Supplementary Material for: Extracorporal Shock Waves Activate Migration, Proliferation and Inflammatory Pathways in Fibroblasts and Keratinocytes, and Improve Wound Healing in an Open-Label, Single-Arm Study in Patients with Therapy-Refractory Chronic Leg Ulcers

Background/Aims: Chronic leg ulcers (CLUs) are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT) has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. Methods: We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. Results: Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regulatory genes were upregulated, and proliferation induced in fibroblasts. This was accompanied by secretion of pro-inflammatory cytokines from keratinocytes, which are known to drive wound healing, and a pro-angiogenic activity of endothelial cells. These observations were transferred “from bench to bedside”, and 60 consecutive patients with 75 CLUs with different pathophysiologies (e.g. venous, mixed arterial-venous, arterial) were treated with ESWT. In this setting, 41% of ESWT-treated CLUs showed complete healing, 16% significant improvement, 35% improvement, and 8% of the ulcers did not respond to ESWT. The induction of healing was independent of patient age, duration or size of the ulcer, and the underlying pathophysiology. Conclusions: The efficacy of ESWT needs to be confirmed in controlled trials to implement ESWT as an adjunct to standard therapy or as a stand-alone treatment. Our results suggest that EWST may advance the treatment of chronic, therapy-refractory ulcers.

Chronic venous leg ulcer (CVLU) is a common and refractory complication of chronic venous diseases, and is clinically characterized by chronic leg ulcers and pain. Matrix metalloproteinases (MMPs) are a kind of highly conservative and zinc-dependent endopeptidases, and can be divided into 6 types according to their substrates and structures. Among the 6 types, collagenases, gelatinases and stromelysins play a key role in the occurrence and development of CVLU. Moreover, the up-regulation of MMP-1,-2,-3,-8 and ratio of MMP-2 to tissue inhibitor of metalloproteinase-2 (TIMP-2) is associated with delayed ulcer healing, whereas the up-regulation of MMP-7,-10,-13 and TIMP-1,-2 facilitates ulcer healing. However, roles of MMP-9 and -12 in venous ulcers are still unclear and need further study. According to functional characteristics of some MMPs, intervention of their composition and functions may provide new therapeutic targets for the treatment of CVLU. Key words: Varicose ulcer; Lower extremity; Matrix metalloproteinases; Extracellular matrix; Wound healing

