In vitro optimisation of topical negative pressure regimens for angiogenesis into synthetic dermal replacements.

BACKGROUND: The use of synthetic dermal replacements (SDRs) in the treatment of large wounds, which have associated morbidity and mortality, has attracted great interest. However, because of poor outcome, SDRs have limited use. The addition of topical negative pressure (TNP) has increased their success, but little research has focused on the underlying mechanisms. This paper studies the in vitro effects of TNP on commonly used SDRs to identify the most effective TNP regimen and optimum SDR for encouraging endothelial cell ingress. METHODS: Endothelial cells were co-cultured in vitro on four SDRs with or without TNP. Negative pressure (125mmHg) was applied intermittently, continuously, for 4h per day, or not at all. Endothelial ingress was measured for each condition. RESULTS: In the collagen controls, cell migration was minimal. Integratrade mark gave the greatest endothelial cell migration (p<0.05, n=3). TNP increased endothelial cell migration, intermittent application being the optimum regimen. CONCLUSIONS: Integratrade mark has an open sponge structure which may account for greater angiogenicity than Allodermtrade mark, Permacoltrade mark and Xenodermtrade mark. In vitro intermittent TNP stimulates the greatest angiogenic response. The majority of clinical studies investigating SDR success with TNP have used continuous regimens; this study suggests a change in clinical practice to intermittent application.

Dermal fibroblasts derived from fetal and postnatal humans exhibit distinct responses to insulin like growth factors.

BACKGROUND: It has been well established that human fetuses will heal cutaneous wounds with perfect regeneration. Insulin-like growth factors are pro-fibrotic fibroblast mitogens that have important roles in both adult wound healing and during development, although their relative contribution towards fetal wound healing is currently unknown. We have compared responses to IGF-I and -II in human dermal fibroblast strains derived from early gestational age fetal (<14 weeks) and developmentally mature postnatal skin to identify any differences that might relate to their respective wound healing responses of regeneration or fibrosis. RESULTS: We have established that the mitogenic response of fetal cells to both IGF-I and -II is much lower than that seen in postnatal dermal fibroblasts. Further, unlike postnatal cells, fetal cells fail to synthesise collagen in response to IGF-I, whereas they do increase synthesis in response to IGF-II. This apparent developmentally regulated difference in response to these related growth factors is also reflected in changes in the tyrosine phosphorylation pattern of a number of proteins. Postnatal cells exhibit a significant increase in phosphorylation of ERK 1 (p44) in response to IGF-I and conversely the p46 isoform of Shc on IGF-II stimulation. Fetal cells however only show a significant increase in an unidentified 100 kDa tyrosine-phosphorylated protein on stimulation with IGF-II. CONCLUSION: Dermal fibroblasts exhibit different responses to the two forms of IGF depending on their developmental maturity. This may relate to the developmental transition in cutaneous wound healing from regeneration to fibrosis.

Synthetic melanin is a model for soluble natural eumelanin in UVA-photosensitised superoxide production.

Studies to UV-irradiate natural eumelanins in vitro have used insoluble pigment obtained by acid hydrolysis, which lacks melanoprotein. Eumelanin synthesised in the presence of a protein is not insoluble, and the insoluble form of melanin from acid hydrolysis may not have the same physicochemical properties as the natural pigment synthesised in vivo in the melanosome. Here we investigated radical production by three natural eumelanins exposed to solar levels of UVA; sepia melanin from Sepia officinalis, and eumelanins isolated from Oriental human and domestic cat hair. UVA irradiation of sepia melanin in solution at pH 4.5 in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave hydroperoxyl and hydroxyl radical-adducts, maximal at 0.6-2.5 mg/ml melanin concentrations. Hydroperoxyl radical production was relatively low in acetate buffer, but detected in aqueous suspensions of sepia melanin. Hair eumelanins were photoreactive with hydroperoxyl radical-adduct production at low concentrations (0.1-0.4 mg/ml melanin). Synthetic pigment after synthesis undergoes photo-oxidation (producing superoxide) at low concentrations (0.3 mg/ml) and its oxidation increases the photoreactivity at higher melanin concentrations. These findings may be physiologically relevant to the properties and function of eumelanin in vivo when it is at low concentration (found in a small proportion of Caucasian melanocytes), and suggest that synthetic melanin has the potential for the basis of a model for natural eumelanin.

