A Prospective, Randomized, Controlled, Clinical Study to Evaluate the Efficacy of High-frequency Ultrasound in the Treatment of Stage II and Stage III Pressure Ulcers in Geriatric Patients

Login toDownload PDF version
Ostomy Wound Manage. 2014;60(8):16–28.
Anna Polak, PT, PhD; Prof. Andrzej Franek, PhD, MSc; Prof. Edward Blaszczak, PhD, MSc; Agnieszka Nawrat-Szoltysik, PT, PhD; Prof. Jakub Taradaj, PhD, PT; Lidia Wiercigroch, PT; Pawel Dolibog, PhD, MSc; Magdalena Stania, PT, PhD; and Prof. Grzegorz Juras, PhD, PT


  International guidelines recommend high-frequency ultrasound (HFUS; MHz) for treating infected pressure ulcers (PUs). A 2-year, prospective, randomized, controlled study was conducted to evaluate how HFUS affects PU healing among 42 geriatric patients treated in four nursing and care centers in Silesia, Poland.

  Participants (age range 71–95 years,) all with wounds that did not respond to previous treatment for at least 4 weeks, were randomly assigned to the treatment group (TG) (20 with 21 PUs, mean age 83.60 ± 5.04 years) or control group (CG) (22 with 23 PUs, mean age 82.59 ± 6.65 years). All patients received standard wound care (SWC); the TG additionally was provided HFUS (1 MHz, 0.5 W/cm2, duty cycle of 20%, 1–3 minutes/cm2; one session per day, 5 days a week). Patients were monitored for 6 weeks or until wounds closed. Percent change in wound surface area (WSA), the Gilman’s parameter, the weekly rate of change in WSA, and the percentage of PUs that improved (ie, decreased in size by at least 50% or closed) were used to compare differences. Data were analyzed using Fisher’s exact test, the Wilcoxon matched pairs test, and the Mann-Whitney U test (P <0.05). Mean baseline WSA and the pretreatment duration of PUs were 15.38 ± 12.92 cm2 and 1.64 ± 0.73 months and 11.08 ± 7.52 cm2 and 2.26 ± 1.42 months in the TG and CG groups, respectively. After 6 weeks of treatment, the WSA of PUs decreased significantly in both groups (P = 0.000069 in the TG and P = 0.0062 in the CG) with significantly greater improvement in the TG (an average of 68.80% ± 37.23% compared with 37.24% ± 57.84%; P = 0.047). The value of the Gilman’s parameter was greater in the TG than in the CG (0.88 ± 0.62 and 0.43 ± 0.50, respectively; P = 0.018). The mean weekly change of WSA was greater in the TG than in the CG but only for Stage II PUs (3.09 ± 2.93 cm2/week and 1.08 ± 1.43 cm2/week; P = 0.045). More Stage II PUs in the TG decreased by at least 50% (11 of 14 = 78.57%) than in the CG (seven of 18 = 38.89%) (P = 0.035). In the TG, seven of 14 (50%) Stage II PUs closed, four of seven (42.86%) Stage III PUs decreased by at least 50%, and one of seven (14.29%) Stage III PUs closed; respective values for the CG are three of 18 (16.67%), three of five (60%,) and zero of five (0%) (P = 0.062, P = 0.999, P = 0.999, respectively). The study showed HFUS therapy can reduce the WSA of PUs regardless of their shape, but further research is necessary, particularly to establish how ultrasound influences the healing of Stage III and Stage IV PUs.

Potential Conflicts of Interest: The project was partly financed from the budget of the Jerzy Kukuczka Academy of Physical Education in Katowice.


  Pressure ulcers (PUs) are a serious condition particularly common among frail elderly people, making the development and implementation of effective therapies an important issue. PU therapies include ensuring adequate nutrition; providing preventive measures, modern dressings, and pharmacotherapy; and surgical procedures.1,2 When a PU does not respond to standard wound care (SWC), international guidelines2 recommend the use of physical modalities such as electrical stimulation, negative pressure wound therapy, ultraviolet radiation, electromagnetic field therapy, or ultrasound (US).

  Human wounds are managed with two types of US: low-frequency (LFUS), using 22.5 to 40 kHz3-5, and high-frequency (HFUS) using 1–3 MHz.5-18 International guidelines2 recommend treating clean recalcitrant Stage III and Stage IV PU with noncontact, LFUS spray (40 kHz). LFUS (22.5, 25, 35 kHz) is recommended as useful for debriding necrotic soft tissue (noneschar); HFUS (MHz) can be used as an adjunct in the treatment of infected PUs.2 These recommendations are based mainly on expert opinions and indirect evidence (studies on humans with chronic wounds other than PUs and animal models). This means sufficiently strong evidence from randomized clinical trials, allowing a reliable evaluation of the therapeutic impact of US on the healing of human wounds (including PUs), has yet to be provided.

  Preclinical research. According to Kloth and Niezgoda’s19 and Lancerotto et al’s20 reviews of in vivo and in vitro studies, mechanical forces generated by HFUS can stimulate signal transduction pathways in tissues and can produce a wide range of cellular effects that influence wound healing.

  The results of in vitro research show HFUS stimulates the activity of macrophages21 and fibroblasts and activates collagen synthesis.22,23 The results of in vivo research with animals show HFUS stimulates cell membrane conductivity24,25 and increases cell calcium concentrations,25 which may result in increased activity of cells important for wounds to heal.

  In in vivo research, Taskan et al26 studied experimental wounds in 16 rats receiving US and 12 rats in a sham US (control) group to determine how HFUS affected the density of polymorphonuclear leukocytes, macrophages, and fibroblasts, as well as collagen arrangement and mast cell biochemical strength. Sonotherapy (0.1 W/cm2, duty cycle of 20%, US frequency not stated) was started on the same day as wounding. A 5-minute US procedure (at 1 minute/cm2) was applied once a day for 7 days. On day 4, the researchers found statistically more macrophages in the wounds of the US group than the sham US group (P <0.05); the number of polymorphonuclear leukocytes and fibroblasts as well as collagen density were comparable. On day 7, the wounds in the US group continued to account for more macrophages (P <0.05) and significantly more fibroblasts (P <0.05) (intergroup differences between the amounts of polymorphonuclear leukocytes and collagen density were not evaluated). Taskan et al’s26 results confirm HFUS is capable of increasing the number of macrophages and fibroblasts in damaged tissue.

