Personne : Auger, François A.
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Université Laval. Département chirurgie
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- PublicationRestreintDynamic mechanical stimulations induce anisotropy and improve the tensile properties of engineered tissues produced without exogenous scaffolding(Elsevier, 2011-09-01) Germain, Lucie; Parenteau-Bareil, Rémi; Larouche, Danielle; Bisson, Francis; Marcoux, Hugo; Bolduc, Stéphane; Auger, François A.; Gauvin, Robert; Bonnet, AdrienMechanical strength and the production of extracellular matrix (ECM) are essential characteristics for engineered tissues designed to repair and replace connective tissues that are subject to stress and strain. In this study, dynamic mechanical stimulation (DMS) was investigated as a method to improve the mechanical properties of engineered tissues produced without the use of an exogenous scaffold, referred to as the self-assembly approach. This method, based exclusively on the use of human cells without any exogenous scaffolding, allows for the production of a tissue sheet comprised of cells and ECM components synthesized by dermal fibroblasts in vitro. A bioreactor chamber was designed to apply cyclic strain to engineered tissues in order to determine if dynamic culture had an impact on their mechanical properties and ECM organization. Fibroblasts were cultured in the presence of ascorbic acid for 35 days to promote ECM production and allow the formation of a tissue sheet. This sheet was grown on a custom-built anchoring system allowing for easy manipulation and fixation of the tissue in the bioreactor. Following the 35 day period, tissues were maintained for 3 days in static culture (SC), or subjected either to a static mechanical stimulation of 10% strain, or a dynamic DMS with a duty cycle of 10% uniaxial cyclic strain at 1 Hz. ECM was characterized by histology, immunofluorescence labeling and Western blotting. Both static and dynamic mechanical stimulation induced the alignment of assessed cytoskeletal proteins and ECM components parallel to the axis of applied strain and increased the ECM content of the tissues compared to SC. Measurement of the tensile mechanical properties revealed that mechanical stimulation significantly increases both the ultimate tensile strength and tensile modulus of the engineered tissues when compared to the non-stimulated control. Moreover, we demonstrated that cyclic strain significantly increases these parameters when compared to a static-loading stimulation and that mechanical stimulation contributes to the establishment of anisotropy in the structural and mechanical properties of self-assembled tissue sheets.
- PublicationRestreintPolyphenols modulate calcium-independent mechanisms in human arterial tissue-engineered vascular media(Mosby, 2007-09-30) Germain, Lucie; Diebolt, Myriam; Labbé, Raymond; Auger, François A.; Laflamme, Karina; Andriantsitohaina, RamarosonBackground: In the present study, an arterial tissue-engineered vascular media (TEVM) was produced from cultured human smooth muscle cells of the umbilical artery and we took advantage of this model to evaluate the regulation of contraction and the signalling pathways of polyphenols in arteries. Methods: Cultured human smooth muscle cells of the umbilical artery were used to produce arterial TEVMs. Contraction experiments were performed to determine intracellular targets involved in the modulation of contraction by polyphenols extract from red wine, Provinols (SEPPIC Groupe Air Liquide, Paris, France). Results: Smooth muscle cells in arterial TEVM displayed a differentiated phenotype as demonstrated by the expression of alpha-smooth muscle actin, a vascular smooth muscle-specific marker, and tissue contraction in response to vasoconstrictor and vasodilator agents. Contractions caused by histamine were associated with an increase in [Ca(2+)](i) and a Ca(2+)-independent signalling pathway. The latter pathway involved mechanisms sensitive to protein kinase C, myosin light chain kinase, and Rho-associated protein kinase inhibitors. The regulation of contraction induced by Provinols shows that treatment of arterial TEVM with this compound significantly decreased histamine-induced contraction. This effect was associated with the inhibition of the Rho-associated protein kinase pathway and the decrease in alpha-smooth muscle actin expression. Conclusion: The use of arterial TEVM, brings new insights into the mechanisms by which polyphenols regulate vascular contraction in the human artery.
- PublicationAccès libreReconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types(Éditeur non identifié, 2010-10-29) Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Audet, Caroline; Auger, François A.; Uwamaliya, Jeanne d'Arc; Proulx, Stéphanie; Carrier, Patrick; Deschambeault, AlexandrePurpose: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. Methods: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. Results: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. Conclusions: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.
