Jessica Jean

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.

Bioengineered Skin: The Self-Assembly Approach

Tissue-engineered skin substitutes represent an innovative therapeutic option for the treatment of burns and skin ulcers as well as a powerful tool for fundamental research. To be efficient, in vitro skin substitutes must closely mimic human skin structures and exogenous material has to be reduced as much as possible. The self-assembly approach is based on the capacity of fibroblasts to create their own extracellular matrix in vitro, which allows the production of cell sheets that are easy to handle. Therefore, a skin substitute devoid of exogenous extracellular matrix proteins and synthetic material is produced, which demonstrates many histological, physico-chemical and mechanical characteristics found in normal human skin in vivo. A particularity of this approach is the possibility to add various other cell types (keratinocytes, melanocytes, adipocytes, endothelial and immunological cells, etc.) according to needs. Furthermore, pathological cells (hypertrophic scar, sclerodermic, tumoral and psoriatic cells) can be used for the production of pathological skin substitutes. The development of these models represents a key component in the fight against such diseases because they can lead to a better understanding of the pathology and to the development of new pharmaceutical therapies.This review will present the need for tissue-engineered skin substitutes, the implication of tissue engineering in the cutaneous field (basic and applied research), the selfassembly approach and its characteristics as well as the actual state of research on healthy and pathological selfassembled skin models.

In Vivo and In Vitro Models of Psoriasis

1.1 Skin The evolution of life in the terrestrial environment required the development of a waterproof integument: the skin (Loden and Maibach, 2006). Skin is an extensive organ covering the entire exterior of the body (Stevens and Lowe, 2005). It provides the primary barrier against chemical and biological external agents and water loss (Hadgraft, 2001). The skin also plays an important role in thermoregulation, sensory perception and vitamin D metabolism (McKay and Leigh, 1995). The skin is composed of three main layers: the epidermis, the dermis and the hypodermis. The epidermis is the protective skin layer in contact with the external environment (Stevens and Lowe, 2005). This skin layer consists mainly of a stratified squamous keratinized epithelium (Junqueira and Carneiro, 2005). The epidermis cells, the keratinocytes, divide in the basal layer and differentiate throughout their migration to the surface. The epidermis is divided in 5 different layers (stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum). The dermis is the feeder layer of the epidermis and provides most of the skins mechanical resistance and elasticity. It is mainly composed of fibroblasts, epidermal appendages, blood vessels, nerves and nerve endings (Stevens and Lowe, 2005). Finally, the hypodermis is the deepest layer of the skin. It varies in size and content, but is usually composed of adipocytes which form the adipose tissue (Stevens and Lowe, 2005). Many severe skin diseases can be observed in human beings such as psoriasis.

Characterization of a psoriatic skin model produced with involved or uninvolved cells

Current knowledge suggests that uninvolved psoriatic skin could demonstrate characteristics associated with both normal and involved psoriatic skins. However, the triggering factor allowing the conversion of uninvolved skin into a psoriatic plaque is not fully understood. To counter this lack of information, we decided to develop pathological skin substitutes produced with uninvolved psoriatic cells, in order to better characterize the uninvolved psoriatic skin. Substitutes were produced using the self‐assembly approach. Macroscopic, immunohistochemical, permeability and physicochemical results showed that involved substitutes had a thicker epidermis, higher cell proliferation, abnormal cell differentiation and a more permeable and disorganized stratum corneum compared with normal substitutes. Various results were observed using uninvolved cells, leading to two proposed profiles: profile 1 was suggested for uninvolved skin substitutes mimicking the results obtained with normal skin substitutes; and profile 2 was dedicated to those mimicking involved skin substitutes in all aspects that were analysed. In summary, uninvolved substitutes of profile 1 had a thin, well‐organized epidermis with normal cell proliferation and differentiation, such as observed with normal substitutes, while uninvolved substitutes of profile 2 showed an inverse trend, i.e. a thicker epidermis, higher cell proliferation, abnormal cell differentiation and a more disorganized and more permeable stratum corneum, such as seen with involved substitutes. The results suggest that uninvolved substitutes could demonstrate characteristics associated with both normal or involved psoriatic skins. Copyright

