Calcium Pyrophosphate Deposition in Nonhuman Primates

Transcription

1 Vet. Pathol. : (984) Calcium Pyrophosphate Deposition in Nonhuman Primates E. D. ROBERTS, G. B. BASKIN, E. WATSON, W. G. HENK, T. C. SHELTON, and M. S. BOWEN Departments of Veterinary Pathology, Veterinary Anatomy and Fine Structure, Louisiana State University, Baton Rouge, LA; Departments of Pathology and Animal Care, Tulane University Delta Regional Primate Research Center, Covington, LA; and Department of Mechanical Engineering, Louisiana State University, Baton Rouge, LA Abstract. Naturally occurring deposition of calcium pyrophosphate has been identified in six rhesus monkeys following acute episodes of trauma and various septicemias. Scanning electron microscopy with energy-dispersive X-ray analytical system and single crystal electron diffraction studies were used to identify the crystals within the articular cartilage. The osteoarthritis grading system was used to determine the degree of cartilage degenerative changes. Calcium pyrophosphate deposition disease (CPDD) is a metabolic disease of articular cartilage associated with a degenerative arthropathy in man? Other crystalline structures have been identified as important etiologic factors of acute and chronic arthropathies such as: urate," hydroxyapatite, lo* and calcium hydrogen phosphate dihydrate* crystals. One case of pyrophosphate crystal deposition in a dog synovial membrane has been reported.' A naturally occurring calcium pyrophosphate deposition arthropathy in a rhesus breeding colony has been identified. The acute form followed acute trauma-septicemias and primarily involved the knee and elbow joints; any joint may be affected, however. Generalized joint disease has been identified in adult animals. The chronic disease was evident in rhesus monkeys as joint rigidity, marked atrophy of the musculature, and a degenerative arthropathy. Preliminary attempts to determine the eti'ology '(mycoplasma, virus, rheumatoid) of the arthropathy were negative. Six naturally occurring cases of calcium pyrophosphate deposition disease in primates are reported and compared to pyrophosphate deposition disease in man. Materials and Methods Knee joints fiom six rhesus monkeys of various ages were positive on scanning electron microscopy for pyrophosphate crystals and are included in this study. A positive sample had pyrophosphate crystals which projected from and integrally 59 involved the articular cartilage. Pertinent clinical history is given in table I. All knees were opened and samples of synovial fluid were collected for bacteriologic aerobic and anaerobic culture. From each knee joint, samples of the synovial membrane, articular surface, and menisci were collected and fixed in % glutaraldehyde in cacodylate buffer, postfixed in % osmium tetroxide, dehydrated, and critical point dried and coated with gold for scanning electron microscopy. For light microscopy, the synovial membrane, and weight bearing and non-weight bearing articular surfaces were fixed in % glutaraldehyde in cacodylate buffer. Electron diffraction studies of each case were conducted after scraping critical point dried cartilage with a scalpel blade over formvar-coated grids. Electron diffraction patterns from known standards of hydroxyapatite, brushite and calcium pyrophosphate, (Sigma Chemical Company, St. Louis, MO.) then were compared to the patterns obtained from the unknown crystals. All articular surface sections for light microscopy were demineralized with decalcifying solution (American Scientific Products, McGraw Park, Ill.) overnight, washed in running water, and processed with a standard paraffin technique. Tissues then were sectioned and stained with hematoxylin and eosin and safranin. All articular surface sections were evaluated with the grading system described by others: The osteoarthritis grading system (table ) allows for structural cartilage changes, changes in cartilage cell morphology, matrix staining for proteoglycan, and reparative attempts of the articular surface. Nasal septum was sectioned from each monkey as a cartilage matrix proteoglycan control for the articular surface. To grade each monkey the weight bearing and nonweight bearing surfaces were evaluated, and two sites from each piece of cartilage (total of four sites) were averaged for the grade in table I. Selected crystal sections were carbon-coated and evaluated

