RESUMO
We have identified mutations in keratins K5 (Arg331Cys) and K14 (Val270Met) in two kinships affected by the dominantly-inherited skin blistering disease, Weber-Cockayne epidermolysis bullosa simplex (EBS-WC). Linkage analysis, DNA sequencing and clinical and ultrastructural analysis are combined to provide the first detailed description of classical EBS-WC. Both phenotypes show similar blistering on trauma, indicating that both mutations compromise the structural resilience of the basal keratinocytes by affecting the keratin cytoskeleton. The location of these mutations in the L12 linker, which bisects the alpha-helical rod region of intermediate filament proteins, identifies another keratin mutation cluster leading to hereditary skin fragility syndromes.
Assuntos
Citoesqueleto/fisiologia , Epidermólise Bolhosa Simples/genética , Queratinas/genética , Mutação , Idade de Início , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Criança , Pré-Escolar , Citoesqueleto/química , Primers do DNA , Feminino , Humanos , Recém-Nascido , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Fenótipo , Pele/patologia , Pele/ultraestruturaRESUMO
BACKGROUND: Epidermolytic ichthyosis (EI) is a skin fragility disorder caused by mutations in genes encoding suprabasal keratins 1 and 10. While the aetiology of EI is known, model systems are needed for pathophysiological studies and development of novel therapies. OBJECTIVES: To generate immortalized keratinocyte lines from patients with EI for studies of EI cell pathology and the effects of chemical chaperones as putative therapies. METHODS: We derived keratinocytes from three patients with EI and one healthy control and established immortalized keratinocytes using human papillomavirus 16-E6/E7. Growth and differentiation characteristics, ability to regenerate organotypic epidermis, keratin expression, formation of cytoskeletal aggregates, and responses to heat shock and chemical chaperones were assessed. RESULTS: The cell lines EH11 (K1_p.Val176_Lys197del), EH21 (K10_p.156Arg>Gly), EH31 (K10_p.Leu161_Asp162del) and NKc21 (wild-type) currently exceed 160 population doublings and differentiate when exposed to calcium. At resting state, keratin aggregates were detected in 9% of calcium-differentiated EH31 cells, but not in any other cell line. Heat stress further increased this proportion to 30% and also induced aggregates in 3% of EH11 cultures. Treatment with trimethylamine N-oxide and 4-phenylbutyrate (4-PBA) reduced the fraction of aggregate-containing cells and affected the mRNA expression of keratins 1 and 10 while 4-PBA also modified heat shock protein 70 (HSP70) expression. Furthermore, in situ proximity ligation assay suggested a colocalization between HSP70 and keratins 1 and 10. Reconstituted epidermis from EI cells cornified but EH21 and EH31 cells produced suprabasal cytolysis, closely resembling the in vivo phenotype. CONCLUSIONS: These immortalized cell lines represent a useful model for studying EI biology and novel therapies.
Assuntos
Linhagem Celular/patologia , Hiperceratose Epidermolítica/patologia , Queratinócitos/patologia , Adolescente , Adulto , Linhagem Celular/efeitos dos fármacos , Transformação Celular Viral , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Temperatura Alta , Humanos , Hiperceratose Epidermolítica/fisiopatologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Queratinas/metabolismo , Masculino , Metilaminas/farmacologia , Modelos Biológicos , Fenótipo , Fenilbutiratos/farmacologia , Estresse Fisiológico , Adulto JovemRESUMO
BACKGROUND: Epidermolysis bullosa simplex (EBS) is a mechanobullous skin fragility disease characterized by cytolysis of basal keratinocytes and intraepidermal blistering often caused by mutations in keratin genes (KRT5 or KRT14). No remedies exist for these disorders presenting a need for development of novel therapies. OBJECTIVES: To identify new genotype-phenotype relationships in vivo and in cultured primary EBS keratinocytes in vitro, and to study the cytoskeletal stabilizing effects of trimethylamine N-oxide (TMAO) in heat-stressed EBS cells. METHODS: Genomic DNA and cDNA samples from three Swedish patients with EBS were analysed for keratin mutations. Primary EBS keratinocyte cultures were established, heat stressed with and without added TMAO, followed by evaluation of cellular fragility. RESULTS: In addition to the previously reported KRT5 mutation (V186L) in one patient, two patients were found to have a novel I183M and recurrent E475G replacements in KRT5. Cultured EBS keratinocytes did not exhibit keratin aggregates or cell loss, except in the patient with the p.I183M mutation who showed 3% aggregates and 2% cell loss. Upon transient heat stress the number of aggregate-containing cells increased to 21%, 27% and 13%, respectively, in the p.I183M, p.E475G and p.V186L mutant cells. Interestingly, pretreatment with TMAO prior to heat stress, dose dependently reduced the number of aggregate-containing cells and cell loss. CONCLUSION: These results revealed a genotype-phenotype correlation in EBS keratinocytes upon heat stress and suggest protein stabilization as a new therapeutic strategy.
