Skin disease’s models
Skin is not only one of the first barriers between the body and the environment but also a common site for local administration of treatments towards skin diseases. The study of the toxicological and permeation profile of drugs in skin disease scenarios is crucial however, the availability of excised human diseased skin is limited mainly due to the increasing regulatory restrictions on the use of (sick) animals and humans.
Models representing heathy skin are a good approach for testing the action of topically applied drugs however, these models do not reflect the skin with the altered morphological and physiological characteristics caused by a disease, a fact that may influence the results and the conclusions of the studies. Accordingly, the development of skin disease’s models as a valuable alternative represents a big challenge and the modification of the existent mimetic models for healthy skin can be considered a possible strategy in order to design mimetics of the altered skin, embracing the diseases’ characteristics. In the last years, many models have been created for the investigation of drugs interaction with skin affected by several disorders such as alterations of skin pigmentation, photodamage (photodermatitis), inflammatory disorders (psoriasis and atopic dermatitis), cutaneous wounds and skin cancer (melanoma) (see references (Abd et al., 2016, Yun et al., 2018, Amelian et al., 2017, Randall et al., 2018) for reviews).
Similarly with healthy skin models, many types of skin models for diseases are found in the literature namely the lipid systems such as the modified skin PVPA membranes. These models can be produced considering different degrees of leakiness in order to potentially represent different degrees of compromised skin (Engesland et al., 2013). Later, the same research group used the altered PVPA membranes presenting reduced barrier functionality to allow the investigation of the permeation of a set of drugs through compromised skin (Engesland et al., 2016). The alterations in the membrane were performed by changing the volume of liposomes in the top layer in order to reduce the thickness of the barrier or by using ethanol in the preparation liposomes to generate different degrees of leakiness (Engesland et al., 2016). More skin disease models have been reported and they can comprise both in vitro cell-based skin substituents and in vivoanimal models, namely based on modified mouse or guinea pig organisms. Research has been focus on the development and application of in vivo models for several diseases such as psoriasis, atopic dermatitis, dermatophytosis and carcinoma, as reviewed in (Abd et al., 2016, Faway et al., 2018, Randall et al., 2018, Bocheńska et al., 2017, Sarkiri et al., 2019, Coricovac et al., 2018). In vivo and in vitromodels developed for skin diseases will be assessed in the following sections according to the type of disease.
Psoriatic models
A substantial number of genetically engineered mice, reviewed in (Abd et al., 2016), was developed to be used as skin diseases models and in particular some of them have been studied as in vivo models of psoriasis (Bocheńska et al., 2017). An example is the development of the epidermal vascular endothelial growth factor (VEGF)-knockout mice which were considered a psoriasis model and were used to identify a specific role for epidermal VEGF in the permeability-barrier homeostasis maintenance (Elias et al., 2008). Knockout mice for c-Jun and JunB proteins exhibiting skin with hallmarks common for psoriasis have been developed, since these factors are important for the differentiation of epithelial cells (Szabowski et al., 2000). Additionally, overexpression of IL-1α cytokine in the murine epidermis leads to increased proinflammatory scenario and thus these mice can be considered an interesting model (Groves et al., 1995). Knockout mice of the IL-1 receptor antagonist exhibited the development of an inflammatory response similar to those verified in human psoriatic skin being considered an useful psoriatic model (Shepherd et al., 2004).
Cell-based in vitro systems have been described in order to simulate compromised skin, and in generally, they are developed in-house by researcher groups. Most models have been described to mimic skin inflammatory diseases, like the approaches developed to simulate psoriasis scenarios, as extensively reviewed in (Bocheńska et al., 2017, Yun et al., 2018). An example is the work published by Chiricozziet al in which a full-thickness skin model closely resemblingin vivo epidermal architecture was used to identify cytokine-responsive genes in psoriasis and the effect of cytokine antagonists (Chiricozzi et al., 2014). Another study described the design of a human psoriatic skin equivalent used to study cytokine-induced gene expression and the effect of different drugs in the disease context (Tjabringa et al., 2008).
Atopic dermatitis models
Regarding atopic dermatitis, one of the most frequently used in vivo model is the NC/Nga mouse (Matsuda et al., 1997, Tanaka et al., 2012, Vestergaard et al., 1999). These animals spontaneously develop skin lesions when housed under conventional conditions, which closely resemble those found in humans. Another in vivo atopic dermatitis model is the flaky tail (ma/maFlgft/ft) mouse which express mutations in the genes involved in the development of the atopic-like skin lesions (LANE, 1972, Moniaga et al., 2010). In addition, histamine H4 receptor-knockout mice were developed to be used as a model for atopic dermatitis and the results obtained in studies using this model pointed out the importance of this receptor as a potential therapeutic target for atopic dermatitis. However, some of these models exhibited distinct atopic dermatitis profiles when compared with that characteristic of the disease in humans, as discussed in (Löwa et al., 2018). Recently, a report described the use of oxazolone-induced hairless mice for the study of a new treatments for atopic dermatitis (Moner et al., 2018).
