Personne : Berthod, François
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Berthod
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François
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Université Laval. Département de chirurgie
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- PublicationRestreintTissue-engineered human skin substitutes developed from collagen-populated hydrated gels : clinical and fundamental applications(Springer, 1998-11-01) Germain, Lucie; Auger, François A.; Rouabhia, Mahmoud; Berthod, François; Goulet, Francine; Moulin, VéroniqueThe field of tissue engineering has opened several avenues in biomedical sciences, through ongoing progress. Skin substitutes are currently optimised for clinical as well as fundamental applications. The paper reviews the development of collagen-populated hydrated gels for their eventual use as a therapeutic option for the treatment of burn patients or chronic wounds: tools for pharmacological and toxicological studies, and cutaneous models for in vitro studies. These skin substitutes are produced by culturing keratinocytes on a matured dermal equivalent composed of fibroblasts included in a collagen gel. New biotechnological approaches have been developed to prevent contraction (anchoring devices) and promote epithelial cell differentiation. The impact of dermo-epidermal interactions on the differentiation and organisation of bio-engineered skin tissues has been demonstrated with human skin cells. Human skin substitutes have been adapted for percutaneous absorption studies and toxicity assessment. The evolution of these human skin substitutes has been monitored in vivo in preclinical studies showing promising results. These substitutes could also serve as in vitro models for better understanding of the immunological response and healing mechanism in human skin. Thus, such human skin substitutes present various advantages and are leading to the development of other bio-engineered tissues, such as blood vessels, ligaments and bronchi.
- PublicationAccès libreLe génie tissulaire au service de la compréhension du vivant(Société des Périodiques Flammarion, 2003-10-15) Germain, Lucie; Auger, François A.; Berthod, François; Goulet, Francine; Moulin, VéroniqueLe génie tissulaire est un nouveau domaine, qui permet l’étude des mécanismes physiologiques du vivant. Il s’agit d’une technologie fondée sur la capacité des cellules vivantes, en présence ou non de biomatériaux, à s’assembler en un tissu tridimensionnel. Elle constitue une voie intéressante ouvrant aux chercheurs la possibilité de considérer les cellules dans un contexte proche de celui retrouvé in vivo. Cet article résume les travaux en génie tissulaire menés par le laboratoire d’organogenèse expérimentale (LOEX) au cours des dernières années, dans le but de comprendre certains des mécanismes physiologiques et pathologiques de l’organisme humain. Ainsi, la cicatrisation cutanée, mais aussi les cellules souches, l’angiogenèse et les interactions cellulaires sont des secteurs ayant profité de l’apport du génie tissulaire.
- PublicationRestreintA tissue-engineered endothelialized dermis to study the modulation of angiogenic and angiostatic molecules on capillary-like tube formation in vitro(Blackwell Scientific, 2003-06-27) Hudon, Valérie.; Germain, Lucie; Black, Annie.; Damour, Odile; Auger, François A.; Berthod, FrançoisBackground: 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.
- PublicationAccès libreWhat is new in mechanical properties of tissue-engineered organs(Springer, 1999-01-01) Germain, Lucie; Auger, François A.; Berthod, François; Goulet, FrancineTissue engineering is a promising new field based on expertise in cell biology, medicine and mechanical engineering. It raises exciting hopes of producing autologous tissue substitutes to replace altered organs. This challenge involves highly specialized technology in order to provide the proper shape to the tissue and promote the maintenance of its native physiological properties. Primary cell populations may lose some of their functional and morphological properties in vitro in the absence of a proper environment. In order to maintain cell integrity, a three-dimensional matrix that mimics the in vivo environment as closely as possible was developed, according to the type of tissue produced [1, 5, 18, 26, 27, 29, 34, 35].
- PublicationRestreintIn vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent(Federation of American Societies for Experimental Biology, 1998-10-01) Germain, Lucie; L'Heureux, Nicolas; Black, Annie.; Auger, François A.; Berthod, FrançoisFor patients with extensive burns, wound coverage with an autologous in vitro reconstructed skin made of both dermis and epidermis should be the best alternative to split-thickness graft. Unfortunately, various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. Our purpose was to develop a vascular-like network inside tissue-engineered skin in order to improve graft vascularization. To reach this aim, we fabricated a collagen biopolymer in which three human cell types—keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells—were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures in a highly differentiated extracellular matrix. Immunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the microvasculature in vivo (von Willebrand factor, Weibel-Palade bodies, basement membrane material, and intercellular junctions). We have developed the first endothelialized human tissue-engineered skin in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate graft revascularization by inosculation of its preexisting capillary-like network with the patient's own blood vessels, as it is observed with autografts. In addition, the ESE turns out to be a promising in vitro angiogenesis model.
- PublicationRestreintComparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis(Elsevier, 2011-06-17) Germain, Lucie; Parenteau-Bareil, Rémi; Gauvin, Robert; Cliche, Simon.; Gariépy, Claude; Berthod, FrançoisCombining bovine collagen with chitosan followed by freeze-drying has been shown to produce porous scaffolds suitable for skin and connective tissue engineering applications. In this study collagen extracted from porcine and avian skin was compared with bovine collagen for the production of tissue engineered scaffolds. A similar purity of the collagen extracts was shown by electrophoresis, confirming the reliability of the extraction process. Collagen was solubilized, cross-linked by adding chitosan to the solution and freeze-dried to generate a porous structure suitable for tissue engineering applications. Scaffold porosity and pore morphology were shown to be source dependant, with bovine collagen and avian collagen resulting into the smallest and largest pores, respectively. Scaffolds were seeded with dermal fibroblasts and cultured for 35 days to evaluate the suitability of the different collagen–chitosan scaffolds for long-term tissue engineered dermal substitute maturation in vitro. Cell proliferation and scaffold biocompatibility were found to be similar for all the collagen–chitosan scaffolds, demonstrating their capability to support long-term cell adhesion and growth. The scaffolds contents was assessed by immunohistochemistry and showed increased deposition of extracellular matrix by the cells as a function of time. These results correlate with measurements of the mechanical properties of the scaffolds, since both the ultimate tensile strength and tensile modulus of the cell seeded scaffolds had increased by the end of the culture period. This experiment demonstrates that porcine and avian collagen could be used as an alternative to bovine collagen in the production of collagen–chitosan scaffolding materials.
