Synovial fluid fetuin-A levels in patients affected by osteoarthritis with or without evidence of calcium crystals

Fetuin-A levels are increased in synovial fluid of osteoarthritic patients with calcium crystals. Sir, Calcium crystals are often associated with OA, although their role in the onset and progression of the disease is still under investigation. Calcium pyrophosphate (CPP) and basic calcium phosphate (BCP) crystals are the two most prevalent types of crystals found in OA and experimental evidence supports their role in inducing inflammation [1]. While CPP is detectable by optical microscopy, BCP is difficult to identify with standard methods because of its submicron dimensions [2]. Fetuin-A (FA) is a secreted multifunctional protein potentially involved in inflammation and in inhibiting ectopic calcifications by forming a soluble fetuin–mineral complex [3]. Some studies have analysed FA levels in OA serum and SF without clearly elucidating its role [4, 5]. The aim of the present study was to evaluate the FA levels in SF of the following three groups of patients (3 males and 21 females) affected by symptomatic knee OA and enrolled according to the inclusion and exclusion criteria previously described [2]: eight patients without crystals, eight patients with CPP and eight patients with BCP. In addition, IL-6 and IL-8 levels were measured in the SF of the three groups. Patients were enrolled at the Rheumatology Unit after providing written informed consent and with the approval of the Padova Hospital Ethical Committee (Comitato Etico per la Sperimentazione Clinica della Provincia di Padova). The presence of crystals in the SF was detected by scanning electron microscopy according to the protocol already described [2]. White blood cells and SF volume were quantified, revealing no statistical differences between the three groups. Patients with CPP and BCP were older compared with patients without crystals {median age 79 years [interquartile range (IQR) 84–75] and 84.5 (89.75–69.25) vs 65 (71.25–61.50); P = 0.0394 and P = 0.0354, respectively}. No differences in BMI were found between groups. Interestingly, FA levels were higher in patients with CPP and BCP compared with subjects without crystals (P = 0.004 and P = 0.035, respectively) (Fig. 1A), suggesting a role of FA in calcium crystals deposition in OA. No correlations were found between Kellgren–Lawrence scoring and SF FA levels. Interestingly, it has been shown that FA cannot bind a minor component of the fetuin–mineral complex, called matrix gla protein, in chondrocytes isolated from OA patients. The loss of the inhibitory mechanism due to the absence of the complex might be responsible for calcium crystal formation in these patients [6]. Impairment of the complex might also explain the increased FA levels in OA SF. FA and inflammatory marker levels in the SF of OA patients with calcium crystals (A) OA patients with crystals exhibit an increase in FA levels in SF compared with OA patients without crystals. (B) Patients with BCP crystals have higher levels of IL-6 compared with patients without crystals. (C) No statistical differences in IL-8 levels are present between the three groups of patients. P-values reported have been calculated by adjusted analysis using age as a covariate. All results are expressed as box‐and‐whisker plots (boxes indicate the 25th and 75th percentiles, whiskers indicate the minimum and maximum values and bars indicate the median). The Shapiro–Wilk test was used to determine whether data were distributed normally and one-way analysis of variance or the Kruskal–Wallis test with Dunn’s multicomparison test were performed depending on the distribution of the data. To test the possible effect of age on the results, a covariate analysis was performed. P-values <0.05 were considered significant. Data were analysed using SPSS version 25 (IBM, Armonk, NY, USA). As far as IL-6 and IL-8 are concerned, we observed that patients with BCP had statistically higher levels of IL-6 compared with patients without crystals (P = 0.043), whereas only a trend was found for CCP compared with controls. No differences were found comparing IL-8 levels between groups (Fig. 1B and C). Therefore the SF of patients with calcium crystals had higher levels of inflammation compared with patients without crystals, confirming the inflammatory in vitro effects of calcium crystals [1]. Nevertheless, no correlation was found between FA levels and inflammatory cytokines in the SF of the three groups. This suggests that FA levels in OA might be a marker of calcium deposition more than inflammation. In conclusion, to the best of our knowledge, this is the first study that has attempted to analyse biomarkers in the SF of patients with different types of calcium crystals. Interestingly, we found increased levels of FA in patients with calcium crystals and IL-6 in the BCP group. The differences between the groups suggest that scientists should pay attention to the presence of calcium crystals for the identification of OA biomarkers in SF. Further research is needed to understand the role of FA in calcium crystal formation in OA in order to develop a suitable treatment to inhibit crystals formation. Funding: This work was supported by an institutional research grant (DOR1784408/17) from the University of Padova. Disclosure statement: The authors have declared no conflicts of interest. E.B., M.F., F.O. and L. Punzi designed the study. E.B., F.O., P.F., L. Peruzzo and L.T. performed the experimental work. E.B., M.F. and F.O. analysed the data. E.B., F.O. and M.F. wrote the manuscript. All authors read and approved the final manuscript.