Margaret A. Hughes

Evidence of oxidative stress in chronic venous ulcers

Reactive oxygen species have been implicated in the impaired healing of chronic leg ulcers but little direct evidence is available. We have observed a significant (p < 0.01) elevation of the allantoin : uric acid percentage ratio, a marker of oxidative stress, in wound fluid from chronic leg ulcers (median 17, range 8–860) compared to both paired plasma (median 2, range 1–8) and acute surgical wound fluid (median 4, range 3–7). However, the allantoin : uric acid percentage ratio did not differ significantly between chronic wounds that healed and those that failed to heal. Neutrophil elastase was elevated 30‐ to 1300‐fold in chronic wound fluid compared to plasma and there was a correlation (r 2 = 0.742) between wound fluid elastase and the allantoin : uric acid percentage ratio. Total antioxidant capacity of wound fluid, as measured with a chemiluminescence assay, did not show a correlation (r 2 = 0.03) with the observed oxidative stress. These observations suggest that conditions of localized oxidative stress, possibly related to neutrophil‐associated production of reactive oxygen species, are present in chronic leg ulcers. It is possible that future therapeutic strategies aimed at reducing oxidative stress, in addition to good standard care, could improve healing rates of chronic wounds. (WOUND REP REG 2003;11:172–176)

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 °C. In initial experiments cells were seeded in six well‐plates, maintained in culture at 33–34 °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 weeks 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.

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 °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.

enhanced Proliferation of Fibroblasts and Endothelial Cells Treated with an Extract of the Leaves of chromolaena odorata (eupolin), an Herbal Remedy for Treating Wounds

&NA; Burns are a major problem in many developing countries. Eupolin ointment is a topical agent used in the treatment of soft‐tissue wounds and burns in Vietnam and is made from an aqueous extract of the leaves of Chromolaena odorata (formerly Eupatorium odoratum). Clinical studies using this extract have shown antimicrobial and anticoagulation effects as well as the promotion of tissue remodeling in the wound healing process. However, the mechanism by which this agent affects cells involved in the wound healing process is unknown. In our research, fibroblasts and endothelial cells, two cell types that play a crucial role in wound healing, were used to investigate some of the effects of Eupolin extract in vitro. Cell growth was estimated by a colorimetric assay at different time intervals. Enhanced growth of fibroblasts and endothelial cells was found at concentrations of 10 &mgr;g/ml and 100 &mgr;g/ml of Eupolin extract. This was particularly evident in medium supplemented with only 0.5% fetal calf serum where the cells were quiescent. Toxicity of the extract to fibroblasts was observed at 250 &mgr;g/ml in Dulbeccos modified Eagles medium/0.5% fetal calf serum, but there was no significant damage at this dose to the endothelial cells. The results of the study demonstrated that Eupolin extract increased fibroblast and endothelial cell growth, and this could explain in part the beneficial clinical effects that have been observed.

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 μg/ml), cell differentiation by higher concentrations (50 to 300 μg/ml), and migration by intermediate concentrations (5 to 60 μg/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.

In vitro studies on the antioxidant and growth stimulatory activities of a polyphenolic extract from Cudrania cochinchinensis used in the treatment of wounds in Vietnam.

The leaves of Cudrania cochinchinensis, a Vietnamese folk remedy, have been suggested as a beneficial agent for wound healing. Animal studies and clinical observations in Vietnam have shown positive wound healing activity. We studied the effects of a polyphenolic extract from the plant on the proliferation of cells in culture and their response to oxidative damage by hydrogen peroxide. Fibroblasts were incubated with different concentrations of the extract in 0.4% fetal calf serum, and proliferation was monitored by a colorimetric assay. Cell damage was induced by exposure to hydrogen peroxide 7 × 10−5 mol/L for 3 hours. The same colorimetric assay was used to assess cell damage and the protective effect of the extract against oxidative damage. The extract at low concentrations (0.1 to 5 µg/ml) had a stimulatory effect on fibroblast growth. The effect was significant by 7 days after the addition of the extract and was strongest at a concentration of 1 µg/ml of extract (p≤ 0.01). The extract at concentrations of 5 or 50 µg/ml protected fibroblasts and endothelial cells against hydrogen peroxide‐induced damage. Pretreatment with the extract or exposure to extract simultaneously with hydrogen peroxide gave only partial protection against oxidative damage, whereas a combination of the two treatments gave complete protection (p≤ 0.005). Stimulation of fibroblast proliferation and protection of cells against destruction by inflammatory mediators may be ways in which the polyphenolic substances from the plant, Cudrania cochinchinensis, contribute to wound healing.

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

Summary Background Linear IgA disease (LAD) is an IgA‐mediated subepidermal immunobullous disease of adults and children, with heterogeneous immunopathology.

The effect of minoxidil analogues and metabolites on the contraction of collagen lattices by human skin fibroblasts

Minoxidil, in addition to its effect on hypertension and hair growth, has been proposed as a potential antifibrotic agent. Minoxidil inhibits the contraction of collagen lattices by human fibroblasts in vitro. However, the mechanism of inhibition is unknown. As minoxidil is metabolised in the body to minoxidil glucuronide and minoxidil sulphate, we investigated the potencies of these metabolites to inhibit collagen lattice contraction. We also studied selected analogues of minoxidil to assess the influence of certain functional groups in the inhibition. The major metabolite, minoxidil glucuronide, proved to be inactive, whereas minoxidil sulphate was considerably more active than minoxidil. In terms of the structural analogues, the substitution of one amino group by a methyl group resulted in loss of the inhibitory activity; removal of the nitroxide oxygen led to stronger inhibition than with minoxidil. Further studies are planned to learn more about a possible role for minoxidil in wound contraction.