective and quantitative. However, experimental design may influence the investigators’ ability to include quantitative methodology for this aspect of assessment of OA.
      • (A)
        When parallel or subsequent outcome assessments do not require aseptic technique, a quantitative macroscopic scoring system using India ink staining and calculation of surface area affected is recommended (See Appendix B).
      • (B)
        When parallel or subsequent assessments require aseptic technique (i.e., explant or cell culture), the following macroscopic scoring system is suggested. A score is assigned to each of four major weight bearing surfaces: medial femoral condyle, lateral femoral condyle, medial tibial plateau, lateral tibial plateau. The score is assigned based on the criteria in Table III and is determined by the most severe pathology noted (Fig. 2).
        Table IIIMacroscopic scoring of cartilage
        Smooth surface – Outerbridge 00
        Slightly fibrillated/roughened surface – Outerbridge 11
        Fibrillated surface with focal partial thickness lesions – Outerbridge 22
        Deep lesions with surrounding damage – Outerbridge 33
        Large areas of severe damage – Outerbridge 44
        Macroscopic cartilage scoring for each compartment, based on original Outerbridge classification (Outerbridge, 1961), and modified from Mastbergen et al Rheumatology 2006.
        Figure thumbnail gr2
        Fig. 2Images showing examples of each category for macroscopic scoring of femoral condyles and tibial plateaus from high-resolution photographs. From left to right starting from a completely smooth surface (panel A) there is increase in fibrillation/roughening of the articular surface of both the weight bearing areas of the femoral condyles and the tibial plateau (see arrows in panels B and C). Subsequently focal partial thickness lesions become visible (arrows in panel C), leading to deeper lesions with surrounding damage as seen in panel D (arrows). Finally large areas of severe damage can be observed (panel E).
        Figure thumbnail gr3
        Fig. 3Representative safranin O fast green-stained photomicrographs providing examples for microscopic grading of cartilage, chondrocyte, and proteoglycan pathology using Table IV, Table V, Table VI. The panels A–E for each of the three characteristics represent the descriptions A–E for each of these three characteristics as described in Table IV, Table V, Table VI indicated by arrows or arches in these figures. The lower row of figures depicts cartilage-bone samples with the bone-cartilage interface visible for use when samples are obtained according to histologic sectioning method A (see ). In this case, integrity of the tidemark and subchondral bone changes can be graded as well according to Table VIII, Table IX. (B) and (C) in this figure refer to .
      When gross macroscopic scoring is used, blinding of scorers is a prerequisite and therefore high-resolution photographs need to be taken for subsequent scoring by multiple independent observers.