Assuntos
Epidermólise Bolhosa Simples/genética , Queratina-5/genética , Queratinócitos/efeitos dos fármacos , Metilaminas/farmacologia , Mutação de Sentido Incorreto , Adulto , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Análise Mutacional de DNA/métodos , Epidermólise Bolhosa Simples/patologia , Feminino , Genótipo , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Queratinócitos/patologia , Masculino , Pessoa de Meia-Idade , Oxidantes/farmacologia , FenótipoRESUMO
BACKGROUND: Hernia surgery, in particular parastomal hernia mesh repair and new techniques for hernia prevention, require novel biomaterials that avoid fibrosis and potential bowel erosion, while retaining adequate strength for their intended purpose. The aim was to evaluate the human host response to an acellular porcine-derived cross-linked collagen implant. METHODS: In a prospective pilot study on prevention of parastomal herniation, 15 patients undergoing loop stoma formation had an implant placed within the anterior abdominal wall. Histopathology and immunohistochemistry were performed to analyse the implant qualitatively and, where appropriate, quantitatively for biocompatibility, degradation, cellular infiltration, neo-extracellular matrix (ECM) formation and neovascularization. RESULTS: At a median of 7 (range 1-8) months, 12 of 15 patients had stoma reversal and 11 implant biopsies were obtained. In biopsies from ten of the 11 patients all responses were limited to the periphery of the implant and native pores. There was a minimal inflammatory response and minimal degradation of the implant. Fibroblastic and neovascular infiltration were noted, as was matrix metalloproteinase 1 activity with organized deposition of host collagen, fibronectin and laminin. CONCLUSION: The collagen implant demonstrated excellent biocompatibility and resistance to degradation in most patients. However, fibrovascular in-growth and ECM deposition were limited. This implant has excellent potential for soft tissue reinforcement.
Assuntos
Colágeno/uso terapêutico , Hérnia/prevenção & controle , Doenças do Íleo/prevenção & controle , Ileostomia/métodos , Íleo/patologia , Estomas Cirúrgicos/patologia , Biópsia , Reação Hospedeiro-Enxerto , Humanos , Imuno-Histoquímica , Projetos Piloto , Estudos Prospectivos , Implantação de Prótese/métodos , Reoperação/estatística & dados numéricos , Resistência à TraçãoRESUMO
Polypyrrole (PPy) is a conducting polymer that may be electrochemically generated with the incorporation of any anionic species, including net-negatively charged biological molecules such as proteins and polysaccharides. In this article, dermatan and chloride-loaded PPy films were prepared on gold sputter-coated coverslips and various skin derived cells were studied on them by electrochemical impedance spectroscopy. Impedance spectra in the frequency range 1-100 kHz were either determined at specific times or impedance was monitored continuously at specific frequencies. An equivalent impedance circuit was fitted to the recorded impedance spectra to obtain parameters whose contributions could be mapped to intracellular and intercellular current pathways, and the membrane properties of cells. Results show cell-induced impedance changes were detected over PPy modified electrodes and were dependent on cell density and type, monitoring frequency, material composition, and treatment. Lower cell densities were detected on PPy when compared with bare gold. Keratinocyte confluence, as determined by impedimetric analysis, was reached more rapidly on PPy than on gold. This was consistent with previous, more cumbersome, biochemical assays. Electrical equivalent circuit analysis provided evidence that the technique may be extended to discriminate cell type because of the intracellular and intercellular resistance, and cell membrane capacitance being related to cell morphology.
