Roxane Pouliot

Characterization of a new tissue-engineered human skin equivalent with hair

SummaryWe designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.

Tissue-engineered skin substitutes: from in vitro constructs to in vivo applications.

The field of skin tissue engineering is a paradigm for the various efforts towards the reconstruction of other tissues and organ substitutes. As skin replacement, this biotechnological approach has evolved from simple cultured autologous epidermal sheets to more complex bilayered cutaneous substitutes. The various types of such substitutes are herein presented with their intended use. However, two integrative characteristics are analysed more specifically because of their critical role: neovascularization and re‐innervation. Furthermore, the in vitro use of these various skin substitutes has shed light on various physiological and pathological phenomena. Thus, not only the in vivo application of these skin substitutes as grafts, but also their in vitro value as skin models, are presented.

Reconstructed human skin produced in vitro and grafted on athymic mice

Background. The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. Methods. The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. Results. After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. Conclusions. This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.

Development of an in vitro psoriatic skin model by tissue engineering

BACKGROUND Psoriasis is a chronic skin disease characterized by a thickening and disorganization of the skins protective barrier. OBJECTIVES This study aims to develop and characterize a novel in vitro psoriatic human skin model produced by tissue engineering. METHODS The self-assembly method, a tissue engineering approach based on the capacity of mesenchymal cells, such as fibroblasts, to create their own extracellular matrix in vitro, was used to create our substitutes. Manipulatable sheets of fibroblasts were superimposed creating a new dermis upon which keratinocytes are seeded, leading to a complete bilayered skin substitute. The characterization of the psoriatic substitutes was performed by macroscopic, histological and immunohistochemical analyses and contrasted to those constructed from healthy cells. RESULTS Macroscopically, the psoriatic substitutes were more white and thicker than the healthy substitutes. The histological analysis of psoriatic substitutes stained with Massons trichrome revealed a characteristic thickening of the epidermal layer seen in psoriatic skin in vivo. Immunohistochemical analysis of the psoriatic substitutes showed, among other things, an overexpression of involucrin and an underexpression of filaggrin and loricrin. CONCLUSION These data suggest that the macroscopic, histological and immunohistochemical characteristics of psoriasis are partially retained in the substitutes, thus providing a good model to investigate the mechanisms of abnormal keratinocyte growth and to study cell-cell interactions.

Physical characterization of the stratum corneum of an in vitro human skin equivalent produced by tissue engineering and its comparison with normal human skin by ATR-FTIR spectroscopy and thermal analysis (DSC)

An in vitro human skin equivalent may be obtained by culturing human keratinocytes on a collagen gel containing fibroblasts. The anchored skin equivalent cultured at the air-liquid interface closely resembles human skin and is acceptable for in vitro percutaneous absorption. However, it is still more permeable than human skin. Since intercellular lipids have been recognized to play an important role in skin permeability, infrared spectroscopy and differential scanning calorimetry were performed on the stratum corneum of bovine or human skin equivalents grown at different days of air-liquid culture. The symmetric and asymmetric CH(2) stretching vibrations suggested that for all days observed, the intercellular lipids were less organized than those in human skin, irrespective of whether bovine or human collagen was used. Different culture conditions were also tested and the medium without serum and no epidermal growth factor at the air-liquid culture showed results significantly more comparable to human skin. Actually, the thermal behavior of in vitro stratum corneum showed transitions at lower temperatures than human skin. The transition around 80 degrees C, in the form of a lipid-protein complex, was absent. These results showed that the structural arrangement of intercellular lipids and their thermodynamic properties hold a crucial role in the barrier function of the stratum corneum.

Antioxidant, toxicological and antiproliferative properties of Canadian polyphenolic extracts on normal and psoriatic keratinocytes.