  Following the degranulation of neutrophils and macrophages, chemotactic agents and growth factors are released, stimulating pericytes, fibroblasts, and endothelial cells (ECs) to produce granulation tissue at the wound site. According to Young and Dyson’s in vitro study27 performed on U937 cells (an unstimulated form of macrophages that can be maintained in vitro), macrophages can be induced by therapeutic levels of US both to release mitogenic factors already resident in their cytoplasm (0.75 MHz US;0.5 W/cm2; 100% over 5 minutes) and to stimulate the cell’s ability to synthesize factors (both 3 MHz and 0.75 MHz US; 0.5 W/cm2; 100% over 5 minutes).

  Doan et al22 studied the influence of HFUS (1 MHz; 0.1, 0.4, 0.7, and 1.0 W/cm2; 20%) on cell proliferation, deoxyribonucleicacid (DNA), collagen and noncollagenous protein (NCP) synthesis, and cytokine and growth factor production (interleukin [IL]-1, IL-6, and IL-8); tumor necrosis factor  (TNF); basic fibroblast growth factor (bFGF); and vascular endothelial growth factor (VEGF) in fibroblasts in vitro. HFUS was found to increase DNA synthesis and cell proliferation in fibroblasts. In the US group, unlike the nonsonicated control group, the most significant results were obtained for 0.7 W/cm2 (P <0.01) and 1.0 W/cm2 (P <0.05). HFUS of 0.1, 0.4, and 0.7 W/cm2 also significantly boosted collagen and NCP synthesis (P <0.01, P <0.05, and P <0.01, respectively). Although HFUS stimulated IL-1, IL-8, and bFGF synthesis in fibroblasts, it did not change the IL-6 and TNF levels. HFUS did not significantly stimulate bFGF but significantly elevated VEGF, most effectively at 0.1 and 0.4 W/cm2 (in both cases P <0.01).

  Restoration of blood supply to tissue with impaired perfusion depends on spontaneous or mediated angiogenesis that includes mechanisms such as stimulation, migration, and proliferation of ECs. Raz et al’s28 in vitro study revealed a higher rate of EC proliferation in the sonicated groups; cell death was not observed. Experiments were performed with frequencies of 0.5, 1.0, 3.5, and 5.0 MHz at an intensity of 1.2 W/cm2. The cultured ECs were sonicated for 15 minutes with either continuous wave (CW) or pulsed wave (PW) mode at a duty cycle of 50%. Cell proliferation was evaluated every 24 hours after US irradiation for 3 days. The authors also examined cases of ECs sonicated at different intensities (eg, 0.8, 1.2, and 1.6 W/cm2) with 1-MHz CW US for 15 minutes. The proliferation rate was found to be independent of both US frequency and intensity. Two days (48 hours) after the 1-MHz CW sonication was applied for 15 minutes, the authors observed a higher rate of proliferation compared with that induced by the PW mode. However, after a longer time (72 hours) from the sonication, the differences in cell proliferation were insignificant for either mode. The authors concluded some type of a threshold to the total acoustic energy transfer to the cells may have been exceeded. Up to a certain level of cell proliferation depends on the total energy transferred to the cells; beyond that point, it is independent of the US mode.

  Demir et al29 performed a randomized, controlled study on the effects of US (0.5 W/cm2; duty cycle 20%; US frequency not stated) and laser (904 nm; 16 Hz; 6 mW average power; 1 J/cm2) treatments on wound (6-cm, full-thickness linear incisions) healing in 124 rats. US was found to promote wound healing in the inflammatory, proliferation, and maturation phases. An US 0.8-cm diameter (1.3 cm2) transductor was used to apply US to a 6-cm long wound for 5 minutes per day over a period of 10 days. The duration of the inflammatory phase decreased in both the laser and US groups compared with the control sham laser and sham US groups (P <0.05); laser treatment was more effective than US (P <0.05). The proliferation phase improved in the US and laser groups owing to higher levels of hydroxyproline and fibroblasts and because of greater stimulation of collagen synthesis than in the control groups (P <0.05). Laser also proved more effective than US with regard to wound breaking strength (P <0.05), which was significantly higher in the treatment group than in the control group (P <0.05). No statistically significant difference was found between the US and laser groups. Collagen density and arrangement were significantly better in the treatment groups than in the control groups (P <0.05), but the treatment groups were not significantly different from each other.

  PU clinical research. Only four clinical studies evaluating the effects of HFUS on PU healing have been published6,11,12,18 (see Table 1). Paul et al’s6 clinical study reported that among 23 spinal cord injury (SCI) patients with PUs, 13 wounds healed, five improved, and five showed equivocal responses. Wounds were treated with an US dosage of 0.5 and 1.0 W/cm2 (frequency and mode not mentioned) for 2 to 4 minutes a day, three times a week for 2 weeks. Unfortunately, very little can be concluded from the results of this study because of its numerous limitations: there was no control group and no information on PU severity, initial wound surface area (WSA), or wound duration.

  The design of Nussbaum et al’s11 randomized, controlled trial (RCT) also makes it difficult to draw conclusions about HFUS. The study compared nursing care alone, nursing care plus laser, and nursing care alternated with HFUS or ultraviolet C (UVC) regarding PU healing in 16 SCI patients with 18 wounds. The authors reported HFUS/UVC treatment had a greater effect on wound healing than nursing care either alone or combined with laser, but it is impossible to draw conclusions on the effect of HFUS treatment alone.

  ter Riet et al12 performed a high-quality RCT in 88 geriatric patients with Stage II through Stage IV PUs treated in two comparative groups — SWC+US group (45 patients) and SWC+sham US group (43 patients). HFUS (3.28 MHz, 0.1 W/cm2 spatial average temporal average, 20% duty cycle, 3 minutes/cm2) was applied five times a week for 12 weeks. A comparison of cumulative incidences of wound closure showed 18 of 45 (40%) PUs in the SWC+US group and 19 of 43 (44%) PUs in the SWC+sham US group were closed. Mean absolute healing rates were 0.18 and 0.13 cm2/week in the SWC+US group and SWC+sham US group, respectively (P = 0.18). Mean WSA reduction was 22.91% and 13.82% in the SWC+US group and SWC+sham US group, respectively (P = 0.10). The authors concluded results do not support the idea HFUS speeds up PU healing.

