Osteogenic Potential of Monosodium Urate Crystals in Synovial Mesenchymal Stem Cells

Abstract

Background and Objectives: Deposits of monosodium urate (MSU) crystals due to increased levels of uric acid (UA) have been associated with bone formation and erosion, mainly in patients with chronic gout. The synovial membrane (SM) comprises several types of cells, including mesenchymal stem cells (SM-MSCs); however, it is unknown whether UA and MSU induce osteogenesis through SM-MSCs. Materials and Methods: Cultures of SM were immunotyped with CD44, CD69, CD90, CD166, CD105, CD34, and CD45 to identify MSCs. CD90+ cells were isolated by immunomagnetic separation (MACS), colony-forming units (CFU) were identified, and the cells were exposed to UA (3, 6.8, and 9 mg/dL) and MSU crystals (1, 5, and 10 μg/mL) for 3 weeks, and cellular morphological changes were evaluated. IL-1β and IL-6 were determined by ELISA, mineralization was assessed by alizarin red, and the expression of Runx2 was assessed by Western blot. Results: Cells derived from SM and after immunomagnetic separation were positive for CD90 (53 ± 8%) and CD105 (52 ± 18%) antigens, with 53 ± 5 CFU identified. Long-term exposure to SM-MSCs by UA and MSU crystals did not cause morphological damage or affect cell viability, nor were indicators of inflammation detected. Mineralization was observed at doses of 6.8 mg/dL UA and 5 μg/mL MSU crystals; however, the differences were not significant with respect to the control. The highest dose of MSU crystals (10 μg/mL) induced significant Runx2 expression with respect to the control (1.4 times greater) and SM-MSCs cultured in the osteogenic medium. Conclusions: MSU crystals may modulate osteogenic differentiation of SM-MSCs through an increase in Runx2.

Keywords: gout; mesenchymal stem cells; monosodium urate crystals; osteodifferentiation; synovial membrane.

Figure 1

Immunophenotyping of SM-derived cells. SM-derived cells showed abundant CD90, CD105, and CD73 (mesenchymal) expression and lower CD117, CD34, CD45 (hematopoietic), CD271 (neural), and CD31 (endothelial) expression. Values represent the mean ± standard deviation of independent experiments (n = 6).

Figure 2

Morphology of SM-MSCs at different concentrations of UA and MSU crystals. Bright-field microscopy of SM-MSCs cultured with UA and MSU crystals. (A) Control, (B) 3 mg/dL UA, (C) 6.8 mg/dL UA, (D) 9 mg/dL UA, (E) 1 µg/mL MSU crystals, (F) 5 µg/mL MSU, (G) 10 µg/mL MSU, and (H) osteogenic medium (20×). Representative images of independent experiments (n = 6). (I) Cell viability increased in cultures treated with 3 and 6.8 mg/dL UA and in cells cultured in osteogenic medium. The data represent the mean ± SD of independent experiments (* p < 0.05 vs. control; ** p < 0.001 vs. control).

Figure 3

Effect of UA and MSU crystals on the formation of calcium nodules. (A) Osteogenic induction control, (B) 3 mg/dL UA, (C) 6.8 mg/dL UA, (D) 9 mg/dL UA, (E) 1 µg/mL MSU crystals, (F) 5 µg/mL MSU crystals, (G) 10 µg/mL MSU, and (H) control. Bright-field microscopy; cell culture with red-stained calcium nodules (20×), representative of independent experiments (n = 6). The formation of calcium nodules was observed to be high in the positive control (osteogenic medium); in the cells cultured in media containing UA and MSU crystals, the effect was null, as was that in the control. (I) Alizarin red staining was quantified by removing the dye with isopropanol. The data represent the mean ± SD of independent experiments (**** p < 0.00001 vs. control +).

Figure 4

Effect of UA and MSU crystals on IL-6 production by SM-MSCs. Quantification of IL-6 in SM-MSCs treated with different stimuli, i.e., UA (3, 6.8, and 9 mg/dL), MSU crystals (1, 5, and 10 µg/mL), osteogenic medium (control +), and control. The data represent the mean ± SD of independent experiments (n = 6) (* p < 0.05 vs. control; ** p < 0.001 vs. control).

Figure 5

Effect of UA and MSU crystals on Runx2 expression. (A) Blot representative of the levels of Runx2 protein expression in relation to actin (internal control). (B) Graph of the densitometric analysis of Runx2 expression in the control group and treatment groups. The values in the columns are the average ± SD of at least 3 independent experiments; Dunnett’s test, ** p < 0.01.