Personne : Gauvin, Robert
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Université Laval. Faculté de médecine
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- PublicationAccès libreOptimisation des substituts cutanés et vasculaires reconstruits par génie tissulaire : étude du comportement biomécanique(2010) Gauvin, Robert; Germain, LucieLes propriétés biomécaniques des tissus produits par génie tissulaire sont une des caractéristiques qui permet d'évaluer leur fonctionnalité. Ces propriétés peuvent dépendre, entre autres, de la méthode de fabrication utilisée lors de l'assemblage des tissus, du type de cellules utilisé lors de la reconstruction des tissus et des facteurs environnementaux cellulaires et tissulaires en présence lors de la culture des tissus. Dans le but de produire des tissus possédant des propriétés optimales pour d'éventuelles applications cliniques, il est important d'analyser et de comprendre le comportement biomécanique des tissus et de développer des méthodes de fabrication qui permettent de les produire de façon fiable et reproductible. L'objectif principal de cette thèse de doctorat était d'optimiser la fabrication des substituts vasculaires et cutanés reconstruits par la méthode d'auto-assemblage, par la compréhension de leur comportement biomécanique. Les travaux ont d'abord été dirigés vers le développement d'une nouvelle méthode de fabrication qui a permis d'augmenter l'efficacité de la production des substituts vasculaires et d'améliorer leurs propriétés mécaniques et viscoélastiques. Par la suite, des travaux ont été réalisés dans le but d'optimiser la production des substituts cutanés. Les résultats obtenus ont démontré qu'il était possible, par de nouvelles méthodes d'assemblage, d'augmenter la surface des substituts cutanés sans influencer leur structure ni leur contractilité. Un autre objectif poursuivi était de déterminer si l'utilisation de diverses sources cellulaires pouvait avoir une incidence sur les caractéristiques structurales et biomécaniques des substituts vasculaires. Ces travaux ont montré que les cellules artérielles et veineuses permettaient de produire des substituts vasculaires adéquats et que ceux produits à partir de cellules artérielles possédaient des propriétés mécaniques et élastiques supérieures à celles de tissus produits à partir de cellules veineuses. Dans le but de poursuivre l'optimisation de la fonctionnalité et des propriétés biomécaniques des tissus reconstruits, des méthodes permettant de stimuler mécaniquement les tissus pendant leur culture ont été développées. L'étude de la structure et des propriétés mécaniques des tissus résultant de cette culture dynamique a démontré une orientation axiale des composantes tissulaires, en plus d'une augmentation de la production des éléments de la matrice extracellulaire et d'une amélioration des propriétés mécaniques des tissus. En conclusion, le développement de ces nouvelles méthodes de fabrication a permis d'optimiser et de mieux définir certaines propriétés biomécaniques des substituts vasculaires et cutanés et a ainsi contribué à en améliorer leur fonctionnalité pour d'éventuelles applications cliniques.
- PublicationRestreintHarvesting the potential of the human umbilical cord : isolation and characterisation of four cell types for tissue engineering applications(S. Karger AG, 2012-09-05) Germain, Lucie; Galbraith, Todd; Hayward, Cindy Jean; Auger, François A.; Gauvin, Robert; Guignard, Rina; Rémy-Zolghadri, Murielle; Fradette, JulieThe human umbilical cord (UC) has attracted interest as a source of cells for many research applications. UC solid tissues contain four cell types: epithelial, stromal, smooth muscle and endothelial cells. We have developed a unique protocol for the sequential extraction of all four cell types from a single UC, allowing tissue reconstruction using multiple cell types from the same source. By combining perfusion, immersion and explant techniques, all four cell types have been successfully expanded in monolayer cultures. We have also characterised epithelial and Wharton’s jelly cells (WJC) by immunolabelling of specific proteins. Epithelial cell yields averaged at 2.3 × 105 cells per centimetre UC, and the cells expressed an unusual combination of keratins typical of simple, mucous and stratified epithelia. Stromal cells in the Wharton’s jelly expressed desmin, α-smooth muscle actin, elastin, keratins (K12, K16, K18 and K19), vimentin and collagens. Expression patterns in cultured cells resembled those found in situ except for basement membrane components and type III collagen. These stromal cells featured a sustained proliferation rate up to passage 12 after thawing. The mesenchymal stem cell (MSC) character of the WJC was confirmed by their expression of typical MSC surface markers and by adipogenic and osteogenic differentiation assays. To emphasise and demonstrate their potential for regenerative medicine, UC cell types were successfully used to produce human tissue-engineered constructs. Both bilayered stromal/epithelial and vascular substitutes were produced, establishing the versatility and importance of these cells for research and therapeutic applications.
- 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.