AIMS OF THE STUDY In a first attempt for establishing the possible utilization of polyphenolic extracts from barks of Canadian wood species in psoriasis treatment, we aimed to study (a) their antioxidant capacity, (b) their toxicological properties on normal human keratinocytes (NHK), and (c) their effect on the growth of normal and psoriatic keratinocytes (PK). MATERIALS AND METHODS Polyphenolic extracts were obtained by 90% ethanolic maceration and hot water extraction (HWE). Scavenging capacity was evaluated towards hydrogen peroxide, hydroxyl, superoxide, nitric oxide and peroxyl radicals. MTT assay and Trypan blue dye exclusion (TBDE) method were used for evaluating the initial toxicity of the most antioxidant extracts on NHK during 24 and 48 h. The effects of extracts on the growth of NHK and PK at non-toxic concentrations were determined after exposure for 48 h. RESULTS Yellow birch extract obtained by maceration (YB(Mac)) and black spruce extract obtained by HWE (BS(HWE)) were determined to have the highest antioxidant capacity, but BS(HWE) was less toxic on NHK. Toxicity of extracts on keratinocyte plasma membrane and mitochondria after 24 h was attributed to their content of hydroxycinnamic acids and proanthocyanidins. BS(HWE) inhibited the growth of NHK and non-lesional PK, but was not selective for lesional PK. CONCLUSION Given that BS(HWE) presented elevated content of total phenols and flavonoids and showed a low toxicity on NHK as well as an adequate chemical reactivity towards different radicals and some antiproliferative properties, it was considered as the most valuable extract obtained from barks of Canadian wood species.

Pathological crosstalk in vitro between T lymphocytes and lesional keratinocytes in psoriasis: necessity of direct cell-to-cell contact

Psoriasis, a chronic autoimmune-related skin disease, involves both immune and non-immune cells like T cells and keratinocytes. This study investigates the regulatory role of T cells–keratinocyte interactions during psoriasis on immune factors production. Cytokines and chemokines were evaluated by multiplex and ELISA assays in an in vitro model of co-culture of keratinocytes with T lymphocytes. Keratinocytes were from psoriatic skin lesions or healthy skin. T lymphocytes were from healthy volunteers. Psoriatic keratinocytes (PKs) alone generated concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, monocyte chemotactic protein (MCP)-1, interferon-γ-induced protein 10 kDa (IP-10) and vascular endothelial growth factor (VEGF) higher than those produced by healthy keratinocytes (HKs). In contrast, IL-1α and IL-Ra production was reduced in PKs. Normal T cells, which had no effect on HKs, increased the production of TNF-α, IL-6, GM-CSF, IL-8, MCP-1 and IP-10 by PKs, but did not influence PK production of IL-1β, IL-1α, IL-Ra and VEGF. The most striking effects were obtained with PK- and IL-2-stimulated T lymphocytes: most of the above cytokines and chemokines were greatly upregulated, except IL-1β and VEGF that were decreased or unchanged, respectively. In addition, fractalkine was overproduced in this latter condition only. Our results indicate (1) a functional interaction between keratinocytes and T lymphocytes that requires a direct cellular contact, and (2) a reciprocal influence that depends on cytokine and chemokine types. In conclusion, lesional keratinocytes from psoriasis vulgaris alter functions of normal T lymphocytes that conversely modulate these keratinocytes.

Concise synthesis of yingzhaosu C and epi-yingzhaosu C by peroxyl radical cyclization. Assignment of relative configuration

Abstract The antimalarial peroxide yingzhaosu C and its diastereoisomer (epi-yingzhaosu C) have been synthesized by means of tandem peroxyl radical cyclization-oxygen entrapment. The relative configuration of yingzhaosu C is assigned as cis (3) on account of NMR data for both diastereoisomers, (2) and (3), and their precursors (8) and (9).

MULTISTEP PRODUCTION OF BIOENGINEERED SKIN SUBSTITUTES: SEQUENTIAL MODULATION OF CULTURE CONDITIONS

SummaryMany studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboïdal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.

Promising New Treatments for Psoriasis

Psoriasis is a chronic, proliferative, and inflammatory skin disease affecting 2-3% of the population and is characterized by red plaques with white scales. Psoriasis is a disease that can affect many aspects of professional and social life. Currently, several treatments are available to help control psoriasis such as methotrexate, ciclosporin, and oral retinoids. However, the available treatments are only able to relieve the symptoms and lives of individuals. The discovery of new immunological factors and a better understanding of psoriasis have turned to the use of immunological pathways and could develop new biological drugs against specific immunological elements that cause psoriasis. Biological drugs are less toxic to the body and more effective than traditional therapies. Thus, they should improve the quality of life of patients with psoriasis. This review describes new psoriasis treatments, which are on the market or currently in clinical trials that are being used to treat moderate-to-severe plaque psoriasis. In addition, this paper describes the characteristics and mechanisms in detail. In general, biological drugs are well tolerated and appear to be an effective alternative to conventional therapies. However, their effectiveness and long-term side effects need to be further researched.