      Microscopic scoring of cartilage alterations

      • (A)
        When osteochondral sections of the entire surface of each major joint area are desired for histologic assessment, a minimum of three sections of tissue should be evaluated for each of the compartments/regions listed below. The sections should be cut to be approximately 1–3 mm apart and span the entire surface of interest. We recommend taking sagittal osteochondral sections which span the entire surface of interest for all four compartments: Lateral femoral condyle, Medial femoral condyle, Lateral tibial plateau, and Medial tibial plateau (see Appendix A describing methods for cartilage sectioning, Approach A). Depending on study purpose and hypothesis, evaluation of patella and trochlear groove may be important.
      • (B)
        When histologic assessment is focused on articular cartilage and/or portions of cartilage or bone are to be used for specific purposes that prohibit approach A (e.g., explant or cell culture, micro-CT of subchondral bone sections), then at least 4 samples of cartilage from fixed pre-defined locations of the weight bearing areas of all four compartments listed above should be sampled and compared to control samples from the same locations in the control knees (see Appendix A describing methods for cartilage sectioning, Approach B).
      For both approaches, the following protocol should be employed:
      • Minimum of two blinded reviewers, independent evaluations.
      • First two below are mandatory, and also recommend including collagen stain and/or polarized light microscopy for all:
        • Hematoxylin and eosin – general architecture and cell features.
        • Toluidine blue or Safranin O fast green – proteoglycan.
        • Picrosirius red (±polarized) – collagen.
      For proteoglycan and picrosirius red staining, it is a prerequisite to stain controls and experimental sections in the same batch or to use an internal control that is stained as a reference in every batch.
      As mentioned, current histologic grading systems use multiple categories to derive a total score for cartilage pathology. Often, only one or two sections are used and only a focal area of the section(s) is assessed. This methodology does not provide a comprehensive assessment of joint pathology. In addition, these grading systems arbitrarily assign values to different degrees of pathology and to a given category without making attempts to “weight” the scores for each category based on relative importance in cartilage pathology. By grading cartilage (and bone) structure using multiple sections which span the entire surface for each major joint compartment and then separately grading the other major categories of cartilage pathology, the stated problems with current cartilage grading are minimized. Cartilage (and bone) structure is suggested as the primary outcome measure as it (1) provides a method for histologically assessing global changes in cartilage integrity as can be done in parallel using gross, arthroscopic, and imaging measures so that subsequent correlations among these assessments of joint health and disease status can be analyzed, (2) allows investigators to consider the entire section of interest and not try to determine an overall score from a focal change, and (3) provides a standardized methodology that can be validated for use across studies, institutions, models, and species.
      The Table IV, Table V, Table VI, Table VII, Table VIII, Table IX provide grading systems for each major category of cartilage and subchondral bone pathology. It may be relevant to choose one or more of these categories, depending on model and study design in order to gain insight into mechanisms of disease and/or specific treatment effects. It is then possible to make comparisons and test for correlations among categories. Certainly, it is also possible to combine some or all scores for a global score as well, but in doing so it is important to realize that you are inadvertently weighting pathology by category score, which may be inappropriate.
      Table IVGrading of cartilage structure (see Fig. 3a for examples)
      Severity of cartilage pathology

      Characteristics
      Area of section affected
      NoneLocal (approx 1/3)Multi-focal (approx 2/3)Global (>2/3)
      ANormal volume, smooth surface with all zones intact0000
      BSurface undulations including fissures in surface/upper zone and/or pannus tissue formation on surface0123
      CFissures to mid zone and/or erosion of surface/upper zone0246
      DFissures that extend to deep zone and/or erosion through mid zone0369
      EFull thickness loss of cartilage04812
      Table VGrading of chondrocyte pathology (See Fig. 3a for examples)
      Severity of chondrocyte pathology

      Characteristics
      Area of section affected
      NoneLocal (approx 1/3)Multi-focal (approx 2/3)Global (>2/3)
      ANormal0000
      BLoss of cells in the surface zone or relative increased density with occasional superficial clusters0123
      CSmall cell clusters (2–4 cells/cluster)
      In any cartilage zone.
      predominate
      0246
      DLarge cell clusters (5 cells/cluster) predominate0369
      ECell loss (necrosis/apoptosis) predominates04812
      In any cartilage zone.
      Table VIGrading of proteoglycan staining (see Fig. 3a for examples)
      Severity of proteoglycan pathology

      Characteristics
      Area of section affected
      NoneLocal (approx 1/3)Multi-focal (approx 2/3)Global (>2/3)
      ANormal0000
      BDecreased proteoglycan content in surface/upper zone0123
      CDecreased proteoglycan content into the mid zone0246
      DDecreased proteoglycan content into the deep zone0369
      EFull depth decrease in proteoglycan content04812
      Table VIIGrading of collagen integrity
      Severity of collagen pathology

