Time Lapse Imaging Techniques for Comparison of Mineralization Dynamics in Primary Murine Osteoblasts and the Late Osteoblast/Early Osteocyte-Like Cell Line MLO-A5

Abstract

Mineralization of bone matrix and osteocyte differentiation occur simultaneously and appear interrelated both spatially and temporally. Although these are dynamic events, their study has been limited to using static imaging approaches, either alone or in combination with chemical and biochemical analysis and/or genetic manipulation. Here we describe the application of live cell imaging techniques to study mineralization dynamics in primary osteoblast cultures compared to a late osteoblast/early osteocyte-like cell line, MLO-A5. Mineral deposition was monitored using alizarin red as a vital stain for calcium. To monitor differentiation into an osteocyte-like phenotype, the calvarial cells were isolated from transgenic mice expressing green fluorescent protein (GFP) driven by an 8-kb dentin matrix protein-1 (Dmp1) promoter that gives osteocyte-selective expression. Time lapse imaging showed that there was a lag phase of 15–20 h after β-glycerophosphate addition, followed by mineral deposition that was rapid in primary osteoblast cultures but more gradual in MLO-A5 cultures. In primary osteoblast cultures, mineral was deposited exclusively in association with clusters of cells expressing Dmp1-GFP, suggesting that they were already differentiating into osteocyte-like cells. In MLO-A5 cells, the first indication of mineralization was the appearance of punctate areas of alizarin red fluorescence of 4–7 μm in diameter, followed by mineral deposition throughout the culture in association with collagen fibrils. A high amount of cell motility was observed within mineralizing nodules and in mineralizing MLO-A5 cultures. These studies provide a novel approach for analyzing mineralization kinetics that will enable us to dissect in a time-specific manner the essential players in the mineralization process.

Near-infrared fluorescent probe traces bisphosphonate delivery and retention in vivo

“…FRFP binding relies on local bone surface area, and removal of tracer probe is reflected in animals undergoing increased bone turnover. Combining fluorescent bisphosphonates with other fluorescent markers of osteoblastic, (72) osteocytic, (73) or osteoclastic (71) markers will facilitate in vivo and histologic imaging studies to investigate models of osteoporosis, osteogenesis imperfecta, and osteonecrosis of the jaw, where drug-cell interactions leading to a positive or negative outcome have yet to be described.…”

Gsα enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice

“…In vitro, expression of osteocytic markers coincides with mineralized nodule formation (41). We therefore examined expression of osteocyte-specific genes in calvarial osteoblasts cultured under osteogenic conditions, and found that after only 1 week in culture, expression of the osteocyte-specific genes Dmp1 and Phex was upregulated in mutant calvarial osteoblasts, coincident with the early appearance of mineralization ( Figure 6H).…”

Extracellular matrix motion and early morphogenesis

“…The tissue dynamics of cartilage and bone accretion, removal and displacement during skeletogenesis remain undocumented. However, with the development of new time-lapse imaging approaches that reveal a dynamic process of cartilage and bone accretion, skeletogenesis is expected to depend on a highly dynamic mesenchymal tissue (Dallas and Veno, 2012;Dallas et al, 2009;Kamel-ElSayed et al, 2015).…”