RESUMEN
BACKGROUND: Ultraviolet radiation (UVR) is responsible for keratinocyte cancers through the induction of mutagenic cyclobutane pyrimidine dimers (CPDs). Many factors influence CPD repair in epidermal keratinocytes, and a better understanding of those factors might lead to prevention strategies against skin cancer. OBJECTIVES: To evaluate the impact of dermal components on epidermal CPD repair efficiency and to investigate potential factors responsible for the dermal-epidermal crosstalk modulating UVR-induced DNA damage repair in keratinocytes. METHODS: A model of self-assembled tissue-engineered skin containing human primary keratinocytes and fibroblasts was used in this study. RESULTS: We showed that CPD repair in keratinocytes is positively influenced by the presence of a dermis. We investigated the secretome and found that the cytokine CXCL5 is virtually absent from the culture medium of reconstructed skin, compared with media from fibroblasts and keratinocytes alone. By modulating CXCL5 levels in culture media of keratinocytes, we have shown that CXCL5 is an inhibitor of CPD repair. CONCLUSIONS: This work outlines the impact of the secreted dermal components on epidermal UVR-induced DNA damage repair and sheds light on a novel role of CXCL5 in CPD repair.
Asunto(s)
Dímeros de Pirimidina , Rayos Ultravioleta , Quimiocina CXCL5 , Daño del ADN , Reparación del ADN , Epidermis , Humanos , Queratinocitos , Piel , Rayos Ultravioleta/efectos adversosRESUMEN
Split-thickness skin autografts (AGs) are the standard surgical treatment for severe burn injuries. However, the treatment of patients with substantial skin loss is limited by the availability of donor sites for skin harvesting. As an alternative to skin autografts, our research group developed autologous self-assembled skin substitutes (SASSs), allowing the replacement of both dermis and epidermis in a single surgical procedure. The aim of the study was to assess the clinical outcome of the SASSs as a permanent coverage for full-thickness burn wounds. Patients were recruited through the Health Canada's Special Access Program. SASSs were grafted on debrided full-thickness wounds according to similar protocols used for AGs. The graft-take and the persistence of the SASS epithelium over time were evaluated. 14 patients received surgical care with SASSs. The mean percentage of the SASS graft-take was 98 % (standard deviation = 5) at 5 to 7 d after surgery. SASS integrity persisted over time (average follow-up time: 3.2 years), without noticeable deficiency in epidermal regeneration. Assessment of scar quality (skin elasticity, erythema, thickness) was performed on a subset of patients. Non-homogeneous pigmentation was noticed in several patients. These results indicated that the SASS allowed the successful coverage of full-thickness burns given its high graft-take, aesthetic outcome equivalent to autografting and the promotion of long-term tissue regeneration. When skin donor sites are in short supply, SASSs could be a valuable alternative to treat patients with full-thickness burns covering more than 50 % of their total body surface area.
Asunto(s)
Quemaduras/terapia , Trasplante de Piel , Piel Artificial , Adulto , Quemaduras/patología , Supervivencia Celular , Elasticidad , Células Epiteliales/patología , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Trasplante Autólogo , Resultado del TratamientoRESUMEN
A major challenge during the engineering of voluminous bone tissues is to maintain cell viability in the central regions of the construct. In vitro prevascularization of bone substitutes relying on endothelial cell bioprinting has the potential to resolve this issue and to replicate the native bone microvasculature. Laser-assisted bioprinting (LAB) commonly uses biological layers of hydrogel, called 'biopapers', to support patterns of printed cells and constitute the basic units of the construct. The self-assembly approach of tissue engineering allows the production of biomimetic cell-derived bone extracellular matrix including living cells. We hypothesized that self-assembled osseous sheets can serve as living biopapers to support the LAB of human endothelial cells and thus guide tubule-like structure formation. Human umbilical vein endothelial cells were bioprinted on the surface of the biopapers following a predefined pattern of lines. The osseous biopapers showed relevant matrix mineralization and pro-angiogenic hallmarks. Our results revealed that formation of tubule-like structures was favored when the cellular orientation within the biopaper was parallel to the printed lines. Altogether, we validated that human osseous cell sheets can be used as biopapers for LAB, allowing the production of human prevascularized cell-based osseous constructs that can be relevant for autologous bone repair applications.
