Basic Calcium Phosphate Crystals Research Articles

Calcium Pyrophosphate and Basic Calcium Phosphate Crystal Arthritis: 2023 in Review

Calcium-containing crystal deposition diseases are extremely common in rheumatology. However, they are under-explored compared to gout or other inflammatory rheumatic diseases. Major advances have been made in 2023 that will undoubtedly stimulate and facilitate research in the field of calcium pyrophosphate (CPP) deposition disease (CPPD): the ACR/EULAR classification criteria for CPPD and a semi-quantitative OMERACT score for ultrasound assessment of the extent of CPP deposition have been validated and published. A large randomized controlled trial compared the efficacy and safety of colchicine and prednisone in acute CPP arthritis. Preclinical studies have elucidated the pro-inflammatory and anti-catabolic effects of basic calcium phosphate (BCP) crystals on mononuclear cells and chondrocytes. The association between osteoarthritis (OA) and IA calcifications has been the subject of several epidemiological publications, suggesting that calcium crystals are associated with a greater risk of progression of knee OA. Research in the field of calcium crystal deposition diseases is active: the areas of investigation for the coming years are broad and promising.

A new ex vivo human model of osteoarthritis cartilage calcification.

Cartilage pathologic calcification is a hallmark of osteoarthritis (OA). Here, we aimed to describe a new ex vivo human model to study the progression of cartilage calcification. Cartilage explants (n = 11), as well as primary chondrocytes (n = 3), were obtained from OA patients undergoing knee replacement. Explants and chondrocytes were cultured in control (NT) or calcification (CM) medium (supplemented with ascorbic acid and β-glycerophosphate). Calcification was evaluated by micro-CT scan at day 0 and 21 in explants, and by Alizarin red staining in chondrocyte monolayers. Raman spectrometry allowed characterization of the crystal type. Interleukin-6 (IL-6) secretion in explant and cell supernatants was measured by ELISA. Finally, matrix degradation was evaluated by Safranin-O staining of explant sections and by glycosaminoglycans (GAG) release in supernatants. Micro-CT scan showed calcifications in all explants at baseline (day 0), which in the CM group increased significantly in number and size after 21 days compared with the NT group. Raman spectrometry revealed that crystals were exclusively basic calcium phosphate crystals (carbonated hydroxyapatite) both in NT and CM. IL-6 secretion was significantly increased in calcifying conditions. Finally, CM significantly increased cartilage catabolism as assessed by decreased Safranin-O staining of tissue explants and increased GAG release in supernatants. CM effects (enhanced calcification, IL-6 secretion and proteoglycans turn-over) were recapitulated in vitro in OA chondrocytes. We have described a new ex vivo human model of cartilage calcification that can summurize the triad of events seen during osteoarthritis progression, i.e. calcification, inflammation, and cartilage degradation. This model will allow the identification of new anti-calcification compounds.

Imaging findings of acute calcific periarthritis, with emphasis on magnetic resonance imaging: pictorial essay.

Acute calcific periarthritis (ACP) is defined as periarticular inflammation associated with intra-articular deposits of hydroxyapatite and other basic calcium phosphate crystals. Patients with ACP present with a sudden onset of pain, together with localized swelling, as well as erythema, tenderness, and reduced range of motion. Familiarity with the clinical and radiological manifestations of ACP aids in the diagnosis and helps differentiate it from other conditions, particularly infectious or inflammatory pathologies such as septic arthritis and gout, thereby reducing the number of unnecessary diagnostic and therapeutic procedures. The objective of this pictorial essay is to illustrate the imaging findings of ACP in various joints, with an emphasis on the findings obtained by magnetic resonance imaging.

Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles.

