Bone Multicellular Unit on a Chip (BMU-Chip) Subjected to Cyclic Mechanical Loading

The skeleton undergoes continuous remodeling to maintain its structural integrity. The basic unit of bone remodeling is the Bone Multicellular Unit (BMU), a highly organized complex of osteocytes, osteoblasts, and osteoclasts that remodels skeletal microarchitecture to adapt to the mechanical demands placed upon the bone. Here, we describe the design and development of the BMU-chip microfluidic platform for the longitudinal investigation of complex interactions between different cell types and their extracellular matrix in response to cyclic mechanical loading. Three-chambered polydimethylsiloxane (PDMS) chips are fabricated using a combination of 3D-printed master molds and soft lithography compatible with real-time, time-lapse, and confocal microscopy methods. The chip is then populated with murine osteocytes (OCY454) suspended in a collagen gel matrix, allowing the self-assembly of three-dimensional networks. Murine preosteoblastic (MC3T3-E1.4) and preosteoclastic (Raw264.7) cells are introduced to a parallel chamber of the device and induced to differentiate in situ, forming the cellular and matrix components of the BMU-chip, which are evaluated in mono- and coculture configurations. Pulsed Unidirectional Fluid Flow Stimuli (PUFFS) are then applied to the osteocyte network via the third parallel chamber. Over a period of up to 31 days of PUFFS stimulation, cells in the devices demonstrated excellent cell viability and lacunocanalicular morphology and expressed cell-specific phenotypic markers as assessed by gene expression and immunofluorescence studies. Throughout the experiment, live-cell fluorescence microscopy was used to study PUFFS-evoked Ca2+ signal propagation through the osteocyte network. These results suggest that the BMU model will be a useful experimental platform for studying key aspects of skeletal mechanoadaptation that cannot be feasibly studied by contemporary methods.