RESUMEN
Over the past two decades a range of 3D models for human skin have been described. Some include native collagen and intrinsic basement membrane proteins and fibroblasts, others are based on xenogeneic collagen or synthetic supports often without fibroblasts. The aim of this study was to look at the influence of media calcium, basement membrane and fibroblasts on the quality of 3D tissue engineered skin produced using human de-epidermized acellular dermis. In this study we deliberately used Euro skin as the source of the donor dermis to examine to what extent this could provide an effective dermal substrate for producing 3D skin for clinical use. Keratinocytes were cultured in the presence and absence of fibroblasts and both with and without basement membrane on decellularized dermis at calcium concentrations ranging from 250µM to 1.6mM over a period of 14 days. Results showed the formation of a well attached epithelium with many of the features of normal skin in the presence of a basement membrane. This was largely independent of the presence of fibroblasts and not greatly influenced by the concentration of calcium in the media. However there was a clear requirement for physiological levels of calcium in the formation of a stratified epithelium in the absence of a basement membrane.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Trasplante de Piel/métodos , Piel Artificial , Ingeniería de Tejidos/métodos , Análisis de Varianza , Membrana Basal/citología , Calcio/administración & dosificación , Calcio/fisiología , Colágeno/metabolismo , Fibroblastos/citología , Humanos , Inmunohistoquímica , Queratinocitos/citologíaAsunto(s)
Quemaduras/complicaciones , Piodermia Gangrenosa/etiología , Administración Tópica , Corticoesteroides/uso terapéutico , Anciano , Antibacterianos/uso terapéutico , Ciclosporinas/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Piodermia Gangrenosa/tratamiento farmacológico , Resultado del TratamientoRESUMEN
The importance of a dermal element when providing permanent wound cover for skin loss has become evident as the shortcomings of pure epidermal grafts are recognized. We are developing a skin composite formed from sterilized human de-epidermized acellular dermis, keratinocytes and fibroblasts with the ultimate aim of using this composite to cover full-thickness excised burn wounds. These composites can be prepared with or without basement membrane (BM) antigens initially present on the dermis. This study investigates the importance of retaining BM antigens on the dermis to the production and appearance of these composites in vitro. Skin composites prepared from dermis with BM antigens either present or absent initially were studied throughout 3 weeks. Composites with BM antigens present initially were significantly better than those initially lacking BM antigens in: (i) the degree of epithelial cell attachment to the underlying dermis (hemidesmosomes were seen only in the former); (ii) the morphology of the epithelial layer; (iii) the consistent presence of collagen IV and laminin and the increasing expression of tenascin; and (iv) the amount of soluble collagen IV and fibronectin detected in the conditioned media. We conclude that an initial BM antigen template is vital in this skin composite model for the attachment and differentiation of the epithelial layer and for the subsequent remodelling of the BM in vitro.
Asunto(s)
Antígenos/inmunología , Membrana Basal/inmunología , Piel Artificial , Células Cultivadas , Matriz Extracelular/inmunología , Fibroblastos/citología , Fibronectinas/metabolismo , Humanos , Inmunohistoquímica , Queratinocitos/citología , Cicatrización de Heridas/inmunologíaRESUMEN
Skin banking was set up in Sheffield in 1991 to provide a readily available source of allograft material to be used both for research purposes and also as a means of providing immediate wound cover for major burns patients. Once skin was available, however, clinical demand for it both within and outside Sheffield, outstripped the resources to run the bank. Logistical difficulties were encountered in the day to day running of the bank. These revolved around shortage of staff available for harvesting, the relative lack of public awareness of skin donation, shortage of banked skin as the bank became more widely known and lack of space and finance to expand. The decision was made to transfer the now established skin bank to the National Blood Service where it now operates with staff and resources dedicated specifically to this purpose. Experience leads to the suggestion that there is a clinical need for allograft skin in the UK which is not being met at the present time. There is a need for dedicated properly resourced skin banks and for the Department of Health to introduce regulation, monitoring and inspection of skin bank facilities in order to safeguard standards.