Calcified aortic valve disease in its final stage leads to aortic valve stenosis, limiting cardiac function. To date, surgical intervention is the only option for treating calcific aortic valve stenosis. This study combined controlled drug delivery by nanoparticles (NPs) and active targeting by antibody conjugation. The chelating agent diethylenetriaminepentaacetic acid (DTPA) was covalently bound to human serum albumin (HSA)-based NP, and the NP surface was modified using conjugating antibodies (anti-elastin or isotype IgG control). Calcification was induced ex vivo in porcine aortic valves by preincubation in an osteogenic medium containing 2.5 mM sodium phosphate for five days. Valve calcifications mainly consisted of basic calcium phosphate crystals. Calcifications were effectively resolved by adding 1-5 mg DTPA/mL medium. Incubation with pure DTPA, however, was associated with a loss of cellular viability. Reversal of calcifications was also achieved with DTPA-coupled anti-elastin-targeted NPs containing 1 mg DTPA equivalent. The addition of these NPs to the conditioned media resulted in significant regression of the valve calcifications compared to that in the IgG-NP control without affecting cellular viability. These results represent a step further toward the development of targeted nanoparticular formulations to dissolve aortic valve calcifications.

Calcium Pyrophosphate and Basic Calcium Phosphate Deposition Diseases: The Year in Review 2022

Calcium-containing crystal deposition diseases are a common cause of pain and disability but remain relatively under-investigated. No drug has been identified that can prevent deposition or effect dissolution of either calcium pyrophosphate (CPP) or basic calcium phosphate (BCP) crystals. In comparison to the field of gout and urate biology, published research in relation to calcium crystal deposition diseases in 2022 was relatively modest in quantity. In CPP deposition (CPPD) disease, progress was made mainly in epidemiology, imaging, surgical management and Gitelman’s syndrome. In relation to BCP crystals, the effect on tenocytes in vitro was explored and results indicate that BCP crystals likely reduce tendon matrix integrity via their interaction with tenocytes. The involvement of calcification in the progression of osteoarthritis (OA) was elegantly demonstrated contributing to further discovery of the process of OA progression. There was a paucity of mechanistic and genetic studies in calcium crystal deposition diseases published in 2022, nor any breakthrough in therapy, showing that there is abundant scope for investigation under these themes in the future.

A functional genomics approach reveals suggestive quantitative trait loci associated with combined TLR4 and BCP crystal-induced inflammation and osteoarthritis

Basic calcium phosphate (BCP) crystalscan activate the NLRP3 inflammasome and are potentially involved in the pathogenesis of osteoarthritis (OA). In order to elucidate relevant inflammatory mechanisms in OA, we used a functional genomics approach to assess genetic variation influencing BCP crystal-induced cytokine production. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers who were previously genotyped and stimulated with BCP crystals and/or lipopolysaccharide (LPS) after which cytokines release was assessed. Cytokine quantitative trait locus (cQTL) mapping was performed. For in vitro validation of the cQTL located in anoctamin 3 (ANO3), PBMCs were incubated with Tamoxifen and Benzbromarone prior to stimulation. Additionally, we performed co-localisation analysis of our top cQTLs with the most recent OA meta-analysis of genome-wide association studies (GWAS). We observed that BCP crystals and LPS synergistically induce IL-1β in human PBMCs. cQTL analysis revealed several suggestive loci influencing cytokine release upon stimulation, among which are quantitative trait locus annotated to ANO3 and GLIS3. As functional validation, anoctamin inhibitors reduced IL-1β release in PBMCs after stimulation. Co-localisation analysis showed that the GLIS3 locus was shared between LPS/BCP crystal-induced IL-1β and genetic association with Knee OA. We identified and functionally validated a new locus, ANO3, associated with LPS/BCP crystal-induced inflammation in PBMCs. Moreover, the cQTL in the GLIS3 locus co-localises with the previously found locus associated with Knee OA, suggesting that this Knee OA locus might be explained through an inflammatory mechanism. These results form a basis for further exploration of inflammatory mechanisms in OA.

Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-β signaling

Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods. Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-β) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors. Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signaling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signaling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression. BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment.

Cartilage calcification in osteoarthritis: mechanisms and clinical relevance.

