Point: Hydroxyapatite Crystal Deposition Is Intimately Involved in the Pathogenesis and Progression of Human Osteoarthritis

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1 Point: Hydroxyapatite Crystal Deposition Is Intimately Involved in the Pathogenesis and Progression of Human Osteoarthritis Geraldine M. McCarthy, MD, and Herman S. Cheung, PhD Corresponding author Geraldine M. McCarthy, MD Department of Medicine, University College Dublin and Mater Misericordiae University Hospital, Eccles St, Dublin 7, Ireland Current Rheumatology Reports 2009, 11: Current Medicine Group LLC ISSN Copyright 2009 by Current Medicine Group LLC The cause of osteoarthritis (OA), the most common form of arthritis, is most likely multifactorial. No drug exists to slow the progression or reverse OA disease progression. Ample data support a key role of calcium-containing crystals, such as hydroxyapatite, in OA pathogenesis. The presence of these crystals, far higher in OA than in any other form of arthritis, correlates with the degree of radiographic degeneration. Calcium-containing crystals have potent biologic effects in vitro that emphasize their pathogenic potential. OA-associated matrix and chondrocyte alterations play an intimate role in the crystal deposition process. A major difficulty has been the lack of a simple technique for crystal identification in affected joints. Enhanced effort is needed to establish calciumcontaining crystals as a therapeutic target in OA, as current data suggest an intimate association in its pathogenesis and progression. Introduction Osteoarthritis (OA) is the most common form of arthritis in humans, currently affecting more than 20 million Americans. Its high prevalence and the frequency of related physical impairment make OA a leading cause of disability, especially with respect to weight-bearing functions. The disease process involves the entire joint, including the cartilage, subchondral bone, ligaments, capsule, synovial membrane, and periarticular muscles. The articular cartilage degenerates with fibrillation, fissures, ulceration, and full thickness loss of the joint surface. Clinically, OA involves symptoms of pain, stiffness, and movement loss. Major signs of OA include bony enlargement, crepitus, joint tenderness (especially at the joint margin), effusion, decreased range of motion, and instability. OA, therefore, is a complex disorder that appears to result from various factors, including genetic predisposition, joint trauma, joint structure/shape, and occupational or recreational use, aggravated by obesity, gender, and hormonal status [1]. To date, no drug has been identified that slows the progression or reverses the OA disease process. The multifactorial nature of OA makes drug discovery exceptionally difficult. Nonetheless, current evidence suggests that deposition of calcium-containing crystals, such as hydroxyapatite, is intimately involved in OA pathogenesis and progression. We suggest that enhanced efforts should be made to pursue calcium-containing crystals as a potential therapeutic target in OA. Clinical Aspects A potentially important, but still inadequately investigated aspect of articular cartilage and other articular tissue is their tendency to form calcium-containing crystals. Crystalline calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are the two most common forms of pathologic articular mineral [2 ]. BCP collectively describes calcium phosphate crystals, including hydroxyapatite (HA), octacalcium phosphate, tricalcium phosphate, and magnesium whitlockite. The most abundant of these crystal species in BCP crystal associated syndromes is HA. BCP crystals frequently deposit in articular tissues, but may also be found in arteries, skin, breast, and other tissues. In the musculoskeletal system, crystals may be found in tendons, intervertebral discs, joint capsule, synovium, and cartilage. In tendons, studies have suggested that dystrophic calcification occurs as a consequence of local trauma, ischemia, and necrosis of tendons. Some evidence suggests that calcifying tendonitis is an active, cell-mediated process in which local vascular and mechanical changes result in focal transformation of