Assuntos
Técnicas Biossensoriais/métodos , Polímeros/metabolismo , Pirróis/metabolismo , Linhagem Celular , Impedância Elétrica , Ouro , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Polímeros/química , Pirróis/química , Fatores de TempoRESUMO
Polypyrrole (PPy) is a conjugated polymer that displays particular electronic properties including conductivity. In biomedical applications, it is usually electrochemically generated with the incorporation of any anionic species including also negatively charged biological macromolecules such as proteins and polysaccharides to give composite materials. In biomedical research, it has mainly been assessed for its role as a reporting interface in biosensors. However, there is an increasing literature on the application of PPy as a potentially electrically addressable tissue/cell support substrate. Here, we review studies that have considered such PPy based conducting polymers in direct contact with biological tissues and conclude that due to its versatile functional properties, it could contribute to a new generation of biomaterials.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Condutividade Elétrica , Polímeros/química , Polímeros/metabolismo , Pirróis/química , Pirróis/metabolismo , Estrutura MolecularRESUMO
Current tissue-culture techniques enable keratinocytes from a small piece of skin to be grown into sheets of epithelium, or cultured keratinocyte grafts, that are suitable for treating wounds. Serial subculture enables rapid expansion of a cell population, such that grafts of a total area equivalent to that of the surface of an adult can be obtained from an initial skin biopsy of approximately 2 cm2 in under one month. In this article, the methods currently used for culturing keratinocytes, the search for a fully functional replacement for the dermal elements of skin, and the prospects for clinical development of these technologies in the near future are discussed.
Assuntos
Transplante de Células , Células Cultivadas , Queratinócitos/citologia , Transplante de Pele , Pele/citologia , Diferenciação Celular , Meios de Cultura , Fibroblastos/citologia , Humanos , Queratinócitos/transplante , Pele/lesões , Transplante Autólogo/métodos , Transplante Homólogo/métodos , CicatrizaçãoRESUMO
Palmoplantar keratoderma of Voerner type (or epidermolytic palmoplantar keratoderma) is an autosomal dominant inherited disorder of keratinization with histologic features of epidermolytic hyperkeratosis. We studied members of two large unrelated kindreds with epidermolytic palmoplantar keratoderma, and biopsy specimens of lesional palmar skin from both families confirmed the histologic changes of epidermolytic hyperkeratosis. Whorls of abnormally aggregated keratin filaments were seen ultrastructurally to be associated with signs of cellular disintegration in spinous and granular cells. Direct sequencing of genomic DNA samples obtained from several members of each family established the substitution of a highly conserved arginine by tryptophan (R162W) in the 1A region of the alpha-helical rod domain of keratin 9. This arginine residue in a highly conserved region of keratins 1 and 10 is affected by disruptive missense point mutations in many patients with bullous ichthyosiform erythroderma. An equivalent position in the sole and palm restricted keratin 9 appears to be the mutation hot spot in epidermolytic palmoplantar keratoderma. To date, R162W is the most prevalent genetic defect reported in this genodermatosis.
Assuntos
Queratinas/genética , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/patologia , Mutação , Composição de Bases , Saúde da Família , Feminino , Humanos , Queratinócitos/ultraestrutura , Queratinas/ultraestrutura , Masculino , Pessoa de Meia-Idade , Pele/patologiaRESUMO
The purpose of this study was to determine the effect of interferon-gamma on keratinocyte major histocompatibility complex class II gene transcription. Transformed human foreskin keratinocytes (SVK14 cells) were incubated with recombinant IFN-gamma in the presence or absence of the protein synthesis inhibitor cycloheximide. Total cellular RNA was extracted from the cells and Northern blot analysis carried out using cDNA probes for all the functional class II genes. We report that 1) there is co-ordinate activation of all the class-II genes; 2) the rate of transcription varies between gene loci after activation; and 3) de novo protein synthesis is required for IFN-gamma activation of class II transcription.