Asunto(s)
Bioimpresión/métodos , Células Endoteliales de la Vena Umbilical Humana/citología , Osteocitos/citología , Ingeniería de Tejidos/métodos , Supervivencia Celular/fisiología , Técnicas de Cocultivo , Humanos , Osteogénesis/fisiologíaRESUMEN
Medical science has vastly improved on the means and methods available for the treatment of wounds in the clinic. The production and use of various types of skin substitutes has led to dramatic improvements in the odds of survival for severely burned patients, but they have also shown promise for many other applications, including cases involving chronic wounds that are not life threatening. Nowadays, more than 20 products are commercially available, more are undergoing clinical trials and a large number of new models are being investigated in various research laboratories worldwide. Many of the current products do not contain any living cells and vary in their capacity to harness the innate capacity of the body to heal itself. Others include living cells, of allogeneic or autologous origin, and are often referred to as 'cellular therapy' or 'tissue-engineered' products. Modifications and improvements are currently investigated that aim at improving the healing potential of those products through the use of recombinant growth factors and additional features such as microvascularization. Fundamental research into wound healing and scar-free regeneration raises the hope that we will eventually be able to restore almost completely the appearance and function of skin after the healing of wounds.
Asunto(s)
Piel Artificial , Ingeniería de Tejidos/métodos , Cicatrización de Heridas , Animales , Quemaduras/patología , Quemaduras/terapia , Humanos , Medicina Regenerativa/métodos , Piel/metabolismo , Piel/patología , Tasa de SupervivenciaRESUMEN
Considering that there is a shortage of organ donor, the aim of tissue engineering is to develop substitutes for the replacement of wounded or diseased tissues. Autologous tissue is evidently a preferable transplant material for long-term graft persistence because of the unavoidable rejection reaction occuring against allogeneic transplant. For the production of such substitutes, it is essential to control the culture conditions for post-natal human stem cells. Furthermore, histological organization and functionality of reconstructed tissues must approach those of native organs. For self-renewing tissues such as skin and cornea, tissue engineering strategies must include the preservation of stem cells during the in vitro process as well as after grafting to ensure the long-term regeneration of the transplants. We described a tissue engineering method named the self-assembly approach allowing the production of autologous living organs from human cells without any exogenous biomaterial. This approach is based on the capacity of mesenchymal cells to create in vitro their own extracellular matrix and then reform a tissue. Thereafter, various techniques allow the reorganization of such tissues in more complex organ such as valve leaflets, blood vessels, skin or cornea. These tissues offer the hope of new alternatives for organ transplantation in the future. In this review, the importance of preserving stem cells during in vitro expansion and controlling cell differentiation as well as tissue organization to ensure quality and functionality of tissue-engineered organs will be discussed, while focusing on skin and cornea.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Uniones Célula-Matriz , Enfermedades de la Córnea/terapia , Matriz Extracelular/fisiología , Células Madre Mesenquimatosas/citología , Enfermedades de la Piel/terapia , Ingeniería de Tejidos/métodos , Adulto , Animales , Células Cultivadas/citología , Córnea/citología , Células Endoteliales/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Recién Nacido , Queratinocitos/citología , Queratinas/fisiología , Células Madre Mesenquimatosas/metabolismo , Ratones , Piel/citología , Piel/crecimiento & desarrollo , Trasplante Autólogo , Vibrisas/citología , Vibrisas/fisiologíaRESUMEN
The invasive properties of cancer cells depend on their intrinsic motile potential and on their ability to breach the endothelial barrier. In the present work, we investigated the mechanisms by which adhesion of colon cancer cells to E-selectin expressed by endothelial cells regulates the barrier function of these cells and modulates transmigration of cancer cells. We found that the stimulation of E-selectin by activating antibodies or the adhesion of HT-29 cells results in an increase in the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases. In turn, the activation of p38 and ERK enhances transendothelial permeability and migration of HT-29 cells. We also obtained evidence suggesting that p38-mediated increase in transendothelial migration of cancer cells depends on a myosin light chain phosphorylation-mediated formation of stress fibres. On the other hand, the activation of ERK by E-selectin modulates the opening of interendothelial spaces by initiating the activation of Src kinase activities and the dissociation of the VE-cadherin/beta-catenin complex. We conclude that activation of E-selectin by adhering cancer cells is an important process that regulates the extravasation of colon cancer cells by initiating p38- and ERK-dependent mechanisms that both contribute to regulate the integrity of the endothelial layer.
