Orriss Isabel R, Key Michelle L, Colston Kay W, Arnett Timothy R
Department of Cell and Developmental Biology, University College London, London, UK.
J Cell Biochem. 2009 Jan 1;106(1):109-18. doi: 10.1002/jcb.21983.
Bisphosphonates are analogues of pyrophosphate, a key physicochemical inhibitor of mineralisation. We examined the direct actions of bisphosphonates on the function of cultured osteoblasts derived from rat calvariae. Treatment with zoledronate, the most potent bisphosphonate studied, reduced osteoblast number at concentrations > or = 100 nM and was strongly toxic at 10 microM, causing a threefold decrease in osteoblast viability after 2 days and a 90% decrease in cell numbers after 14 days. In control osteoblast cultures on plastic, abundant formation of 'trabecular' mineralised bone matrix nodules began after 10 days. Continuous exposure to zoledronate inhibited bone mineralisation at concentrations as low as 10 nM. Pamidronate and clodronate exerted similar effects but at higher doses > or = 1 and > or = 10 microM, respectively). Short-term or intermittent exposure of osteoblasts to zoledronate and pamidronate (1-10 microM) was sufficient to inhibit bone mineralisation by > or = 85%. Zoledronate but not pamidronate or clodronate also strongly inhibited osteoblast alkaline phosphatase activity at concentrations > or = 100 nM and soluble collagen production at concentrations > or = 1 microM. We additionally studied the effects of zoledronate on osteoblasts cultured on dentine, a bone-like mineralised substrate, observing similar inhibitory effects, although at concentrations 10-100-fold higher; this shift presumably reflected adsorption of zoledronate to dentine mineral. Thus, zoledronate blocked bone formation in two ways: first, a relatively non-toxic, selective inhibition of mineralisation at concentrations in the low nanomolar range and second, a cytotoxic inhibition of osteoblast growth and function at concentrations > or = 1 microM. Although no data are available on the bisphosphonate concentrations that osteoblasts could be exposed to in vivo, our results are consistent with earlier observations that bisphosphonates may inhibit bone formation.
双膦酸盐是焦磷酸盐的类似物，焦磷酸盐是矿化作用的一种关键物理化学抑制剂。我们研究了双膦酸盐对源自大鼠颅骨的培养成骨细胞功能的直接作用。用唑来膦酸（研究的最有效的双膦酸盐）处理，在浓度≥100 nM时会减少成骨细胞数量，在10 μM时具有强烈毒性，导致2天后成骨细胞活力降低三倍，14天后细胞数量减少90%。在塑料上培养的对照成骨细胞中，10天后开始大量形成“小梁”矿化骨基质结节。持续暴露于唑来膦酸会在低至10 nM的浓度下抑制骨矿化。帕米膦酸和氯膦酸也有类似作用，但分别在更高剂量（≥1 μM和≥10 μM）时出现。成骨细胞短期或间歇性暴露于唑来膦酸和帕米膦酸（1 - 10 μM）足以抑制骨矿化≥85%。唑来膦酸但不是帕米膦酸或氯膦酸在浓度≥100 nM时也强烈抑制成骨细胞碱性磷酸酶活性，在浓度≥1 μM时抑制可溶性胶原蛋白的产生。我们还研究了唑来膦酸对在牙本质（一种骨样矿化基质）上培养的成骨细胞的影响，观察到类似的抑制作用，尽管浓度要高10 - 100倍；这种变化可能反映了唑来膦酸吸附到牙本质矿物质上。因此，唑来膦酸通过两种方式阻止骨形成：第一，在低纳摩尔范围内的浓度下对矿化进行相对无毒的选择性抑制；第二，在浓度≥1 μM时对成骨细胞生长和功能进行细胞毒性抑制。尽管没有关于成骨细胞在体内可能接触到的双膦酸盐浓度的数据，但我们的结果与早期观察结果一致，即双膦酸盐可能抑制骨形成。
