Personne : Guillemette, Maxime.
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Département de physique, de génie physique et d'optique, Faculté des sciences et de génie, Université Laval
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- PublicationAccès libreRôle de la microcirculation et du microenvironnement sur la fonctionnalité de substituts vasculaires reconstruits par génie tissulaire(2009) Guillemette, Maxime.; Veres, Teodor; Auger, François A.Les cellules et la matrice extracellulaire composant les tissus humains ont une architecture tridimensionnelle organisée leur conférant des propriétés propres à leur physiologie. La plupart des substituts créés par génie tissulaire ne possèdent pas cette organisation architecturale leur permettant d'être physiologiquement comparable aux tissus à remplacer. Cela s'avère particulièrement crucial lors de la conception de substituts vasculaires où les propriétés mécaniques ont une importance primordiale afin de résister à la pression artérielle, mais aussi la compliance nécessaire pour ne pas induire une athérosclérose postimplantation due à une trop grande rigidité. De plus, les présents modèles vasculaires conçus par génie tissulaire n'intègrent pas la microcirculation dans les parois vasculaires. Les vasa vasorum, capillaires de l'adventice, servent à nourrir et oxygéner les parois des vaisseaux sanguins et ont probablement un rôle clé dans l'intégration dans les tissus avoisinants des substituts vasculaires. À ces fins, nous avons conçu un biomatériau pouvant être microstructuré afin de reproduire une topographie similaire à celle composant le micro environnement des tissus. Les polymères thermoplastiques permettent la production à grande échelle de substrats avec une nanotopographie de surface permettant l'alignement des cellules et de la matrice extracellulaire. Nous avons fait la démonstration que ces substrats permettent l'auto-assemblage tridimensionnel des cellules et de la matrice extracellulaire suivant un angle précis, lequel correspond à la même organisation physiologique intrinsèque du tissu et varie en fonction du type cellulaire en culture. Pour les cellules musculaires lisses vasculaires composant la média, nous avons montré que cette organisation se traduit par une augmentation de plus de 100% en résistance mécanique. D'autre part, une nouvelle technique de culture cellulaire pour les adventices vasculaires a permis de créer un réseau de capillaires in vitro s'apparentant au vasa vasorum. Ces substituts vasculaires ont été implantés en sous-cutané dans un modèle animal permettant de démontrer la rapidité d'intégration des tissus pré-vascularisés (48h) en comparaison avec des tissus non-vascularisés (141). Ces améliorations aux substituts vasculaires leurs confèrent une importante ressemblance anatomique et physiologique avec les vaisseaux utilisés notamment pour les pontages des artères soit coronaires ou périphériques, les rapprochant ainsi d'une utilisation en clinique.
- PublicationRestreintTissue-Engineered Vascular Adventitia with Vasa Vasorum Improves Graft Integration and Vascularization Through Inosculation(2010-05-05) Guillemette, Maxime.; Perron, Cindy; Germain, Lucie; Labbé, Raymond; Auger, François A.; Gauvin, RobertTissue-engineered blood vessel is one of the most promising living substitutes for coronary and peripheral artery bypass graft surgery. However, one of the main limitations in tissue engineering is vascularization of the construct before implantation. Such a vascularization could play an important role in graft perfusion and host integration of tissue-engineered vascular adventitia. Using our self-assembly approach, we developed a method to vascularize tissue-engineered blood vessel constructs by coculturing endothelial cells in a fibroblast-laden tissue sheet. After subcutaneous implantation, enhancement of graft integration within the surrounding environment was noted after 48 h and an important improvement in blood circulation of the grafted tissue at 1 week postimplantation. The distinctive branching structure of end arteries characterizing the in vivo adventitial vasa vasorum has also been observed in long-term postimplantation follow-up. After a 90-day implantation period, hybrid vessels containing human and mouse endothelial cells were still perfused. Characterization of the mechanical properties of both control and vascularized adventitia demonstrated that the ultimate tensile strength, modulus, and failure strain were in the same order of magnitude of a pig coronary artery. The addition of a vasa vasorum to the tissue-engineered adventitia did not influence the burst pressure of these constructs. Hence, the present results indicate a promising answer to the many challenges associated with the in vitro vascularization and in vivo integration of many different tissue-engineered substitutes
