Personne : Pouliot, Roxane
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Pouliot
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Roxane
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Université Laval. Faculté de pharmacie
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- PublicationRestreintReconstructed human skin produced in vitro and grafted on athymic mice(Ovid, 2002-06-15) Li, Hui; Germain, Lucie; Xu, Wen; Larouche, Danielle; Juhász, Julianna; Auger, François A.; Pouliot, RoxaneBackground. The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. Methods. The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. Results. After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. Conclusions. This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.
- PublicationAccès libreUsing infrared and raman microspectroscopies to compare ex vivo involved psoriatic skin with normal human skin(SPIE, 2015-06-17) Lefèvre, Thierry; Auger, Michèle; Laroche, Gaétan; Leroy, Marie; Pouliot, RoxanePsoriasis is a chronic dermatosis that affects around 3% of the world’s population. The etiology of this autoimmune pathology is not completely understood. The barrier function of psoriatic skin is known to be strongly altered, but the structural modifications at the origin of this dysfunction are not clear. To develop strategies to reduce symptoms of psoriasis or adequate substitutes for modeling, a deep understanding of the organization of psoriatic skin at a molecular level is required. Infrared and Raman microspectroscopies have been used to obtain direct molecular-level information on psoriatic and healthy human skin biopsies. From the intensities and positions of specific vibrational bands, the lipid and protein distribution and the lipid order have been mapped in the different layers of the skin. Results showed a similar distribution of lipids and collagen for normal and psoriatic human skin. However, psoriatic skin is characterized by heterogeneity in lipid/protein composition at the micrometer scale, a reduction in the definition of skin layer boundaries and a decrease in lipid chain order in the stratum corneum as compared to normal skin. A global decrease of the structural organization is exhibited in psoriatic skin that is compatible with an alteration of its barrier properties.
- PublicationRestreintHuman epithelial stem cells persist within tissue-engineered skin produced by the self-assembly approach(Mary Ann Liebert, 2013-02-15) Germain, Lucie; Goyer, Benjamin; Larouche, Danielle; Kim, Dong Hyun; Paquet, Claudie; Fugère, Claudia.; Dunnwald, Martine; Robitaille, Hubert; Sauvé, Sarah; Desgagné, Maxime; Fradette, Julie; Lavoie, Amélie; Beauparlant, Annie.; Pouliot, RoxaneTo adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TESs) must ultimately contain and preserve functional epithelial stem cells (SCs). It is therefore essential that a maximum of SCs be preserved during each in vitro step leading to the production of TESs such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial SCs within the human TESs made by the self-assembly approach, slow-cycling cells were identified using 5′-bromo-2′-deoxyuridine (BrdU) in the three-dimensional construct. A subset of basal epithelial cells retained the BrdU label and was positive for the SC-associated marker keratin 19 within TESs after a chase of 21 days in culture post-BrdU labeling. Moreover, keratinocytes harvested from TESs gave rise to SC-like colonies in secondary monolayer subcultures, indicating that SCs were preserved within TESs. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on SCs, human epithelial cells were extracted from skin samples, amplified in culture, and used to produce TESs, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial SCs in monolayer culture and in TESs remained constant before and after cryopreservation. Further, the functionality of these substitutes was demonstrated by successfully grafting human TESs on athymic mice for 6 months. We conclude that human epithelial skin SCs are adequately preserved upon human tissue reconstruction. Thus, these TESs produced by the self-assembly approach are suitable for clinical applications.
- PublicationRestreintA microfluidic approach to micromembrane synthesis for complex release profiles of nanocarriers(Royal Society of Chemistry, 2020-02-14) Nan, Jia; Greener, Jesse; Rosella, Erica; Kleitz, Freddy; Juère, Estelle; Pouliot, RoxanePhysically crosslinked microscale biomembranes synthesized from pure chitosan are designed and demonstrated for pH-triggered release of embedded functionalized mesoporous silica nanoparticles. Nanoparticle-loaded membranes are formed in a microfluidic channel at the junction between accurately controlled co-flowing streams to achieve highly tuneable membrane properties. After formation, the loaded membranes remain stable until contact with physiological acidic conditions, resulting in controlled nanoparticle release. Furthermore, nanoparticle-loaded membranes with complex layered architectures are synthesized using different flow schemes, thus enabling customized nanoparticle release profiles. These novel materials are well-suited for integration within small medical devices as well as off-chip applications.
- PublicationAccès libreA comparative study between human skin substitutes and normal human skin using Raman microspectroscopy(Minerals, Metals and Materials Society, 2014-02-12) Labbé, Jean-François; Jean, Jessica; Auger, Michèle; Ouellet, Marise; Laroche, Gaétan; Leroy, Marie; Pouliot, Roxane; Lefèvre, ThierryResearch in the field of bioengineered skin substitutes is motivated by the need to find new dressings for people affected by skin injuries (burns, diabetic ulcers), and to develop adequate skin models to test new formulations developed in vitro. Thanks to advances in tissue engineering, it is now possible to produce human skin substitutes without any exogenous material, using the self-assembly method developed by the Laboratoire d’Organogénèse Expérimentale. These human skin substitutes consist of a dermis and a stratified epidermis (stratum corneum and living epidermis). Raman microspectroscopy has been used to characterize and compare the molecular organization of skin substitutes with normal human skin. Our results confirm that the stratum corneum is a layer rich in lipids which are well ordered (trans conformers) in both substitutes and normal human skin. The amount of lipids decreases and more gauche conformers appear in the living epidermis in both cases. However, the results also show that there are fewer lipids in the substitutes and that the lipids are more organized in the normal human skin. Concerning the secondary structure of proteins and protein content, the data show that they are similar in the substitutes and in the normal human skin. In fact, the epidermis is rich in α-keratin, whereas the dermis contains mainly type I collagen.