Background: People living with chronic leg ulcers frequently experience moderate to severe wound-related pain with the highest level of pain occurring at dressing change. Wound- related pain is not always able to be alleviated by oral analgesics alone. Persistent poorly- controlled leg ulcer pain physiologically impacts wound healing and prevents timely, effective wound management strategies being implemented which can negatively impact wound healing and health-related quality of life (HRQoL). Topical agents such as morphine gel and ibuprofen foam are used as primary dressings as a strategy for managing chronic leg ulcer pain. Studies investigating ibuprofen foam have shown significant improvement in chronic leg ulcer pain compared to standard care. There is insufficient evidence to suggest morphine gel is effective for chronic leg ulcer pain. Topical local anaesthetics, in particular the eutectic mixture of local anaesthetics (EMLA®), have been used for decades to relieve pain associated with debridement of chronic leg ulcers. However, their effectiveness as a primary dressing for managing chronic wound-related pain is yet to be evaluated. Since there was no known evidence regarding the effectiveness of EMLA® as a primary dressing for painful chronic leg ulcers to relieve wound-related pain and its associated impact on wound healing and HRQoL this feasibility study was conducted. Study aims: The primary aim of this study was to assess the processes, resources, management and scientific aspects of the study to ensure implementation of a larger study is feasible and to generate data that could be used for future sample size calculations. Study feasibility was assessed using the following criteria for determining success: recruitment of at least 80% of eligible patients within 12 months; retention of 80% of participants during the study period and achieving at least 80% adherence to the intervention protocol. Secondary aims were to investigate the effectiveness of the daily topical application of EMLA® as a primary dressing to painful chronic leg ulcers as a pain-relieving strategy and the associated impact on wound healing and HRQoL; and whether improvement in pain levels is associated with reduced need for oral analgesia, particularly opiates. Design: A pilot, parallel group, non-blinded, superior, randomised, controlled trial. There was a 4-week intervention period and a 12-week study period. Setting: Six procedure clinics located in a public community nursing service Central Coast, New South Wales, Australia. Participants: Participants (n = 60) were adult patients with painful chronic leg ulcers of varied aetiology. The preliminary screening criteria included a chronic leg ulcer of more than six weeks duration; pain relieving medications required to manage wound-related pain; and, the participant was able to be treated in a community nursing clinic. Intervention: EMLA® was applied to the chronic leg ulcers daily for four weeks as a primary dressing followed by standard care. Data collection: Feasibility data were collected on human resource requirements, number of home visits, use of consumables and study management including ease of administering data collection instruments. Wound-related pain was measured at baseline and each dressing change; chronic leg ulcer surface areas and HRQoL were measured at baseline, weeks 4 and 12. Outcomes: Feasibility: Although all proposed participants were recruited (n=60) the recruitment rate was lower than expected and it is possible some eligible patients were missed during the screening process. Fifty-four participants remained in the study until completion for a 90% retention rate. Intervention fidelity was influenced by resource availability and participant factors such as increased wound-related pain. Data generated from the primary clinical outcome wound- related pain, was used to calculate the sample size for a larger study. Given two-sided significance of 0.05, a power of 0.8%, effect size 0.45 +/- 0.3 and variability/standard deviation of 2.2 +/- 0.2, 274 to 306 participants will be required for a larger randomised controlled trial. The sizes have been adjusted upwards based on an estimated dropout rate of 10%. However, an effect size of 0.45 is not clinically meaningful so a difference of at least two pain scores on the 11-point pain intensity Numerical Rating Scale used in this study is suggested as a clinically important difference. Recalculation using an effect size of two estimated that 52 participants would be needed to detect a clinically meaningful difference between the treatment and control groups based on pain as the primary outcome. Assuming a dropout of 10%, this figure was adjusted upwards to 58. Wound-related pain: Mean pain scores were similar between the two groups at baseline (p = 0.84). During dressing change, mean pain scores across the 4-week intervention period were significantly lower in the intervention compared to the control group (intervention group: Mean (SD) 3.39 (2.16); control group: Mean (SD) 4.82 (2.27), p = 0.02). Mean pain scores after dressing change were also significantly lower for the intervention group over the 4-week intervention period (intervention group: Mean (SD) 2.71(1.94); control group: Mean (SD) 3.92 (2.03), (p = 0.03). Wound healing and HRQoL: During the intervention period there was no significant difference in wound sizes between groups (intervention group - Median (cm2): 2.4, IQR: 1.3 – 12.7 v control group- Median (cm2): 5.0, IQR: 2.5-9.9; p = 0.05). Mean HRQoL scores for all subscales at baseline, weeks 4 and 12 were similar between groups except for Wellbeing, which was significantly higher in the intervention group at the end of the 4-week intervention period (intervention group - Mean 52.41, SD 24.50 vs. control group- Mean: 38.15, SD 21.25; p = 0.03, d = .62). Conclusion: It is feasible to conduct a larger multisite RCT following modifications to the study protocol. The study findings suggest that daily applications of EMLA® as a primary dressing reduces wound-related pain during and after dressing change, do not inhibit wound healing and may improve a person’s well-being.

1. Co-chaired this panel2. University of South Florida, Tampa, FL3. Healthpoint Ltd., Fort Worth, TX4. University of California, San Francisco, CA5. University of Texas Medical Branch, Galveston, TX6. University of Cardiff, Cardiff, Wales, UK7. University of Pennsylvania, Philadelphia, PA8. Private practice, Warren, PA9. Private practice, Tamarac, FL10. University of Pittsburgh, Pittsburgh, PA11. St. Louis University, St. Louis, MO, and12. Washington University, St. Louis, MO

In cold plasma medicine, anti-inflammatory, anti-itch, antimicrobic, ultravio - let, and other therapeutic modalities are combined within one treatment. Two types of cold plasma can be discerned: direct (dielectric barrier discharge (DBD)) and indirect plasma. DBD generates a low-temperature plasma under atmospheric pressure. The PlasmaDerm VU-2010 device is a noninvasive active medical intervention that does not come into direct contact with skin. For our medical application, a nonequilibrium, weakly ionized, physical DBD plasma is generated by the application of high voltages across small gaps; the electrode is covered by a dielectric. The skin itself acts as the second electrode. Chronic leg ulcers are a major problem among the elderly. The prevalence corresponds to 2-4% of the population. Eighty percent of chronic leg ulcers are caused by varicosis. In general, 3 phases of wound healing (cleaning of the wound ground, granulation, and epithelialization) can be discerned as disturbed in chronic venous leg ulcers. Wound debridement, modern wound dressings, and compression hosiery comprise methods of standard care. Despite these measures, leg ulcers often persist. Addi- tional plasma treatment may have the potential to facilitate wound healing by disinfection, stimulation of tissue regeneration and microcirculation, and acidification of the wound envi- ronment. We are currently conducting an ongoing clinical trial with the PlasmaDerm VU-2010 device to assess the safety, applicability, and efficacy of plasma treatment for chronic venous leg ulcers. So far, no adverse effects of plasma treatment have been reported, pointing toward a positive outcome of our study.

Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization. To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence. Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6). Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine. Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.

Slough in chronic venous leg ulcers may be associated with delayed healing. The purpose of this study was to assess larval debridement in chronic venous leg ulcers and to assess subsequent effect on healing. All patients with chronic leg ulcers presenting to the leg ulcer service were evaluated for the study. Exclusion criteria were: ankle brachial pressure indices <0.85 or >1.25, no venous reflux on duplex and <20% of ulcer surface covered with slough. Participants were randomly allocated to either 4-layer compression bandaging alone or 4-layer compression bandaging + larvae. Surface areas of ulcer and slough were assessed on day 4; 4-layer compression bandaging was then continued and ulcer size was measured every 2 weeks for up to 12 weeks. A total of 601 patients with chronic leg ulcers were screened between November 2008 and July 2012. Of these, 20 were randomised to 4-layer compression bandaging and 20 to 4-layer compression bandaging + larvae. Median (range) ulcer size was 10.8 (3-21.3) cm(2) and 8.1 (4.3-13.5) cm(2) in the 4-layer compression bandaging and 4-layer compression bandaging + larvae groups, respectively (Mann-Whitney U test, P = 0.184). On day 4, median reduction in slough area was 3.7 cm(2) in the 4-layer compression bandaging group (P < 0.05) and 4.2 cm(2) (P < 0.001) in the 4-layer compression bandaging + larvae group. Median percentage area reduction of slough was 50% in the 4-layer compression bandaging group and 84% in the 4-layer compression bandaging + larvae group (Mann-Whitney U test, P < 0.05). The 12-week healing rate was 73% and 68% in the 4-layer compression bandaging and 4-layer compression bandaging + larvae groups, respectively (Kaplan-Meier analysis, P = 0.664). Larval debridement therapy improves wound debridement in chronic venous leg ulcers treated with multilayer compression bandages. However, no subsequent improvement in ulcer healing was demonstrated.

Low-dose ultrasound seems to be an effective method to enhance wound healing, particularly in chronic venous leg ulcers. The aim of our investigation was to examine the effect of 30 kHz low-dose ultrasound in local treatment of chronic venous leg ulcers, when added to conventional therapy of outpatients. Twenty-four patients with chronic ulcerations of the leg due to chronic venous insufficiency were randomised in placebo-controlled parallel groups in a single-blind clinical study. Patients were randomised to conventional therapy with topical application of hydrocolloid dressings and compression therapy or conventional therapy with additional ultrasound treatment for 12 weeks. The ultrasound treatment consisted of 10 min of footbathing, with application of 30 kHz continuous ultrasound 100 mW/cm2 three times a week. The ulcer area was measured by planimetry, using a millimeter grid before treatment and after 2, 4, 6, 8, 10 and 12 weeks of therapy. The ulcer radius and the daily ulcer radius reduction were calculated. Colour photographs of the ulcers were taken under standard conditions at the same time. After each treatment local findings and side effects were recorded. After 12 weeks of treatment the control group showed a mean decrease of 16.5% in the ulcerated area. In contrast the mean ulcerated area decreased by 55.4% in the ultrasound group (p<0.007). The daily ulcer reduction in the ultrasound-treated patients was 0.08 mm+/-0.04 mm and in the placebo patients 0.03 mm+/-0.03 mm. Patients recorded only minor side-effects such as a tingling feeling and occasionally pinhead-sized bleeding in the ulcer area. The application of low-frequency and low-dose ultrasound is a helpful treatment option in chronic venous leg ulcers, especially if they do not respond to conventional ulcer treatment.

The possible impact of nutritional status on healing and course of disease in patients with chronic wounds is widely suggested, however, most data are based on small groups of patients with no control group and minor afflictions. Clear diagnostic strategies are missing. To analyse in detail the nutritional status of chronic wound patients relative to healthy controls based on a large patient population. We screened a group of 50 patients for their nutritional status based on body mass index (BMI), the Mini-Nutritional Assessment (MNA), and Nutritional Risk Screening (NRS), as well as additional laboratory investigations. Twenty-five patients suffered from chronic venous leg ulcers and were compared with a matching control group of 25 patients with acute surgical wounds. Patients with chronic venous leg ulcers showed significantly higher BMI, hyperhomocysteinaemia, and higher levels of serum copper but significantly lower levels of vitamin B6, B9 and C, as well as a significantly lower level of zinc. Vitamin D deficiency was present in both groups, however, severe vitamin D deficiency was present only in the leg ulcer group. Mobility was significantly reduced in patients with leg ulcers. Ulcer patients are often obese but suffer from qualitative malnutrition, including a lack of vitamin D, which might be explained by reduced mobility and being housebound. Hypoalbuminaemia, as a sign of protein deficiency, was observed significantly more often in patients with chronic leg ulcers, irrespective of wound area or wound duration.

