Microfluidic Platform to Investigate the Role of Osteocytes on the Mechanical Regulation of Bone Metastasis

Middleton, Kevin 1 ;  Mei, Xueting (Christina) 2 ;  Shim, Dongsub 2 ;  Devadas, Deepika 2 ;  Ma, Yu-Heng (Vivian) 1 ;  Young, Edmond 1, 2 ;  You, Lidan 1, 2

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Mechanical and Industrial Engineering, University of Toronto

INTRODUCTION:  Bone metastasis is a common outcome of breast cancer. Once metastasized to bone, the outcome of the patient significantly worsens. However, it is unclear what initiates causes this metastasis, and animal models to study this process are difficult to perform. In recent years, microfluidic systems to investigate the role that the bone environment has on bone metastases. We have previously demonstrated that mechanically stimulated osteocytes regulate bone metastases using traditional in vitro techniques; however, these experiments have significant limitations. To minimize these limitations, we are developing a microfluidic platform that, for the first time, allows for mechanical stimulation to bone cells. Using this platform, we then investigate how mechanically stimulated osteocytes regulate bone metastasis.

METHODS: Our microfluidic device consists of 2 channels separated by side channels that allow for diffusive signal transport. To stimulate the osteocytes, we connect a tube to the osteocyte channel, and use a stepper motor to compress/decompress the tube to generate fluid flow. To quantify the shear stress developed, we utilized PIV. To validate osteocytes could be mechanically stimulated we seeded MLO-Y4 cells in the device, and stained them with a calcium indicator. We applied flow to the cells and quantified the cellular calcium flux. To prepare model blood vessels, we loaded one channel with a hydrogel, and used viscous finger patterning to form a lumen. We then used fluorescent microbeads and confocal microscopy to validate the formation of a lumen. We assessed the diffusability of our gel by loading a fluorescent model of VEGF (40 kDa dextran solution) into one channel, and imaging the evolving fluorescent gradient at different time points. To demonstrate the formation of intercellular junctions between endothelial cells we seeded HUVECs in the lumen. The HUVECs were then fixed and fluorescently stained for VE-cadherins, after which they were imaged using confocal microscopy.

RESULTS and DISCUSSION: Using our custom built microfluidic pump, we measured peak oscillatory shear stresses of 2 Pa in our device, which is well within the physiological range of bone. When exposed to flow, we observed significant calcium responses in approximately 45% of the osteocytes, with a mean response of 2.8x the maximum baseline response. This suggests that we can successfully stimulate cells in our device. Confocal imaging demonstrated that we are able to form consistent lumens within our device. We also demonstrated the formation of a chemical gradient through our gel, which was able to achieve equilibrium within 5 minutes of dye loading. Finally, fluorescent imaging of endothelial cells showed the formation of VE-cadherins. This suggests that endothelial cells were able to form intercellular and physiologically relevant adherens junctions.