Fx 5000 compression system

Before loading, the cell/alginate constructs were placed within the 5 mm diameter foam ring of Biopress™ compression plate wells (Flexcell international Corporation), and 4 mL F-12 media with 10% FBS was added to each well. Dynamic unconfined compression was applied by a computer-controlled Flexcell^® FX-5000™ Compression system (Flexcell International Corporation) as described in the manufacturer's manual (www.flexcellint.com). The compression testing regimen consisted of a sinusoidal strain from 0 kPa to 20 kPa amplitude at 0.5 Hz as indicated (Figure 1A). Control cell/alginate constructs were maintained under uncompressed conditions. After compressive stimulation, 3D cell culture constructs were washed with phosphate buffered saline (PBS; Sigma), and a 1-mm thickness sample was vertically cut from each construct to observe HDAC4 location by confocal laser scanning microscope. The remaining cell/alginate constructs were collected to evaluate the HDAC4 protein, metabolic and biosynthetic activities of chondrocytes.

Differentiated C2C12 cells were either stretched at 15% biaxial stretch for 6 hours using the Flexcell^® FX-5000™ Compression System (Flexcell International Corporation, Hillsborough, NC), or used as controls (non-stretched) and incubated in parallel to experimental cells without stretch (non-stretched cells). The stretched differentiated C2C12 cells were then unloaded by stopping the stretch and allowing the cells to incubate for another 12 hours. Media was collected at baseline, or after 6 hours of stretch, and 1, 3, 6, and 12 hours after the cessation of stretch. Replicate control samples and cells undergoing cessation of stretch were maintained under static conditions with no applied cyclic strain. In parallel, second experimental group of differentiated C2C12 cells were stimulated with 10% FBS for 6 hours to induce hypertrophy (experimental controls were incubated in parallel without FBS).