Asunto(s)
Permeabilidad Capilar , Movimiento Celular , Neoplasias del Colon/metabolismo , Selectina E/metabolismo , Endotelio Vascular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Anticuerpos Biespecíficos/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Adhesión Celular , Células Cultivadas , Neoplasias del Colon/patología , Endotelio Vascular/enzimología , Activación Enzimática , Células HT29 , Humanos , Invasividad Neoplásica , Unión Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Fibras de Estrés/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , beta Catenina/metabolismoRESUMEN
The potential of natural dietary polyphenols in the treatment of vascular diseases originating from veins has been suggested in the literature. However, the mechanisms involved to explain the effects of polyphenols are not yet elucidated. Therefore, the aim of this study was to investigate the mechanisms by which polyphenols from red wine (Provinols) modulated contraction in human veins. We took advantage of a human model previously reported as a new tool for pharmacological research, using tissue-engineered techniques allowing the production of vascular media based exclusively on human smooth muscle cells. Thus human tissue-engineered vascular media (TEVM) were produced with cells originating from umbilical cord vein. TEVM were treated with either vehicle or Provinols. Results showed that treatment of TEVM with Provinols significantly potentiated the contractile responses induced by histamine and bradykinin. The potentiating effect of Provinols was not associated with an enhancement of histamine-induced increase in cytosolic calcium; rather, it implied the presence of a Ca(2+)-independent signaling pathway. Pharmacological studies indicated that action of Provinols took place at the level of phospholipase A(2)-Rho-kinase pathway and was associated with an enhancement of myosin light chain kinase activity. These results, obtained using the human TEVM, bring new insights to explain the regulation of venous contraction by polyphenols.
Asunto(s)
Calcio/fisiología , Flavonoides/farmacología , Contracción Muscular/fisiología , Fenoles/farmacología , Venas Umbilicales/fisiología , Señalización del Calcio/fisiología , Células Cultivadas , Medios de Cultivo , Histamina/farmacología , Humanos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Polifenoles , Venas Umbilicales/efectos de los fármacosRESUMEN
We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro.
Asunto(s)
Bronquios/citología , Fibroblastos/fisiología , Mucosa Respiratoria/fisiología , Ingeniería de Tejidos/métodos , Cilios/fisiología , Medios de Cultivo , Gelatinasas , Humanos , Inmunohistoquímica , Laminina/metabolismo , Microscopía Electrónica de Rastreo , Mucosa Respiratoria/ultraestructura , Tretinoina/fisiologíaRESUMEN
BACKGROUND: Because angiogenesis is a major feature of different physiological and pathological situations, the identification of factors that stimulate or inhibit this process and the elucidation of their mechanisms of action are most certainly of clinical relevance. We have produced a new model of endothelialized reconstructed dermis that promotes the spontaneous formation of a human capillary-like network and its stabilization in vitro for a period longer than 1 month. OBJECTIVES: The aim of this work was to describe the three-dimensional structure of the capillary-like network. Thereafter we strove to study, quantitatively and qualitatively, the influence of angiogenic and angiostatic drugs on capillary-like tube (CLT) formation in vitro in the model. METHODS: The endothelialized dermis was prepared by coculturing two human cell types, dermal fibroblasts and umbilical vein endothelial cells, in a collagen sponge biomaterial. RESULTS: The visualization by confocal microscopy of the tubes present in the model showed that the endothelial structures were not cord-like but rather CLTs with well-defined lumina. Moreover, these tubes were organized in a complex network of branching structures. When angiogenic factors (vascular endothelial growth factor 10 ng mL-1 or basic fibroblast growth factor 10 ng mL-1) were added to the model, 1.8 and 1.4 times more capillaries, respectively, were observed, whereas the addition of progesterone (10 microg x mL(-1)) reduced by 2.4 times the number of tubes compared with the control. CONCLUSIONS: These results suggest that this model is a highly efficient assay for the screening of potentially angiogenic and angiostatic compounds.