Pathological calcification of cartilage is a hallmark of osteoarthritis (OA). Calcification can be observed both at the cartilage surface and in its deeper layers. The formation of calcium-containing crystals, typically basic calcium phosphate(BCP) and calcium pyrophosphate dihydrate (CPP)crystals, is an active, highly regulated and complex biological process that is initiated by chondrocytes and modified by genetic factors, dysregulated mitophagy or apoptosis, inflammation and the activation of specific cellular-signalling pathways. The links between OA and BCP deposition are stronger than those observed between OA and CPP deposition. Here, we review the molecular processes involved in cartilage calcification in OA and summarize the effects of calcium crystals on chondrocytes, synovial fibroblasts, macrophages and bone cells. Finally, we highlight therapeutic pathways leading to decreased joint calcification and potential new drugs that could treat not only OA but also other diseases associated with pathological calcification.

Basic calcium phosphate crystals induce the expression of extracellular matrix remodelling enzymes in tenocytes.

Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability. In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability. BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability. BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes.

CALCIFICATION AND PROTEOGLYCAN SULFATION IN OSTEOARTHRITIS AND CHONDROCALCINOSIS

Purpose: Osteoarthritis (OA) and chondrocalcinosis (CC) are common arthropathies associated with the accumulation of calcium-containing crystals in the cartilage and synovial membrane. Basic calcium phosphate (BCP) crystals are present in OA, whereas calcium phosphate dehydrate (CPPD) crystals are a characteristic of CC. Further, BCP crystals have been linked to Wnt signaling and hypertrophic differentiation of chondrocytes, whereas CPPD crystals may be responsible for chondrocyte senescence. Despite their association with different types of calcium crystals, OA and CC share similar phenotypes including a progressive degeneration and calcification of articular cartilage.

CHARACTERIZATION OF THE CALCIFICATIONS IN THE POSTERIOR HORN OF HUMAN MENISCUS VIA RAMAN MICROSPECTROSCOPY

Purpose: Calcifications in joint soft tissues, such as meniscus and articular cartilage, are closely associated with osteoarthritis (OA) progression and could act as a potential target for disease-modifying agents in OA therapy. Basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) crystals are the two most common forms of calcium crystals found in these tissues. Moreover, within the CPPD crystals, two different polymorphs - monoclinic and triclinic - have been identified in arthritic patients.

Кристаліндукований остеоартрит: роль кристалів основного фосфату кальцію

Кристали основних фосфатів кальцію вже давно асоціюються з патогенезом остеоартриту. Основні фосфати кальцію з великою частотою визначаються в синовіальній рідині і суглобовому хрящі при цьому захворюванні. У той час як осадження основних фосфатів кальцію розглядається багатьма вченими як наслідок прогресуючого перебігу остеоартриту, з’явилися суттєві докази того, що ці кристали можуть виступати активними патогенними медіаторами остеоартриту. «Кристали основних фосфатів кальцію» — загальний термін для опису декількох видів фосфатів кальцію, у тому числі карбонатапатиту, гідроксиапатиту, три- і октакальцієвих фосфатів, кристалів вітлокіта магнію. Кристали основних фосфатів кальцію патогенетично пов’язані з остеоартритом, кальцинуючим тендинітом, гострим кристалічним артритом та атеросклерозом. Кристали кальцію визначаються в синовіальній рідині в 65 % випадків остеоартриту колінного суглоба, вони з високою частотою виявляються в гіаліновому хрящі при остеоартриті, а також у 100 % пацієнтів у суглобовому хрящі при проведенні операції тотального ендопротезування. Кристали основних фосфатів кальцію також були виявлені в синовіальній рідині та синовіальній мембрані пацієнтів із легким, помірним і тяжким ураженням колінного суглоба при артроскопії. Основні фосфати кальцію проявляють множинні біологічні ефекти in vitro, у тому числi здатність стимулювати мітогенез, продукцію простагландинів, цитокінів і матриксних металопротеїназ у різних типах клітин, включаючи синовіоцити, хондроцити і макрофаги. Кристали основних фосфатів кальцію також обумовлюють запалення при остеоартриті шляхом прямої взаємодії з вродженою імунною системою. У цьому огляді обговорюються останні досягнення в цій галузі і підбиваються підсумки сучасного розуміння ролі кристалів основних фосфатів кальцію в патогенезі остеоартриту. Погляди на роль кристалів основних фосфатів кальцію в патогенезі остеоартриту продовжують розширюватися, проте вже сьогодні існує достатньо експериментальних і клінічних доказів найважливішої ролі кальційумісних кристалів у розвитку цього захворювання і прогресуючій дегенерації суглобового хряща.

