A prospective pilot study of ultrasound therapy effectiveness in refractory venous leg ulcers

Abstract

Venous insufficiency is the most common cause of leg ulcers in the United States. Venous leg ulcers cost the health care system billions of dollars annually, and healing rates are less than 70% with standard of care; therefore, new therapies are needed to increase healing times and minimize associated costs. Non contact ultrasound therapy has been used to treat a variety of chronic wounds including venous leg ulcers, and it is thought that ultrasound has an effect on decreasing the bacterial count in wounds, although the exact mechanism of action of ultrasound is yet to be determined. We conducted an open labelled pilot study of 10 refractory venous ulcers of large size to determine the effect of non contact ultrasound on wound closure, bacterial counts, expression of inflammatory cytokines and pain reduction. We lacked a sham control group but we compared the baseline and end of treatment assessments and noted the differences. We found a significant reduction in wound area (P = 0·0039) over the 4‐week treatment period. We also found a decline in individual and total bacterial counts; however, these differences were not significant. For all patients, there was also a trend toward reduced inflammatory cytokine expression compared with baseline levels; however, this reduction did not reach statistical significance. Interestingly, there was a correlation between healing and change in cytokine expression, which showed statistically significance for tumour necrosis factor (TNF)‐αP = 0·0395, IL‐1a P = 0·0351, IL‐6 P = 0·0508, IL‐8 P = 0·0990. Pain as measured by the visual analogue scale (VAS) was reduced from 4 at the baseline to 2·7 by the end of the study. In conclusion, we found that patients treated with ultrasound therapy and compression therapy show clinical improvement over the course of 4 weeks and had a decrease in inflammatory cytokines, bacterial counts and pain.

INTRODUCTION

Venous ulcers are the most common cause of leg ulcer with approximately 70% of leg ulcers due to venous insufficiency (1). The refractory nature of venous leg ulcers is associated with many economic and quality of life considerations. For example, the expenses of treating venous leg ulcers are about 2% of the health care budget in some European countries (2), and recently, we reported that the presence of chronic wounds puts patients at increased risk of death (3). It has been shown that long duration and large size of the venous ulcers impact the prognosis of wound healing and determines whether a patient is likely to heal with compression therapy alone or if the patient may need adjuvant therapies (4). While compression therapy is the gold standard for treating venous leg ulcerations, compliance issues exist. Moffatt et al. found rates of non compliance that ranged from 2% to 42% of patients enrolled in randomized controlled trials, with greater degree of non compliance in real‐world patients, ranging from 9·7% to 80% (5). Adjuvant therapies have been developed to speed healing of venous ulcers but there is a need for better and cost effective approaches.

The use of non contact ultrasound has been used to treat various types of chronic wounds including venous leg ulcers. Although the mechanism of action of ultrasound therapy is not clearly known, it has been proposed to work through reduction of bacterial bioburden 6, 7, 8 and mechanical debridement.

Our study evaluated refractory venous leg ulcers during treatment with non contact ultrasound therapy. The primary objectives were to determine the change in wound size and change in bacterial burden over a 4‐week treatment course. We also evaluated the change in gene expression for various inflammatory cytokines and explored relationships between wound size, bacteria burden and inflammation in those wounds that healed and did not heal. Change in pain as reported by the patient was also recorded by use of the visual analogue scale (VAS).

MATERIALS AND METHODS

Participants and study visit

After Institutional Review Board approval, we recruited patients with a refractory venous leg ulcer, defined by presence of at least 6 months, at least 5 cm² in size and also the failure to improve in the previous 30 days with supervised multilayered compression bandages (4). Venous insufficiency was confirmed clinically and with non invasive venous studies. Patients with an ankle–brachial index >0·7, albumin >2·1 mg/dl and pre‐albumin >5 mg/dl were included. Exclusion criteria were the use of topical antibiotics, systemic anticoagulation, diagnosed deep vein thrombosis within the last 6 weeks, malignancy in the wound bed or the aetiology of the wound was not venous.

Patients were seen three times a week for 4 weeks. The duration of each therapy depended on the size of the wound, the minimum treatment time was 3 minutes for a wound of <10 cm² and the treatment time increased 1 minute for every 10 cm² increase in wound size. Wounds were covered with a foam dressing plus a four layered elastic compression system. The wound size was captured by measuring the greatest length × width. VAS score for pain was completed, and wounds were photographed every week. Biopsies (2 mm) for quantitative culture and inflammatory cytokine measurements from the wound bed were obtained at baseline and at the end of the study, unless the wound was healed.

