Yes / The use of biological macromolecules like quince seed mucilage (QSM), as the common curative practice has a long history in traditional folk medicine to cure wounds and burns. However, this gel cannot be applied on exudative wounds because of the high water content and non-absorption of infection of open wounds. It also limits cell-to-cell interactions and leads to the slow wound healing process. In this study to overcome these problems, a novel QSM-based hybrid scaffold modified by PCL/PEG copolymer was designed and characterized. The properties of this scaffold (PCL/QSM/PEG) were also compared with four scaffolds of PCL/PEG, PCL/Chitosan/PEG, chitosan, and QSM, to assess the role of QSM and the combined effect of polymers in improving the function of skin tissue-engineered scaffolds. It was found, the physicochemical properties play a crucial role in regulating cell behaviors so that, PCL/QSM/PEG as a smart/stimuli-responsive bio-matrix promotes not only human-adipose stem cells (h-ASCs) adhesion but also supports fibroblasts growth, via providing a porous-network. PCL/QSM/PEG could also induce keratinocytes at a desirable level for wound healing, by increasing the mechanobiological signals. Immunocytochemistry analysis confirmed keratinocytes differentiation pattern and their normal phenotype on PCL/QSM/PEG. Our study demonstrates, QSM as a differentiation/growth-promoting biological factor can be a proper candidate for design of wound dressings and skin tissue-engineered substrates containing cell.

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Yes / Fabrication of extracellular matrix (ECM)-like scaffolds (in terms of structural-functional) is the main challenge in skin tissue engineering. Herein, inspired by macromolecular components of ECM, a novel hybrid scaffold suggested which includes silk/hyaluronan (SF/HA) bio-complex modified by PCP: [polyethylene glycol/chitosan/poly(ɛ-caprolactone)] copolymer containing collagen to differentiate human-adipose-derived stem cells into keratinocytes. In followed by, different weight ratios (wt%) of SF/HA (S1:100/0, S2:80/20, S3:50/50) were applied to study the role of SF/HA in the improvement of physicochemical and biological functions of scaffolds. Notably, the combination of electrospinning-like and freeze-drying methods was also utilized as a new method to create a coherent 3D-network. The results indicated this novel technique was led to ~8% improvement of the scaffold's ductility and ~17% decrease in mean pore diameter, compared to the freeze-drying method. Moreover, the increase of HA (>20wt%) increased porosity to 99%, however, higher tensile strength, modulus, and water absorption% were related to S2 (38.1, 0.32 MPa, 75.3%). More expression of keratinocytes along with growth pattern similar to skin was also observed on S2. This study showed control of HA content creates a microporous-environment with proper modulus and swelling%, although, the role of collagen/PCP as base biocomposite and fabrication technique was undeniable on the inductive signaling of cells. Such a scaffold can mimic skin properties and act as the growth factor through inducing keratinocytes differentiation.

Estudou-se a adição de hidrolisado de colágeno à rações com caseína nas proporções de 0%, 25%, 50%, 65%, 75% e 100%. As rações com 25% e 65% de hidrolisado protéico foram acrescidas com prolina e aminoácidos essenciais (tirosina, triptofano, metionina e leucina). As rações continham 10% e 20% de proteína. Determinou-se o coeficiente de eficácia protéica, coeficiente de digestibilidade, composição centesimal, aminograma e avaliou-se histologicamente o fígado e rim e a pele. Doseou-se o teor de hidroxiprolina da pele dos animais testados. Verificou-se que: a adição de 25% de hidrolisado de colágeno à caseína não promoveu modificação significativa no valor biológico da ração. A adição de prolina em ração com 25% de colágeno para ratos em crescimento não demonstrou alteração do peso dos animais. Entretanto com adição de 65% de colágeno houve queda no peso dos animais em relação ao controle. O figado dos animais estudados (quando utilizadas rações com 10% de proteína), não demonstrou alteração significativa em relação às diferentes adições de colágeno. Quando a ração foi acrescida de 50% e 65% de hidrolisado de colágeno, o doseamento de hidroxiprolina na pele aumentou e os cortes histológicos de tecido cutâneo apresentaram ausência de hipoderme. / We studied hidrolised collagen in association with casein at the proportions of 0%, 25%, 50%, 65%, 75% and 100%. We concluded that 25% addition of collagen, do not change significantly nutritional value. Higher additions in hidrolised collagen, on diets, demostrated higher content of hidroxiproline and lower in adipose tissue in cutaneous tissue.

