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\begin{document}

\title{Mimicking skin: healthy and disease models for transdermal permeation
studies}



\author[1]{Tânia Moniz}%
\author[1]{Sofia Lima}%
\author[1]{Salette Reis}%
\affil[1]{REQUIMTE LAQV Porto}%


\vspace{-1em}



  
  \date{June 16, 2024}


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\selectlanguage{english}
\begin{abstract}
Skin drug delivery is an emerging route in the drug development, due to
its great advantages, thus leading to an urgent need to understand the
behaviour of active pharmaceutical ingredients into/through the skin.
This knowledge is crucial in the early stages of product design and
development. Yet, given the skin barrier properties as one of the first
body's natural defence systems, it can act as an obstacle to the
successful outcome of a skin drug therapy. To unravel the mechanisms
underlying this barrier, reductionist strategies have designed several
models with different levels of complexity and integrity, using
non-biological and biological components. Besides the detail of
information and resemblance to the in vivo Human skin that each in vitro
model offers, the technical and economic efforts required should be
considered when selecting the most adequate model for the intended
research. This review provides an outline of the most commonly applied
skin models, including healthy and diseased conditions, lab developed
systems and commercialized models, their advantages and limitations and,
also an overview of the new trends in skin engineered models.%
\end{abstract}%



\sloppy


\textbf{}\emph{}

\section*{Contents}

{\label{contents}}

\textbf{Introduction}

In the last decades, the study and development of mimetic skin models
have been a hot topic of discussion mainly due to the rising of ethical
questions and the establishment of new rules regarding the prohibition
of animal testes. The need of new and efficient skin mimetic models
remains crucial namely for pharmaceutical, cosmetic and toxicological
purposes.

The safety and toxicity of new products and formulations must be
assessed prior human application according to the European Union (EU)
guidelines and to the Organisation for Economic Cooperation and
Development (OECD) guidelines for testing dangerous ingredients for the
skin (Kandarova et al., 2004, OECD, 2015, Fentem, 1999, Fentem and
Botham, 2004, Worth et al., 1998).

Efficient methods for development and rationalization of drug
formulations aiming topical application demand specific skin models
capable of estimating the properties and the most suitable drug
formulation. After the identification of important penetration and
permeation properties (wanted and unwanted) for the drug formulations,
its optimization becomes achievable (Flaten et al., 2015).

In line with this facts, several mimetic skin models have been developed
and some are already available in the market, while the research
continues for further improvements in their quality, complexity and
mimetic properties. Different biomimetic materials have been used from
the silicone membranes or poly(dimethylsiloxane) (PDMS) models to human
cell cultures as well as different technologies to simulate the highly
complex and stratified structure of the human skin.

The big challenges in the development of skin substitutes are related
with: a) the need of reproducibility in the results obtained using these
models; b) their capacity to better mimic the multitude of human skin
structure and functions and c) the development of cost-effective skin
membrane models (Sarkiri et al., 2019, Flaten et al., 2015). The main
focus of the present review is to summarize the currently reported
healthy and disease skin mimetic models, discuss the characteristics and
applications of these proposals and to identify the new trends in skin
engineering.

\par\null

\textbf{Human skin -- structure and functions}

The skin is the major organ of the human body having a surface area
of\emph{at ca} 2 m\textsuperscript{2}, representing approximately 10\%
of the body mass for adults (Lee et al., 2006, Ng and Lau, 2015, Sofia
A. Costa Lima., 2018). Skin is a physical barrier in the interface
between the body and the external environment and constitutes a
first-line defence entity for protection of the body, controlling what
may enter and exit in the body. Moreover, skin is composed by a network
of cells and matrix elements providing multifaceted functions such as
the prevention of the body\selectlanguage{ngerman}´s dehydration, protection of the body against
infectious agents or ultraviolet radiation, thermoregulation, sensation
and synthesis of vitamin D (Prausnitz et al., 2012, Sofia A. Costa
Lima., 2018, Lee et al., 2006).

The organization of the skin consists of three major layers hypodermis,
dermis and epidermis. The type of structure, cellular composition and
major components of the three skin layers are summarized in Figure 1 (A
and B), and these topics are discussed in more detail on the following
subsections.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} --\textbf{Structure of human skin. A)} Schematic
representation; \textbf{B)} Characteristics of the three skin layers.

\textbf{Epidermis}

Epidermis constitutes the outermost barrier between the organism and the
external environment and is itself organized in four layers
the\emph{stratum basale, stratum spinosum, stratum granulosum}
and\emph{stratum corneum} (\emph{SC} ) (Figure 1A). A complementary
layer,\emph{stratum lucidum} can be found on the palm and sole of the
foot, which is responsible for the thickened skin. Nevertheless,
the\emph{stratum lucidum} is often considered the lower part of
the\emph{SC} but not an individual epidermal layer. There are also hair
follicles and sweat ducts that cross different skin layers (Ng and Lau,
2015).

