The experimental set-up used for all USWF exposures is depicted in . A plastic exposure tank (36 × 20 × 18 cm) was filled with degassed, deionized water at room temperature. The acoustic source consisted of a 1 MHz unfocused transducer, fabricated from a 2.5 cm diameter piezoceramic disk. The transducer was mounted on the bottom of the water tank. The signal driving the transducer was generated by a waveform generator (Model 33120A, Hewlett Packard, Palo Alto, CA, USA), RF power amplifier (Model 2100L, ENI, Rochester, NY, USA), and an attenuator (Model 837, Kay Elemetrics Corp., Lincoln Park, NJ, USA). Samples were contained within the wells of a modified silicone elastomer-bottomed cell culture plate (BioFlex® culture plates, FlexCell International Corporation, Hillsborough, NC, USA). These sample holders were mounted to a three-axis positioner (Series B4000 Unislide, Velmex Inc., East Bloomfield, NY, USA) to allow precise control over their location within the sound field. The air interface above the samples was used as the acoustic reflector to generate an USWF within the sample volume.
Schematic of the Experimental Set-up and Sample Holders used for USWF Exposures
Sample Holder Preparation
The BioFlex® culture plates used as sample holders for our investigations are depicted in . They were modified from the manufacturer’s form by reducing the diameter of 3 wells per plate from 4 cm to 1 cm using Sylgard® 184 silicone elastomer (Dow Corning Corporation, Midland, MI, USA). Through this modification, the diameter of the sample was comparable in size to the −6 dB beam width at the exposure location. The two-part silicone elastomer was mixed in a 10:1 ratio as recommended by the manufacturer’s instructions. The solution was degassed at room temperature using a vacuum chamber (Model 5830, National Appliance Company, Portland, OR, USA) and was subsequently poured around 1 cm diameter Teflon® mandrels (Dupont, Wilmington, DE, USA) that were placed at the center of the 3 wells of interest. Following curing of the silicone elastomer at 20°C for 48 hr, the mandrels were carefully removed to leave a 1 cm diameter sample space within 3 wells of each BioFlex® culture plate ().
The acoustic attenuations of the silicone elastomer well bottom of the BioFlex® plates, the Sylgard® 184 silicone elastomer, and standard tissue culture polystyrene (Corning/Costar, Cambridge, MA, USA) were measured using an insertion loss technique. Using the water tank set-up, each material was inserted into the acoustic path between the unfocused 2.5 cm diameter, 1 MHz transducer and a hydrophone (either a bilaminar PVDF membrane hydrophone (Marconi Research Center, Chelmsford, England) or a needle hydrophone (Model HNC-0400, Onda Corporation, Sunnyvale, CA, USA)). Peak positive and peak negative pressure amplitudes were measured using the hydrophone and a digital oscilloscope (Model 9310AM, LeCroy, Chestnut Ridge, NY, USA) in the presence and absence of each material for various source amplitudes. The thickness of each material was measured using calipers. The acoustic attenuation coefficient (in dB/MHz/cm) was calculated for each material.
The acoustic absorption coefficient of Sylgard® 184 silicone elastomer was measured using a thermocouple technique. Briefly, a 50 μm copper-constantan thermocouple was embedded in a sample of Sylgard® 184 silicone elastomer. Using the water tank set-up, the active element of the embedded thermocouple was positioned at the focus of a 1 MHz transducer fabricated from a 3.8 cm diameter plane, piezoceramic disk cemented to the back of a plano-concave lens. A laboratory thermometer (Model BAT-4, Bailey Instruments Co. Inc., Saddle Brook, NJ, USA) and digital oscilloscope were used to monitor the thermocouple output for various pulsing parameters and exposure amplitudes. For each exposure condition, the initial rate of temperature rise in the sample and the spatial peak temporal average intensity (Ispta) were measured and used to calculate the absorption coefficient. The calculated absorption coefficients from each exposure condition were averaged to determine the acoustic absorption coefficient (in dB/cm) of Sylgard® 184 silicone elastomer at 1 MHz.
Temperature changes in the collagen/cell samples were also monitored during USWF exposure using a 50 μm copper-constantan thermocouple. Thermocouple output was monitored using a digital laboratory thermometer (Model BAT-12, Physitemp Instruments Inc., Clifton, NJ, USA), sensitive to changes of 0.1°C, over the duration of USWF exposures.
