**1. Introduction**

#### **1.1. Skin**

The integumentary system is the largest and heaviest organ of the body [1, 2]. This organ is divided into three distinct layers: the epidermis (superficial layer), the dermis (intermediate layer), and the hypodermis (deepest layer). Its main function is to protect the body from external aggressions, such as chemical, mechanical, thermal, microbial, and UV rays [3, 4]. It is therefore a physical, biological, and immunological barrier. The epidermis, the outer layer of the skin, predominantly ensures this barrier function by a constant renewal of keratinocytes, the epidermal cells. Keratinocytes differentiated into five layers: from the *stratum basale* (*stratum germinativum*), in which skin stem cells are found, to the *stratum spinosum*, *stratum granulosum*, *stratum lucidum*, and *stratum corneum*, which is the outer layer where keratinocytes have lost their nuclei and are completely keratinized [5]. Keratinocytes from this layer, also called corneocytes, will gradually detach to cause the phenomenon called desquamation. The highly regulated process of differentiation involves specific proteins to maintain this epidermal structure, and deregulation of these proteins expression can induce skin pathology such as psoriasis.

example the antioxidant and antiproliferative potentials, and the toxicology of molecules or extracts [16, 17], to study the mechanism of action of compounds [18] and to perform percutaneous absorption studies, and thus study the permeability of the skin, the diffusion rate, and

Optimization of the Self-Assembly Method for the Production of Psoriatic Skin Substitutes

http://dx.doi.org/10.5772/intechopen.79843

197

Many approaches are used to obtain animal models as representative as possible to the human pathology. Spontaneous mutations, like the homozygous asebia, xenotransplantation, like the severe combined immunodeficient mice (SCID) and the athymic nude mouse, and genetic models, such as the CD18 hypomorphic mice model, the K14/TGF-α, and the involucrin/INF-γ, have been used over the years to study psoriasis but all of them displayed some limitations [21–23]. Animal models are mostly used to study specific aspects of the pathology.

There are two types of models: monolayer models (dermal or epidermal) and bilayer substitutes. Monolayer models use only one cell type, keratinocytes or fibroblasts, and will be used to study a specific characteristic or to understand the role of a certain cell type in pathologies such as psoriasis. However, these models exclude interactions between different cell types. Bilayer models displayed two layers of skin: dermis and epidermis, which allow the study of skin complexity more representatively. The challenge of skin engineering is to reproduce the complexity and the functionalities of a pathological skin. There are different *in vitro* psoriatic skin models, which include interesting pathological features. Various pathological bilayer skin models were developed using a collagen gel as dermal equivalent. Most of these studies involve pathological keratinocytes seeded on a dermis made of collagen and fibroblasts [24, 25], but there are also studies where a full-thickness psoriatic skin biopsy is incorporated into the dermal equivalent [26]. These models have been useful to better understand the disease and the interactions between fibroblasts and keratinocytes [26, 27]. However, the main disadvantage of these models is the use of an exogenous material, which does not represent exactly the properties of the human dermis. To counter the use of exogenous material such as collagen, some research teams have used de-epidermized dermis to produce their psoriatic skin models [28–30]. Although these equivalents demonstrate several psoriatic features, the use of these models for pharmaceutical studies would require an excessive amount of skin biopsies. Thus, a pathological model free of exogenous material that can generate many samples at a time is

Our team has developed a psoriatic skin model based on a self-assembly method, which is free of exogenous material [31]. This model has been characterized towards its permeability, lipid organization and response to antipsoriatic drugs [32, 33]. This basic model has also been improved by the addition of other cell types such as endothelial cells in order to reproduce the angiogenesis observed *in vivo* [34]. These studies have confirmed that our psoriatic skin substitute model produced according to the self-assembly approach maintained many characteristics of the disease including the presence of a disorganized and thicker epidermis compared with normal skin substitutes [31]. This self-assembly approach allows the understanding of

The development of a representative animal model can be expensive.

the site of action of compounds [19, 20].

still required for pharmaceutical research.

