Structure-Dependent Effects of Bisphosphonates on Inflammatory Responses in Cultured Neonatal Mouse Calvaria

Bisphosphonates (BPs) are classified into two groups, according to their side chain structures, as nitrogen-containing BPs (NBPs) and non-nitrogen-containing BPs (non-NBPs). In this study, we examined the effects of NBPs and non-NBPs on inflammatory responses, by quantifying the inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), in cultured neonatal mouse calvaria. All examined NBPs (pamidronate, alendronate, incadronate, risedronate, zoledronate) stimulated lipopolysaccharide (LPS)-induced PGE2 and NO production by upregulating COX-2 and iNOS mRNA expression, whereas non-NBPs (etidronate, clodronate, tiludronate) suppressed PGE2 and NO production, by downregulating gene expression. Additionally, [4-(methylthio) phenylthio] methane bisphosphonate (MPMBP), a novel non-NBP with an antioxidant methylthio phenylthio group in its side chain, exhibited the most potent anti-inflammatory activity among non-NBPs. Furthermore, results of immunohistochemistry showed that the nuclear translocation of NF-κB/p65 and tyrosine nitration of cytoplasmic protein were stimulated by zoledronate, while MPMBP inhibited these phenomena, by acting as a superoxide anion (O2−) scavenger. These findings indicate that MPMBP can act as an efficacious agent that causes fewer adverse effects in patients with inflammatory bone diseases, including periodontitis and rheumatoid arthritis.

pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis

We examined the effects of HCO3 − and CO2 acidosis on osteoclast-mediated Ca2+ release from 3-day cultures of neonatal mouse calvaria. Ca2+ release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H+ to culture medium (HCO3 − acidosis). For example, addition of 4 meq/l H+ reduced pH from 7.12 to 7.03 and increased Ca2+ release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15–16 meq/l added H+, were comparable to the maximal Ca2+ release elicited by 1,25-dihydroxyvitamin D3[1,25(OH)2D3; 10 nM], parathyroid hormone (10 nM), or prostaglandin E2 (1 μM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from ∼7.1 to ∼7.3. CO2 acidosis was a less effective stimulator of Ca2+ release than HCO3 −acidosis over a similar pH range. Ca2+ release stimulated by HCO3 − acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, ∼400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO3 − acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO3 − acidosis, however, reduced numbers of osteoclast-like cells by ∼50%, whereas 1,25(OH)2D3 treatment caused increases of ∼75%. The results suggest that HCO3 − acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.