  In Maeshige et al’s pilot study,18 SWC+HFUS (1–3 MHz, 0.5 W/cm2, 20% duty cycle, 10 minutes a day, 5 days a week) was applied to three Stage III PUs and one Stage IV PU. Over 4 weeks, three PUs markedly decreased in size at a rate of 0.09 to 0.41 cm2/week. In the control group, SWC+sham US was applied to three Stage III to Stage IV PUs; not one PU decreased markedly in size. The author concluded the outcomes of the pilot study pointed to the positive effect of sonotherapy on the healing of Stage III and Stage IV PUs and provided grounds for further research.


  A prospective, randomized, controlled clinical study was designed specifically to test the hypothesis that HFUS administered as part of an interdisciplinary wound care program can improve the healing of Stage II and Stage III PUs in a geriatric population at high risk of PU development.

Methods and Procedures

  Ethical approval. The study was approved by the Bioethics Commission of the Jerzy Kukuczka Academy of Physical Education in Katowice (Resolution no. 10/2010 of 11 March 2010).

  Study enrollment. Patients with PUs screened for the study were the residents of four nursing and care centers in Silesia, Poland between January l, 2009 and September 30, 2011. Study eligibility was determined by the patient’s attending physician based on the following criteria: older than 70 years of age, presence of a Stage II or Stage III PU of at least 1.0 cm2 located on the trunk or in the buttock region, persisting for a minimum of 4 weeks. Patients with two PUs were eligible, and both PUs were evaluated for healing progress; patients with more than two PUs were deemed ineligible. Patients with deep, tunneling, necrotic wounds likely to involve osteomyelitis and requiring surgical intervention; neoplasm; lymphatic system diseases; central nervous system demyelinating diseases; or cirrhosis of the liver also were not eligible for study participation.

  Patient demographic information was obtained from standardized interviews with the patients, additional examinations of the patients, and from the history of concomitant diseases available in their medical documentation. Patients’ physical and mental conditions, activity, mobility, and incontinence were assessed using the Norton scale (a score <14 indicated a high risk of PU development). To assess the possibility of friction and shear and wound moisture, as well as sensory perception, physical activity, and mobility, the Braden Scale was applied (a score <16 pointed to a high risk of PU development). Patients’ nutritional status was identified by means of the Nutritional Risk Score (NRS—2002).30 Wound severity at enrollment was assessed according to National Pressure Ulcer Advisory Panel and European Pressure Ulcer Advisory Panel2 criteria: Stage II ulcers = partial-thickness loss of the dermis presenting as a shallow open ulcer with a red pink wound bed, without slough; Stage III ulcers = full-thickness tissue loss; subcutaneous fat may be visible but bone, tendon or muscle are not exposed.

  Allocation to main groups/randomization. After the selected patients or their legal guardians gave written consent to take part in the study, patients were randomly divided into the treatment group (TG) or control group (CG). The main investigator in charge of patients’ allocation to groups had 50 envelopes, each containing a piece of paper marked with A (TG) or B (CG). The envelopes were opened one-by-one in the presence of a physiotherapist, and the patient was directed to the appropriate study group.

  SWC program administered to both groups. All patients received treatment to prevent the development of additional PUs. Pressure-redistribution surfaces, devices, and pillows were provided as needed. A nurse repositioned patients who could not move unaided at least every 2 hours. Persons who could change position were asked to do so in order to relieve pressure on the ulcer area as often as they could.

  Blood analysis was performed to screen for nutritional status markers and metabolic disorders such as anemia (iron deficiency anemia or anemia of chronic disease), thyroid dysfunction, impaired glycemic control, dehydration, protein deficit, and hypoalbuminemia.

  Wounds were regularly assessed by the attending physician throughout the period of the study to select topical treatments to appropriately address moisture control, bacterial burden, and debridement needs; microbiological culture and sensitivity testing were provided. A comprehensive, interdisciplinary assessment was conducted by a team consisting of a physician, a nurse, a physical therapist, and a dietitian to develop a SWC program addressing the specific demands of each participant— eg, nutritional intervention, optimization of the wound dressing protocol, and incontinence management. The clinician caregivers were blinded to participant group.

  Patients in both groups received similar standard topical care, selected to address the needs of individual patients and to promote moist interactive healing. Wounds were first cleansed with 0.9% sodium chloride, potassium permanganateoroctenidine/phenoxyethanol; the ulcer base then was covered with a dressing. Wound dressings (regardless of the group) included nonadherent gauze pads, dressings moistened with 0.9% sodium chloride, hydrogel, solcoseryl, and calendulae anthodium extractum. If wound debridement was needed or infection was suspected, fibrinolysin/deoxyribonuclease, colistinum, and sulfathiazolumnatricum (only in the CG) were additionally administered.

  All immobilized patients received low-molecular-weight heparin (enoxaparin) as a standard therapy. Patients with elevated leukocyte levels were treated with antibiotics selected following microbiological culture and sensitivity testing of the PU swab.

  US. The TG was administered SWC in conjunction with HFUS. To generate the acoustic beam, the Intelect Advanced device (Chatanooga Group, Holbrook, NY USA) was used. The periwound and wound areas were stimulated through sterile US gel (Aquasonic, Parker, Fairfield, NJ USA). The effective radiating area (ERA) of the transducer was 4.0 cm2. The beam nonuniformity ratio was maximum 5:1.

  Pulsed-wave US of 1 MHz and a duty cycle of 20% were selected (pulse duration was 2 minutes and the interval between pulses was 8 minutes). The spatial average temporal peak intensity (SATP) was 0.5 W/cm2, and the spatial average temporal average intensity (SATA) — obtained by averaging intensity values over the “on” and “off” periods — was 0.1 W/cm2. When the pulsed-wave US is applied, the maximum intensity (SATP) occurs during the pulse and is zero when the sound is off. In this study, pulsed-wave US was used with SATP of 0.5 W/cm2 and a duty cycle of 20%, so SATA was 0.1 W/cm2.