Estudou-se a adição de hidrolisado de colágeno à rações com caseína nas proporções de 0%, 25%, 50%, 65%, 75% e 100%. As rações com 25% e 65% de hidrolisado protéico foram acrescidas com prolina e aminoácidos essenciais (tirosina, triptofano, metionina e leucina). As rações continham 10% e 20% de proteína. Determinou-se o coeficiente de eficácia protéica, coeficiente de digestibilidade, composição centesimal, aminograma e avaliou-se histologicamente o fígado e rim e a pele. Doseou-se o teor de hidroxiprolina da pele dos animais testados. Verificou-se que: a adição de 25% de hidrolisado de colágeno à caseína não promoveu modificação significativa no valor biológico da ração. A adição de prolina em ração com 25% de colágeno para ratos em crescimento não demonstrou alteração do peso dos animais. Entretanto com adição de 65% de colágeno houve queda no peso dos animais em relação ao controle. O figado dos animais estudados (quando utilizadas rações com 10% de proteína), não demonstrou alteração significativa em relação às diferentes adições de colágeno. Quando a ração foi acrescida de 50% e 65% de hidrolisado de colágeno, o doseamento de hidroxiprolina na pele aumentou e os cortes histológicos de tecido cutâneo apresentaram ausência de hipoderme. / We studied hidrolised collagen in association with casein at the proportions of 0%, 25%, 50%, 65%, 75% and 100%. We concluded that 25% addition of collagen, do not change significantly nutritional value. Higher additions in hidrolised collagen, on diets, demostrated higher content of hidroxiproline and lower in adipose tissue in cutaneous tissue.

Yes / A major challenge in the production of 3D tissue engineered skin is the recreation of the basement membrane region to promote secure attachment and yet segregation of keratinocytes from the dermal substitute impregnated with fibroblasts. We have previously shown that simple electrospun scaffolds provide fibres on which the cells attach, proliferate, and self-sort into epithelium and dermis. In a development of this in this study tri-layered scaffolds were then electrospun from poly L-lactic acid and poly hydroxybutyrate-co-hydroxyvalerate. In these a central layer of the scaffolds comprising nano-porous/nano-fibrous poly hydroxybutyrate-co-hydroxyvalerate fibres was interwoven into the bulk micro-porous poly L-lactic acid microfibers to mimic the basement membrane. Keratinocytes and fibroblasts seeded onto these scaffolds and cultured for 2 weeks showed that neither cell type was able to cross the central nano-porous barrier (shown by SEM, and fluorescence monitoring with CellTracker™) while the micro-fibrous poly L-lactic acid provided a scaffold on which keratinocytes could create an epithelium and fibroblasts could create a dermal substitute depositing collagen. Although cells did not penetrate this barrier the interaction of cells was still evident-essential for epithelial development.