2 Calcium Pyrophosphate: Nonhuman Primates 59 Table I. Six rhesus monkeys with calcium pyrophosphate deposition, disease Monkey Age/Sex History Total Mankin grade Adult/M Trauma (severe, chronic) 8 Adult/M Arthropathy (severe, chronic), negative culture, diarrhea, debilitation Adult/M Chronic anemia, pneumonia, ulcerative colitis 4 4 Adult/F Arthropathy (severe) 9 5 Adult/F Arthropathy (chronic in knees and elbows), negative culture, emaciation yearsp Extreme debilitation 9 with an energy dispersive elemental X-ray analysis system (EDAX 9, Philips Electronic Instruments, Prairie View, Ill). Results Pyrophosphate deposition disease occurred in all ages of monkeys following periods of stress. Stress was defined as acute trauma which followed prolonged diarrhea or septicemias from various causes. All knee joints were negative for bacteria. Gross joint lesions varied from no change to moderate osteoarthrosis (fig. ) with the remaining monkeys having intermediate articular surface involvement. Synovial fluid in all knee joints was scanty. Table. Osteoarthritis grading system used to evaluate rhesus knee ioints I. Structure b. Surface irregularities c. Pannus and surface irregularities d. Clefts to transitional zone e. Clefts to radial zone f. Clefts to calcified zone g. Complete disorganization. Cells b. Diffuse hypercellularity c. Cloning d. Hypocellularity. Safranin- staining b. Slight reduction c. Moderate reduction d. Severe reduction e. No dye noted IV. Tidemark integrity a. Intact b. Crossed by blood vessels Total Mankin made Mankin grade Pyrophosphate crystals were concentrated in the articular cartilage, and were evident from the articular surface on scanning electron microscopy evaluation (fig. ),as well as from cartilage sections evaluated with compensated polarizing light. Crystals were clustered; this suggested perilacunar space localization. The crystals were flat and slate-like and occasional tophi were observed (fig. ). Surrounding cartilage matrix always had a loss of ground substance which was evidenced by bare surface collagen fibrils. A diffuse pattern of crystal involvement was identified in one monkey (fig. 4). Articular cartilage evaluation is shown in table I. Most crystals were found in acute joint inflammatory processes where there was insufficient time for the development of chronic joint changes. Chronic degena erative changes were observed in four monkeys. The most advanced case scored on the osteoarthrosis grading system and was characterized by a complete loss of safranin matrix staining and had decreased cellularity of the cartilage with cloning (fig. 5), fibroplasia, and vascularization of the frictional surface. A score below 4 was considered normal articular surface. The synovial membrane contained crystals en&&- ded in the surface (fig. 6) with mild inflammatory involvement of the subsynovial cell layer. Microscopically, the synovial membrane changes consisted of mild villous hyperplasia with the lining synovial cells concentrated near the tips of the villi. Ififlammatory infiltrate primarily consisted of neutrophils (fig. 7) mixed with fibrin and an occasional macrophage. The degree of inflammation was related directly to the quantity of crystals observed with scanning electron microscopy. Crystals evaluated with the energy dispersive X-ray analytical system were composed of an equal ratio of calcium and phosphorus. A surface map of the relative surface concentration suggested a greater concentration of phosphorus ions on the surface (fig. 8). Electron diffraction studies demonstrated a pattern

3 594 Roberts, et al. Fig. : Distal end of femur with marginal ulceration and erosion (arrow). Bar = cm. Fig. Articular cartilage surface with clumping of crystals corresponding to chondrocyte perilacunar location. Bar = Fig. : Articular surface; scanning electron micrograph. Tophus of slate-like calcium pyrophosphate crystals. Bar = 4 pm. Fig. 4: Calcium pyrophosphate crystals distributed throughout articular surface. Bar = pm. Fig. 5 Advanced osteoarthrosis from rhesus monkey knee joint with calcium pyrophosphate deposition disease. Chondro- cyte cloning, loss of matrix staining with safranin, disorganization of frictional surface. Safranin, hematoxylin counterstain. Fig. 6: Synovial membrane; scanning electron micrograph. Numerous pyrophosphate crystals embedded in membrane surface. Bar = 4 pm.

4 Calcium Pyrophosphate: Nonhuman Primates 595 similar to the control pyrophosphate crystals (fig. 9). Brushite and hydroxyapatite both had a hexagonshaped crystal diffraction pattern. Fig. 7: Acute synovitis in knee of monkey with calcium pyrophosphate crystals. Numerous pyrophosphate crystals (arrow) in exudate; compensated polarized light. Discussion Calcium pyrophosphate deposition disease of articular structure has been identified in a rhesus breeding colony. Acute generalized diseases such as trauma and septicemia all may have resulted in a greater metabolic turnover of the articular cartilage with subsequent pyrophosphate crystal deposition. The naturally occurring disease of the rhesus parallels the disease in man where subsequent crystal deposition is associated with many diseases such as trauma,i- l4 septicemias, and various metabolic endocrine diseases?. 6* l Crystal deposition may represent the final COl'nmOn mode of expression of various metabolic disturbances of the cartilage cell.i6. 86 b c 9a 96 Fig.8: Topographic (energy dispersive X-ray analysis) mapping of calcium (a) and phosphorus (b.) ions of calcium pyrophosphate crystal from naturally occurring pyrophosphate arthropathy. Darker portions represent higher ion concentration. Fig. 9: Electron diffraction patterns. a. Reference calcium pyrophosphate. 6. Naturally occurring calcium pyrophosphate crystal.

