RESUMO
Missense mutations in the 3' end of the p63 gene are associated with either RHS (Rapp-Hodgkin syndrome) or AEC (Ankyloblepharon Ectodermal defects Cleft lip/palate) syndrome. These mutations give rise to mutant p63alpha protein isoforms with dominant effects towards their wild-type counterparts. Here we report four RHS/AEC-like patients with mutations (p.Gln9fsX23, p.Gln11X, p.Gln16X), that introduce premature termination codons in the N-terminal part of the p63 protein. These mutations appear to be incompatible with the current paradigms of dominant-negative/gain-of-function outcomes for other p63 mutations. Moreover it is difficult to envisage how the remaining small N-terminal polypeptide contributes to a dominant disease mechanism. Primary keratinocytes from a patient containing the p.Gln11X mutation revealed a normal and aberrant p63-related protein that was just slightly smaller than the wild-type p63. We show that the smaller p63 protein is produced by translation re-initiation at the next downstream methionine, causing truncation of a non-canonical transactivation domain in the DeltaN-specific isoforms. Interestingly, this new DeltaDeltaNp63 isoform is also present in the wild-type keratinocytes albeit in small amounts compared with the p.Gln11X patient. These data establish that the p.Gln11X-mutation does not represent a null-allele leading to haploinsufficiency, but instead gives rise to a truncated DeltaNp63 protein with dominant effects. Given the nature of other RHS/AEC-like syndrome mutations, we conclude that these mutations affect only the DeltaNp63alpha isoform and that this disruption is fundamental to explaining the clinical characteristics of these particular ectodermal dysplasia syndromes.
Assuntos
Anormalidades Múltiplas/genética , Códon sem Sentido , Displasia Ectodérmica/genética , Proteínas de Membrana/genética , Anormalidades da Boca/genética , Biossíntese de Proteínas , Anormalidades Múltiplas/metabolismo , Adolescente , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Criança , Pré-Escolar , Displasia Ectodérmica/metabolismo , Feminino , Humanos , Queratinócitos/metabolismo , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Anormalidades da Boca/embriologia , Anormalidades da Boca/metabolismo , Alinhamento de Sequência , Transcrição Gênica , Ativação TranscricionalRESUMO
Psoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the keratinocytes or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult keratinocytes after air exposure and stimulation by keratinocyte growth factor and epidermal growth factor displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-2, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin keratinocytes were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult keratinocytes were stimulated with psoriasis-associated cytokines [tumor necrosis factor-alpha, interleukin (IL)-1alpha, IL-6, and IL-22] or combinations thereof, strong expression of hBD-2, SKALP/elafin, CK16, IL-8, and tumor necrosis factor-alpha was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro.
Assuntos
Técnicas de Cultura de Células/métodos , Queratinócitos/citologia , Psoríase , Pele , Engenharia Tecidual/métodos , Adulto , Biomarcadores/análise , Expressão Gênica , Humanos , Imuno-Histoquímica , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Porous PEGT/PBT implants with different physico-chemical characteristics were evaluated to identify its potential as biodegradable and biofunctional soft tissue filler. Implants (50 x 10 x 5 mm3) were implanted subcutaneously in mini-pigs and tissue response, tissue volume generated and its consistency were assessed quantitatively with a 52 weeks follow-up. The absence of wound edema, skin irritation, and chronic inflammation demonstrated biocompatibility of all implants evaluated. The hydrophobic implants induced the mildest foreign body response, generated highest amount of connective tissue and demonstrated a decrease in copolymer MW of 34-37% compared to 90% decrease of the hydrophilic implants. The rate and extent of copolymer fragmentation seems to be the determining factor of success of soft tissue augmentation using porous PEGT/PBT copolymer implants.
