Damage to our tissues occurs daily and must be repaired by the body in a timely manner in order to prevent infection and restore tissue integrity. Many cell types are involved in the healing process, but it is the cells of the stroma that are largely responsible for rebuilding fibrous tissue, which provides structure and support for all other cell types during healing. This dissertation focuses on stromal tissue repair, the rebuilding of fibrous tissue by fibroblasts following injury. Specifically, I focus on 1) models to study wound healing in vitro, and the specific biological processes of healing that each model captures, 2) the response of engineered stromal microtissues to different methods of injury, namely laceration and laser ablation, and the subsequent clearance and rebuilding of the extracellular matrix by fibroblasts, and 3) how different types of stromal cells and extracellular matrix proteins contribute to tissue repair in vitro.

The ultraviolet (UV) spectrum has been known to cause damage to skin in varying degrees. UVB radiation (290-320 nm) in particular, has been proven to be highly mutagenic and carcinogenic in many animal experiments compared to either UVA or UVC. The alarming rate of increase in skin cancer incidence has prompted many investigators to pursue other alternatives to sunscreens including changes in lifestyle habits and dietary consumption in order to boost our efforts in tackling this widespread disease. Previous studies employing confocal reflectance, MTT assay and histology suggest that milk phospholipids may possess protective properties against UVB-mediated damage but the molecular mechanism for this effect remains unclear. This study aims to evaluate changes in cell morphology, apoptosis and p21 expression in tissue engineered epidermis to increase our understanding of the mechanisms behind the potential protective effects of milk phospholipids against UV-induced photodamage. Human skin tissue equivalents were incubated in either 1% milk phospholipid solution or maintenance media then exposed to 120 mJ/cm2 dose of 300 nm UVB after 24 hours. The upregulation of p21 protein in response to DNA damage was measured with Western blot and immunofluorescence microscopy and markers for apoptosis and hyperplasia were examined 24 hours after irradiation. Results revealed that p21 levels and the amount of apoptotic markers such as fragmented DNA and nuclear condensation were significantly reduced in UV-exposed tissues pre-incubated with milk phospholipids compared to levels seen in both the positive control and UV-exposed skin tissue not incubated with milk phospholipids. This decrease in p21 expression may imply a reduction in DNA damage 24 hours after UV exposure or a decrease in acquired photodamage at the outset. Milk phospholipid incubation however, induced an increase in epidermal thickening with or without UV exposure, which may imply induction of a protective mechanism to enhance the barrier properties of skin.

La peau et son vieillissement sont un enjeu de santé publique. Les modèles expérimentaux disponibles pour l'étude du vieillissement cutané restent perfectibles. Dans ce contexte, nos objectifs étaient simultanément d'utiliser les modèles de peaux reconstruites (PR) développés dans notre laboratoire afin i) de mieux comprendre les mécanismes du vieillissement cutané, ii) de démontrer l'efficacité et le mécanisme d'action du sélénium comme « anti âge » et enfin iii) de les faire évoluer en utilisant le support poreux ou auto-assemblé avec des fibroblastes du même donneur prélevés à des âges différents. Ainsi, le modèle de PR cultivé sur une longue période a montré une surexpression du microARN miR30-a par RT qPCR dans les PR « âgées » avec une altération de la fonction barrière mesurée par la perte insensible en eau et une perturbation de la différenciation terminale (baisse d'expression de la loricrine et de l'involucrine). Avec le même modèle in vitro, nos résultats démontrent que la supplementation en sélénium retarde la sénescence des kératinocytes souches. Cette efficacité passe non pas par un effet antioxydant comme attendu mais par l'activation de leur adhésion à la lame basale, qui participe à les conserver souche et donc à préserver le renouvellement épidermique. Enfin, nous avons eu la chance exceptionnelle de préparer des PR avec des fibroblastes provenant d'un donneur unique prélevé à 36 et 72 ans. Les résultats immunohistologiques montrent que l'âge induit une augmentation de l'expression de l'élastine et de la fibrilline ainsi que leur co-expression. L'augmentation de LTBP1 et aSMA suggère que cette augmentation inattendue est due à une dérégulation de la voie TGF-ß et une différenciation des fibroblastes en myofibroblastes. En conclusion l'utilisation de différents modèles de PR a permis d'explorer les mécanismes conduisant au vieillissement cutané et de démontrer l'efficacité du sélénium comme anti âge / Skin and its aging is a public health issue. In vitro skin models available for the study aging remain perfectible. In this context, our objectives were simultaneously to use skin equivalent (SE) developed in our laboratory i) to better understand mechanisms of skin aging, ii) to demonstrate the effectiveness of selenium as “anti-aging” and finally iii) to improve SE using the porous or scaffold free model with fibroblasts from the same donor at different ages. Thus, the model of SE mimicking senescence showed an overexpression of microRNA miR30-a by RT qPCR in old SE with an alteration of the barrier function measured by the transepidermal water loss and a deficiency of epidermal terminal differentiation (decreased expression of loricrin and involucrin). With the same SE model, our results demonstrate that selenium supplementation delays the senescence of keratinocytes stem cells. This effectiveness does not involve antioxidant effect as expected but the activation of their adhesion to the basement membrane, which participates in preserving stemness and epidermal renewal. Finally, we had the opportunity to prepare SE with fibroblasts from a single donor at 36 and 72 years old. The histological results show that age induces an increase in the expression of elastin and fibrillin as well as their co-expression. The increase of LTBP1 and aSMA suggests that this unexpected increase is due to deregulation of the TGF-ß pathway and fibroblasts differentiation into myofibroblasts. In conclusion, the use of different models of SE helps us to explore some mechanisms leading to skin aging and to demonstrate the efficacy of selenium as “anti-aging”