5 596 Roberts, et al. Pyrophosphate crystals were identified in the perilacunar and matrix spaces by compensated polarized light microscopy and scanning electron microscopy. Crystal analysis demonstrated equal quantities of calcium and phosphorus with an electron diffraction pattern (fig. 9) of calcium pyrophosphate articular surface crystal. Tophi were formed in several monkeys-sometimes corresponding to the perilacunar location of the crystals. The location and concentration of the crystals suggested a primary disease of hyaline cartilage. The cause-effect relationship between joint cartilage calcification and the degree of osteoarthritis has not been established. Absence of a suitable animal model has prevented evaluation of this phase of the disease. In man, a high percentage of persons over 6 yeas of age have pyrophosphate crystals in their articular cartilage without apparent joint symptoms. The disease in man is associated with the intraarticular shedding of the crystals which results in an acute joint symptom.*7*8 These symptoms refer to an acute neutrophilic inflammatory process elicited by the shed crystals. The character of the inflammatory infiltrate in primates is similar, however, the intracellular crystal ass~ciation ~ has not been well established at this time. The best diagnostic method for observing the crystals is scanning electron microscopy since the crystals are insoluble in the reagents normally used. Polarized light may be used to identify the crystals in cartilage and synovial membrane. Joint fluid aspirates which are normally used to diagnose the crystals were very difficult to obtain in these primates since small quantities of synovial fluid were present. Direct smears of synovium can be made and stained with alizarin red and evaluated using compensated polarized light microscopy.5 The naturally occurring disease identified in rhesus monkeys can serve as an animal model for this important arthropathy. Whether the crystals are primary or secondary disease-producing factors has not been determined in any species. Preliminary incidence studies suggest that the rhesus commonly forms articular calcium pyrophosphate crystals. This model should facilitate studies to define the metabolic condition that leads to crystal formation and its role in the pathogenesis of the arthropathy. References BENNETT, R.M.; LEHR, J.R.; MCCARTY, D.J.: Crystal shedding and acute pseudogout. Arthritis Rheum 9:9-97, 976 DODDS, W.J.; STEINBACH, H.L.: Primary hyperparathyroidism and articular cartilage calcification. Am J Roentgenol , 968 FAURE, G.; NETTER, P.; MALAMAN, B.; STEINMETZ, J.: Monocrystalline calcium hydrogen phosphate dihydrate in destructive arthropathies of chondrocalcinosis. Lancet 4, FAURE, G.; NETTER, P.; MALAMAN, B.; STEINMETZ, J.; DUHEILLE, J.; GAUCHER, A.: Scanning electron microscopic study of microcrystals implicated in human rheumatic diseases. Scan Electron Microsc : 6-76, 98 5 GIBSON, J.P.; ROENIGK, W.J.: Pseudogout in a dog. J Am Vet Med Assoc 6:9-95, 97 6 GRAHAME, R.; SUTOR, J.D.; MITCHENER, M.B.: Crystal deposition in hyperparathyroidism. Ann Rheum Dis :597-6, 97 7 KOHN, N.N.; HUGHES, R.E.; MCCARTY, D.J.: The significance of calcium phosphate crystal in the synovial fluid of arthritic patients: The pseudogout syndrome.. Identification of crystals. Ann Intern Med 5&78-745, 96 8 KOZIN, F.; MCCARTY, D.J.: Protein absorption to monosodium urate, calcium pyrophosphate, dihydrate, and silica crystals. Arthritis Rheum 9:4-48,976 9 MCCARTY, D.J.: Calcium pyrophosphate dihydrate crystal deposition disease. Arthritis Rheum 9:75-85, 975 MCCARTY, D.J.; HOGAN, J.M.; GAITER, R.A.; GROSSMAN, M.: Studies on pathological calcification in human cartilage. I. Prevalence and types of crystal deposits in the menisce of two hundred fifteen cadavers. J Bone Joint Surg [Am] 48:9-5, 966 MCCARTY, D.J.; KOZIN, F.: An overview of cellular and molecular mechanisms in crystal induced inflammation. Arthritis Rheum 8: , 975 MCCARTY, D.J.; SILCOX, D.C.; COE, F.; JACOBELLI, S.; REISS, E.; GENANT, A.; ELLMAN, M.: Diseases associated with calcium pyrophosphate dihydrate crystal deposition, a controlled study. Am J Med , 974 MANKIN, H.J.; DORFMAN, H.; LIPPIELLO, L.; ZARINS, A.: Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. J Bone JoinrSurg [Am] 5-57, 97 4 MARKEL, S.F.; HART, W.R.: Arthropathy in calcium pyrophosphate dihydrate crystal deposition disease. Pathologic study of cases. Arch Pathol Lab Med 6:59-5, 98 5 PAUL, H.; REGINATO, A.J.; SCHUMACHER, H.R.: Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum 6: 9-, 98 6 RUSSELL, R.G.G.: Metabolism of inorganic pyrophosphate (PPi). Arthritis Rheum 9: , SCHUMACHER, H.R.: Ultrastructural finding in chrondrocalcinosis and pseudogout. Arthritis Rheum 9:4-45, WOODARD, J.C.; SHIELDS, R.P.; ALDRICH, H.C.; CARTER, R.L.: Calcium phosphate deposition disease in great danes. Vet Pathol 9: , 98 Request reprints from Dr. E. D. Roberts, Department of Veterinary Pathology, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 78 (USA).