Evaluation of hypochlorous acid washes in the treatment of chronic venous leg ulcers.

OBJECTIVE: Hypochlorous acid (HOCl) is a highly microbiocidal agent active against bacteria, viruses and fungi. Using quantitative microbiology, preliminary studies showed it achieved an appreciable reduction in the bacterial burden in chronic venous leg ulcers. The study aimed to determine whether it has a role as an additional treatment for chronic venous ulcers that have not healed with conventional treatment. METHOD: On the basis of previous reports we designed a study in which patients acted as their own controls, in that only patients who failed to achieve a 44% reduction in wound size with standard treatment (compression bandaging) received HOCl washes. RESULTS: Of 30 patients admitted to the study, 10 achieved a 44% ulcer reduction after three weeks of standard treatment. In addition to the standard compression treatment, the remaining 20 patients were given HOCl washes over 12 weeks. Of the 20 ulcers, nine (45%) healed and five (25%) reduced in size by over 60%. All patients became free of pain. CONCLUSION: These findings confirm the clinical efficacy of treating venous leg ulcers with hypochlorous washes. Use of HOCl washes as an adjunctive therapy for recalcitrant venous leg ulcers appreciably increases healing and rapidly relieves pain.

Using cycloidal vibration to heal venous leg ulcers: a cost-analysis based on retrospective data.

Using cycloidal vibration to stimulate the circulation to enhance healing can significantly reduce treatment costs. For the patient, the benefits include faster healing times, a better quality of life and a marked reduction in pain.

How echographic image analysis of venous oedema reveals the benefits of leg elevation.

OBJECTIVE: Venous insufficiency, leading to venous oedema, is a key pathogenic factor for the non-healing of venous leg ulcers. This study aimed to evaluate the effects of leg elevation on venous oedema. METHOD: Ten patients aged 44-89 years (median: 61) with leg oedema had high-frequency B-mode ultrasound scanning and digital image analysis before and after three to four hours of leg elevation. The echographic image analysis system was used, where oedema is represented by the hypoechogenic part of the image--that is, the total number or density of low echogenic pixels (LEPs) in a particular area. RESULTS: Compared with pre-elevation, the volume of the lower leg decreased by 2.9% +/- 0.6 (138 cm3 +/- 39) after three to four hours' elevation (p < 0.05). After elevation, the LEPs in the upper, middle and lower sites of the limb decreased by 8.8%, 15.6% and 17.3% respectively, reaching statistical significance (p < 0.05) in the lower site. The ratio of LEPs in the upper and lower dermis in the upper, middle and lower sites decreased by 30.3%, 45.8% and 22.5% respectively. This was significant in both the middle and lower sites (p < 0.01). After elevation dermal thickness increased by 0.047 mm, 0.194 mm and 0.232 mm respectively. This change was statistically significant in the middle (p < 0.05) and lower sites of the limb (p < 0.01). CONCLUSION: LEPs are a sensitive marker of dermal oedema and its effects. Leg elevation is extremely effective in reducing oedema, even if only for three to four hours.

Effect of glucose concentration on the growth of normal human dermal fibroblasts in vitro.

OBJECTIVE: Glucose requirements increase in tissue repair as glucose is an energy source for cell proliferation and the formation of extracellular matrix components. Glucose concentrations in leg ulcer wound fluid are lower than in normal human serum, with a median of 0.7 mM (range: 0.3-1.2 mM). This study investigated the effect of such low concentrations on the growth of fibroblasts in vitro. METHOD: Fibroblasts from 50-year-old and 87-year-old subjects were used. Growth in medium with various concentrations of glucose was determined by a colorimetric assay and microphotography. RESULTS: Up to day 6, there were minimal differences in growth, but after day 6 a clear dose-dependent increase in growth was observed. In the lower dose range (up to 2.3 mM), growth was highly dose-dependent (r2 = 0.981), with an increase of 1.8 mM stimulating cell growth by day 10 up to 163% of the controls (p < 0.0001). Concentrations of 5.5-25.5 mM glucose stimulated cell growth by day 10 to about 210% of the controls (p < 0.0001), with little difference between these concentrations. CONCLUSION: These results suggest that glucose enhancement to cells involved in healing might serve as an adjunctive treatment for chronic wounds.