2 142 Crystal Arthritis I rarely seen in joints damaged by inflammatory arthritis, such as rheumatoid arthritis. No known therapy prevents or removes BCP crystals from joints or interferes specifically with the biological effects of BCP crystals. In the past few years, however, major advances in the understanding of the biologic effect of crystals and the signal transduction pathway of crystal-induced cell activation have offered a unique opportunity to examine the role of crystal in OA and cartilage degeneration [6]. Figure 1. Role of basic calcium phosphate (BCP) crystals in joint degeneration. MMP matrix metalloproteases; PG prostanoids. tendinous tissues into fibrocartilaginous material containing chondrocytes. This is followed by local deposition of HA crystals within extracellular matrix-vesicle like structures derived from these chondrocytes [2 ]. Concurrence of BCP crystal and OA is well-established [2 ]. Growing evidence from the study of OA in which BCP crystals can be found in most affected joints and closely correlate with the extent of joint destruction suggests a pathogenic role in driving disease. The incidence of BCP crystals in synovial fluid (SF) from patients with knee OA is at least 30% to 60%. Calcium-containing crystals, including CPPD and hydroxyapatite-like BCP crystals, are found in 60% of SF from unselected patients during knee replacement for OA [3]. It has been suggested recently that many OA fluids contain clusters of BCP crystals that are too small or too few in number to be identified by conventional techniques [4]. Ample data support the role of BCP crystals in cartilage degeneration. The presence BCP crystals correlates strongly with the severity of radiographic OA, and larger joint effusions are seen in affected knee joints when compared with joint fluid from OA knees without crystals. Therefore, the presence of CPPD and BCP crystals predict severe radiologic damage and rapid progression of arthritis [2 ]. The pathogenic potential of BCP crystals is further emphasized by their association with a distinctive type of destructive arthropathy of the shoulder described in elderly individuals, known as large joint destructive arthritis/milwaukee Shoulder Syndrome [5]. Although the basis of cartilage damage by crystals has been the subject of numerous investigations, there are ongoing controversies concerning the relationship between calcium-containing crystals and OA and whether the crystals cause damage or are present as a result of joint damage. If BCP crystals were present purely as a result of joint damage, they should be readily identifiable in other causes of joint destruction, as well as in OA. However, BCP crystals are Biologic Effects In vitro studies of BCP crystal induced cell activation support the active role of BCP in OA pathogenesis. BCP crystals have many biologic effects, including the ability to induce mitogenesis, as well as matrix metalloproteases (MMP) and prostaglandin synthesis, in human fibroblasts, synoviocytes, and chondrocytes (Fig. 1). At concentrations found in pathologic human joint fluids, these crystals exert biological effects on cultured cells in a manner similar to growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and serum. BCP crystals stimulate fibroblast, synoviocyte, and chondrocyte mitogenesis in vitro [7]; stimulate the production of prostaglandin (PGE 2 ) via the phospholipase A 2 /cyclooxygenase (COX) pathway [8]; activate phospholipase C and inositol phospholipid hydrolysis [9]; induce the expression of the proto-oncogenes, c-fos and c-myc, [7]; and induce the synthesis and secretion of MMP 1, 3, 8, 9, and 13 [10 12] and downregulation of tissue inhibitor of metalloproteinases (TIMP) [11]. In contrast to other mitogenic and growth factors, BCP crystal elicited signal transduction pathways have not been completely studied. However, some component molecules involved in calcium-containing crystal signal transduction mechanisms have been identified. Thus, BCP particles have been shown to activate synovial fibroblasts and articular chondrocytes, inducing cellular proliferation and MMP secretion through various intracellular signaling pathways, including protein kinase C (PKC), extracellular signal-related kinase 1/2 (ERK1/2), mitogen-activated protein kinase (MAPK), nuclear factorκb, and activator protein 1 [13,14]. Limited evidence from studies with murine macrophages have also demonstrated their ability to interact with BCP crystals in vitro, resulting in DNA synthesis and cytokine production [15]. More recent investigation into BCP crystal-induction of PGE 2 indicate that BCP crystals have the unusual ability to augment PGE 2 production through induction of both COX-1 and COX-2 in OA synovial fibroblasts (OASF) [16 ]. Intra-lysosomal BCP crystal dissolution and activity of the PKC and ERK1/2 signal transduction pathways are required for BCP crystal induced COX-1 upregulation. These data add to the evidence suggesting that the constitutive COX-1/inducible COX-2 concept is an oversimplification when considering drug therapies and suggest that nonselective COX inhibition may be preferable to COX-2 selective inhibition in BCP crystal associated OA.

