Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines

Chronic venous ulcer (CVU) represents a dreaded complication of chronic venous disease (CVD). The onset of infection may further delay the already precarious healing process in such lesions. Some evidences have shown that matrix metalloproteinases (MMPs) are involved and play a central role in both CVUs and infectious diseases. Two groups of patients were enrolled to evaluate the expression of MMPs in infected ulcers and the levels of inflammatory cytokines as well as their prevalence. Group I comprised 63 patients (36 females and 27 males with a median age of 68·7 years) with infected CVUs, and group II (control group) comprised 66 patients (38 females and 28 males with a median age of 61·2 years) with non‐infected venous ulcers. MMP evaluation and dosage of inflammatory cytokines in plasma and wound fluid was performed by means of enzyme‐linked immunosorbent assay test; protein extraction and immunoblot analysis were performed on biopsied wounds. The first three most common agents involved in CVUs were Staphylococcus aureus (38·09%), Corynebacterium striatum (19·05%) and Pseudomonas aeruginosa (12·7%). In this study, we documented overall higher levels of MMP‐1 and MMP‐8 in patients with infected ulcers compared to those with uninfected ulcers that showed higher levels of MMP‐2 and MMP‐9. We also documented higher levels of interleukin (IL)‐1, IL‐6, IL‐8, vascular endothelial growth factor and tumour necrosis factor‐alpha in patients with infected ulcers with respect to those with uninfected ulcers, documenting a possible association between infection, MMP activation, cytokine secretions and symptoms. The present results could represent the basis for further studies on drug use that mimic the action of tissue inhibitors of metalloproteinases in order to make infected CVU more manageable.     

Hyperhomocysteinaemia and chronic venous ulcers

Chronic venous ulceration (CVU) is the major cause of chronic wounds of lower extremities, and is a part of the complex of chronic venous disease. Previous studies have hypothesised that several thrombophilic factors, such as hyperhomocysteinaemia (HHcy), may be associated with chronic venous ulcers. In this study, we evaluated the prevalence of HHcy in patients with venous leg ulcers and the effect of folic acid therapy on wound healing. Eighty‐seven patients with venous leg ulcers were enrolled in this study to calculate the prevalence of HHcy in this population. All patients underwent basic treatment for venous ulcer (compression therapy ± surgical procedures). Patients with HHcy (group A) received basic treatment and administered folic acid (1·2 mg/day for 12 months) and patients without HHcy (group B) received only basic treatment. Healing was assessed by means of computerised planimetry analysis. The prevalence of HHcy among patients with chronic venous ulcer enrolled in this study was 62·06%. Healing rate was significantly higher (P < 0·05) in group A patients (78·75%) compared with group B patients (63·33%). This study suggests a close association, statistically significant, between HHcy and CVU. Homocysteine‐lowering therapy with folic acid seems to expedite wound healing. Despite these aspects, the exact molecular mechanisms between homocysteine and CVU have not been clearly defined and further studies are needed.     

Study on the efficacy of surgery of the superficial venous system and of compression therapy at early stages of chronic venous disease for the prevention of chronic venous ulceration

The mainstay of treatment of chronic venous ulceration (CVU), as also suggested by current treatment guidelines for chronic venous disease (CVD), is represented by surgery and compression therapy for which there is strong evidence of their role in clinically relevant improvement in wound healing and also in the reduction of CVU recurrence, but no information is available as to whether or not these treatments provide effective protection from the onset of CVU. In our study, we have followed, for a median time of 13 years, a total of 3947 patients with CVD at classes C2–C3 of CEAP classification, treated with our treatment protocol (surgery and compression therapy) in order to track the natural history of these patients with regards to CVU development. We identified four groups of patients: 2354 patients (59·64%) (Group A) fully adherent to protocols; 848 patients (21·48%) (Group B) fully adherent to surgery and non‐compliant to compression therapy; 432 patients (10·95%) (Group C) fully adherent to compression therapy and non‐compliant to surgery; and 313 patients (7·93%) (Group D) non‐compliant to either treatments. Regardless of compliance to treatments, the ulcer development rates were very similar between groups (range: 3·23–4.79%), with no statistical significance (P = 0·1522). Currents treatments used in the early stages of CVD appear to have no effects to progression to CVU. Additional longitudinal studies are required to confirm these findings.     

