Substitut cutané produit par génie tissulaire : aspects reliés à l'allogénicité, la pigmentation et l'élastine

Les brûlures profondes et étendues sont, de loin, le traumatisme le plus grave auquel un être humain peut faire face. La rapidité de couverture de ces plaies avec des pansements, surtout de nature biologique (p. ex. peau de cadavre), d’abord temporaire, et ensuite impérativement permanente, aura une influence déterminante sur le taux de survie des patients. À date, de nombreuses technologies ont été utilisées telles que les greffes pleines épaisseurs de peau provenant d’un site donneur sain, la culture d’épiderme autologue (CEA) et les substituts synthétiques. Cependant, ces technologies ont pour principale limite, respectivement, leur biodisponibilité, leur manque de propriété mécanique et leur manque d’activité biologique. Ces éléments ont ainsi mené au développement de substituts biologiquement actifs parmi lesquels les plus prometteurs possèderont une base autologue, l’épiderme, reposant sur une matrice, allogénique, xénogénique ou biosynthétique. Toutefois, la majorité de ces substituts ne pourra être utilisée que temporairement à cause de leur potentiel immunogénique résultant de la présence d’antigènes étrangers. Les technologies de production de substituts cutanés peuvent être divisées en deux approches distinctes, soit de type « Top-down », soit de type « Bottom-up ». La technologie la plus courante ainsi que la plus utilisée aujourd’hui est celle de type « Top-down ». Les substituts produits par cette approche se basent sur des matrices artificielles structurées élaborées via deux classes majeures de biomatériaux incluant (1) les biopolymères du sang comme la fibrine, et du tissu mou comme le collagène et la fibronectine ; (2) les polysaccharides, comme l’alginate, le chitosan et les glycosaminoglycanes, incluant le hyaluronan. L’approche de type « Bottom-up », quant à elle, se base sur la capacité des cellules à synthétiser et assembler leur propre matrice extracellulaire. Dans ce domaine, le laboratoire d’organogénèse expérimentale (LOEX) se positionne en tant que pionnier dans une méthode de production de substitut dite « d’auto-assemblage ». Cette méthode repose sur la capacité de l’acide ascorbique à promouvoir l’assemblage matriciel sécrété par les fibroblastes dermiques. Toutefois, les substituts obtenus par cette méthode présenteront (1) un temps de production long, car l’assemblage matriciel représentera à lui seul les 2/3 de la technique d’un total d’environ 8 à 9 semaines, (2) un manque de photoprotection de par l’absence de pigmentation, c’est à-dire sans ajout de cellules pigmentogènes (mélanocytes), et (3) peu ou pas de réseau de fibres élastiques, essentiel aux propriétés mécaniques cutanées. Afin de répondre aux problématiques identifiées, nous avons entrepris de concevoir plus rapidement des substituts cutanés pigmentés, intégrant un réseau de fibres élastiques selon la méthode d’autoassemblage. Pour ce faire, nous avons investigué la possibilité d’utiliser un derme reconstruit par « auto-assemblage » de type allogénique combiné à un épiderme syngénique. Cette modification au protocole qui implique initialement une reconstruction dermique et épidermique autologue à 100 % permettra d’éliminer l’étape de production dermique et ainsi réduire le temps global de production des substituts cutanés bilamellaires. L’esthétisme et la fonctionnalité des substituts ont été évalués par l’ajout de différentes densités de mélanocytes lors de la reconstruction de l’épiderme. Enfin, parallèlement à cette étude, nous avons investigué sur la faible présence du réseau de fibres élastiques dans nos substituts cutanés afin de proposer une technique de production modifiée permettant d’induire l’élastogenèse, le processus de formation de ce réseau. Plus spécifiquement, de l’aldostérone, une hormone minéralocorticoïde, et son inhibiteur compétitif, la spironolactone, ont été ajoutés dans le milieu de culture cellulaire en tout temps lors de la production des substituts cutanés afin de stimuler la voie de l’ « insulin-like growth factor I » (IGF-I) et ainsi l’élastogenèse. En résumé, mon projet de doctorat a permis de mettre en évidence : (1) la survie des fibroblastes allogéniques dermiques pour au moins huit semaines après greffes sur un modèle murin immunocompétent ; (2) qu’un seuil minimal de 200 mélanocytes par mm2 à la reconstruction de l’épiderme avait la capacité d’induire une pigmentation homogène après greffe, ainsi qu’une photoprotection comparable à une densité de 1500 mélanocytes par mm2 ; (3) que l’ajout d’aldostérone et de spironolactone augmente le nombre de fibres élastiques dans nos substituts cutanés et améliore les propriétés mécaniques par la diminution de la rétraction et l’augmentation de l’élasticité. De plus, l’ajout de