3 Point: Hydroxyapatite Crystal Deposition Is Involved in OA Pathogenesis/Progression I McCarthy and Cheung I 143 Furthermore, BCP crystal induction of MMP-13 expression has been investigated in some detail and may involve the ERK1/2 and p38 MAPK pathways and activation of NFκB; this upregulation of MMP-13 may contribute to the accelerated cartilage breakdown in BCP crystal associated OA. Exogenous PGE 2 downregulated BCP crystal induced MMP-13 expression but upregulated MMP-3 expression, mediated in an E-prostanoid (EP)2/EP4/cyclic AMP dependent manner, suggesting that PGE 2 may have beneficial and deleterious effects in BCP crystal associated OA [17 ] Similarly, BCP crystals can increase prostacyclin production and upregulate expression of the prostacyclin receptor in OASF. The potential of prostacyclin to influence BCP crystal stimulated responses was supported by the effects of the prostacyclin analogue, iloprost. Iloprost diminished BCP crystal stimulated prostacyclin receptor upregulation, upregulated microsomal prostaglandin E2 synthase 1 (mpges1) and downregulated MMP-13 expression in BCP crystal stimulated OASF [18 ]. These data demonstrate the potential involvement of prostacyclin in BCP crystal associated OA and suggest that inhibition of PGE 2 synthesis with nonsteroidal anti-inflammatory drugs may also have both deleterious and beneficial effects in BCP crystal associated OA. The ultimate biological effects of calcium-containing crystals on cells in vivo are an increase in MMP synthesis and secretion and increased mitogenesis. These effects appear to correlate with calcium deposition disease in vivo. The increased production of matrix-degrading MMPs by synoviocytes results in articular damage and degeneration and the release of additional crystals from the surrounding tissue, whereas mitogenesis leads to an increase in synoviocytes, manifested by synovial lining hypertrophy, that generate more MMPs [2]. Mechanisms of Deposition BCP crystals and CPPD crystals frequently coexist clinically. Much about the development of CPPD and BCP crystals in OA is poorly understood, however. Laboratory studies supporting the hypothesis that OA causes or worsens calcium crystal formation typically back one of two hypotheses. The first hypothesis is that the damaged extracellular matrix in OA cartilage facilitates matrix mineralization by reducing inhibitors or increasing stimulants of pathologic mineralization in the normally unmineralized articular cartilage matrix. The second hypothesis is that aging, injury, or OA produces changes in the chondrocyte that force it to assume a hypertrophic phenotype. These altered chondrocytes then promote mineralization of their surrounding matrix [19]. Histologic studies show a loss of proteoglycans and disruption of collagen fibril formation around calcium crystals. Similar matrix changes occur in OA. High levels of osteopontin, calcium, and phosphate, and other mineralpromoting factors are found in OA cartilage. It is likely that normal articular cartilage matrix contains mineralization inhibitors whereas OA matrix contains less large proteoglycans, disrupted collagen fibrils, and increased quantities of proteins that promote mineralization [20]. Phenotypic changes in chondrocytes from OA cartilage facilitate calcium crystal formation. Chondrocytes can undergo terminal differentiation and assume characteristics similar to those of hypertrophic chondrocytes that produce bone mineral during epiphyseal growth. These chondrocytes display increased alkaline phosphatase activity and higher levels of annexins and type X collagen. Mineral formation occurs in the pericellular matrix and is facilitated by small chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) [21]. Cartilage matrix changes that occur with OA, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization [22 ]. These features are not seen in chondrocytes from normal healthy articular cartilage, but are well-described in cartilage containing BCP or CPPD crystals. Inorganic pyrophosphate (PPi) is generated from exogenous adenosine triphosphate (ATP) by various chondrocytes ectoenzymes, and complexes with ambient calcium to form CPPD crystals [23 ]. Inorganic phosphate can also be generated by chondrocyte ectoenzymes. When phosphate rather than PPi predominates, BCP mineral is generated. Therefore, local dysregulation of extracellular PPi/inorganic phosphate (Pi) homeostasis appears to underlie calcium crystal deposition. Other features of hypertrophic chondrocytes found in OA that might promote calcium crystal formation include alterations in the putative pyrophosphate transporter, ANK [24]. Levels of activity of transglutaminase enzymes, also a marker of chondrocyte hypertrophy, are increased in OA cartilage [25]. These enzymes contribute to calcium crystal formation. Osteopontin may play an important role in facilitating CPPD crystal formation in articular cartilage [26 ]. Osteopontin is a matricellular protein found in increased quantities in the pericellular matrix of osteoarthritic cartilage, in the pericellular matrix of chondrocytes adjacent to CPPD deposits, and near active transglutaminase. Osteopontin stimulates ATP-induced CPPD crystal formation by chondrocytes in vitro. This effect is augmented by osteopontin s incorporation into extracellular matrix by transglutaminase enzymes, is only modestly affected by its phosphorylation state, and is inhibited by integrin blockers. Finally, growth factors, such as the bone morphogenic proteins and transforming growth factor-β, may contribute to mineralization and cartilage degeneration by promoting phenotypic changes in chondrocytes [27]. BCP Crystal Identification Although BCP crystals are common, particularly in OA, their presence in SF is recognized infrequently because