Asunto(s)
Trasplante de Piel , Bancos de Tejidos/organización & administración , Obtención de Tejidos y Órganos/organización & administración , Bancos de Sangre/economía , Bancos de Sangre/organización & administración , Quemaduras/cirugía , Documentación , Inglaterra , Administración Financiera/organización & administración , Educación en Salud , Recursos en Salud/economía , Recursos en Salud/legislación & jurisprudencia , Recursos en Salud/organización & administración , Recursos en Salud/normas , Necesidades y Demandas de Servicios de Salud , Humanos , Cuerpo Médico/economía , Cuerpo Médico/organización & administración , Administración en Salud Pública/legislación & jurisprudencia , Administración en Salud Pública/normas , Investigación , Bancos de Tejidos/economía , Bancos de Tejidos/legislación & jurisprudencia , Bancos de Tejidos/normas , Donantes de Tejidos , Obtención de Tejidos y Órganos/economía , Obtención de Tejidos y Órganos/legislación & jurisprudencia , Obtención de Tejidos y Órganos/normas , Trasplante Homólogo , Reino UnidoRESUMEN
Local regulation of alpha1-antitrypsin (alpha1-AT) may have importance in maintenance of the protease-antiprotease balance in the microenvironment of inflammatory cells. We therefore studied whether lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNFalpha) affect the pericellular concentration of alpha1-AT in human peripheral blood mononuclear cells (PBMC). PBMC taken from normal healthy volunteers were treated with LPS, IL-1beta, and TNFalpha, and the concentration of human alpha1-AT in conditioned supernatants was measured. When compared with unstimulated control supernatants (147 +/- 19 ng/ml), LPS (439 +/- 66 ng/ml; p < or = 0.001), IL-1beta (263 +/- 37 ng/ml; p < or = 0.01), and TNFalpha (316 +/- 59 ng/ml; p < or = 0.05) induced a 2- to 3-fold increase of alpha1-AT. Up-regulation of alpha1-AT protein correlated with an increase in alpha1-AT mRNA, suggesting a simultaneous increase in alpha1-AT synthesis. Despite the increase in alpha1-AT concentration, functional antiprotease activity could not be detected. Furthermore, protease activity was present in all samples, with the amount of activity being inversely related to the amount of alpha1-AT measured in supernatants. These findings suggest that local inflammatory conditions up-regulate alpha1-AT production by monocytes which complex with a protease derived from the PBMC population.
Asunto(s)
Endopeptidasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Interleucina-1/fisiología , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Monocitos/enzimología , Factor de Necrosis Tumoral alfa/fisiología , alfa 1-Antitripsina/metabolismo , Northern Blotting , Western Blotting , Humanos , Inmunohistoquímica , Monocitos/inmunología , alfa 1-Antitripsina/análisis , alfa 1-Antitripsina/genéticaRESUMEN
Composites of human de-epidermised acellular dermis and normal adult human keratinocytes and fibroblasts were examined for the ability of cells to contract these composites. Image analysis of the outline of the composites showed that, in this model, keratinocytes alone or in the presence of fibroblasts caused highly significant contraction (of the order of 25% by day 12). There was no significant contraction of the dermis with fibroblasts alone or in the absence of cells. The presence or absence of basement membrane antigens did not influence the effect of keratinocytes on dermal contraction. Analysis of the conditioned media from these composites showed that the greatest fibronectin production was seen with fibroblasts alone in the presence of basement membrane. Keratinocytes alone produced little fibronectin irrespective of the presence of the basement membrane. If keratinocytes were present with fibroblasts, however, then fibronectin production was significantly reduced both in the presence and absence of the basement membrane, indicating that keratinocytes modify dermal fibroblast extracellular matrix production. This study shows that while keratinocytes and fibroblasts are clearly influencing each other's activity in this human skin composite model, under the circumstances we describe it is the keratinocyte and not the fibroblast which causes contraction of the human de-epidermised acellular dermis.
