Bioflex plate

Chondrocytes were isolated from the ventral parts of the rib cages of 6-d-old newborn mice (C57Bl/6) and cultured in F-12 media with 10% fetal bovine serum (FBS) as previously described [20 (link)–22 (link)]. Briefly, the 6-d-old mice were killed with CO₂, and the ventral parts of the rib cages were treated with 3% collagenase D (Roche, cat. no. 11 088 882 001). The chondrocytes were seeded at a density of 1×10⁵ cells/cm² and cultured at 37°C in Ham’s F-12 medium with 10% FBS in a thermal incubator under 5% CO₂. The medium of the chondrocyte culture was refreshed every other day.
Reaching 100% confluency, chondrocytes were subcultured into six-well BioFlex^® plates (25 mm diameter, Flexcell International Corporation, Hillsborough, NC) at a density of 45% confluency. After cultured for 12 hours, the culture medium was refreshed, and chondrocytes were incubated with adenoviral vectors containing GFP-HDAC4 for 24 hours at a multiplicity of infection (MOI) of 30. After 48 hours, transfection efficiency was confirmed with observation of the expression of GFP in infected chondrocytes by using Nikon E800 fluorescence microscope (Nikon, Melville, NY, USA).

BMSCs were seeded at a density of 3.0 × 10⁵ cells/cm² on collagen I-coated 6-well BioFlex plates (Flexcell International, Burlington, NC, USA) and then incubated in ɑ-MEM until reaching 95% cell confluence. Subsequently, culture plates were subjected to cyclic tensile strain using iStrain with extension rates of 9%, 12%, and 15% at a frequency of 0.3 Hz for 12 h or subjected to cyclic tensile strain with a 12% extension rate at a frequency of 0.3 Hz for 6 h, 12 h, 24 h, 36 h, or 48 h. The BMSCs were then subjected to microscopic observation, immunohistochemistry staining, or RNA and protein extraction.

Cells were passaged to DMEM containing 10% FBS six-well BioFlex plates coated with collagen type I (Flexcell) for 24 h to form a confluent monolayer. Then the medium was replaced by DMEM without FBS for 6 h before stretch. A Flexcell FX-5000TM Tension System (Flexcell International Corporation, Burlington, CA, USA) was used to order cyclic mechanical stretch. Cells were stretched at a frequency of 1HZ with 20% amplitude and a 1:1 stretch:relaxation ratio for 15 min, 2 h, 6 h, 12 h and 24 h. Both the tension system and the control plates were placed in the incubator at 37 °C, with 5% CO₂.

For the in vitro experiment, hPDLCs and TG-derived neurons were seeded onto collagen І-coated six-well BioFlex® plates (Flexcell Int. Corp., Hillsborough, NC, USA). After the cells reached 70% to 80% confluence, the culture medium was replaced with α-MEM containing 1% FBS. Cells were then subjected to tensile strain with tensity of 10% and frequency of 0.5 Hz for different time (1 h, 3 h, 6 h, 12 h, 24 h) using a Flexcell® Tension Plus™ FX-5000™ system (Flexcell Int. Corp., Hillsborough, NC, USA). Cells plated on BioFlex® plates but not subjected to stretch served as controls.

Two bioreactors [12 (link)] were used in parallel for the experiments, both using the same vibration pattern per experiment. The frequency ranges of the generated patterns were designed to include the fundamental frequencies of the male and female voice [13 (link)] (100–135 Hz and 200–250 Hz, respectively). The vibration pattern sound files were created using Audacity software (version 2.2.2, audacityteam.org, registered trademark of Dominic Mazzoni). Spectrums of membrane displacements of BioFlex® plates (Flexcell International Corporation, Burlington, NC, US) were measured using laser Doppler vibrometry as previously described [12 (link)].

Keratinocytes were plated in six‐well, flexible silicone rubber BioFlex™ plates coated with type‐I collagen at 2 × 10⁵ cells/mL. Cyclic mechanical tension with a 0.5‐Hz sinusoidal curve at 10% elongation was applied using an FX‐5000™ Tension Plus unit (Flexcell, Hillsborough, NC, USA). Cells were harvested immediately after mechanical stretching was complete. Control cells were cultured in the same plates in the same incubator without the application of tension.