Tophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts. Conditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay. In MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs. Factors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

中文翻译：

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尿酸单钠晶体刺激巨噬细胞分泌的因子促进成骨细胞的促炎状态：痛风骨侵蚀的潜在间接机制

痛风石是常见于痛风影响关节骨侵蚀部位的病变。痛风石包含由含有巨噬细胞和其他免疫细胞的软组织包围的尿酸单钠 (MSU) 晶体核心。先前的研究发现，MSU 晶体直接降低了成骨细胞的活力和功能。本研究的目的是确定 MSU 晶体对成骨细胞的间接、巨噬细胞介导的影响。将用 MSU 晶体培养的 RAW264.7 小鼠巨噬细胞系的条件培养基添加到 MC3T3-E1 小鼠成骨细胞系中。将来自用 MSU 晶体培养的 THP-1 人单核细胞系的条件培养基添加到原代人成骨细胞 (HOB) 中。通过 von Kossa 染色评估基质矿化。基因表达通过实时PCR确定，通过酶联免疫吸附试验测定分泌因子的浓度。在成骨培养基中培养 13 天的 MC3T3-E1 细胞中，成骨细胞标记基因 Col1a1、Runx2、Sp7、Bglap、Ibsp 和 Dmp1 的表达被来自 MSU 晶体刺激的 RAW264.7 巨噬细胞的条件培养基抑制。第 21 天 MC3T3-E1 培养物的矿物染色证实了成骨细胞分化的抑制。在 HOB 培养物中，用来自 MSU 晶体刺激的 THP-1 单核细胞的条件培养基孵育 20 小时对成骨细胞基因表达的影响不太一致。在与来自 MSU 晶体刺激的巨噬细胞/单核细胞的条件培养基一起孵育的 MC3T3-E1 和 HOB 中诱导了编码环氧合酶 2 和 IL-6 的基因的表达以及促炎介质 PGE2 和 IL-6 的分泌。然而，抑制 HOBs 中 cyclooxygenase-2 活性和 PGE2 分泌表明该途径在介导 HOBs 中 MSU 晶体的间接影响中不起主要作用。由 MSU 晶体刺激的巨噬细胞分泌的因子会减弱成骨细胞的分化并诱导成骨细胞中促炎介质的表达和分泌。我们认为受痛风影响的关节中的骨侵蚀是由 MSU 晶体的直接和间接影响共同作用的结果。