Ultrasound therapy

We used the MIST Therapy® System (Celleration Inc., Eden Prairie, MN) as ultrasound therapy; it delivers ultrasound acoustic waves through a sterile saline steam. This system comprises a generator that has an electric input; it is connected to a transducer cable that is assembled with the treatment head or applicator, which has the ultrasound control button. Adapted to the end of this applicator, there is a saline water bottle. The applicator has a leading edge from which the saline mist comes from. The treatment time depended on the size of the wound that is set in the entry screen in the machine, for example for a wound size 10–20 cm² the irrigation time will be 4 minutes. The same physician operated the machine every time and placed the leading edge about 2 cm away from the wound.

Wound biopsies

Punch biopsies (2 mm) were taken twice during the study, once before first treatment and once right after for last treatment in week 4 unless the wound was healed. After the biopsies were taken, biopsies were stabilized by immediate immersion in RNA later stabilization solution (Qiagen Inc., Valencia, CA) before processing in the laboratory.

Tissue digestion, cell lysis and RNA purification

Stabilized biopsies were removed aseptically from the stabilization solution and incubated in Proteinase K solution [120 µl of buffer (30 mM Tris, 10 mM EDTA (ethylenediaminetetraacetic acid), 1% SDS (sodium dodecyl sulfate), pH 8) + 30 µl of Proteinase K (Qiagen Inc.)] for 30 minutes at 37° C for extracellular matrix degradation. Tissue was then digested in lysis buffer [containing mercaptoethanol and included in ‘RNeasy mini kit' (Qiagen Inc.)], and total RNA was purified from each sample individually. Manufacturer's suggested protocol was used from this point on including on‐column DNA digestion with RNase free DNase (Qiagen Inc.). RNA was eluted in 100 µl of nuclease free water and adjusted to 5 ng/ µl final RNA concentration.

Real‐time onestep RT‐PCR reactions

All reactions were conducted in a final volume of 20 µl containing 20 ng of total RNA. Reactions contained 1× concentration of Bio‐Rad iScript Onestep reverse transcriptase‐polymerase chain reaction (RT‐PCR) mix for use with Taqman probes (Bio‐Rad Inc., Hercules CA) and 1× of the appropriate primer/probe mix (Applied Biosystems Inc., Foster City, CA).

Expression analysis and normalization

Samples were assessed for the expression of inflammatory and anti‐inflammatory cytokines involved in local acute phase reactions, cellular chemotaxis and activation. Specifically, studied genes were interleukin (IL)‐1α, IL‐6, IL‐8, IL‐10, IL‐11, tumour necrosis factor (TNF)‐α and vascular endothelial growth factor (VEGF). For accurate normalization of target genes, the relative quantity of the expression of the target genes was divided by the relative quantity of the expression of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) from the same sample.

Statistical analyses

Descriptive statistics were completed for wound size change, bacterial count and cytokine expression from baseline to last follow‐up, we present values for means and standard deviations. P values were calculated to assess the significance in the changes that we observed. Pearson's correlation was completed for wound size change and cytokine expression, correlation coefficient and P values were obtained for each cytokine in relation with wound size change. A P value <0·05 was considered to be statistically significant.

Visual analogue scale

We assessed pain every week using a VAS, a validated tool which has been routinely used to measure pain (9). Study subjects were asked to draw a perpendicular line along a continuous horizontal line between two end‐points being the first point 0 (0 = no pain) and the second point 10 (10 = worst pain ever experienced). The score where the perpendicular line intersected was recorded and was used to assess the level of pain.

RESULTS

Study group

Totally 11 patients were screened and coded VLU‐01 to VLU‐11. One subject failed screening because the patient had an active wound infection and started antibiotic therapy. Ten patients were enrolled and nine completed 4 weeks. Ten patients were evaluated for wound area; one patient, healed at week 3 and end of study biopsy was not obtained for that week. Nine patients were evaluable for bacterial count. There were seven of nine samples of sufficient quality from the tissue biopsy to process the cytokine analysis as two samples had insufficient volume to be processed. Clinical assessments were documented with photography and improvement was found in all subjects (Figure 1).

Figure 1.

Clinical presentation of venous leg ulcers baseline and post treatment with ultrasound therapy.

Wound area

Overall, wound size mean area was 38·3 cm² at the baseline, and it was reduced to 29·0 cm² at last follow‐up visit. Overall, there was a statistically significant reduction (45% mean reduction) (P = 0·0039, Figure 2).

Figure 2.

Wound size at baseline and post treatment with ultrasound therapy. Area by length × width showed a percentage reduction of 42% between baseline and end of the study P = 0·0039.

Bacterial load

Nine sets of samples, one per patient corresponding to baseline and end of the study tissue biopsies were evaluated, as one subject healed and no end of study sample was available, Staphylococcus aureus was the most prevalent species, being present in 9/9 samples, Pseudomona aeruginosa was found in 6/9 samples and Kocuria kristinae was present in 5/9 samples. We found that these last two bacteria had a reduction in their mean but this was not statistically significant in this group of patients. The overall bacterial profile was unchanged over the trial period (Figure 3).