Assuntos
Implantes Absorvíveis , Materiais Biocompatíveis/química , Tecido Conjuntivo/cirurgia , Tecido Conjuntivo/ultraestrutura , Regeneração Tecidual Guiada/instrumentação , Poliésteres/química , Polietilenoglicóis/química , Animais , Materiais Biocompatíveis/efeitos adversos , Desenho de Equipamento , Análise de Falha de Equipamento , Regeneração Tecidual Guiada/efeitos adversos , Regeneração Tecidual Guiada/métodos , Estudos Longitudinais , Teste de Materiais , Poliésteres/efeitos adversos , Poliésteres/uso terapêutico , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/uso terapêutico , Porosidade , Suínos , Porco MiniaturaRESUMO
Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression.
Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Psoríase/tratamento farmacológico , Sesquiterpenos/farmacologia , Animais , Antineoplásicos/química , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Avaliação Pré-Clínica de Medicamentos , Dysidea/química , Elafina/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Interleucina-8/genética , Interleucina-8/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinas/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Psoríase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sesquiterpenos/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: The antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism. RESULTS: Human LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1. CONCLUSION: Although Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , HIV-1/efeitos dos fármacos , Lentivirus/efeitos dos fármacos , Proteínas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Anti-Infecciosos/toxicidade , Peptídeos Catiônicos Antimicrobianos/toxicidade , Catelicidinas , Linhagem Celular , Vetores Genéticos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Cocos Gram-Positivos/efeitos dos fármacos , Cocos Gram-Positivos/crescimento & desenvolvimento , HIV-1/fisiologia , Humanos , Lentivirus/fisiologia , Luciferases/genética , Luciferases/metabolismo , Peptídeos/farmacologia , Peptídeos/toxicidade , Proteínas/toxicidade , Transdução GenéticaRESUMO
Engineering tissues in bioreactors is often hampered by disproportionate tissue formation at the surface of scaffolds. This hinders nutrient flow and retards cell proliferation and tissue formation inside the scaffold. The objective of this study was to optimize scaffold morphology to prevent this from happening and to determine the optimal scaffold geometric values for connective tissue engineering. After comparing lyophilized crosslinked collagen, compression molded/salt leached PEGT/PBT copolymer and collagen-PEGT/PBT hybrid scaffolds, the PEGT/PBT scaffold was selected for optimization. Geometric parameters were determined using SEM, microcomputed tomography, and flow permeability measurements. Fibroblast were seeded and cultured under dynamic flow conditions for 2 weeks. Cell numbers were determined using CyQuant DNA assay, and tissue distribution was visualized in H&E- and Sirius Red-stained sections. Scaffolds 0.5 and 1.5 mm thick showed bridged connected tissue from top-to-bottom, whereas 4-mm-thick scaffolds only revealed tissue ingrowth until a maximum depth of 0.6-0.8 mm. Rapid prototyped scaffold were used to assess the maximal void space (pore size) that still could be filled with tissue. Tissue bridging between fibers was only found at fiber distances < or =401 +/- 60 microm, whereas filling of void spaces in 3D-deposited scaffolds only occurred at distances < or =273 +/- 55 microm. PEGT/PBT scaffolds having similar optimal porosities, but different average interconnected pore sizes of 142 +/- 50, 160 +/- 56 to 191 +/- 69 microm showed comparable seeding efficiencies at day 1, but after 2 weeks the total cell numbers were significantly higher in the scaffolds with intermediate and high interconnectivity. However, only scaffolds with an intermediate interconnectivity revealed homogenous tissue formation throughout the scaffold with complete filling of all pores. In conclusion, significant amount of connective tissue was formed within 14 days using a dynamic culture process that filled all void spaces of a PEGT/PBT scaffolds with the following geometric parameters: thickness 1.5-1.6 mm, pore size range 90-360 microm, and average interconnecting pore size of 160 +/- 56 microm.