The most intrinsic layer of the epidermis, the \emph{stratum basale} is
composed by different types of cells: keratinocytes, melanocytes, Merkel
cells and Langerhans cells, which participate in the immune response.
The following layer, the \emph{stratum spinosum} , is composed by
keratinocytes. The third layer, \emph{stratum granulosum} , consisting
of several rows of keratinocytes and is a reservoir of ceramides which
are relevant lipids for the constitution of the outermost layer,
the\emph{stratum corneum} (Sofia A. Costa Lima., 2018, Lee et al.,
2006). Together, the three most intrinsic layers of the epidermis form
the denominated ``viable epidermis'' (Ng and Lau, 2015). The viable
epidermis is generally 50--100 \selectlanguage{greek}µ\selectlanguage{english}m thick and contains no blood vessels
and sensory nerve endings (Ng and Lau, 2015, Prausnitz et al., 2012).
Keratinocytes originate from the \emph{stratum basale} and expose
oneself to progressive differentiation while migrating to the
\emph{SC}they are necessary to maintain the \emph{SC} by replacing lost
corneocytes and \emph{SC} lipids during desquamation (Ng and Lau, 2015).
The viable epidermis is considerably hydrophilic (\textgreater{} 50\%)
in the opposite to \emph{SC} which is lipophilic and contains \emph{at
ca.} 10\% of water (Forster et al., 2009).

Due to the importance of the \emph{SC} for the permeation of drugs and
the protection of the skin, the structure and functions of this layer
will be more extensively discussed in this review in the following
subsection.

Stratum corneum

The \emph{stratum corneum} is typically 10-20 \selectlanguage{greek}µ\selectlanguage{english}m thick and is composed
by 10--25 layers of dead mature keratinocytes, denominated as
corneocytes, which are obtained from terminally differentiated
keratinocytes during the passage from granular layers to \emph{SC} .
This process is characterised by loss of nuclei and cytoplasmic
organelles, flattening and elongation of the keratinocytes. The
continuous shedding of corneocytes from the skin surface is called
desquamation (Ng and Lau, 2015) and it balances proliferating
keratinocytes that constitute the\emph{stratum basale} . The cells
migration through the epidermis to the surface takes approximately
fourteen days (Ovaere et al., 2009). Morphological dimension of the
corneocytes are about 0.2 \selectlanguage{greek}µ\selectlanguage{english}m thick and 40--60 \selectlanguage{greek}µ\selectlanguage{english}m wide (Ng and Lau, 2015,
Agache, 2004). Their membrane is 0.015 to 0.020 \selectlanguage{greek}µ\selectlanguage{english}m thick and includes
involucrin and keratolinin as structural proteins (Pouillot et al.,
2008).

The composition of \emph{SC} is generally 20\% of lipids and 70\% of
insoluble keratins (Walters and Roberts, 2002) and the organization of
the layer is usually compared with the ''bricks and mortar'' model
(Figure 2A). The bricks are the corneocytes and the extracellular matrix
analogous is compared to the mortar in a brick wall, which is composed
of lipids (stacked lipid bilayers that surround the corneocytes) like
the cement holding the bricks together (Prausnitz et al., 2012, Uchida
and Park, 2016, Menton and Eisen, 1971).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} -- \emph{Stratum corneum} \textbf{structure and
organization:} \textbf{A)} ``Bricks and mortar'' Two- model (adapted
from (Prausnitz et al., 2012)); \textbf{B)} \emph{Stratum
corneum}structure: cornified envelope (blue); corneocyte lipid envelope
(CLE, black); corneodesmosome (brown). \textbf{C)}Lamellar membrane
structure with intercellular lipids. Insert, electron micrograph: Murine
skin was fixed in Karnovsky's fixative, and post-fixed with 1 \% aqueous
osmium tetroxide, containing 1.5 \% potassium ferrocyanide (adapted from
Uchida (Uchida and Park, 2016)).

The interior of the corneocytes is filled with keratin filaments
embedded in a matrix mainly consisted of filaggrin (filament aggregating
histidine-rich protein) and its breakdown products. These amino acids
together with certain ions, such as chloride, sodium, lactate and urate
form the natural moisturizing factors (Ovaere et al., 2009). These
substances are endogenous humectants that keep the hydration of the
\emph{SC} on an adequate level. It is important for skin elasticity and
permits regularly function for hydrolytic enzymes of desquamation
(Fowler, 2012). Reduced levels of filaggrin and properly natural
moisturizing factors are caused by mutations of the filaggrin gene
connected with atopic dermatitis, ichthyosis vulgaris, psoriatic skin,
ichthyosis and general xerosis (dry skin) (Uchida and Park, 2016,
Fowler, 2012). The cornified envelope (CE) that encloses the Corneocytes
is formed by structural proteins crosslinked by transglutaminases
(Prausnitz et al., 2012). CE has a stable stiff property to withstand
mechanical barrier stress (Figure 2B) and is surrounded by a layer of
corneocyte lipid envelope (CLE) (Uchida and Park, 2016). This CLE is a
support to form lamellar membrane structures and controls exit of
hydrophilic agents from corneocytes, but the roles of CLE in the
\emph{SC} remain totally understood (Uchida and Park, 2016).

The extracellular space in the \emph{SC} is filled by lipid-enriched
layers (Uchida and Park, 2016) which are composed by ceramides
(45--50\%), cholesterol (25\%), cholesterol sulphate (5\%) and free
fatty acids (FFAs) (10--15\%) (Ng and Lau, 2015, Uchida and Park, 2016,
Madison, 2003). The lipids form lamellar membranes that stretch out in a
horizontal direction parallel to the corneocytes (Figure 2C). In
addition, corneodesmosomes are found in the lamellar membrane structure.
They are modified desmosomes including corneodesmosin, a structural
protein that determines corneocyte adhesion (Ovaere et al., 2009).
Corneodesmosomes consist of desmoglein-1 and desmocollin-1 connected to
other corneocytes, intensify cohesion and regulate desquamation by their
pH-dependent degradation (Uchida and Park, 2016).