Asunto(s)
Capilares/crecimiento & desarrollo , Piel/irrigación sanguínea , Ingeniería de Tejidos/métodos , Factores de Crecimiento Endotelial/fisiología , Factor 2 de Crecimiento de Fibroblastos , Humanos , Péptidos y Proteínas de Señalización Intercelular/fisiología , Linfocinas/fisiología , Microscopía Confocal/métodos , Neovascularización Fisiológica/fisiología , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularAsunto(s)
Ingeniería de Tejidos/métodos , Ingeniería de Tejidos/tendencias , Animales , Prótesis Vascular/efectos adversos , Prótesis Vascular/normas , Prótesis Vascular/tendencias , Humanos , Piel/metabolismo , Piel/ultraestructura , Piel Artificial/normas , Piel Artificial/tendencias , Ingeniería de Tejidos/instrumentación , Procedimientos Quirúrgicos Vasculares/efectos adversos , Procedimientos Quirúrgicos Vasculares/métodos , Procedimientos Quirúrgicos Vasculares/tendenciasRESUMEN
Wound closure of epithelial tissues must occur efficiently to restore rapidly their barrier function. We have developed a tissue-engineered wound-healing model composed of human skin keratinocytes and fibroblasts to better understand the mechanisms of reepithelialization. It allowed us to quantify the reepithelialization rate, which was significantly accelerated in the presence of fibrin or platelet-rich plasma. The reepithelialization of these 6 mm excisional wounds required the contribution of keratinocyte proliferation, migration, stratification, and differentiation. The epidermis regenerated progressively from the surrounding wound margins. After 3 days, the neoepidermis showed a complete spectrum of changes. Near the wound margin, the differentiation of the neoepidermis (keratins 1/10, filaggrin, and loricrin) and regeneration of the dermoepidermal junction (laminin 5 and collagen IV) were more advanced than toward the wound center, where the proliferative index was significantly increased. The spatial distribution of keratinocytes distinguished by particular features suggests two complementary mechanisms of reepithelialization: 1) the passive displacement of the superficial layers near the wound margin that would rapidly regenerate a barrier function and 2) the crawling of keratinocytes over each other at the tip of the progressing neoepidermis. Therefore, this study brings a new perspective to long-standing questions concerning wound reepithelialization.
Asunto(s)
Epitelio/crecimiento & desarrollo , Cicatrización de Heridas/fisiología , Moléculas de Adhesión Celular/análisis , Células Cultivadas , Colágeno Tipo IV/análisis , Células Epidérmicas , Epidermis/crecimiento & desarrollo , Epidermis/ultraestructura , Epitelio/química , Epitelio/ultraestructura , Femenino , Fibroblastos/citología , Proteínas Filagrina , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Queratinocitos/citología , Masculino , Microscopía Electrónica , Piel/citología , Piel/crecimiento & desarrollo , Piel/ultraestructura , KalininaRESUMEN
Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).