      Characteristics

      (Based on intensity of picrosirius red staining and/or polarized light evaluation)
      Area of section affected
      NoneLocal (approx 1/3)Multi-focal (approx 2/3)Global (>2/3)
      ANormal0000
      BLoss of integrity of surface/upper zone0123
      CLoss of integrity of surface/upper and mid zones0246
      DLoss of integrity of surface/upper, mid, and deep zones0369
      Table VIIIGrading of tidemark (If using full thickness osteochondral sections – recommended) (Fig. 3b)
      Tidemark integrityArea of section affected
      NoneLocal (approx 1/3)Multi-focal (approx 2/3)Global (>2/3)
      AIntact and distinct0000
      BNot consistent or distinct (loss and/or duplication)0123
      CLoss of tidemark which is crossed by blood vessels0246
      Table IXGrading of subchondral bone plate (If using full thickness osteochondral sections – recommended)
      Subchondral bone plate changeArea of section affected
      NoneLocal (approx 1/3)Multi-focal (approx 2/3)Global (>2/3)
      AIntact with normal thickness (300 microns)0000
      BMild increase in thickness (>300–<450 μm)0123
      CModerate increase in thickness (450–<750 μm)0246
      DMarked increase in thickness (>750 μm), subchondral pseudocysts, and/or marrow fibrosis0369
      This grading system is designed to be comprehensive and specific. As described above, multiple sections should be evaluated and the entirety of the section should be evaluated for each category (table) used. When a section includes several local areas of pathology or local and multi-focal pathology, then the scores are added to derive a total score for the section. The score is based on the most severe pathology seen in each area of the section you are evaluating based on the descriptors provided in each table.
      Examples: 1/3 of section scored B+1/3 scored C+1/3 scored D=1+2+3=6
      1/3 of section scored B+2/3 scored D=1+6=7
      2/3 of section scored B+1/3 scored C=2+2=4
      1/3 of section scored E+rest of section normal=4+0=4
      Entire section scored B=3=3

      Macroscopic scoring of synovial alterations

      An overall view on the synovial tissue as large as possible, at least including the tissue below the patella and the medial and lateral sides of the joint capsule from patellar ligament to lateral and medial attachments should be scored by a minimum of two, blinded observers based on the scoring system in Table X (Fig. 4).
      Table XMacroscopic scoring of synovial pathology
      Gross CharacteristicsScore
      Normal – opal white, semitranslucent, smooth, with sparse well defined blood vessels0
      Slight – focal involvement, slight discoloration, visible proliferation/fimbriation/thickening, notable increase in vascularity1
      Mild – diffuse involvement, slight discoloration, visible proliferation/fimbriation/thickening, notable increase in vascularity2
      Moderate – diffuse involvement, severe discoloration, consistent notable proliferation/fimbriation/thickening, moderate vascularity3
      Marked – diffuse involvement, severe discoloration, consistent and marked proliferation/fimbriation/thickening, diffuse hypervascularity4
      Severe – diffuse involvement, severe discoloration, consistent and severe proliferation/fimbriation/thickening, thickening to the point of fibrosis, and severe hypervascularity5
      Figure thumbnail gr4
      Fig. 4Gross (A) and arthroscopic (B) images showing examples of each score (0–5) for synovial pathology according to .

      Microscopic scoring of synovial alterations (Grading of synoviopathy)

      A minimum of three sections of tissue should be evaluated from medial, axial, and lateral compartments of each joint. The sections should be representative of the entire tissue (Table XI, Fig. 5).
      • Minimum of two blinded reviewers, independent evaluations.
      • Use H&E stain.
      Table XIMicroscopic grading of synovial changes
      Severity of pathologyArea of section affected
      Lining cells characteristicsNoneLocal (approx 1/3)Multi-focal (approx 2/3)Global (>2/3)
      A1–2 layers of cells0000
      B3–6 layers of cells0123
      C>6 layers of cells0246
      Lining characteristics
      ANo villous hyperplasia0000
      BShort villi0123
      CFinger-like hyperplasia0246
      Cell infiltration characteristics
      ANo cellular infiltration0000
      BMild to moderate inflammatory cell infiltrates including small lymphoid follicles0123
      CMarked, diffuse inflammatory cell infiltrates including large lymphoid follicles0246
      Figure thumbnail gr5
      Fig. 5Representative hematoxylin and eosin-stained photomicrographs providing examples for microscopic grading of synovial pathology using . The panels A-C for each of the three characteristics represent the descriptions A–C for each of these three characteristics as described in indicated by arrows or arches in these figures.