Assuntos
Materiais Biocompatíveis , Tecido Conjuntivo , Derme , Fibroblastos/fisiologia , Polímeros , Engenharia Tecidual , Reatores Biológicos , Células Cultivadas , Colágeno , Fibroblastos/citologia , Humanos , Engenharia Tecidual/métodosRESUMO
Human skin equivalents (HSEs) were engineered using biodegradable-segmented copolymer PEGT/PBT as a dermal scaffold. As control groups, fibroblast-populated de-epidermized dermis, collagen, fibrin and hybrid PEGT/PBT-collagen matrices were used. Two different approaches were used to generate full-thickness HSE. In the 1-step approach, keratinocytes were seeded onto the fibroblast-populated scaffolds and cultured at the air-liquid (A/L) interface. In the 2-step approach, fully differentiated epidermal sheets were transferred onto fibroblast-populated scaffolds and cultured at the A/L. In a 1-step procedure, keratinocytes migrated into the porous PEGT/PBT scaffold. This was prevented by incorporating fibroblast-populated collagen into the pores of the PEGT/PBT matrix or using the 2-step procedure. Under all experimental conditions, fully differentiated stratified epidermis and basement membrane was formed. Differences in K6, K16, K17, collagen type VII, laminin 5 and nidogen staining were observed. In HSE generated with PEGT/PBT, the expression of these keratins was higher, and the deposition of collagen type VII, laminin 5 and nidogen at the epidermal/matrix junction was retarded compared to control HSEs. Our results illustrate that the copolymer PEGT/PBT is a suitable scaffold for the 2-step procedure, whereas the incorporation of fibroblast-populated collagen or fibrin into the pores of the scaffold is required for the 1-step procedure.
Assuntos
Fibroblastos/citologia , Fibroblastos/fisiologia , Queratinócitos/citologia , Queratinócitos/fisiologia , Poliésteres/química , Polietilenoglicóis/química , Pele Artificial , Engenharia Tecidual/métodos , Materiais Biocompatíveis/química , Diferenciação Celular , Divisão Celular , Tamanho Celular , Células Cultivadas , Técnicas de Cocultura/métodos , Proteínas da Matriz Extracelular/metabolismo , Humanos , Teste de Materiais , Pele/citologia , Fenômenos Fisiológicos da PeleRESUMO
The engineering of dermal skin substitutes, using autologous fibroblasts, requires high seeding efficiencies, a homogeneous cell distribution in the scaffolds, and optimal culture conditions. Dynamic seeding in spinner flasks was used to seed and subsequently culture fibroblasts in three-dimensional scaffolds. Several seeding and culture variables were investigated. Simulation of medium movement with microspheres showed that three different regions existed in medium (outer, middle, and inner), where overall particle movement was different. In the middle region the flow was turbulent and scaffolds were best placed in this region. After fibroblast seeding, methylene blue staining and scanning electron microscopy analysis of the scaffolds showed that at a low stirring speed (20 rpm) fibroblasts attached mainly onto the upper part of the scaffold, and at 40 and 60 rpm fibroblasts attached and spread throughout the scaffolds. Measurements of total DNA content per scaffold showed that lower stirring speeds (20 and 40 rpm) resulted in significantly higher cell-seeding efficiencies (20 rpm, 99.8 +/- 11.3%; 40 rpm, 93.8 +/- 10.5%) compared with 60 rpm (85.9 +/- 5.3%). Seeding kinetics were comparable for all three speeds investigated. In subsequent studies, 40 rpm was chosen for seeding. Using initial cell numbers ranging from 0.3 x 10(6) to 1.5 x 10(6) fibroblasts per scaffold, seeding efficiencies higher than 85% were consistently found (n = 4). The culture of fibroblast-seeded scaffolds at different stirring speeds (10-80 rpm) showed that stirring speeds higher than 10 rpm significantly stimulated fibroblast proliferation and glycosaminoglycan and collagen deposition as compared with 10 rpm. After 21 days, scaffolds cultured at 80 rpm showed significantly more collagen deposition as compared with those maintained at lower speeds. In conclusion, to achieve high seeding efficiencies, uniform fibroblast distribution and tissue formation in a three-dimensional scaffold, fibroblasts can be dynamically seeded at 40 rpm and subsequently cultured at a stirring speed of 60-80 rpm in spinner flasks. This flexible system shows that it is feasible to tissue engineer autologous dermal substitutes in a clinically acceptable time frame.