Doxycycline speeds up healing of chronic venous ulcers

Venous ulcers are common, with an overall prevalence of up to 2% in the general population of western countries, and have significant socioeconomic impact. Matrix metalloproteinases (MMPs) are involved in the alteration of extracellular matrix that could lead to venous ulceration. Sixty‐four patients with venous ulcers were recruited in a 22‐month period. All patients were subjected to the most appropriate treatment considering also the patient's wishes (compression therapy followed or not by vein surgery). Patients were randomised into two groups of 32 persons in each (groups A and B). Patients of group A in addition to the basic treatment, described above, received the administration of oral low doses of doxycycline 20 mg b.i.d. for 3 months, whereas patients of group B received basic treatment only. Healing was assessed by means of direct ulcer tracing with computerised planimetry. Group A showed a higher healing rate compared with group B. In group B, the lower healing rate was related to higher levels of MMP‐9; neutrophil gelatinase‐associated lipocalin and vascular endothelial growth factor, documented in plasma; wound fluid and biopsies executed and compared between both groups. Pharmacological treatments, as doxycycline administration, which by means of its immunomodulatory and anti‐inflammatory actions, through the inhibition of MMP, could improve extracellular matrix functioning and represent a possible solution to support wound healing.     

Effectiveness of prostaglandin E1 in patients with mixed arterial and venous ulcers of the lower limbs

Mixed arterial and venous ulcers of the lower limbs are present in around 15–30% of patients with chronic venous ulcers (CVUs) and are considered difficult‐to‐heal wounds. The aim of this study was to evaluate the results of the treatment of mixed arterial and venous ulcers of the lower limbs with prostaglandin E1 (PGE1) infusion. This study was carried out in 48 consecutive patients. Patients who showed intolerability to PGE1, and patients with peripheral neuropathy, blood or systemic diseases, malignancy and acute wound infections or necrotic tissue on the wound bed were excluded. The patients were separated at random into two main groups: group I (25 patients) received standard treatment and PGE1 infusion. Group II (23 patients) received only standard treatment. Pre‐treatment data indicated the area of ulceration. The number of healed ulcers and the variation in the area of ulceration were considered as endpoints. The endpoints were noticed after 120 days from the beginning of treatment. Healing occurred in 80% of limbs of group I and in 52·2% of limbs of group II patients. The average reduction in area was 92% versus 60% in patients of group I and II, respectively. During the whole treatment period, the incidence of adverse events was 8% in group I: there was one case of headache and one case of headache and hypotension combined. No side effects were recorded in patients of group II. In conclusion, PGE1 infusion is a determinant in the reduction of the healing time of mixed ulcers of the lower limbs.     

Chronic venous leg ulcers are associated with high levels of metalloproteinases‐9 and neutrophil gelatinase‐associated lipocalin

Venous ulcers are related to dysfunctions in extracellular matrix. Both matrix metalloproteinases (MMP) and neutrophil gelatinase‐associated lipocalin (NGAL) could play a role in the healing process in patients with chronic venous ulcers. We evaluated the role of MMP‐9 and NGAL in the healing process in venous ulceration. We performed an open‐label, parallel groups, single clinical center study. Patients with chronic venous leg ulcers represented the test group (Group I), whereas patients without chronic ulcers represented the control group (Group II). In Group I plasma and wound fluid samples were collected at the time of admission, at the time of the surgery, and at the follow‐up, while ulcer tissues were taken at the time of the surgery. In Group II, plasma and wound fluid were collected at admission and at the time of the surgery, whereas skin tissues were collected at the time of the surgery. Enzyme‐linked immunosorbent assay test was used to evaluate the levels of MMP‐9 and NGAL in plasma and wound fluid, whereas Western blot analysis was performed to estimate the expression of MMP‐9 and NGAL in tissues. Enzyme‐linked immunosorbent assay tests revealed significantly higher levels of MMP‐9 and NGAL in both plasma and wound fluid of patients with ulcers compared to patients without ulcers (p < 0.01). Moreover, Western blot analysis documented an increased expression of MMP‐9 and NGAL in biopsy tissue of patients with ulcers compared to patients without ulcers (p < 0.01). In conclusion MMP‐9 and NGAL may correlate with the clinical course of venous ulcers.     