Ceramides are mainly responsible for the skin renewal process (Mizutani
et al., 2009) and play a critical role for the lamellar organization of
this layer barrier (Prausnitz et al., 2012). At least 14 different
classes of ceramides have been isolated from the human epidermis
(Meckfessel and Brandt, 2014, Jurek et al., 2019, Sofia A. Costa Lima.,
2018, Mojumdar et al., 2016). Each ceramide molecule contains a polar
head group from sphingoid nature (sphingosine, phytosphingosine,
6-hydroxysphingosine or dihydrosphingosine) and two hydrocarbon chains
derived from a fatty acid, a sphingoid or fatty acid ester moiety.

Cholesterol is the second most prevalent lipid by weight in the\emph{SC}
(Zbytovsk\selectlanguage{ngerman}á et al., 2008, Pouillot et al., 2008, Prausnitz et al., 2012).
The increment of cholesterol concentration in the lipid bilayers
correlates with the diminution of the membrane thickness and density,
and at the same time with an extension of the membrane surface area.
Cholesterol fluidizes the \emph{SC} lipid bilayers at skin temperature
(Ng and Lau, 2015, Zbytovská et al., 2008).

The content of FFAs is 10 -- 15\% of the \emph{SC} lipids and they
consist mainly of very-long-chain species with \selectlanguage{english}[?]18 carbon atom and are
mostly saturated, like ceramides.

In addition to lipid species, cholesterol sulphate is typically at 2--5
\% weight ratio. Its potential functions are to help in the formation of
the lipid lamellae and stabilization of the \emph{SC} by inhibiting
enzymatic degradation of corneodesmosomes (Prausnitz et al., 2012, Ng
and Lau, 2015).

The majority of lipids is synthetized by keratinocytes in \emph{stratum
granulosum} and packed in lamellar bodies. These organelles deliver
their lipid content in the extracellular space by fusion with the plasma
membrane of keratinocytes of the \emph{stratum granulosum} (Pouillot et
al., 2008). Any changes in the concentrations of these main lipid
components can damage barrier integrity that mediates normal barrier
function.

These lipid structures prevent the excessive loss of water from the
organism and block the entry of most topically administered drugs that
have high molecular weight and low lipophilicity. Thereby, this barrier
represents a great challenge for drug delivery across the skin, intended
either for local effects or for systemic therapy (Groen et al., 2008).

Concerning the functions of \emph{SC} , it is established that this
layer is responsible for the barrier and immune functions, namely
preventing excessive water loss, maintaining body temperature,
preventing the entry of xenotoxic chemicals and allergens as well as the
invasion of microbes. Its functions also comprise the protection of the
epidermis from oxidative stress and from mechanical stress.

The epidermal permeability barrier prevents intake and uptake of
compounds. The \emph{SC} barrier functions may be partially connected to
its low hydration of 15\%--20\% and its very high density (1.4
g/cm\textsuperscript{3} in the dry state) (Walters and Roberts, 2002).
The body temperatures maintenance and water balance are regulated by
blocking surplus evaporation of water from nucleated layers. One of the
basic properties of this barrier is the prevention of penetration of
allergens, microbial pathogens and xenotoxic chemicals (Uchida and Park,
2016). As a general rule, a compound with molecular weight larger than
500 Dalton cannot pass through the \emph{SC} (Bos and Meinardi, 2000).

The epidermis has a pH gradient: the extracellular pH stays neutral
between the \emph{stratum granulosum} and \emph{SC} , where it becomes
more acidic to ca. 4.5 at the outer \emph{SC} layer. Acidification
enables antimicrobial activity and regulates, by enzymatic activity
performed by proteases, the formation and desquamation of the epidermal
barrier. The use of alkaline or neutral soaps lead to the increase of
the \emph{SC} pH, which conducts to untimely cleavage of
corneodesmosomes, decline in \emph{SC} cohesion and following impairment
of the epidermal barrier. The pH changes are reported to matter in the
pathogenesis of skin diseases such as atopic dermatitis, acne vulgaris
and \emph{Candida albicans} infections (Ovaere et al., 2009). For
example, with pH below 5.5, the growth of \emph{Pseudomonasacne} ,
\emph{Staphylococcus epidermidis} and a problematic microbial pathogen,
\emph{Staphylococcus aureus} , are inhibited (Uchida and Park, 2016).
The permanent bacterial flora in the skin presents a comprehensive
ecosystem. \emph{Staphylococcus} and \emph{Micrococcus}strains and
\emph{Diphtheroid bacilli} are the main part of nonpathogenic
microflora. They consume the sebum like basic nutrient and confine skin
colonization by potentially pathogenic organisms (Pouillot et al.,
2008).

