Models of wound healing: an emphasis on clinical studies.

BACKGROUND: The healing of wounds has always provided challenges for the medical community whether chronic or acute. Understanding the processes which enable wounds to heal is primarily carried out by the use of models, in vitro, animal and human. It is generally accepted that the use of human models offers the best opportunity to understand the factors that influence wound healing as well as to evaluate efficacy of treatments applied to wounds. OBJECTIVES: The objective of this article is to provide an overview of the different methodologies that are currently used to experimentally induce wounds of various depths in human volunteers and examines the information that may be gained from them. METHODS: There is a number of human volunteer healing models available varying in their invasiveness to reflect the different possible depth levels of wounds. RESULTS: Currently available wound healing models include sequential tape stripping, suction blister, abrasion, laser, dermatome, and biopsy techniques. The various techniques can be utilized to induce wounds of variable depth, from removing solely the stratum corneum barrier, the epidermis to even split-thickness or full thickness wounds. CONCLUSION: Depending on the study objective, a number of models exist to study wound healing in humans. These models provide efficient and reliable results to evaluate treatment modalities.

A plant oil-containing pH 4 emulsion improves epidermal barrier structure and enhances ceramide levels in aged skin.

OBJECTIVE: Xerosis is a serious problem among the very old. It is a dermatological challenge caused by significant alterations in stratum corneum (SC) function and structure. Two negative changes in aged skin are (i) the enhanced skin surface pH and (ii) the altered SC lipid content, composition and ordering. METHODS: Therefore, we investigated the way in which an acidic skin care product with different plant oils affects SC function, structure and lipid profile in older subjects with dry skin. Before and after a 3-week application period, different biophysical measurements were performed: transepidermal water loss, SC hydration and skin surface pH. In addition, the SC lipid matrix was evaluated by analysis of the intercellular lipid lamellae and the SC lipid profile. RESULTS: After treatment, a significant increase in lipid lamellae in the intercellular space of the SC was observed in the area treated with the test product compared to the untreated area. Furthermore, the ceramide level was found to be increased, although ceramides were not provided by the acidic test formulation. CONCLUSION: In summary, topical application of a pH 4.0 product containing plant oils improves epidermal barrier formation and SC lipid ordering and ratio in aged dry skin.

Dexpanthenol modulates gene expression in skin wound healing in vivo.

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1ß, CYP1B1, CXCL1, CCL18 and KAP 4-2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts.

p53 labeling index in assessing the efficacy of a sunscreen in protection against UV-induced damage.

BACKGROUND: Sunscreen efficacy is currently evaluated by the estimation of the sun protection factor (SPF). Our objective was to determine the role of the p53 labeling index in assessing the effectiveness of a sunscreen in the prevention of UV-induced DNA damage. METHODS: A broad-spectrum sunscreen (SPF15) was applied to two of four test areas on the backs of 20 volunteers. Two test areas (with and without prior sunscreen application) were then irradiated with two minimal erythemal doses (MEDs) of solar-simulated UV radiation (290-400 nm). At 24 h, suction blisters were raised from all four test areas using a vacuum technique, and immunohistochemical staining for p53 was performed. RESULTS: In normal epidermis, nonirradiated without sunscreen, p53 was almost undetectable [0.5% positive cells per field (PC/F)]. In UV-exposed skin without sunscreen, p53 expression was 10.4% PC/F, whereas, in skin treated with sunscreen before UV irradiation, p53 expression was 0.3% PC/F. The mean positive labeling indices (x10(3)/microm) for UV-exposed skin with and without sunscreen were 0.002 and 0.059, respectively (P < 0.001). CONCLUSIONS: In vivo assessment of the p53 labeling index using the suction blister method can serve as a sensitive endpoint in the evaluation of the effectiveness of a sunscreen in the prevention of UV-induced DNA damage.

Guidelines for visualization of cutaneous blood flow by laser Doppler perfusion imaging. A report from the Standardization Group of the European Society of Contact Dermatitis based upon the HIRELADO European community project.

This report reviews how to set up a laser Doppler perfusion imaging system intended for visualization of skin blood perfusion, capture images and evaluate the results obtained. A brief summary of related papers published in the literature within the areas of skin irritant and allergy patch testing, microdialysis and skin tumour circulation is presented, as well as early applications within other fields such as diabetology, wound healing and microvascular research.

Comparison of three techniques for evaluating skin erythemal response for determination of sun protection factors of sunscreens: high resolution laser Doppler imaging, colorimetry and visual scoring.