Hypertrophic scar cells fail to undergo a form of apoptosis specific to contractile collagen-the role of tissue transglutaminase.

Failure of apoptosis has been postulated to cause the hypercellularity and thus excess scar-tissue formation of hypertrophic scars (HTS). Here, we have examined the susceptibility of fibroblasts derived from normal or HTS to apoptosis induced during collagen-gel contraction, a wound-healing model. Normal scar (NS) fibroblasts underwent significant apoptosis (>40% total) in contractile collagen, whereas apoptosis was not detected in HTS cells. This inability was specific to apoptosis induced by contractile collagen because apoptosis could be induced using diverse modalities. Since chronic fibrotic tissue is known to be excessively cross-linked, we next examined whether collagen matrix that had been conditioned by HTS fibroblasts became refractory to enzymatic breakdown and indeed, found that it is resistant to breakdown by both collagenase D and matrix metalloproteinase-2. Newly formed extracellular matrix is stabilized by the enzyme, tissue transglutaminase, which we demonstrated to be overexpressed by HTS fibroblasts in vivo and in vitro. Reducing tissue transglutaminase activity in collagen gels containing HTS fibroblasts permitted induction of apoptosis on gel contraction, whereas increasing enzymic activity in NS cell-containing gels completely abrogated collagen-contraction-induced-apoptosis. Together, these observations show that HTS fibroblasts exhibit resistance to a specific form of apoptosis elicited by contraction of collagen gels, and that this phenomenon is dependent on excess activity of cell surface tissue transglutaminase.

The prevalence of interferon-alpha transcription defects in malignant melanoma.

The type I interferons, interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta), are situated on the short arm of chromosome 9, specifically 9p21-22. This locus lies very close to an area that is deleted or rearranged in nearly half of all melanomas tested. The identification of 9p rearrangements in both melanoma precursor lesions (dysplastic naevi) and primary lesions has implicated the 9p locus in the early stages of melanoma development. Recent evidence has demonstrated that metastatic melanoma cell lines have a specific loss of IFN-alpha gene expression, a defect that appears to occur at the level of transcription. In this study, we examined the expression of IFN-alpha in cell lines isolated from the various stages of melanoma progression, with a view to determine the prevalence of the IFN-alpha transcription defects exhibited by malignant melanoma, and to assess whether the loss of IFN-alpha expression was particular to a certain stage of melanoma progression. We showed that all the melanoma cell lines tested (n=20) demonstrated an inability to express IFN-alpha, a defect that was reflected in the apparent inactivity of the IFN-alpha promoter. These defects were found to occur in cells isolated from early melanomas, lending support to the hypothesis that IFN-alpha has a role in the aetiology of malignant melanoma.

Sunscreens inadequately protect against ultraviolet-A-induced free radicals in skin: implications for skin aging and melanoma?

Sunscreens are employed to mitigate the adverse effects of sunlight on skin but are primarily designed to prevent ultraviolet-B-associated burning and damage. The increasingly recognized role of ultraviolet A in aging, and possibly melanoma, highlights the need to include ultraviolet A screens; however, validation remains difficult. We have used a novel method to establish the efficacy of sunscreens, by measuring ultraviolet-A-induced free-radical production (thought to contribute towards ultraviolet-A-related aging and malignant change). Electron spin resonance spectroscopy was used to detect free radicals directly in human Caucasian skin during irradiation with levels of ultraviolet comparable to solar intensities. Using this system the protection afforded by three high factor sunscreens (sun protection factor 20+) that claim ultraviolet A protection was examined. Each sunscreen behaved similarly: at recommended application levels (> or = 2 mg per cm2) the ultraviolet-induced free radicals were reduced by only about 55%, and by about 45% at 0.5-1.5 mg per cm (0.5 mg per cm2 reported for common usage). A "free-radical protection factor" calculated on the basis of these results was only 2 at the recommended application level, which contrasts strongly with the erythema-based sun protection factors (mainly indicative of ultraviolet B protection) quoted by the manufacturers (20+). The disparity between these protection factors suggests that prolonged sunbathing (encouraged by use of these creams) would disproportionately increase exposure to ultraviolet A and consequently the risk of ultraviolet-A-related skin damage.

