Treatment with allopurinol and oxypurinol promotes osteoblast differentiation and increases bone formation

Allopurinol and, and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They act by inhibiting xanthine oxidase an enzyme in the purine degradation pathway that converts xanthine to uric acid. This oxygen-dependent reaction also results in the generation of superoxide free radicals. The aim of this investigation was to examine the effect of allopurinol and oxypurinol on osteoblast differentiation and function. Rat calvarial osteoblasts were isolated by trypsin/collagenase digestion and cultured for up to 14 days with 2mM β-glycerophosphate, 50 μg/ml ascorbate and 10 nM dexamethasone. The effect of allopurinol and oxypurinol on bone formation, cell number, cell viability, gene expression and enzyme activity was investigated in differentiating (day 7) and/or mature bone-forming osteoblasts (day 14). Xanthine oxidase expression and activity were detected in both differentiating and mature cells. Although mRNA expression remained relatively constant, xanthine oxidase activity decreased over time with cultures of mature osteoblasts displaying reduced levels of uric acid (20% decrease, P<0.001). Treatment with both allopurinol and oxypurinol (0.1–1 μM) reduced xanthine oxidase activity by up to 30% (P<0.001) in differentiating and mature osteoblasts. At these concentrations, allopurinol and oxypurinol increased bone formation ~fold and ~threefold respectively (P<0.001). Cell number and viability were unaffected. Both drugs increased alkaline phosphatase (TNAP) activity up to 50% in differentiating osteoblasts (P<0.001) and 65% (P<0.01) in mature osteoblasts. The expression of key osteoblast marker genes was investigated using qPCR. Osteocalcin and TNAP mRNA expression was increased, fivefold and twofold respectively. In contrast expression of NPP1, the enzyme responsible for the generation of the mineralisation inhibitor, pyrophosphate, was decreased fivefold. Col1α1 mRNA expression was unaffected. Our data suggest that inhibition of xanthine oxidase activity promotes osteoblast differentiation, leading to increased bone formation.