  The same frequency (1 MHz) was selected in several other studies where US significantly improved the healing of venous leg ulcers (VLUs)5,8,13-16 and PUs.18 In all clinical studies,7-9,11-18 the pulsed mode of HFUS was used and the duty cycle was usually 20%.5,12-16,18 The current researchers’ decision to use 0.5 W/cm2 SATP was determined by the results of earlier preclinical and clinical studies. In in vitro study, Reher et al31 established the best intensity to promote cytokine release from monocytes, fibroblasts, and osteoblasts was 0.1 or 0.4 W/cm2. In a controlled study on induced wounds in Yucatan pigs, Byl et al32,33 found 1 MHz US with intensity of 0.5 W/cm2 (20% duty cycle) increased hydroxyproline synthesis, collagen deposition, wound breaking strength, and wound closure. The authors of several clinical studies5,8,13-16,18 concluded SATP of 0.5 W/cm2 contributed to a significant decrease in VLU area and a significantly faster healing rate.13 Moreover, the use of 0.5 W/cm2 SATP and 20% pulsed mode eliminated the thermal effects.19

  In the current study, patients were provided US for 1 to 3 minutes per cm2 of ulcer area (ie, 1 to 3 minutes per US probe area): 1 minute in week one, 2 minutes in week two, and 3 minutes between week three and the end of treatment. These times are consistent with the times used in other clinical studies, which range between 1 and 5 minutes/cm2.5-18

  Patients received HFUS once a day, 5 days in a week. The authors of other studies who also found HFUS to improve the healing of PUs used the same frequency of sessions.18

  Before and after each procedure, the US transducer was sterilized in a disinfectant solution. PUs were thoroughly cleansed with 0.9% sodium chloride solution in preparation for the procedure and then covered with the aforementioned dressings immediately afterwards.

  The healing progress of ulcers receiving SWC and SWC+US was monitored for 6 weeks or until wounds closed, whichever occurred first.

  Outcome measures.
  Primary outcome measures. Researchers sought to determine absolute average change in WSA (cm2) after treatment in relation to its baseline in both groups (showing how effective treatment was in particular groups); and the percentage change/decrease in WSA after 6 weeks of intervention with SWC and US+SWC (to compare changes in PU surface area between the groups).

  Secondary outcome measures. The Gilman’s parameter34,35 was calculated to ensure the comparability of healing progress regardless of wound shape. Wound healing rates in terms of average weekly change in wound area (cm2/week) were calculated to compare the groups. Also, the percentage of PUs where WSA was significantly smaller at the end of week 6 of intervention (by at least 50%), healed completely, or increased (WSA greater than the baseline) was determined. Additionally, patients were observed for the possible occurrence of negative effects.

  Data collection. Two measurements were performed in each group to establish each individual patient’s WSA (cm2): immediately before treatment and at the end of week 6. For PUs that closed before the end of week 6, the date of closure was recorded. Clinicians performing the measurements were blinded to participant’s group.

  WSA was determined using the same method employed in several previous clinical trials.13-16,36 Wound contours were copied using transparent film sheets then measured with the planimeter to establish the surface area of each wound. A digitizer (Mutoh Kurta XGT, Altek, Digitizer Technology Company, LLC, Redmond, WA USA) connected to a personal computer (C-GEO v. 4.0 Nadowski, Poland) was used to process and store resulting data. Measurement errors were addressed similar to the authors’ previous study.36

  The formulas used to calculate percentage reduction in WSA against the baseline, the weekly mean absolute healing rates (cm2/week), and the Gilman’s parameter are presented in Table 2.

  Statistical analysis. The statistical evaluation was performed by computer analysis using Statistica software (version 8.0, StatSoft Polska Sp. z o.o.). Patient characteristics were analyzed for normality of distribution using the Shapiro-Wilk W-test. Because in some cases the distribution was found not to be normal, the results of the experiment were verified with the nonparametric tests. The distributions of the characteristics also were tested for skewness, kurtosis, and modality. In all cases, skewness and kurtosis were smaller than 4 and the distributions were unimodal, so a mean and a standard deviation were adopted as a measure of central value and dispersion.

  The distribution homogeneity of patient characteristics was evaluated in both groups with the Fisher test for independence and the Mann-Whitney U test. The mean wound surface areas before and after treatment in particular groups were compared using the Wilcoxon signed-rank test. The Mann-Whitney U test was used to compare mean percentage changes in wound areas, the values of the Gilman’s parameter, and mean weekly change in WSA between the groups.The healing rates of Stage II and Stage III wounds that significantly improved (decreasing by at least 50%), closed, or worsened (their WSA exceeded its initial value) at the end of intervention (at 6 week) were calculated with the Fisher test.

  The level of significance in all statistical tests performed was P  0.05.


  Participant enrollment. Between January 1, 2009 and September 30, 2011, 85 patients with PUs were screened; 45 met the study inclusion/exclusion criteria. The remaining patients were excluded because they were <70 years old (nine patients) or their PU was too small in area (<1.0 cm2) or too short in duration (<1 month) (15 patients). Eight patients were in poor health: deep, tunneling, necrotic wounds likely involving osteomyelitis or surgical intervention were assessed in three patients, one had cirrhosis of the liver, two had a foot PU, one had neoplastic disease, and one refused to participate (see Figure 1).

  Three of the 45 patients selected dropped out before the minimum, 4-week treatment period because of deterioration in health and refusal to participate in the experiment (TG = one; CG = one) or death (CG = one).

  Patient and wound characteristics.
  Sample characteristics. The demographic and wound characteristics of the 42 patients that completed the experiment are presented in Tables 3 and 4. Participants comprised 36 (85.71%) women and six (14.28%) men, age range 71–95 years. Five patients (three in the TG and two in the CG) were obese (body mass index [BMI]>30), and three (all in the control group) were underweight (BMI <19).

  The risk of PU development assessed with the Norton Scale and the Braden Scale was <14 and <16, respectively, for all patients. Thirty-two patients (76.19%) were immobile or their mobility was severely limited (1–2 points on the Norton Scale). With regard to mental status, most patients (37; 88.09%) were in a stupor or confused (1–2 points on the Norton Scale).

  General atherosclerosis was diagnosed in 35 patients (83.33%), diabetes in 17 (40.48%), and paresis due to cerebral strokes in 12 (28.57%). Four patients (7.55%) affected by cerebral strokes also had diabetes mellitus.

  The patients had a total of 44 PUs, which ranged in size from 2.31 cm2 to 43.68 cm2 and included 32 Stage II (72.73%) and 12 Stage III (27.27%). Measurement errors caused by different wound shapes ranged from 2.7% (for PUs 60–70 cm2) to 37.9% (for PUs <1 cm2). Forty PUs (90.90%) were located in the buttock and hip region (ischial tuberosity, sacrum, coccyx, greater femoral trochanter) and four (9.09%) on the trunk. Two patients (4.54%) had two PUs each. PU duration was 1 to 6 months; most PUs (33; 75%) had been present <3 months.

