Personne : Germain, Lucie
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Université Laval. Département de 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.
- PublicationRestreintPromotion of growth and differentiation of rat ductular oval cells in primary culture(Waverly Press, 1988-01-15) Germain, Lucie; Noël, Micheline; Marceau, Normand; Gourdeau, HenrietteOval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to alpha-fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45:673-681, 1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and polyclonal antibodies directed against cytokeratins of Mr 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS7) and normal hepatocytes (HES6) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES6 antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. The procedure yielded about 8 x 10(7) cells, of which 95% expressed CK39, CK52, and BDS7, 84% gamma-glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [3H]thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 mM pyruvate, 0.2 mM aspartate, 0.2 mM serine, 1 mM tyrosine, 1 mM proline, 1 mM phenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 microM bilirubin, 10 ng/ml cholera toxin, 1 microM dexamethasone, 2.5 micrograms/ml insulin, 50 mM beta-mercaptoethanol, and 5 micrograms/ml transferrin (medium MX)], the labeling index increased to around 30% and the level of cell fusion greatly decreased. The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor.
- PublicationAccès libreDes Canadiens « précurseurs de la recherche sur les cellules souches hématopoïétiques » lauréats du prix Lasker(EDK, 2006-02-15) Germain, Lucie; Larouche, Danielle; Paquet, ClaudieÀ la fin de l'année 2005, le prestigieux prix Lasker pour la recherche médicale fondamentale a été décerné à deux scientifiques canadiens, les Docteurs Ernest A. McCulloch et James E. Till, qui se sont illustrés par leurs travaux portant notamment sur la capacité des cellules souches à s’auto-renouveler et à se différencier en cellules pouvant présenter de multiples phénotypes.
- 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.
- PublicationRestreintTransplantation of a tissue-engineered corneal endothelium reconstructed on a devitalized carrier in the feline model(Association for Research in Vision and Ophthalmology, 2009-01-17) Germain, Lucie; Bensaoula, Thouria; Audet, Caroline; Nada, Ossama; Uwamaliya, Jeanne d'Arc; Proulx, Stéphanie; Devaux, Angèle; Allaire, Guy; Brunette, Isabellepurpose. To evaluate the functional outcome of tissue-engineered corneal endothelium reconstructed on a devitalized carrier and transplanted in the living feline model. methods. Eighteen healthy adult cats underwent full-thickness corneal transplantation. In 11 animals, the donor cornea was reconstructed from cultured allogeneic feline corneal endothelial cells seeded on the denuded Descemet’s membrane of a devitalized human cornea. The reconstructed corneal endothelium was cultured for 2 weeks before transplantation. Five control animals received autologous (n = 1), allogeneic (n = 3), or human xenogeneic (n = 1) native cornea. Two other control animals were grafted with the devitalized carrier only (no cells). Animals were observed daily by slit lamp until euthanatization on day 7. Postmortem analysis included optical coherence tomography (OCT), alizarin red staining, histology, fluorescence microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). results. Nine of the 11 reconstructed corneal endothelial grafts and all five native (autologous, allogeneic, xenogeneic) control grafts were clear and thin 7 days after grafting. In contrast, the two control grafts consisting of the carrier only (without endothelium) remained thick and opaque. Alizarin red staining, histology, SEM, and TEM showed that the transplanted reconstructed endothelium maintained a normal morphology and ultrastructure and expressed the function-related proteins Na+/K+-ATPase α1, Na+/HCO3, and ZO-1. conclusions. This study provides evidence for the short-term (7-day) anatomic and functional success of corneal transplantation with a tissue-engineered corneal endothelium reconstructed on a devitalized carrier.
- PublicationRestreintTGF-beta receptor expression on human keratinocytes : a 150 kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with type I and type II receptors(Wiley, 1998-09-15) Tam, Betty Y. Y.; Germain, Lucie; Philip, AnieKeratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-β has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-β receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-β receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-β1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-β receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-β1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-β receptors on keratinocytes, supporting the heterotetrameric model of TGF-β signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-β by markedly downregulating all four TGF-β binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with the TGF-β signalling receptors suggests that this GPI-anchored protein may modify TGF-β signalling in human keratinocytes.