Sensory testing in patients with chronic venous leg ulcers using a 10 g Owen Mumford monofilament.

OBJECTIVE: Chronic venous insufficiency in the lower legs may directly contribute to tissue damage, and there is increasing evidence that neuronal damage may be involved. This study was carried out to determine the feasibility of using a simple neuropathy screening test in the community to diagnose sensory neuropathy in the feet of patients with chronic venous insufficiency. METHOD: We tested 15 randomly selected patients with documented chronic venous insufficiency with the 10 g Owen Mumford monofilament, in order to ascertain objectively sensory blunting. RESULTS: Forty-seven per cent of the patients had normal sensation in the foot of the ulcerated leg and 53% showed some degree of neuropathy. A recent study comparing the performance of commercially available 10 g monofilaments showed that the Owen Mumford filament was one of the most accurate, with 100% buckling within +/- 1 g of 10 g. CONCLUSION: This preliminary study indicates that the 10 g monofilament is an easy method of testing the sensory status of patients with chronic venous insufficiency in the community.

The cellular origins of the linear IgA disease target antigens: an indirect immunofluorescence study using cultured human keratinocytes and fibroblasts.

BACKGROUND: Linear IgA disease (LAD) is an IgA-mediated subepidermal immunobullous disease of adults and children, with heterogeneous immunopathology. Objectives To investigate to what extent the cellular origins of the target antigens account for the heterogeneity of the immune response in LAD. METHODS: Forty-nine adult and 33 childhood LAD sera were studied. Immunofluorescence was carried out to determine the expression of the LAD antigens by normal human keratinocytes, fibroblasts and mixed cultures of keratinocytes and fibroblasts. Immunoblotting was performed to determine the localization of the LAD target antigens in tissue extracts (48 adult and 31 childhood sera) and cell extracts (21 adult and 10 childhood sera). RESULTS: Thirty-one adult and 13 childhood LAD sera bound proteins expressed by human keratinocytes; of these sera, 15 adult and four childhood LAD sera also recognized proteins expressed by fibroblasts. A single adult serum was positive on fibroblasts alone. Seventeen adult and 20 childhood sera were negative on both cell types. There was a modest increase (9%) in the detection of the IgA autoantibodies on keratinocytes and fibroblasts grown together in mixed culture. Immunoblotting showed that the LAD target antigens could be detected in cell as well as in tissue extracts. CONCLUSIONS: Our results have shown that normal human keratinocytes and fibroblasts in culture express the LAD target antigens. LAD sera (with a single exception) bound antigens expressed by keratinocytes alone or by both keratinocytes and fibroblasts. The principal pattern of expression in keratinocytes was cytoplasmic, similar to that demonstrated by polyclonal antibodies to the 180-kDa bullous pemphigoid antigen (BP180). This reflects the pivotal role of BP180 in LAD. The finding that LAD antigens are expressed by both human keratinocytes and fibroblasts in culture may explain the heterogeneity of the target antigens, and may be a contributory factor in the immunopathology of the disease.

Effect of intermittent radiant warming on proliferation of human dermal endothelial cells in vitro.

OBJECTIVE: This study investigated the effect of Warm-Up therapy on the proliferation of human microvascular dermal endothelial cells. METHOD: Endothelial cells from an adult subject were seeded in six-well plates and placed in an incubator at 32.5 degrees C. The following day Warm-Up dressings were placed over the plates, with or without warming cards. Cards set at 38 degrees C or 42 degrees C raised the temperature in the medium to maxima of 34.5 degrees C and 37.5 degrees C respectively. Units were switched on daily for three one-hour periods. Cell numbers were counted by haemocytometer. RESULTS: Maximum stimulation of endothelial cell proliferation occurred under the 38 degrees C card, with cells numbering 135-158% of the controls (p < 0.05). the 42 degrees C card also stimulated cells (110-155%) but this did not reach statistical significance. CONCLUSION: The accelerated proliferation of microvascular dermal endothelial cells achieved by intermittent radiant warming may have contributed to the increase in granulation tissue reported previously in our clinic.