Assuntos
Fibroblastos/metabolismo , Ácidos Ftálicos , Polietilenoglicóis , Polietilenotereftalatos/análogos & derivados , Polímeros , Pele Artificial , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Derme , Cinética , Microscopia Eletrônica de Varredura , Microesferas , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
In this study in vitro and in vivo functions were compared between cultured dermal equivalents produced with human fibroblasts isolated either from papillary dermis or adipose tissue of the same donors. Papillary dermal fibroblasts had a normal spindle cell shape; in contrast, adipose tissue fibroblasts had a stellate cell shape, actin stress fibers containing alpha-smooth muscle actin, multiple narrow extensions at their edges, and longer focal adhesion plaques. After dynamic culture for 14 days in PEGT/PBT carrier scaffolds, cell numbers between the two cell sources were comparable, but tissue morphology was different between the cultured groups. In addition, papillary fibroblasts had deposited significantly more glycosaminoglycans (214 +/- 15 versus 159 +/- 21 microg, p < 0.001) and a lower amount of collagen (49 +/- 14 versus 111 +/- 25 microg of hydroxyproline, p < 0.001) than had adipose fibroblasts. Moreover, the latter constructs were significantly more contracted than the papillary fibroblast-cultured constructs (78 +/- 6 versus 96 +/- 3%, p < 0.001). In comparison with the influence of cultured dermal equivalents on wound healing, the transplantation of five groups (control acellular carrier, papillary fibroblast-seeded construct, adipose fibroblast-seeded construct, papillary fibroblast-cultured construct, and adipose fibroblast-cultured construct) to full-thickness wounds on the backs of athymic mice showed clear differences in angiogenesis and tissue ingrowth after 10 days, and in reepithelialization after 21 days. After 10 days, the level of vascular ingrowth in the carrier (von Willebrand staining) for the five groups was as follows: adipose fibroblast-cultured > papillary fibroblast-cultured = adipose fibroblast-seeded > papillary fibroblast-seeded > acellular carrier. After 21 days, only the acellular carriers were not vascularized and the papillary fibroblast-seeded constructs were not completely vascularized. Complete wound reepithelialization (92 +/- 12%) was observed only in the group treated with adipose cultured constructs. Wound contraction was not observed. Staining for HLA-ABC and alpha-smooth muscle actin showed that human fibroblasts had survived and that adipose fibroblasts continued to express the actin isoform. These results showed not only stimulation of skin repair when fibroblasts were present in the carrier, but also significant positive effects of the deposited extracellular matrix (ECM) in the carrier. In addition, the adipose fibroblast-seeded construct, and especially the adipose fibroblast-cultured construct, significantly stimulated angiogenesis and reepithelialization when compared with their corresponding papillary fibroblast constructs. Apparently, tissue source or fibroblast phenotype and the presence of ECM play a crucial role in the stimulation of (impaired) healing and engineering of dermal equivalents.