      Microscopic scoring and micro-CT analysis of bone alterations

      Table IX provides the mechanism for assessing subchondral bone when included for evaluation. Bone morphometry can be performed when histologic sectioning method A (see Appendix A) is chosen as has been described. When histologic sectioning method B (Appendix A) is chosen, micro-CT analyses of bone architecture can be performed. Three-dimensional subchondral plate thickness, trabecular bone volume fraction, three-dimensional trabecular thickness, structure model index, and connectivity density can be calculated and expressed in objective, quantitative measures for each of the four joint compartments as well as for areas in the metaphysis as a control. These parameters have been demonstrated to change with development of OA in humans as well as dogs and other animal models of OA
      • Sniekers Y.H.
      • Intema F.
      • Lafeber F.P.
      • van Osch G.J.
      • van Leeuwen J.P.
      • Weinans H.
      • et al.
      A role for subchondral bone changes in the process of osteoarthritis; a micro-CT study of two canine models.
      • Boyd S.K.
      • Muller R.
      • Leonard T.
      • Herzog W.
      Long-term periarticular bone adaptation in a feline knee injury model for post-traumatic experimental osteoarthritis.
      • Intema F.
      • Sniekers Y.H.
      • Weinans H.
      • Vianen M.E.
      • Yocum S.A.
      • Zuurmond A.M.
      • et al.
      Similarities and discrepancies in subchondral bone structure in two differently induced canine models of osteoarthritis.
      . Both methods allow for incorporation of subchondral bone changes in a “whole organ” approach to assessment of OA, as well as correlation of these data to other clinically relevant outcome measures such as MRI. The authors strongly recommend this whole organ approach when using canine models of OA for comparative and translational research.

      Microscopic scoring of meniscal alterations

      Meniscal scoring and grading systems:

      Macroscopic

      Meniscal pathology is scored for each zone (anterior (cranial), middle, and posterior (caudal) thirds) of each meniscus (medial and lateral) according to the criteria outlined in Table XII:
      Table XIIMacroscopic scoring of meniscal changes
      Severity of meniscal pathology characteristicsArea of section affected
      Anterior 1/3Middle 1/3Posterior 1/3
      ANone000
      BFibrillation only111
      CIncomplete tear or tears222
      DComplete tear or tears333
      EComplete disruption of structure (maceration of tissue)444
      Scores from all sections are added to derive a total severity score for each meniscus with a maximum score of 12. Individual scores for each section can also be used depending on study purpose and hypothesis.