Figure 3.

Bacterial count change from tissue biopsies of baseline and post treatment with ultrasound therapy. Comparison between baseline and end of study: S. aureus P‐value = 0·8203. P. aeruginosa P‐value = 0·8432. K. kristinae P‐value = 0·81.

Inflammatory cytokines profile

Gene expression analysis of inflammatory cytokines changed from baseline to the last follow‐up. TNFα and IL‐1 that are potent inflammatory cytokines had a marked decrease from the first to the last assessment. Cytokines such as IL‐6, IL‐8, IL‐11 and VEGF also decreased from the baseline to the end of study biopsies, IL‐10 increased during the course of treatment (Figure 4).

Figure 4.

Change in cytokines expression in venous leg ulcers from baseline and post treatment with ultrasound therapy. Comparison before and after MIST therapy in cytokine expression, look at the trend to decrease in the cytokines except for IL‐10.

Correlation between healing and cytokine expression change

Correlation analysis between change in wound area and change in cytokine expression from baseline and final assessment was performed, and a negative correlation was found, this correlation showed increased healing as cytokine expression decreased. This correlation showed to be statistically significant for TNFα, IL‐1a, IL‐6, IL‐8, IL‐11 and VEGF (Table 1).

Table 1.

Correlation between healing as measured in change in wound area and change in cytokine variables ^*

Change variable (SAS variable name)	Pearson correlation
Cytokines	r	P‐value
TNFα	−0·96 (4)	0·0395
IL‐1a	−0·84 (6)	0·0351
IL‐6	−0·81 (6)	0·0508
IL‐8	−0·73 (6)	0·0990
IL‐11	−0·76 (6)	0·0816
VEGF	−0·78 (6)	0·0660

IL, interleukin; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.

^*Pearson correlation was made to determine the relation of inflammatory cytokines and change in the wound area after MIST treatment and significance of this change.

Change in pain before and after treatment with MIST therapy

All 10 patients completed the VAS every week and experienced decrease in pain. The median VAS score was 4·0 at baseline and 2·7 at end of the study (Table 2).

Table 2.

Role of cytokines studied in wound healing and their changes from baseline to end of study after treatment with ultrasound therapy

Cytokine	Role in wound healing	Baseline expression	End of study expression
IL‐1	Inflammation. Activation of fibroblasts, keratinocytes and macrophages. Neutrophil attraction.	High	Decreased
IL‐6, IL‐8	Inflammation. Leucocyte attraction. Showed to be elevated in chronic wound healing and decrease when healing occurs 10, 11.	High	Decreased
IL‐10	Cytokine regulation by decrease of IL‐1, IL‐6 and decrease cytokine release by T helper cells 12, 13, 14.	Low	Increased
IL‐11	Acute‐phase reactant proteins. Present in chronic inflammation (15).	High	Decreased
VEGF	Vasculogenesis. It is increased in chronic wound healing probably to contribute with vascular permeability and because of accelerated degradation by metalloproteinases, found in impaired wound healing (16).	High	Decreased
TNF	Inflammation. Has been reported to be increased in non healing wounds versus healing wounds (17), and its levels have been correlated to the chronicity of the wound (18).	High	Decreased
	Also systemic levels of TNF have been found in wound patients with subsequent decrease after healing (19).

IL, interleukin; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.

DISCUSSION

We assessed the effectiveness of non contact ultrasound therapy in large chronic, refractory venous ulcers that have not responded to treatment with multi‐layered compression bandages. We found a statistically significant reduction in wound size from baseline to end of the study (P = 0·0039) (Figure 2).

Using ultrasound therapy, a prior retrospective analysis found a reduction in the median wound area from 2·5 to 0·6 cm² and increased percentage of granulation tissue from 32% before ultrasound therapy to 46% after ultrasound therapy (20). Another retrospective study showed that 30% of 41 patients treated with ultrasound had complete healing and granulation tissue increased from 26% at the beginning of the study to 80% at the end of the study (21). Ultrasound therapy plus standard of care has also been shown to be effective in ischaemic limbs improving the rate of healing compared with standard of care alone (63% versus 29%; P < 0·001) after 12 weeks of treatment (22). Therefore, the results of our study are consistent with previous reports that show the benefit of using ultrasound therapy, with the particularity that we used a group of wounds that are considered hard to heal.

Controversy still exists regarding the significance of bacteria in non healing wounds. While healing occurs despite the presence of microorganisms (23), it is possible that the density of microorganisms, how bacteria exist or the presence of specific pathogens, is critical to determine whether or not a wound will heal (24). High microbial burden leads to the presence of neutrophils in the wound which perpetuates an inflammatory environment with release of cytotoxic enzymes, free oxygen radicals and matrix metalloproteinases 25, 26.