Healing venous ulcers with cycloidal multidirectional vibration therapy.

OBJECTIVE: This preliminary study combined compression bandaging with cycloidal vibration to determine whether this would enhance the rate of healing of venous leg ulceration. METHOD: Twenty-one patients with venous ulceration were enrolled into a 12-week trial. The vibration device was used three times daily for 30 minutes on each occasion, along with compression bandaging. RESULTS: Ulcers of 13 patients (62%) healed within 12 weeks. The remaining eight patients completed the 12-week study with a 31-90% reduction in ulcer size. A reduction in pain was observed in 17 out of 21 patients (81%). Ultrasound measurements showed reduced fluid content in the upper dermis in patients whose ulcers had healed and in others whose ulcers were improving. CONCLUSION: This preliminary study shows that gentle cycloid vibration, combined with standard compression bandaging, enhances the healing rate of venous ulcers and helps to relieve pain.

Effect of a lipopolysaccharide from E. coli on the proliferation of fibroblasts and keratinocytes in vitro.

Studies previously conducted in our laboratory have shown that an extract from the leaves of Chromo-laena odorata is mitogenic for human skin fibroblasts and keratinocytes. However, lipopolysaccharides, sometimes present in plant extracts, can also play a role in cell growth and might have been responsible for or contributed to the mitogenic activity observed. The present study aimed to investigate whether a lipopolysaccharide would have any effect on the proliferation of human fibroblasts and keratinocytes. Cells were seeded in 96-well plates and concentrations from 0.0 to 5.0 microg/mL of lipopolysaccharide in basal or growth medium were added. Cell growth was determined over a period of 10 days using a colorimetric assay. Lipopolysaccharide at concentrations between 0.05 microg/mL and 0.5 microg/mL in the growth medium significantly stimulated fibroblast proliferation after incubation for more than 6 days. In basal medium, more than 8 days of incubation was needed for significant stimulation of growth. Lipopolysaccharide stimulation of keratinocytes was evident at 0.5 microg/mL by day 3 in basal medium and by day 5 in growth medium. Although the lipopolysaccharide did stimulate cell growth it did so only at higher concentrations than were present in our plant extracts and to a lesser degree.

Effects of an aqueous extract from the leaves of Chromolaena odorata (Eupolin) on the proliferation of human keratinocytes and on their migration in an in vitro model of reepithelialization.

Eupolin ointment, prepared from the leaves of Chromolaena odorata, has been shown to promote the healing of soft tissue wounds and burns in Vietnam. However, the mechanism by which this agent affects cells involved in the wound healing process is unknown. Cultured human keratinocytes were used in this study to investigate the effects of the Eupolin extract in vitro on processes involved in wound reepithelialization. Keratinocyte proliferation was monitored by a colorimetric assay and migration by the closure of a denuded area scratched in a confluent monolayer. Human keratinocyte proliferation was stimulated by low concentrations of the extract (from 0.1 to 5 microg/ml), cell differentiation by higher concentrations (50 to 300 microg/ml), and migration by intermediate concentrations (5 to 60 microg/ml). The increased proliferation and migration of human keratinocytes observed in vitro might explain, in part, the beneficial effects that have been observed in the clinic.

Effects of Buddleja globosa leaf and its constituents relevant to wound healing.

An aqueous extract of Buddleja globosa leaves, used traditionally in Chile for wound healing, was tested for the ability to stimulate growth of fibroblasts in vitro and for antioxidant activity in the same fibroblast cell system challenged with hydrogen peroxide. Low concentrations of the extract gave an increase in fibroblast growth which was not statistically significant but cytotoxicity was observed at concentrations greater than 50 microg/ml. The extract showed strong antioxidant effect and fractionation led to the isolation of three flavonoids and two caffeic acid derivatives, each of which was shown to contribute to the antioxidant effect at concentrations below 10 microg/ml. These activities would accelerate the healing of wounds.

Simple biochemical markers to assess chronic wounds.