Assuntos
Antígenos HLA-DP/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Interferon gama/farmacologia , Queratinócitos/citologia , Linhagem Celular Transformada , Cicloeximida/farmacologia , Humanos , Masculino , Biossíntese de Proteínas , Transdução de Sinais/efeitos dos fármacos , Vírus 40 dos Símios/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Mutations in distinct sites of epidermal keratins, in particular in the helix initiation and termination regions, cause human genodermatoses due to faulty intermediate filament formation. Extension of this observation to human hereditary hair and nail diseases includes population analyses of human hair keratin genes for natural sequence variations in the corresponding sites. Here we report on a large-scale genotyping of the short helix termination region (HTR) of the human type I cortical hair keratins hHa1, a3-I, and a3-II, and the cuticular hair keratin hHa2. We describe two polymorphic loci, P1 and P2, exclusively in the cuticular hHa2 gene, both creating dimorphic protein variants. P1 is due to a C to T mutation in a CpG element leading to a threonine to methionine substitution; P2 concerns a serine codon AGT that also occurs as an asparagine coding variant AAC. A third polymorphism, P3, is linked with a C to T point mutation located at the very beginning of intron 6. The three polymorphic sites are clustered in a 39-nucleotide sequence of the hHa2 gene. Both allelic frequency calculations in individuals of different races and pedigree studies indicate that the two-allelic hHa2 variants resulting from P1 and P2 occur ubiquitously in a ratio of about 1:1 (P1) and 2:1 (P2) respectively in our survey, and are clearly inherited as Mendelian traits. A genotype carrying both mutations simultaneously on one allele could not be detected in our sampling, and there was no association of a distinct allelic hHa2 variant with the known ethnic form variations of hairs. Sequence comparisons of the HTR of hHa2 with those of other type I hair keratins including the hHa2-ortholog from chimpanzee provide evidence that the P1- and P2-linked mutations must have occurred very early in human evolution and that the two P2-associated codon variants may be the result of two independent point mutations in an ancestral AGC serine codon. These data describe natural polymorphisms in the HTR of a member of the keratin multigene family.
Assuntos
Cabelo/química , Queratinas/genética , Polimorfismo Genético , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon/genética , DNA/genética , Primers do DNA/genética , Evolução Molecular , Feminino , Frequência do Gene , Genótipo , Humanos , Íntrons , Queratinas/química , Masculino , Dados de Sequência Molecular , Família Multigênica , Pan troglodytes/genética , Linhagem , Mutação Puntual , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Bullous congenital ichthyosiform erythroderma is a human hereditary skin disorder in which suprabasal keratinocytes rupture. Recent reports have implicated keratins K1 and K10 in this disease. Here we describe four diverse keratin mutations that are all significantly associated with this disease. Two of these are in the helix 1A subdomain of the type II keratin 1, giving a serine-to-proline substitution in codon 185 and an asparagine-to-serine substitution in codon 187. In the analogous region of type I keratin 10, an arginine-to-proline and an arginine-to-serine transition in codon 156 have been identified. All four mutations create restriction fragment length polymorphisms that were used exclude the mutations from 120 normal chromosomes. Insertional polymorphism (in the V2 subdomains of the non-helical tails of K1 and K10) was excluded as the cause of the phenotypic heterogeneity observed within one family.
Assuntos
Hiperceratose Epidermolítica/genética , Queratinas/genética , Mutação/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/análise , DNA/genética , Feminino , Humanos , Queratinas/análise , Masculino , Microscopia Eletrônica , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de RestriçãoRESUMO
Apligraf consists of bovine collagen dermis seeded with allogeneic male fibroblasts and keratinocytes. It is been shown to promote healing, but the length of persistence and pathological features have not been characterized previously in acute wounds. Forty-eight deep dermal wounds were created and Apligraf, a split-skin graft (SSG), or a dressing was applied. Biopsies of wounds were taken for immunohistochemical analysis and polymerase chain reaction was performed to detect the Y chromosome from Apligraf cells in 14 female wounds. Male allogeneic DNA was detected in wounds for the first 4 weeks. All subsequent time points were negative apart from one biopsy at 6 weeks. The wounds took 4-9 weeks to heal, with the Apligraf exhibiting no features of engraftment. This was in contrast to the rapid healing seen in the SSG control group. Histology revealed a more intense cellular infiltrate, but less vascularization below Apligraf compared with controls. Evidence of an epidermal-mesenchymal interaction was observed. This is the first article to elucidate the survival of Apligraf allogeneic cells in acute wounds in immunocompetent human subjects for up to 6 weeks and demonstrates that in the management of acute surgical wounds, Apligraf has a role only as a temporary biological dressing.