  TG. The TG included 20 patients, 17 women and three men, mean age 85 (range 74–94) years, mean body mass 67.65 (range 45–120) kg. Three patients were obese (BMI >30), but none was underweight (BMI <19). Communication with 18 patients (90%) was very difficult or impossible; 14 patients (70%) could not change position by themselves. Nineteen patients (95%) had general atherosclerosis, nine (45%) had diabetes, and four (20%) had paresis caused by cerebral strokes. Two patients (10%) were diagnosed with both diabetes and cerebral strokes.

  The TG had a total of 21 PUs — 19 (90.48%) in the buttock region and two (9.52%) on the trunk; 14 (66.67%) were Stage II and seven (33.33%) Stage III. Pretreatment PU duration averaged 1.25 (range 1–3) months; most (18; 85.71%) were present <3 months. One patient (5%) had two PUs (both monitored for healing).

 CG. The control group included 22 patients — 19 women, three men — mean age 84 (range 71–95) years, mean body mass 60 (range 40–95) kg. Two patients were obese (BMI >30) and three were underweight (BMI <19). Logical communication with 19 patients (86.36%) was very difficult or impossible, 18 (81.82%) needed assistance to change position, 16 (72.73%) had general atherosclerosis, eight (36.36%) had diabetes, and eight (36.36%) had paresis caused by cerebral strokes. Two patients (13.64%) had both diabetes and a history of cerebral stroke.

 The CG had a total of 23 PUs — 18 (78.26%) Stage II and five (21.74%) Stage III. One patient (4.35%) had two PUs (both were monitored for healing). Most ulcers (21, 91.30%) were located in the buttock and hip regions, and two (8.7%) were on the trunk. Pretreatment average PU duration was 2.26 (range 1–6) months; most (15; 65.22%) were present < 3 months.

  Measured variables at baseline were not statistically significant between the two groups (see Table 3 and Table 4).

  PU outcomes by group. After 6 weeks of treatment, the mean WSA of PUs was significantly smaller in both groups, decreasing in the TG from 15.38 ±12.92 cm2 at baseline to 6.16 ± 8.26 cm2 after treatment (P = 0.000069) and in the CG from 11.08 ± 7.52 cm2 to 8.28 ± 8.79 cm2 (P =0.0062; see Table 5).

  Comparison of wound outcomes by group (see Table 6). The WSA of PUs decreased significantly more in the TG (68.80% ± 37.23%) than in the CG (37.24% ± 57.84%; P = 0.047). The Gilman’s parameter calculated for Stage II to Stage III PUs was significantly greater for the TG (0.88 ± 0.62) than for the CG (0.43 ± 0.50; P = 0.018).

  In absolute terms, the weekly decrease in the surface area of Stage II to Stage III PUs was faster in the TG (2.63 ± 2.49 cm2/week) than in the CG (1.52 ± 2.02 cm2/week), but the difference was not statistically significant (P = 0.07). However, calculations performed for different stage wounds showed the mean absolute healing rate for Stage II PUs was greater in the TG than in the CG (3.09 ± 2.93 cm2/week and 1.08 ± 1.43 cm2/week, respectively; P = 0.045). In the CG, the mean weekly healing rate for Stage III PUs was higher than in the TG (3.44 ± 3.27 cm2/week and 1.70 ± 0.82 cm2/week, respectively), but the difference was not statistically significant (P = 0.65). It should be noted this result was obtained with a very small sample of Stage III PUs (only seven in the TG and five in the CG).

  The percentage of Stage II PUs by which the WSA decreased following treatment by at least 50% was greater in the TG (11 of 14, 78.57%) than in the CG (seven of 18, 38.89%) (P = 0.035). The percentage of Stage II PUs that closed also was greater in the TG (seven of 14, 50%) than in the CG (three of 18; 16.67%) but the difference was not statistically significant (P = 0.062) (see Figures 2 and 3).

  In the TG, the WSA of four of seven (42.86%) Stage III PUs decreased by at least 50% versus three of five (60%) in the CG. Only one of seven (14.29%) Stage III PUs closed in the TG and none of five (0%) in the CG. In neither case were the differences statistically significant (P = 0.999).

  One PU increased in size in the TG over the 6 weeks of the experiment compared with three in the CG (P = 0.61).


  The results of this study confirmed the hypothesis HFUS can enhance the healing of PUs. The treatment of Stage II and Stage III PUs using HFUS or SWC proved effective in both groups of patients, but ulcers receiving HFUS along with SWC decreased more in size. The values of the Gilman’s parameter showed PUs treated with US+SWC decreased more regardless of their shape than those treated with SWC alone. The weekly healing rates of PU treated with SWC+HFUS were better than of ulcers receiving SWC alone, but the difference was statistically significant only for Stage II PUs; after 6 weeks of treatment, more Stage II PU treated with SWC+HFUS than Stage II PUs receiving only SWC decreased in surface area by at least 50%.

  Current findings are consistent with the studies of Paul et al6 and Maeshige et al18 that established the concurrent use of HFUS and SWC promotes PU healing. ter Riet et al12 also used HFUS in combination with SWC to treat PUs but did not find differences in healing over SWC alone. However, the PU in the ter Riet et al12 study were small (42.2% were <1 cm2), and 55.6%, varied in area from 1.1 to 10 cm2, unlike in the current study where 42.86% of PUs measured in that range. Also, seven5,7,8,13-16 of 10 clinical studies5,7-10,13-17 involving VLUs noted the positive effect of HFUS.

  In the current study, HFUS was applied to 20 patients with a total of 21 PUs. Only ter Riet et al12 used HFUS to treat a larger group of PU patients (N = 45). Paul et al6 included 23 patients, but his clinical experiment was carried out without a control group. In other studies, HFUS was applied in small groups of patients — five in Nussbaum et al11 and three in Maeshige et al.18

  Current study patients ranged in age from 71 to 94 years (mean 83.60 in the TG and 82.59 in the CG). Most patients suffered typical geriatric comorbidities and could not change position on the bed without help. The patients in the ter Riet et al12 and Meashige et al18 studies were similar in age to the current study population (mean 82, range 79–87 years and mean 84, range 76–92 years, respectively). Nussbaum et al11 administered HFUS to SCI patients with a mean age of 42.2 (range 26–59) years. Paul et al6 also treated SCI patients, but their research report omits their ages.

