Equipment. The LFU generator used in this experiment operates at a frequency of 35 kHz, with an adjustable treatment intensity of up to approximately 2.0 W/cm2 (Arobella Medical LLC, Qoustic Wound Therapy System™ Minnetoka, MN). The treatment delivered in this experiment utilized 100% power output at an intensity of 2.0 W/cm2. The intensity was calibrated using a proprietary laser interferometer that characterizes the displacement, frequency, and frequency waveform of the Qoustic Qurette™ applicator tip. In addition, acoustic output pressure, intensity, and power parameters were measured in an anechoic chamber and/or hydrophone tank to confirm accomplishment of desired output characterization (ie, intensity and power versus distance and acoustic field shape).
Saline jet irrigation was delivered through an orifice inside a metal curette capable of producing a distal displacement from 0–75 µm depending on the percent power setting. The LFU energy was produced through the transduction of 60-cycle wall current into mechanical energy via a titanium alloy transducer using piezoelectric elements made of lead zirconate titanate.
Mechanical energy was produced as the transducer resonated to create an axial oscillation of the curette that radiates ultrasonically both through the scoop shape as well as off the distal end of the curette. This dual action acts to fragment tissue as well as focus ultrasound energy propagated toward the treatment area through the saline jet via the scoop shape. Sterile normal saline was connected to a port on the hand piece and exited the curette as a saline jet, which serves both to irrigate and cleanse the wound of tissue fragments, debris, exudate, and other matter as well as to provide a coupling medium for the transmission of ultrasonic energy directly or through the scoop shape.
The hand piece, with its treatment curette probe, was used to deliver LFU in a zigzag fashion across the fibroblast cultures at a calibrated distance by placing the probe directly adjacent to the lip of the culture dish.
Cell culture. Normal adult human dermal fibroblasts (HDFa; American Type Culture Collection [ATCC], Manassas, VA) were cultured in complete fibroblast basal medium (FBM) comprised of FBM (ATCC) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Inc, Lawrenceville, GA), GlutaMAX ITM (2 mM) (Life Technologies, Grand Island, NY); penicillin and strepytomycin (Sigma Aldrich, St. Louis, MO), 100 U/mL and 100 µg/mL, respectively; and phenol red (2 µM) (Sigma). All cultures were maintained in a humidified 5% CO2 incubator at 37˚ C.
Scratch wound assay. HDFa were seeded in 35-mm tissue culture dishes and grown until approximately 90% confluence, so cells were elongated without contact inhibition at the time of the scratch wound assay. By using a model of subconfluence, singular fibroblastic migration patterns could be observed.15
All cultures were scratched with a sterile 200-µL plastic pipette tip. A smooth, straight scratch was made through the monolayer of cells by holding the culture dish at a 45˚ angle and allowing the weight of the pipette to rest on the back of the hand. Cells then were treated with 35 kHz LFU or saline only (control). Cells treated with the LFU were sprayed with saline in a slow zigzag sweeping motion for 10 seconds. Saline was removed from both ultrasonic-treated and control cells by aspirating with a pipette. Complete FBM (1 mL) was added to the cells in a slow drip down the side of the culture dish. Culture dishes were positioned on an Olympus Ix70 inverted or a Zeiss Axiovert inverted microscope and magnified 10X. Additional complete FBM (1 mL) was added to the culture dish, and the cells were maintained at 37˚ C and 5% CO2. Scratch images were taken at approximately 9-second intervals during a 48-hour period.
Two observers blinded to study or control cultures viewed the images taken at 8-hour increments. For each image collected at 8, 16, 24, 32, 40, and 48 hours, the number, length, and angle of the HDFa cells entering the scratch wound were recorded. Length was determined by measuring the distance between the 2 points at maximal distance on the outer edge of the cell. Average length of the cells entering the scratch wound for each culture dish was calculated and reported.
Morphological observations. Images taken at 8, 16, 24, 32, 40, and 48 hours using the Olympus Ix70 inverted or a Zeiss Axiovert inverted microscope and magnified 10X were analyzed for morphological changes including cell shape, packing, and apparent length.
Data collection and statistical analysis. A 2-tailed, paired t-test was used to compare migration rates of treated and control fibroblasts (untreated groups). Comparison of the angulation of fibroblasts entering the artificially created wounds was performed using a 2-tailed, Fisher’s exact test. All graphs were created and statistical analyses were performed using GraphPad Prism 6.00 for Windows (GraphPad Software, Inc, San Diego, CA). Observational analysis of fibroblast morphological changes was performed using the microscopic videography images collected for the migration pattern analysis.
To compare the rate of closure for untreated and LFU-treated scratch wound assays, the average width of a scratch wound remaining at 3 locations for each wound at 0, 8, 16, and 24 hours was recorded. The percentage of width of a scratch wound remaining at 8, 16, and 24 hours for each wound was calculated. The percentages of width of scratch wounds remaining for untreated and LFU-treated cells were compared at 8, 16, and 24 hours using an unpaired t-test statistical analysis. A total of 5 separate experiments were conducted with 2 treatment groups (5 untreated and 5 ultrasound-treated). One of the ultrasound treatment groups was excluded due to insufficient CO2 in the incubator as the result of a line leakage.
The number and length of untreated and LFU-treated cells were compared at each 8-hour increment using an unpaired t-test statistical analysis. The number of HDFa cells aligned at 0˚ to 22˚ from (relatively parallel to) the wound and the number of cells oriented at a 23˚ to 45˚ angle, a 46˚ to 67˚ angle, and a 68˚ to 90˚ angle from (more perpendicular to) the wound were counted. The number of cells oriented at a 0˚ to 22˚ angle from (relatively parallel to) the wound and the number of cells oriented at a 23˚ to 90˚ angle from (more perpendicular to) the wound for both control (untreated) and LFU-treated HDFa cells at each 8-hour increment were compared using a 2-tailed Fisher’s exact test statistical analysis.