A multicenter, randomized, controlled study was carried out with two groups: the study group undergoing debridement using ULSC-12D (Ul-tech, Uiwang, Korea) and the control group undergoing debridement using SONOCA 180 (Söring, Quickborn, Germany). It was conducted at three medical institutes from February to November 2013. A total of 30 patients with pressure ulcers were recruited and enrolled according to the inclusion and exclusion criteria (Table 1). In detail, patients with a stage 2 or greater pressure ulcer sized at least 3 cm² requiring debridement were included, but patients with signs of cellulitis or other comorbidities hindering proper assessment were excluded. Cognitive diseases that induce an inappropriate response and spinal diseases that induce a lack of sensitivity were examples of such comorbidities. Except for simple dressing changes, patients with wounds treated in 2 weeks were also excluded. The patients were randomly allocated to two groups with 15 patients per group.

All the patients were evaluated for intra-procedural pain and post-procedural reduction of bacterial load and necrotic tissue. The procedures were done without anesthesia. Pain was assessed on a visual analog scale. Both bacterial load and the extent of necrotic tissue were measured before and 30 minutes after debridement. Bacterial load was measured using 5×5 cm nitrocellulose membrane filters gently pressed to the center of the wound and immediately inserted into a Columbia Blood Agar plate. Bacterial load was measured by counting the number of individual colonies on the plates after incubating at 37°C for 16 hours. The extent of necrotic tissue was measured using OpSite Flexigrid and Visitrak (Smith and Nephew, London, UK).

The clinical trial was approved by the institutional review board and ethics committee of Seoul National University Bundang Hospital, Samsung Medical Center, and Hanyang University Hospital. It was registered at ClinicalTrials.gov (Identifier: NCT02007824). Informed consent was obtained for all patients.

Human skin fibroblast culture was established using explants from the dermis of adult foreskin. Fibroblasts were cultured in complete Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Sigma Chemical Company, Saint Louis, MO, USA), and 1X Antibiotic-Antimycotic (Gibco Life Technologies, Grand Island, NY, USA). The cells were subcultured when approximately 90% confluent. Primary endothelial cells were obtained from human umbilical veins, subcultured, and maintained in a 0.2% w/v gelatin-coated 25 cm² culture flask with complete McCoy’s 5A medium (Flow Laboratories, McLean, VA, USA) supplemented with 20% FBS, 50 mg/mL endothelial cell growth supplement (Collaborative Research, Bedford, MA, USA), and 100 mg/mL heparin (Sigma Chemical Company) until reaching confluence, within 4 to 5 days. Endothelial cells were seeded in a 6-well plate coated with 0.2% w/v gelatin. Cells were cultured in a humidified incubator at 37°C and 5% CO₂ and used at passages 3 to 6 for all experiments. For all in vivo assays, there were three experimental groups: treated with ULSC-12D, treated with SONOCA 180, and untreated (control).

The WST-8 cell proliferation assay was performed on each fibroblast group. When reduced by intracellular dehydrogenase, water-soluble WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt] forms a yellowish product (formazan), which is used to measure the number of viable cells.

Collagen synthesis was measured by an immunofluorescence technique using a monoclonal anti-collagen antibody. The anti-collagen antibody was obtained by immunizing mouse splenocytes with human collagen. The anti-collagen antibody was labeled with a fluorescent marker and collagen synthesis was measured in each group of human fibroblasts after ultrasound exposure.

Growth factor expression was measured by reverse transcription polymerase chain reaction. Expression levels of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and granulocyte macrophage colony stimulating factor (GM-CSF) were measured in each group of cultured fibroblasts after exposure to each device.

Nitric oxide is a highly reactive free radical gas and oxidizer, which remains stored in tissues as nitrate or nitrite. A colorimetric Griess reaction was used to quantify the combined concentration of nitrate and nitrite. Quantitative analysis of nitric oxide production was performed in each endothelial cell group after ultrasound exposure.

The mean and standard deviation (SD) for each set of measurements was calculated. The results were analyzed using one-sample t-tests and one-way analysis of variance. Results were considered significant if the significance level (P-value) was lower than 0.05.

The reduction in bacterial counts after debridement was 96.3%±6.7% (mean±SD) in patients treated with ULSC-12D, and 91.6%±16.2% in patients treated with SONOCA 180. The extent of the necrotic tissue was reduced 27.1%±8.1% (mean± SD) post-debridement with ULSC-12D, and 15.1%± 18.2% with SONOCA 180. The differences between groups did not reach statistical significance, however there was a trend toward improved outcomes using ULSC-12D. The average intra-procedural pain assessment score was 5.4 in the ULSC-12D group, and 4.8 in the SONOCA 180 group. This difference was also statistically insignificant, suggesting both devices have comparable efficacy. No significant adverse event was reported in either group during the study (Table 2).

The proliferation rate of fibroblasts was 122.48%±7.11% (mean±SD) in the ULSC-12D group, 110.38%±2.46% in the SONOCA 180 group, and 98.23%±2.47% in the control group. The groups treated with ultrasound were significantly more proliferative than the control group, but the difference between the two ultrasound treatment modality groups was not significant. The collagen synthesis of fibroblasts was 14.53±1.94 μg/mL (mean±SD) in the ULSC-12D group, 6.23±1.48 μg/mL in the SONOCA 180 group, and 1.53±0.22 μg/mL in the control group. The ULSC-12D-treated fibroblasts showed significantly higher collagen production than the other two groups. Expression of bFGF also increased significantly in ULSC-12D group, but there was no significant change in the expression of PDGF or GM-CSF or any growth factors tested in other groups.

Nitric oxide production of endothelial cells was 3.90±0.63 μM (mean±SD) in the ULSC-12D group, 3.32±0.27 μM in the SONOCA 180 group and 2.65±0.25 μM in the control group. Ultrasound treatment significantly enhanced nitric oxide production by epithelial cells compared to the control group, and the effects of ULSC-12D and SONOCA 180 were equivalent (Table 3, Fig. 3).