Assuntos
Procedimentos Cirúrgicos Dermatológicos , Matriz Extracelular/fisiologia , Fibroblastos/transplante , Transplante de Pele/métodos , Pele/lesões , Engenharia Tecidual/métodos , Cicatrização/fisiologia , Tecido Adiposo/patologia , Animais , Adesão Celular , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Células Cultivadas , Fibroblastos/classificação , Fibroblastos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Especificidade de Órgãos , Pele/patologia , Pele Artificial , Resultado do Tratamento , Ferimentos Penetrantes/patologia , Ferimentos Penetrantes/cirurgiaRESUMO
Poly(ether ester) block-copolymer scaffolds of different pore size were implanted into the dorsal skinfold chamber of balb/c mice. Using intravital fluorescent microscopy, the temporal course of neovascularization into these scaffolds was quantitatively analyzed. Three scaffold groups (diameter, 5 mm; 220-260 thickness, microm; n = 30) were implanted. Different pore sizes were evaluated: small (20-75 microm), medium (75-212 microm) and large pores (250-300 microm). Measurements were performed on days 8, 12, 16, and 20 in the surrounding normal tissue, in the border zone, and in the center of the scaffold. Standard microcirculatory parameters were assessed (plasma leakage, vessel diameter, red blood cell velocity, and functional vessel density). The large-pored scaffolds showed significantly higher functional vessel density in the border zone and in the center (days 8 and 12) compared with the scaffold with the small and medium-sized pores. These data correlated with a larger vessel diameter and a higher red blood cell velocity in the large-pored scaffold group. Interestingly, during the evaluation period the microcirculatory parameters on the edge of the scaffolds returned to values similar to those found in the surrounding tissue. In the center of the scaffold, however, neovascularization was still active 20 days after implantation. Plasma leakage and vessel diameter were higher in the center of the scaffold. Red blood cell velocity and functional vessel density were 50% lower than in the surrounding tissue. In conclusion, the dorsal skinfold chamber model in mice allows long-term study of blood vessel growth and remodeling in porous biomedical materials. The rate of vessel ingrowth into poly(ether ester) block-copolymer scaffolds is influenced by pore size and was highest in the scaffold with the largest pores. The data generated with this model contribute to knowledge about the development of functional vessels and tissue ingrowth into biomaterials.
Assuntos
Éteres , Neovascularização Fisiológica/fisiologia , Próteses e Implantes , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Microscopia de Fluorescência , Polímeros , Técnica de Janela Cutânea , Gravação de VideoteipeRESUMO
The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2-3 mm3) and with volumes tissue ratios > or =2 ml/g tissue. For incubations < or =10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions < or =10 h (collagenase 2700-3900 Mandl U/ml, neutral protease 5100-10,000 caseinase U/ml, and clostripain 35-48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350- 3000 U/ml, neutral protease 2550-7700 U/ml, and clostripain 18-36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1 x 10(4) cells/cm2. These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.
Assuntos
Fibroblastos/citologia , Pele Artificial , Transplante Autólogo , Fibroblastos/metabolismo , Humanos , Colagenase Microbiana/metabolismo , Pele/citologia , Engenharia Tecidual/métodosRESUMO
Although dermal substitution is a main topic of current wound healing research, there is a paucity of clinical trials with a long-term clinical and histopathological evaluation. A clinical trial was conducted to perform an intra-individual comparison of conventional treatment (split-thickness autograft) to a collagen/elastin dermal substitute in combination with an autograft. Promising results with this substitute were obtained with respect to dermal organisation and scar elasticity in animal studies and clinical trials with a short-term follow-up. Twenty-nine of the 42 pairs of the burn wounds and 28 of the 44 pairs of the scar reconstructions enrolled in the study were biopsied after 1 year. Promising but not statistically significant differences were found between substituted groups and control groups for epidermal thickness, basement membrane maturation, rete ridges (P=0.055), fibroblasts, myofibroblasts, inflammatory cells, vessels and extracellular matrix maturation. An objective and accurate technique, Fourier analysis, was used to evaluate collagen bundle orientation and packing. However, no statistically significant differences were obtained for these parameters. This microscopic evaluation provided no convincing evidence for a long-term effectiveness of a dermal substitute despite promising data over a short-term in in vitro and in vivo studies with the same material.