- PublicationRestreintA computer-controlled apparatus for the characterization of mechanical and viscoelastic properties of tissue-engineered vascular constructs(Springer, 2011-01-25) Germain, Lucie; Larouche, Danielle; Auger, François A.; Gauvin, Robert; Lévesque, PhilippeTissue-engineered blood vessels can be partly characterized by analyzing their mechanical properties using burst pressure testing, compliance measurement, creep and cyclic testing. Studying these parameters provides information on the capability of a fabrication method to produce tissue-engineered blood vessels (TEBV) and allow for the optimization of their resistance and viscoelastic properties. This study presents the design and fabrication of an apparatus allowing accurate and reliable measurements of the mechanical properties of tissue-engineered vascular constructs. A computer-controlled system was designed to monitor pressure and diameter variations of vascular constructs submitted to hydrostatic loading. The system was programmed to control the motorized portion of the setup and allow simultaneous data acquisition, analysis and real-time display. Data acquisition cards allow for synchronous monitoring of pressure and diameter of the constructs through a pressure transducer and a CCD camera. Image analysis and pressure data computation resulted in compliance, creep and dynamic characterization of the tested tissues. This experimental setup succeeded in measuring the burst pressure, compliance, creep and cyclic behavior of tissue-engineered vascular media (TEVM), adventitia (TEVA) and a combination of a media and an adventitia (TEVMA) reconstructed by the self-assembly method. Our apparatus has proven to be a precise and reliable tool for the characterization of the mechanical properties of vascular constructs.
- 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.
- PublicationRestreintRestoration of the transepithelial potential within tissue-engineered human skin in vitro and during the wound healing process in vivo(Mary Ann Liebert, 2010-08-05) Plante, Michel; Germain, Lucie; Roberge, Charles; Auger, François A.; Gauvin, Robert; Dubé, Jean; Goulet, Daniel; Rochette-Drouin, Olivier; Bourdages, Michel; Lévesque, Philippe; Moulin, VéroniqueNormal human epidermis possesses a transepithelial potential (TEP) that varies in different parts of the body (10–60 mV). The role of TEP in normal epidermis is not yet identified; but after skin injury, TEP disruption induces an endogenous direct current electric field (100–200 mV/mm) directed toward the middle of the wound. This endogenous electric field could be implicated in the wound healing process by attracting cells, thus facilitating reepithelialization. However, little is known on the restoration of the TEP during human skin formation and wound healing. In this study, the variations in TEP and Na+/K+ ATPase pump expression during the formation of the epithelium were investigated in vitro using human tissue-engineered human skin (TES) reconstituted by tissue engineering and in vivo with a porcine wound healing model. Results showed that TEP undergoes ascending and decreasing phases during epithelium formation in TES as well as during wound repair within TES. Similar results were observed during in vivo reepithelialization of wounds. The ascending and decreasing TEP values were correlated with changes in the expression of Na+/K+ ATPase pump. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Taken together, these results suggest that the variations in the expression of Na+/K+ ATPase pump over time and across epidermis would be a determinant parameter of the TEP, dictating a cationic transport during the formation and restoration of the epidermis. Therefore, this study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process. This might have important future medical applications regarding the treatment of chronic wound healing.
- PublicationRestreintConsiderations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro(Butterworth-Heinemann, 2010-12-03) Germain, Lucie; Larouche, Danielle; Paquet, Claudie; Fugère, Claudia.; Genest, Hervé; Auger, François A.; Gauvin, Robert; Têtu, Félix-Andre; Bouchard, Maurice; Roy, Aphonse; Fradette, Julie; Lavoie, Amélie; Beauparlant, Annie.The treatment of severely burned patients has benefited from the grafting of skin substitutes obtained by expansion of epithelial cells in culture. The aim of this study was to evaluate whether the anatomic site chosen for harvesting skin had an impact on the quality of the derived cell cultures. Considering that hair follicles contain epithelial stem cells, we compared hairy skin sites featuring different densities and sizes of hair follicles for their capacity to generate high quality keratinocyte cultures. Three anatomic sites from adult subjects were compared: scalp, chest skin and p-auricular (comprising pre-auricular and post-auricular) skin. Keratin (K) 19 was used as a marker to evaluate the proportion of stem cells. Keratinocytes were isolated using the two-step thermolysin and trypsin cell extraction method, and cultured in vitro. The proportion of K19-positive cells harvested from p-auricular skin was about twice that of the scalp. This K19-positive cell content also remained higher during the first subcultures. In contrast to these in vitro results, the number of K19-positive cells estimated in situ on skin sections was about double in scalp as in p-auricular skin. Chest skin had the lowest number of K19-positive cells. These results indicate that in addition to the choice of an adult anatomic site featuring a high number of stem cells in situ, the quality of the cultures greatly depends on the ability to extract stem cells from the skin biopsy
