In Vitro Biomaterial Particle Cytotoxicity Investigated With the Help of ...

In Vitro Biomaterial Particle Cytotoxicity Investigated With the Help of Polystyrene Beads V. Olivier, J-L. Duval and M-D. Nagel Domaine Biomatériaux-Biocompatibilité, UMR CNRS 6600, Université de Technologie de Compiègne, BP 20529, 60205 Compiègne, France

Introduction In vitro experiments to study cytotoxicity from biomaterial particles mimicking wear debris of joint replacement components represent useful models. Macrophage, fibroblast and osteoblast reactions toward particles have already been documented. However, mixture of particle sizes and shapes makes difficult to correlate a harmful process for cell with one particle criterion only. The aim of our work was to obtain a more comprehensive understanding of particle sizedependent toxicity on fibroblasts. Preliminary studies using particles sized from alumina powders in contact with L929 murine fibroblasts showed a dose-dependent cytotoxicity [1]. Nevertheless, our experimental conditions seemed not entirely satisfactory for the following reasons : particle shape parameter could not be excluded (differences were significant), size distributions were not monodispersed and particles easily aggregated. Polystyrene beads, ruling out shape criterion and being monodispersed, seemed then to be suitable to complete and interpret results gained with alumina particles. Materials and methods We used two populations of polystyrene (PS) beads (0.45 and 3.53 µm mean diameter) from Estapor® Microspheres. L929 cells were seeded at 104/cm2 in MEM supplemented with 10% FBS. The numbers and sizes of cells after 24, 48 and 72 hours of culture with each population of particles were mesured with a Coulter® Multisizer. Cytotoxicity was confirmed at 24h through different assays : trypan blue exclusion, lactatedeshydrogenase release (LDH) and neutral red absorbance. Induction of apoptosis was tested with terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labelling (TUNEL). Environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM) were used to precise cell morphological alteration and visualise particles internalisation. Results No significant difference between experimental cultures with 0.45µm beads and control were found for the numeration as well as for cell sizes. On the contrary, 3.5µm PS beads appeared to be dosedependently cytotoxic, and mean cell size increased with concentration. No increase of the number of apoptotic cells was found in comparison to the negative

control, while necrosis was attested by the loss of membrane integrity shown by trypan blue and LDH assays. TEM observations confirmed both 0.45 and 3.5 µm bead internalisation by fibroblasts, but 0.45µm beads appeared to be grouped in cytoplasmic vesicles while 3.5µm ones were dispersed (Fig. 1)

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b Fig. 1. Transmission electron micrographs of L929 fibroblasts cultured for 48h with 0.5mg/ml of 0.45µm (a) or 3.5µm (b) PS beads Conclusions We evidenced a strictly size-dependent cytoxicity on fibroblasts by necrosis from internalised PS particles. Therefore using PS beads appears to be attractive to investigate further the mechanisms by which cells can react to particle internal presence. Acknoledgements This work was supported by a grant from the Conseil régional de Picardie, France. References 1. Duval et al., (1999) 15th European Conference on Biomaterials, ESB 99.