Literatura científica selecionada sobre o tema "Ex-Vivo perfused skin"

In order to avoid in vivo experiments and to gain information about the suitability of surrogates for skin replacement, Franz-type diffusion cell experiments were conducted by using three ibuprofen-containing formulations (cream, gel and microgel) on bovine split-skin samples and cellophane membranes. Moreover, ex vivo examinations were performed on the isolated perfused bovine udder, to study the comparability of in vitro and ex vivo experimental set-ups. Depending on the formulation, noticeable differences in the permeation of Ibuprofen occurred in vitro (udder skin) and ex vivo (isolated perfused bovine udder), but not in the cellophane membrane. The rates of ibuprofen permeability (cream > gel > microgel) and adsorption into the skin (gel > microgel > cream) varied with the formulation, and were probably caused by differences in the ingredients. Furthermore, different storage conditions and seasonal variation in the collection of the skin samples probably led to differences in the amounts of ibuprofen adsorption apparent in the isolated bovine udder and udder skin. In vitro diffusion experiments should be preferred to experiments on isolated organs with regard to the costs involved, the throughput, and the intensity of labour required, unless metabolism of the drug in the skin, or cell–cell interactions are of particular interest.

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.

Detailed knowledge related to the morphology, anatomy, physiology, and pathology of the canine mammary gland is scarce. Mammary tissue undergoes massive changes instructed by hormones multiple times within the lifespan of every bitch, affecting its appearance. To address the ductal system’s appearance and to present how different our findings regarding the canine mammary gland are in comparison with the available literature, we obtained cadaveric specimens after euthanasia and mastectomy. All bitches were euthanised due to poor prognosis for their recovery from maladies unrelated to mammae. Using intraductal cannulation ex vivo, milk- or fluid-yielding ducts were perfused using VasQtec (polyurethane resin), which revealed casts, i.e., imprints of ducts and glandular structures in situ. We observed primary, vertically positioned ducts that ascended throughout the teat and continued branching to secondary, tertiary, etc., horizontally positioned ducts, which drained mammary gland lobes under the skin located close to the abdominal wall. The ascendant teat canal could be split into two and could be connected to gland alveoli or end blind. Alveolar formations were located along ducts and ductules in bigger and/or smaller clusters. This study is the first to generate a 3D image of canine ducts and glandular tissue using an intraductal approach.

AbstractObjectiveExisting methods to evaluate skin care products suffer limitations. This is the case for ex vivo skin explants, a first‐choice 3D model. While essential to analyse mid‐ to long‐term biological effects, this classical model hinders assessing microrelief variations. To circumvent these limitations, we developed an ex vivo PERFused EXplant setup (Perfex) that maintains the outer skin surface in the open air, closely mirroring physiological conditions.MethodsA custom‐designed reservoir enables perfusing the dermal side of explants with buffered, temperature‐controlled medium, while the epidermis is subjected to “normal” conditions. Skin tension and characteristics of the stratum corneum, microrelief, histology and immunohistology (collagen types I and III, elastin and fibrillin‐1) were analysed and compared to those of explants maintained under conventional conditions or in vivo skin. The effects of skin care formulas intended to induce short‐ and/or mid‐ to long‐term effects were also assessed.ResultsSkin explants maintained with the Perfex setup exhibit characteristics (firmness, elasticity, hydration and barrier function) closer to those of in vivo skin than with conventional conditions. Moreover, Perfex‐maintained explants present no alteration in histology after 7 days and slight variation in the expression of key protein markers. Microrelief characteristics also remain mostly stable over 7 days. Formula applications corroborate that skin tensor‐containing products primarily induce short‐term changes in the microrelief, while those with biologically active ingredients mainly lead to mid‐ to long‐term effects on the histology and expression of molecular markers. Furthermore, maintaining skin explants with a physiologically relevant skin surface enabled analysing the relationship between microrelief and key markers, showing that fibrillin‐1 is the protein most correlated with microrelief characteristics.ConclusionsThe Perfex setup allows for similar preservation of skin explant histology and key protein expression as the conventional system, yet it maintains a skin surface close to that of in vivo skin. Therefore, it is valuable to analyse both the short‐ and mid‐ to long‐term impacts of skin care formulas and better comprehend their effects. The Perfex system also offers a new tool for investigating fundamental questions, such as the link that can exist between dermal proteins and skin surface properties.