Assuntos
Queimaduras/cirurgia , Procedimentos Cirúrgicos Dermatológicos , Transplante de Pele , Adulto , Queimaduras/patologia , Feminino , Seguimentos , Análise de Fourier , Sobrevivência de Enxerto , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Procedimentos de Cirurgia Plástica , Pele/patologia , Pele Artificial , Fatores de TempoRESUMO
Tenascin-X is a large extracellular matrix protein that is widely expressed in connective tissues during development and in the adult. Genetically determined deficiency of tenascin-X causes the connective tissue disease Ehlers-Danlos syndrome. These patients show reduced collagen density and fragmentation of elastic fibers in their skin. In vitro studies on the role of tenascin-X in elastic fiber biology are hampered because monolayers of fibroblasts do not deposit tenascin-X and elastic fibers into the extracellular matrix. Here, we applied an organotypic culture model of fibroblasts and keratinocytes to address this issue. We investigated the deposition of tenascin-X and elastin into skin-equivalent in vitro and also in vivo after transplantation onto immunodeficient mice. Whereas tenascin-C and fibrillin-1 were readily expressed in the skin-equivalents before transplantation, tenascin-X and elastin were not present. Three weeks post-grafting, a network of elastin was observed that coincided with the appearance of tenascin-X. At the ultrastructural level, microfibrils were observed, some of which were associated with elastin. Transplanted skin-equivalents containing tenascin-X-deficient fibroblasts showed deposition of immunoreactive elastin in similar quantities and distribution as those containing control fibroblasts. This suggests that tenascin-X is important for the stability and maintenance of established elastin fibers, rather than for the initial phase of elastogenesis. Thus, the transplantation of reconstructed skin on nude mice allows the study of tenascin-X and elastin expression and could be used as a model system to study the potential role of tenascin-X in matrix assembly and stability.
Assuntos
Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patologia , Tecido Elástico/patologia , Tenascina/deficiência , Tenascina/metabolismo , Transplante Heterólogo , Animais , Células Cultivadas , Colágeno/metabolismo , Colágeno/ultraestrutura , Síndrome de Ehlers-Danlos/genética , Tecido Elástico/ultraestrutura , Fibroblastos/metabolismo , Géis , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Nus , Microfibrilas/ultraestrutura , Modelos Genéticos , Transplante de Pele , Tenascina/ultraestrutura , Fatores de TempoRESUMO
Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether fibroblasts can substitute growth factors needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different fibroblast numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of fibroblasts the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of fibroblasts keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of fibroblasts showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3 weeks culture at the air-liquid interface, the proliferation index decreased irrespective of the number of fibroblasts present within the dermal matrix to levels observed also in native epidermis. Keratin 10 was localized in all viable suprabasal cell layers irrespective of the absence or presence of fibroblasts. Keratin 6 was downregulated with increasing numbers of fibroblasts, and keratins 16 and 17 were absent in fibroblast-populated matrices. The expression of involucrin or transglutaminase 1 showed a similar pattern as for the keratins. Irrespective of the number of fibroblasts incorporated into DED, the expression of alpha(3), alpha(6), beta(1), and beta(4) integrin subunits was upregulated. In fibroblast-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte growth factor. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with fibroblasts.
Assuntos
Células Epidérmicas , Epiderme/fisiologia , Fibroblastos/metabolismo , Queratinócitos/fisiologia , Pele Artificial , Divisão Celular , Movimento Celular , Separação Celular , Células Cultivadas , Derme/citologia , Fator de Crescimento Epidérmico/metabolismo , Humanos , Imuno-Histoquímica , Integrina alfa3/metabolismo , Integrina alfa6/metabolismo , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Queratina-10 , Queratinócitos/metabolismo , Queratinas/metabolismo , Morfogênese , Fator de Crescimento Derivado de Plaquetas/metabolismo , Precursores de Proteínas/metabolismo , Subunidades Proteicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transglutaminases/metabolismoRESUMO
In the present study, we compared the use of autologous versus allogeneic fibroblasts in dermal skin substitutes in a porcine wound model. The allogeneic fibroblast populations were isolated from female and a male pig (allo-1, - 2 and - 3) and the controls, autologous fibroblasts, from female graft-recipient pigs (control). The histocompatibility of the three donor pigs with the recipient pigs was determined with a mixed lymphocyte reaction. In two pigs, full-thickness wounds were treated with the fibroblast-seeded dermal substitutes (n = 5 per animal) and immediately overgrafted with meshed split-skin autografts. After 6 weeks, wound contraction was measured by planimetry and scar formation was scored. At 2, 4, and 6 weeks biopsies were taken and evaluated for the presence of inflammatory reactions, myofibroblasts, and scar formation. The mixed lymphocyte reaction of both recipient pigs showed the highest responses on peripheral blood mononuclear cells of the allo-3 donor pig, and was low or negative for allo-1 and allo-2. In all "allogeneic" wounds, more inflammatory cells were observed over time along with inflammatory foci consisting of a mix of lymphocytes and granulomatous cells. After 4 weeks, myofibroblasts were absent in the control wounds, whereas in "allogeneic" wounds, myofibroblasts colocalized with inflammation foci. The final scar tissue of the "allogeneic" wounds showed granulating areas with thin, immature collagen bundles. In contrast, the control wounds showed a dermal tissue with mature collagen bundles organized randomly like in normal skin. The wounds treated with allo-3 fibroblasts showed in both pigs a significant increase in scar formation and wound contraction when compared with control wounds. In conclusion, for optimal restoration of dermal skin function with minimal scar formation, skin substitutes containing autologous fibroblasts are preferred over skin substitutes with allogeneic fibroblasts.
