Bone Abstracts

Osteoclasts are the major bone resorbing cells, essential for bone turnover and development. Human osteoclasts can be generated in vitro by stimulation of peripheral blood mononuclear cells (PBMCs) with M-CSF and RANK-L. Seeding fully differentiated osteoclasts onto mineralized surfaces facilitates the analysis of molecular interactions between the osteoclast and the mineralized matrix.

Currently, a widely used protocol for harvesting osteoclasts from tissue-culture plastic involves trypsinization and subsequent scraping of the osteoclasts into suspension. Trypsinization and scraping may affect processes such as initial attachment on the bone and initiation of resorption. Therefore, there is a need for an alternative harvesting method. A potential technique involves the use of culture plastics grafted with a temperature-responsive polymer. These culture plastics are cell adhesive at 37 °C. At room temperature (RT), the PIPAAm chains become extended, resulting in the release of adherent cells.

Human PBMCs were differentiated towards osteoclasts on temperature-responsive culture plastic (Thermo Scientific Nunc Up-Cell™). When multinucleated osteoclasts had formed, cells were released into suspension by incubation at RT. As a control, osteoclasts were harvested from traditional culture plastic by trypsinization and scraping.

The resulting cell suspensions were reseeded on bovine cortical bone slices. The cells were cultured on bone for a maximum of 48 h. Osteoclasts were detected by staining for TRAcP. Actin rings were visualized by FITC-conjugated phalloidin and the nuclei by DAPI. In order to analyze the osteoclastic resorption activity, cells were removed from the bone slices and resorption pits were revealed by WGA-lectin.

The results showed that multinucleated viable osteoclasts could be harvested from temperature-responsive culture plastic by temperature reduction. The obtained osteoclasts quickly attached to the bone surface and showed formation of actin rings (<15 min). Osteoclasts retrieved from temperature-responsive culture plastic showed significantly higher resorption activity compared to osteoclasts harvested by trypsinization and scraping.