From varices to venous ulceration: the story of chronic venous disease described by metalloproteinases

Chronic venous disease (CVD) and its most frightening complication, chronic venous ulceration (CVU), represent an important socioeconomic burden in the western world. Metalloproteinases have been identified in the pathogenesis of several vascular diseases such as venous problems. The aim of this study was to evaluate a broad range of metalloproteinases, such as matrix metalloproteinases (MMPs), ADAMs (a disintegrin and metalloproteinases) and ADAMTSs (a disintegrin and metalloproteinases with thrombospondin motifs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) and a related protein, neutrophil gelatinase‐associated lipocalin (NGAL), in patients with CVD in order to correlate their serum levels with each stage of the disease. We performed a multicenter open‐label study that comprised the enrolment of 541 patients with CVD of clinical stages C1–C6, (178 males, 363 females; mean age 57·29, median age 53·72, age range 29–81); 29 subjects without CVD were included in this study (9 males and 20 females; mean age 54·44, median age 50, age range 28–84) as the control group. Enzyme‐linked immunosorbent assay (ELISA) was performed for measuring serum levels of proteases and related proteins. The study found that the serum elevation of MMP‐2, ADAMTS‐1 and ADAMTS‐7 appeared to be correlated with the initial stages of CVD, whereas the serum elevation of MMP‐1, MMP‐8, MMP‐9, NGAL, ADAM‐10, ADAM‐17 and ADAMTS‐4 was particularly involved in skin change complications. This study showed that each stage of CVD may be described by particular patterns of metalloproteinases, and this may have therapeutic implications in discovering new targets and new drugs for the treatment of CVD.     

PredyCLU: a prediction system for chronic leg ulcers based on fuzzy logic; part I – exploring the venous side

Chronic leg ulcers (CLUs) are a common occurrence in the western population and are associated with a negative impact on the quality of life of patients. They also cause a substantial burden on the health budget. The pathogenesis of leg ulceration is quite heterogeneous, and chronic venous ulceration (CVU) is the most common manifestation representing the main complication of chronic venous disease (CVD). Prevention strategies and early identification of the risk represent the best form of management. Fuzzy logic is a flexible mathematical system that has proved to be a powerful tool for decision‐making systems and pattern classification systems in medicine. In this study, we have elaborated a computerised prediction system for chronic leg ulcers (PredyCLU) based on fuzzy logic, which was retrospectively applied on a multicentre population of 77 patients with CVD. This evaluation system produced reliable risk score patterns and served effectively as a stratification risk tool in patients with CVD who were at the risk of developing CVUs.     

Healing and Recurrence Rates Following Ultrasound-guided Foam Sclerotherapy of Superficial Venous Reflux in Patients with Chronic Venous Ulceration

ObjectivesTo determine healing and recurrence rates following ultrasound-guided foam sclerotherapy (UGFS) of superficial venous reflux (SVR) in patients with healed (clinical, etiologic, anatomic and pathophysiologic (CEAP) classification, C5) and open (C6) chronic venous ulceration (CVU).MethodsBetween 1 March 2005 and 31 December 2009, 130 consecutive patients (132 limbs, 49 CEAP C5, 83 C6) of median age 70 (interquartile range (IQR) 56–76) years underwent UGFS as part of their treatment for CVU.ResultsThe median (IQR) follow-up time was 16 (12–32) months. One C6 patient moved abroad 1 week after UGFS and was lost to follow-up. Healing was observed in 67/82 (82%) remaining C6 patients at a median (IQR) of 1 (1–2) month following their first UGFS treatment. In 49 limbs originally treated for C5 disease, and in 67 limbs treated for C6 that healed following UGFS, there were five recurrent ulcers during the follow-up period, giving a 4.9% Kaplan–Meier estimate of recurrence at 2 years. In legs treated for C6 and C5 disease, the median (IQR) ulcer-free periods were 22 (IQR 9–32) and 14 (IQR 8–36) months, respectively.DiscussionHealing rates following UGFS for CVU are comparable to those reported after surgery but recurrence may be lower. UGFS is a safe, clinically effective and, thus, highly attractive minimally invasive alternative to surgery in patients with C5 and C6 disease.     

