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.

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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.

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...

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.

Depuis plus d’une cinquantaine d’années, de formidables avancées ont été initiées dans le domaine de l’ingénierie tissulaire cutanée menant à la reconstruction in vitro de substituts de peau. La plupart sont des substituts dermiques destinés à être utilisés comme aide à la cicatrisation des plaies aigües et chroniques en complément des traitements de greffes conventionnels ainsi que pour l’augmentation des tissus mous. Bien qu’un nombre croissant de patients aient pu bénéficier de ces matrices dermiques, leur application clinique reste encore restreinte, en raison de leur coût élevé mais également à cause de résultats cicatriciels parfois peu satisfaisants. Par conséquent, il reste un défi de taille, celui de développer des substituts dermiques stimulant activement la cicatrisation, présentant un faible coût de production, sans propriétés antigéniques et possédant des propriétés mécaniques adaptées. Dans ce cadre, les hydrogels à base de fibrine constituent des candidats prometteurs, en particulier en raison du rôle central de cette protéine dans la cicatrisation. Le principal inconvénient est qu’à concentration physiologique, ces hydrogels sont faibles mécaniquement, ce qui les rend difficilement manipulables. L’objectif de cette thèse a été la mise au point ainsi que la caractérisation de différents hydrogels destinés à être utilisés comme substituts dermiques. Ces derniers présentent l’avantage d’associer les propriétés biologiques de la fibrine avec les propriétés mécaniques d’un polymère synthétique, le polyéthylène glycol dans une architecture de réseaux interpénétrés de polymères (RIP). Les résultats obtenus ont permis : - de confirmer les propriétés physico-chimiques des RIP développés initialement par nos collaborateurs de l’université de Cergy-Pontoise, - de valider en trois étapes (in vitro, ex vivo puis in vivo) la biocompatibilité de ces nouvelles matrices, destinées à être utilisées comme supports de culture 2D et pour l’augmentation des tissus mous, - d’élaborer et de caractériser des matrices macroporeuses, optimisées pour la culture 3D de fibroblastes de dermes humains. / Over the past five decades, we assisted in extraordinary advances in the field of skin tissue engineering which led to the in vitro reconstruction of a wide range of skin substitutes. Most of them are dermal substitutes: Their clinical application ranges from treating acute and chronic wounds to soft tissue augmentation. Although increasing numbers of patients have been treated with dermal substitutes, their clinical application has been limited by their substantial cost and some poor healing outcomes. Hence, there is still a challenge to produce a dermal substitute which enhance sufficiently wound healing. To this end, the substitute should exhibit suitable properties for enabling the repair process. Other requirements such as excellent biocompatibility, minimal antigenicity, ease to handle and cost-effective production are also essential. In this context, fibrin hydrogels constitute promising candidates for skin tissue engineering since fibrin fibers form a physiological and provisional backbone during wound healing. However, the poor mechanical properties of fibrin-based hydrogels at physiological concentration are an obstacle to their use. In this study, our aim was to design and characterize mechanically reinforced fibrin-based hydrogels by combining the intrinsic properties of a fibrin network with the mechanical features of a polyethylene glycol network using an interpenetrating polymer network (IPN) architecture. They are intended to be used as dermal scaffolds. The results obtained in this thesis: - Confirmed the suitable physico-chemical properties of IPN, first developed by our partner of the University of Cergy-Pontoise. - Validated their biocompatibility using a three-step approach (in vitro, ex vivo and in vivo assays). - Led to the synthesis and characterization of a new type of fibrin-based macroporous matrices, optimized for 3D dermal fibroblast culture.