BACKGROUND/PURPOSE: Sun protection factor (SPF) measurement is based on the determination of the minimal erythema dose (MED). The ratio of doses required to induce a minimal erythema between product-treated and untreated skin is defined as SPF. The aim of this study was to validate the conventionally used visual scoring with two non-invasive methods: high resolution laser Doppler imaging (HR-LDI) and colorimetry. Another goal was to check whether suberythemal reactions could be detected by means of HR-LDI measurements. MATERIALS AND METHODS: Four sunscreens were selected. The measurements were made on the back of 10 subjects. A solar simulator SU 5000 (m.u.t., Wedel, Germany) served as radiation source. For the visual assessment, the erythema was defined according to COLIPA as the first perceptible, clearly defined unambiguous redness of the skin. For the colorimetric determination of the erythema, a Chromameter CR 300 (Minolta, Osaka, Japan) was used. The threshold for the colorimetry was chosen according to the COLIPA recommendation as an increase of the redness parameter delta a* = 2.5. For the non-contact perfusion measurements of skin blood flow, a two-dimensional high resolution laser Doppler imager (HR-LDI) (Lisca, Linköping, Sweden) was used. For the HR-LDI measurements, an optimal threshold perfusion needed to be established. RESULTS: For the HR-LDI measurements basal perfusion +1 standard deviation of all basal measurements was found to be a reliable threshold perfusion corresponding to the minimal erythema. Smaller thresholds, which would be necessary for detection of suberythemal responses, did not provide unambiguous data. All three methods, visual scoring, colorimetry and HR-LDI, produced similar SPFs for the test products with a variability of < 5% between methods. The HR-LDI method showed the lowest variation of the mean SPF. Neither of the instrumental methods, however, resulted in an increase of the sensitivity of SPF determination as compared with visual scoring. CONCLUSION: Both HR-LDI and colorimetry are suitable, reliable and observer-independent methods for MED determination. However, they do not provide greater sensitivity and thus do not result in lower UV dose requirements for testing.

SLS-irritated human skin shows no correlation between degree of proliferation and TEWL increase.

It is well known that cutaneous irritants influence epidermal proliferation but the pathogenesis is poorly understood. Recent investigations have shown that the skin barrier integrity influences the proliferation of the basal keratinocytes. Our question was whether the proliferating activity of keratinocytes is indeed regulated by the degree of skin barrier damage or by a direct toxic action of the irritant on the keratinocytes. Therefore various degrees of skin irritation were induced by the application of 0.1%, 0.5% and 2% sodium lauryl sulphate (SLS) solution to the forearm skin of six healthy volunteers. This experiment was performed to evaluate the relationship between SLS concentration and epidermal proliferation. In a second experiment another 14 volunteers were treated with a single SLS concentration (0.5%) to look for interindividual differences in the patterns of skin reaction and susceptibility to the irritant. Skin barrier function was evaluated by measurements of transepidermal water loss (TEWL) before and after irritation. Punch biopsies were taken after 96 h from exposed areas and from unexposed normal skin. Dividing keratinocytes were identified immunocytochemically using three different monoclonal antibodies: PCNA, MIB 1 and KiS1. Exposure to SLS resulted in concentration-dependent increases in both TEWL and epidermal proliferation. However, no significant correlation could be found between the degree of hyperproliferation and the TEWL changes. The results suggest that epidermal proliferation is modulated by a direct interaction of the surfactant with the keratinocytes and/or by release of mediators rather than the consequence of a barrier disturbance.

Determination of acitretin in the skin, in the suction blister, and in plasma of human volunteers after multiple oral dosing.

Several HPLC methods for quantification of acitretin and its 13-cis isomer in biological fluids have been described. Only limited data are available on determination of this drug in skin samples. Our objective was to improve the sensitivity and selectivity of existing methods to measure drug in small skin samples from humans treated with acitretin. With a new optimized mobile phase [methanol: acetonitrile (7:3, v/v), purified water with 1.5% (v/v) acetic acid, mixed in a 85:15 ratio (v/v)] and a new internal standard (arotinoid ethyl sulfone), a limit of quantification of 1 ng/g tissue was reached. Nine male volunteers were given an oral daily dose of 50 mg acitretin for up to 28 days. Blood and skin samples (punch and shave biopsies, suction blister skin, and fluid) were taken at various time points during and after treatment. Drug concentration and metabolism in plasma and skin samples appeared to be linked in that the trans-isomer concentration was always higher than the cis-isomer concentration during dosing and 3 h after the last dose. However, 7 and 14 days after the last dose in plasma and in all tissue samples (except the shave biopsy), the all-trans-acitretin concentration rapidly decreased and approached the detection limit. In the shave biopsy, the all-trans-acitretin concentration remained higher than the 13-cis-acitretin concentration. Furthermore, the elimination of two isomers from the shave biopsy was delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

[Penicillamine-induced elastosis perforans serpiginosa]. / Penicillamin-induzierte Elastosis perforans serpiginosa.

Elastosis perforans serpiginosa is a rare disease with variable etiology. We report on a 33-year-old male patient in whom the disease was induced by long-term treatment with the chelating agent D-penicillamine.

[Hibernoma of the forehead. A rare tumor of brown fatty tissue in an unusual site]. / Hibernom der Stirn. Ein seltener Tumor des braunen Fettgewebes in ungewöhnlicher Lokalisation.

We report a hibernoma located on the forehead in a 52-year-old female patient. The hibernoma is a rare tumour derived from brown adipose tissue. Location on the forehead is unusual and has not been reported before.

In vivo skin penetration of acitretin in volunteers using three sampling techniques.