Assuntos
Colágeno/uso terapêutico , Fibroblastos/patologia , Sobrevivência de Enxerto/fisiologia , Queratinócitos/patologia , Pele Artificial , Ferimentos Penetrantes/patologia , Ferimentos Penetrantes/terapia , Órgãos Bioartificiais , Curativos Biológicos , Células Cultivadas , Feminino , Fibroblastos/transplante , Humanos , Queratinócitos/transplante , Masculino , Resultado do TratamentoRESUMO
The treatment of extensive burn injuries has been enhanced by the development of artificial skin substitutes. Integra Artificial Skin, an acellular collagen-glycosaminoglycan (C-GAG) dermal equivalent requires a two-stage grafting procedure. However, preseeding the C-GAG dermal equivalent with cultured fibroblasts and keratinocytes, with the aim of performing a single-stage grafting procedure, may be beneficial in terms of replacing the requirement for traditional split-skin grafts. In this comparative in vitro study, the interactions of cultured human dermal fibroblasts and epidermal keratinocytes in Integra Artificial Skin in comparison to cadaver deepidermalized dermis (DED) was investigated. An increase in cell proliferation and migration in the C-GAG dermal equivalent was observed over time. Cocultures of fibroblasts and keratinocytes on both dermal equivalents showed positive expression of proliferation, differentiation, and extracellular matrix (ECM) protein markers. Organization of keratinocytes in the epidermal layers of DED composites were better compared to the C-GAG composites. Deposition of ECM proteins was enhanced in the presence of keratinocytes in both dermal equivalents. Results demonstrate that in vitro the C-GAG dermal equivalent is biocompatible for cell attachment, migration, proliferation, and differentiation. Preseeding Integra Artificial Skin with cultured autologous fibroblasts and keratinocytes for in vivo application, as a single-stage grafting procedure, warrants testing. A better clinical outcome may be achieved as shown by our in vitro results of the coculture composites.
Assuntos
Órgãos Artificiais , Transplante de Pele , Pele , Humanos , Engenharia TecidualRESUMO
A number of previous studies have documented an increase in cyclic adenosine monophosphate-phosphodiesterase activity in patients with atopic dermatitis. We have studied the activity of this enzyme in keratinocytes cultured from patients with atopic dermatitis compared to keratinocytes from non-atopic controls. Results show that these pure preparations of keratinocytes express only one form of cyclic adenosine monophosphate phosphodiesterase with a Km of 1.9 microM, and a Vmax of 17 pmol/min/mg of substrate converted. We conclude that cyclic adenosine monophosphate-phosphodiesterase activity in cultured keratinocytes from patients with atopic eczema does not differ from controls.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Dermatite Atópica/enzimologia , Queratinócitos/enzimologia , Células Cultivadas , Dermatite Atópica/patologia , Humanos , Queratinócitos/patologiaRESUMO
AIMS: To establish the structural changes that occur in deep surgical wounds engrafted with allogeneic sheets, their time course and inter-relation. METHODS: Deep surgical wounds following shave excision of tattoos (down to deep dermis/subcutaneous fat) were treated with sheets of sex mismatched allogeneic keratinocytes in 19 patients and then biopsied weekly until wound healing was complete. More superficial surgical wounds--that is, 20 standard skin graft donor sites, were biopsied at seven to 10 days (all healed) following application of keratinocyte allografts. All biopsy specimens were examined with a large panel of monoclonal antibodies to keratins, envelope proteins, basement membrane components, and to extracellular matrix components. RESULTS: The hyperproliferative keratin pair K6/16 was expressed in all wounds, for up to six weeks in keratinocyte grafted deep wounds, and up to six months in split thickness skin grafted wounds. CONCLUSIONS: Keratins 6 and 16 have not been detected in normal skin, although the relevant mRNA has. This raises the possibility of regulation at a post-transcriptional level allowing a rapid response to injury with cytoskeletal changes that may aid cell migration. This keratin pair offers the most sensitive marker for altered epidermis following wounding.
