Tania J. Phillips

A study of the impact of leg ulcers on quality of life: Financial, social, and psychologic implications

BACKGROUND Leg ulcers affect probably 2.5 million persons in the United States, and their prevalence is likely to rise as the population ages. They cause considerable disability, and the cost of treating these chronic wounds is enormous. OBJECTIVE The purpose of this study was to assess the financial, social, and psychologic implications of leg ulcers. METHODS Data were collected by standardized personal interviews with 73 patients with chronic leg ulcers. The interview covered several domains that were selected to determine the impact of a leg ulcer on overall quality of life. RESULTS A significant number of patients had moderate to severe symptoms, principally pain, related to the leg ulcer. Eighty-one percent believed that their mobility was adversely affected by the ulcer; the dominant predictor of impaired mobility was swelling of the leg (p < 0.001). For younger, working patients, leg ulceration was correlated with time lost from work (p < 0.001), job loss (p < 0.01), and adverse effects on finances (p < 0.02). Fifty-eight percent of patients found caring for the ulcer burdensome. There was a strong correlation between time spent on ulcer care and feelings of anger and resentment. Sixty-eight percent of patients reported that the ulcer had a negative emotional impact on their lives, including feelings of fear, social isolation, anger, depression, and negative self-image. CONCLUSION Leg ulcers pose a substantial threat to a variety of dimensions of a patients quality of life.

Treatment of skin ulcers with cultured epidermal allografts

Thirty-six skin ulcers in 23 patients were treated with cultured allogeneic epidermal sheets derived from neonatal foreskin. In 73% of ulcers, there was complete healing within 8 weeks, with a mean healing time of 3.3 weeks. In the other 27%, there was reduction in ulcer size of 35% to 93% by 8 weeks after grafting. In 30 painful ulcers, pain was markedly relieved within 24 hours of grafting. The healing pattern suggested that the cultured epidermal sheets acted by stimulation of host keratinocytes to divide and migrate rather than by permanent acceptance of the allograft. Of the 26 ulcers that healed within 8 weeks, 23 (88.5%) remained healed for follow-up periods of 10 to 18 months (mean 13.7 months), with an overall mean duration of healing of 13 months.

Fibroblasts cultured from venous ulcers display cellular characteristics of senescence

PURPOSE A well-recognized characteristic of venous ulcers is impaired healing. Fibroblasts cultured from venous ulcers (wound-fb) have been shown to have reduced growth rates and are larger than normal fibroblasts (normal-fb) from the ipsilateral limb. Reduced growth capacity and morphologic changes are 2 well-known traits of cellular senescence. Other molecular changes are overexpression of matrix proteins, such as cellular fibronectin (cFN), and enhanced activity of beta-galactosidase at pH of 6.0 (senescence associated beta-Gal, or SA-beta-Gal). Senescence, an irreversible arrest of cell proliferation with maintenance of metabolic functions, may represent in vivo aging and thus may be related to impaired healing. METHODS Cultured normal-fb and wound-fb from 7 venous ulcer patients (average age, 51 years) were obtained by taking punch biopsies of the perimeter of the ulcer and from the ipsilateral thigh of the same patient. Growth rates, SA-beta-Gal activity, and level of cFN protein (immunoblot) and message (Northern blot) were measured. RESULTS In all patients, wound-fb growth rates were significantly lower than those of normal-fb (P =.006). A higher percentage of SA-beta-Gal positive cells were found in all wound-fb (average, 6.3% vs. 0.21%; P =.016). The level of cFN, was consistently higher in all wound-fb tested. Also, in 4 patients, the level of cFN messenger RNA (mRNA) was increased. CONCLUSION Fibroblasts cultured from venous ulcers exhibited characteristics associated with senescent cells. Accumulation of senescent cell in ulcer environment may be associated with impaired healing.

Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers.

About 600,000 people in the United States are estimated to be affected by venous ulcers. The cornerstone of care of chronic venous ulcers involves the application of compression bandages. Other therapies include treatment of associated infection, treatment for edema and inflammation, and debridement when necessary. Repifermin, a recombinant human KGF‐2 (fibroblast growth factor‐10), exerts a proliferative effect on epithelial cells, in vitro and in vivo, and has been shown to accelerate wound healing in several experimental animal models. A randomized, double‐blind, parallel‐group, placebo‐controlled, multicenter study was conducted to evaluate the safety and efficacy of topical repifermin treatment, for 12 weeks, in the healing of chronic venous ulcers in 94 patients. Repifermin was shown to accelerate wound healing, with significantly more patients achieving 75% wound closure with repifermin than with placebo. The treatment effect appeared more marked for a subgroup of patients with initial wound areas ≤ 15 cm2 and wound ages of ≤ 18 months. A longer duration of treatment (e.g., 26 weeks) may allow better differentiation of the benefit of repifermin compared with placebo, particularly with respect to complete wound closure. The safety assessment showed that repifermin was well tolerated.

