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.