Can leg ulcer fibroblasts phenotype be influenced by human amniotic membrane extract?

Prevalence of leg ulcer in general population is important and new efficient treatments are now needed, especially for chronic leg ulcers. Human amniotic membrane (HAM) can be used as an alternative treatment for recalcitrant leg ulcers. The aim of this study is to investigate the effects of a HAM extract on normal fibroblasts (NF) and ulcer fibroblasts (UF). NF and UF were obtained from biopsies by explants technique. HAM extract was used at 10 µg of total proteins per ml. Single patient-matched NF and UF were compared, without or with HAM extract. Studied parameters were proliferation rate, retraction of free-floating lattices, alpha smooth muscle actin expression by flow cytometry, and synthesis of elastin, glycosaminoglycans (GAGs), pro-collagen I, MMP-1 and TIMP-1. Our results show that UF had a specific phenotype compared to NF: low proliferation, high expression of alpha-SM actin and high synthesis of MMP-1, TIMP-1 and elastin. HAM extract significantly increased the synthesis of GAGs, pro-collagen I and MMP-1 in NF and decreased retraction of free lattices. HAM extract transiently increased UF proliferation, slowed down lattices retraction and decreased elastin synthesis. In conclusion, HAM extract has little effect on UF for the parameters studied and NF are more responsive than UF. However, clinical beneficial effect of HAM application on leg ulcers was previously observed and might rather be related to an action on keratinocytes and/or a modulation of the highly inflammatory environment of these chronic wounds.

Development of an in vitro fibrin clot model to evaluate fibrinolytic agents for wound care application.

OBJECTIVE: To describe an in vitro fibrin clot model that could reliably assess the fibrinolytic activity of enzymatic debriding agents for wound care application. METHOD: A model of a fibrin clot was reconstructed in vitro by mixture of human fibrinogen and (alpha)-thrombin supplemented with factor XIII. These clots were then treated with enzymatic ointments. Fibrinolytic activity was investigated by measuring D-dimer levels, using an automated immunoturbidimetric Liatest D-dimer assay. RESULTS: Collagenase and papain-urea ointments demonstrated fibrinolytic activity which was macroscopically visible. Their effect was identical on the in vitro reconstructed fibrin clot and ex vivo collected wound fibrin clot; collagenase and papain-urea both induced a complete degradation and dissolution of both fibrin clots after 24 hours of treatment. This was associated with an increase in D-dimer concentration. CONCLUSION: This reconstructed fibrin clot in vitro model has the potential to predict the efficacy of fibrinolytic agents and therefore appears to be a suitable model for in vitro assays. DECLARATION OF INTEREST: This study was supported by a grant from URGO Laboratory.

Assessment of the efficacy of a new formulation for plantar wart mummification: new experimental design and human papillomavirus identification.

Cutaneous warts are caused by infection of the epidermis with human papillomavirus (HPV). Cryotherapy using liquid nitrogen is one of the most common local treatments. In this study, we used a novel ex vivo approach to compare the efficacy of a new product with conventional liquid-nitrogen cryotherapy by studying epidermal histology and assessing the presence of HPV types 1 and 2 DNA in plantar warts. The studied formulation, which acts by tissues mummification, is a combination of nitric acid, organic acids and metallic salts. We found that, similar to liquid nitrogen, the studied product induced alterations in the wart structure. In addition, unlike liquid nitrogen, this product also reduced the amount of HPV DNA. The results suggest that there is a poor correlation between the histological response and the antiviral efficacy of standard wart treatment.

[Human amniotic membrane in the management of chronic venous leg ulcers]. / La membrane amniotique humaine dans la prise en charge des ulcères de jambe veineux chroniques.

BACKGROUND: The aim of our paper is to examine changes in the use of human amniotic membrane for venous leg ulcers through clinical studies and to present different models of tissue engineering employing human amniotic membrane for the purpose of future therapeutic use in wound healing. MATERIALS AND METHODS: This review is based on information obtained from a PubMed search using the following keywords "Amnion"[Mesh] AND "Leg Ulcer"[Mesh]; "Amnion"[Mesh] AND "Dermatology"[Mesh]. The selected articles are in English or French and deal with the sole use of human amniotic membrane in venous leg ulcers alone. RESULTS: Human amniotic membrane has a positive impact on chronic venous leg ulcers by promoting granulation tissue formation, reducing fibrosis and inducing re-epithelialisation. Nevertheless, further randomized clinical studies are needed. At the same time, tissue engineering models using human amniotic membrane may help to reduce wound healing time, thereby creating renewed interest in the use of human amniotic membrane. CONCLUSION: Considering its properties and the clinical studies analysed, human amniotic membrane could be useful in venous leg ulcer care.

Comparing the contractile properties of human fibroblasts in leg ulcers with normal fibroblasts.

