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)α, 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)-κB pathway plays a role in the macrophage TNFα 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 TNFα 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-κB pathway was demonstrated by time-dependent IκBα degradation and sensitivity to inhibitors of IκBα degradation. To test whether smaller particles were intrinsically more bioactive, their mitogenic activity on fibroblast proliferation was examined. This showed close correlation between TNFα secretion and crystal-induced fibroblast proliferation (P = 0.007). In conclusion, the ability of hydroxyapatite crystals to stimulate macrophage TNFα secretion depends on NF-κB activation and is inversely related to particle and pore size, with crystals of 1–2 μm diameter and pore size of 10–50 Å the most bioactive. Microscopic calcific 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.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Carlstrom D.
        • Engfeldt B.
        • Engstrom A.
        • Ringertz N.
        Studies on the chemical composition of normal and abnormal blood vessel walls. I. Chemical nature of vascular calcified deposits.
        Lab Invest. 1953; 2: 325-335
        • Schmid K.
        • McSharry W.O.
        • Pameijer C.H.
        • Binette J.P.
        Chemical and physicochemical studies on the mineral deposits of the human atherosclerotic aorta.
        Atherosclerosis. 1980; 37: 199-210
        • Fitzpatrick L.A.
        • Severson A.
        • Edwards W.D.
        • Ingram R.T.
        Diffuse calcification in human coronary arteries: association of osteopontin with atherosclerosis.
        J Clin Invest. 1994; 94: 1597-1604
        • Guo W.
        • Morrisett J.D.
        • DeBakey M.E.
        • Lawrie G.M.
        • Hamilton J.A.
        Quantification in situ of crystalline cholesterol and calcium phosphate hydroxyapatite in human atherosclerotic plaques by solid-state magic angle spinning NMR.
        Arterioscler Thromb Vasc Biol. 2000; 20: 1630-1636
        • Jin H.
        • Ham K.
        • Chan J.Y.
        • et al.
        High resolution three-dimensional visualization and characterization of coronary atherosclerosis in vitro by synchrotron radiation X-ray microtomography and highly localized X-ray diffraction.
        Phys Med Biol. 2002; 47: 4345-4356
        • Nadra I.
        • Mason J.C.
        • Philippidis P.
        • et al.
        Proinflammatory activation of macrophages by basic calcium phosphate crystals via protein kinase C and MAP kinase pathways: a vicious cycle of inflammation and arterial calcification?.
        Circ Res. 2005; 96: 1248-1256
        • Tintut Y.
        • Patel J.
        • Parhami F.
        • Demer L.L.
        Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway.
        Circulation. 2000; 102: 2636-2642
        • Wexler L.
        • Brundage B.
        • Crouse J.
        • et al.
        Coronary artery calcification: pathophysiology, epidemiology, imaging methods, and clinical implications. A statement for health professionals from the American Heart Association. Writing Group.
        Circulation. 1996; 94: 1175-1192
        • Weiss R.M.
        Another calcium paradox?.
        Arterioscler Thromb Vasc Biol. 2001; 21: 1561-1562
        • Shaw L.J.
        • Raggi P.
        • Schisterman E.
        • Berman D.S.
        • Callister T.Q.
        Prognostic value of cardiac risk factors and coronary artery calcium screening for all-cause mortality.
        Radiology. 2003; 228: 826-833
        • Thompson G.R.
        • Partridge J.
        Coronary calcification score: the coronary-risk impact factor.
        Lancet. 2004; 363: 557-559
        • Virmani R.
        • Burke A.P.
        • Kolodgie F.D.
        • Farb A.
        Pathology of the thin-cap fibroatheroma: a type of vulnerable plaque.
        J Interv Cardiol. 2003; 16: 267-272
        • Virmani R.
        • Burke A.P.
        • Farb A.
        • Kolodgie F.D.
        Pathology of the vulnerable plaque.
        J Am Coll Cardiol. 2006; 47: C13-C18
        • Fujii K.
        • Carlier S.G.
        • Mintz G.S.
        • et al.
        Intravascular ultrasound study of patterns of calcium in ruptured coronary plaques.
        Am J Cardiol. 2005; 96: 352-357
        • Cheung H.S.
        • Devine T.R.
        • Hubbard W.
        Calcium phosphate particle induction of metalloproteinase and mitogenesis: effect of particle sizes.
        Osteoarth Cartilage. 1997; 5: 145-151
        • McCarthy G.M.
        • Augustine J.A.
        • Baldwin A.S.
        • et al.