- PublicationRestreintPhysical characterization of the stratum corneum of an in vitro human skin equivalent produced by tissue engineering and its comparison with normal human skin by ATR-FTIR spectroscopy and thermal analysis (DSC)(ScienceDirect, 1999-08-18) Germain, Lucie; Juhász, Julianna; Auger, François A.; Tremblay, Nathalie; Pouliot, RoxaneAn in vitro human skin equivalent may be obtained by culturing human keratinocytes on a collagen gel containing fibroblasts. The anchored skin equivalent cultured at the air-liquid interface closely resembles human skin and is acceptable for in vitro percutaneous absorption. However, it is still more permeable than human skin. Since intercellular lipids have been recognized to play an important role in skin permeability, infrared spectroscopy and differential scanning calorimetry were performed on the stratum corneum of bovine or human skin equivalents grown at different days of air-liquid culture. The symmetric and asymmetric CH(2) stretching vibrations suggested that for all days observed, the intercellular lipids were less organized than those in human skin, irrespective of whether bovine or human collagen was used. Different culture conditions were also tested and the medium without serum and no epidermal growth factor at the air-liquid culture showed results significantly more comparable to human skin. Actually, the thermal behavior of in vitro stratum corneum showed transitions at lower temperatures than human skin. The transition around 80 degrees C, in the form of a lipid-protein complex, was absent. These results showed that the structural arrangement of intercellular lipids and their thermodynamic properties hold a crucial role in the barrier function of the stratum corneum.
- PublicationRestreintMultistep production of bioengineered skin substitutes : sequential modulation of culture conditions.(2000-02-01) Germain, Lucie; Noël, Patricia; Juhász, Julianna; Auger, François A.; Guignard, Rina; Goulet, Francine; Tremblay, Nathalie; Pouliot, RoxaneMany studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboïdal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.
- PublicationRestreintHow to achieve early vascularization of tissue-engineered skin substitutes(Mary Ann Liebert, 2010-01-01) Germain, Lucie; Auger, François A.; Berthod, François; Pouliot, RoxaneBackground: The coverage of deep and extensive burns with autologous tissue-engineered skin is a promising strategy to improve the cosmetic aspect and functionality of the skin, compared to the transplantation of simple epithelial sheets. Indeed, a dermal compartment could markedly help to prevent hypertrophic scar formation and to strengthen the dermal–epidermal junction while increasing skin suppleness and pliability. The Problem: The thickness of the dermis could be a limitation to the survival of the tissue after transplantation, since its vascularization can take up to 2 weeks to occur through neovascularization. This delay could lead to graft necrosis. Basic/Clinical Science Advances: To overcome this problem, the reconstruction of a preformed network of branching capillaries in the dermis before grafting has proven to be an efficient solution in connecting to the host's vasculature in less than 4 days after grafting. The formation of this capillary-like network is achieved by the coculture of human fibroblasts and endothelial cells in a collagen sponge for a 1-month in vitro maturation period. The successful inosculation process between human capillaries and the host's vasculature was demonstrated after grafting onto nude mice. Clinical Care Relevance: In addition to autologous epithelial sheets and split-thickness autografts, this endothelialized reconstructed skin made of the patient's own cells could be a valuable additional strategy to permanently cover deep wounds. Conclusion: The reconstruction in tissue-engineered organs of a capillary-like network made of the patient's own cells before grafting is a promising approach to promote their early vascularization.
- PublicationAccès libreMarkers for an In vitro skin substitute(Artech House, 2018-02-01T16:41:46Z) Germain, Lucie; Jean, Jessica; Larouche, Danielle; Berthod, François; Pouliot, Roxane; Maguire, Tim; Novik, EricThe tissue engineering self-assembly approach allows the production of skin substitutes comprising both the dermis and epidermis, using methods promoting the secretion and organization of a dense extracellular matrix by skin cells. In a reconstructed epidermis, all cellular layers of the native tissue are present. An evaluation of the expression and localization of a number of specific protein markers revealed that the self-assembled, tissue-engineered skin substitute shares some common features with normal human skin, such as the expression of Ki-67, keratins 10 and 14, filaggrin, involucrin, transglutaminase, DLK, a3-integrin subunit, laminin-S, and collagens I, II, 1V, and VII. At the ultrastructural level, many differentiation markers can be observed, including desmosomes, as well as an organized basement membrane presenting hemidesmosomes, lamina densa, and lamina lucida. In this chapter, protocols to generate skin substitutes by the self-assembly approach will be presented and the methods including the labeling of the principal skin differentiation markers by immunofluorescence will be examined.
- PublicationRestreintCharacterization of a new tissue-engineered human skin equivalent with hair(springerLink, 1999-06-01) Michel, Martine; Germain, Lucie; Xu, Wen; L'Heureux, Nicolas; Auger, François A.; Pouliot, RoxaneWe designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.