- PublicationAccès libreAlignment of cells and extracellular matrix within tissue-engineered substitutes(Intech, 2013-01-01) Guillemette, Maxime.; Germain, Lucie; Bourget, Jean-Michel; Veres, Teodor; Auger, François A.Most of the cells in our body are in direct contact with extracellular matrix (ECM) compo‐ nents which constitute a complex network of nano-scale proteins and glycosaminoglycans. Those cells constantly remodel the ECM by different processes. They build it by secreting dif‐ ferent proteins such as collagen, proteoglycans, laminins or degrade it by producing factors such as matrix metalloproteinase (MMP). Cells interact with the ECM via specific receptors, the integrins . They also organize this matrix, guided by different stimuli, to generate pat‐ terns, essential for tissue and organ functions. Reciprocally, cells are guided by the ECM, they modify their morphology and phenotype depending on the protein types and organization via bidirectional integrin signaling [2-4]. In the growing field of tissue engineering , control of these aspects are of the utmost importance to create constructs that closely mimic native tis‐ sues. To do so, we must take into account the composition of the scaffold (synthetic, natural, biodegradable or not), its organization and the dimension of the structure. The particular alignment patterns of ECM and cells observed in tissues and organs such as the corneal stroma, vascular smooth muscle cells (SMCs), tendons, bones and skeletal mus‐ cles are crucial for organ function. SMCs express contraction proteins such as alpha-smoothmuscle (SM)-actin, desmin and myosin  that are essential for cell contraction . To result in vessel contraction, the cells and ECM need to be organized in such a way that most cells are elongated in the same axis. For tubular vascular constructs, it is suitable that SMCs align in the circumferential direction, as they do in vivo [7, 8]. Another striking example of align‐ ment is skeletal muscle cells that form long polynuclear cells, all elongated in the same axis. Each cell generates a weak and short contraction pulse but collectively, it results in a strong, long and sustained contraction of the muscle and, in term, a displacement of the member. In the corneal stroma, the particular arrangement of the corneal fibroblasts (keratocytes) and ECM is essential to keep the transparency of this tissue [9-13]. Tendons also present a pecu‐ liar matrix alignment relative to the muscle axis. It gives a substantial resistance and excep‐ tional mechanical properties to the tissue in that axis [14, 15]. Intervertebral discs , cartilage , dental enamel , and basement membrane of epithelium are other examples of tissues/organs that present peculiar cell and matrix organization. By reproducing and controlling those alignment patterns within tissue-engineered substitutes, a more physiolog‐ ical representation of human tissues could be achieved. Taking into account the importance of cell microenvironment on the functionality of tissue engineered organ substitutes, one can assume the importance of being able to customise the 3D structure of the biomaterial or scaffold supporting cell growth. To do so, some methods have been developed and most of them rely on topographic or contact guidance. This is the phenomenon by which cells elongate and migrate in the same axis as the ECM. Topographic guidance was so termed by Curtis and Clark  to include cell shape, orientation and movement in the concept of contact guidance described by Harrison  and implemented by Weiss [21, 22]. Therefore, if one can achieve ECM alignment, cells will follow the same pattern. Inversely, if cells are aligned on a patterned culture plate, the end result would be aligned ECM deposition . The specific property of tissues or materials that present a variation in their mechanical and structural properties in different axis is called anisotropy. This property can be evaluated ei‐ ther by birefringence measurements [24, 25], mechanical testing in different axis , immu‐ nological staining of collagen or actin filaments  or direct visualisation of collagen fibrils using their self-fluorescence around 488 nm [27, 28]. Several techniques have been recently developed to mimic the specific alignment of cells within tissues to produce more physiologically relevant constructs. In this chapter, we will describe five different techniques, collagen gel compaction, electromagnetic field, electro‐ spinning of nanofibers, mechanical stimulation and microstructured culture plates.
- PublicationRestreintMicrostructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication(John Wiley & Sons, Inc, 2016-04-28) Guillemette, Maxime.; Tondreau, Maxime; Laterreur, Véronique; Germain, Lucie; Miville-Godin, Caroline; Ruel, Jean; Mounier, Maxence; Tremblay, Catherine; Labbé, Raymond; Bourget, Jean-Michel; Veres, Teodor; Auger, François A.; Gauvin, RobertIn the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs.
