CPP crystals arise within enzyme-rich vesicles located in the pericellular matrix of chondrocytes within the extracellular milieu of synovial fluid. These vesicles are designated as articular cartilage vesicles (ACVs). ACVs are enriched with the enzymes nucleotide pyrophosphohydrolase (NTPPH) and tissue nonspecific alkaline phosphatase (TNAP), which are implicated in the biosynthesis of CPP crystals. Inorganic phosphate (Pi) is characterized by its low energy state and comprises a singular phosphate ion (PO₄^3–). It plays a pivotal role in cellular energy transfer and is an integral component of the structure of adenosine triphosphate (ATP). PPi is composed of two phosphate groups linked by a high-energy bond (P₂O₇^4–). It is essential for the biosynthesis of ribonucleic acid (RNA) and deoxyribose nucleic acid (DNA). PPi undergoes hydrolysis to yield Pi via an enzymatic reaction. An imbalance between PPi and Pi within the cellular environment presents a significant risk factor for the crystallization of CPP, with an elevated extracellular PPi (ePPi) ratio being instrumental in the precipitation of CPP crystals. In the context of synovial fluid, PPi is present in both fibrocartilage and hyaline cartilage [1]. Extracellular ATP serves as the primary precursor of PPi synthesized by chondrocytes, with its production being modulated by the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Chondrocytes subjected to mechanical stress exhibit an elevated synthesis of ATP, which concomitantly enhances the production of PPi. The ePPi generated by chondrocytes acts as an inhibitor of calcium mineralization. The accumulated ePPi interacts with calcium ions to facilitate the formation of CPP crystals [2]. TNAP is integral to the hydrolysis of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) while concurrently modulating Pi levels through the hydrolysis of PPi to Pi. Ecto-5'-nucleotidase (CD73) facilitates the hydrolysis of AMP to Pi, and its deficiency is correlated with an elevation in TNAP enzyme activity (Figure 1). Transforming growth factor-beta (TGF-β), growth factors, and cytokines significantly influence the synthesis of PPi. TGF-β initiates the production of PPi by upregulating the expression levels of ENPP1 and the PPi transport regulator [progressive ankylosis protein (ANKH)]. Furthermore, TGF-β contributes to the augmentation of PPi, exhibiting effects analogous to those of epidermal growth factor (EGF) [2]. It is widely recognized that mutations in the ANKH gene, which are situated within the locus located on chromosome 5p, designated as the CCAL2 locus, correlate significantly with joint deterioration. The ANKH protein functions as a transmembrane protein and is hypothesized to enhance extracellular CPP crystal formation by facilitating the passage of PPi across the cellular membrane in the context of CPPD disease [2]. ACVs are small vesicles found in the pericellular matrix of the articular cartilage. These vesicles carry enzymes for ATP and PPi metabolism and play a role in CPP crystal formation by affecting cartilage mineralization [2].