Asunto(s)
Colágeno/metabolismo , Tejido Elástico/fisiología , Epidermis/fisiología , Matriz Extracelular/fisiología , Trasplante de Piel , Animales , Células Cultivadas , Tejido Conectivo/metabolismo , Tejido Conectivo/fisiología , Dermis , Tejido Elástico/metabolismo , Elastina/metabolismo , Células Epidérmicas , Epidermis/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibrilinas , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Queratinocitos/citología , Queratinocitos/fisiología , Masculino , Ratones , Ratones Desnudos , Proteínas de Microfilamentos/metabolismo , Neovascularización Fisiológica , Piel/irrigación sanguínea , Piel/citología , Piel/metabolismo , Factores de Tiempo , Trasplante HeterólogoRESUMEN
One of the differences between fetal and adult skin healing is the unique ability of fetal wounds to heal without contracture and scar formation. Studies have shown that the ratio between the three isoforms of TGFbeta is different in adult and fetal wounds. Thus, we analyzed the capacity of adult and fetal human skin fibroblasts to contract collagen gels after stimulation with TGFbeta isoforms. In control medium, fetal fibroblasts had a contractile capacity similar to that of adult fibroblasts. However, the growth capacity of fetal fibroblasts was completely inhibited, in contrast to adult fibroblasts. When cells were treated with TGFbeta, fetal fibroblasts showed an inhibition of their contractile capacity whereas adult fibroblasts further contracted gels. The contractile response was similar for all isoforms of TGFbeta although TGFbeta3 always had the strongest effect. We considered that the regulation of cell contractile capacity by TGFbeta may be dependent on receptor expression for this cytokine, on myofibroblast differentiation of the cells, or in cell links with matrix. Since TGFbeta receptor analysis did not show differences in receptor affinity, we studied the expression of alpha-smooth muscle (SM) actin, a fibroblast contractile marker and of three integrins, the cell surface receptors specific of the attachment of the fibroblasts with collagen matrix. We observed that the expression of alpha-SM actin and alpha3 and beta1 integrin subunits was increased when TGFbeta was added to the medium of adult fibroblasts whereas the levels of the alpha1 and alpha2 subunits were unchanged. In contrast, fetal fibroblasts treated with TGFbeta showed a decrease of alpha1, alpha2, and beta1 integrin expression but no change in alpha3 integrin and in alpha-SM actin expression. These results indicate that intrinsic differences between fetal and adult fibroblasts might explain their opposite responses to TGFbeta stimuli. The variations in their alpha-SM actin and integrin expression patterns represent potentially important mechanisms used by fetal fibroblasts to regulate their response to cytokines, and likely contribute to the resultant differences in the quality of wound repair.
Asunto(s)
Feto/citología , Fibroblastos/citología , Piel/citología , Cicatrización de Heridas/fisiología , Actinas/análisis , Adulto , Factores de Edad , Antígenos CD/análisis , Western Blotting , Cicatriz/patología , Fibroblastos/química , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Humanos , Integrina alfa1 , Integrina alfa2 , Integrina alfa3 , Integrina beta1/análisis , Integrinas/análisis , Radioisótopos de Yodo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1 , Factor de Crecimiento Transformador beta2 , Factor de Crecimiento Transformador beta3RESUMEN
Our method for producing tissue-engineered blood vessels based exclusively on the use of human cells, i.e., without artificial scaffolding, has previously been described (1). In this report, a tissue-engineered vascular media (TEVM) was specifically produced for pharmacological studies from cultured human vascular smooth muscle cells (VSMC). The VSMC displayed a differentiated phenotype as demonstrated by the re-expression of VSMC-specific markers and actual tissue contraction in response to physiological stimuli. Because of their physiological shape and mechanical strength, rings of human TEVM could be mounted on force transducers in organ baths to perform standard pharmacological experiments. Concentration-response curves to vasoconstrictor agonists (histamine, bradykinin, ATP, and UTP) were established, with or without selective antagonists, allowing pharmacological characterization of receptors (H1, B2, and P2Y1, and pyrimidinoceptors). Sustained agonist-induced contractions were associated with transient increases in cytosolic Ca2+ concentration, suggesting sensitization of the contractile machinery to Ca2+. ATP caused both Ca2+ entry and Ca2+ release from a ryanodine- and caffeine-sensitive store. Increased cyclic AMP or cyclic GMP levels caused relaxation. This human TEVM displays many of functional characters of the normal vessel from which the cells were originally isolated, including contractile/relaxation responses, cyclic nucleotide sensitivity, and Ca2+ handling mechanisms comparable to those of the normal vessel from which the cells were originally isolated. These results demonstrate the potential of this human model as a versatile new tool for pharmacological research.