Assuntos
Fibroblastos/transplante , Pele Artificial , Animais , Matriz Extracelular , Feminino , Tecido de Granulação/patologia , Imuno-Histoquímica , Teste de Cultura Mista de Linfócitos , Masculino , Modelos Animais , Suínos , Transplante Autólogo , Transplante Homólogo , CicatrizaçãoRESUMO
The recovery of skin function is the goal of each burn surgeon. Split-skin graft treatment of full-thickness skin defects leads to scar formation, which is often vulnerable and instable. Therefore, the aim of this study was to analyze wound healing and scar tissue formation in acute full-thickness wounds treated with clinically available biopolymer dermal regeneration templates. Full-thickness wounds (3 x 3 cm) on both flanks of Gottingen mini pigs (n= 3) were treated with split-thickness skin graft alone or in combination with a 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) cross-linked-collagen scaffold, Integra, or a polyethyleneglycol terephthalate-polybutylene terephthalate (PEGT/PBT) scaffold. The wounds (n= 12 per group) were examined weekly for six weeks to evaluate graft take, contraction (planimetry), and cosmetic appearance. Histologic samples taken after one and six weeks were used to assess scaffold angiogenesis, biocompatibility, and scar tissue quality. In all wounds, one week postwounding graft take was between 93 and 100 percent. The control wound, treated with split-skin graft, showed little granulation tissue formation, whereas the EDC-collagen treated wounds showed two to three times more granulation tissue formation. The collagen scaffold was completely degraded within one week. The Integra and PEGT/PBT scaffolds showed angiogenesis only through two-thirds of the scaffold, which resulted in loss of integrity of the epidermis. Only basal cells survived, proliferated, and regenerated a fully differentiated epidermis within three weeks. Granulation thickness was comparable to collagen scaffold-treated wounds. After six weeks, control wounds showed a wound contraction of 27.2 +/- 6.1 percent, Integra-treated wounds 34.6 +/- 6.4 percent, collagen scaffold-treated wounds 38.1 +/- 5.0 percent, and PEGT/PBT scaffold-treated wounds 54.5 +/- 3.9 percent. The latter wounds had significantly more contraction than wounds of other treatment groups. Microscopically, the control and collagen scaffold-treated wounds showed an immature scar tissue that was two times thicker in the EDC-collagen treated wounds. The Integra-treated wounds showed nondegraded collagen scaffold fibers with partly de novo dermal tissue formation and partly areas with giant cells and other inflammatory cells. The PEGT/PBT scaffold was almost completely degraded. Scaffold particles were phagocytosized and degraded intracellularly by clusters of macrophages. The scar tissue was in the early phase of ECM remodeling. In conclusion, this study showed that the rate of dermal tissue formation and scarring is influenced by the rate of scaffold angiogenesis, degradation, and host response induced by the scaffold materials.