  Patients in the current study had Stage II and Stage III PU. Other studies addressed PUs from Stage II to Stage IV.12,18 In the current study, patients were diagnosed with PUs in the buttock region (ischia tuberosity, sacrum, coccyx), and trunk. Nussbaum et al11 and Maeshige et al18 treated PUs in the buttock region and legs. In ter Riet et al’s study,12 PUs were located on patient’s trunk. Paul et al6 did not report PU locations.

  Because geriatric patients in the current were affected by concomitant conditions, increasing the risk of PU development and impeding their healing, their wounds were not expected to heal completely over the 6 weeks of intervention. Therefore, the primary outcome of wound healing was anticipated to be a percentage decrease in WSA against baseline. The authors of other clinical studies evaluating wound treatment efficacy5,10-16,37 used the same approach to assess wound size changes. In an epidemiological report38 that compared various methods of expressing wound size changes, percent decrease in wound size consistently was recommended as the reliable endpoint in clinical trials. This parameter accounts for large variances in actual wound size, normalizes data based on the initial wound size, and is known to be a strong predictor of ultimate wound closure.

  In the HFUS group in the current study, the baseline area of Stage II and Stage III PUs (15.38 cm2 on average) decreased 68.8% after 6 weeks of treatment. In ter Riet et al’s study,12 after 12 weeks Stage II to Stage IV PUs treated with HFUS decreased in area by 22.91%; the result was not significantly different from the control group (13.81%; P = 0.10). Authors of other studies6,18 who applied HFUS to treat PU did not state percentage changes in WSA.

  However, researchers who applied HFUS to treat VLUs found significant decreases in the surface area. In a controlled, clinical study, Callam et al8 treated VLUs (14.5 cm2 at the baseline) for 12 weeks and found WSA decreased 91% in the HFUS+SWC group (versus 73% in the SWC group; P = 0.02); and Roche and West,7 in a controlled, clinical trial, showed a 35.4% reduction in WSA (32.51 cm2 at the baseline) in the HFUS+SWC group after 4 weeks of treatment (9.7% in the control group; 23.62 cm2 at the baseline; P <0.01).

  The Gilman’s parameter showed HFUS+SWC decreased PU surface area more than SWC alone, regardless of wound size. Other authors who applied HFUS to treat VLUs made the same observation.15,16 Percentage changes in WSA and Gilman’s parameter values obtained in the current study and by other authors indicate that HFUS effectively reduces WSA.

  Additionally, HFUS may help speed WSA decrease. In the current study, HFUS applied in conjunction with SWC raised the healing rate of Stage II PUs to 3.09 cm2/week, compared with 1.08 cm2/week using SWC alone. Maeshige et al18 found the healing rate of Stage III and Stage IV PUs treated with HFUS+SWC ranged from 0.09 to 0.41 cm2/week and was faster than in the case of SWC alone. Unfortunately, he did not mention how fast PU changed in the control group (SWC+sham HFUS). ter Riet’s study12 did not confirm HFUS significantly changes PU healing rate (0.18 cm2/week in the HFUS+SWC group versus 0.31 cm2/week in the sham HFUS+SWC group; P = 0.09), but Franek et al13 found HFUS improves the healing rate of VLUs; in his study, VLUs treated with HFUS+SWC (1 MHz; 0.5 W/cm2; 20%) healed at a rate of 19.2% a week, whereas in the SWC group the rate was 9.6% (P <0.05). Further clinical research is necessary to determine if HFUS can accelerate wound healing rates, particularly in Stage III and Stage IV PUs.

  The chronic wound healing process is complicated and subject to endogenous and exogenous factors at each stage, so it is difficult to predict when a wound may close. In the current study, seven (50%) of 14 Stage II PUs and one of seven Stage III PUs treated with HFUS+SWC closed and 11 (78.57%) of 14 Stage II PUs and four of seven Stage III PUs decreased in size by at least 50%. These posttreatment observations suggest HFUS therapy must be applied longer than 6 weeks for Stage II and Stage III PUs in this patient population in order to achieve a 50% decrease or close. In a RCT with VLUs, Olyaie et al5 reported all wounds healed after an average of 8.50 months (SD 2.17), 6.86 months (SD 2.04), and 6.65 (SD 1.59) months in the SWC, HFUS+SWC, and LFUS+SWC groups, respectively (P = 0.001); the baseline sizes of VLUs in these groups were 9.60 cm2, 9.86 cm2, and 10.01 cm2, respectively. No studies indicating how long HFUS should be applied for Stage II to Stage IV PUs to heal completely have yet been published.

  Methodology of HFUS research. The authors of most studies found HFUS to have a positive effect on wound healing,5-8,11,13-16,18 but some failed to document ceratin aspects of therapy.9,10,12,17 In the current study, patients received 1 MHz HFUS. Kloth and Niezgoda19 recommend using 1 MHz to treat tissue located deeper than 1–2 cm from skin surface and 3 MHz frequency when the treatment focuses on superficial tissue within 1–2 cm of skin surface or on places where bone is close to skin surface. In the current study, PUs did not tunnel to the bone, negating the need to use 3 MHz frequency. Most authors of clinical studies also choose one frequency (1 or 3 MHz) to treat all wounds; 1 MHz frequency was used in several studies where US significantly improved the healing of VLUs5,8,13-16 and PUs.18 Maeshige et al,18 who applied 3 MHz to PUs located close to the bone, is the only author to have used both frequencies.

  Like other researchers, current study authors used pulsed mode US with SATP 0.5 W/cm2 and duty cycle 20% (therefore, the SATA was 0.1 W/cm2). These US parameters prevented the occurrence of thermal effects.19 The same parameters were successfully used by Maeshige et al18 who treated PUs and by Franek et al13,14 and Taradaj et al15,16 who managed VLUs. Franek et al13 additionally compared the effects of 0.5 W/cm2 and 1 W/cm2 SATP (1 MHz; a duty cycle of 20% in both cases), concluding the former helped wounds heal significantly faster than 1 W/cm2 (P = 0.0001) and SWC administered to the control group (P = 0.008). Kloth and Niezgoda19 likewise recommend US intensity of 0.5 W/cm2 (duty cycle of 20%), particularly in the reparative and early remodelling phases of wound healing. This recommendation is founded on the results of in vivo studies with animals obtained by Byl et al32,33 in which US intensity of 0.5 W/cm2 (duty cycle of 20%) increased hydroxyproline level, collagen deposition, wound breaking strength, and wound closure. Reher et al’s31 in vitro study asserts the best intensity to promote cytokine release from monocytes, fibroblasts, and osteoblasts is 0.1 or 0.4 W/cm2. Maeshige et al39 observed in vitro that US intensities of 0.1, 0.5, and 1 W/cm2 (duty cycle of 20%) promote alpha-smooth muscle actin and transforming growth factor-beta 1 expression in human dermal fibroblasts, which may substantially improve the healing of chronic wounds.