Purinergic receptors are part of a signaling system for keratinocyte proliferation, differentiation, and apoptosis in human fetal epidermis.

We have investigated the expression of P2X5, P2X7, P2Y1, and P2Y2 receptor subtypes in 8- to 11-wk-old human fetal epidermis in relation to markers of proliferation (proliferating cell nuclear antigen (PCNA) and Ki-67), keratinocyte differentiation (cytokeratin K10 and involucrin), and markers of apoptosis (TdT-mediated dUTP nick end labeling (TUNEL) and anti-caspase-3). Immunohistochemistry showed that each of the four receptors was expressed in spatially distinct zones of the developing epidermis: P2Y1 receptors were found in the basal layer, P2X5 receptors were predominantly in the basal and intermediate layers, and both P2Y2 and P2X7 receptors were in the periderm. Colocalization experiments suggested different functional roles for these receptors. P2Y1 receptors were found in fetal keratinocytes positive for PCNA and Ki-67, suggesting a role in proliferation. P2X5 receptors double labeled with differentiated fetal keratinocytes that were positive for cytokeratin K10, suggesting a role in differentiation. P2X7 receptors colocalized with anti-caspase-3 antibody and were also expressed in periderm cells positive for TUNEL, suggesting a role in periderm cell apoptosis. P2Y2 receptors were found only in periderm cells and may have a role in chloride and fluid secretion into the amniotic fluid.

Purinergic receptors are part of a functional signaling system for proliferation and differentiation of human epidermal keratinocytes.

We investigated the expression of P2X5, P2X7, P2Y1 and P2Y2 receptor subtypes in normal human epidermis and in relation to markers of proliferation (PCNA and Ki-67), keratinocyte differentiation (cytokeratin K10 and involucrin) and markers of apoptosis (TUNEL and anticaspase-3). Using immunohistochemistry, we showed that each of the four receptors was expressed in a spatially distinct zone of the epidermis, suggesting different functional roles for these receptors. Functional studies were performed on primary cultures of human keratinocytes and on explanted rat skin, where different P2 receptor subtype agonists and antagonists were applied to cultured keratinocytes or injected subcutaneously into the skin, respectively. An increase in cell number was caused by low doses of the nonspecific P2 receptor agonist ATP, the P2Y2 receptor agonist UTP (p<0.001), and the P2Y1 receptor agonist 2MeSADP (p<0.05). There was a significant decrease in cell number as a result of treatment with the P2X5 receptor agonist ATPgammaS (p<0.001) and the P2X7 receptor agonist BzATP (p<0.001). Suramin caused a significant block in the effect of 100 microm ATP (p<0.01) and 1000 microm ATP (p<0.001) on cell number. These results imply that different purinergic receptors have different functional roles in the human epidermis with P2Y1 and P2Y2 receptors controlling proliferation, while P2X5 and P2X7 receptors control early differentiation, terminal differentiation and death of keratinocytes, respectively.

Expression of purinergic receptors in non-melanoma skin cancers and their functional roles in A431 cells.

