The osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances that occur during bone remodeling, caused by hormone perturbations or alterations in mechanical loading, can induce bone disease as osteoporosis. Due to limited understanding of the underlying mechanisms, current therapies for osteoporosis cannot adequately address this imbalance because current studies of osteocytes rely on conventional cell culture that cannot recapitulate local in vivo microenvironments for the lack of control of the spatial/temporal distribution of cells and biomolecules. Microfluidics is the science and technology of microscale fluid manipulating and sensing and can help fill this gap. We used a microfluidic device to enable the culture of osteocyte-like cells (MLO-Y4 and MLO-A5) in a 3D fashion. Osteocytes were cultured in a perfused and 160 μm high channel and embedded in a bone-like extracellular matrix: osteocytes were embedded in a matrigel- and collagen-based hydrogel enriched with nanostructured hydroxypatite crystals (HA-NP) to mimic bone. To set up the best combination of matrigel enriched with Type I collagen we used fluorescent microspheres and confocal analysis. To evaluate the viability and the expression of osteocytic markers, we used live-dead assay amd immunofluorescent staining and confocal analysis combined with automated quantification. For mineralization, we performed alizarin red staining.Introduction and Objective
Materials and Methods
