Personne : Grenier, Guillaume.
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Département de chirurgie, Université Laval
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- PublicationRestreintRecent optimization of a tissue engineered blood vessel : the LOEX experience(Excerpta Medica Foundation, 2004-05-01) Grenier, Guillaume.; Germain, Lucie; Auger, François A.; Rémy-Zolghadri, Murielle; Laflamme, KarinaCreating a blood vessel by tissue engineering is one of the most demanding goals in tissue engineering. Our laboratory developed, using the self-assembly approach, the first completely biological tissue engineered blood vessel (TEBV) constituted of living human cells in the absence of any synthetic or exogenous material. The phenotypic and functional variations of smooth muscle cell (SMC) are of paramount importance in TEBV reconstruction. Thus, the phenotype and extracellular matrix (ECM) production of SMC were studied along the whole sequence of TEBV production. The functional and mechanical properties can be greatly enhanced by active cell orientation in the ECM. Accordingly, the method of preparing living tissue engineered sheets was modified to obtain an optimal alignment of SMC before rolling them into a tubular form. These results have allowed us to create a better TEBV.
- PublicationRestreintA truly new approach for tissue engineering : the LOEX self-assembly technique(SpringerLink, 2002-01-01) Grenier, Guillaume.; Germain, Lucie; Auger, François A.; Rémy-Zolghadri, MurielleTissue engineering has created several original and new avenues in the biomedical sciences. There is ongoing progress, but the tissue-engineering field is currently at a crossroads in its evolution; the validity of this technique is weIl established. Thus, new clinical applications must appear rapidly, within a few years, so that it will have a true impact on patient care. The self-assembly approach of the Laboratoire d'Organogénèse Expérimentale (LOEX) should be at the forefront.
- PublicationRestreintMechanical loading modulates the differentiation state of vascular smooth muscle cells(Mary Ann Liebert, 2006-11-24) Bergeron, François; Grenier, Guillaume.; Germain, Lucie; Labbé, Raymond; Auger, François A.; Guignard, Rina; Baker, Kathleen; Rémy-Zolghadri, MurielleThe cause underlying the onset of stenosis after vascular reconstruction is not well understood. In the present study, we evaluated the effect of mechanical unloading on the differentiation state of human vascular smooth muscle cells (hVSMCs) using a tissue-engineered vascular media (TEVM). hVSMCs cultured in a mechanically loaded three-dimensional environment, known as a living tissue sheet, had a higher differentiated state than cells grown on plastic. When the living tissue sheet was detached from its support, the release of the residual stress resulted in a mechanical unloading and cells within the extracellular matrix (ECM) dedifferentiated as shown by downregulation of differentiation markers. The relaxed living tissue sheet can be rolled onto a tubular mandrel to form a TEVM. The rolling procedure resulted in the reintroduction of a mechanical load leading to a cohesive compacted tissue. During this period, cells gradually redifferentiated and aligned circumferentially to the tubular support. Our results suggest that differentiation of hVSMCs can be driven by mechanical loading and may occur simultaneously in the absence of other cell types. The extrapolation of our results to the clinical context suggests the hypothesis that hVSMCs may adopt a proliferative phenotype resulting from the mechanical unloading of explanted blood vessels during vascular reconstruction. Therefore, we propose that this mechanical unloading may play an important role in the onset of vascular graft stenosis.
- PublicationRestreintAdventitia contribution in vascular tone : insights from adventitia-derived cells in a tissue-engineered human blood vessel(Federation of American Societies for Experimental Biology, 2006-04-12) Grenier, Guillaume.; Germain, Lucie; Pouliot, Stéphanie; Labbé, Raymond; Roberge, Charles; Auger, François A.; Baker, Kathleen; D’Orléans-Juste, Pedro; Rémy-Zolghadri, Murielle; Laflamme, KarinaWhether the adventitia component of blood vessels directly participates in the regulation of vascular tone remains to be demonstrated. We have recently developed a human tissue-engineered blood vessel comprising the three tunicae of a native blood vessel using the self-assembly approach. To investigate the role of the adventitia in the modulation of vascular tone, this tissue-engineering method was used to produce three vascular constructs from cells explanted and proliferated from donor vessel tunicae 1) an adventitia + a media, or only 2) an adventitia, or 3) a media. The vasoconstriction responses of these 3 constructs to endothelin, the most potent vasopressor known up-to-date, as well as to nonselective and selective agonists and antagonists, were compared. The adventitia contracted to endothelin-1, -2, whereas the media and the media+adventitia contracted to all three endothelins. Endothelin-induced contraction of the adventitia was dependent on ETA receptors, whereas that of the media and the adventitia+media was ETA and ETB receptor-dependent. RT-PCR studies corroborated these results. SNP induced a dose-dependent relaxation of the three tissue constructs. We also demonstrated that the endothelin-converting enzyme, responsible for the formation of the active endothelin peptides, was present and functional in the adventitia. In conclusion, this is the first direct demonstration that the adventitia has the capacity to contract and relax in response to vasoactive factors. The present study suggests that the adventitia of a blood vessel could play a greater role than expected in the modulation of blood vessel tone.