- PublicationAccès libreElectric potential across epidermis and its role during wound healing can be studied by using an in vitro reconstructed human skin(S-N Publications, 2012-02-28) Plante, Michel; Germain, Lucie; Roberge, Charles; Auger, François A.; Gauvin, Robert; Dubé, Jean; Rochette-Drouin, Olivier; Goulet, Daniel; Lévesque, Philippe; Bourdages, Michel; Moulin, VéroniqueBackground : After human epidermis wounding, transepithelial potential (TEP) present in nonlesional epidermis decreases and induces an endogenous direct current epithelial electric field (EEF) that could be implicated in the wound re-epithelialization. Some studies suggest that exogenous electric stimulation of wounds can stimulate healing, although the mechanisms remain to be determined. The Problem : Little is known concerning the exact action of the EEF during healing. The mechanism responsible for TEP and EEF is unknown due to the lack of an in vitro model to study this phenomenon. Basic Science Advances : We carried out studies by using a wound created in a human tissue-engineered skin and determined that TEP undergoes ascending and decreasing phases during the epithelium formation. The in vitro TEP measurements over time in the wound were corroborated with histological changes and with in vivo TEP variations during porcine skin wound healing. The expression of a crucial element implicated in Na+ transport, Na+/K+ ATPase pumps, was also evaluated at the same time points during the re-epithelialization process. The ascending and decreasing TEP values were correlated with changes in the expression of these pumps. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Further, inhibition of the pump activity induced a significant decrease of the TEP and of the re-epithelization rate. Clinical Care Relevance : A better comprehension of the role of EEF could have important future medical applications regarding the treatment of chronic wound healing. Conclusion : This study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process.
- PublicationRestreintMinimal contraction for tissue-engineered skin substitutes when matured at the air–liquid interface(John Wiley & Sons, 2013-06-03) Germain, Lucie; Larouche, Danielle; Auger, François A.; Marcoux, Hugo-Bastien; Gauvin, Robert; Guignard, RinaThe structural stability of skin substitutes is critical to avoid aesthetic and functional problems after grafting, such as contractures and hypertrophic scars. The present study was designed to assess the production steps having an influence on the contractile behaviour of the tissue-engineered skin made by the self-assembly approach, where keratinocytes are cultured on tissue-engineered dermis comprised of fibroblasts and the endogenous extracellular matrix they organized. Thus, different aspects were investigated, such as the assembly method of the engineered dermis (various sizes and anchoring designs) and the impact of epithelial cell differentiation (culture submerged in the medium or at the air–liquid interface). To evaluate the structural stability at the end of the production, the substitutes were detached from their anchorages and deposited on a soft substrate, and contraction was monitored over 1 week. Collected data were analysed using a mathematical model to characterize contraction. We observed that the presence of a differentiated epidermis significantly reduced the amount of contraction experienced by the engineered tissues, independently of the assembly method used for their production. When the epidermis was terminally differentiated, the average contraction was only 24 4% and most of the contraction occurred within the first 12 h following deposition on the substrate. This is 2.2-fold less compared to when the epidermis was cultured under the submerged condition, or when tissue-engineered dermis was not overlaid with epithelial cells. This study highlights that the maturation at the air–liquid interface is a critical step in the reconstruction of a tissue engineered skin that possesses high structural stability
- PublicationAccès libreIn vivo remodeling of fibroblast-derived vascular scaffolds implanted for 6 months in rats(Hindawi, 2016-11-24) Tondreau, Maxime; Laterreur, Véronique; Germain, Lucie; Vallières, Karine; Ruel, Jean; Tremblay, Catherine; Bourget, Jean-Michel; Auger, François A.; Gauvin, Robert; Lacroix, Dan.There is a clinical need for tissue-engineered small-diameter (<6 mm) vascular grafts since clinical applications are halted by the limited suitability of autologous or synthetic grafts. This study uses the self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold (FDVS) that can be available off-the-shelf. Briefly, extracellular matrix 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 decellularized by immersion in deionized water. The FDVSs were implanted as an aortic interpositional graft in six Sprague-Dawley rats for 6 months. Five out of the six implants were still patent 6 months after the surgery. Histological analysis showed the infiltration of cells on both abluminal and luminal sides, and immunofluorescence analysis suggested the formation of neomedia comprised of smooth muscle cells and lined underneath with an endothelium. Furthermore, to verify the feasibility of producing tissue-engineered blood vessels of clinically relevant length and diameter, scaffolds with a 4.6 mm inner diameter and 17 cm in length were fabricated with success and stored for an extended period of time, while maintaining suitable properties following the storage period. This novel demonstration of the potential of the FDVS could accelerate the clinical availability of tissue-engineered blood vessels and warrants further preclinical studies.
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