Chronic leg ulceration may often be accompanied and exacerbated by the presence of oedema, a common health problem that can cause ulceration, which if left untreated can require amputation. Current clinical consensus indicates compression as the recommended treatment for oedema. While cardiac function is also known to influence oedema, it is rarely considered a parameter of wound management. This article presents a case study of a patient with oedema and ulceration, who avoided a scheduled amputation when lesions healed following the optimisation of cardiac function. The patient had right-sided heart failure with preserved ejection fraction. After fluid drainage, the ulcers began to heal. The case raises the question of cardiac optimisation of leg ulcer patients. This case highlights the potential role of cardiac management in leg ulcer treatment and raises an important question: should cardiac optimisation be a greater consideration in the care of patients with leg ulcers? The prevailing emphasis on compression therapy may benefit from reassessment, with particular attention to the role of cardiac function in wound healing.

Forty-two patients (10 males and 32 females) with 52 chronic leg ulcers were treated with sheets of cultured allogeneic keratinocytes. Sixty-five % of the ulcers healed completely and the healing rate differed between various diagnostic groups. The best results were obtained in patients with venous ulcers and wounds with mixed etiology, whereas less improvement was observed with ischaemic ulcers. Rheumatic ulcers also responded well in combination with oral corticosteroids. The overall impression was that the grafting procedure markedly enhanced wound healing.

Objective To investigate the effect of low-energy extracorporeal shock wave (ESW)treatment on collagen deposition and transforming growth factor-β1 (TGF-β1) expression in excisional wound of diabetic rats,and the possible mechanism of ESW promoting wound healing.Methods Chronic diabetic wound models were made in 64 6-8-week-old male SD rats weighing (220 ±20) g,and randomly divided into the ESW treatment group and the diabetic control group.Normal wound models were made in 32 SD rats and were used as the normal control group.In ESW treatment group,wound was treated by ESW with 0.11 mJ/mm2,1.5 Hz and 500 pulses one day post-wounding.In diabetic control group and normal control group,wound was only given coupling fluid and received no ESW treatment.Wound closing rate was calculated at 3rd,7th,14th and 21st day after treatment.Wound specimens were harvested to observe histological changes,hydroxyproline contents,density of collagen fibers and TGF-β1 expression.Resuits At different time points after treatment,collagen deposition,the relative density of collagen and hydroxyproline content were decreased in the diabetic control group as compared with those in the normal control group (P ＜ 0.05).At the same time,TGF-β1 expression was significantly decreased in the diabetic control group (P ＜ 0.05).After ESW treatment,fibroblasts and granulation tissue formation were increased,and collagen fibers density and hydroxyproline content in the wound were also increased simultaneously (P ＜ 0.05),and there was no significant difference in collagen fibers density and hydroxyproline content between the ESW group and normal control group (P ＞0.05).In addition,TGF-β1 expression at different time points was increased (P ＜ 0.05) and began to decline at 14th day in the ESW treatment group as compared with that in the diabetic control group.Conclusion The reduced collagen deposition is a major contributor of impaired diabetic wound healing and ESW treatment may increase the expression of TGF-β1,fibroblast migration or proliferation,collagen formation and hydroxyproline content within the wound tissue,and thus accelerate granulation tissue formation and wound healing process. Key words: Diabetes ; Wound; Collagen ; Transforming growth factor-β1

Dramatic improvement of wound closure in nonhealing chronic leg ulcers after topical application of an antitumor necrosis factor-alpha monoclonal antibody (infliximab)

Aim To investigate the effectiveness of a new community nursing model of care for clients with chronic leg ulcers in terms of levels of pain and ulcer healing. Method A randomised controlled trial comparing the new model of care with standard community nursing care was conducted with a sample of 56 clients with chronic venous leg ulcers, 28 clients in the intervention group and 28 in the control group. Data were collected on admission to the study and at 12 weeks from admission. Results Significant improvements in levels of pain and ulcer healing were found in the intervention group receiving the new model of care. Conclusion Results from this study have implications for health professionals providing care for clients with chronic leg ulcers. Authors

To investigate the effectiveness of a new community nursing model of care for clients with chronic leg ulcers in terms of levels of pain and ulcer healing. A randomised controlled trial comparing the new model of care with standard community nursing care was conducted with a sample of 56 clients with chronic venous leg ulcers, 28 clients in the intervention group and 28 in the control group. Data were collected on admission to the study and at 12 weeks from admission. Significant improvements in levels of pain and ulcer healing were found in the intervention group receiving the new model of care. Results from this study have implications for health professionals providing care for clients with chronic leg ulcers.