OBJECTIVE: The tissue contraction phenomenon associated with wound healing is of prime importance for wound closure. Contractile properties of human fibroblasts from chronic venous leg ulcers were compared with those of normal fibroblasts using in vitro models. METHOD: Biopsies were taken from the uninvolved skin of the thigh, the epithelialised ulcer edge and the non-epithelialised ulcer centre in four patients (average age: 78 years). Fibroblasts were obtained by an explant technique and expanded in vitro in Dulbecco's Modified Eagle's Medium supplemented with 10% foetal calf serum and used for the assays at their fourth passage. Intracellular alpha-smooth muscle actin expression (alphaSM-actin) was studied by immunofluorescence labelling of cells cultured in monolayer. Contractile properties were evaluated using three-dimensional collagen lattices. RESULTS: Fibroblasts from the ulcer centre were the richest cells in actin filaments. Both populations of venous ulcer fibroblasts contracted more rapidly and to a greater extent than normal fibroblasts. The peak contractile forces developed by fibroblasts from the ulcer centre and the ulcer edge were 30% and 18% greater than normal fibroblasts respectively. CONCLUSION: Some functions of fibroblasts, in particular the generation of contractile forces and the formation of cytoplasmic actin filaments, seem not to be affected in chronic venous ulcers. DECLARATION OF INTEREST: This study was supported by the Fondation Coloplast pour la Qualite de la Vie of France.

Comparison of different wound dressings on cultured human fibroblasts and collagen lattices.

OBJECTIVE: We compared the effects on cultured human fibroblasts of a new non-adhesive wound dressing, Urgotul, with five other wound dressings. Urgotul is a hydrocolloid dressing; the comparator dressings included impregnated gauze and modern wound dressings. METHOD: Cultures in monolayer were used to study the morphology and growth of fibroblasts. The Bell model of cultured dermis equivalents was used to investigate myofibroblast differentiation. These cultures were labelled a-SM actin and F-actin. RESULTS: Two of the tested dressings induced cytotoxic effects. They were found to inhibit cell growth (greater than 60%) and to disturb cell shape and cytoskeletal differentiation. Urgotul and the remaining three dressings showed no effect on proliferation. However, some of them modified fibroblast morphology and affected F-actin distribution. CONCLUSION: Depending on their nature and components, wound dressings may respect or affect fibroblast behaviour in vitro (proliferation, morphology and a-SM actin and F-actin distribution). The significance of these in vitro observed findings require further investigations.

Assessment of collagen lattice thickness by B-scan echography.

BACKGROUND/AIMS: Collagen lattices are an in vitro dermal equivalent that has led to the development of an original model of dermal tissue. Fibroblasts cultured in three-dimensions in a collagen matrix differentiate similarly to in vivo. New technological performances in ultrasonic imaging can now provide precise measurements of tissue thickness with good resolution. The aim of this study was to assess, by B-scan echography, the correlation between collagen lattice thickness and various collagen and cell concentrations. METHODS: Three concentrations of human dermal fibroblasts (F1 = 8.10(5)C/mL, F2 = 16.10(5)C/mL, F3 = 32.10(5)C/mL) and three concentrations of rat tail collagen (C1 = 2 mg mL(-1), C2 = 3 mg mL(-1), C3 = 4 mg mL(-1)) were prepared for five different kinds of collagen lattices: F(2)C(1), F(2)C(2), F(2)C(3), F(1)C(1) and F(3)C(1) (n = 5 per case). Ultrasonic imaging was performed on day 0, 4, 6, 10, 12 and 14 using a Dermcup 2020 scanner. The scans measured thickness in the centre and periphery of the lattice. RESULTS: The collagen lattice echogenicity was similar to a dermis in vivo. For each assessment, the collagen lattice thickness increased until day 12 and then stabilized. The lattice was thicker when the cellular concentration was higher, (at day 14: F(1C1) = 0.66 mm, F(2C1) = 0.86 mm, F(3C1) = 1.21 mm). The collagen concentration did not significantly influence lattice thickness. CONCLUSION: Collagen lattice thickness increased with retraction time and cellular concentration.

[Mechanical behavior of fibroblasts included in collagen lattices]. / Comportement mécanique de fibroblastes de vergetures inclus dans des lattices de collagène.

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin. Excessive steroid activity, genetic and mechanical factors and inherited defects of connective tissues are the most frequent causes of this disease. Fibroblasts derived from women presenting striae distensae lesions were included into collagen gels to study their mechanical behavior: capacity to contract free-floating lattices and to produce isometric force in tense lattices. To measure the retracted lattice diameter, the culture dishes were placed on a transparent metric scale. An isometric force system was used to study quantitatively the forces developed during lattice contraction. alpha 2 beta 1 integrins expression (transmembrane receptors) was evaluated by flux cytometry. Striae distensae fibroblasts contract collagen gels slower than normal human fibroblasts but the final contraction is similar. They produce a greater isometric force which is associated with enhanced alpha 2 beta 1 integrins expression. By their mechanical properties, striae distensae fibroblasts appear as a different population from normal fibroblasts.