mélanocytes a aussi permis d’améliorer les propriétés mécaniques des peaux reconstruites par leur effet positif encore mal compris sur l’élastogenèse.Vast deep burns injuries are, by far, the worst trauma that a human being can experience. The time necessary for the coverage of thermal wounds with bandages, especially of a biological nature (e.g. cadaveric skin), at first temporary, and then permanently, influences the survival rate of patients. Over the years, numerous technologies were used to cover the skin wounds. Among them, we note split- and full-thickness skin grafts from a spared donor site, as well as cultured epithelial autografts (CEAs) and synthetic substitutes. However, these technologies showed some issues as the bioavailability of donor tissue or mechanical properties or biological activities. Indeed, it is the cellular and the extracellular matrix component that have a direct impact on the wound healing and on the longterm graft survival rate. In this way, new biologically active skin substitutes were developed. The most promising reconstructed skins to date are composed of an autologous epidermis associated with an extracellular matrix either allogeneic or xenogeneic or biosynthetic. However, the majority of these substitutes could only be used as temporary because of their immune inflammatory risk resulting from the presence of allogeneic or xenogeneic cells. Tissue-engineered technologies can be split in two approaches, either "Top-Down" or "Bottom-up". The most current as well as the most represented technology today is the "Top-Down" approach. Substitutes produced by this approach are based on structured artificial matrices developed via the major classes of biomaterials including (1) blood’s biopolymers such as fibrin, and components of the soft tissue such as collagen or fibronectin and (2) polysaccharides, as alginates, chitosan and glycosaminoglycan, including the hyaluronan. Alternatively, the "bottom-up" approach is based on the fibroblast’s capacity to synthesize and assemble their own extracellular matrix. In this field, the Laboratory of Organogenesis Experimental (LOEX) is a pioneer in a method of production of skin substitute called the "self-assembly" method. This method is based on the capacity of ascorbic acid to promote the extracellular matrix assembly secreted by fibroblasts. However, the histology and mechanical properties of these tissue-engineered skin substitutes, which are similar to native skin, (1) are quite long to produce, essentially because of the extracellular matrix assembly which takes twothird of the time of the whole production (2) lack of photoprotection, since pigment-producing cells called melanocytes are not added in the standard production and (3) the elastic fiber network, essential to insure their mechanical properties, is rudimentary. To address the identified problems, we designed a faster way to produce pigmented skin substitutes, integrating an elastic fiber network by tissue engineering according to the “self-assembly” method. To do so, we investigated the possibility of using an allogeneic reconstructed dermis associated with a syngeneic epidermis. This new method would allow eliminating two-third of the production time of the dermal part initially requested in the “on demand” production that was initially 100 % autologous. Estheticism and its functionality were evaluated by the addition of various densities of melanocytes during the epidermis reconstruction. Finally, at the same time, we addressed the weak presence of the elastic fiber network in our reconstructed skin. Indeed, we modified our production method to stimulate the elastogenesis process. More specifically, aldosterone, a mineralocorticoid hormone, and its competitive inhibitor, spironolactone, were added in the culture media during the skin substitute production in order to stimulate the insulin-like growth factor I (IGF-I) pathway and so the elastogenesis process. To conclude, my PhD project allowed to highlight: (1) the immunological tolerance of the allogeneic dermis with a long-term survival of their cellular components over the eight-week period, investigated in an immunocompetent murine model ; (2) that a minimal threshold of 200 melanocytes per mm2 in the epidermis reconstruction had the capacity to lead a homogeneous pigmentation after the transplant, as well as a photoprotection comparable to a 1500 melanocytes per mm2 density ; (3) that the addition of aldosterone and spironolactone increased the number of elastic fibers in our skin substitutes and improved the mechanical properties by the decrease of the shrinkage and the increase of the elasticity. Furthermore, the addition of melanocyte also increased the mechanical properties of the skin substitute. This positive impact on elastogenesis by melanocytes is poorly understood