      Microscopic

      Medial and lateral menisci are each divided into anterior (cranial), middle, and posterior (caudal) thirds: three sections from each third are used for evaluation (Table XIII, Fig. 6). The following protocol should be employed:
      • Minimum of two blinded reviewers, independent evaluations.
      • Mandatory two stains, recommend including collagen stain and or polarized light microscopy for all:
        • Hematoxylin and eosin – general architecture and cell features.
        • Toluidine blue or Safranin O fast green – proteoglycan.
        • Picrosirius red and/or polarized – collagen.
      Table XIIIMicroscopic grading of meniscal changes
      CategoryScore
      Tissue Architecture-Tissue LossAnterior 1/3Middle 1/3Posterior 1/3
      Normal000
      Minimal disruption111
      Moderate disruption with loss of tissue222
      Complete loss of tissue architecture, >50% loss333
      Total Tissue Architecture-Tissue Loss Score
      Cell and Matrix (PG and Collagen) Content and MorphologyAnterior 1/3Middle 1/3Posterior 1/3
      Normal000
      Minimal alterations in cell and matrix content and morphology111
      Moderate alterations in cell and matrix content and morphology222
      Severe loss/disruption of cells, PG, and collagen333
      Total Cell and Matrix Score
      Proliferative ResponseAnterior 1/3Middle 1/3Posterior 1/3
      None000
      Minimal proliferation of cells at synovial-meniscal junction111
      Proliferation of cells at synovial junction and extending into tissue or along surfaces222
      Marked proliferation of cells involving majority of remaining tissue333
      Total Proliferative Response Score
      TOTAL SCORE
      Figure thumbnail gr6
      Fig. 6– Photomicrographs of menisci showing (A) normal safranin O fast green-stained meniscus at low magnification, (B) safranin O fast green-stained meniscus at low magnification showing grade three changes in all three categories, (C) safranin O fast green-stained meniscus at high magnification showing grade three tissue architecture-tissue loss change, (D) safranin O fast green-stained meniscus at high magnification showing grade three cell and matrix change, (E) safranin O fast green-stained meniscus at high magnification showing grade three proliferative response change.
      Scores from all sections are added to derive a total score for each category for each meniscus. A total score for each meniscus can then be derived by adding all category scores. Individual scores for each section can also be used depending on study purpose and hypothesis.

      Evaluation of sources of variability

      In order to validate the reliability of this histologic assessment system, we enlisted experts (six individuals with training and experience in histologic assessment of articular tissues) and non-experts (four individuals with training and experience in histologic assessment, but not of articular tissues) to review images and use the system to score each image after reading the instructions for scoring. Images were divided into three categories: (1) Cartilage (n=28 images using Table IV, Table V, Table VI), (2) Osteochondral (n=17 images using Table IV, Table V, Table VI, Table VIII, Table IX, Table IIITable VIII, Table IX), and (3) Synovium (n=31 images using Table XI). A total score was determined for each reviewer in each category. Scores were analyzed in each category using Repeated Measures ANOVA on Ranks to determine significant differences in scores among observers, Spearman Rank Order Correlation test to assess the strength of correlations among observers, and a weighted Kappa test to assess inter-observer agreement. For the correlations, we considered r>0.4 to moderately strong and r>0.7 to be strong. For the weighted kappa test, we considered k>0.2 to be fair agreement, k>0.4 to be moderate agreement, k>0.6 to be good agreement, and k>0.8 to be very good agreement (Altman DG (1991) Practical statistics for medical research. London: Chapman and Hall).
      Each statistical analysis method indicated that the scoring systems were reliable when performed by experts. No significant differences in scores were noted among experts in any category. However, some significant (p<0.001) differences in scores were noted between experts and non-experts and among non-experts, most notably in the Osteochondral category. Correlation analyses revealed that Cartilage category scoring was very similar among all observers with all observer comparisons having significant (p<0.001) moderate to strong (r=0.56–0.95) positive correlations. For Osteochondral category scoring, all experts had significant (p<0.001) strong (r=0.79–0.96) positive correlations, while only weak correlations (r<0.4) were seen for expert to non-expert and non-expert to non-expert comparisons. For Synovium category scoring, all observers scores had significant (p<0.001) strong (r=0.7–0.92) positive correlations. Similar findings were seen for weighted Kappa analyses. For Cartilage category scoring, inter-observer agreement ranged from fair (k=0.48) for non-expert to non-expert comparison to very good (k=0.87) for expert to expert comparison. For Osteochondral category scoring, inter-observer agreement ranged from slight (k=0.27) for non-expert to non-expert comparison to good (k=0.68) for expert to expert comparison. For Synovium category scoring, inter-observer agreement was in the good range (k=0.64–0.80) for all comparisons. Taken together, these data suggest this histologic assessment system is reliable in terms of producing consistent and repeatable scores for cartilage, osteochondral, and synovial tissue status among observers with expertise and experience in articular tissue histology. The data also suggest that non-experienced observers using the system for the first time can effectively assess tissues to acceptable levels of agreement with experts.