Asunto(s)
Ingeniería Biomédica/métodos , Contracción Muscular/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Adenosina Trifosfato/farmacología , Bradiquinina/farmacología , Calcio/metabolismo , Células Cultivadas , Técnicas de Cultivo/métodos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Histamina/farmacología , Humanos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Cultivo de Órganos/métodos , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Venas Umbilicales/citología , Uridina Trifosfato/farmacología , Vasoconstrictores/farmacologíaRESUMEN
Tissue engineering is progressing rapidly. Bioengineered substitutes are already available for experimental applications and some clinical purposes such as skin replacement. This review focuses on the development of reconstructed human cornea in vitro by tissue engineering. Key elements to consider in the corneal reconstruction, such as the source for epithelial cells and keratocytes, are discussed and the various steps of production are presented. Since one application of this human model is to obtain a better understanding of corneal wound healing, the mechanisms of this phenomenon as well as the function played both by membrane-bound integrins and components from the extracellular matrix have also been addressed. The analysis of integrins by immunohistofluorescence labelling of our reconstructed human cornea revealed that beta(1), alpha(3), alpha(5), and alpha(6) integrin subunits were expressed but alpha(4) was not. Laminin, type VII collagen and fibronectin were also detected. Finally, the future challenges of corneal reconstruction by tissue engineering are discussed and the tremendous applications of such tissue produced in vitro for experimental as well as clinical purposes are considered.
Asunto(s)
Ingeniería Biomédica , Córnea , Córnea/metabolismo , Lesiones de la Cornea , Matriz Extracelular/metabolismo , Humanos , Integrinas/metabolismo , Cicatrización de Heridas , Heridas y Lesiones/fisiopatologíaRESUMEN
Many studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboidal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.
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Queratinocitos/citología , Piel Artificial , Animales , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Cromatografía Líquida de Alta Presión , Colorantes , Medio de Cultivo Libre de Suero , Técnicas de Cultivo/métodos , Humanos , Queratinocitos/ultraestructura , Queratolíticos/farmacología , Ratones , Microscopía Electrónica , Tretinoina/farmacologíaRESUMEN
In human skin, large burned surfaces heal using two concomitant phenomena: re-epithelialization and dermal neoformation. Numerous studies report the role of interactions between keratinocytes and fibroblasts, but the relationship between wound healing myofibroblasts and keratinocytes is not clear, even though these two cell types coexist during healing. We investigated the influence of myofibroblasts on keratinocyte growth and differentiation using an in vitro skin model. A histological study was performed to determine the speed and quality of epithelialization. When the dermis was populated with fibroblasts, a continuous epidermis was formed in 7-10 days. In contrast, with wound healing myofibroblasts or without cell in dermis, the complete reepithelialization never occurred over the 10-day period studied. After 7 further days of epidermal differentiation, histology showed an epidermis more disorganized and expression of basement membrane constituents was reduced when wound healing myofibroblasts or no cells were added in the dermis instead of fibroblasts. These results suggest that wound healing myofibroblasts are not efficient to stimulate keratinocyte growth and differentiation. Treatment of fibroblasts with TGFbeta1 induced an increase of epidermal cell differentiation as seen when myofibroblasts were present. However, this cytokine did not change re-epithelialization rate and induced an increase of basement membrane matrix deposition in opposition to myofibroblasts. Thus, TGFbeta1 action is not sufficient to explain all the different keratinocyte reactions towards fibroblasts and wound healing myofibroblasts. Our conclusion is that myofibroblasts seem to have a limited role in the re-epithelialization process and might be more associated with the increased extracellular matrix secretion.
Asunto(s)
Fibroblastos/fisiología , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas/fisiología , Membrana Basal/fisiología , Diferenciación Celular , Células Cultivadas , Dermis/fisiología , Células Epiteliales/fisiología , Epitelio/fisiología , Humanos , Inmunohistoquímica , Queratinocitos/fisiología , Músculo Liso/citología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
We designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.