CPP crystals elicit a stimulation of the innate immune system, subsequently provoking an inflammatory response. Chondrocytes serve as the repository for CPP crystals within the pericellular matrix vesicles. Indeed, the synthesis of PPi by chondrocytes constitutes a normal physiological process. When the equilibrium between Pi and PPi is perturbed in favor of PPi, the formation of CPP crystals occurs, potentially leading to the development of CPPD, which may manifest as arthritis [2]. Two types of crystals have been detected in the synovial fluid of CPPD patients. They have been described as monoclinic (needle-shaped) and triclinic (rectangular rods). Small size (due to easier phagocytosis) and monoclinic crystals (larger area to interact with inflammatory mediators) have been reported to be more inflammatory [5]. It is known that CPP crystals stimulate synovial fibroblasts, activate the release of matrix metalloproteinase-8 (MMP-8) and interleukin-6 (IL-6), and cause inflammation [6]. CPP crystals activate synovial cells by direct phagocytosis or by interaction of the cell membrane and crystals [7]. Macrophages play an active role in synovial inflammation. Phagocytosis of insoluble CPP crystals by macrophages causes lysosomal rupture, release of reactive oxygen species (ROS) in the body, potassium release, ATP release, an increase in nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3), and activation of the inflammasome complex [8]. NLRP3 is sensitive to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and activates caspase-1. Caspase-1 initiates the inflammatory cascade by increasing IL-1β production [9]. The inflammatory cytokine that is the major cause of the inflammatory process is IL-1β. In addition, CPP crystals activate the inflammasome via nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) and trigger the release of IL-1β [10, 11]. Nevertheless, CPP crystals possess the capacity to directly activate Toll-like receptors (TLRs), predominantly TLR2 and TLR4, as well as nucleotide-binding oligomerization domain-like receptors (NLRs) located on the cellular membrane, thereby instigating an inflammatory reaction through the activation of NF-κB [9]. The engagement of CPP crystals with the cellular membrane culminates in the activation of intracellular signaling cascades, including extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase (JNK), and other MAPKs, along with the induction of genes that facilitate the activation of transcription factors such as activator protein 1 and NF-κB. The secretion of IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and pro-IL-1β is significantly augmented [5] (Figure 2). CPP crystals have also been reported to cause inflammation via neutrophil extracellular traps (NETs) formation [6].

Age constitutes the principal risk factor for CPPD. Given that OA is a pathological condition whose prevalence escalates with advancing age, it is posited that a frequent correlation exists between OA and CPPD. Empirical studies utilizing the US have indicated that both the leukocyte count and the prevalence of synovitis are significantly elevated in patients with CPPD in comparison to those with OA. It is hypothesized that the presence of CPPD may expedite the deterioration of OA. Furthermore, it has been documented that secondary OA predominantly manifests in the scaphotrapeziotrapezoid joint as a consequence of CPPD [17]. Previous joint trauma increases the risk of CPPD. It has been reported that the prevalence of CPPD increases, especially in patients who have undergone meniscus surgery [12]. A case-control study reported that knee joint malalignment, such as varus deformity, was a risk factor for the development of CPPD at an early age [18].

Gitelman syndrome represents a renal tubular disorder characterized by a deficiency in magnesium levels. Magnesium functions as an inhibitor of the TNAP enzyme, which catalyzes the hydrolysis of PPi to Pi. This deficiency leads to the accumulation of CPP, particularly within the axial skeleton. Furthermore, chronic renal insufficiency exacerbates the likelihood of CPPD. Additional conditions such as primary hyperparathyroidism, hereditary hemochromatosis, hypophosphatasia, familial hypocalciuric hypocalcemia, and a range of other metabolic disorders are also recognized as being associated with CPPD [12].

In a particular investigation, it was documented that 30% of individuals afflicted with hemochromatosis exhibited chondrocalcinosis, and it was noted that this condition demonstrated a positive correlation with serum age, ferritin, and parathyroid hormone concentrations [19].

Although familial CPPD attributable to genetic predisposition is infrequently observed, instances of polyarticular involvement and disease manifestation prior to the age of 55 may occur in affected individuals. Empirical data suggest that mutations in the ANKH gene, along with other genetic loci such as CCAL1, are correlated with familial chondrocalcinosis. Furthermore, there exists substantiation that mutations within the ANKH gene augment the production of ePPi, and this genetic alteration has been implicated in familial CPPD. A research investigation conducted in the United Kingdom identified the E490del mutation in the ANKH gene as being associated with chondrocalcinosis and the onset of premature OA [20]. The human homeostatic iron regulatory protein (HFE) gene mutation has been reported to be associated with CPPD with polyarticular involvement [21]. Mutations in the TNFRSF11B gene, which encodes osteoprotegerin (OPG), have been found to be associated with OA-associated chondrocalcinosis [22].

It is imperative that serum calcium, ferritin, magnesium, phosphorus, alkaline phosphatase, and thyroid-stimulating hormone assessments be requisitioned for patients diagnosed with CPPD at a relatively early age, taking into account the concomitant conditions that may coexist. Furthermore, should the situation warrant, these individuals ought to be evaluated for potential genetic mutations.

