Bernhardt Schöpf, 2004
Vetsuisse-Fakultät Universität Zürich, Pferdeklinik, Musculoskeletal Research Unit
Introduction and goal of the study: Superparamagnetic iron oxide nanoparticles (SPN) coated with dextran, polyvinyl alcohol (PVA) and other substances were shown to be biocompatible and non-toxic in cells of the musculoskeletal system in previous experiments. The goal of this study was to compare different histological methods for the most reliable detection of SPN in synovial cells (SZ), chondrocytes (CZ) and osteoblasts (OB) after incubation in vitro. This was seen as preliminary tests for later in vivo studies, where the biocompatibility, toxicity and mainly the characteristics of their elimination behaviour were to be tested in experimental animals.
Materials and methods: Different iron oxide particles with dextran-, biotin-, avidin-, streptavidin-, gold- and PVA-coatings were used for incubation on cell cultures. SZ, CZ and OB were incubated on six-well-plates and three chamber slides in different concentrations (2.5, 1.25, 0.625 mg/ml) and for different time periods (3, 12, 24, 48 hours). Thereafter, histochemical stainings, such as Pearls Blue and Turrnbulls reaction, direct and indirect immunofluorescence using dextran coated SPN functionalised with avidine or biotin, electron microscopy (TEM) and “Mapping Local Susceptibility” were applied for particle detection within the cells. A semi-quantitative score system following a descriptive evaluation was used to assess the fraction of dead cells, single particle structure, omission of the nucleus, intraplasmatic particles and cell morphology.
Results: In preliminary experiments, the three SPN: dextran-avidin (10nm; Liquid Research), dextran-avidin (130nm; Micromod) and dextran-avidin (250nm; Micromod) could be reliably detected in cell cultures, and thus, were further used for the main experiments. The D-avidine coated, 250nm particles (particle 3) proved to be the most suitable for detection within the cells. As for the cultivation technique the method where cells were incubated on top of three chamber slides proved to be the most reliable, fastest and most effective. Pearls blue-, the Turnbulls staining and the TEM were the most reliable methods for the chosen SPN detection in cells.
Discussion and conclusion: SPN can reliably be detected in cells of the musculoskeletal system in vitro using special iron stainings for Fe2+, Fe3+, direct or indirect immunhistochemical methods using antibodies against avidine-coated particles and TEM.Deutsche Version