\emph{SC} plays an important role in the innate immunity, which is
related with the presence of antimicrobial peptides such as
cathelicidin, dermcidin, RNase7, S100A7/psoriasin and defensins (Uchida
and Park, 2016). They have revealed potent antimicrobial activities
against a wide spectrum of microbes, including gram-negative and
gram-positive bacteria, fungi and some viruses. Cathelicidin
antimicrobial peptide (CAMP) is inducible with infection, injury or
inflammatory response and stimulates the production of a signal lipid,
sphingosine-1-phosphate (S1P) under stress conditions and activate
vitamin D receptor (VDR). Also, defensins are classified in three
subfamilies, \selectlanguage{greek}α-, β-, \selectlanguage{english}and \selectlanguage{greek}θ-\selectlanguage{english}defensin. They are inducible peptides in
epidermis in response to microbial infection, inflammation. and
differentiation (Uchida and Park, 2016). Adaptive immunity in
the\emph{SC} is associated with the availability of urocanic acid.
The\emph{trans} isomer of urocanic acid is generated from histidine
(principally from NMF) by histidase but can be converted to
the\emph{cis} isomer through exposure to ultraviolet (UV)
radiation.\emph{Cis} -urocanic acid binds to the serotonin
{[}5-hydroxytryptamine (5-HT){]} receptor to eliminate immune function
(Egawa et al., 2010). Urocanic acid is an epidermal major chromophore,
which works as a powerful endogenic UV absorbent. Most lower UV
wavelengths (UVB = 280--315 nm) are absorbed in the epidermis, but
longer wavelengths (UVA = 315--340 nm) get to the dermis. The bulk
amounts of proteins, lipids, and nucleotides has individually low potent
chromophores, but they can form the UV barrier together (Uchida and
Park, 2016). The \emph{SC} is constantly exposed to oxidants, including
UV light, chemical oxidants and air pollutants. \selectlanguage{greek}α- \selectlanguage{english}and \selectlanguage{greek}γ-\selectlanguage{english}Tocopherol,
ascorbic acid and glutathione are the chief hydrophobic antioxidants of
\emph{SC} , providing the lipid bilayers stability and safeguarding from
lipid peroxidation (Pouillot et al., 2008).

\textbf{Dermis}

The dermis is the bigger layer of the skin, with a thickness of
approximately 1--2 mm and provides important physical properties, namely
flexibility, elasticity and tensile strength. It is an integrated system
of fibrous and connective tissue, composed by collagenous and elastin
fibbers, accommodating epidermally derived appendages (hair follicles,
nails, sebaceous glands and sweat glands) and sensory nerve endings,
lymphatic vessels and blood capillaries. Thus, metabolic exchanges
between the epidermis and the blood systems may occur as well as
clearance of cell metabolic products and penetrated foreign agents. The
dermis contains resident cells (e.g., fibroblasts and mast cells) and
cells from the immune system, including macrophages and dermal dendritic
cells.

The thermal barrier, energy depositary and protection from physical
stroke are mainly connected with adipose tissue associated with collagen
fibbers, found in the lower reticular dermis layer. The water content
reaches 70\%, favouring hydrophilic drug uptake. Below the reticular
dermis layer, the fibrous connective tissue transitions to the adipose
tissue of the hypodermis. This is mainly constituted of adipocytes
interconnected by collagen fibbers, forming a thermal barrier able to
store energy and protect from physical shock (Sofia A. Costa Lima.,
2018, Forster et al., 2009).

\textbf{Hypodermis}

The subcutaneous layer or hypodermis is the innermost layer of the skin
and consists of fat cells. However, this layer can be absent in some
thin skin, for example on the eyelid. The hypodermis is between the skin
and the subjacent tissues of the body, such as muscles and bones. Larger
lymphatic and blood vessels are standing in this layer. Consequently,
the major functions of the hypodermis are insulation, mechanical
integrity and support and conductance of the vascular and neural signals
of the skin (Alkilani et al., 2015, Ng and Lau, 2015).

\textbf{Drug skin penetration routes}

The skin is an attractive site for delivery of drugs and cosmetics. But
normal skin is a serious barrier to drug absorption, which is why
pharmacologists and cosmetologists became interested in the development
of new drugs, formulations and ways of delivery.

Drugs can be administrated through the skin providing a local action
(topical administration) or a systemic effect, reaching the bloodstream.
The types of administration of drugs through the skin can be organized
in many ways, depending of the criteria selected. A possible
classification can be performed considering the invasive and
non-invasive nature of the different routes. Regarding invasive routes
of administration, they can be categorized as the routes in which the
drugs can enter through the skin by needle injections (subcutaneous,
intramuscular or intravenous routes) and those that consist in the
implantation of a device. In the subcutaneous route, the needle is
inserted into fatty tissue thus reaching the bloodstream. This type of
administration is usually used for the administration of many protein
drugs (eg. insulin) as they are destroyed in the digestive tract.
Intramuscular route is considered preferably over subcutaneous route for
the administration of larger volumes of drugs. In the intravenous route,
the drug is administrated directly into a vein and is used to give a
drug in a rapid and in a well-controlled manner the drug is delivered
immediately to the bloodstream. Drugs can also be administrated by
implantation of a device which is inserted under the skin. This type of
administration is probably the less commonly used and is usually
considered for the delivery of the drugs by controlled release along the
time for longer periods .

Considering the non-invasive methods, there are four possible routes of
drug penetration across the skin: intercellular, intrafollicular,
transcellular and polar (Pouillot et al., 2008), as depicted in Figure
3A. Sometimes, the diffusion through the skin appendages (e.g., fair
follicles, sebaceous glands and sweat glands) is classified as
appendageal route (Prausnitz et al., 2004, Ng and Lau, 2015).
Intercellular and transcellular ways are considered transepidermal
pathways.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} -- \textbf{Skin Drug Delivery.} \textbf{A)} Pathways
into the skin for transdermal drug delivery: a) Transcellular pathway
(penetration through the corneocytes); b) Intercellular pathway
(penetration between the corneocytes through the intercellular lipids);
c) Intrafollicular pathway (penetration through the hair follicles); d)
Polar pathway (penetration through the polar pores); adapted from (Sofia
A. Costa Lima., 2018); \textbf{B)} Types of drugs entrance routes
through the skin.