Assuntos
Procedimentos Cirúrgicos Dermatológicos , Queratinócitos/transplante , Transplante de Pele , Tatuagem , Cicatrização/fisiologia , Membrana Basal/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Queratinas/metabolismo , Masculino , Mucinas/metabolismo , Período Pós-Operatório , Precursores de Proteínas/metabolismo , Pele/metabolismoRESUMO
Extracts of cultured human keratinocytes and fibroblasts were assayed for nerve growth factor-like immunoreactivity (NGF) by a specific enzyme-linked immunoabsorbant assay. NGF levels were higher in primary cultured keratinocytes than in freshly isolated keratinocytes or culture through multiple passages. Viral transformation of keratinocytes with the human papilloma virus (HPV16) significantly increased NGF levels, whilst transformation with the simian virus (SV40), which induces simple epithelial differentiation, reduced the concentration of NGF. Passaged epidermal keratinocytes contained more than twice as much NGF as did passaged fibroblasts. Oral keratinocytes and fibroblasts, and psoriatic fibroblasts, all from high turnover tissues, did not contain significantly different levels of NGF in culture than dermal keratinocytes or fibroblasts. Foetal fibroblasts contained five times as much NGF as did adult fibroblasts. These results suggest that basal keratinocytes are a major but not sole source of NGF in human skin, and that NGF may play a role in human skin development.
Assuntos
Transformação Celular Viral , Feto/metabolismo , Fatores de Crescimento Neural/metabolismo , Pele/metabolismo , Adulto , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Papillomaviridae/fisiologia , Psoríase/metabolismo , Psoríase/patologia , Vírus 40 dos Símios/fisiologia , Pele/citologiaRESUMO
BACKGROUND: Keratinocyte grafting can be used to treat acute traumatic and chronic non-healing wounds. The keratinocyte sheets are fragile and clinical "take" is difficult to assess, especially as activated keratinocytes secrete many growth factors, which have effects on wound healing apart from take. We have developed animal models of grafting that allow us to examine factors influencing autologous keratinocyte graft take. Results show clearly that pretreatment of the wound bed with viable dermis greatly increases the take of keratinocyte grafts. DATA SOURCES: International literature. CONCLUSIONS: As a greater understanding of the complex interactions of cell and matrix evolve, so will potential therapeutic maneuvers, not just in the field of cultured keratinocyte grafts, but clearly in that of benign tumors, for example, keloids, and that of oncology. There is now overwhelming evidence of the requirement for a dermal substitute for cultured keratinocyte autografts, and the sheet complexity of the situation demands that this should approximate live human dermis as closely as possible. The stumbling blocks relate to avoiding the risks of viral transmission, tissue matching of host and donor, providing early epithelial cover, and improving delivery systems for fragile keratinocyte grafts.
Assuntos
Queratinócitos/transplante , Ferimentos e Lesões/terapia , Animais , Técnicas de Cultura , Modelos Animais de Doenças , Matriz Extracelular , Humanos , Transplante de Pele/tendências , Transplante de TecidosRESUMO
The aim of this project was to reconstruct human skin from glycerol-preserved dermis and layers of cultured keratinocytes for use in the treatment of deep burns and ulcers. Glycerol-preserved cadaver skin from the Euro Skin Bank was treated with Dispase II or PBS, under various conditions, to find the best method of retaining the physical structure of the dermis while removing the epidermis and remnants of dead dermal cells which might provoke an allogeneic reaction in a graft recipient. Monoclonal antibodies LH39 and LH7.2, with specificity for basement membrane determinants, showed that treatment with Dispase II resulted in separation of the epidermis from the dermis with concomitant loss of all cellular elements from the dermal layer (as judged by H and E staining). However, when sheets of cultured keratinocytes were applied to the treated dermis and cultured for several days, the keratinocytes attached and regenerated a new basement membrane.