- PublicationRestreintMechanical properties of endothelialized fibroblast-derived vascular scaffolds stimulated in a bioreactor(Elsevier BV, 2015-03-06) Tondreau, Maxime; Laterreur, Véronique; Germain, Lucie; Vallières, Karine; Ruel, Jean; Tremblay, Catherine; Bourget, Jean-Michel; Auger, François A.; Gauvin, Robert; Lacroix, Dan.There is an ongoing clinical need for tissue-engineered small-diameter (<6 mm) vascular grafts since clinical applications are restricted by the limited availability of autologous living grafts or the lack of suitability of synthetic grafts. The present study uses our self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold that can then be available off-the-shelf. Briefly, scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and then decellularized by immersion in deionized water. Constructs were then endothelialized and perfused for 1 week in an appropriate bioreactor. Mechanical testing results showed that the decellularization process did not influence the resistance of the tissue and an increase in ultimate tensile strength was observed following the perfusion of the construct in the bioreactor. These fibroblast-derived vascular scaffolds could be stored and later used to deliver readily implantable grafts within 4 weeks including an autologous endothelial cell isolation and seeding process. This technology could greatly accelerate the clinical availability of tissue-engineered blood vessels.
- PublicationRestreintDéfis et perspectives de la médecine régénératrice cardiovasculaire(John Libbey Eurotext, 2008-01-01) Germain, Lucie; D’Orléans-Juste, Pedro; Labbé, Raymond; Auger, François A.Le présent chapitre sera consacré aux diverses méthodes de génie tissulaire ayant trait à la reconstruction des vaisseaux sanguins (in vitro) avec une visée clinique (in vivo). Toutefois, nous dédierons quelques lignes à l’utilisation de ces substituts vasculaires comme modèle in vitre pour des études parfois très pointues et complexes, dans les applications suivantes : physiologie, pathophysiologie, pharmacologie et toxicologie. Ainsi, un tour d’horizon non exhaustif des travaux de la reconstruction vasculaire au plan mondial s’accompagnera de notre expérience unique au Laboratoire d’Organogenèse EXpérimentale (LOEX). En effet, notre groupe est l’un des rares, sinon le seul, groupes de recherche à effectuer en parallèle des travaux en génie tissulaire tant sur les microvaisseaux (capillaires) que les artères de petit calibre (s 5 mm) [5, 6]. Ces deux aspects vasculaires du génie tissulaire répondent à deux impératifs cliniques. En premier lieu, les micro-vaisseaux permettent d’entrevoir une vascularisation préimplantatoire des organes reconstruits. Ainsi, les espoirs de survie de divers substituts seraient grandement améliorés puisqu'il s’agit là d’une des principales pierres d’achoppement de ce domaine. En second lieu, la création de vaisseaux cultivés de petits calibres répond à un besoin clinique, tels des pontages cardiaques et infrapoplités où les prothèses synthétiques sont inutilisables en raison d’une fréquence plus élevée de thrombose. Ainsi, le cahier de charge de ces substituts vasculaires (SV) obtenus par génie tissulaire est très exigeant comme démontre le Tableau I. Enﬁn, notons, dans un registre entièrement différent, que notre programme de recherche sur la reconstruction des valves cardiaques se poursuit actuellement .
- PublicationRestreintInosculation of tissue-engineered capillaries with the host's vasculature in a reconstructed skin transplanted on mice.(Blackwell, 2005-02-17) Hudon, Valérie.; Germain, Lucie; Tremblay, Pierre-Luc; Auger, François A.; Berthod, FrançoisThe major limitation for the application of an autologous in vitro tissue-engineered reconstructed skin (RS) for the treatment of burnt patients is the delayed vascularization of its relatively thick dermal avascular component, which may lead to graft necrosis. We have developed a human endothelialized reconstructed skin (ERS), combining keratinocytes, fibroblasts and endothelial cells (EC) in a collagen sponge. This skin substitute then spontaneously forms a network of capillary-like structures (CLS) in vitro. After transplantation to nude mice, we demonstrated that CLS containing mouse blood were observed underneath the epidermis in the ERS in less than 4 days, a delay comparable to our human skin control. In comparison, a 14-day period was necessary to achieve a similar result with the non-endothelialized RS. Furthermore, no mouse blood vessels were ever observed close to the epidermis before 14 days in the ERS and the RS. We thus concluded that the early vascularization observed in the ERS was most probably the result of inosculation of the CLS network with the host's capillaries, rather than neovascularization, which is a slower process. These results open exciting possibilities for the clinical application of many other tissue-engineered organs requiring a rapid vascularization.
