Background/Purpose:  Imaging
and histology studies have identified monosodium urate (MSU) crystals present
in subchondral bone in erosive gout, suggesting that
these crystals could interact with osteocytes.  Osteocytes are the most abundant cell
population within bone (>95% of the cellular component of the adult
skeleton) and play a central role in regulating bone remodeling.  The aim of
this study was to investigate the effects of MSU crystals on osteocyte
viability.

Methods: MSU crystals were prepared by
recrystallization of uric acid.  The
murine immortalized osteocyte MLO-Y4 cell line was used in these assays; cells
were cultured on plastic (2D) or in 3mg/mL type I collagen gels (3D).  MSU crystals or soluble urate
(0.01-0.5mg/mL) were added to the cells.  After 24 hours, cells were washed and MSU
crystals or urate completely removed.  Cell viability was assessed using MTT and
alamarBlue assays.  Viability was
assessed in the 2D cultures 24 hours after addition of MSU crystals, and both
24 and 48 hours after addition of MSU crystals in the 3D cultures.  Different sizes of MSU crystals were prepared
by sonication.  Basic calcium
phosphate, calcium pyrophosphate and aluminum crystals were tested using the
same methods in the 3D cultures.

Results: In the 2D cultures at the 24 hour time point,
0.1-0.5mg/mL MSU crystals reduced MLO-Y4 cell viability by ~70% (P<0.001 vs.
no MSU).  In the 3D collagen gel
cultures at the 24 hour time point, 0.3-0.5mg/mL (but not lower concentrations)
MSU crystals reduced MLO-Y4 cell viability by ~30-40% (P<0.001 vs. no MSU) (Figure).
 In the 3D collagen gel cultures at the
48 hour time point, 0.1mg/mL MSU crystals reduced MLO-Y4 viability (P<0.01
vs. no MSU), and culture with the higher MSU crystal concentrations
(0.3-0.5mg/mL) resulted in further reduction of MLO-Y4 cell viability by
~60-70% (P<0.001 vs. no MSU) (Figure).  Different sizes of MSU crystals (5.6-16.2mm) reduced MLO-Y4 cell viability in a similar
manner.  The effect on cell
viability was specific to MSU crystals, as soluble urate and other types of
crystals did not reduce MLO-Y4 cell viability.

Conclusion: MSU crystals inhibit osteocyte viability.  Direct crystal-cell contact is not
necessarily required to induce this effect.  These data suggest that osteocytes may
play a role in bone erosion in gout.

Figure: MSU
crystals reduce the viability of MLO-Y4 osteocyte-like cells in 3D cultures.  Viability
was assessed using the alamarBlue assay 24 and 48 hours after the addition of
MSU crystals.  Data are pooled from
three biological repeats and are presented as mean (SEM); two-way ANOVA (P<0.0001)
with post hoc Dunnett’s test
*p<0.01, **p<0.001 versus control (no MSU crystals) for the relevant time
point.