Biocompatibility and functionality of a tissue-engineered living corneal stroma transplanted in the feline eye

PURPOSE. Corneal tissue shortage has become a major concern worldwide, which has motivated the search for alternative solutions to eye bank human eyes for corneal transplantation. Minimally invasive lamellar transplantation and tissue engineering may offer new opportunities for the rehabilitation of diseased corneas. The aim of this study was to evaluate the biocompatibility and functionality of stromal lamellar grafts tissue-engineered (TE) in vitro and transplanted in vivo in the cornea of a feline model. METHODS. The corneal stromas were engineered in culture from corneal stromal cells using the self-assembly approach, without the addition of exogenous material or scaffold. Eight healthy animals underwent two intrastromal grafts in one eye and the contralateral eye was used as a control. Animals were followed with slit-lamp ophthalmic examination, corneal esthesiometry and optical coherent tomography. Confocal microscopy, immunofluorescence, histology, and transmission electron microscopy (TEM) were performed at 4 months. RESULTS. Four months after transplantation, the TE-stromal grafts were transparent, functional, and well tolerated by the eye. All grafts remained avascular, with no signs of immune rejection, despite a short course of low-dose topical steroids. Corneal sensitivity returned to preoperative level and reinnervation of the grafts was confirmed by confocal microscopy and immunofluorescence. Histology and TEM of the TE-grafts showed a lamellar stromal structure with regular collagen fibril arrangement. CONCLUSIONS. These results open the way to an entirely new therapeutic modality. Intracorneal filling using a biocompatible, transparent, and malleable TE-stroma could be the basis for multiple types of novel therapeutic options in corneal interventional surgery

Comparative Capitalism without Capitalism, and Production without Workers: The Limits and Possibilities of Contemporary Institutional Analysis

The aim of this paper is to consider the extent to which the comparative capitalism literature fully reflects the available empirical evidence in its attempts to model different versions of capitalism and, in particular, whether it adequately captures the roles of diverse stakeholders within the capitalist system. In doing so, particular attention is accorded to the varieties of capitalism literature, business systems theory and regulation theory. In addition, there is reflection in the paper on whether any strand of the literature is able to deal effectively with the recent economic crisis and systemic change. It is argued that more attention needs to be devoted to exploring the structural causes of change and the marginalization of the interests of key social groupings, most notably workers, from the process of institutional redesign

HIV Replication Increases the Mitochondrial DNA Content of Plasma Extracellular Vesicles

Extracellular vesicles (EVs) and their cargo have been studied intensively as potential sources of biomarkers in HIV infection; however, their DNA content, particularly the mitochondrial portion (mtDNA), remains largely unexplored. It is well known that human immunodeficiency virus (HIV) infection and prolonged antiretroviral therapy (ART) lead to mitochondrial dysfunction and reduced mtDNA copy in cells and tissues. Moreover, mtDNA is a well-known damage-associated molecular pattern molecule that could potentially contribute to increased immune activation, oxidative stress, and inflammatory response. We investigated the mtDNA content of large and small plasma EVs in persons living with HIV (PLWH) and its implications for viral replication, ART use, and immune status. Venous blood was collected from 196 PLWH, ART-treated or ART-naïve (66 with ongoing viral replication, ≥20 copies/mL), and from 53 HIV-negative persons, all recruited at five HIV testing or treatment centers in Burkina Faso. Large and small plasma EVs were purified and counted, and mtDNA level was measured by RT-qPCR. Regardless of HIV status, mtDNA was more abundant in large than small EVs. It was more abundant in EVs of viremic than aviremic and control participants and tended to be more abundant in participants treated with Tenofovir compared with Zidovudine. When ART treatment was longer than six months and viremia was undetectable, no variation in EV mtDNA content versus CD4 and CD8 count or CD4/CD8 ratio was observed. However, mtDNA in large and small EVs decreased with years of HIV infection and ART. Our results highlight the impact of viral replication and ART on large and small EVs’ mtDNA content. The mechanisms underlying the differential incorporation of mtDNA into EVs and their effects on the surrounding cells warrant further investigation

