Effect of particle size on hydroxyapatite crystal-induced tumor necrosis factor alpha secretion by macrophages

Macrophages may promote a vicious cycle of inflammation and calcification in the vessel wall by ingesting neointimal calcific deposits (predominantly hydroxyapatite) and secreting tumor necrosis factor (TNF)alpha, itself a vascular calcifying agent. Here we have investigated whether particle size affects the proinflammatory potential of hydroxyapatite crystals in vitro and whether the nuclear factor (NF)-kappa B pathway plays a role in the macrophage TNF alpha response. The particle size and nano-topography of nine different crystal preparations was analyzed by X-ray diffraction, Raman spectroscopy, scanning electron microscopy and gas sorbtion analysis. Macrophage TNF alpha secretion was inversely related to hydroxyapatite particle size (P = 0.011, Spearman rank correlation test) and surface pore size (P = 0.014). A necessary role for the NF-kappa B pathway was demonstrated by time-dependent I kappa B alpha degradation and sensitivity to inhibitors of I kappa B alpha degradation. To test whether smaller particles were intrinsically more bioactive, their mitogenic activity on fibroblast proliferation was examined. This showed close correlation between TNF alpha secretion and crystal-induced fibroblast proliferation (P = 0.007). In conclusion, the ability of hydroxyapatite crystals to stimulate macrophage TNF alpha secretion depends on NF-kappa B activation and is inversely related to particle and pore size, with crystals of 1-2 mu m diameter and pore size of 10-50 angstrom the most bioactive. Microscopic calcitic deposits in early stages of atherosclerosis may therefore pose a greater inflammatory risk to the plaque than macroscopically or radiologically visible deposits in more advanced lesions. (C) 2007 Elsevier Ireland Ltd. All rights reserved.