Acoustic Field Measurements
Using the water tank set-up, axial and transaxial spatial distributions of pressure from the 1 MHz, 2.5 cm diameter unfocused transducer were measured under USWF exposure conditions in both the presence and absence of the sample holder. The Onda needle hydrophone, connected to a three-axis positioner, and a digital oscilloscope were used to measure the acoustic pressure. The sample holder was placed in the far-field with the well bottoms situated at an axial distance of 12.2 cm from the transducer. Axial spatial distributions of pressure were measured through a 0.5 cm distance below the air interface in 0.1 mm intervals. The 0.5 cm distance approximates the height of the collagen samples used in our investigations. Transaxial spatial distributions of pressure were measured at an axial distance of 12.2 cm from the transducer in 0.1 mm intervals. A sinusoidal pulse of 50 μs duration was employed and peak positive pressures were measured for each position.
Acoustic Field Calibrations
Prior to each experiment, the acoustic field was calibrated using either the Marconi membrane hydrophone or the Onda needle hydrophone under traveling wave conditions. Hydrophones were calibrated regularly using the steel sphere radiometer technique (Dunn et al. 1977
). Acoustic pressure was measured in the far-field at an axial distance of 12.2 cm from the transducer (where samples were located during USWF exposure). Coordinates from the exposure site to a fixed pointer were determined using the three-axis positioner and were used to position the center of the lower, left-hand well of the sample holder at the exposure site (bottom of the well was 12.2 cm from the transducer). Some water was removed from the tank such that the sample holder was located at the exposure site without full submersion.
Fibronectin-null mouse embryonic myofibroblasts (obtained from Dr. Jane Sottile, University of Rochester) were used for all experiments. These cells do not produce fibronectin and have been adapted to grow under serum-free conditions (Sottile et al. 1998
). Cells were routinely cultured in a 1:1 mixture of AimV (Invitrogen, Carlsbad, CA, USA) and Cellgro (Mediatech, Herndon, VA, USA) on tissue culture dishes pre-coated with collagen type-I. These media do not require serum supplementation. Thus, no source of fibronectin is present during routine culture. On the day of USWF exposure, fibronectin-null cells were harvested from monolayer culture by treatment with 0.08% trypsin (Invitrogen) and 0.5 mM EDTA in PBS. Trypsin activity was neutralized with 2 mg/ml soybean trypsin inhibitor (STI; Sigma, St. Louis, MO, USA). Cells were washed one time with 1 mg/ml STI in PBS and were then resuspended in a 1:1 mixture of AimV/Cellgro.
Collagen Solution Preparation
A neutralized type-I collagen solution was prepared on ice by mixing collagen type-I (isolated from rat tail tendons (Windsor et al. 2002
)) with 2X concentrated Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) and 1X DMEM containing HEPES so that the final mixture consisted of 0.8 mg/ml collagen and 1X DMEM (Hocking et al. 2000
). Both the 1X and 2X DMEM media were degassed in a vacuum chamber for 30 min under sterile conditions prior to incorporation into the collagen mixture.
Fibronectin-null cells were added to aliquots of neutralized type-I collagen solutions on ice at various final concentrations immediately prior to USWF exposure. Aliquots (400 μl) of the collagen/cell solution were then loaded into two of the 1 cm diameter Sylgard® 184 silicone elastomer molded wells of the BioFlex® plate. For “no-cell” samples, an equal volume of AimV/Cellgro was added in place of fibronectin-null cells and aliquots were loaded into a third well. The collagen/cell solution in the left-hand well of each plate was exposed to a 1 MHz, continuous wave USWF for 15 min at room temperature. The two other samples in the plate (right-hand side) served as sham control wells that were treated exactly as the exposed sample but were not exposed to the USWF. The 15 min exposure duration was sufficient to promote collagen polymerization at room temperature. Following USWF exposure, collagen gels were incubated for 1 hr at 37°C and 8% CO2 to allow for complete collagen polymerization. An equal volume (400 μl) of DMEM was then added to wells containing collagen gels. In some experiments, collagen/cell and collagen/no-cell solutions were incubated for 1 hr at 37°C and 8% CO2 in the sample holders to allow collagen polymerization before USWF exposure.
