Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro.

BACKGROUND: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. METHODS: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). RESULTS AND CONCLUSION: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.

Calcium phosphate apatites with variable Ca/P atomic ratio II. Calcination and sintering.

The calcination and natural sintering of calcium deficient hydroxyapatite powders Ca(10-x)(PO4)(6-x)(HPO4)x(OH)(2-x) (with 0 < or = x < or = 1) were studied. For temperature below 700 degrees C, particle coalescence occurs without densification. The particle coalescence is associated with a reduction of the specific surface area. This surface decreases all the more as the Ca/P molar ratio of the powder is small. The mechanism agrees with a transfer of matter occurring by superficial diffusion, which is enhanced by the augmentation of vacancies in the apatitic structure (i.e. by a decrease of the Ca/P ratio). The sintering of compacted powders begins at 700 degrees C. At the same temperature, the calcium deficient hydroxyapatites dissociate into biphasic mixtures of hydroxyapatite and tricalcium phosphate. The sintering is slowed down when the content of TCP in the biphasic mixture increases. In parallel, the grain size increases. This result relates to the augmentation of the coalescence of particles at low temperature.

Calcium phosphate apatites with variable Ca/P atomic ratio I. Synthesis, characterisation and thermal stability of powders.

Single phased apatitic calcium phosphate powders Ca(10-x)(PO4)(6-x)(HPO4)x(OH)(2-x) with Ca/P molar ratio ranging from 1.5 to 1.667 (0 < or = x < or = 1) were synthesised using wet method. Outside this compositional range the powders were biphasic mixtures composed of a calcium phosphate apatite and CaHPO4 (Ca/P < 1.5) or Ca(OH)2 (Ca/P > 1.667). Temperature and pH of synthesis were the preponderant parameters for the control of the precipitate composition. The precise determination of the chemical composition requires the use of several complementary techniques and thermal treatments of powders. These techniques include high resolution and high temperature X-ray diffractometry and FTIR spectroscopy and show that very small variations of the Ca/P molar ratio of the powder lead to great changes in powder composition and characteristics after thermal treatment.

Calcium phosphate apatites with variable Ca/P atomic ratio III. Mechanical properties and degradation in solution of hot pressed ceramics.

Calcium deficient hydroxyapatite powders Ca(10-x)(PO4)(6-x)(HPO4)x(OH)(2-x) (0 < or = x < or = 1) were hot pressed to produce dense hydroxyapatite-tricalcium phosphate (HAP/TCP) biphasic materials. Ceramics hot pressed at 1000 degrees C were composed of an homogeneous distribution of the HAP and beta-TCP grains with an average size of 0.2 microm. Grain growth was observed for TCP loading > 70 wt%. The strength exhibited a maximum of sigma(f) = 150 MPa for 90/10 (w/w) HAP/TCP and it dropped for smaller and greater amounts of TCP. This value was twice that of pure HAP. When placed in Ringer's solution, only the surface of biphasic compounds was degraded after 60 days of immersion with a preferential etching of the TCP phase. After hot pressing at 1200 degrees C, grain growth was observed and the mechanical properties were decreased. This was explained by the allotropic transformation alpha/beta of TCP.

About the use of stoichiometric hydroxyapatite in compression - incidence of manufacturing process on compressibility.

Literature concerning calcium phosphates in pharmacy exhibits the chemical diversity of the compounds available. Some excipient manufacturers offer hydroxyapatite as a direct compression excipient, but the chemical analysis of this compound usually shows a variability of the composition: the so-called materials can be hydroxyapatite or other calcium phosphates, uncalcined (i.e. with a low crystallinity) or calcined and well-crystallized hydroxyapatite. This study points out the incidence of the crystallinity of one compound (i.e. hydroxyapatite) on the mechanical properties. Stoichiometric hydroxyapatite is synthesized and compounds differing in their crystallinity, manufacturing process and particle size are manufactured. X-Ray diffraction analysis is used to investigate the chemical nature of the compounds. The mechanical study (study of the compression, diametral compressive strength, Heckel plots) highlights the negative effect of calcination on the mechanical properties. Porosity and specific surface area measurements show the effect of calcination on compaction. Uncalcined materials show bulk and mechanical properties in accordance with their use as direct compression excipients.