ABSTRACTIntroductionThe aim of this study was to evaluate ex vivo perfusion (EVP) for vascularized composite tissue preservation. We hypothesized that EVP could maintain allografts in near‐physiologic conditions for ≥ 24 h.MethodsTwenty superior epigastric artery perforator‐based abdominal flaps were procured from 10 Yorkshire pigs. Flaps were preserved for 12 h (n = 5) or 24 h (n = 5) using EVP with an oxygenated colloid solution containing HBOC‐201 oxygen carrier (HbO2 Therapeutics, Souderton, PA). Contralateral flaps were cold storage controls (4°C) (n = 10). Hemodynamics, temperature, gases, metabolites, electrolytes, indocyanine green (ICG) angiography, and weight were analyzed. Biopsies were taken every 6 h for histology.ResultsIschemia time was 16 ± 6 min. There were no significant differences between the 12 h EVP and 24 h EVP groups at perfusion end for the following parameters: MAP (p = 0.63), pH (p = 0.77), pO2 (p = 0.20), venous pCO2 (p = 0.22), lactate (p = 0.28), creatine kinase (p = 0.89), or myoglobin (p = 0.95). Electrolytes were also comparable at perfusion termination: sodium (p = 0.31), potassium (p = 0.61), and calcium (p = 0.29).After 12 h, flaps in the 24 h EVP group demonstrated a significant weight decrease (−4.5% [−4.6%, −4.1%] weight change), compared to the 12 h EVP group (1.2% [−1.1%, 1.6%] weight change) (p = 0.03). At perfusion end, weight did not differ from baseline between the 24 h EVP (2.3% [0%, 3%]) and 12 h EVP groups (p = 0.37). SCS flap weight was unchanged from baseline at 12 h (p = 0.954) and 24 h (p = 0.616). ICG revealed well‐perfused flaps. H&E staining revealed preserved skin architecture without histopathological changes.ConclusionEVP preserved flaps for 24 h within physiologic parameters and without development of edema. EVP may extend preservation time for VCAs.

Abstract Topically applied therapies must not only be effective at the molecular level but also efficiently access the target site which can be on milli/centimetre-scales. This bottleneck is particularly inhibitory for peptide and nucleic acid macromolecule drug delivery strategies, especially when aiming to target wounded, infected, and poorly perfused tissues of significant volume and geometry. Methods to drive fluid-flow or to enhance physical distribution of such formulations after local administration in accessible tissues (skin, eye, intestine) would be transformative in realizing the potential of such therapeutics. We previously developed a technology termed Glycosaminoglycan (GAG)-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding peptides and cell penetrating peptides (CPPs) in the form of nanoparticles. Herein, we demonstrate that the most simplistic GET formulation is relatively poor in diffusing into tissue matrix (tested in collagen scaffolds). Changing nanoparticle physicochemical properties can enhance penetration, however the use of a pressure differential, generating fluid-flow significantly enhances effective gene delivery over milli/centimetre scales. We adapted clinically used pressure systems to administer both negative (Negative pressure (NP) wound therapy; NPWT) and positive pressures (PP; Insufflator). Pressure differences generated enhanced distribution, and we were able to show for the first-time localized gene transfer in vitro in cell scaffolds and enhanced transfection of ex vivo skin explants. The ability to simply control intra-tissue localization of gene delivery on milli/centimetre scales using pressure application will facilitate new drug delivery strategies for accessible tissues. Importantly site-specific enhancement of penetration and activity of novel nanotechnologies and gene therapeutics could be transformative for future regenerative medicine strategies. Graphical Abstract