We investigated the use of purinergic receptors as a new treatment modality for nonmelanoma skin cancers. Purinergic receptors, which bind adenosine 5'-tri-phosphate, are expressed on human cutaneous keratinocytes. Previous work in rat and human epidermis suggested functional roles for purinergic receptors in the regulation of proliferation, differentiation, and apoptosis. Immunohistochemical analysis of frozen sections in human basal cell carcinomas and squamous cell carcinomas for P2X5, P2X7, P2Y1, P2Y2, and P2Y4 receptors was performed, accompanied by detailed analysis of archive material of tumor subtypes in paraffin sections. Functional studies were performed using a human cutaneous squamous cell carcinoma cell line (A431), where purinergic receptor subtype agonists were applied to cells and changes in cell number were quantified via a colorimetric assay. Immunostaining in paraffin sections was essentially the same as that in frozen sections, although more detail of the subcellular composition was visible. P2X5 and P2Y2 receptors were heavily expressed in basal cell carcinomas and squamous cell carcinomas. P2X7 receptors were expressed in the necrotic center of nodular basal cell carcinomas and in apoptotic cells in superficial multifocal and infiltrative basal cell carcinomas, and squamous cell carcinomas. P2Y1 receptors were only expressed in the stroma surrounding tumors. P2Y4 receptors were found in basal cell carcinomas but not in squamous cell carcinomas. P2X5 receptors appear to be associated with differentiation. The P2X7 receptor agonist benzoylbenzoyl-adenosine 5'-triphosphate and high concentrations of adenosine 5'-triphosphate (1000-5000 microM) caused a significant reduction in A431 cell number (p<0.001), whereas the P2Y2 receptor agonist uridine 5'-triphosphate caused a significant amount of proliferation (p<0.001). We have demonstrated that non-melanoma skin cancers express functional purinergic receptors and that P2X7 receptor agonists significantly reduce cell numbers in vitro.

An experimental and theoretical model for solar UVA-irradiation of soluble eumelanin: towards modelling UVA-photoreactions in the melanosome?

A model is developed for the UVA-irradiation of soluble eumelanin exposed to levels of irradiation comparable to sunlight. Radical production was determined in soluble dl- and l-dopa melanins exposed to solar levels of UVA, using electron spin resonance spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Steady-state concentrations of DMPO-O(2)H(.-), which increased up to 0.3 mg/ml melanin, and then declined above 0.3 mg/ml, were detected at pH 4.5. The kinetic model incorporated the photosensitizing and radical-scavenging reactions of eumelanin, and assumed semiquinone radical reduction of oxygen to be fast compared to disproportionation. The model is consistent with experimental data for melanin concentrations <0.1 mg/ml; but >0.1 mg/ml melanin is consistent only with data at raised oxygen tension. The rate-constant for reaction of the melanin semiquinone-radical and oxygen is estimated to be 10(3) mol(-1)dm(3)s(-1). In this model, where DMPO competes with melanin for HO(2)(.-), at ambient oxygen levels, eumelanin exposed to solar levels of UVA photosensitizes superoxide at concentrations <0.3 mg/ml melanin, and is increasingly stable towards oxidation when >0.3 mg/ml concentration. Eumelanin could have a negligible screening effect <0.1 mg/ml and very strong screening >1 mg/ml. This model would be biologically relevant if soluble forms of eumelanin were shown to exist in vivo, and is potentially useful for studies of the photochemistry and photophysics of eumelanin and phaeomelanin and to explore the effects of metal-ions, proteins and lipids in a model system.

Cutaneous scarring: a clinical review.

Cutaneous scarring can cause patients symptoms ranging from the psychological to physical pain. Although the process of normal scarring is well described the ultimate cause of pathological scarring remains unknown. Similarly, exactly how early gestation fetuses can heal scarlessly remains unsolved. These questions are crucial in the search for a preventative or curative antiscarring agent. Such a discovery would be of enormous medical and commercial importance, not least because it may have application in other tissues. In the clinical context the assessment of scars is becoming more sophisticated and new physical, medical and surgical therapies are being introduced. This review aims to summarise some of the recent developments in scarring research for non-specialists and specialists alike.

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles.

