Background/Purpose:   Bone erosion in gout is strongly associated with tophi; lesions comprising of inflammatory cells surrounding collections of monosodium urate (MSU) crystals.  Osteocytes are the most abundant cell population within bone and are important regulators of bone remodeling.  The aim of this study was to investigate the direct effects of MSU crystals and indirect effects of MSU crystal-induced inflammation on osteocyte function.

Methods:   For direct in vitro assays, MSU crystals were added to MLO-Y4 osteocyte-like cells cultured in type I collagen gels (3D) for 1h, 6h and 24h.  For indirect in vitro assays, RAW264.7 macrophage-like cells were cultured with or without MSU crystals (0.5mg/mL) for 24h and supernatants were harvested and filtered for conditioned media preparation.  The conditioned media (40%) was then added to the 3D MLO-Y4 cultures for 1h, 6h and 24h. All changes in MLO-Y4 gene expression were examined using real-time PCR and analyzed using two-way ANOVA with post hoc Sidak’s tests.  The relationship between osteocytes, MSU crystals and macrophages in erosive gout was examined by polarizing light microscopy and CD68 immunohistochemistry in joint samples obtained from cadaveric donors with crystal-proven gout.

Results:   In the direct in vitro assays, culture with MSU crystals alone did not alter the expression of osteocyte-related genes or inflammatory mediator gene expression (P>0.1 for all).  In contrast, addition of conditioned media from MSU crystal-exposed RAW264.7 cells to MLO-Y4 cultures led to a ~2-4-fold increase in expression of E11 and connexin43 at the 6h and 24h time-points compared to control conditioned media (P<0.0001).  RANKL expression was also increased at 6h and 24h (P<0001, Figure) and OPG expression was reduced at the 6h time-point by ~2-fold (P=0.007).  In addition, expression of inflammatory genes was upregulated, including TNF-a (~4-fold at 1h), IL-1b (~5-fold at 6h and 24h), IL-8 (>500-fold at all time-points), IL-11 (>200-fold at 6h and 24h), IL-6 and cyclooxygenase-2 (Figure) (P≤0.001 for all).  In histological analysis of cadaveric joint samples affected by gout, numerous CD68+ macrophages and MSU crystals were identified in proximity to osteocytes within bone.            

Conclusion: MSU crystals do not directly influence osteocyte-related gene expression.  However, interactions between MSU crystals and immune cells in the joint may promote osteocyte expression of pro-resorptive and inflammatory mediators, which can have important consequences for local bone remodeling.  These indirect effects on osteocyte function may contribute to bone erosion in gout.

Figure: Mean (SEM) changes in relative expression levels of RANKL, COX-2 and IL-6 in MLO-Y4 cells cultured with 40% conditioned media from MSU crystal-exposed RAW264.7 cells or control RAW264.7 cells (no MSU). Post hoc Sidak’s test, *p<0.05, **p<0.01 and ***p<0.001 vs control conditioned media.