In vivo evaluation and imaging of a bilayered self-assembled skin substitute using a decellularized dermal matrix grafted on mice

As time to final coverage is the essence for better survival outcome in severely burned patients, we have continuously strived to reduce the duration for the preparation of our bilayered self-assembled skin substitutes (SASS). These SASS produced in vitro by the self-assembly approach have a structure and functionality very similar to native skin. Recently, we have shown that a decellularized dermal matrix preproduced by the self-assembly approach could be used as a template to further obtain self-assembled skin substitute using a decellularized dermal template (SASS-DM) in vitro. Thus, the production period with patient cells was then reduced to about 1 month. Herein, preclinical animal experiments have been performed to confirm the integration and evolution of such a graft and compare the maturation of SASS and SASS-DM in vivo. Both tissues, reconstructed from adult or newborn cells, were grafted on athymic mice. Green fluorescent protein-transfected keratinocytes were also used to follow grafted tissues weekly for 6 weeks using an in vivo imaging system (IVIS). Cell architecture and differentiation were studied with histological and immunofluorescence analyses at each time point. Graft integration, macroscopic evolution, histological analyses, and expression of skin differentiation markers were similar between both skin substitutes reconstructed from either newborn or adult cells, and IVIS observations confirmed the efficient engraftment of SASS-DM. In conclusion, our in vivo graft experiments on a mouse model demonstrated that the SASS-DM had equivalent macroscopic, histological, and differentiation evolution over a 6-week period, when compared with the SASS. The tissue-engineered SASS-DM could improve clinical availability and advantageously shorten the time necessary for the definitive wound coverage of severely burned patients

Biocompatibility and Functionality of a Tissue-Engineered Living Corneal Stroma Transplanted in the Feline Eye

PURPOSE. Corneal tissue shortage has become a major concern worldwide, which has motivated the search for alternative solutions to eye bank human eyes for corneal transplantation. Minimally invasive lamellar transplantation and tissue engineering may offer new opportunities for the rehabilitation of diseased corneas. The aim of this study was to evaluate the biocompatibility and functionality of stromal lamellar grafts tissue-engineered (TE) in vitro and transplanted in vivo in the cornea of a feline model. METHODS. The corneal stromas were engineered in culture from corneal stromal cells using the self-assembly approach, without the addition of exogenous material or scaffold. Eight healthy animals underwent two intrastromal grafts in one eye and the contralateral eye was used as a control. Animals were followed with slit-lamp ophthalmic examination, corneal esthesiometry and optical coherent tomography. Confocal microscopy, immunofluorescence, histology, and transmission electron microscopy (TEM) were performed at 4 months. RESULTS. Four months after transplantation, the TE-stromal grafts were transparent, functional, and well tolerated by the eye. All grafts remained avascular, with no signs of immune rejection, despite a short course of low-dose topical steroids. Corneal sensitivity returned to preoperative level and reinnervation of the grafts was confirmed by confocal microscopy and immunofluorescence. Histology and TEM of the TE-grafts showed a lamellar stromal structure with regular collagen fibril arrangement. CONCLUSIONS. These results open the way to an entirely new therapeutic modality. Intracorneal filling using a biocompatible, transparent, and malleable TE-stroma could be the basis for multiple types of novel therapeutic options in corneal interventional surgery