*1.3.1. In vivo models*

*1.3.2. In vitro models*

#### **1.2. Psoriasis**

Psoriasis is an erythematous-squamous dermatosis touching both men and women. This chronic skin pathology affects 2–3% of the world's population [6, 7], which correspond to approximately 125 M people [8]. This pathology is characterized by a hyperproliferation and an abnormal differentiation of keratinocytes resulting in reddish and whitish plaques [5]. At a cellular level, histopathological characteristics consist of acanthosis, parakeratosis, hyperkeratosis, agranulose, and papillomatosis [9, 10]. The disease's etiology is still unknown. However, environmental and immune factors, as well as genetic predispositions, would act together to trigger psoriasis [10, 11]. This disease seriously affects the quality of life of patients due to the appearance of their skin and the side effects of drugs. Existing treatments cause many severe side effects such as nephrotoxicity, hepatotoxicity, immunosuppression, teratogenicity, and no curable treatments have been found [12–14]. Moreover, several comorbidities may be related to psoriasis, such as major cardiac events, type 2 diabetes, and psoriatic arthritis [6].

#### **1.3.** *In vivo* **and** *in vitro* **psoriatic skin models**

The skin is a complex organ. Thus, the production of representative and reproducible skin models is a constant challenge. The use of *ex vivo* human skin biopsies would be more convenient, since with skin biopsies, it is possible to observe the mechanisms and interactions of the human skin. However, because of skin donor availability and inter-individual variability, the use of *ex vivo* biopsies is not practical, and thus, the development of new models is important. Over the years, there has been a lot of progress in the field of tissue engineering [15]. Tissue engineering of the skin is used for various clinical applications and in fundamental research such as for drug development. Now, with the development and optimization of *in vivo* and *in vitro* models, it is possible to research new treatments for a skin disease by studying, for example the antioxidant and antiproliferative potentials, and the toxicology of molecules or extracts [16, 17], to study the mechanism of action of compounds [18] and to perform percutaneous absorption studies, and thus study the permeability of the skin, the diffusion rate, and the site of action of compounds [19, 20].

#### *1.3.1. In vivo models*

**1. Introduction**

The integumentary system is the largest and heaviest organ of the body [1, 2]. This organ is divided into three distinct layers: the epidermis (superficial layer), the dermis (intermediate layer), and the hypodermis (deepest layer). Its main function is to protect the body from external aggressions, such as chemical, mechanical, thermal, microbial, and UV rays [3, 4]. It is therefore a physical, biological, and immunological barrier. The epidermis, the outer layer of the skin, predominantly ensures this barrier function by a constant renewal of keratinocytes, the epidermal cells. Keratinocytes differentiated into five layers: from the *stratum basale* (*stratum germinativum*), in which skin stem cells are found, to the *stratum spinosum*, *stratum granulosum*, *stratum lucidum*, and *stratum corneum*, which is the outer layer where keratinocytes have lost their nuclei and are completely keratinized [5]. Keratinocytes from this layer, also called corneocytes, will gradually detach to cause the phenomenon called desquamation. The highly regulated process of differentiation involves specific proteins to maintain this epidermal structure, and deregulation of these proteins expression can induce skin pathology such

Psoriasis is an erythematous-squamous dermatosis touching both men and women. This chronic skin pathology affects 2–3% of the world's population [6, 7], which correspond to approximately 125 M people [8]. This pathology is characterized by a hyperproliferation and an abnormal differentiation of keratinocytes resulting in reddish and whitish plaques [5]. At a cellular level, histopathological characteristics consist of acanthosis, parakeratosis, hyperkeratosis, agranulose, and papillomatosis [9, 10]. The disease's etiology is still unknown. However, environmental and immune factors, as well as genetic predispositions, would act together to trigger psoriasis [10, 11]. This disease seriously affects the quality of life of patients due to the appearance of their skin and the side effects of drugs. Existing treatments cause many severe side effects such as nephrotoxicity, hepatotoxicity, immunosuppression, teratogenicity, and no curable treatments have been found [12–14]. Moreover, several comorbidities may be related to psoriasis, such as major cardiac events, type 2 diabetes, and psoriatic arthritis [6].