  In the studies analyzed, US was applied to the wound area or periwound area for 1 minute,8,9 3 minutes,12 or 5 minutes/cm2.11 Kloth and Niezgoda19 recommend applying US to wound area two times as large as the surface of the applicator for 5 minutes (approximately 2.5 minutes per 1 cm2 of wound area). No guidelines specify how long a wound should be sonicated. In the current study, US was applied for 1–3 minutes/cm2 of wound area, which falls in the range of sonication times used by other researchers. In week 1, US was applied for 1 minute/cm2, in week 2 for 2 minutes/cm2, and between week 3 and the end of treatment for 3 minutes/cm2. The results of Raz et al’s28 in vitro study indicate US effects depend on total energy delivered to tissue. The same dose of US delivered to tissue may bring about stronger or weaker results depending on whether the delivery time is longer or shorter. The current authors chose to start with shorter sonication times and gradually lengthen them only if wound deterioration was not observed (eg, strong inflammatory reactions). If no negative symptoms were noted, procedures were extended from 1 to 3 minutes/cm2 for all patients.

  Patients in the current study received HFUS once a day, 5 times a week. The same approach was used in other studies. Interestingly, a positive effect of HFUS on wound healing treatments was observed in all studies where it was administered at least 3 times a week,5-7,11,13-16,18; where the patients were treated once17 or twice a week,10 improved wound healing was not observed.


  This study lacked patient blinding and the nonapplication of sham US to the control group, which limit the overall applicability of its findings. In addition, patients were recruited from four different medical centers, increasing the likelihood of different overall PU care.


  A prospective, randomized, controlled clinical study has shown that US, delivered at 1 MHz, 0.5 W/cm2 SATP, duty cycle of 20%, applied to Stage II and Stage III PU for 1–3 minutes per 1 cm2 of wound area once a day, five times a week, can reduce WSA more effectively than standard care alone. This conclusion applies to wounds of all shapes. These findings are consistent with the results of other research in which HFUS was found to promote wound healing. Additional clinical research to identify how HFUS contributes to the healing, particularly of Stage III and IV PUs, is warranted. Clinical studies also should aim to establish the US frequency, intensity, and duration to establish guidelines for protocols utilizing US therapy.

Dr. Polak is an Assistant Professor, Chair of Physical Therapy, Academy of Physical Education, Katowice, Silesia; and Assistant Professor, Department of Physical Therapy, Katowice School of Economics, Katowice, Poland. Prof. Franek is a Professor of Medical Sciences and Head of the Department of Biophysics; and Prof. Blaszczak is a statistical analyst and Professor, Department of Medical Biophysics, Medical University of Silesia, Katowice, Poland. Dr. Nawrat-Szoltysik is an assistant, Department of Physiotherapy in Disorders of Nervous and Locomotor System, Academy of Physical Education, Katowice; and a physiotherapist in a skilled nursing home, Ruda Slaska, Poland. Prof. Taradaj is a Head of the Department of Physical Therapy, Academy of Physical Education, Katowice; and Professor, Institute of Physical Therapy, Public School of Medicine, Opole, Poland. Ms. Wiercigroch is a physiotherapist and an assistant in Department of Biophysics; and Dr. Dolibog is an Assistant Professor, Department of Medical Biophysics, Medical University of Silesia, Katowice. Dr. Stania is a physiotherapist and an assistant, Department of Physical Therapy; and Prof. Juras is a statistical analyst and Head of the Department of Motor Behavior, Academy of Physical Education, Katowice. Please address correspondence to: Anna Polak, PT, PhD, Academy of Physical Education, Physical Therapy, Mikolowska 72A, Katowice, Silesia 40-065, Poland; email: a.polak@awf.katowice.pl; polanna@op.pl.


1. Consortium for Spinal Cord Medicine Clinical Practice Guidelines. Pressure ulcer prevention and treatment following spinal cord injury: a clinical practice guideline for health-care professionals. J Spinal Cord Med. 2001;24(suppl 1):S40–S101.

2. European Pressure Ulcer Advisory Panel and National Pressure Ulcer Advisory Panel. Treatment of pressure ulcers: Quick Reference Guide. Washington DC: National Pressure Ulcer Advisory Panel; 2009. Available at: www.npuap.org. Accessed August 1, 2014.

3. Kavros SF, Schenck EC. Use of noncontact low-frequency ultrasound in the treatment of chronic foot and leg ulcerations: a 51-patient analysis. J Am Podiatr Med Assoc. 2007;97(2):95–101.

4. Conner-Kerr T, Alston G, Stovall A, Vernon T, Winter D, Meixner J, et al. The effects of low-frequency ultrasound (35 kHz) on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. Ostomy Wound Manage. 2010;56(5):32–43.

5. Olyaie M, Rad FS, Elahifar MA, Garkaz A, Mahsa G. High-frequency and noncontact low-frequency ultrasound therapy for venous leg ulcer treatment: a randomized, controlled study. Ostomy Wound Manage. 2013;59(8):14–20.

6. Paul BJ, Lafratta CW, Dawson AR, Baab E, Bullock F. Use of ultrasound in the treatment of pressure sores in patients with spinal cord injury. Arch Phys Med Rehabil. 1960;41:438–440.

7. Roche C, West J. A controlled trial investigating the effect of ultrasound on venous ulcers referred from general practitioners. Physiotherapy. 1984;70(12):475–477.

8. Callam MJ, Harper DR, Dalle JJ, Ruckley CV, Prescott RJ. A controlled trial of weekly ultrasound therapy in chronic leg ulceration. Lancet. 1987;2(8552):204-206.

9. Lundeberg T, Nordstrom F, Brodda-Jansen G, Eriksson SV, Kjartansson J, Samuelson UE. Pulsed ultrasound does not improve healing of venous ulcers. Scand J Rehab Med. 1990;22(4);195-197.

10. Eriksson SV, Lundeberg T, Malm M. A placebo controlled trial of ultrasound therapy in chronic leg ulceration. Scand J Rehab Med. 1991;23(4):211–213.