4 144 Crystal Arthritis I of the lack of a simple, reliable test for detection [28]. Polarized light microscopy, which effectively identifies monosodium urate and CPPD crystals, is unable to detect BCP crystals, which are too small to be resolved by light microscopy ( nm). The larger BCP aggregates have been detected by alizarin red S staining, but this method is difficult to interpret and also stains other calcium containing particulates. More specific techniques for BCP crystal identification include radiograph diffraction, scanning, or transmission electron microscopy with energy dispersive analysis, electron microprobe, Raman spectroscopy, atomic force microscopy, and a binding assay using [14C]ethane-1- hydroxy-1,1-diphosphonate. Unfortunately, these methods typically are unavailable or too costly for the handling of routine clinical specimens [28]. Therefore, progress in understanding the role of BCP crystals in OA has been hampered by difficulties in identification. Substantial progress has been made in the past year in adopting new approaches to BCP crystal identification. One is a fast, sensitive technique that detects and isolates BCP crystals by binding them to magnetic beads. Micrometer-sized magnetic beads were modified with bisphosphonates (BP) using a simple amide-forming reaction between a carboxyl-functionalized bead and an amine-bp derivative. BPs are known to bind selectively to calcium phosphate and have been previously used to treat osteoporosis. The bisphosphonate-modified superparamagnetic (BPSM) beads can extract calcium phosphate crystals from biological fluid by attaching to the calcium atoms on the surface of the crystals, thus forming a complex that can be easily removed from the fluid using a magnet. The viability of the technique was established by using a model system comprising a biological fluid spiked with synthetic calcium phosphate crystals. The technique was then used to test the fluid of patients with suspected osteoarthritis, and confirmed the presence of calcium phosphate crystals. The technique could lead to improved diagnosis and characterization of osteoarthritis and other diseases involving BCP crystal deposition [29 ]. Because tetracyclines stain calcium phosphate mineral in bone, a study was performed to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in SF [30 ]. After oxytetracycline staining, synthetic and native BCP crystals in SF appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or monosodium urate crystals or other particulates. Oxytetracycline staining had fewer falsepositive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid. Finally, synchrotron Fourier transform infrared spectroscopy (sftir) readily and accurately identified CPPD and BCP crystals in a porcine model of crystal formation, as well as in fresh SFs and will be a useful research tool to study articular crystals [31 ]. Animal Models To a limited extent, animal models have been used to investigate calcium-containing crystal deposition diseases. For example, the effects of chronic calcium pyrophosphate dihydrate (CPPD) synovitis on the development of OA lesions were investigated using a lapine model of experimental OA [32]. The data demonstrated a worsening effect of chronic CPPD crystal-induced synovitis on experimental OA and support a possible role for CPPD microcrystalline inflammation in the progression of OA lesions in clinical CPPD crystal deposition disease. No similar investigation has been performed in an animal model using BCP crystals. The murine progressive ankylosis (MPA) model was subsequently used to evaluate phosphocitrate (PC), a potential therapy for BCP crystal deposition disease [33]. PC is the only agent examined to date that blocks the deleterious biologic effects of BCP crystals and also prevents calcification [34,35]. MPA is a manifestation of an autosomal recessive mutation that produces an inflammatory joint disorder associated with intra-articular BCP crystal deposition and culminates in fusion of the joints. Mice with MPA were treated with PC in vivo to determine its effect on disease progression. Clinically, histologically, and microradiographically, there were significant differences in disease progression and severity between the PC-treated and control mice. PC appeared to inhibit disease progression in MPA when given before disease was established. However, this animal model was subsequently dropped from consideration as a model for BCP crystal deposition disease, as it more closely resembled reactive arthritis than OA. Finally, a guinea pig OA model was identified in which meniscal calcification, consistent with BCP, appeared to correlate with age and disease progression. All the mineral deposits in Hartley guinea pigs are BCP crystal (calcium phosphate ratio, 1.54). Moreover, crystals isolated from the meniscus cannot be dissolved by peptidylprolyl isomerase. After weekly treatment of this animal model for 3 months with a new, more potent formulation of PC involving salt and calcium (CaNaPC), the content of calcification in menisci and cartilage degeneration was examined. As a control, Cheung et al. [36 ] also evaluated whether similar CaNaPC treatment had a therapeutic effect in a hemi-meniscectomy model with no known crystal involvement. Meniscal calcification correlated with the cartilage degeneration in this animal model. CaNaPC treatment led to significant reduction of calcium deposits and arrested OA disease progression. Similar CaNaPC treatment had no effect in the hemi-meniscectomy model in which articular calcification does not occur [36 ]. These results support hypotheses that calcification in the form of BCP plays an important role in OA disease progression and that CaNaPC is a potential therapeutic agent for human chondrocalcinosis and BCP deposition disease. These data also suggest that this is a useful animal model for examining the role of calcification in OA.