The role of calcium crystals and their effect on osteoarthritis pathogenesis.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive degeneration of articular cartilage. Due to its high prevalence and limited treatment options, OA has become one of the most disabling diseases in developed countries. In recent years, OA has been recognized as a heterogenic disease with various phenotypes. Calcium crystal-related endotypes, which are defined by either a distinct functional or pathobiological mechanism, are present in approximately 60% of all OA patients. Two different calcium crystals can accumulate in the joint and thereby calcify the cartilage matrix, which are basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) crystals. The formation of these crystals depends mainly on the balance of phosphate and pyrophosphate, which is regulated by specific proteins controlling the pyrophosphate metabolism. Dysregulation of these molecules subsequently leads to preferential formation of either BCP or CPP crystals. BCP crystals, on the one hand, are directly associated with OA severity and cartilage degradation. They are mostly located in the deeper cartilage layers and are associated with chondrocyte hypertrophy. CPP crystal deposition, on the other hand, is a hallmark of chondrocalcinosis and is associated with aging and chondrocyte senescence. Therefore, BCP and CPP crystals are associated with different chondrocyte phenotypes. However, BCP and CPP crystals are not mutually exclusive and can coexist in OA, creating a mixed endotype of OA. Both crystals clearly play a role in the pathogenesis of OA. However, the exact impact of each crystal type on either driving the disease progression or being a result of chondrocyte differentiation is still to be elucidated.

Chondrocytes From Osteoarthritic and Chondrocalcinosis Cartilage Represent Different Phenotypes.

Basic calcium phosphate (BCP)-based calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease and hypertrophic differentiation of chondrocytes. Chondrocalcinosis (CC) is associated with calcium pyrophosphate dihydrate (CPPD) deposition disease in the joint inducing OA-like symptoms. There is only little knowledge about the effect of CPPD crystals on chondrocytes and the signaling pathways involved in their generation. The aim of this study was to investigate the chondrocyte phenotype in CC cartilage and the effect of CPPD crystals on chondrocytes. Cartilage samples of patients with CC, patients with severe OA, and healthy donors were included in this study. The presence of CC was evaluated using standard X-ray pictures, as well as von Kossa staining of cartilage sections. OA severity was evaluated using the Chambers Score on cartilage sections, as well as the radiological Kellgren–Lawrence Score. Patients with radiologically detectable CC presented calcification mainly on the cartilage surface, whereas OA patients showed calcification mainly in the pericellular matrix of hypertrophic chondrocytes. OA cartilage exhibited increased levels of collagen X and matrix metalloproteinase 13 (MMP13) compared with CC and healthy cartilage. This observation was confirmed by qRT-PCR using cartilage samples. No relevant influence of CPPD crystals on hypertrophic marker genes was observed in vitro, whereas BCP crystals significantly induced hypertrophic differentiation of chondrocytes. Interestingly, we observed an increased expression of p16 and p21 in cartilage samples of CC patients compared with OA patients and healthy controls, indicating cellular senescence. To investigate whether CPPD crystals were sufficient to induce senescence, we incubated chondrocytes with BCP and CPPD crystals and quantified senescence using β-gal staining. No significant difference was observed for the staining, but an increase of p16 expression was observed after 10 days of culture. Primary chondrocytes from CC patients produced CPPD crystals in culture. This phenotype was stabilized by mitomycin C-induced senescence. Healthy and OA chondrocytes did not exhibit this phenotype. BCP and CPPD crystals seem to be associated with two different chondrocyte phenotypes. Whereas BCP deposition is associated with chondrocyte hypertrophy, CPPD deposition is associated with cellular senescence.