The intercellular pathway involves the passage of the drugs through the
lipid matrix that occupies the intercellular spaces of the corneocytes
and is usually the preferred route for lipophilic substances. Otherwise,
the transcellular pathway, also known as the intracellular pathway,
occurs through the successive skin layers and dead cells and allows the
transport of hydrophilic or polar substances. The transappendageal
pathway uses the different skin appendages to enter through the skin.
Various sweat glands, hair follicles and pores opening to the outer
surface of the skin via their ducts can be used as a possible way for
the entrance of drugs. These polar pores are located between cells and
encircled by polar lipids, which make small holes in \emph{SC} (Alkilani
et al., 2015, Sofia A. Costa Lima., 2018). Hence, it was considered an
inessential pathway for drug penetration but nowadays, current research
suggests that hair follicles and sweat glands may present an alternate
pathway for a diffusing molecule (Uchechi et al., 2014). In the polar
pathway, the penetration of the drugs occurs through the polar pores
available in the skin.

When the drugs are able to penetrate deep in the skin, from the surface
through the various layers, this type of penetration is called
``transdermal drug delivery'' (TDD). The drug firstly pass through
the\emph{SC} and then permeates via the viable epidermis and dermis by
diffusion. After reaching the dermal layer, the drug becomes available
for the uptake into the systemic circulation (Alkilani et al., 2015).
TDD has advantages namely over hypodermic injections as the drugs
(usually administrated in patches) can be applied only one time and
released for a longer period of time (with no need of additional
applications), is almost pain-free and doesn't lead to the generation of
dangerous medical waste such as needles and syringes. Furthermore,
transdermal devices can be self-administered, and the administration can
be easily stopped in case of need by removal of the patch (Van Gele et
al., 2011).

The different types of drugs entrance routes through the skin and their
classification are summarized in Figure 3B.

\textbf{Healthy skin mimetic models}

Despite de fact that the \emph{in vivo} human skin is the most realistic
and gold standard experimental model for the investigation of drugs
interaction with the skin, the use of this model is not always possible
mainly because of the ethical concerns, regulatory issues, laboratory
facilities and the potential risk associated to the eventual toxic
effects of the drugs (Van Gele et al., 2011). Moreover, the results
obtained by the use of human \emph{ex vivo} models present significant
variability because samples are usually obtained from different
anatomical places of the same donor, different donors and have
unpredictable character depending on the different subjects or different
age groups (Flaten et al., 2015). These facts reinforce the need of
alternative models able to better mimic the real scenario of drug
interaction with the skin and concomitantly allowing reproducible
results (Abd et al., 2016).

In this section, an overview of the existent \emph{ex vivo} and \emph{in
vitro} mimetic skin models will be given.

\emph{Ex vivo} \textbf{human and animal models}

During long time, the main way for the preclinical research of new drugs
and for the optimization of topical drug formulations was the
investigation considering the use of \emph{ex vivo} skin mimetic models.
The literatures describe two main groups of \emph{ex vivo} models
obtained from human or animal organisms (see references (Abd et al.,
2016, Flaten et al., 2015) for reviews).

Human skin is absolutely the most suitable model for study TDD (Ruela et
al., 2016). The skin samples used in \emph{ex vivo} permeation assays
can be obtained from different origins namely from plastic surgeries,
amputations or cadavers and in generally the skin excerpts can be
collected from different organs, such as the abdomen, back, leg or
breast (Schaefer et al., 2008). Different membrane types can be obtained
by using human skin excerpts for further use in drug permeation studies.
Full-thickness skin models, in which the excisions containing connective
tissue and subcutaneous fat and consists of all layers below, including
the dermis, are reported as useful model to test different drugs and
formulations (Abd et al., 2016, Cross et al., 2003, Manca et al., 2014,
Junyaprasert et al., 2012, Dragicevic-Curic et al., 2008,
Dragicevic-Curic et al., 2010, Elmoslemany et al., 2012, Bragagni et
al., 2012, Cal, 2006, Sahle et al., 2014, Gaur et al., 2013, Marimuthu
et al., 2012).

\emph{Ex vivo} epidermal membranes models are also used for permeation
experiments and those models are obtained from thermal treatment of
full-thickness skin (immersion in hot water) (Junyaprasert et al., 2012,
Kligman and Christophers, 1963) or by chemical action namely by the use
of different reagents such as ethylenediaminetetraacetic acid, ammonia
and enzymes (Cross et al., 2003) in order to separate the membrane at
the dermal--epidermal junction. Other methodologies using human
dermatomed skin (Dragicevic-Curic et al., 2010, Dubey et al., 2007,
Clares et al., 2014, Marepally et al., 2013) or dermatopharmacokinetic
method in which tape stripping is used to remove \emph{SC} layers have
been described (reviewed in (Abd et al., 2016)).

More recently, abdomen skin samples from patients who underwent
abdominoplasty are used as skin models (Ternullo et al., 2018). Many
examples report the use of human \emph{ex vivo} skin models (reviewed in
references (Flaten et al., 2015, Abd et al., 2016)), as the
investigation of the dermal uptake and percutaneous penetration of some
organophosphate esters in a human skin \emph{ex vivo} model (Frederiksen
et al., 2018). In another study, the effect of some nanoemulsions
containing alpha-tocopherol was evaluated in skin wounds either in cell
lines and using \emph{ex vivo} human biopsies samples (Bonferoni et al.,
2018).