La surexposition aux rayonnements ionisants est actuellement une préoccupation croissante des cliniciens. Lors d'une exposition externe, la peau est le premier tissu lésé. Or, il n'existe pas d'outil fiable qui permette de diagnostiquer l'atteinte tissulaire. L'objectif de ce travail est donc de montrer la possibilité d'utiliser une méthode non invasive pour l'aide in vivo au diagnostic et au pronostic du syndrome cutané d'irradiation aigüe. La première partie de ce travail concerne le choix de la méthode d'investigation. L'interaction entre une lumière cohérente et un milieu diffusant engendre un phénomène d'interférence appelé speckle. Une analyse fréquentielle classique sur le champ de speckle est complétée par une approche stochastique pour en extraire des paramètres caractérisant les figures de speckle. Dans la deuxième partie le protocole expérimental a été testé afin de mieux comprendre le comportement des paramètres en fonciton de quelques propriétés physiques de milieux diffusants synthétiques. Cette étude a révélé que certains des paramètres du speckle étaient plus influencés par les gros diffuseurs (Mie) alors que d'autres l'étaient par les plus petits (Rayleigh). La troisième partie concerne l'application de cette méthode in vivo au syndrome cutané d'irradiation aigüe chez le porc. L'analyse des résultats acquis lors du suivi de plusieurs animaux montre la possibilité de discriminer les zones irradiées des zones saines plusieurs semaines avant l'apparition des premiers signes cliniques. Enfin, pour comprendre les résultats obtenus sur la brûlure radiologique, nous avons confronté l'ensemble des résultats physiques et ceux obtenus par les analyses histologiques. / Overexposure to ionizing radiation is now a growing concern of clinicians. In case of external exposure, the skin is the first tissue exposed. However, there are no tools that can diagnose pathological tissue. The objective of this work is to demonstrate the possibility of using a non-invasive method for in vivo diagnosis and prognosis of acute cutaneous radiation syndrome. The first part of this work concerns the choice of the investigation method. Interaction of coherent light and scattering medium creates a phenomenon called speckle. A classical frequential analysis on the spekle field is supplemented by a stochastic approach to extract parameters characterizing speckle patterns. In the second part, the experimental setup has been tested in order to understand the parameters behavior in function of some physical properties of synthetic scattering media. The study revealed that some of the speckle parameters were more influenced by big scatterers (Mie) while others were by the smallest (Rayleigh). The third part concerns the in vivo application of this method on acute cutaneous radiation syndrome in pigs. Analysis of the results gained during the monitoring of several animals showeb the ability to discriminate between irradiated and healthy zones several weeks before apparition of firsts clinical signs. Finally, in order to understand the results on the radiological burn, we have confronted all physical results and those obtained by histological analyses.

Platelet derivatives are an attractive source of natural growth factors and they are widely used in various tissue engineering and regenerative medicine applications. The aim of this study was to optimize cell culture conditions using platelet lysate and to develop platelet-functionalized fibrous scaffolds as a controlled drug delivery system for native growth factors. Fibrous scaffolds were prepared by electrostatic and centrifugal spinning of PCL and they were functionalized by the platelets by surface adhesion or their encapsulation using emulsion spinning techniques. The cell culture study determined the 7% platelet lysate to be the optimum concentration as a medium supplement in keratinocyte and fibroblast culture. Additionally, following surface adhesion of the platelets to PCL electrospun nanofibres, the platelets were activated due to their contact with the nanofibre nanotopography, resulting in formation of fibrin network. Fibrin served as a reservoir of the growth factors, prolonging the half-time of EGF release to 1.7 days. Such platelet-functionalized samples fostered proliferation of keratinocytes, fibroblasts and melanocytes. Furthermore, adhesion of platelets to centrifugally spun nanofibrous scaffolds resulted in almost two-fold increase in the amount of immobilized platelet-derived...

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

Yes / Creation of functional skin substitutes within a clinically acceptable time window is essential for timely repair and management of large wounds such as extensive burns. The aim of this study was to investigate the possibility of fabricating skin substitutes via a bottom-up nanofiber-enabled cell assembly approach and using such substitutes for full-thickness wound repair in nude mice. Following a layer-by-layer (L-b-L) manner, human primary skin cells (fibroblasts and keratinocytes) were rapidly assembled together with electrospun polycaprolactone (PCL)/collagen (3:1 w/w, 8% w/v) nanofibers into 3D constructs, in which fibroblasts and keratinocytes were located in the bottom and upper portion respectively. Following culture, the constructs developed into a skin-like structure with expression of basal keratinocyte markers and deposition of new matrix while exhibited good mechanical strength (as high as 4.0 MPa by 14 days). Treatment of the full-thickness wounds created on the back of nude mice with various grafts (acellular nanofiber meshes, dermal substitutes, skin substitutes and autografts) revealed that 14-day-cultured skin substitutes facilitated a rapid wound closure with complete epithelialization comparable to autografts. Taken together, skin-like substitutes can be formed by L-b-L assembling human skin cells and biomimetic nanofibers and they are effective to heal acute full-thickness wounds in nude mice.