As an alternative to in vivo models, some in vitro atopic dermatitis mimetic models were described (reviewed in (Löwa et al., 2018, Randall et al., 2018, Huet et al., 2018) such as in the work from Pendaries and co-workers (Pendaries et al., 2014) where a 3D reconstructed human epidermis model was designed and used to investigate filaggrin expression in the epidermis of atopic patients and showing that downregulation occurs and can justify some of the disease related alterations. Other study reports the design and characterization of a compromised reconstructed epidermis model mimicking atopic dermatitis scenario (Rouaud-Tinguely et al., 2015). A multicell-type 3D model to mimic atopic dermatitis which includes human foreskin fibroblasts, human keratinocytes, memory-effector (CD45RO+) T cells, collagen type I and fibronectin was also reported (Engelhart et al., 2005). To study the effect of the exposure to UV light to the formation of wrinkles and discoloration process, a full-thickness skin model that mimics photodermatitis disease has been used (Kuchler et al., 2011).
Dermatophytosis models
A skin disease model for dermatophytosis was reported by Cambieret al. using an experimental mouse model for the study of the fungal infections in the skin (Cambier et al., 2014). Other experimental models were designed to study dermatophytosis and to evaluate the efficacy of potential antifungal treatments, as reviewed in (Faway et al., 2018). For instance, the antifungal effect of terbinafine in a reconstructed tissue have been described (Rashid et al., 1995) and another study regarding Candida albicans (Green et al., 2004) and reconstructed epidermis has demonstrated the potential of this type of models for the control of dermatophytosis. In addition, an in vitro model of dermatophytosis using arthroconidia and reconstructed feline epidermis was developed in order to investigate the efficacy of a set of antifungal molecules (Tabart et al., 2008).
Skin cancer models
Concerning on skin cancer mimetic models, several mouse models have been used to mimic melanoma and other common skin cancers, like squamous cell carcinoma and basal cell carcinoma (extensively reviewed in (Abd et al., 2016, Coricovac et al., 2018)). The work performed by Burns and co-works described an example of a squamous cell carcinoma mouse model in which the skin of SKH1 hairless mice is exposed to UVB irradiation and used to study the potential activity of anti-carcinoma drugs (Burns et al., 2013). Cozzi et al (Cozzi et al., 2013), Singh et al(Singh et al., 2015) and Wang et al (Wang et al., 2013) used the same mouse model to investigate the effect of different drugs in the treatment of squamous cell carcinoma. Other mouse models were developed to assess the effect of antitumor signalling inhibitors for the pathways of basal cell carcinoma (Filocamo et al., 2016, Tang et al., 2011), while xenograft models were applied in the evaluation of the activity of potential anti-melanoma drugs (Schroder et al., 2016, Chen et al., 2012, Yu et al., 2016), and a hairless mouse model which spontaneously develops cutaneous malignant melanoma has been reported (Thang et al., 2012).
Moreover, chimeric models are reported in the literature in which living human skin is transplanted onto the skin of severe combined immunodeficient (SCID) mice allowing the study of the effect of drugs in living human skin. Kundu-Raychaudhuri and co-workers have used this approach to develop a human psoriatic model to study a potential treatment for psoriasis (Kundu-Raychaudhuri et al., 2014). Likewise, SCID mouse–human melanoma models were described for the inspection of different cancer targets and therapies as reported in (Salton et al., 2015, Yue et al., 2015).
Design of an in vitro skin cancer mimetic models may be complex as it includes the incorporation of various tumour entities in a 3D skin system to resemble cell-cell and cell-ECM interactions (Marconi et al., 2018). As representative examples, Li et al described a 3D human skin reconstructed model which includes cultured melanocytic cells (Li et al., 2011), while melanoma cells (A375), normal human-derived epidermal keratinocytes, normal human-derived dermal fibroblasts and collagen type I were assembled to simulate a metastatic melanoma (Mohapatra et al., 2007); and later on, Commandeur and co-workers proposed a skin squamous carcinoma mimetic model with squamous carcinoma cell lines (SCC12B2 and SCC13 cell lines), and normal human-derived epidermal keratinocytes, normal human-derived dermal fibroblasts and collagen type I (Commandeur et al., 2012). Moreover, Vörsmann et al designed a human 3D melanoma model, which includes primary keratinocytes and fibroblasts embedded into a collagen I scaffold and different types of cancer cell lines such as SBCL2 (RGP), WM-115 (VGP) 451-LU (MM) cells, in order to mimic in vivo tumour environment, and showed in vivo -like responses (Vorsmann et al., 2013).
Commercially available skin disease models
In addition to in-house developed systems, some disease’s models are already commercially available, such as MelanoDerm®, Melanoma®, Psoriasis® and “Psoriasis Like” products. These models can be applied to the screening of new drugs, as reviewed in (Amelian et al., 2017).