Effect of oxidised regenerated cellulose/collagen matrix on proteases in wound exudate of patients with chronic venous ulceration

Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) modifies wound microenvironments by binding and inactivating excess levels of proteases such as elastase, plasmin and gelatinases in wound exudates. To compare levels of the gelatinases matrix metalloproteinase 2 (MMP-2), elastase and plasmin in wound exudates collected from chronic venous insufficiency patients with venous leg ulcers treated with either an ORC/collagen matrix or a standard control therapy. During a 12-week treatment period, wound exudate samples were obtained from a control group of 10 patients treated with a hydrocolloid dressing and a treatment group of 17 patients treated with a combination of ORC/collagen matrix and hydrocolloid dressing. On admission and days 5, 14 and every subsequent 14th day, ulcers were photographed to determine healing rate and changes in ulcer appearance, and MMP-2 concentration and the gelatinase, elastase and plasmin activities were analysed from wound exudates. The patients treated with ORC/collagen matrix showed a significant decrease in elastase, plasmin and gelastinase activity as compared with the control group, with no significant difference in the MMP-2 concentrations between the two groups. The results show a significant and immediate reduction in protease activity in wound exudates from venous leg ulcers treated with ORC/collagen.     

Effects of a new nutraceutical substance on clinical and molecular parameters in patients with chronic venous ulceration

Pathophysiological events involved in the onset of chronic venous ulceration (CVU) are inflammation, activation of polymorphonucleates (PMNs) and secretion of proteases such as matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) that is a support for vascular and tissutal wall. MMPs, neutrophil gelatinase‐associated lipocalin (NGAL) and inflammatory cytokines are overexpressed in CVUs and they could play a central role in pathophysiological mechanisms of skin lesion and delayed wound healing. Bioflavonoids, such as diosmin and other compounds, appear to have several provessel function activities including anti‐inflammatory, antioxidant and phlebotonic effects and are widely used in the treatment of chronic venous disease (CVD)‐related problems. In this article, we evaluated the effects of Axaven^®, a new nutraceutical on both clinical and molecular parameters in patients with CVUs. During the study period, 83 patients with CVUs of both sexes were enrolled and divided into two groups: group A (treated group): 25 females and 19 males (median age is 67·7 years) received standard treatment (compression therapy and surgical correction of superficial venous incompetence) + Axaven^® once a day for 8 months as adjunctive treatment. Group B (control group): 24 females and 15 males (median age is 65·2 years) were treated only with basic treatment according to their clinical conditions. In our study, the administration of Axaven^® in patients with CVUs was able to decrease inflammatory cytokines, MMPs and NGAL, inducing an improvement of both symptoms with an increase of the speed of wound healing.     

Low molecular weight heparin improves healing of chronic venous ulcers especially in the elderly

Venous ulcers are common, especially in the elderly, accounting for more than 50% of all lower extremity ulcers with important socioeconomic problems. Improving extracellular matrix functioning, by heparin administration, seems to be a way to support wound healing. A total of 284 patients with venous ulcers were recruited in a 4‐year period. All patients were subjected to the most appropriate treatment after considering their preference (compression therapy followed or not by vein surgery). Patients were randomised into two groups of 142 persons in each (group A and group B as cases and controls, respectively). Patients of group A, in addition to the basic treatment as described earlier, received administration of nadroparin 2850 IU/0.3 ml through subcutaneous injection once a day for 12 months, whereas group B patients received basic treatment alone. Healing was assessed by means of direct ulcer tracing with computerised planimetry. Group A showed a healing rate of 83·80% at 12 months, whereas that of group B was 60·56%. Results by age group surprisingly showed that the group of older patients took the most advantage from long‐term treatment with low molecular weight heparin; this group also had lowest recurrence rate.     

Contact sensitivity in patients with venous leg ulcer: A multi‐centric Indian study

Venous leg ulcers are the most common form of non‐healing leg ulcers. They are subjected to treatments such as topical medications, dressings, and compression therapies. This can lead to exposure to a number of allergens with subsequent sensitisation and contact dermatitis of the regional skin. This may contribute to the poor ulcer healing. To detect the various contact sensitisers in patients with venous leg ulcers through patch testing, patients from 6 centres across India with venous leg ulcers of longer than 6 weeks duration were enrolled for the study. They were patch tested using a special parch test kit with 27 antigens. A total of 172 patients were included in the study; 82 (48.2%) tested positive for at least 1 antigen. Among them, polyvalent sensitisation was noted in 71% of patients. Wood tar mix (10.4%) and the framycetin (8.7%) were the most common allergens. There is a high frequency of allergic sensitisation to various ingredients of topical therapies used in the venous ulcer management, which may interfere with wound healing. Avoiding them can help obtain a better therapeutic outcome.     