Assuntos
Glicerol , Queratinócitos/transplante , Transplante de Pele , Preservação de Tecido , Células Cultivadas , Endopeptidases , Células Epidérmicas , Humanos , Queratinócitos/citologia , Pele/citologia , Bancos de Tecidos , Transplante HomólogoRESUMO
Autologous cultured keratinocytes grafted onto full thickness wounds take poorly, and any epidermal cover that is produced is unstable. However, a more stable epidermis has been reported when keratinocytes are grafted onto a dermal surface. We have developed a skin grafting model using a polytetrafluoroethylene (PTFE) skin graft chamber in the domestic pig. The chambers isolate individual wounds and prevent epithelial migration from the wound edge. Dermal grafts were prepared by enzymatic separation of the epidermis from split skin to leave a de-epidermalized dermis (DED). Full thickness wounds were initially grafted with autologous DED and, subsequently, 7 days later with cultured autologous keratinocytes. The wounds were biopsied serially over 6 weeks, and processed for histology, immunocytochemistry and electron microscopy. Clinically, the grafts were seen to mature over the 6-week period. In 14/20 wounds, 40-72 per cent of the wound areas (as assessed by image analysis) had acquired epidermal cover at day 14. In 6/20 wounds, the epidermal cover was 0-27 per cent. The skin surface was stable at day 21. At this time electron microscopy and immunohistochemistry demonstrated a continuous, well formed basement membrane. The epidermis was initially acanthotic, but was histologically mature by day 14. Surprisingly, the dermal grafts were broken down by day 14. However, a neodermis formed beneath all areas with epithelial cover 21 days after grafting the keratinocytes. In this model, we have demonstrated the advantage of providing a dermal bed for cultured keratinocytes.
Assuntos
Queratinócitos/transplante , Transplante de Pele , Ferimentos e Lesões/cirurgia , Animais , Membrana Basal/patologia , Células Cultivadas , Epiderme/patologia , Queratinócitos/citologia , Suínos , Transplante Autólogo , Ferimentos e Lesões/patologiaRESUMO
The aim of this study was to study innervation and angiogenesis in response to grafts of dermis and cultured keratinocytes using immunohistochemical techniques. In a porcine model, fresh autologous de-epidermalized dermis and cultured autologous keratinocytes were combined using a two-stage technique, to produce keratodermal grafts. Wounds were encased within skin graft chambers that prevented the influence of the surrounding skin. As grafts contracted, a peripheral rim of granulation tissue became exposed, allowing us to compare the wound bed beneath grafts with that beneath the raw granulating surface. Grafts were studied for 6 weeks. Angiogenesis was studied using antisera to von Willebrand factor to detect endothelial cells. Nerve growth was studied using antisera to S-100, a Schwann cell marker, and to four axonal markers: protein gene product 9.5, C-flanking peptide of neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide. In kerato-dermal grafts (n = 28), organization of blood vessels and nerve growth occurred only beneath areas with epidermal cover as compared with the surrounding granulation tissue. Initially, the immunoreactivity to von Willebrand factor was high, but in areas with epidermal cover it assumed a more orderly pattern with fewer blood vessels. Innervation was first detected by S-100 immunoreactivity seen at 1 to 2 weeks, closely followed by that to protein gene product 9.5 and much later to calcitonin gene-related peptide. C-flanking peptide of neuropeptide Y and vasoactive intestinal peptide immunoreactivity were detected in the wound depth surrounding large blood vessels at 4 to 6 weeks. In control wounds that had been either grafted with de-epidermalized dermis alone (n = 10) or allowed to granulate (n = 10), persistently there was high immunoreactivity to von Willebrand factor but minimal immunoreactivity to the neural markers. In conclusion, kerato-dermal grafts become innervated, and beneath their surface there is also vascular organization to resemble normal skin. Keratinocytes themselves may influence angiogenesis and innervation, as both processes failed to occur beneath granulating areas.