Etretinate and acitretin are given orally to treat psoriasis and various keratinization disorders. Acitretin, the main active metabolite of etretinate, has the pharmacokinetic advantage of being rapidly eliminated, but it shares etretinate's toxicologic profile. Thus a topical delivery of acitretin with no or reduced systemic adverse effects is desirable. To characterize the therapeutic potential of topically delivered acitretin, we quantitatively assessed its percutaneous penetration in healthy human volunteers. Additionally, three skin sampling techniques, the punch biopsy, the shave biopsy, and the suction blister technique, were validated to quantitate acitretin in the skin. The results suggest that topical delivery of acitretin renders skin concentrations which exceed those reported after oral administration of etretinate or acitretin. However, because of possible interlaminate drug contamination, drug localization within a particular skin compartment cannot be determined.

Effect of sodium lauryl sulfate-induced skin irritation on in vivo percutaneous penetration of four drugs.

The influence of sodium lauryl sulfate-induced irritant contact dermatitis on in vivo percutaneous penetration was investigated for four 14C-labeled compounds with diverse physicochemical properties: hydrocortisone (HC), indomethacin (IM), ibuprofen (IB), and acitretin (AC). Hairless guinea pigs were pretreated for 24 h with either 0.5% sodium lauryl sulfate (SLS) to induce irritant contact dermatitis or with water (controls). Twenty-four hours after pretreatment, 450 microliters saturated solutions of HC, IM, IB, or AC in isopropylmyristate were applied to the pretreated skin for 24 h. Systemic absorption was determined by urinary and fecal excretion of compounds. Drug concentrations in stratum corneum (obtained by tape cellophane stripping after decontamination of the application site) and in epidermis/dermis (punch biopsy) were also investigated. Systemic absorption of topically applied drugs (as evaluated by urinary and fecal excretion) in SLS-irritated skin was significantly increased for HC (factor 2.6) followed by IB (1.9 times) and IM (1.6 times) but not increased for AC. However, drug concentrations in the viable epidermis and dermis were 70% lower in SLS-irritated than normal skin for HC, but not different for IB, IM, and AC. Thus, the influence of the state of the skin (irritant dermatitis versus healthy) on percutaneous penetration was different for diverse drugs. The general assumption that percutaneous penetration and drug tissue concentrations were higher in diseased versus healthy skin was not found to be true in our irritated-skin model.

Enhanced in vivo-lipid peroxidation associated with halothane hepatotoxicity in rats.

To study the role of lipid peroxidation in halothane-induced hepatic damage, ethane exhalation by rats exposed to 1% halothane for 1 hour was determined under normoxic (21% O2) and hypoxic (6% O2) conditions. The effects of microsomal enzyme induction by phenobarbital and/or glutathione depletion on this parameter of in vivo lipid peroxidation were studied. To assess the degree of liver damage, serum activities of liver specific enzymes (glutamate-pyruvate-transaminase, GPT, and sorbitol dehydrogenase, SDH) were measured 3 hrs after the end of exposure. Besides, liver content of thiobarbituric acid-reactive material was estimated as a further parameter of lipid peroxidation. Enhanced rates of lipid peroxidation over basal levels were only seen under conditions leading to hepatic damage, i.e. phenobarbital induction and hypoxia. The highest rate of lipid peroxidation was observed after depletion of hepatic glutathione in addition to microsomal enzyme induction and hypoxia. Deferrioxamine, diethyldithiocarbamate and (+)-catechin inhibited in vivo lipid peroxidation, but only (+)-catechin suppressed halothane-hepatoxicity. These results indicate that halothane-induced hepatic damage is associated with an enhanced rate of lipid peroxidation, but this might not be the only mechanism of halothane toxicity.

Halothane hepatotoxicity in glutathione depleted rats.

Experimental models for halothane hepatotoxicity require microsomal enzyme induction by phenobarbital or triiodo-thyronine pretreatment and hypoxic conditions. The role of GSH in the metabolism of halothane, however, is still unclear. We therefore pretreated male rats with phorone to deplete hepatic GSH, phenobarbital as a microsomal enzyme inducer and exposed them to halothane 1% for 4 h under hypoxia (10% O2). Increases in serum enzyme activities of alanine aminotransferase (GPT) and sorbitol dehydrogenase (SDH) were observed 24 and 48 h later. Histomorphological examinations showed centrilobular hepatic necrosis. In GSH-depleted rats the increments of serum enzyme activities and histomorphological alterations were significantly aggravated as compared with controls. In this model (+)-catechin protected against halothane-induced hepatotoxicity as evidenced by reduced serum enzyme elevations and morphological alterations whereas diethyldithiocarbamate failed to exert any protective effects. Free fluoride concentrations in plasma was used as an index of the non-oxidative defluorination of halothane. Increased plasma fluoride levels were observed under conditions which evoked hepatotoxicity but did not correlate with the protective effect of (+)-catechin. Our experimental data indicate that glutathione might be involved in the non-oxidative metabolic pathways of halothane. Furthermore, (+)-catechin seems capable of protecting against the direct toxic effect of halothane metabolites resulting from the reductive pathways.