Reduced growth of dermal fibroblasts from chronic venous ulcers can be stimulated with growth factors.

PURPOSE Although the slow healing rate of venous ulcers is well known, the underlying defect in the healing process is not well understood. The purpose of this study was to examine the cellular characteristics of fibroblasts taken from venous ulcers (wound-fb) and compare them with the fibroblasts of normal tissue (normal-fb). METHODS Biopsy specimens were obtained from wound margins and normal tissue of the upper thigh in each patient. Dermal fibroblasts were isolated from explant cultures in Dulbeccos modified Eagles medium supplemented with 10% calf serum. These cells were then plated at 1000 cells per plate, and total cells per plate were counted over time so that growth curves could be generated. In further experimentation, media was supplemented with additional calf serum (20%, 30%, 40%, 50%) and growth factors (epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta) in an attempt to stimulate growth. RESULTS Two major differences were noted: (1) normal-fb replicated more rapidly than wound-fb; and (2) the morphologic features of wound-fb were different. Normal-fb were compact and tapered, with well-defined nuclear morphologic features. Wound-fb were larger and polygonal in shape, with less-uniform nuclear morphologic features. Additional calf serum in tissue culture media enhanced normal-fb growth but had no effect on wound-fb. Supplementation of media with growth factors stimulated the growth of wound-fb. Statistically significant differences were noted at day 10 and 14 with basic fibroblast growth factor supplementation (p = 0.02 and 0.0001, respectively) and at day 14 with epidermal growth factor (p = 0.008). Although interleukin-1 beta stimulated cell growth in five of six patients, the differences observed were not statistically significant. CONCLUSIONS Our data demonstrate that wound-fb proliferate at a slower rate and are morphologically distinct from normal-fb. These characteristics are typical of aged or senescent cells. This decreased growth can be stimulated by growth factors basic fibroblast growth factor, epidermal growth factor, and interleukin-1 beta. Slowed growth may be partially responsible for the defect in healing of venous stasis ulcers. Furthermore, we believe that in some patients ulcer healing may be improved by exogenous provision of specific growth factors.

Serial surgical debridement: A retrospective study on clinical outcomes in chronic lower extremity wounds

This investigation was conducted to determine if a correlation exists between wound healing outcomes and serial debridement in chronic venous leg ulcers (VLUs) and diabetic foot ulcers (DFUs). We retrospectively analyzed the results from two controlled, prospective, randomized pivotal trials of topical wound treatments on 366 VLUs and 310 DFUs over 12 weeks. Weekly wound surface area changes following debridement and 12‐week wound closure rates between centers and patients were evaluated. VLUs had a significantly higher median wound surface area reduction following clinical visits with surgical debridement as compared with clinical visits with no surgical debridement (34%, p=0.019). Centers where patients were debrided more frequently were associated with higher rates of wound closure in both clinical studies (p=0.007 VLU, p=0.015 DFU). Debridement frequency per patient was not statistically correlated to higher rates of wound closure; however, there was some minor evidence of a positive benefit of serial debridement in DFUs (odds ratio—2.35, p=0.069). Our results suggest that frequent debridement of DFUs and VLUs may increase wound healing rates and rates of closure, though there is not enough evidence to definitively conclude a significant effect. Future clinical research in wound care should focus on the relationship between serial surgical wound debridement and improved wound healing outcomes as demonstrated in this study.

Wound chronicity and fibroblast senescence – implications for treatment

A proportion of chronic wounds fail to heal in response to standard therapy. For venous leg ulcers, a correlation exists between longer duration before treatment initiation and poor healing response to compression therapy. Differences identified between the healing wound microenvironment and that of the non healing chronic wound suggests that many potential mechanisms exist to impair healing. One contributory mechanism may be inhibition of fibroblast proliferation and induction of a stress‐induced premature senescence phenotype by the continuing inflammation found in chronic wounds. Senescent fibroblasts exhibit an extracellular matrix degradative phenotype that contributes to wound chronicity. Accumulation of greater than 15% senescent fibroblasts has been described as a threshold beyond which wounds become hard to heal. The ratio of senescent : non senescent cells is therefore critical to determining response to treatment, and adjunctive therapies that modulate this ratio in favour of non senescent cells are likely to enhance therapeutic healing rates. A number of tissue‐engineered dermal replacements contain non senescent fibroblasts and can donate cells to the wound environment additional to releasing growth factors and reversing the antiproliferative activity of chronic wound exudate. Recognition of the role of fibroblast senescence in wound chronicity may allow for identification of those wounds that will respond positively to these products.