5 Point: Hydroxyapatite Crystal Deposition Is Involved in OA Pathogenesis/Progression I McCarthy and Cheung I 145 The definitive animal experiment to prove that BCP crystals play a pathogenic role in OA would require that BCP crystals, injected intra-articularly, be shown to induce or accelerate joint degeneration. Response to Dr. Pritzker s Article Dr. Pritzker believes that BCP crystals may be present in end-stage OA, but are present in insufficient amounts to contribute significantly to OA pathogenesis or progression. His rationale is expressed, firstly, in the context of SF observations. He notes that BCP crystals are undetectable by conventional microscopy and therefore require special techniques for identification. This fact is well recognized and efforts are being made to address this limitation [29 31]. However, he draws the mistaken conclusion that difficulty in detection means that small quantities only are present, when clearly quantification cannot be successfully achieved without a satisfactory identification method. He states that the search for apatite crystals is a well-known exercise among rheumatologists. It is indeed well-known, but rarely practiced. In a comprehensive literature review, Swan et al. [38] pointed to poor standards worldwide in synovial analysis in the diagnosis of joint disease and suggested that rationalization of SF tests with improved quality control should be immediate priorities. It is noted correctly that the search for crystals in SF is usually futile without special techniques. However, it is not true that crystals are found only in very advanced disease. Crystals have also been found in mild and moderate OA [3]. Additional studies are badly needed to determine the prevalence of BCP crystals in mild and moderate OA, especially with the development of improved technology to identify and quantify BCP crystals in SF. Dr. Pritzker has observed that in aqueous solutions without the presence of matrix proteins and when concentrations of PPi and Pi, relative to each other are controlled, the likely mineral type can be determined [39]. Furthermore, when matrix vesicles (MV) were isolated from 17-day-old chicken embryo growth plate cartilages and subjected to mineralization in the presence of various Pi/PPi ratios, the mineral type could be determined by the ratio [40]. Derfus et al. [41] showed that MV isolated from normal human cartilage could generate pathologically relevant crystal phases in vitro, as assessed by sftir. In a biomineralization assay, mineral consistent with BCP was produced in the absence of ATP, whereas mineral consistent with CPPD was produced in the presence of ATP. Their logical conclusion was that matrix integrity and substrate availability may be crucial factors in the control of pathologic biomineralization in vivo. This may explain why, in fact, BCP and CPPD frequently coexist in vivo. In some studies, the two occur more commonly than either occurs alone. This conundrum has been discussed [24]. Dr. Pritzker discusses the association of CPPD and OA and states that radiographic chondrocalcinosis is seldom related to BCP crystal deposition. On the contrary, with the aid of modern technology, including digital contact radiography and field-emission scanning electron microscopy (FESEM) equipped with energy-dispersive radiograph (EDX), mineralization consistent with HA was found in the cartilages of all 88 patients undergoing total knee replacement. Radiographic chondrocalcinosis was seen in only 26% of these patients [42]. These results suggest that pathologic cartilage mineralization is far more common than previously appreciated and emphasizes the power of modern technology to help resolve unsolved problems in this area. Dr. Pritzker makes anecdotal reference to his observations based on histology of more than 10,000 joints removed at arthroplasty and describes changes noted in osteonecrosis, which include deposits of BCP in synovium, with largely intact cartilage. In these cases, BCP is unlikely to have originated from bone. Dr. Pritzker unfortunately does not inform us of his methods for BCP identification. He discusses the potential of exposed bone as a source of BCP in OA but suggests that evidence is lacking for BCP shedding from bone into the synovial space and contributing to OA. This is despite reference to Nalbant et al. [43], in which sequential studies of