—Laflamme, K., Roberge, C. J., Grenier, G., Rémy-Zolghadri, M., Pouliot, S., Baker, K., Labbé, R., D’Orléans-Juste, P., Auger, F. A., Germain, L. Adventitia contribution in vascular tone: insights from adventitia-derived cells in a tissue-engineered human blood vessel. the wall of a blood vessel is composed of three tunicae: intima, media, and adventitia (1)⤻ . The innermost tunica, known as the intima, includes a single layer of endothelial cells lining the vessel lumen and the internal elastic lamina membrane. The middle tunica, termed media, is mainly composed of vascular smooth muscle cells (VSMCs) in an extracellular matrix (ECM) and corresponds to the muscular portion of the blood vessel, whereas the tunica adventitia is mainly composed of vascular fibroblasts (VFs) and ECM. It is well accepted that the media of a blood vessel is responsible for the vasomotor tone control by contracting and relaxing in response to different hormonal factors released, for example, by the endothelial cells of the intima (2)⤻ . The adventitia, on the other hand, has long been thought to mainly serve as a structural support for the media, its main contribution to vascular compliance being controlled by autonomous perivascular innervation (1)⤻ . Interestingly, recent studies suggest that the adventitia influences vascular function (3⤻ 4⤻ 5⤻ 6⤻ 7)⤻ . Nonetheless, whether the adventitia can directly participate in the regulation of vasomotor tone of blood vessels still remains to be demonstrated. The lack of appropriate technical procedures to separate the adventitia tunica from the other components of a native blood vessel (stripping) has prevented direct investigations on the possible role of that layer in the regulation of vasomotor tone. For example, the stripping method used in these procedures can result in the injury of the media tunica and does not permit us to obtain functional adventitia isolated from a native blood vessel (6)⤻ . We have recently developed, using the self-assembly technique, a human tissue-engineered blood vessel (TEBV) composed of the layers representing the three tunicae found in a native blood vessel (8)⤻ . In the present study, we took advantage of the self-assembly method to produce three independent vascular constructs from amplified VSMCs and VFs isolated from the same human saphenous vein biopsy. The first vascular construct was composed of only an adventitia (TEVA), a second vascular construct contained only a media (TEVM), and the third contained a media and an adventitia (TEVMA). These three vascular models (TEVA, TEVM, and TEVMA) were reconstructed to investigate the role of the adventitia in the modulation of vascular tone by comparing each of these vascular construct responses to endothelin, the most powerful vasopressor agent known to date (9)⤻ . Studies in humans have demonstrated the importance of endothelin in the maintenance of vascular tone (10)⤻ and blood pressure (11)⤻ . Three endogenous isoforms of endothelin have been discovered, endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) (12)⤻ . ET binds two different receptor subtypes: endothelin A (ETA) receptors, which have a higher affinity for ET-1 and ET-2 than ET-3, and endothelin B (ETB) receptors, which have equal affinity for ET-1, ET-2, and ET-3 (13)⤻ . The endothelin receptors (ETA and ETB) implicated in the observed responses to the peptide were also investigated in our three different vascular constructs. In the present study, all of the vascular constructs tested responded to endothelin, although a heterogeneity in the response was observed. Indeed, all three vascular constructs tested contracted to ET-1 and ET-2, but only TEVMA and TEVM responded to ET-3. Furthermore, endothelin-induced contraction of TEVA was found to be dependent on the presence of ETA receptors, while both ETA and ETB receptors were present and functional on TEVMA and TEVM. Finally, the three types of vascular constructs tested also had the capacity of vasodilating in response to a relaxing agent such as sodium nitroprusside (SNP). Our results show that the adventitia may play a greater role than expected in the maintenance of vascular tone and compliance.
- 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.
- PublicationRestreintTissue reorganization in response to mechanical load increases functionality(2005-02-28) Bergeron, François; Langelier, Ève.; Grenier, Guillaume.; Germain, Lucie; Larouche, Danielle; Dupuis, Daniel; Rancourt, Denis; Auger, François A.; Gauvin, Robert; Baker, Kathleen; Rémy-Zolghadri, MurielleIn the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are recognized as being of the utmost importance. The present study was designed to evaluate the effects of static mechanical forces on the functionality of the produced tissue constructs. Living tissue sheets reconstructed by the self-assembly approach from human cells, without the addition of synthetic material or extracellular matrix (ECM), were subjected to mechanical load to induce cell and ECM alignment. In addition, the effects of alignment on the function of substitutes reconstructed from these living tissue sheets were evaluated. Our results show that tissue constructs made from living tissue sheets, in which fibroblasts and ECM were aligned, presented higher mechanical resistance. This was assessed by the modulus of elasticity and ultimate strength as compared with tissue constructs in which components were randomly oriented. Moreover, tissue-engineered vascular media made from a prealigned living tissue sheet, produced with smooth muscle cells, possessed greater contractile capacity compared with those produced from living tissue sheets that were not prealigned. These results show that the mechanical force generated by cells during tissue organization is an asset for tissue component alignment. Therefore, this work demonstrates a means to improve the functionality (mechanical and vasocontractile properties) of tissues reconstructed by tissue engineering by taking advantage of the biomechanical forces generated by cells under static strain.