Le tissu adipeux de l’Homme est un réel organe dynamique dont l’intérêt en chirurgie est grandissant. Sa composante sous cutanée, dense en adipocytes, se compose d’un stroma riche en cellules souches mésenchymateuses. Les ADSC (cellules souches mésenchymateuses dérivées du tissus adipeux ou Adipose-derived Stem cells) peuvent en être isolées au laboratoire après digestion enzymatique, ou mécaniquement (émulsification mécanique et nanofat), les rendant disponibles immédiatement au bloc opératoire. La pluripotence des ADSC en fait un atout majeur pour leur utilisation en médecine régénératrice.La chirurgie plastique est amenée à réparer des défects pouvant, entre autres, affecter les tissus nerveux périphérique, osseux et cutané. Ainsi, la lipoaspiration, geste chirurgical effectué quotidiennement au bloc opératoire, offre la possibilité de disposer de lipoaspirat issu du tissu adipeux sous cutané, potentiellement riche en ADSC. Ainsi, après avoir réalisé une revue de la littérature recensant 51 marqueurs phénotypiques depuis 2006, 5 marqueurs différenciants se démarquaient depuis les dernières recommandations de 2019 : CD34-, CD45-, CD73+, CD90+ et CD105+. Alors, nous avons appliqué cette nouvelle stratégie à une cohorte de 21 patients en analysant la cellularité en ADSC de la Fraction Vasculaire Stromale (SVF) selon la technique, l’âge, le sexe, la localisation et l’indice de masse corporelle. Ensuite, la culture cellulaire avait pour but de confirmer la fonctionnalité des ADSC en comparant la production d’IL-6 et de TNF- dans 4 sous populations issus de lipoaspirats. Les variations phénotypiques secondaires à l’amplification cellulaire étaient observées. L’objectif suivant était d’user de la pluripotence des ADSC dans 3 indications : différenciation neurogène, ostéogénique et cutanée.Ainsi, le potentiel neurogénique des ADSC contenus dans la SVF isolée mécaniquement (nanofat) était étudié dans la repousse nerveuse. Pour cela, un défect de nerf sciatique chez le rat était reconstruit à l’aide d’un neurotube de chitosan, associé ou non à l’adjonction d’ADSC. L’environnement favorable à une repousse nerveuse médiée par l’ajout de nanofat était objectivé, entre autres, par analyses histologiques et immunohistochimiques (PGP9.5 et protéine S100).Ensuite, la différenciation ostéoblastique était étudiée après culture cellulaire 3D des ADSC sur une matrice osseuse commerciale. La production d’hydroxyapatite et d’ostéocalcine supportaient la preuve d’une différenciation ostéogénique des ADSC.Enfin, nous avons développé un modèle d’étude de peau humaine isolée et perfusée viable pendant 5 jours, support d’études futures entre ADSC et matrice dermique équivalente. Ainsi, une revue de la littérature étudiait les caractérisations biomécaniques de ce type de biomatériaux (electrospinnés) comme substitut cutané
Human adipose tissue is a truly dynamic organ with an increasing interest in surgery. Its subcutaneous component, dense in adipocytes, is composed of a stroma rich in mesenchymal stem cells. Adipose-derived stem cells (ADSCs) can be isolated from this stroma after enzymatic digestion in the laboratory, or mechanically (mechanical emulsification and nanofat), making them immediately available at the operating room. Moreover, t ADSCs pluripotency makes them a major asset for use in regenerative medicine.Plastic and reconstructive surgery is required to repair defects that can affect peripheral nerve, bone and skin tissue. Liposuction, a surgical procedure performed daily at the operating room, offers the possibility of using liposuction from subcutaneous adipose tissue, which is potentially rich in ADSCs. So, after carrying out a literature review listing 51 phenotypic markers since 2006, 5 different markers stood out since the latest 2019 recommendations: CD34-, CD45-, CD73+, CD90+and CD105+. We therefore took up this new strategy in a cohort of 21 patients by analyzing the cellularity of Stromal Vascular Fraction (SVF) in ADSC according to technique, age, sex, location and body mass index. Cell culture was then used to confirm ADSC functionality by comparing IL-6 and TNF-a production in 4 sub-populations derived from lipoaspirates. Phenotypic variations secondary to cell amplification were observed. The next objective was to use the pluripotency of ADSCs in 3 indications: neurogenic, osteogenic and cutaneous differentiation.The neurogenic potential of ADSCs contained in mechanically isolated SVF (nanofat) was studied in nerve regrowth. A