We investigated the expression of P2X(5), P2X(7), P2Y(1) and P2Y(2) receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X(5), P2Y(1) and P2Y(2) receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y(1) receptors in the outer root sheath and bulb, P2X(5) receptors in the inner and outer root sheaths and medulla and P2Y(2) receptors in living cells at the edge of the cortex/medulla. P2X(7) receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y(1) receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X(5) receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y(2) receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.

A role for TGF-beta1-induced cellular responses during wound healing of the non-scarring early human fetus?

Early human fetuses regenerate cutaneous wounds perfectly without scarring. However, transforming growth factor-beta1 (TGF-beta1), the cytokine linked with scarring in mature tissue, is also present during fetal wound repair, albeit transiently. We present a comparison of response to TGF-beta1 by fibroblasts derived from early human fetal skin (non-scarring) and their mature (scarring) postnatal counterparts, which revealed that although fetal fibroblasts do indeed differentiate into myofibroblasts, this response is altogether more rapid and short-lived. Fetal fibroblasts also failed to exhibit the TGF-beta1-induced increase in collagen (mRNA and protein) demonstrated by their postnatal counterparts. Fetal cells exhibited a comparatively short-lived or rapid phosphorylation of several components of the TGF-beta1 signaling pathways: Smad2/3 and c-Jun N-terminal kinase. Unlike quiescent postnatal fibroblasts, quiescent fetal fibroblasts also phosphorylated extracellular signal-regulated kinases in response to TGF-beta1. These altered responses to TGF-beta1 may well contribute to the transition between perfect regeneration and scar formation seen during development.

Retrospective study of the association between hypertrophic burn scarring and bacterial colonization.

Although the association between hypertrophic burn scarring and infection is well described, an association with colonization has not been established. This retrospective study sought to determine whether a significant association between hypertrophic scarring and bacterial colonization exists. Details from the case notes of all patients seen in our institution's burns unit over a two-year period were recorded and the incidence of hypertrophic scarring and burn bacterial colonization was noted. A total of 127 scars were recorded, and of these, 51 were hypertrophic and 76 nonhypertrophic. The incidence of bacterial colonization in the hypertrophic scar group was 88%, an association that achieved significance (P < .05) in comparison with nonhypertrophic scars (27%). This association holds true for individual organisms such as Staphylococcus aureus and Escherichia coli and for all burn depths and healing times. This study suggests that burn wound bacterial colonization may be more important than previously believed and perhaps suggests that striving toward an aseptic burn wound environment may reduce the incidence of hypertrophic scarring.

Differential gene expression in response to transforming growth factor-beta1 by fetal and postnatal dermal fibroblasts.

The multipotent growth factor transforming growth factor (TGF)-beta1 is consistently linked with fibrosis and scarring. The perfect (scarless) healing of cutaneous wounds in early gestational age fetuses is proposed to be due to this tissue's predominance of the TGF-beta3 isoform over the profibrotic TGF-beta1 and 2. Nevertheless, TGF-beta1 is present during wound healing in the early fetus and recently we demonstrated that relevant intracellular signaling pathways are activated (albeit transiently) on TGF-beta1 stimulation. This study aimed to determine whether TGF-beta1 has different effects on gene transcription in human fetal (<14 weeks) vs. human postnatal dermal fibroblasts, using real-time polymerase chain reaction. The regulation pattern of a number of TGF-beta response genes differed dramatically between the two cell sources. The typical autocrine loop of TGF-beta1 autoinduction did not occur in fetal fibroblasts and genes that are normally up-regulated, connective tissue growth factor and collagen type I were actually down-regulated. Furthermore, other response genes responded in a delayed fashion (TGF-beta3) compared with that seen in the more developmentally mature postnatal fibroblasts. Finally, genes unaltered by TGF-beta stimulation in postnatal cells, TGF-beta2 and collagen III, were up-regulated in fetal cells. These developmentally related differences in fibroblast response to TGF-beta1 may influence wound-healing outcome, i.e., perfect regeneration or fibrosis.