fluid from patients with knee OA showed clearly that some patients with OA had no evidence of SF HA at onset, but crystals appeared with disease progression. Perhaps at least some of such HA crystal originated from exposed bone [43]. However, if HA deposition is simply a consequence of bone exposure resulting from cartilage wear, how can the lack of HA deposition in inflammatory arthritides such as rheumatoid arthritis, a destructive arthropathy with gross erosion of cartilage and bone, be explained? All evidence points to a unique association between OA and HA deposition, an area that badly needs intellectual investment. Dr. Pritzker refers to Muehleman et al. [44], in which data from ankle joints examined at autopsy were reported. This article clearly states that the presence of either monosodium urate or CPPD strongly correlated with cartilage lesions in the talus. Unfortunately, HA was not studied in this work, making it somewhat irrelevant to the current debate. In his conclusion, Dr. Pritzker reiterates his concept that difficulty in crystal identification equates to small quantities. He correctly states that no study has convincingly shown that HA crystals affect the extensive pathologic processes observed in OA. This emphasizes the urgent need for appropriate studies [45]. Fam et al. [32] showed convincingly that intraarticular CPPD could aggravate induced OA in an animal model. Similar work is lacking, but urgently needed, in the case of BCP crystals. Recent in vitro observations of the biological effects of BCP crystals has attracted the attention of the cardiology community so that the potential pathogenic role of similar BCP deposits in atherosclerotic vessels is currently under investigation [37,46 48]. One can only hope that investigators in areas related to rheumatology take a page from the cardiology book and

6 146 Crystal Arthritis I intensify their studies of BCP, a potential pathogenic mediator that, after all, appears unique to OA. Modern technology should allow improved identification and quantitation. Consideration of the importance of tumor necrosis factor, as well as other cytokines present in minute concentrations, as a pathogenic mediator in rheumatoid arthritis has revolutionized management of this potentially devastating disease. Patients with OA deserve similar effort. Conclusions Despite ongoing effort and expense worldwide, an effective, safe disease modifier for OA remains elusive. Given the complexity of the disorder, it may never be possible to identify a single therapy to treat all cases effectively. We suggest that there exists, at the very least, a large subset of patients with OA in whom BCP crystal deposition plays a substantial role in disease progression and that the effects of such crystal deposition represent a potential therapeutic target in the management of these OA cases. The attention of a greater number of investigators, in disciplines relevant to rheumatology, is necessary to realize the full potential of this approach to OA management. The interest of other disciplines, such as cardiology, has already been captured. The body of literature from rheumatology has given direction to investigators interested in the biological effects of calcification in vascular disease, an area that has been, to date, less well explored [37]. Disclosure Dr. McCarthy has been on the advisory board for Wyeth Pharmaceuticals and Bristol Myers-Squibb. Dr. Cheung reported no conflicts of interest relevant to this article. References and Recommended Reading Papers of particular interest, published recently, have been highlighted as: Of importance Of major importance 1. Brandt K, Lohmander LS, Doherty M: Pathogenesis of osteoarthritis. In Osteoarthritis. Edited by Brandt K, Doherty M, Lohmander LS. Oxford: Oxford University Press; 1998: Molloy ES, McCarthy GM: Calcium crystal deposition disease update in pathogenesis and manifestations. Rheum Dis Clin North Am 2006, 32: This is a useful review of clinical implications of the biological effects of both CPPD and BCP crystals and their relevance for therapeutic intervention. 3. Derfus BA, Kurian JB, Butler JJ, et al.: The high prevalence of pathologic calcium crystals in pre-operative knees. J Rheumatol 2002, 29: Swan A, Chapman B, Heap P, et al.: Submicroscopic crystals in osteoarthritic synovial fluids. 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