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A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro
Published on: June 16, 2022
Direct and indirect effects of functionalised fluorescence-labelled nanoparticles on human osteoclast formation and
Andrea Tautzenberger1, Ludwika Kreja, Anke Zeller
1Institute of Orthopaedic Research and Biomechanics, Centre of Musculoskeletal Research, University of Ulm, Helmholtzstrasse 14, 89081 Ulm, Germany. andrea.tautzenberger@uni-ulm.de
Abstract:
Recently, it was demonstrated that phosphonate-functionalised nanoparticles were successfully taken up by mesenchymal stem cells without influencing their viability and differentiation capacity, suggesting that they may provide a promising basis for the development of nanoparticles for drug delivery or cell labelling. The present study aimed to investigate the effects of these nanoparticles on osteoclast formation and activity as well as on the inflammatory response of osteoclasts and osteoblasts. The intracellular uptake of the particles by human osteoclasts and osteoblasts was demonstrated by confocal laser scanning microscopy, transmission electron microscopy and fluorescence microscopy. The expression of tartrate-resistant acid phosphatase, carboanhydrase II, cathepsin K, calcitonin receptor and osteoclast-specific vacuolar proton pump subunit TCIRG1 as well as actin ring formation were not significantly altered in osteoclasts by particle treatment, as demonstrated by cytochemical staining and immunostaining. Active calcium phosphate resorption by osteoclasts was also not significantly influenced by the particles. The expression and secretion of pro-inflammatory cytokines (IL-6, IL-1β) by osteoclasts and osteoblasts and the expression of osteoclast-regulating genes (M-CSF, OPG, RANKL) in osteoblasts were similarly not significantly affected. In conclusion, phosphonate-functionalised nanoparticles did not affect osteoclast formation and activity either directly or indirectly, suggesting that they could provide a promising tool for the development of particle-based treatments for anti-resorptive therapies.