- PublicationRestreintA full spectrum of functional tissue-engineered blood vessels : from macroscopic to microscopic(Springer, 2003-01-01) Grenier, Guillaume.; Germain, Lucie; Auger, François A.; Rémy-Zolghadri, MurielleTissue engineering has created several original and new avenues of investigation in biology (Auger et al., 2000). This new domain of research in biotechnology was introduced in the l980$ as a life-saving procedure for burn patients. The successful engrai‘tment of autologous living epidermis was the first proof of concept of this powerful approach. From the efforts in this ﬁeld, two schools of thought emerged. A ﬁrst one is the seeding of cells into various gels or scatTolds in which the cells secrete and/or reorganize the surrounding extracellular matrix (ECM), and a second one, the coaxing of cells onto the secretion of an abundant autologous ECM, thus creating their own environment in the absence of any exogenous material. This latter methodology, which we called the “self assembly approach,” takes advantage of the ability of cells to recreate in vitro tissue-like structures when appropriately cultured (Auger et al., 2000). The conditions entail particular media composition and adapted mechanical straining ol‘ these three-dimensional structures. Our own experience with the culture of autologous epidermal sheets gave us some insight in the property of cells to recreate such in rim: tissue-like structures. This expertise led us to develoP tissue-engineered structures on the basis ol‘ the following two concepts: the living substitutes that we created have no artiﬁcial biomaterial, and the ECM is either a biological one repopulated by the ceiis or an ECM neosynthesized by the cells themselves. Such living substitutes have distinct advantages because of their cellular composition that confer to them superior physiologicai characteristics when implanted into the human body, that is, their ability to renew themselves over time and their healing property if they are damaged. Moreover, the presence of autologous cells in the living reconstructed tissue should facilitate its interactions with the surrounding host environment. Here, we describe our own experience in the reconstruction of a full spectrum of blood vessels by tissue engineering: macroscopic and microscopic. We applied the self-assembly approach with some impressive results to the reconstruction of a small-diameter blood vessel and the use of a cell-seeded scaffold leading to the formation of capillary-like structures in a full-thickness skin. The following highlights the major points for the generation of these organs.
- PublicationRestreintInfluence of initial collagen and cellular concentrations on the final surface area of dermal and skin equivalents : a box-behnken analysis(Springer, 1990-10-01) LeDuy, Anh; Germain, Lucie; Rompré, Pierre; Defrance-Bouvard, Véronique; López Valle, Carlos Antonio; Auger, François A.; Thibault, J.Our laboratory has been involved in finding optimal conditions for producing dermal and skin equivalents. As an original approach, a Box-Behnken experimental design was used to study the effects of the initial collagen and fibroblast concentrations and the initial gel thickness on the contraction of dermal and skin equivalents. The final surface area of dermal equivalent varied significantly with the initial concentration of collagen and fibroblast, whereas the initial thickness of gel had no appreciable effect on the contraction of the dermal equivalent. When keratinocytes were grown on these dermal equivalents they produced a very severe contraction, to an extent that all skin equivalents had a similar final surface area. This severe contraction was independent of collagen and fibroblast concentrations. Models for the prediction of the final percentage contraction of dermal and skin equivalents as a function of the initial concentration of collagen, the logarithm of fibroblast concentration, and the initial gel thickness were obtained and analyzed. Keratinocytes grown at the lowest seeding density did not contract the equivalents. However, histologic analysis has shown an incomplete coverage by these cells of the equivalents. The extensive contraction of the skin equivalent presenting adequate morphology is a major drawback toward its clinical utilization for burn wound coverage.
- PublicationRestreintRole and innocuity of Tisseel ®, a tissue glue, in the grafting process and in vivo evolution of human cultured epidermis(Churchill Livingstone, 2005-04-14) Germain, Lucie; Auger, François A.; López Valle, Carlos Antonio; Guignard, RinaCultured epidermal sheets are currently used for burn wound treatment but reported results on graft take are variable. This study was designed to evaluate the role and influence of Tisseel®, a fibrin glue, in the take of cultured human epidermal sheets in an athymic mouse model. On days 4, 10 and 21 post-grafting, histology, electron microscopy and immunofluorescence staining confirmed the presence of a human epithelium and the development of a basement membrane. Tisseel® was detectable on day 4 only, but overall treated and untreated grafts were similar. The use of Tisseel® enhanced the mechanical stability of these fragile grafts, increased the percentage of graft take, and its innocuity on thein vivo evolution of cultured epidermal sheets was demonstrated. For these reasons, we think that Tisseel® may be advantageous in a clinical setting.
- PublicationRestreintMorphological changes of human skin cells exposed to a DC electric field in vitro using a new exposure system(John Wiley & Sons, 2001-08-01) Plante, Michel; Germain, Lucie; Rancourt, Denis; Bourdages, Michel; Auger, François A.; Goulet, Daniel; Méthot, Stéphane; Moulin, VéroniqueThe human skin contains a physiological battery that could be implicated in the healing process, by creating an endogenous electric field. Skin cells undergo morphological changes in response to an external DC electric field (EF). We found that fibroblasts reorient their cell bodies in a manner perpendicular to the EF direction, for normal and above physiological intensities. Actin and tubulin filaments (cytoskeleton proteins) follow the same pattern of reorientation. Keratinocytes tend to elongate in the same direction, although to a lesser extent. The study of the response of human skin cells to an external EF is a first step toward a better understanding of the mechanisms involved in wound healing and eventually toward the improvement of wound repair.