Characterization of Tissue-Engineered Posterior Corneas Using Second- and Third-Harmonic Generation Microscopy

<div><p>Three-dimensional tissues, such as the cornea, are now being engineered as substitutes for the rehabilitation of vision in patients with blinding corneal diseases. Engineering of tissues for translational purposes requires a non-invasive monitoring to control the quality of the resulting biomaterial. Unfortunately, most current methods still imply invasive steps, such as fixation and staining, to clearly observe the tissue-engineered cornea, a transparent tissue with weak natural contrast. Second- and third-harmonic generation imaging are well known to provide high-contrast, high spatial resolution images of such tissues, by taking advantage of the endogenous contrast agents of the tissue itself. In this article, we imaged tissue-engineered corneal substitutes using both harmonic microscopy and classic histopathology techniques. We demonstrate that second- and third-harmonic imaging can non-invasively provide important information regarding the quality and the integrity of these partial-thickness posterior corneal substitutes (observation of collagen network, fibroblasts and endothelial cells). These two nonlinear imaging modalities offer the new opportunity of monitoring the engineered corneas during the entire process of production.</p></div

Immune Cells Release MicroRNA-155 Enriched Extracellular Vesicles That Promote HIV-1 Infection

The hallmark of HIV-1 infection is the rapid dysregulation of immune functions. Recent investigations for biomarkers of such dysregulation in people living with HIV (PLWH) reveal a strong correlation between viral rebound and immune activation with an increased abundance of extracellular vesicles (EVs) enriched with microRNA-155. We propose that the activation of peripheral blood mononuclear cells (PBMCs) leads to an increased miR-155 expression and production of miR-155-rich extracellular vesicles (miR-155-rich EVs), which can exacerbate HIV-1 infection by promoting viral replication. PBMCs were incubated with either HIV-1 (NL4.3Balenv), a TLR-7/8 agonist, or TNF. EVs were harvested from the cell culture supernatant by differential centrifugation, and RT-qPCR quantified miR-155 in cells and derived EVs. The effect of miR-155-rich EVs on replication of HIV-1 in incubated PBMCs was then measured by viral RNA and DNA quantification. HIV-1, TLR7/8 agonist, and TNF each induced the release of miR-155-rich EVs by PBMCs. These miR-155-rich EVs increased viral replication in PBMCs infected in vitro. Infection with HIV-1 and inflammation promote the production of miR-155-rich EVs, enhancing viral replication. Such autocrine loops, therefore, could influence the course of HIV-1 infection by promoting viral replication

Immune Cells Release MicroRNA-155 Enriched Extracellular Vesicles That Promote HIV-1 Infection

The hallmark of HIV-1 infection is the rapid dysregulation of immune functions. Recent investigations for biomarkers of such dysregulation in people living with HIV (PLWH) reveal a strong correlation between viral rebound and immune activation with an increased abundance of extracellular vesicles (EVs) enriched with microRNA-155. We propose that the activation of peripheral blood mononuclear cells (PBMCs) leads to an increased miR-155 expression and production of miR-155-rich extracellular vesicles (miR-155-rich EVs), which can exacerbate HIV-1 infection by promoting viral replication. PBMCs were incubated with either HIV-1 (NL4.3Balenv), a TLR-7/8 agonist, or TNF. EVs were harvested from the cell culture supernatant by differential centrifugation, and RT-qPCR quantified miR-155 in cells and derived EVs. The effect of miR-155-rich EVs on replication of HIV-1 in incubated PBMCs was then measured by viral RNA and DNA quantification. HIV-1, TLR7/8 agonist, and TNF each induced the release of miR-155-rich EVs by PBMCs. These miR-155-rich EVs increased viral replication in PBMCs infected in vitro. Infection with HIV-1 and inflammation promote the production of miR-155-rich EVs, enhancing viral replication. Such autocrine loops, therefore, could influence the course of HIV-1 infection by promoting viral replication