      Discussion

      The dog is a commonly used and appropriate model for translational and comparative study of all aspects of OA based on animal size; anatomical, disease mechanism, and clinical similarities to humans; and response to treatments. In addition, OA occurs in dogs in very similar ways to that seen in humans as a result of traumatic, degenerative, and overuse etiologies. Historically, histologic evaluation of articular cartilage pathology associated with OA has been used as the “gold standard” for determining the presence, extent, and severity of disease in all species. Subjective scoring systems have served to provide a mechanism for histologic degree of OA among subjects, treatment groups, studies, and with respect to historical data sets. However, to the authors’ knowledge, these scoring systems have not been critically assessed for reliability or validity, and have some perceived shortcomings including lack of versatility, comprehensive assessment of the joint, or standardization. Therefore, we attempted to develop, optimize, and critically assess a histologic evaluation system to address these shortcomings and provide a more optimal methodology for study of OA in dogs.
      To address perceived shortcomings, this assessment system was developed to provide a more global assessment of the joint. We addressed global joint assessment by including assessment of synovium, subchondral bone, and meniscus; by including gross scoring of tissues; and recommending multi-location assessments of each tissue. As such, we provided two recommended protocols for tissue processing because of the recognized variation among investigators with respect to additional outcome measures and requirements for aseptic processing. In addition, we provided versatility and the potential for relative “weighting” of various types of pathology by separating categories of pathologic change into separate grading tables. This allows investigators to assess the joints with relevance to the hypothesis being tested, while still allowing for combined scores for total or global joint assessment for comparison among time points, studies, and centers. In order to allow for these comparisons to be most accurate and informative, we recommend a standardized methodology for grading tissues which we feel helps to address issues regarding the problem of evaluating the worst area vs the best area vs the average area. Lastly, we critically assessed the reliability of this system for its ability to consistently measure tissue pathology accurately among multiple observers.
      The proposed system for assessment of canine tissues appears to provide an effective method for global assessment of articular pathology in OA. The system is versatile, comprehensive, and reliable and appears to have advantages over other scoring systems for progress in OA research.

      Disclosures

      Sub-Coordinator James L. Cook is employed by the University of Missouri, Columbia, Missouri, USA.

        Committee Members.

      • -
        Floris Lafeber is employed by University Medical Centre Utrecht, Utrecht, The Netherlands.
      • -
        Keiichi Kuroki is employed by the University of Missouri, Columbia, Missouri, USA.
      • -
        Denise Visco is employed by Merck and Co. Inc. Rahway, NJ 07065.
      • -
        Jean-Pierre Pelletier is employed by the Medical School of the University of Montreal, Montréal, Québec, Canada.
      • -
        Loren Schulz is employed by the University of Missouri, Columbia, Missouri, USA.
      Coordinator Thomas Aigner is employed by the University of Leipzig.

      Conflict of interest

      No author has any conflict of interest related to this work.

      Acknowledgements

      No external sources of funding were provided for this work except that the printing costs were supported by an unrestricted educational grant to OARSI by Bayer, Expanscience, Genzyme, Lilly, MerckSerono, Novartis, Pfizer, SanofiAventis, Servier, and Wyeth. The work performed was not influenced at any stage by the support provided.
      Validation study participants: Bridget Garner, S.C. Mastbergen, M.J.G. Wenting, Christelle Boileau (Montreal, Canada), Stephan Soeder (Leipzig).
      External experts: K. Brandt (Indianapolis, USA), JM Williams (Chicago, USA), Cathy Carlson (Minnesota, USA), Chris Little (Australia), Ted Oegema (Chicago, USA), R. Altmann (Agua Dulce, US), Kenneth Pritzker (Toronto, Canada).

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