US is a non-invasive method that is more sensitive than radiography in detecting crystal structures in the joint area. It can be used as a guide in synovial fluid aspiration [29].

The OMERACT initiative documented the US methodology utilized for the diagnosis of CPPD in the knee joint, revealing a sensitivity of 91% and a specificity of 54%. Nonetheless, it was elucidated that both sensitivity and specificity exhibited variability contingent upon the specific anatomical region under examination. The article indicated that sensitivity reached as high as 87% in the medial meniscus, whereas specificity attained a maximum of 92% in the hyaline cartilage of the medial condyle. Consequently, it was posited that a comprehensive assessment of both the medial meniscus and the hyaline cartilage of the medial epicondyle should be conducted in conjunction. Furthermore, the study reported the absence of a statistically significant difference in synovial inflammation between patients with OA and those with concurrent CPPD and OA when utilizing the US technique [40]. In a study examining the prevalence of CPPD in patients with knee pain, they reported that CPPD deposition was detected in fibrocartilage at a younger age than in hyaline cartilage. In the same study, they found fibrocartilage involvement at a rate of 46.7% and hyaline cartilage involvement at a rate of 23.3% in those over the age of 80 [14]. In the literature, studies comparing the US technique with conventional radiography have reported that US is more sensitive [41, 42]. CPP deposits located within the articular cartilage may be subject to displacement due to joint mobility, and US represents a beneficial modality for evaluating such displacement. The efficacy of the ultrasonographic technique is contingent upon the proficiency of the operator and is unable to differentiate between CPP dihydrate crystals and BCP crystals [38].

CT represents a superior modality for the assessment of deep joints and yields more precise outcomes compared to traditional radiographic techniques. CT serves as an efficacious tool for the differential diagnosis of crowned dens syndrome, particularly in cases characterized by an acute onset of cervical and shoulder discomfort. The detection of linear calcific deposits within the transverse ligament of the atlas, which exhibit a radiographic density less than that of cortical bone, constitutes a hallmark involvement pattern associated with crowned dens syndrome. The manifestation of linear or punctate opacities is observed with diminished density (< 300 Hounsfield Units) utilizing this imaging methodology. It is noteworthy that CPP and BCD crystals remain indistinguishable via CT [4]. DECT represents an advanced imaging modality that facilitates the distinction between CPPD and BCP crystals through the application of a specific color coding system associated with the crystals. DECT effectively identifies both linear and punctate calcifications present within fibrocartilage or hyaline cartilage matrices. Such calcifications exhibit enhanced detectability when utilizing imaging techniques characterized by reduced thickness and lower density (< 300 Hounsfield Units) in contrast to cortical bone structures [43]. In one study, it was reported that the distinction between CPP and BCP could be made with 90% sensitivity using the DECT technique [44].

The analysis of synovial fluid ought to be conducted from the appropriate anatomical region utilizing US guidance. The identification of calcium CPP crystals through polarized light microscopy (PLM) is regarded as the gold standard approach for the diagnosis of CPPD, exhibiting a sensitivity and specificity of 95%. The crystalline morphology serves as the primary criterion for diagnosis, with no established threshold pertaining to their quantity. CPP crystals exhibit a morphology akin to a rectangle characterized by slender parallel extensions, and demonstrate birefringence in PLM. Research has indicated that CPP crystals may remain intact for an extended duration of 24 to 48 hours at ambient temperature [45]. Pastor et al. [46] reported that CPP crystals in the synovial fluid could be detected at 100% in heparin and 89% in EDTA at room temperature in 24 hours and at 89% in heparin and 83% in EDTA at refrigerated temperatures. Crystals can sometimes be found inside vacuoles in a phagocytosed form and may not show birefringence, which may lead to false positive results. Therefore, synovial fluid analysis should be performed by experienced clinicians [47].