Effect of pathogenic crystals on the production of pro- and anti-inflammatory cytokines by different leukocyte populations

PurposeTo evaluate cytokine production in vitro by different types of leukocytes stimulated with monosodium urate (MSU), calcium pyrophosphate (CPP) and basic calcium phosphate (BCP) crystals. Material and methodsPolymorphonuclear cells (PMN), monocytes and lymphocytes, isolated from healthy volunteer blood, were stimulated for different time periods with increasing MSU, CPP or BCP crystal concentrations. IL-1β, IL-8, IL-6, CCL2, IL-1Ra and TGFβ1 were determined by ELISA. ResultsExposure of PMN to different crystals resulted in a moderate IL-8 and IL-1Ra release. Stimulation of monocytes induced a significant production of all the cytokines evaluated. The highest levels of IL-1β, IL-6, CCL2 and IL-8 were observed with MSU at 0.5 mg/ml, CPP at 0.01–0.05 mg/ml and BCP at 1 mg/ml after 18–48 h and then decreased. At the same crystal concentrations, IL-1Ra and TGFβ1 increased until the end of the experiment. Treatment of lymphocytes with different crystals did not induce cytokine release. ConclusionThis study demonstrates that PMN, monocytes and lymphocytes from the same donor respond differently after stimulation with MSU, CPP or BCP crystals, depending on the dose and the time of exposure. Crystals induce a rapid increase of pro-inflammatory cytokines, whereas longer time is required to release high levels of anti-inflammatory cytokines.

Imaging of crystalline arthropathy in 2020.

Crystal-related arthropathies are the result of crystal deposition in joint and periarticular soft tissues. Identification of urate crystals is mandatory to distinguish gout from other crystalline arthropathies, including calcium pyrophosphate dihydrate and basic calcium phosphate crystal deposition diseases. ACR/EULAR classification criteria for gout included dual-energy computed tomography and ultrasound with equal impact to the final score. Different diagnostic strengths of these imaging modalities depend on disease duration and scanned anatomic site. While ultrasound has been indicated as the first-choice imaging technique, especially in the early stages of the disease, dual-energy computed tomography has shown to be highly specific, allowing the detection of crystal deposits in anatomic sites not accessible by ultrasound, such as the spine. At the spinal level, MRI findings are usually nonspecific. Finally, there is preliminary evidence that at the knee, dual-energy computed tomography may discriminate calcium pyrophosphate dihydrate from basic calcium phosphate crystal deposits.

BCP crystals promote chondrocyte hypertrophic differentiation in OA cartilage by sequestering Wnt3a

ObjectiveCalcification of cartilage with basic calcium phosphate (BCP) crystals is a common phenomenon during osteoarthritis (OA). It is directly linked to the severity of the disease and known to be...

Dual-energy computed-tomography-based discrimination between basic calcium phosphate and calcium pyrophosphate crystal deposition in vivo.

Background:Dual-energy computed tomography (DECT) is being considered as a non-invasive diagnostic and characterization tool in calcium crystal-associated arthropathies. Our objective was to assess the potential of DECT in distinguishing between basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) crystal deposition in and around joints in vivo.Methods:A total of 13 patients with calcific periarthritis and 11 patients with crystal-proven CPPD were recruited prospectively to undergo DECT scans. Samples harvested from BCP and CPP calcification types were analyzed using Raman spectroscopy and validated against synthetic crystals. Regions of interest were placed in BCP and CPP calcifications, and the following DECT attenuation parameters were obtained: CT numbers (HU) at 80 and 140 kV, dual-energy index (DEI), electron density (Rho), and effective atomic number (Zeff). These DECT attenuation parameters were compared and validated against crystal calibration phantoms at two known equal concentrations. Receiver operating characteristic (ROC) curves were plotted to determine the highest accuracy thresholds for DEI and Zeff.Results:Raman spectroscopy enabled chemical fingerprinting of BCP and CPP crystals both in vitro and in vivo. DECT was able to distinguish between HA and CPP in crystal calibration phantoms at two known equal concentrations, most notably by DEI (200 mg/cm3: 0.037 ± 0 versus 0.034 ± 0, p = 0.008) and Zeff (200 mg /cm3: 9.4 ± 0 versus 9.3 ± 0, p = 0.01) analysis. Likewise, BCP calcifications had significantly higher DEI (0.041 ± 0.005 versus 0.034 ± 0.005, p = 0.008) and Zeff (9.5 ± 0.2 versus 9.3 ± 0.2, p = 0.03) than CPP crystal deposits with comparable CT numbers in patients. With an area under the ROC curve of 0.83 [best threshold value = 0.0 39, sensitivity = 90. 9% (81.8, 97. 7%), specificity = 64.6% (50.0, 64. 6%)], DEI was the best parameter in distinguishing between BCP and CPP crystal depositions.Conclusion:DECT can help distinguish between crystal-proven BCP and CPP calcification types in vivo and, thus, aid in the diagnosis of challenging clinical cases, and in the characterization of CPP and BCP crystal deposition occurring in osteoarthritis.