- PublicationAccès libreMarkers for an In vitro skin substitute(Artech House, 2018-02-01T16:41:46Z) Germain, Lucie; Jean, Jessica; Larouche, Danielle; Berthod, François; Pouliot, Roxane; Maguire, Tim; Novik, EricThe tissue engineering self-assembly approach allows the production of skin substitutes comprising both the dermis and epidermis, using methods promoting the secretion and organization of a dense extracellular matrix by skin cells. In a reconstructed epidermis, all cellular layers of the native tissue are present. An evaluation of the expression and localization of a number of specific protein markers revealed that the self-assembled, tissue-engineered skin substitute shares some common features with normal human skin, such as the expression of Ki-67, keratins 10 and 14, filaggrin, involucrin, transglutaminase, DLK, a3-integrin subunit, laminin-S, and collagens I, II, 1V, and VII. At the ultrastructural level, many differentiation markers can be observed, including desmosomes, as well as an organized basement membrane presenting hemidesmosomes, lamina densa, and lamina lucida. In this chapter, protocols to generate skin substitutes by the self-assembly approach will be presented and the methods including the labeling of the principal skin differentiation markers by immunofluorescence will be examined.
- PublicationRestreintHow to achieve early vascularization of tissue-engineered skin substitutes(Mary Ann Liebert, 2010-01-01) Germain, Lucie; Auger, François A.; Berthod, François; Pouliot, RoxaneBackground: The coverage of deep and extensive burns with autologous tissue-engineered skin is a promising strategy to improve the cosmetic aspect and functionality of the skin, compared to the transplantation of simple epithelial sheets. Indeed, a dermal compartment could markedly help to prevent hypertrophic scar formation and to strengthen the dermal–epidermal junction while increasing skin suppleness and pliability. The Problem: The thickness of the dermis could be a limitation to the survival of the tissue after transplantation, since its vascularization can take up to 2 weeks to occur through neovascularization. This delay could lead to graft necrosis. Basic/Clinical Science Advances: To overcome this problem, the reconstruction of a preformed network of branching capillaries in the dermis before grafting has proven to be an efficient solution in connecting to the host's vasculature in less than 4 days after grafting. The formation of this capillary-like network is achieved by the coculture of human fibroblasts and endothelial cells in a collagen sponge for a 1-month in vitro maturation period. The successful inosculation process between human capillaries and the host's vasculature was demonstrated after grafting onto nude mice. Clinical Care Relevance: In addition to autologous epithelial sheets and split-thickness autografts, this endothelialized reconstructed skin made of the patient's own cells could be a valuable additional strategy to permanently cover deep wounds. Conclusion: The reconstruction in tissue-engineered organs of a capillary-like network made of the patient's own cells before grafting is a promising approach to promote their early vascularization.
- PublicationRestreintHair follicles guide nerve migration in vitro and in vivo in tissue-engineered skin(Elsevier, 2011-03-03) Gingras, Marie; Germain, Lucie; Parenteau-Bareil, Rémi; Larouche, Danielle; Gagnon, Vicky; Berthod, François
- PublicationRestreintMultiple applications of tissue-engineered human skin(Thieme, 2001-01-01) Carlos, A.; Germain, Lucie; Auger, François A.; López Valle, Carlos Antonio; Berthod, François; Goulet, Francine; Moulin, VéroniqueThe progress in tissue engineering has lead to the development of tri-dimensional tissues that can be used in vitro for various applications. Different methods have been designed to produce reconstructed dermis or skin in vitro. This chapter describes the human skin models and substitutes with respect to the evolution of their complexity as well as some of their potential applications. Dermal fibroblasts or myofibroblasts included in floating collagen gels produce useful wound healing models. Bi-layered human skin constructs comprising both the dermis and the epidermis could serve. for fundamental (eg. cell-matrix interactions) or applied (e.g. dermatoabsorption) studies. Another skin substitute is produced by seeding keratinocytes on fibroblasts. cultured in a collagen-chondroitin 4-6 sulfates and. chitosan sponge. The addition of endothelial cells to this model lead to the formation of capillary-like structure in the dermis. Finally, a method of human reconstructed skin production by the "auto-assembly" approach is presented. This model is developped from cells that produce their own extracellular matrix. No synthetic material or exogenous matrix is added. Thus, it could be completely autologous. Tissue engineered skin is an attractive tissue for gene therapy. Cells could be transplanted safely in vitro, evaluated for gene expression before their incorporation in reconstructed tissue and grafting in vivo. Of particular importance will be skin stem cells that have a long term regeneration potential and that can he cultured in vitro. The progress accomplished in tissue. engineering of skin is now applied to the reconstruction of other tissues and more complex organs such as ligaments, bronchi, bladder, cornea and blood vessels. These tissues could provide therapeutic alternatives in organ transplantation as well as models for varions in vitro applications.