rat sciatic nerve defect was reconstructed using a chitosan neurotube, with or without the addition of ADSCs. The favorable environment for nerve regrowth mediated by the addition of nanofat was assessed by histological and immunohistochemical analyses (PGP9.5 and S100 protein).Osteoblastic differentiation was then studied after 3D cell culture of ADSCs on commercially available bone matrix. Production of hydroxyapatite and osteocalcin supported evidence of osteogenic differentiation.Finally, we developed an ex vivo model of isolated and perfused human skin viable for 5 days, to support future studies between ADSCs and dermal matrix equivalent. A review of the literature looked at the biomechanical characterizations of such electrospun biomaterials as skin substitutes

Les modèles organotypiques tels que les explants de peau humaine sont les modèles les plus complexes et parmi les plus représentatifs de la peau in vivo existants à ce jour pour tester l’efficacité ou l’innocuité de molécules d’intérêts thérapeutiques au stade des études pré-cliniques. Cependant, l’absence de circulation sanguine et lymphatique dans ces modèles reste une limite importante dans l’homéostasie du tissu, notamment pour prédire les réponses de la peau à un traitement. De plus, les échanges en nutriments et en oxygène n’étant possibles que par diffusion, la durée de vie de ces modèles reste limitée. Différentes stratégies ont été mises en place afin de contrôler les mécanismes de transports moléculaires au sein de tissus biologiques. La microfluidique offre un fort potentiel pour contrôler la convection et la diffusion permettant l’échange de composés dans ces modèles de peau.L’objectif de ce projet est de développer, caractériser et valider un modèle de peau humaine ex vivo perfusé. Le but de cette perfusion intra-tissulaire est de favoriser les échanges de nutriments, d’oxygène ou de médicaments, mais également d’améliorer l’élimination des déchets métaboliques.Le premier objectif de mes travaux a consisté à mettre en place un flux intra-tissulaire dans un explant de peau humaine, et à développer un procédé permettant de maintenir l’explant perfusé en culture pendant plusieurs jours. Pour cela, un dispositif poreux a été implanté dans le derme du modèle ex vivo de peau humaine NativeSkin, développé par la société Genoskin, puis relié à un système microfluidique permettant l’infusion de composés au sein du tissu.Le deuxième objectif a consisté à développer des méthodes d’analyse de la diffusion de composés dans des explants de peau. Quatre méthodes ont été développées : l’évaluation macroscopique et qualitative de la diffusion à l’aide d’un colorant, l’étude de la diffusion en temps réel par radiographie à rayons X, l’étude de la diffusion en trois dimensions par tomographie à rayons X, et enfin l’analyse de la diffusion de dextrans fluorescents de différents poids moléculaires, sur coupes histologiques. Un modèle numérique permettant de simuler la diffusion dans le modèle de peau a également été développé sur le logiciel COMSOL, permettant de prédire le profil de diffusion d’un composé.Le troisième et dernier objectif a consisté à déterminer les paramètres de perfusion permettant une bonne diffusion des composés dans l’explant de peau, sans toutefois endommager le tissu. Une première série d’expériences (8 donneurs) a été réalisée sur des modèles perfusés à flux constant (2,5µL/min) avec du milieu de culture, pendant 10 jours. Les résultats ont montré qu’à l’issue de la culture, les modèles de peau ne présentent pas d’altération de la viabilité cellulaire ni de l’intégrité du tissu, avec un maintien de la prolifération et du métabolisme cellulaire. Cependant, la caractérisation de la diffusion dans le modèle a démontré un manque de reproductibilité dans les expériences, avec d’importantes variabilités inter et intra-donneurs. De plus, la perfusion de dextrans de différents poids moléculaires a démontré que la diffusion de composés de hauts poids moléculaires était limitée. Afin de pallier ces limites, nous avons proposé une nouvelle méthode de perfusion basée sur une modulation de la pression au sein du dispositif. L’application d’une légère surpression au sein du dispositif poreux permet d’améliorer la reproductibilité et l’efficacité des échanges moléculaires au sein de l’explant.Les résultats obtenus positionnent le modèle FlowSkin ainsi développé comme un nouvel outil pertinent pour évaluer l’efficacité ou la toxicité de molécules administrées par voie intraveineuse, directement sur de la peau humaine. De plus, la perfusion de transporteurs d’oxygène via ce système pourrait permettre de prolonger la durée de vie et donc d’améliorer encore la pertinence du modèle de peau ex vivo