Cell Viability Assay
Thiazolyl blue tetrazolium bromide (MTT) was used to assess cell viability (Mosmann 1983
). At various time points after USWF exposure, collagen gels were incubated with 5.3 mM MTT (USB Corporation, Cleveland, OH, USA) for 4 hr at 37°C and 8% CO2
. Gels were then digested with 0.77 mg/ml collagenase (from Clostridium histolyticum
, type-I, Sigma) and formazan crystals were dissolved using acidified isopropanol (0.04 N HCl). Absorbance measurements at 570 nm and 700 nm (background) were determined using a spectrophotometer. MTT absorbance was calculated by subtracting background absorbance values and non-specific reduction of MTT in no-cell gels from the 570 nm readings. There was a linear relationship between cell number and MTT absorbance. This assay is sensitive to differences of 5000 cells and greater (data not shown).
Collagen Gel Contraction Assays
The extent of collagen gel contraction was determined using two established methods. For volumetric
gel contraction assays, collagen gels were scored around their edges to form free-floating gels. After an additional 20 hr of incubation at 37°C and 8% CO2
, the gels were removed from the wells and weighed (Model B303, Mettler Toledo, Columbus, OH, USA). Volumetric collagen gel contraction was calculated as a decrease in gel weight as compared to the control, no-cell gel weight (Hocking et al. 2000
). For radial
gel contraction assays, collagen gel diameters were measured using a 10X inspection microscope equipped with a calibrated eyepiece micrometer. Two measurements were recorded for each gel and averaged to calculate gel diameter. Investigators measuring diameters were blinded to exposure conditions. Radial collagen gel contraction was calculated as a decrease in gel diameter as compared to the original gel diameter of 1 cm (Tingstrom et al. 1992
Soluble Fibronectin Binding
Fibronectin-null cells in suspension (2×107
cell/ml) were incubated with 100 μg/ml of Alexa Fluor® 488-labeled human, plasma-derived fibronectin (FN-488; labeled according to manufacturer’s instructions) in the presence of 1 mM MnCl2
for 30 min at room temperature (Akiyama and Yamada 1985
; Mastrangelo et al. 1999
). Cells were washed twice with AimV/Cellgro to remove unbound fibronectin and were then added to neutralized type-I collagen solutions and exposed to an USWF as described above. In other experiments, 10 μg/ml of FN-488 was added to neutralized type-I collagen solutions in the absence of cells and exposed to an USWF as described above.
One hour after USWF exposure, cell-embedded collagen gels were examined using an Olympus IX70 inverted microscope (Center Valley, PA, USA) with a 4X phase-contrast objective and were photographed using a digital camera (Spot RT Slider, Model 2.3.1, Diagnostic Instruments Inc., Sterling Heights, MI, USA). FN-488 was visualized using epifluorescence microscopy. Gels were flipped on their side to visualize cell bands through the height of the cylindrical sample. For volumetric collagen gel contraction experiments, gels were imaged after obtaining weight data. Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA) was used to measure the linear distance between fibronectin-null cell bands within collagen gels. Pixel distance was converted to micron values using a micrometer calibration. A total of 10 distances were measured on each of 20 different images collected from 3 different experiments.
To visualize type-I collagen fibers, cell-embedded collagen gels were examined using second-harmonic generation microscopy (Freund and Deutsch 1986
; Roth and Freund 1979
; Williams et al. 2005
). One hour after USWF exposure, gels were fixed in 4% paraformaldehyde for 1 hr at room temperature. Second-harmonic generation microscopy was performed using an Olympus Fluoview 1000 AOM-MPM microscope equipped with a 25X, 1.05 NA water immersion lens (Olympus). Samples were illuminated with 780 nm light generated by a Mai Tai HP Deep See Ti:Sa laser (Spectra-Physics, Mountain View, CA, USA) and the emitted light was detected with a photomultiplier tube using a bandpass filter with a 390 nm center wavelength (Filter FF01-390/40-25, Semrock, Inc., Rochester, NY, USA). Fibronectin-null cells were simultaneously visualized using a second bandpass filter with a 519 nm center wavelength (Filter BA 495-540 HQ from MPFC1, Olympus) by exploiting the intrinsic auto-fluorescence of cells (Monici 2005
). Cell-embedded collagen gels were photographed using a CMOS digital camera (Moticam 1000, Motic, China).
Data are presented as the mean ± SEM. Statistical comparisons between USWF-exposed and sham experimental conditions were performed using either the Student’s t test for paired samples or one-way analysis of variance in GraphPad Prism software (La Jolla, CA, USA). Differences were considered significant for p values < 0.05.