Chronic Humoral Rejection of Human Kidney Allografts Is Associated with MMP‐2 Accumulation in Podocytes and its Release in the Urine

Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase‐2 (MMP‐2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP‐2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP‐2 staining was detected by immunohistochemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP‐2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non‐CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP‐2 correlated with higher levels of proteinuria in both CHR and non‐CHR patients. Longitudinal analysis indicated that increase in urine MMP‐2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP‐2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP‐2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR.     

Primary cutaneous diffuse large B‐cell lymphoma presenting as chronic non‐healing ulcer

Primary cutaneous diffuse large B‐cell lymphoma is an uncommon and aggressive lymphoproliferative disorder with a rapid growth rate and dismal prognosis. We present the case of a 91‐year‐old female with an unusual manifestation of primary cutaneous diffuse large B‐cell lymphoma, mimicking other more prevalent diseases like chronic non‐healing venous ulceration. Dermatopathologic evaluation rendered the correct diagnosis. A discussion of this rare presentation is important for clinician consideration to prevent misdiagnosis and prolongation of proper management in patients with chronic non‐healing leg ulcers.     

Dystrophic calcification as a cause for non healing leg ulcers

Despite advances in molecular biology and a repertoire of other therapeutic options, chronic venous leg ulcers remain a significant problem within our society. There are various reasons, both local and systemic, which contribute to the non healing nature of such wounds. Among them, dystrophic calcification (DC) or calcified deposits within the ulcer bed, although rare, is an overlooked and a seldom reported cause. In the presence of DC, wound healing cannot proceed through a timely and orderly manner resulting in a non healing ulcer. In this article, we discuss the aetiology, pathophysiology and the management options of this rarely reported condition. We also report their clinical prognosis using a series of patients with venous ulcers complicated by DC leading to difficulties in healing.     

Skin grafting for the treatment of chronic leg ulcers – a systematic review in evidence‐based medicine

Skin grafting is one of the most common surgical procedures in the area of non‐healing wounds by which skin or a skin substitute is placed over a wound to replace and regenerate the damaged skin. Chronic leg ulcers are an important problem and a major source of expense for Western countries and for which many different forms of treatment have been used. Skin grafting is a method of treatment that decreases the area of chronic leg ulcers or heals them completely, thus improving a patient's quality of life. Skin grafting is an old technique, rediscovered during the first and second world wars as the main treatment for wound closure. Nowadays, skin grafting has a pivotal role in the context of modern wound healing and tissue regeneration. The aim of this review was to track and to analyse the specific outcomes this technique achieved, especially in the last decade, in relation to venous, arterial, diabetic, rheumatoid and traumatic leg ulcers. Our main findings indicate that autologous split‐thickness skin grafting still remains the gold standard in terms of safety and efficacy for chronic leg ulcers; skin grafting procedures have greater success rates in chronic venous leg ulcers compared to other types of chronic leg ulcers; skin tissue engineering, also supported by genetic manipulation, is quickly expanding and, in the near future, may provide even better outcomes in the area of treatments for long‐lasting chronic wounds.     

Multiplexed analysis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compression therapy

Elevated matrix metalloproteinases (MMP) levels have been implicated in the pathogenesis of chronic venous insufficiency ulcers. Quantitative measurements of a broad range of MMP proteins in human tissue treated with compression bandaging have not been reported. The goal of this study was to determine the expression of a wide range of proteases in untreated venous leg ulcer tissue and the changes in these levels after 4 weeks of high‐strength compression therapy. Twenty‐nine limbs with new or untreated chronic venous insufficiency and leg ulceration received therapy for 4 weeks with sustained high compression bandaging. Biopsies were obtained from healthy tissue and from ulcerated tissue before and after therapy. A novel multiplexed protein assay was used to measure multiple MMPs in a single sample. MMP protein activity, TIMP protein levels, and gene expression levels were also addressed. MMP1, 2, 3, 8, 9, 12, and 13 protein levels were elevated in ulcer tissue compared with healthy tissue. MMP8 and 9 were highly expressed in ulcer tissue. MMP3, 8, and 9 significantly decreased following treatment. Reduction in the levels of MMP1, 2, and 3 was associated with significantly higher rates of ulcer healing at 4 weeks. We conclude that compression therapy results in a reduction of the pro‐inflammatory environment characterizing chronic venous ulcers, and ulcer healing is associated with resolution of specific elevated levels of protease expression.     