The skin is a complex organ. Thus, the production of representative and reproducible skin models is a constant challenge. The use of *ex vivo* human skin biopsies would be more convenient, since with skin biopsies, it is possible to observe the mechanisms and interactions of the human skin. However, because of skin donor availability and inter-individual variability, the use of *ex vivo* biopsies is not practical, and thus, the development of new models is important. Over the years, there has been a lot of progress in the field of tissue engineering [15]. Tissue engineering of the skin is used for various clinical applications and in fundamental research such as for drug development. Now, with the development and optimization of *in vivo* and *in vitro* models, it is possible to research new treatments for a skin disease by studying, for

**1.1. Skin**

196 Cell Culture

as psoriasis.

**1.2. Psoriasis**

**1.3.** *In vivo* **and** *in vitro* **psoriatic skin models**

Many approaches are used to obtain animal models as representative as possible to the human pathology. Spontaneous mutations, like the homozygous asebia, xenotransplantation, like the severe combined immunodeficient mice (SCID) and the athymic nude mouse, and genetic models, such as the CD18 hypomorphic mice model, the K14/TGF-α, and the involucrin/INF-γ, have been used over the years to study psoriasis but all of them displayed some limitations [21–23]. Animal models are mostly used to study specific aspects of the pathology. The development of a representative animal model can be expensive.

#### *1.3.2. In vitro models*

There are two types of models: monolayer models (dermal or epidermal) and bilayer substitutes. Monolayer models use only one cell type, keratinocytes or fibroblasts, and will be used to study a specific characteristic or to understand the role of a certain cell type in pathologies such as psoriasis. However, these models exclude interactions between different cell types. Bilayer models displayed two layers of skin: dermis and epidermis, which allow the study of skin complexity more representatively. The challenge of skin engineering is to reproduce the complexity and the functionalities of a pathological skin. There are different *in vitro* psoriatic skin models, which include interesting pathological features. Various pathological bilayer skin models were developed using a collagen gel as dermal equivalent. Most of these studies involve pathological keratinocytes seeded on a dermis made of collagen and fibroblasts [24, 25], but there are also studies where a full-thickness psoriatic skin biopsy is incorporated into the dermal equivalent [26]. These models have been useful to better understand the disease and the interactions between fibroblasts and keratinocytes [26, 27]. However, the main disadvantage of these models is the use of an exogenous material, which does not represent exactly the properties of the human dermis. To counter the use of exogenous material such as collagen, some research teams have used de-epidermized dermis to produce their psoriatic skin models [28–30]. Although these equivalents demonstrate several psoriatic features, the use of these models for pharmaceutical studies would require an excessive amount of skin biopsies. Thus, a pathological model free of exogenous material that can generate many samples at a time is still required for pharmaceutical research.

Our team has developed a psoriatic skin model based on a self-assembly method, which is free of exogenous material [31]. This model has been characterized towards its permeability, lipid organization and response to antipsoriatic drugs [32, 33]. This basic model has also been improved by the addition of other cell types such as endothelial cells in order to reproduce the angiogenesis observed *in vivo* [34]. These studies have confirmed that our psoriatic skin substitute model produced according to the self-assembly approach maintained many characteristics of the disease including the presence of a disorganized and thicker epidermis compared with normal skin substitutes [31]. This self-assembly approach allows the understanding of pathological skin complexity through the possibility of: (1) dissecting step by step the mechanisms of skin pathologies according to which kinds of cells are present in the model at that time and/or (2) using various cell combinations such as healthy fibroblasts and healthy keratinocytes, which can be compared with healthy fibroblasts and pathological keratinocytes. Although the self-assembly method is very effective for the reconstruction of substitutes used in basic mechanisms studies, it required an optimization of its original protocol to consider a productive capacity of it in the pharmaceutical industry. Thus, the aim of this work was to improve the original self-assembly method to allow the reconstruction of more reproducible psoriatic skin substitutes that could be used for pharmacological testing.