We investigated the potential for the biochemical analysis of chronic wound fluid to predict healing using simple and widely available analytes in an out-patient clinic setting. Wound fluid was collected from 12 patients attending a leg ulcer clinic and analyzed for a variety of analytes, including lactate, total protein, and albumin. Twelve weeks after collection the wound was assessed for healing (defined as complete healing or greater than 50% reduction in wound size). The median total protein (44.3 +/- 8.8 g/l) and albumin (25.0 +/- 2.3 g/l) concentrations in exudate collected from four healing wounds were significantly higher (p < 0.05) than in exudate from eight nonhealing wounds (median total protein 29.7 +/- 7.6 g/l, median albumin 17.0 +/- 4.3 g/l). No significant difference was observed for lactate. A second specimen of wound fluid was collected from four of the patients (three nonhealing and one healing). The protein analysis confirmed the pattern observed for the first collection: nonhealing wounds had total protein and albumin which remained low compared to healing wounds. No wound with an exudate albumin of less than 20 g/l healed. Both total protein and albumin are stable analytes which can be easily measured in any laboratory and may offer a simple biomarker of healing in chronic wounds.

Stimulation of fibroblast growth in vitro by intermittent radiant warming.

A number of clinical studies have suggested that radiant heat improves the healing of selected acute and chronic wounds. The purpose of this study was to investigate in vitro the effect of intermittent radiant heating on the growth of human skin fibroblasts using a radiant heat-producing dressing with a designated temperature of 38 degrees C. In initial experiments cells were seeded in six well-plates, maintained in culture at 33-34 degrees C, and warmed daily for three cycles of 1 hour with 1.5 hour intervals. Changes in cell growth and metabolism were determined in sets of triplicate wells by cell counts and a colorimetric assay before and after one week's treatment. After eight days the number of cells in the radiant heat-treated group was 30% higher and the metabolic activity 47%- 90% higher than in the control group. In quiescent fibroblasts which had been maintained for four weeks in low-serum medium, the warming regime completely prevented the decrease in cell number observed in control cells. Our findings suggest that the stimulation of cell proliferation induced by intermittent heating in vitro may indicate a possible mechanism contributing to in vivo effects.

Investigating plant-based medicines for wound healing with the use of cell culture technologies and in vitro models: a review.

INTRODUCTION: Cell culture and molecular technologies are basic yet sophisticated research tool used to investigate plant-based medicine for wound healing. METHODS: Cell viability and proliferation assay is used to determine whether there are any positive effects and to discover what is the limiting cytotoxic concentration in vitro. The scratch technique, fibroblast-populated collagen lattices and aortic rings embedded gels are used as the in-vitro models of wound re-epithelialization, contraction and angiogenesis. The immunofluorescence, immunoblotting and organotypic culture can be used to detect expression of specific proteins that are modulated by plant extracts during the wound healing process. MAIN FINDINGS: Given the dynamics of the wound healing process, cell culture and molecular technologies are advantageous in providing us with detailed studies and analysis of each intricate process. CONCLUSION: The scientific approaches for the study of traditional plant-based remedies for wound healing will provide us an important platform for rigorous testing and evaluation of their clinical efficacy based on accepted rules of evidence.

Effects of chronic wound fluid on the bioactivity of platelet-derived growth factor in serum-free medium and its direct effect on fibroblast growth.

The fate of biologically active proteins applied to chronic wounds is almost totally unknown. Growth factors may be degraded by proteases, which are produced by both inflammatory and skin cells and by resident bacteria. However, there has been little work on the effect of chronic wound fluid on the activity of growth factors. A bioassay method has been chosen to examine the effect of incubation of platelet-derived growth factor with chronic wound fluid from leg ulcers on the in vitro growth of human dermal fibroblasts. Human dermal fibroblasts were cultured in serum-free medium, and a dose-response curve for proliferation in response to platelet-derived growth factor was obtained. Wound fluid was collected under occlusive dressings from five patients with chronic leg ulcers. Platelet-derived growth factor was incubated with chronic wound fluid at 37 degrees C for 4 hours, and the reactions arrested by snap freezing. The resultant solutions were tested for their ability to promote fibroblast proliferation. A colorimetric assay was used to monitor changes in the platelet-derived growth factor mitogenicity. The results showed that, in our standard culture conditions, chronic wound fluid always stimulated fibroblast proliferation, and, in most cases, incubation of platelet-derived growth factor with chronic wound fluid increased the stimulation compared with that produced by platelet-derived growth factor or chronic wound fluid alone.