P2X(5) and P2X(7) receptors in human warts and CIN-612 organotypic raft cultures of human papillomavirus infected keratinocytes.

Purinergic receptors, which bind adenosine 5'-triphosphate (ATP), are expressed on human cutaneous keratinocytes and in squamous cell carcinomas. Studies on normal human epidermis and primary keratinocyte cultures have suggested that P2X(5) receptors are likely to be involved in keratinocyte differentiation and P2X(7) receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of keratinocytes. P2X(7) receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2X(5) and P2X(7) receptors was performed on paraffin sections of normal human skin, warts, raft cultures of normal human keratinocytes and raft cultures of CIN 612 cells, a model of keratinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2X(5) receptors. A similar pattern was seen in the CIN 612 raft cultures. Both P2X(5) and P2X(7) receptors were found in the nuclei of koilocytes, abnormal keratinocytes characteristic of human papillomavirus infection. P2X(5) and P2X(7) receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death.

An investigation to optimize angiogenesis within potential dermal replacements.

BACKGROUND: Acute and chronic wounds are costly and invariably expose significant structures. Surgical reconstruction causes donor-site morbidity, scarring, and the need for intensive care. Reconstruction using an artificial dermis avoids donor sites, but available collagen-based solutions are susceptible to poor take. Using an in vitro angiogenic assay, the authors investigated dermal matrices for potential inclusion in a second-generation proangiogenic synthetic dermal replacement. METHODS: Human placental endothelial cells were cocultured on Cytodex beads (Pharmacia Biotech) and plated in eight different extracellular matrix gels (collagen, fibrin, four glycosaminoglycans, vitronectin, and fibronectin), with or without stimulation from two soluble angiogenic factors. Three different cell lines were used, with 30 beads per condition. Cellular invasion into gels was calculated using Sigma Scan computer software, and statistical comparisons were made. RESULTS: The authors found that fibrin provided greatest stimulus for endothelial invasion, with invasion in fibrin inhibited by collagen in a concentration-dependent fashion. Invasion by alternative extracellular matrix components and soluble angiogenic factors was far less than that in fibrin. CONCLUSIONS: The authors identified that extracellular matrices can provide greater angiogenic potential than soluble angiogenic factors. Fibrin provided a better proangiogenic scaffold than collagen. This could well be used to encourage blood vessel ingress and eventual take of a second-generation proangiogenic synthetic dermal replacement.

Differences in production of melanin radicals by 694 nm ruby laser and UVA radiation.

BACKGROUND AND OBJECTIVES: The 694 nm ruby laser is used clinically for hair removal and the mechanism is predominantly photo thermal via melanin targeting. We investigated 694 nm laser-irradiation of human hair, and laser-irradiation of synthetic dopa melanin to establish whether photolysis and oxygen radical production is also contributory, and which may have side effects. STUDY DESIGN/MATERIALS AND METHODS: Ultraviolet-A (UVA) irradiation of melanin was used as a positive control for radical production. Laser- and UVA-irradiated hair samples, and synthetic dopa melanin in media of different viscosity, were analyzed using electron spin resonance spectroscopy, and compared. The spin trap 5,5-dimethyl- 1-pyrroline N-oxide (DMPO) was used to probe laser-irradiated dopa melanin for superoxide radical production. RESULTS: Comparable to UVA, laser-irradiation of hair increased the signal-intensity of the intrinsic melanin radical. UVA-induced radicals decay rapidly; however, laser-induced radicals decayed slowly and did not fully revert to original levels after 24 hours. Laser-induced radicals were increasingly stable with viscosity of the medium. Superoxide radicals were detected using DMPO in UVA- but not laser-irradiated synthetic dopa-melanin at pH 4.5. CONCLUSIONS: Laser-irradiation of melanin does not result in oxygen radical formation; however, a paramagnetic species, long-lived in rigid media, is detected which is worth further investigation.