- PublicationRestreintTissue engineering of human cornea(CRC Press, 2014-03-27) Guillemette, Maxime.; Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Auger, François A.; Gaudreault, Manon.; Proulx, Stéphanie; Carrier, Patrick; Chirila, TraianThe cornea is a well-organized tissue composed of three cell types (epithelial, stromal and endothelial cells), each having an important role for its functionality. This chapter will address different tissue engineering approaches to the reconstruction of either partial or full-thickness living corneal substitutes that can be used either as in vitro models for woundhealing studies, or in vivo, eventually replacing the donor cornea for transplantation in humans. Isolation of the proper cells, followed by appropriate culture conditions, and assembly into a three-dimensional tissue construct, are the first steps required for producing a functional corneal substitute.
- PublicationRestreintSurface topography induces 3D self-orientation of cells and extracellular matrix resulting in improved tissue function(RSC Pub., 2009-01-15) Guillemette, Maxime.; Cui, Bo; Germain, Lucie; Roy, Emmanuel; Veres, Teodor; Auger, François A.; Giasson, Claude J.; Gauvin, Robert; Esch, Mandy B.; Carrier, Patrick; Deschambeault, Alexandre; Dumoulin, Michel; Toner, MehmetThe organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same non-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology.
- PublicationAccès libreRecent advances in the development of tissue-engineered vascular media made by self-assembly(Elsevier, 2013-06-05) Guillemette, Maxime.; Laterreur, Véronique; Germain, Lucie; Ruel, Jean; Miville Godin, Caroline; Bourget, Jean-Michel; Mounier, Maxence; Veres, Teodor; Auger, François A.; Gauvin, RobertThere is a lack of an optimal transplant material for small calibre blood vessels. This could be overcome by tissue engineering. The optimal construct is to be derived from autologous cells and present mechanical resistance comparable to the gold standard, autologous vessels such as the internal mammary artery or the saphenous vein. Our laboratory has developed the self-assembly approach to produce tissue sheets that can be rolled into such vessel substitutes. Over the years, many improvements have been made to the technique to facilitate smooth muscle cell culture and to produce vascular media substitutes with higher circumferential mechanical resistance.
- PublicationRestreintMechanical properties of tissue-engineered vascular constructs produced using arterial or venous cells(Mary Ann Liebert, Inc. Publishers, 2011-04-02) Guillemette, Maxime.; Germain, Lucie; Galbraith, Todd; Larouche, Danielle; Aubé, David; Marcoux, Hugo; Hayward, Cindy Jean; Bourget, Jean-Michel; Auger, François A.; Gauvin, RobertThere is a clinical need for better blood vessel substitutes, as current surgical procedures are limited by the availability of suitable autologous vessels and suboptimal behavior of synthetic grafts in small caliber arterial graft ( < 5 mm) applications. The aim of the present study was to compare the mechanical properties of arterial and venous tissue-engineered vascular constructs produced by the self-assembly approach using cells extracted from either the artery or vein harvested from the same human umbilical cord. The production of a vascular construct comprised of a media and an adventitia (TEVMA) was achieved by rolling a continuous tissue sheet containing both smooth muscle cells and adventitial fibroblasts grown contiguously in the same tissue culture plate. Histology and immunofluorescence staining were used to evaluate the structure and composition of the extracellular matrix of the vascular constructs. The mechanical strength was assessed by uniaxial tensile testing, whereas viscoelastic behavior was evaluated by stepwise stress-relaxation and by cyclic loading hysteresis analysis. Tensile testing showed that the use of arterial cells resulted in stronger and stiffer constructs when compared with those produced using venous cells. Moreover, cyclic loading demonstrated that constructs produced using arterial cells were able to bear higher loads for the same amount of strain when compared with venous constructs. These results indicate that cells isolated from umbilical cord can be used to produce vascular constructs. Arterial constructs possessed superior mechanical properties when compared with venous constructs produced using cells isolated from the same human donor. This study highlights the fact that smooth muscle cells and fibroblasts originating from different cell sources can potentially lead to distinct tissue properties when used in tissue engineering applications.