The treatment of ambulatory venous ulcer patients with warming therapy.

The standard treatment for ambulatory patients with venous ulcers is compression therapy. The aim of the present study was to develop a warming regimen to treat venous ulcers, which could be easily used by patients in their home or work environment. Five patients with a mean age of 66 years (51-80) who had venous ulcers for an average of 8 months (3-13) were treated with zip-up compression stockings (gradient compression 40 mmHg at the ankle) and a warming dressing. The latter was controlled by the patient to warm the ulcer to 38 degrees C for 1 hour three times daily. Warming therapy was carried out for 2 weeks and patients' ulcers were monitored for healing for 12 weeks. In all but one of the patients following warming therapy, there was marked increase in granulation tissue as well as a decrease in pain. Four of the five patients completely healed during the 12-week period. In conclusion, this study has demonstrated that warming therapy can be used by ambulatory patients with venous ulcers in conjunction with compression therapy. A randomized prospective study is in progress.

Effect of burn healing liquid on keratinocyte and fibroblast proliferation and on collagen lattice contraction.

OBJECTIVES: To investigate the effect of Burn Healing Liquid (BHL) on the proliferation of keratinocytes and fibroblasts and to explore the potential effect of BHL on fibroblast contraction. METHODS: Human keratinocytes and dermal fibroblasts were cultured in media containing serial dilutions of BHL followed by cell proliferation determination assessed with MTT (3-[4, 5-dimehtylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay at different time intervals. The in vitro collagen lattice contraction model was utilized for determining the contractility of fibroblasts cultured in BHL containing medium. RESULTS: The 1:10(7) dilution of BHL enhanced the growth of both keratinocytes and fibroblasts whereas the 1:10 dilution increased the growth of keratinocytes only. Collagen lattice contraction was inhibited dose-dependently by BHL and such an inhibition could be reversed by switching BHL containing medium to normal medium containing 10% fetal calf serum. CONCLUSION: BHL enhances the growth of both keratinocytes and fibroblasts and reversibly inhibits the fibroblast contraction in collagen lattice.

Topical tamoxifen--a potential therapeutic regime in treating excessive dermal scarring?

Abnormal dermal scarring which affects a large number of people is aesthetically disfiguring and can be functionally disabling. Existing medical and surgical strategies to prevent or to treat scars are frequently disappointing and more effective therapies are needed. Tamoxifen, which has been used extensively in the treatment of breast cancer over the last 20 years has recently been shown to inhibit the proliferation of fibroblasts cultured from keloid biopsies. Successful treatment of retroperitoneal fibrosis and desmoid tumours with tamoxifen has also been reported. We have investigated the potential of tamoxifen as an inhibitor of wound contraction, using fibroblast-populated collagen lattices as an in vitro model. From these studies we postulate that tamoxifen may have potential clinical significance in the treatment of abnormal scarring. Normal adult human skin fibroblasts were embedded within type I collagen, then medium either with or without addition of tamoxifen was added to the collagen lattices. Lattice diameters were measured at intervals to assess the influence of tamoxifen on the lattice contraction. The reversibility of the inhibitory effect of tamoxifen on lattice contraction was investigated by 'washing out the tamoxifen' at different time-points. To visualise changes in the morphology of fibroblasts MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was added to the lattices. Tamoxifen at 1 and 5 microM had no significant influence on lattice contraction but higher concentrations of 50 and 100 microM completely inhibited contraction. At intermediate concentrations from 10 to 20 microM the degree of lattice contraction was dose-dependent. The reversibility of the inhibition was both dose- and time-dependent. Both the inhibition of contraction and the reversibility of inhibition appeared to correlate with changes in fibroblast morphology. The dose- and time-dependent inhibition of contraction by fibroblasts suggests that tamoxifen could be investigated as a novel potential therapeutic agent in treating abnormal dermal scarring.