        Molecular mechanism of basic calcium phosphate crystal-induced activation of human fibroblasts. Role of nuclear factor kappab, activator protein 1, and protein kinase c.
        J Biol Chem. 1998; 273: 35161-35169
        • Reuben P.M.
        • Sun Y.
        • Cheung H.S.
        Basic calcium phosphate crystals activate p44/42 MAPK signal transduction pathway via protein kinase Cmicro in human fibroblasts.
        J Biol Chem. 2004; 279: 35719-35725
        • Grandjean-Laquerriere A.
        • Laquerriere P.
        • Guenounou M.
        • Laurent-Maquin D.
        • Phillips T.M.
        Importance of the surface area ratio on cytokines production by human monocytes in vitro induced by various hydroxyapatite particles.
        Biomaterials. 2005; 26: 2361-2369
        • Grandjean-Laquerriere A.
        • Tabary O.
        • Jacquot J.
        • et al.
        Involvement of toll-like receptor 4 in the inflammatory reaction induced by hydroxyapatite particles.
        Biomaterials. 2007; 28: 400-404
        • Bett J.A.S.
        • Christner L.G.
        • Hall W.K.
        Studies of the hydrogen held by solid XII hydroxyapatite catalysts.
        J Am Chem Soc. 1967; 89: 5535-5541
        • Landis R.C.
        • Yagnik D.R.
        • Florey O.
        • et al.
        Safe disposal of inflammatory monosodium urate monohydrate crystals by differentiated macrophages.
        Arth Rheum. 2002; 46: 3026-3033
        • McCarthy G.M.
        • Mitchell P.G.
        • Cheung H.S.
        The mitogenic response to stimulation with basic calcium phosphate crystals is accompanied by induction and secretion of collagenase in human fibroblasts.
        Arth Rheum. 1991; 34: 1021-1030
        • Gonzalez O.
        • Smith R.L.
        • Goodman S.B.
        Effect of size, concentration, surface area, and volume of polymethylmethacrylate particles on human macrophages in vitro.
        J Biomed Mater Res. 1996; 30: 463-473
        • Habibovic P.
        • Yuan H.
        • van d V.
        • Meijer G.
        • van Blitterswijk C.A.
        • de G.K.
        3D microenvironment as essential element for osteoinduction by biomaterials.
        Biomaterials. 2005; 26: 3565-3575
        • Huang J.
        • Best S.M.
        • Bonfield W.
        • et al.
        In vitro assessment of the biological response to nano-sized hydroxyapatite.
        J Mater Sci Mater Med. 2004; 15: 441-445.
        • Tintut Y.
        • Patel J.
        • Territo M.
        • Saini T.
        • Parhami F.
        • Demer L.L.
        Monocyte/macrophage regulation of vascular calcification in vitro.
        Circulation. 2002; 105: 650-655
        • Shioi A.
        • Katagi M.
        • Okuno Y.
        • et al.
        Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages.
        Circ Res. 2002; 91: 9-16
        • Jeziorska M.
        • McCollum C.
        • Woolley D.E.
        Calcification in atherosclerotic plaque of human carotid arteries: associations with mast cells and macrophages.
        J Pathol. 1998; 185: 10-17
        • Kolodgie F.D.
        • Burke A.P.
        • Farb A.
        • et al.
        The thin-cap fibroatheroma: a type of vulnerable plaque: the major precursor lesion to acute coronary syndromes.
        Curr Opin Cardiol. 2001; 16: 285-292
        • Lin T.C.
        • Tintut Y.
        • Lyman A.
        • Mack W.
        • Demer L.L.
        • Hsiai T.K.
        Mechanical response of a calcified plaque model to fluid shear force.
        Ann Biomed Eng. 2006; 34: 1535-1541
        • Tearney G.J.
        • Yabushita H.
        • Houser S.L.
        • et al.
        Quantification of macrophage content in atherosclerotic plaques by optical coherence tomography.
        Circulation. 2003; 107: 113-119
        • Jang I.K.
        • Tearney G.J.
        • MacNeill B.
        • et al.
        In vivo characterization of coronary atherosclerotic plaque by use of optical coherence tomography.
        Circulation. 2005; 111: 1551-1555
        • van der Meer F.J.
        • Faber D.J.
        • Baraznji Sassoon D.M.
        • Aalders M.C.
        • Pasterkamp G.
        • van Leeuwen T.G.
        Localized measurement of optical attenuation coefficients of atherosclerotic plaque constituents by quantitative optical coherence tomography.
        IEEE Trans Med Imaging. 2005; 24: 1369-1376