The proliferative capacity of neonatal skin fibroblasts is reduced after exposure to venous ulcer wound fluid: A potential mechanism for senescence in venous ulcers

PURPOSE We have previously shown that fibroblasts cultured from venous ulcers display characteristics of senescence and have reduced growth rates. Susceptibility of young fibroblasts to the microcirculatory changes associated with venous ulcers, such as macrophage trapping and activation, could explain the prevalence of senescent fibroblasts in these wounds. METHODS We tested the in vitro effect of venous ulcer wound fluid (VUWF), as well as pro-inflammatory cytokines known to be present in VUWF (TNF-alpha, IL-1beta, and TGF-beta1), on neonatal foreskin fibroblasts (NFFs). NFF growth rates, cellular morphology, and senescence-associated beta-galactosidase (SA-beta-Gal) activity were determined in the presence or absence of VUWF and the above cytokines. VUWF TNF-alpha concentration and the effect of anti-TNF-alpha antibody on VUWF inhibitory activity were determined in samples obtained from four patients with venous ulcers. RESULTS NFF growth rates were significantly reduced by VUWF (42,727 +/- 6301 vs 3902 +/- 2191 P =. 006). TNF-alpha also significantly reduced NFF growth rates in a dose-dependent manner (P =.01). No significant growth-inhibitory activity was seen for IL-1alpha or TGF-beta. Incubation with VUWF significantly increased the percentage of SA-beta-Gal-positive fibroblasts in vitro on culture day 12 (P =.02). TNF-alpha and TGF-beta1 had similar effects. TNF-alpha was detected in all VUWF tested, with a mean of 254 +/- 19 pg/mL. CONCLUSION These data suggest that the venous ulcer microenvironment adversely affects young, rapidly proliferating fibroblasts such as NFFs and induces fibroblast senescence. Pro-inflammatory cytokines such as TNF-alpha and TGF-beta1 might be involved in this process. The role of other unknown inhibitory mediators, as well as pro-inflammatory cytokines, in venous ulcer development and impaired healing must be considered.

Early healing rates and wound area measurements are reliable predictors of later complete wound closure

This study was undertaken to determine if healing rates are reliable early predictors of ultimate complete wound closure in venous leg ulcers and diabetic foot wounds. We conducted a retrospective analysis of 306 venous leg ulcers and 241 diabetic foot ulcers enrolled in two large controlled, prospective, randomized pivotal trials to compare topical wound treatments, to determine whether certain early markers of healing could be correlated with later total wound closure. Two‐sided tests at 95% confidence demonstrated that wound margin advance, initial healing rate, percent wound surface area reduction, and wound healing trajectories (all p<0.001) were powerful predictors of complete wound healing at 12 weeks. Wounds with poor healing progress by these criteria at 4 weeks were highly likely to remain unhealed after 8 additional weeks of treatment. Analysis of the diabetic foot ulcers and venous leg ulcers subgroups separately demonstrated consistent statistical test results with high significance; similarly, the results remained valid independent of the topical treatment used. The early prediction of eventual wound healing or nonhealing using early healing rates may enable more efficient triage of patients to advanced healing technologies. We believe that these surrogate markers are robust predictors of healing regardless of wound etiology and that they merit wider use in clinical trials and routine patient care.

Cultured epidermal autografts and allografts: A study of differentiation and allograft survival

Cultured epidermal sheets were examined before and at various times after grafting on skin ulcer beds. Before grafting, the sheet consisted of four to five layers of keratinocytes with incomplete differentiation. Ten days after grafting, graft recipient sites showed compact hyperkeratosis, a normal-appearing epidermis, and a flat dermoepidermal junction. At 6 months, the stratum corneum had a basket-weave appearance but the dermoepidermal junction remained flat. Monoclonal antibodies to keratins 14 and 10 showed normal basal and suprabasal localization, respectively. Electron microscopy showed a normal basement membrane with anchoring fibrils. LH7:2, a monoclonal antibody that binds to the type VII collagen molecule, stained the dermoepidermal junction in all biopsy specimens. AE-1, an antibody that stains suprabasal cells in hyperproliferative skin, was expressed suprabasally for up to 12 weeks after healing (16 weeks after grafting), but expression was confined to the basal layer at 18 weeks after healing (6 months after grafting). Anti-involucrin staining was found in the deeper layers of the epidermis up to 12 weeks after healing (16 weeks after grafting) but had receded to a normal distribution in upper spinous and granular layers at 18 weeks (6 months after grafting). Overall, the histologic patterns observed in recipient sites during the first 4 months after grafting resembled those observed for 10 to 14 days in newly healed epidermis and in hyperproliferative states such as psoriasis. In four sex-mismatched graft sites, specimens were reacted with a biotinylated probe to the Y chromosome by in situ hybridization. Lack of Y chromosome-positive cells suggested that host keratinocytes had replaced the allografts. Multilocus DNA analysis in one patient confirmed this observation. Our data suggest that an altered state of epithelial maturation persists for several months after culture grafting, with restoration of the normal pattern by 6 months. No differences were detected between autografted and allografted sites.