Organotypic models as human skin explants are the most complex and among the most representative of in vivo skin existing today to test the efficacy or the safety of molecules of therapeutic interest during preclinical studies. However, the loss of vascularization and lymphatic system in these models remains a major limitation in tissue homeostasis that impedes the prediction of skin responses to a treatment. In addition, exchanges of nutrients and oxygen being limited to diffusion, models lifetime is limited. Different strategies have been implemented to study and improve mass transport mechanism in such models. Microfluidics offers a great potential to control diffusion and convection mechanisms that permit molecular exchanges in skin models.The objective of this project is to develop, characterize and validate an ex vivo perfused human skin model. The purpose of this intra-tissue infusion is to promote the exchanges of nutrients, oxygen or drugs, but also to improve metabolic waste elimination.The first objective of my work consisted in implementing an intra-tissue flow in a human skin explant, and in setting up a process to maintain the perfused model in culture for several days. To this end, a porous device was implanted in the dermis of the ex vivo human skin model NativeSkin, developed by the company Genoskin. The implantable device is then connected to a microfluidic system allowing the infusion of compounds within the tissue.The second objective was to develop analysis methods of the diffusion of compounds in skin explants. Four methods have been developed: macroscopic and qualitative evaluation of the diffusion using a dye, the study of the diffusion in real time by X-ray radiography, the study of the diffusion in three dimensions by X-ray tomography, and finally the analysis of the diffusion of fluorescent dextrans of different molecular weights, on histological sections. A numerical model allowing to simulate the diffusion in the skin model has also been developed using COMSOL software, allowing to predict the diffusion profile of a compound.The third and last objective of my work was to determine perfusion parameters allowing efficient molecular exchanges of compounds in the skin explant, but without damaging the tissue. A first series of experiments (8 donors) was carried out on models perfused with a constant flow-rate (2.5 µL/min) with culture medium, for 10 days. The results showed that at the end of the culture, skin models did not show any alteration in cell viability or tissue integrity, with maintenance of cell proliferation and metabolism. However, diffusion characterization in the model demonstrated a lack of reproducibility in the experiments, with significant inter and intra-donor variability. In addition, the infusion of different molecular weights dextrans has demonstrated that the mass transport of high molecular weight compounds was limited through the implantable device. We demonstrated that the control of the fluid pressure is critical and that imposing a pulsatile injection with slight overpressures improves the efficiency and reproducibility of the molecular species delivery and collection in the explant.These results have shown the potential of the developed FlowSkin model as a new tool to study the efficacy or toxicity of intravenously administered drugs directly onto human skin. In addition, the combination of FlowSkin with perfusion of oxygen carriers offers unique opportunities to extend the lifetime and further improve the relevance of such ex vivo skin model