The use of skin perfusion models, a surgically prepared portion of skin
including subcutaneous fatty tissue with assured continuous vascular
circulation is reported (Ternullo et al., 2017a, Ternullo et al.,
2017b), to test different drugs, namely nanoparticle formulations. The
use of this model is considered a promising strategy since they present
benefits over \emph{in vitro} models, as they overcome the existence of
only epidermis and part of the dermis and the lack of a vascular system
as verified in the most commonly used \emph{in vitro} models (Ternullo
et al., 2017a, Ternullo et al., 2017b).

Regarding animal \emph{ex vivo} models, pig skin models are the most
relevant because of the multiple anatomical, physiological and
histological similarities with the human skin such as the dermal/
epidermal thickness ratio, epidermal thickness, similarity in hair
follicle and blood vessel density in the skin and content of
\emph{SC}ceramides, dermal collagen and elastin (Abd et al., 2016). The
pig skin is easily obtained as a waste from animals slaughtered for
food. Amongst the different parts of the pig body, the central outside
part of the porcine ear has been the mostly recommended due to the
analogy with human skin layers (Meyer et al., 2006). Variability of
permeability in different samples of pig skin also takes place. The pig
ear skin permeability is comparable with human skin. In fact, studies
showed a good correlation especially for lipophilic substances.
Furthermore, the age of the animal influences the permeability of the
drugs, however most of literature does not specify the age of animal
(reviewed in (Flaten et al., 2015)).

Many different drugs and formulations such as liposomes, nanoparticles
and microemulsions have been studied using \emph{ex vivo} pig skin
models. Amongst the number of studies available, some reports describe
the evaluation of the permeation of liposomes containing different drugs
in excised pig ear (Scognamiglio et al., 2013, Knudsen et al., 2012,
Gillet et al., 2011). Other studies tested the permeation of different
nanoparticles in pig ear models (Gomes et al., 2014, Pople and Singh,
2011, \selectlanguage{polish}Ş\selectlanguage{english}enyi\selectlanguage{polish}ğ\selectlanguage{english}it et al., 2010). Most recently, new formulations including
a transferosomal gel were tested using pig ear skin as an \emph{ex
vivo}model for the study of the transdermal permeation and delivery of
the drug (Das et al., 2017). The use of excerpts from other pig skin
regions, namely from abdomen (Nagelreiter et al., 2013) and dorsum
(Hathout et al., 2010) is also described. Furthermore, newborn pig skin
excisions are used as skin models for evaluation of topical drug
formulations (Cilurzo et al., 2007).

In addition to the pig skin models, several other animals are used
namely primates, mice, rats, guinea pigs, rabbits, bovines (udder) and
snakes (shed skin). However, these models require ethical permissions.
Since 2009, the use of animals for collection of toxicological data for
cosmetic ingredients has been prohibited in the EU (76/768/EEC, February
2003) (Van Gele et al., 2011).

Mainly due to the fact that primate research is highly restricted and
very expensive, skin of rodents (mice, rat and guinea pigs) is sometimes
considered for permeation studies, due to its high availability, small
size and quite low price. There are available many hairless strains
which can be advantageous for this type of studies (Abd et al., 2016).
Amongst rodents, rat skin is most like human skin however many studies
pointed out the fact that its skin is more permeable than human skin
(Barber et al., 1992, Chowhan and Pritchard, 1978, Hughes and Edwards,
2010, Schmook et al., 2001, van Ravenzwaay and Leibold, 2004). Yet, a
study has shown that hairless mouse skin is an inadequate model for
assessing the effects of the skin penetration enhancers (Bond and Barry,
1988).

Shed snake skin was considering as well as a useful model to mimic human
skin and it can be obtained without killing the animal however it lacks
hair follicles (Rigg and Barry, 1990, Itoh et al., 1990, Wonglertnirant
et al., 2012, Kumpugdee-Vollrath et al., 2013). Additionally, udders
from slaughtered cows are used as an \emph{ex vivo} animal model and
studies involving the comparison of this model with porcine skin has
confirmed that both models are well correlated, thus enabling its use
for studies regarding topical administration of drugs (Netzlaff et al.,
2006).

The several \emph{ex vivo} animal models mainly differ in the thickness
of \emph{SC} , hair density, number of corneocyte layers, hydration,
lipid profile and morphology which may constitutes several advantages
and limitations of each model. The most relevant features are summarized
in Table 1 (Flaten et al., 2015).

\textbf{Table 1} -- Characteristic of \emph{ex vivo} skin models in
relation to human skin.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
\emph{Ex vivo} Skin model\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Advantages\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Disadvantage\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Human excerpts\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Gold-standard model\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Ethical permission High inter- and intra-variability than with porcine
ear skin Storage stability lack\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Pig (ear)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
No ethical restrictions Availability (waste from slaughter) Similarity
with human skin\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Age of animal influences skin thickness Hair follicles (removal may
conduce to skin damage) Storage stability lack\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Newborn pig\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Similar thickness of the \emph{SC}\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Higher number of hairs than in humans Variability due to the use of
different anatomical sites Storage stability lack\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Mouse / Rat\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Small size Uncomplicated handling Hairless species available\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Ethical restrictions Lower thickness High permeability High density of
hair follicles Removal of hairs (skin damage)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Guinea pig\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Similar permeability to human skin Hairless species available\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Ethical restrictions High density of hair follicles Removal of (skin
damage)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Rabbit\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Ears as waste from slaughter Similar permeability to guinea pig\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Ethical restrictions High density of hair follicles Hair follicles
(removal may conduce to skin damage)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Shed snake\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Single animal provides repeated sheds Multiple samples from one animal
Storage at room temperature\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Absence of hair follicles Different skin metabolism Absence of living
epidermis and dermis\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Bovine udders\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Availability (easily obtained waste from slaughter) Multiple samples
from one animal\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Weaker barrier to some drugs than pig skin Storage stability lack\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Adapted from (Flaten et al., 2015).