MelanoDerm® composition includes normal human-derived epidermal keratinocytes and normal human melanocytes and it has been used for the screening of the effect of topically applied agents to prevent UVB-induced DNA damage (Passeron et al., 2009, Li et al., 2011). Melanoma® represents a full-thickness skin cancer model consisting of human malignant melanoma cells (A375), normal human-derived epidermal keratinocytes and normal human-derived dermal fibroblasts and the use of this model has been described for the investigation of some potential active anti-melanoma drugs (Li et al., 2011, Ma et al., 2008). The composition of the commercially available Psoriasis® model includes normal human-derived epidermal keratinocytes and psoriatic dermal fibroblasts, expressing psoriasis-specific markers and releasing psoriasis-specific proinflammatory cytokines and the model allows the study of the psoriasis biology phenomena and to screening of anti-psoriasis drugs . “Psoriasis Like” consists in normal human-derived epidermal keratinocytes cultured in a special medium to induce a diseased psoriatic phenotype, namely the destabilization of the epidermis (Desmet et al., 2017).
These commercially available models can represent a valuable alternative to in-house developed systems probably leading to more reproducible results. However, their price and the low shelf storage time are some of the possible disadvantages of these approaches. Nevertheless, these alternatives may be useful in the understanding of the role of several skin diseases as well in the evaluation of new targets and potential treatments for some skin disorders.
New trends in skin models engineering
Despite the great developments done concerning in vitro lipid- or cell-based models, the demand for new and more realistic human skin models still remains. Thus, and following the most recent advances in 3D bioprinting technology, production of bioprinted skin has been reported for skin engineering field. Bioprinting has been used for the fabrication of several tissues and organ models, and skin is not an exception (reviewed in (Randall et al., 2018, Yun et al., 2018, Weinhart et al., 2019, Satpathy et al., 2018, Tarassoli et al., 2018)). Bioprinting is now considered a promising fabrication method to produce skin equivalents as it allows the obtention of multilayered and multicellular system.
The production of bioprinted skin using these new approaches comprises a computer-controlled deposition of skin cells and matrix polymers following spatially controlled patterns, thus controlling the architecture of the skin model with high reproducibility and therefore revealing great potential to mimic this human organ (Randall et al., 2018).
Complex human skin models with appropriate cell compositions and matrix structure could be biofabricated through different 3D printing techniques such as electrospinning, microextrusion, ink-jet printing and laser-assisted bioprinting (reviewed in (Yu et al., 2019, Randall et al., 2018, Yun et al., 2018)). The selection of the most adequate printing technique is usually determined by the type of the biomaterials chosen for the mimetic model. The variety of available printing technologies has provided multiple options to fine tune the structure of the model according to the desired application.
In particular, with the electrospinning technique, different voltages are applied to the polymer solution in order to generate filaments which are therefore deposited into a surface. In microextrusion printing, the polymer solution passes through a needle, is deposited layer-by-layer on the platform and is possible to assemble multiple layers by controlling the needle movement. Alternatively, ink-jet printing methods allow the dropwise deposition of the bioink and the droplets can be generated considering temperature or pressure variations. Laser-assisted bioprinting approaches comprise the use of a laser beam is which is pulsed on top of the donor layer containing the desired bioink formulation and thus leading to the creation of bioink droplets which are further deposited in the acceptor surface. The fine-tune of the laser position allow the construction of a model with a of the desired pattern (Yu et al., 2019).
In the last years, many studies reported the successful use of this method to obtain skin mimetic models. As an example, a direct cell printing method was used to produce multilayered models containing fibroblasts, keratinocytes and a collagen-based hydrogel as the structural components to mimic skin layers (Lee et al., 2014). In another study, the authors considered the use of a mixture of collagen/ fibroblasts as the bioink (i.e. a substance composed by living cells and/or polymers that can be used for 3D printing of the models) and the consecutive deposition of melanocytes and keratinocytes to obtain functional skin constructs (Min et al., 2018). Koch et al . reported the development of a model in which keratinocytes and fibroblasts were embedded in a collagen/Matrigel® matrix and results have shown that cells were able to express connexins, pan-cadherin and laminin (Koch et al., 2012).
In addition to bioprinting technics, other complex next-generation skin models are reported in the literature, namely regarding microfluidic technology, the called “skin-on-a-chip” devices (Rademacher et al., 2018, van den Broek et al., 2017, Zhang et al., 2018, Sriram et al., 2018). Skin-on-a-chip systems comprise the growing of different cells at a microscale environment using a microfluidic culture device in which is possible a dynamic perfusion and controlled ventilation, thus presenting many advantages namely in epidermal morphogenesis and differentiation. However, due to the high costs and technical requirements associated with microfluidics devices, the use of this new approaches is still limited and thus reinforcing the need of further investigations in this field in order to optimize these methods and allow the overcoming of their disadvantages.
In summary, the production of readily accessible and reproducible constructs for use in research laboratories, with high durability and at a low price is still aimed. Some studies have already reported the first steps considering these challenges (reviewed in (Abaci et al., 2017)) however it is expected that further studies in this field of research can solve the existent drawback of the available models.