Excessive metalloproteinases degrade the extracellular matrix, may inhibit cell migration and prevent wound closure. As a result, bacterial proliferation and colonization may retard wound healing (27). Debridement addresses bacteria by removing foreign material and devitalized tissue. In traumatic wounds, debridement is advised to be implemented early as well as prophylactic antibiotic treatment to impede microbial proliferation (28). We found both presence of bacteria and high levels of inflammatory cytokines, while other options might exist to address this clinical scenario, ultrasound therapy resulted in wound size reduction and reduction in inflammatory cytokines.

Non contact, low‐frequency ultrasound therapy is thought to promote healing in chronic wounds by cleansing and debriding devitalized tissue, removing bacteria, fibrin and exudate. Two processes have been proposed as mechanisms of action: surface cavitation (creation and dissipation of tiny bubbles in the tissues) and acoustic microstreaming (movement of fluids along acoustic boundaries, such as cell membranes) (29).

Serena et al. (30) conducted a series of experiments with ultrasound therapy. They found that ultrasound penetrated into both wounded and intact skin in an ex vivo pig model by tracing the presence of a dye that was used in the irrigation preparation. They also performed in vitro experimentation on bacteria survival and showed that 33% of P. aeruginosa, 40% of Escherichia coli and 27% of Enterococcus faecalis disappeared after one‐time ultrasound treatment. Additionally in pigs with induced wounds, compared with silver dressings ultrasound therapy reduced bacterial counts, except for P. aeruginosa. Finally, patients with stage III pressure ulcers, less than 2 weeks of non contact ultrasound treatment reduced pre‐treatment bioburden of 4 × 10⁷ to 2 × 10⁷ (30).

Among other reported effects that ultrasound therapy produces are changes in molecular expression, increased blood flow, vascular permeability, angiogenesis, increase in collagen content and alignment and growth factors release 8, 31, 32, 33, 34, 35. Local alterations in levels of pro‐inflammatory cytokines have been identified in non healing leg ulcers indicating a possible implication in wound chronicity and a physiologic prognostic indicator of wound healing (36). This may be useful for stratifying venous leg ulcers according to their potential to heal. Non healing wounds have persistent elevated levels of pro‐inflammatory cytokines, high levels of proteases and low levels of protease inhibitors. This affects the growth and viability of cell types, leads to impaired mitogenic activity 37, 38, 39, 40 and subsequent impaired healing.

Kuo et al. (41) showed increased blood perfusion after ultrasound therapy in a diabetic rat model plus histological findings of reduced pro‐inflammatory reaction and increased presence of VEGF, eNOS (endothelial nitric oxide synthase) and PCNA (proliferating cell nuclear antigen). We found (Table 2) the baseline expression of pro‐inflammatory cytokines within our sample of recalcitrant venous leg ulcers was high and decreased in such expression after non contact ultrasound therapy. IL‐1, IL‐6, IL‐8, IL‐11, TNFα and VEGF were higher in the baseline biopsy than in the end of study biopsy, whereas IL‐10 was lower in the baseline biopsy that it was after therapy with non contact ultrasound (Figure 4).

We found a relationship between cytokine levels and healing, measured by reduction in wound area. Thus (Table 1), a decrease in cytokine levels was associated with an increase in healing. This association was statistically significant for IL‐1 and TNF‐α. Although the association for the other cytokines was not significant, it showed a trend towards significance (P‐values >0·05 and <0·1). Our results support that healing improves as the excessive inflammatory process, that may inhibit healing, subsides. We also found data consistent with others' work that ultrasound therapy reduces wound‐related pain 42, 43. Our patients showed reduction in pain and an overall, non significant, difference in VAS score from 4·0 at baseline to 2·7 at the end of the 4‐week treatment (Table 3).

Table 3.

Change in pain scale from baseline to end of study after treatment with ultrasound therapy [Mean ± standard deviation (median) (minimum ‐ maximum)]

Gene expression	Follow‐up		Change variables
	Baseline, N = 10	End, N = 10	Magnitude, N = 10	P‐value
VAS	4·0 ± 2·8 (2·7)	2·7 ± 1·8 (3·0)	−1·3 ± 3·3 (−1·3)	0·2754
	(0·5–8·2)	(0·6–5·0)	(−6·8–3·4)

VAS, visual analogue scale.

In conclusion, we found wound size reduction and decreased inflammatory cytokines expression were correlated despite a non significant decrease in bacterial count. We also found decreased pain after treatment with non contact ultrasound and compression therapy in refractory venous leg ulcers.