Assuntos
Cicatriz/patologia , Pele Artificial , Ferimentos e Lesões/patologia , Ferimentos e Lesões/cirurgia , Animais , Bandagens , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Masculino , Curativos Oclusivos , Probabilidade , Distribuição Aleatória , Fatores de Risco , Sensibilidade e Especificidade , Fenômenos Fisiológicos da Pele , Transplante de Pele/métodos , Suínos , Cicatrização/fisiologiaRESUMO
Currently, the use of cultured epithelial autografts as an alternative to split-thickness skin autografts for coverage of full-thickness wounds is limited due to fragility of the sheet and variability in the outcome of healing. This could be circumvented by the transfer of proliferating keratinocytes, instead of differentiated sheets, to the wound bed and the "in vivo" regeneration of epidermis. The aim of this study was to achieve re-epithelialization on experimental full-thickness wounds in the pig using a porous, synthetic carrier seeded with proliferating keratinocytes. Porcine keratinocytes were isolated by enzymatic digestion and cultured in Optimem basal medium with mitogens. In a full-thickness wound model, carriers with different seeding densities were transplanted upside down onto the wound bed. Keratinocytes were labeled using a fluorescent red membrane marker, PKH-26 GL. Transfer of keratinocytes and re-epithelialization were recorded macroscopically and histologically. On day 4 after transplantation, transfer of fluorescently labeled keratinocytes was shown by their presence in the granulation tissue. An immature epidermis, as well as epithelial cords and islands, formed as early as day 8. At day 12 a stratified epidermis and wound closure were established and epithelial cysts were formed by differentiation of epithelial islands. Wounds treated with seeding densities as low as 50,000 cells/cm(2) showed wound closure within 12 days, whereas wounds treated with 10,000 cells/cm(2) or the nonseeded (acellular) carriers did not show complete re-epithelialization before day 17 after treatment. This study showed that porcine keratinocytes, transplanted "upside down" in experimental full-thickness wounds using a synthetic carrier, continued to proliferate and started to differentiate, enabling the formation of a new epidermis in a time frame of 12 days.
Assuntos
Bandagens , Transplante de Células/métodos , Queratinócitos/transplante , Tela Subcutânea/lesões , Ferimentos Penetrantes/terapia , Animais , Técnicas de Cultura de Células , Feminino , Poliésteres/uso terapêutico , Polietilenoglicóis/uso terapêutico , Polietilenotereftalatos/análogos & derivados , Polietilenotereftalatos/uso terapêutico , Suínos , Fatores de Tempo , Resultado do Tratamento , CicatrizaçãoRESUMO
In human dermis, collagen bundle architecture appears randomly organized, whereas in pathological conditions, such as scar tissue and connective tissue disorders, collagen bundle architecture is arranged in a more parallel fashion. Histological examination by one or two observers using polarized light is the most common method to determine collagen orientation. The hypothesis on which this study is based is that an objective image analysis technique, Fourier analysis, would improve the reliability (are the measurements reproducible?) and the accuracy (does the method measure what it is supposed to measure?) of collagen orientation assessment, compared with observer ratings. Fourier analysis was applied to 271 images of scar tissue and normal skin that were acquired by confocal laser-scanning microscopy. Observers rated the same areas using polarized light as well as the confocal microscopy images. Computer images consisting of different types of ellipses were generated with a fixed orientation. Observers and Fourier analysis evaluated the images to evaluate accuracy. The inter-observer reliability was acceptable when at least three observers rated polarized light images (r > 0.69), whereas two observers were sufficient for rating confocal microscopy images (r > 0.71). Fourier analysis correlated better with observer ratings of confocal microscopy images (r = 0.69) than with polarized light microscopy images (r = 0.42). Fourier analysis was more accurate than four observers for the evaluation of the 'true' orientation for almost all types of computer-generated images. For the first time it is shown that Fourier image analysis is suitable for the morphometry of dermal collagen orientation and leads to a superior measurement of collagen orientation compared with subjective histological evaluation by several experts. If an evaluation is performed by conventional light microscopy, at least three observers are required to attain an acceptable inter-observer reliability.