Osteoarthritis-associated basic calcium phosphate crystals alter immune cell metabolism and promote M1 macrophage polarization

A number of studies have demonstrated that molecules called 'alarmins' or danger-associated molecular patterns (DAMPs), contribute to inflammatory processes in the OA joint. Metabolic reprogramming of immune cells, including macrophages, is emerging as a prominent player in determining immune cell phenotype and function. The aim of this study was to investigate if basic calcium phosphate (BCP) crystals which are OA-associated DAMPs, impact on macrophage phenotype and metabolism. Human monocyte derived macrophages were treated with BCP crystals and expression of M1 (CXCL9, CXCL10) and M2 (MRC1, CCL13)-associated markers was assessed by real-time PCR while surface maturation marker (CD40, CD80 & CD86) expression was assessed by flow cytometry. BCP induced metabolic changes were assessed by Seahorse analysis and glycolytic marker expression (hexokinase 2(HK2), Glut1 and HIF1α) was examined using real-time PCR and immunoblotting. Treatment with BCP crystals upregulated mRNA levels of CXCL9 and CXCL10 while concomitantly downregulating expression of CCL13 and MRC1. Furthermore, BCP-treated macrophages enhanced surface expression of the maturation makers, CD40, CD80 and CD86. BCP-treated cells also exhibited a shift towards glycolysis as evidenced by an increased ECAR/OCR ratio and enhanced expression of the glycolytic markers, HK2, Glut1 and HIF1α. Finally, BCP-induced macrophage activation and alarmin expression was reduced in the presence of the glycolytic inhibitor, 2-DG. This study not only provides further insight into how OA-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for calcium crystal-related arthropathies.

Basic Calcium Phosphate Crystals Induce Osteoarthritis-Associated Changes in Phenotype Markers in Primary Human Chondrocytes by a Calcium/Calmodulin Kinase 2-Dependent Mechanism.

Chondrocytes in osteoarthritis undergo a phenotype shift leading to increased production of cartilage-degrading enzymes. There are similarities between the phenotype of osteoarthritic chondrocytes and those of growth plate chondrocytes. Hydroxyapatite can promote chondrocyte differentiation in the growth plate. Basic calcium phosphate (BCP) crystals (which consist of hydroxyapatite, octacalcium apatite and tricalcium phosphate) are frequently found in osteoarthritic joints. The objective of this study was to determine whether BCP crystals induce disease-associated changes in phenotypic marker expression in chondrocytes. Primary human chondrocytes isolated from macroscopically normal cartilage were treated with BCP for up to 48h. Expression of indian hedgehog (IHH), matrix metalloproteinase 13 (MMP13), interleukin-6 (IL-6) and type X collagen (COLX) were higher, and expression of sry-box 9 (SOX9) lower, in BCP-treated chondrocytes (50µg/mL) compared to untreated controls. COLX protein was also present in BCP-treated chondrocytes. Intracellular calcium and levels of phosphorylated and total calcium/calmodulin kinase 2 (CaMK2) were elevated following BCP treatment due to BCP-induced release of calcium from intracellular stores. CaMK2 inhibition or knockdown ameliorated the BCP-induced changes in SOX9, IHH, COLX, IL-6 and MMP13 expression. BCP crystals induce osteoarthritis-associated changes in phenotypic marker expression in chondrocytes by calcium-mediated activation of CaMK2. The presence of BCP crystals in osteoarthritic joints may contribute to disease progression.