12 M. M. Saunders To first analyze the effect of mechanical stimulation and particulate debris on osteocytes, only the osteocyte layer of the LOC was used. The fully cured PDMS osteocyte layer was sealed with a PDMS membrane on top and a slab of PDMS (2 mm thick) on the bottom. The main channel of the LOC was coated with collagen type I by flowing the solution through the outside access channels. After 1 h the channel was rinsed with Dulbecco’s phosphate-buffered saline. The central access channels were then used to fill the entire chip with Minimum Essential Medium (MEM) α supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Optimizing osteocyte feeding in the LOC: MLO-Y4 osteocytes were seeded within the main channel of the chip at a density of 104 cells/cm2 and were given 4 h to adhere to the surface. Assuming a similar culture medium consumption rate as osteocytes grown within a T-25 flask, it was determined that the medium within the main channel should subsequently be replaced with medium from the reservoirs every 3 h. The feeding protocol was fully automated using the linear actuators to drive fluid movement. Chips with a reservoir depth of 3.5 mm contained sufficient culture medium to maintain the cells for 24 h. Every 24 h the medium in the reservoirs was manually replaced. To address the concern of excessive evaporation of culture medium through the PDMS that would result in the formation of air bubbles in the main channel, a humidity chamber was designed and 3D printed to house the chip within the laboratory incubator. This chamber fit on top of a standard 6-well culture plate that was filled with sterile dH2O. Volumetric flow rates used during the automated portion of the feeding protocol were analyzed to balance osteocyte viability with minimal stimulatory effects. A flow rate of 14.9 µl/h (lower feed rate) was initially tested, which generated a wall shear stress two orders of magnitude less than the lowest value reported in literature that is known to stimulate osteocytes. At this flow rate, osteocyte proliferation rates were substantially less than that of cells grown in a 96-well plate. By increasing the flow rate to 29.8 µl/h (higher feed rate), proliferation rates were comparable to that of cells grown in the 96-well plate (Fig. 3). Additionally, cells displayed typical dendritic morphology as shown for 4 and 72 h (scale bars represent 100 µm); rhodamine phalloidin and DAPI stains at 72 h revealed the presence of an organized cytoskeleton (scale bar represents 50 µm). Based upon osteocytic iNOS production, the higher feed rate was utilized for subsequent studies. Fig. 3 Osteocytes in the LOC channels maintained viability with cellular proliferation more responsive to a higher feeding rate (29.8 µl/h). At the higher feeding rate, proliferation was comparable to that observed under identical feeding conditions in the 96-well plate. Validating experimental flow rates: Micro-particle image velocimetry (µPIV) was used to validate fluid flow rates within the osteocyte culture chamber (Fig. 4). A chip was filled with 0.02% solution of fluorescent polystyrene tracer particles
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