\emph{In vitro} \textbf{membrane models}

In order to overcome the disadvantages of \emph{ex vivo} human and
animal models, mainly related with the availability and the ethical
problems, numerous efforts have been done to develop alternative skin
equivalents based on \emph{in vitro} approaches. \emph{In vitro} skin
alternative models may consist either on mimetic membranes (non-lipid
and lipid systems) or cell cultures (Abd et al., 2016, Flaten et al.,
2015, Berben et al., 2018, Sink\selectlanguage{ngerman}ó et al., 2014, Faller, 2008). The
existent models and their applications will be reviewed in the following
sections.

The first studies regarding the development of \emph{in vitro} skin
equivalents developed in mid 70s included the use of normal human
keratinocytes (NHKs) as a model for skin irritancy (Rheinwald, 1989).
The cell culture of NHKs begins with a small piece of human skin (about
0.5-1 cm\textsuperscript{2}) obtained from surgery after separation and
specific treatment, that grows easily in culture medium. It allows the
use of a large number of cells, leading to the opportunity for wide
ranging toxicity screening tests with many substances. This model shows
good ability for testing hydrophilic compounds however presenting less
capability for the evaluation of poorly water soluble compounds and
complex formulations (reviewed in (Ponec, 1992)).

The model was improved with its application in membranes, which support
the NHKs during growing and forming the called reconstructed human
epidermis. More complex models mimicking the full thickness skin
consisting of the fibroblast populated collagen matrices (dermis
equivalent) and an epidermal cover composed by NHKs (Van Gele et al.,
2011).

Nowadays there are different commercially available models of the human
epidermis or full thickness skin. These models will be reviewed with
more detail in section 3.2.3.

Additionally, other acellular \emph{in vitro} models have been
described: a) non-lipid-based models like the silicone membranes or
poly(dimethylsiloxane) (PDMS) models (Oliveira et al., 2011) and b)
lipid-based models like Parallel Artificial Membrane Permeability Assay
(PAMPA) (Sinko et al., 2012, Sinkó et al., 2014) and Phospholipid
Vesicle-based Permeation Assay (PVPA) systems, as simpler alternative
approaches (Engesland et al., 2013).

\textbf{Non-lipid-based models}

The first studies regarding the use of non-lipid-based skin models date
from 1970 and report the use of silicone membranes to study the release
of salicylic acid (Nakano and Patel, 1970). Later, some studies reported
the use of different microporous membranes based on pure cellulose
acetate, cellulose and polysulfone to investigate the permeation of
hydrocortisone from two commercial creams (Shah et al., 1989). Other
synthetic membranes considering polysulfone, cellulose mixed esters,
polytetrafluoroethylene and polypropylene in their composition were
described and used to study the nitroglycerin release from commercial
ointments (Wu et al., 1992). More recently, some studies describe the
interaction of many drugs and vehicles with skin models of PDMS or with
silicone membranes skin mimetic models (Dias et al., 2007, Watkinson et
al., 2009a, Watkinson et al., 2009b, Watkinson et al., 2011, Oliveira et
al., 2011, Oliveira et al., 2010, Oliveira et al., 2012).

These skin equivalents are simple models with great applicability to
test a basic diffusion mechanisms however they present some
disadvantages namely the lack of similarity with the human skin and they
are not much useful in the study of the permeability of hydrophilic
compounds despite the good results obtained for lipophilic drugs (Abd et
al., 2016, Miki et al., 2015).

Another study reports a new and improved model based on a membrane
impregnated with a polymer of PDMS and PEG 6000 and some preliminary
results elucidating the permeation of drugs in the aqueous solutions
were described by Miki and co-workers (Miki et al., 2015).

\textbf{Lipid-based models}

\textbf{Parallel Artificial Membrane Permeability Assay}

The lipid-based skin equivalents appeared as valuable alternatives to
non-lipid-based models, improving its complexity and the ability to
mimic the human skin, namely the \emph{SC} layer.

A first report dates from 1989 and describes the preparation of model
membranes, using \emph{SC} lipids, to study the permeability of drugs in
the skin. The authors consider ceramides, cholesterol, FFA and
cholesteryl sulphate in its composition (Abraham and Downing, 1989). The
selected lipid composition agrees to those found in the human
\emph{SC}layer and thus it constitutes an advantage for this model since
it better represents the layer in terms of its lipid composition.
However, the most consistent studies started in 1998 when Kansy and
co-workers reported the first PAMPA model as a tool for rapid
determination of passive membrane permeability of drugs (Kansy et al.,
1998). This system included a mimetic membrane from hydrophobic filter
coated with phosphatidylcholine dissolved in \emph{n} -dodecane as a
membrane barrier, which differentiates the donor and acceptor parts.
Even though this model was oriented for testing the transcellular
intestinal permeability, it was the precursor of the following models,
especially those developed to mimic the human skin.

