Small Vessel Replacement by Human Umbilical Arteries With Polyelectrolyte Film-Treated Arteries In Vivo Behavior

ObjectiveThe aim of this study was to evaluate the patency of human umbilical arteries treated with polyelectrolyte multilayers (PEMs) after rabbit implantation.BackgroundThe development of small-caliber vascular substitutes with high patency after implantation remains a real challenge for vascular tissue engineering.MethodsCryopreserved human umbilical arteries were enzymatically de-endothelialized and the luminal surfaces were coated with poly(styrene sulfonate)/poly(allylamine hydrochloride) (PSS/PAH) multilayers. The PEM-untreated arteries and PEM-treated rabbit carotids were used as graft control. The native rabbit carotids were bypassed by grafts.ResultsThe Doppler ultrasound evaluation, performed in vivo, showed that all PEM-treated grafts remained patent during the full experimental period, whereas after only 1 week, no blood circulation was detected in untreated arteries. Scanning electron microscopy and histological graft examination showed pervasive thrombus formation on the luminal surface of untreated arteries after 1 week and clean luminal surface for treated arteries for at least up to 12 weeks. The arterial wall cells were identified through alpha-smooth muscle actin αυδ platelet endothelial cell adhesion molecule-1 expression. The smooth muscle cells positive to alpha-smooth muscle actin were identified in adventitia and media and the endothelial cells positive to platelet endothelial cell adhesion molecule in intima. Von Kossa reaction didn't reveal any calcium salt deposits on the wall arteries, suggesting a good wall remodelling with no sign of graft rejection.ConclusionsThe in vivo evaluation of human umbilical arteries treated with PSS/PAH multilayers demonstrated a high graft patency after 3 months of implantation. Such modified arteries could constitute a useful option for small vascular replacement

O2 Level Controls Hematopoietic Circulating Progenitor Cells Differentiation into Endothelial or Smooth Muscle Cells

BACKGROUND:Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium. METHODOLOGY/PRINCIPAL FINDINGS:Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O2) or normoxic (21% O2) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3(rd) passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability. CONCLUSION/SIGNIFICANCE:We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells

Influence des conditions de culture sur la différenciation de progéniteurs vasculaires en vue de l'obtention d'un substitut vasculaire autologue

L augmentation de la fréquence des pathologies vasculaires va créer ces prochaines années, des besoins importants en substituts vasculaires de petits calibres. L idéal, à ce jour, reste l utilisation de vaisseaux autologues réduisant les risques infectieux et immunologiques mais d usage limité ce qui est à l origine de l ingénierie cellulaire. L identification des progéniteurs vasculaires est d un intérêt majeur dans ce domaine. Ces cellules d origine autologues sont douées d une grande capacité de prolifération et d un potentiel de différenciation en cellules vasculaires (cellules endothéliales (CE) et cellules musculaires lisses (CML)) mais la difficulté réside dans le choix du recouvrement de la surface du support de culture qui favorise leur adhésion et prolifération Dans ce travail, nous avons choisi les films multicouches de polyélectrolytes et mesuré leur impact sur le comportement de ces cellules progénitrices en fonction de différentes conditions de culture (normoxie ou hypoxie). Nous avons montré dans un premier temps, que ces films multicouches de polyélectrolytes permettaient d accélérer la différenciation de ces cellules en CE matures. Nous avons également montré que ces mêmes cellules cultivées en hypoxie étaient capables de se différencier en cellules contractiles stables dans le temps, présentant un phénotype comparable à celui des CML matures. L association de ces résultats additionnés aux avantages apportés par des feuillets détachables est à la base de la construction d un substitut vasculaire autologue de petit calibre, composé de CE et de CML issues d un même pool de cellules, mais cultivées dans des conditions différentes.The increase of vascular pathologies is going to create these next years, important needs in vascular substitutes of small calibres. The gold standard, this day, remains the use of autologous vessels reducing the infectious and immunological risks, but of limited custom, which is at the origin of tissue engineering. The identification of vascular progenitor cells is of major interest in vascular engineering. These autologous cells present a high capacity of proliferation and a potential of differentiation in vascular cells (endothelial cells (EC) and smooth muscle cells (SMC)) but the difficulty lies in the choice of the coated surface of culture which facilitates their adhesion and proliferation. In this work, we chose polyelectrolytes multilayer films and measured their impact on these vascular progenitor cells in various conditions of culture (normoxia or hypoxia). We showed at first, that these polyelectrolytes multilayer films allowed to accelerate the differentiation of these cells into matures EC. We also showed that these same cells cultivated under hypoxic conditions were able to differentiate into stable and contractile cells, presenting a phenotype comparable to the mature SMC. The association of these results added to the advantages brought by detachable sheet is the basis of the construction of an autologous small calibre vascular substitute consisting of EC and SMC differentiated from the same pool of cells, but cultivated in different conditions.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

An Inflamed and Infected Reconstructed Human Epidermis to Study Atopic Dermatitis and Skin Care Ingredients

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential

Phenotype stability under hypoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under hypoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Confocal microscopic observations showed positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that more than 80% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§)PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control, (#) PEMs <i>versus</i> collagen. (§,* and #: <i>p</i><0.05 and §§§ and ***: <i>p</i><0.001).</p

Morphological aspect of differentiated cell.

<p>Optical phase contrast microscopy visualization of differentiated cells seeded on type I collagen (A, B) and polyelectrolyte multilayer films (PEMs) (C, D) until confluence under normoxic (A, C) and hypoxic (B, D) environment. Objective×20, scale bar 55 µm. The morphological examination of the confluent cells showed cobblestone shape (A, C) in normoxia and a spindle like (B, D) shape in hypoxia.</p

Phenotype stability under normoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under normoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Microscopical observations show positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that about 90% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§) PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control. (§ and *: <i>p</i><0.05, §§ and **: <i>p</i><0.01, and *** <i>p</i><0.001).</p

Vascular cell phenotype characterization.

<p>The endothelial cell were characterized by the expression of specific markers: CD31 (A–D) and von Willebrand Factor (E–H) and the smooth muscle cells by the expression of contractile markers: α- Smooth Muscle Actin (α-SMA: E–H), Smooth Muscle Myosin Heavy Chain (SM-MHC: I–L) and Calponin (M–P). Images were obtained by confocal microscopy observation at cell confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)) and cultivated under normoxic and hypoxic conditions. Objective×40, NA = 0.8, scale bars 75 µm. The figure showed the positive expression of specific ECs markers for cell differentiated under normoxic environment and positive expression of specific contractile SMCs markers for cell differentiated under hypoxic environment.</p