Asunto(s)
Contractura/patología , Matriz Extracelular/patología , Fibronectinas/biosíntesis , Queratinocitos/fisiología , Piel/patología , Adulto , Membrana Basal/fisiología , Comunicación Celular , Técnicas de Cultivo de Célula , Femenino , Fibroblastos/fisiología , Humanos , Trasplante de Piel , Solubilidad , Cicatrización de HeridasRESUMEN
Cytoplasmic antineutrophil cytoplasmic antibodies (cANCA) that accompany the neutrophilic vasculitis seen in Wegener's granulomatosis (WG), are directed against proteinase-3 (PR-3), a serine proteinase which is located in azurophilic granules of neutrophils and monocytes. PR-3, when expressed on the surface of TNFalpha-primed neutrophils, can directly activate neutrophils by complexing cANCA and promoting concomitant Fcgamma receptor (FcgammaR) cross-linking. Although the neutrophil's pathogenic role in WG has been studied, the role of the monocyte has not been explored. The monocyte, with its ability to release cytokines and regulate neutrophil influx, also expresses PR-3. Therefore, the monocyte may play a significant role in WG via the interaction of surface PR-3 with cANCA, inducing cytokine release by the monocyte. To test this hypothesis, monocytes were studied for PR-3 expression and for IL-8 release in response to cANCA IgG. PBMC obtained from healthy donors displayed dramatic surface PR-3 expression as detected by immunohistochemistry and flow cytometry in response to 0. 5-h pulse with TNFalpha (2 ng/ml). Purified monoclonal anti-PR-3 IgG added to TNFalpha-primed PBMC induced 45-fold more IL-8 release than an isotype control antibody. Furthermore, alpha 1-antitrypsin (alpha1-AT), the primary PR-3 antiprotease, inhibited the anti-PR-3 induced IL-8 release by 80%. Importantly, Fab and F(ab')2 fragments of anti-PR-3 IgG, which do not result in Fcgamma receptor cross-linking, do not induce IL-8 release. As a correlate, IgG isolated from cANCA positive patients with WG induced six times as much PBMC IL-8 release as compared to IgG isolated from normal healthy volunteers. Consistent with PR-3 associated IL-8 induction, alpha1-AT significantly inhibited this effect. These observations suggest that cANCA may recruit and target neutrophils through promoting monocyte IL-8 release. This induction is mediated via Fcgamma receptor cross-linking and is regulated in part by alpha1-AT.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Granulomatosis con Poliangitis/inmunología , Interleucina-8/metabolismo , Monocitos/metabolismo , Receptores de IgG/metabolismo , Serina Endopeptidasas/metabolismo , alfa 1-Antitripsina/farmacología , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Reactivos de Enlaces Cruzados/farmacología , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunohistoquímica , Monocitos/efectos de los fármacos , Mieloblastina , Serina Endopeptidasas/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Evidence that oxygen free radicals may be contributory to further tissue damage in the events following cutaneous thermal injury supports a role for interventional therapy using antioxidants. However, previous work has relied almost entirely on animal-based models with little clinical information available. Also, methods used to support an oxidative role in thermal injury have relied almost exclusively upon the use of lipid peroxidation studies. Further work substantiating a contributory role of free radicals is therefore required using additional methodology before considering antioxidant therapy aimed at retarding tissue damage. We investigated general oxidative damage to protein in burn blister fluid by quantifying the protein carbonyl levels from 11 patients admitted with superficial or partial thickness burns. Total antioxidant capacity was also assessed, together with measurement of protein and the antioxidants uric acid and bilirubin. Data were compared with values obtained for serum in healthy volunteers. Following thermal injury, burn blister fluid protein carbonyl level was increased by almost 50 per cent (P = 0.005) compared with normal serum. Antioxidant scavenging capacity, protein and bilirubin were all significantly reduced, but uric acid unaltered compared with control values. The present data support a role for oxidative damage in cutaneous thermal injury.