Antipsoriatic Drug Development: Challenges and New Emerging Therapies

Psoriasis is a chronic recurring skin disorder affecting up to 2% of the worlds population. Psoriatic lesions are generally visible, leading to significant emotional and social disabilities for patients. In the context of psoriasis, the orchestrated interplay between activated T cells, antigen-presenting cells and keratinocytes leads to the release of proinflammatory cytokines, chemokines and chemical mediators responsible for the perpetuation of this disease. Even though some therapies are available for psoriasis treatment, there is still no cure for this skin disorder and psoriatic patients are significantly unsatisfied, as demonstrated by recent worldwide surveys. Unlike other diseases, psoriasis does not have a generally accepted animal model, which complicates the successful introduction of new antipsoriatic drugs into clinical phases of development. Moreover, psoriasis affects infants, children and elderly patients which require long-term therapies. Thus, the development of new therapeutic approaches should consider multiple factors such as efficacy, dosing frequency, route of administration, toxicity as well as co-morbidities of patients. This article analyzes current challenges for the antipsoriatic drug development and reviews recent patent applications gathered from 2000 to 2011 for psoriasis treatment. Additionally, future perspectives for antipsoriatic drug development are summarized.

A 3D-psoriatic skin model for dermatological testing: The impact of culture conditions

Background Inadequate representation of the human tissue environment during a preclinical screen can result in inaccurate predictions of compound effects. Consequently, pharmaceutical investigators are searching for preclinical models that closely resemble original tissue for predicting clinical outcomes. Methods The current research aims to compare the impact of using serum-free medium instead of complete culture medium during the last step of psoriatic skin substitute reconstruction. Skin substitutes were produced according to the self-assembly approach. Results Serum-free conditions have no negative impact on the reconstruction of healthy or psoriatic skin substitutes presented in this study regarding their macroscopic or histological appearances. ATR-FTIR results showed no significant differences in the CH2 bands between psoriatic substitutes cultured with or without serum, thus suggesting that serum deprivation did not have a negative impact on the lipid organization of their stratum corneum. Serum deprivation could even lead to a better organization of healthy skin substitute lipids. Percutaneous analyses demonstrated that psoriatic substitutes cultured in serum-free conditions showed a higher permeability to hydrocortisone compared to controls, while no significant differences in benzoic acid and caffeine penetration profiles were observed. Conclusions Results obtained with this 3D-psoriatic skin substitute demonstrate the potential and versatility of the model. It could offer good prediction of drug related toxicities at preclinical stages performed in order to avoid unexpected and costly findings in the clinic. General significance Together, these findings offer a new approach for one of the most important challenges of the 21st century, namely, prediction of drug toxicity.

Pigmented Skin Models: Understand the Mechanisms of Melanocytes

Skin is composed of three layers: epidermis, dermis and hypodermis. The epidermis is organized into five layers in which there are different cell types. The most important cell type in the epidermis is the keratinocytes which constitute approximately 95 % of the total epider‐ mal cells. Melanocytes, Langerhans cells, Merkel cells and inflammatory cells form the remaining 5 % [1]. Among these cells, the melanocytes, which are dendritic cells, are the second most important cell type in the epidermis. They have the capacity to synthesize melanin, a skin pigment. Melanocytes are not only found in the skin, but can also be observed in hair, eyes, ears and central nervous system (Table 1) [2-3]. Their different localization gives them different functions in the organism, but they all keep a common function: melanogenesis [1].

Psoriatic Skin Models: A Need for the Pharmaceutical Industry

Skin is composed of three layers: epidermis, dermis and hypodermis (Sugihara et al., 1991). Epidermis is divided into five layers namely, stratum basale, spinosum, granulosum, lucidum, and corneum (Bragulla & Homberger, 2009, Nagarajan et al., 2009). The differentiation process implies that keratinocytes are transformed through the different cell layers to reach their complete maturation in the stratum corneum (Harding, 2004). In this process, various proliferation and differentiation markers are expressed in a well-orchestrated sequence of events (Fig. 1). When the differentiation process is negatively affected, skin pathologies such as psoriasis can appear (Rashmi et al., 2009, Karlsson et al., 2004).