Effects of alumina and zirconium dioxide particles on arachidonic acid metabolism and proinflammatory interleukin production in osteoarthritis and rheumatoid synovial cells.

We describe a model which can be used for in vitro biocompatibility assays of biomaterials. We studied the in vitro response of human osteoarthritis or rheumatoid arthritis fibroblast-like synoviocytes to Al2O3 or ZrO2 particles by analysing the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) and the metabolism of arachidonic acid via lipoxygenase and cyclo-oxygenase pathways. Our results show that, in these cells and under our experimental conditions, Al2O3 and ZrO2 did not significantly modify the synthesis of IL-1 and IL-6 or the metabolism of arachidonic acid.

Toughening characterization in alumina platelet-hydroxyapatite matrix composites.

Fracture toughness of Al2O3 platelet-reinforced hydroxyapatite (HAP) ceramics was investigated using the Vickers' indentation technique. The geometrical anisotropy of alumina platelets induces an anisotropic toughening. The efficiency of reinforcing mechanisms remains maximum for a crack propagating with an angular deviation inferior to 30 degrees around the direction perpendicular to alumina disc faces. This is assumed to result from a crack deflection mechanism which induces a favorable contribution of mode II failure. A small effect of hydroxyapatite grain size becomes noticeable in the direction parallel to alumina disc faces. The toughening depends on the size and volume content of alumina platelets. Large size platelets provoke a spontaneous microcracking of the HAP matrix which is detrimental to the mechanical reliability, whereas small platelets lead to a strong toughening. The results relate to the intensity of thermoelastic residual stresses within the matrix around alumina inclusions.

Interactions of hydroxyapatite and fluorapatite particles on human osteoarthritis type B synoviocytes: effects on interleukin-1 alpha levels and lipoxygenase pathways.

We investigated the effects of the biomaterials hydroxyapatite (HAP) and fluorapatite (FAP) on cultured human osteoarthritis type B synoviocytes by analyzing interleukin-1 alpha (IL-1 alpha) production and arachidonic acid metabolism via lipoxygenase pathways. A portion of opsonized particles was endocytosed and was found in numerous phagolysosomes in human synoviocyte cytoplasms. The present study demonstrates that HAP and FAP calcined at 700 degrees C induced a decrease in IL-1 alpha production but markedly decreased the synthesis of lipoxygenase products after 1-month incubation with the particles. This model will allow us to study the possible inflammatory response (arachidonic acid metabolism, proinflammatory cytokines) that can be induced by any biomaterials used in orthopedics.

Osteoclast adhesion and activity on synthetic hydroxyapatite, carbonated hydroxyapatite, and natural calcium carbonate: relationship to surface energies.

This study investigates the adhesion, cytoskeletal changes, and resorptive activity of disaggregated rat osteoclasts cultured on polished slices of three biomaterials: crystalline synthetic hydroxyapatite (HA), carbonated hydroxyapatite (C-HA), and natural calcium carbonate (C). The surface chemistry of each substrate was defined by X-ray diffraction and IR spectroscopy, surface wettability by the dispersive, and the polar components of the surface energies. Osteoclast adhesion was modulated by the polar component of the surface energy: fewer (p < 0.01) osteoclasts adhered to C-HA (97 +/- 20/slice, surface energy 9 +/- 5 mJ/m2) than to HA (234 +/- 16/slice, surface energy 44 +/- 2 mJ/m2) or to C (268 +/- 37/slice, surface energy 58 +/- 0.5 mJ/m2). Actin rings, which are the cytoskeletal structure essential for resorption, developed on all three materials. The area of the actin ring, which is resorbed by local acidification, and the osteoclast area, which reflects osteoclast spreading, were both greater in osteoclasts cultured on HA and C-HA than in those cultured on C. C was resorbed, but HA and C-HA were not. Thus, the surface energy plays an essential role in osteoclast adhesion, whereas osteoclast spreading may depend on the surface chemistry, especially on protein adsorption and/or on newly formed apatite layers. Resorption may be limited to the solubility of the biomaterial.