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

On entend par substitut dermo-épidermique un épiderme reconstruit à la surface d'un derme équivalent composé de fibroblastes cultivés classiquement dans un biomatériau support, souvent à base de collagène poreux ou sous forme de gel. Ce support possède ses propres propriétés biomécaniques, influant sur la réponse biomécanique globale des peaux reconstruites, nous nous sommes donc intéressés à un modèle de peau reconstruite sans support, dans lequel le derme équivalent est « auto-assemblé » par les fibroblastes néosynthétisant leur propre matrice extracellulaire (MEC). Notre premier objectif a été d'optimiser et de caractériser ce modèle auto-assemblé en termes de structure, de reproductibilité et de fonctionnalité. Notre second objectif a été d'adapter aux peaux reconstruites in vitro (PR) des outils traditionnellement utilisés pour des études in vivo, pour explorer leurs propriétés biophysiques et biomécaniques. Ces outils permettent une exploration morphologique à des résolutions différentes avec l'échographie, la tomographie à cohérence optique (OCT) et la microscopie confocale à balayage et une exploration fonctionnelle des propriétés biomécaniques des PR par cutométrie. Ces données biophysiques ont ensuite été analysées par rapport aux résultats en histologie, immunohistologie et microscopie électronique à transmission. La cinétique de culture du modèle auto-assemblé sur un temps prolongé a montré la grande stabilité de l'épiderme et le remodelage continuel de la MEC avec notamment l'augmentation des fibres de collagène et d'élastine. Au temps de culture de référence sélectionné, correspondant à l'obtention de la différenciation terminale de l'épiderme, nous avons démontré la reproductibilité des épaisseurs de l'épiderme et du derme en histologie et en OCT, de la maturité de l'épiderme et de la jonction dermo-épidermique et de l'expression dermique de l'élastine colocalisée avec la fibrilline. Sur le plan fonctionnel, nous avons démontré la fonction barrière de l'épiderme via l'imperméabilité du stratum corneum et des jonctions serrées / A skin equivalent consist of a epidermis reconstructed on the top of a dermis equivalent classically composed of fibroblasts cultured into a biomaterial scaffold which is often a collagen gel or sponge. This scaffold hold its own mechanical properties, influencing the global skin equivalent biomechanical response, so we choose to develop a scaffold-free skin equivalent (SFSE), based on the ability of fibroblasts to synthezise their own extracellular matrix. Our first objective was to optimize and characterize the structure, the reproducibility and functionality of this scaffold-free model. Our second goal was to adapt biophysical and biomechanical tools classically used for in vivo evaluation to in vitro skin equivalents. Their morphology was explored with different resolutions using echography, optical coherence tomography (OCT) and laser scanning microscopy whereas biomechanical functionality was evaluate by a suction test, the cutometry. This biophysical data were compared to more classical histological, immununohistological and transmission electronic microscopy results. The long-term culture of the scaffold-free model showed the good stability of epidermis and the continuous remodelling of MEC with notably an increase of collagen and elastin fibers. We selected a reference culture time, corresponding to the complete terminal differentiation of epidermis. At this culture time, we showed the epidermis and dermis thickness reproducibity in histology and OCT, the constant epidermis and dermo-epidermal junction maturity and the dermal expression of elastin, colocalized with fibrillin. The barrier function of epidermis was also demonstrated via stratum corneum and tight junctions impermeability

Development of methods for studying drug delivery systems is of great significance for the improvement of topical formulations. Active compounds for topical drug delivery are often formulated into gels and creams, that can be applied onto skin surfaces. It is important to know the extent of the permeability of the active compounds, in order to determine the medical effect. This study examines the possibilities of using an animal-free skin equivalent for penetration and permeation experiments, i.e. a silk scaffold integrated with viable human dermaland epidermal cells. Mammalian cell culturing together with silkconstruct formation, constituted the upstream bioprocess and acquisition of the skin equivalents. Permeability of fluorescently labelled Bovine Serum Albumin and Sodium Fluorescein salt was assessed, using a Franz- cell setup incorporated with the skin equivalents. Furthermore, fluorescence analysis and SDS-PAGE was performed on the collected samples, along with cryosectioning and image analysis of the skin equivalents. The results indicate variations in tissue integrity, leading to both high and low permeability. Fluorescence intensity can be correlated with the amount of sample liquid passing through. The model is still under development, hence more research is needed to draw a conclusion regarding the cellular composition of the skin equivalents, and how it influences permeability. / NextBioForm