Asunto(s)
Antioxidantes/análisis , Vesícula/metabolismo , Proteínas Sanguíneas/metabolismo , Quemaduras/metabolismo , Peroxidación de Lípido , Adolescente , Adulto , Bilirrubina/análisis , Líquidos Corporales/metabolismo , Quemaduras/sangre , Niño , Preescolar , Femenino , Radicales Libres/análisis , Radicales Libres/metabolismo , Humanos , Masculino , Oxidación-Reducción , Valores de Referencia , Estadísticas no Paramétricas , Ácido Úrico/análisisRESUMEN
In recent years, an improved understanding of the pathogenesis of sepsis, along with an explosion in the biotechnology industry, has led to the development of a variety of agents with potential to interdict the pathogenesis of sepsis at many points. This article reviews the rationale, efficacy and shortcomings of these immunotherapeutic agents as they relate to the management of human septic shock.
Asunto(s)
Infecciones por Bacterias Gramnegativas/terapia , Inmunoterapia/métodos , Sepsis/terapia , Animales , Citocinas/antagonistas & inhibidores , Endotoxinas/antagonistas & inhibidores , Infecciones por Bacterias Gramnegativas/etiología , HumanosAsunto(s)
Dispositivos de Expansión Tisular , Vagina/cirugía , Adulto , Femenino , Humanos , Cirugía Plástica , Mallas Quirúrgicas , Vagina/anomalíasRESUMEN
STUDY OBJECTIVE: To evaluate two methods of augmenting venous filling as potential aids to i.v. cannulation. DESIGN: Comparative study of cross-sectional areas of preselected antecubital fossa veins. SETTING: Vascular laboratory of a British university teaching hospital. PARTICIPANTS: Thirty healthy, normotensive, adult volunteers. INTERVENTIONS: Vessel cross-sectional areas measured noninvasively using a color flow duplex ultrasound scanner. Measurements were taken at rest, after application of a venous tourniquet, with tourniquet and Esmarch bandage, and with tourniquet and Rhys-Davies exsanguinator. RESULTS: Mean (+/- SD) cross-sectional area at rest was 0.18 +/- 0.094 cm2 and after tourniquet was 0.28 +/- 0.14 cm2. Application of an Esmarch bandage produced an increase to 0.33 +/- 0.14 cm2, and Rhys-Davies exsanguinator produced an increase to 0.32 +/- 0.15 cm2 (P < .0001 for all comparisons except Rhys-Davies exsanguinator versus Esmarch bandage). CONCLUSION: Application of either the Esmarch bandage or the Rhys-Davies exsanguinator caused significant filling of antecubital fossa veins in excess of that produced by a venous tourniquet alone in normovolemic, normotensive volunteers.
Asunto(s)
Cateterismo Periférico/métodos , Codo/irrigación sanguínea , Torniquetes , Adulto , Vendajes , Humanos , Masculino , Ensayo de Materiales , Persona de Mediana Edad , Ultrasonografía Doppler en Color , Venas/diagnóstico por imagen , Venas/fisiologíaRESUMEN
More than 2 million upper limb injuries occur each year in Britain. Information about the precise sequence of events leading up to an accident may help prevention of similar injuries by alteration of the work environment or through safety education. The first impression of the cause of an accident may not identify the key event triggering it. The Merseyside Accident Information Model (MAIM; Manning, 1987) provides a structured questionnaire, based on a menu-led computer programme. This not only allows the first unforeseen event to be identified from the history, but is also the basis of a database. Pooled data can then be analyzed. We present an analysis of 100 consecutive cases presenting to one Plastic Surgery Unit.