Healing of chronic venous ulcer with topical sevoflurane

The present study assesses the efficacy and safety of topical sevoflurane in chronic venous ulcers and its impact on analgesia and healing. This retrospective study included 30 patients older than 65 years with painful refractory chronic venous ulcers. Patients were treated with topical sevoflurane prior to the usual ulcer cleaning every 2 days for a period of 1 month. Treatment was initiated with a saline solution, followed by direct irrigation of 1 ml of liquid sevoflurane over the wound size, which was measured as cm². Sevoflurane had a fast, intense and long‐lasting analgesic effect. Latency time ranged from 2 to 7 minutes (3·9 ± 1·5 minutes), and duration varied from 8 to 18 hours (12 ± 2·9). The mean ulcer size was 8·4 ± 9·7 cm². There was a progressive decrease in size in all patients, with a mean size of 4·2 ± 5·4 cm² at the end of the study. There were no adverse systemic effects. Local adverse effects were mild and transient, including pruritus, erythema and heat. Topical sevoflurane is a new, efficient and safe therapeutic alternative in painful chronic venous ulcers, refractory to usual analgesic treatment. It can improve the ulcer‐healing process that shortens the cicatrisation period.     

A pilot study evaluating non‐contact low‐frequency ultrasound and underlying molecular mechanism on diabetic foot ulcers

Non‐contact low‐frequency ultrasound (NCLF‐US) devices have been increasingly used for the treatment of chronic non‐healing wounds. The appropriate dose for NCLF‐US is still in debate. The aims of this pilot study were to evaluate the relationship between dose and duration of treatment for subjects with non‐healing diabetic foot ulcers (DFUs) and to explore the correlation between wound healing and change of cytokine/proteinase/growth factor profile. This was a prospective randomised clinical study designed to evaluate subjects with non‐healing DFUs for 5 weeks receiving standard of care and/or NCLF‐US treatment. Subjects were randomly assigned to one of the three groups: application of NCLF‐US thrice per week (Group 1), NCLF‐US once per week (Group 2) and the control (Group 3) that received no NCLF‐US. All subjects received standard wound care plus offloading for a total of 4 weeks. Percent area reduction (PAR) of each wound compared with baseline was evaluated weekly. Profiles of cytokines/proteinase/growth factors in wound fluid and biopsied tissue were quantified to explore the correlation between wound healing and cytokines/growth factor expression. Twelve DFU patients, 2 (16·7%) type 1 and 10 (83·3%) type 2 diabetics, with an average age of 58 ± 10 years and a total of 12 foot ulcers were enrolled. Average ulcer duration was 36·44 ± 24·78 weeks and the average ABI was 0·91 ± 0·06. Group 1 showed significant wound area reduction at weeks 3, 4 and 5 compared with baseline, with the greatest PAR, 86% (P < 0·05); Groups 2 and 3 showed 25% PAR and 39% PAR, respectively, but there were no statistically significant differences between Groups 2 and 3 over time. Biochemical and histological analyses indicated a trend towards reduction of pro‐inflammatory cytokines (IL‐6, IL‐8, IL‐1β, TNF‐α and GM‐CSF), matrix metalloproteinase‐9 (MMP‐9), vascular endothelial growth factor (VEGF) and macrophages in response to NCLF‐US consistent with wound reduction, when compared with control group subjects. This proof‐of‐concept pilot study demonstrates that NCLF‐US is effective in treating neuropathic diabetic foot ulcers through, at least in part, inhibiting pro‐inflammatory cytokines in chronic wound and improving tissue regeneration. Therapeutic application of NFLU, thrice (3) per week, renders the best wound area reduction.