11. Nussbaum EL, Biemann I, Mustard B. Comparison of ultrasound/ultraviolet-C and laser for treatment of pressure ulcers in patients with spinal cord injury. Phys Ther. 1994;74(9):812–825.

12. ter Riet G, Kessels AGH, Knipschild P. A randomized clinical trial of ultrasound in the treatment of pressure ulcers. Phys Ther. 1996;76(12):1301–1311.

13. Franek A, Chmielewska D, Brzezinska-Wcislo L, Slezak A, Blaszczak E. Application of various power densities of ultrasound in the treatment of leg ulcers. J Dermatol Treat. 2004;15(6):379–386.

14. Franek A, Krol P, Chmielewska D, Blaszczak E, Polak A, Kucharzewski M, Taradaj J. The venous ulcer therapy in use of the selected physical methods (Part 2) —the comparison analysis. Pol Mer Lek. 2006:20(120):691-695.

15. Taradaj J, Franek A, Cierpka L, Brzezinska-Wcislo L, Blaszczak E, Polak A, et al. Early and long-term results of physical methods in the treatment of venous leg ulcers: randomized controlled trial. Phlebology. 2011;26(6): 237–245.

16. Taradaj J, Franek A, Brzezinska-Wcislo L, Cierpka L, Dolibog P, Chmielewska D, et al. The use of therapeutic ultrasound in venous leg ulcers: a randomized, controlled clinical trial. Phlebology. 2008;23(4):178-183.

17. Watson JM, Kang’ombe AR, Soares M, Chuang LH, Worthy G, Bland JM, et al. Use of weekly, low dose, high frequency ultrasound for hard to heal venous leg ulcers: the VenUS III randomized controlled trial. BMJ. 2011;342:d1092.

18. Maeshige N, Fujiwara H, Honda H, Yoshikawa Y, Terashi H, Usami M, et al. Evaluation of the combined use of ultrasound irradiation and wound dressing on pressure ulcers. J Wound Care. 2010;19(2):63–68.

19. Kloth LC, Niezgoda JA. Ultrasound for wound debridement and healing. In: McCulloch JM, Kloth LC, eds. Wound Healing. Evidence-based Management, 4th Ed. Philadelphia, PA: FA Davis Company;2010:545–575.

20. Lancerotto L, Orgill DP. Mechanoregulation of angiogenesis in wound healing. Adv Wound Care. In press;2014.

21. Young SR, Dyson M. Macrophage responsiveness to therapeutic ultrasound. Ultrasound Med Biol. 1990;16(8):809–816.

22. Doan N, Reher P, Meghji S, Harris M. In vitro effects of therapeutic ultrasound on cell proliferation, protein synthesis, and cytokine production by human fibroblasts, osteoblasts, and monocytes. J Oral Maxillofac Surg. 1999;57(4):409–419.

23. Zhou S, Schmelz A, Seufferleins T, Li Y, Zhao J, Bachem MG. Molecular mechanisms of low intensity pulsed ultrasound in human skin fibroblasts. J Bio Chem. 2004;279(52):54463–54469.

24. Dinno MA, Crum LA, Wu J. The effect of therapeutic ultrasound on electrophysiological parameters of frog skin. Ultrasound Med Biol. 1989;15(5):461–470.

25. Al-Karmi AM, Dinno MA, Stoltz DA, Crum LA, Matthews JC. Calcium and the effects of ultrasound on frog skin. Ultrasound Med Biol. 1994;20(1):73–81.

26. Taskan I, Ozyazgan I, Tercan M, Kardas HY, Balkanli S, Saraymen R, et al. A comparative study of the effect of ultrasound and electrostimulation on wound healing in rats. Plast Reconstr Surg. 1997;100(4):966–972.

27. Young SR, Dyson M. The effect of therapeutic ultrasound on angiogenesis. Ultrasound in Med Biol. 1990;16(3):261–269.

28. Raz D, Zaretsky U, Einav S, Elad D. Cellular alterations in cultured endothelial cells exposed to therapeutic ultrasound irradiation. Endothelium. 2005;12(4):201–213.

29. Demir H, Yaray S, Kirnap M, Yaray K. Comparison of the effects of laser and ultrasound treatments on experimental wound healing in rats. J Rehabil Res Dev. 2004;41;5(5):721–728.

30. Kondrup J, Rasmussen HH, Hamberg O, Stanga Z, Ad hoc ESPEN Working Group. Nutritional risk screening (NRS 2002): a new method based on an analysis of controlled clinical trials. Clin Nutr. 2003;22(3):321–326.

31. Reher P, Doan N, Bradnock B, Meghji S, Harris M. Effect of ultrasound on the production of IL-8, basic FGF and VEGF. Cytokine. 1999;11(6):416–423.

32. Byl NN, McKenzie AL, West JM, Whitney JD, Hunt TK, Scheuenstuhl HA. Low-dose ultrasound effects on wound healing: a controlled study with Yacatan pigs. Arch Phys Med Rehabil. 1992;73(7):656-564.

33. Byl N, McKenzie A, Wong T, West J, Hunt TK. Incisional wound healing: a controlled study of low and high dose ultrasound. JOSP. 1993;18(5):619–628.

34. Gilman TH. Parameter for measurement of wound closure. WOUNDS. 1990;2(3):95–101.

35. Gilman TH. Comparing healing rates across studies is the vision, but first, a correct equation please! Ostomy Wound Manage. 1995;41(1):6–7.

36. Franek A, Kostur R, Polak A, Taradaj J, Szlachta Z, Blaszczak E, et al. Using high-voltage electrical stimulation in the treatment of recalcitrant pressure ulcers: results of a randomized, controlled clinical study. Ostomy Wound Manage. 2012;58(3):30–44.

37. Houghton PE, Campbell KE, Fraser CH, Harris C, Keast DH, Potter PJ, et al. Electrical stimulation therapy increases rate of healing of pressure ulcers in community-dwelling people with spinal cord injury. Arch Phys Med Rehabil. 2010;91(5):669-678.

38. Cardinal M, Eisenbud DE, Philips T, Harding K. Early healing rates and wound area measurements are reliable predictors of later complete wound closure. Wound Repair Regen. 2008;16(1):19-22.

39. Maeshige N, Terashi H, Aoyama M, Torii K, Sugimoto M, Usami M. Effect of ultrasound irradiation on -SMA and TGF-1 expression in human dermal fibroblasts. Kobe J Med Sci. 2010;56(6):E242-E252.