The first PAMPA model for skin penetration estimation was published by
Ottaviani and co-workers in 2006 (Ottaviani et al., 2006) however this
proposal is, in fact, a non-lipid-based model which incorporates
silicone oil and isopropyl myristate as membrane components. This model
was used to study the permeation of a large variety of drugs and
vehicles (Dobri\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al., 2014, Markovic et al., 2012, Karadzovska and
Riviere, 2013, Karadzovska et al., 2013).

In the same year, Loftsson and co-workers described a novel skin
substitute membrane based on a hydrated semi-permeable cellophane
membrane and a lipophilic membrane of pure \emph{n} -octanol in a
nitrocellulose matrix. This model was used to study the permeation of
different cyclodextrin formulations and the results have shown that the
drug permeation patterns of the different formulations were similar to
those previously observed for biological membranes (Loftsson et al.,
2006).

In 2012, Sinko \emph{et al} . designed the skin-PAMPA (Sinko et al.,
2012), which consist of ceramides analogues called the synthetic
ceramides or certramides, as substitution for the native ceramides found
in \emph{SC} . Certramides are cheaper alternatives to natural ceramides
with the potential to prolong the storage time. This skin-PAMPA model
was used in the skin permeability studies and exhibited poor correlation
with skin epidermis, however it presented a good correlation with full
thickness skin (Sinko et al., 2012). Even though its composition intents
to simulate mainly the \emph{SC} , in some cases, skin-PAMPA can even be
a valuable alternative to replace models that use real human skin.
Moreover, it constitutes an easy, quick and cost-effective research tool
to study the permeation of pharmaceutical and cosmetic ingredients.

More recently, a modified version of the skin-PAMPA was reported
(Tsinman and Sink\selectlanguage{ngerman}ó, 2013) and used to evaluate the skin permeation of
different some ibuprofen-containing formulations. The developed system
was able to distinguish between the different types of formulations the
results correlate well with those found in permeation studies using
human epidermis as a mimetic system.

Several other reports can be found in the literature reporting the use
of PAMPA models to investigate drugs' permeability in the skin (Markovic
et al., 2012, Dobri\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al., 2014, Faller, 2008, K\selectlanguage{ngerman}öllmer et al., 2019,
Alvarez-Figueroa et al., 2011, Vizserálek et al., 2015, Wu et al., 2019,
Luo et al., 2016, Zhang et al., 2019, Balázs et al., 2016). The vast
number of reports considering the use of this type of mimetic models to
study the interaction and permeation of many different bioactive
ingredients highlights the large spectra of applicability of PAMPA
approaches and points out the ability of these barriers to predict the
permeation of drugs in the human skin, despite of the more simplistic
composition of these systems.

\textbf{Phospholipid Vesicle-based Permeation Assay}

The original PVPA (PVPA\textsubscript{o}) was introduced as a model for
screening the intestinal permeability and is composed of a consistent
coat of liposomes deposited on a filter support acting as biological
barrier (Flaten et al., 2006b, Flaten et al., 2006a). Later, by changing
the lipid composition of the liposomes used to produce the permeation
barrier, a new PVPA model was developed aiming to mimic the
\emph{SC}barrier of the skin (Engesland et al., 2013). In this model,
liposomes are located within the pores and on the surface of the
membranes (Flaten et al., 2006a). Thereafter, other modified versions
have been reported (Berben et al., 2018, Engesland et al., 2013,
Engesland et al., 2015, Engesland et al., 2016, Ma et al., 2017, Palac
et al., 2014, Zhang et al., 2016).

Mainly, two skin PVPA models, presenting different lipid composition,
for estimating skin penetration have been described: a) PVPAc -
liposomes made of egg phosphatidylcholine (EPC) (77.1\%, w/w) and
cholesterol (22.9\%, w/w) (Engesland et al., 2013) and b) PVPAs - lipid
mixture of EPC (50\%, w/w), ceramide (27.5\%, w/w), cholesterol (12.5\%,
w/w), FFA (7.5\%, w/w) and cholesteryl sulfate (2.5\%, w/w) (Engesland
et al., 2013).

The permeability of different compounds was evaluated in these PVPA skin
models and the results were compared with reported permeabilities using
animal skin models (rat, cattle, dog and pig) and with estimated\emph{in
silico} values. The PVPA permeability data mainly corresponded with the
literature values of the animal skin penetration assays and the\emph{in
silico} values, with the exception of flufenamic acid that showed a
relatively lower permeation (Engesland et al., 2013).

Later, the PVPAs model was examined with diverse formulations made of
different types of liposomes containing diclofenac sodium salt. The
results showed a rising permeation ranking of diclofenac sodium from
liposomal formulations correlating with the physicochemical parameters
of the liposomal vehicle. The permeability of diclofenac increased in
the availability of the penetration enhancers (Palac et al., 2014). The
PVPA model was further optimized considering a complex skin PVPA
containing all the classes of lipids found in the \emph{SC} , and the
penetration enhancing effect of menthol was investigated for a set of
active compounds with different physicochemical properties (Ma et al.,
2017).

The previously described PVPA skin models were studied in comparison
with a reconstructed human skin model (EpiSkin\textsuperscript{®}). The
permeability results indicate that the PVPA model has the ability to
distinguish between the liposomal formulations and drug solutions, as
opposed to EpiSkin\textsuperscript{®}. PVPA models were better than
EpiSkin\textsuperscript{®} concerning their potential to determinate the
influence of the formulations on the drug permeability which could be
used in drug development at early stage. Moreover, PVPA barriers
revealed straightforward, effectiveness, economical and long storable
properties (Engesland et al., 2015).

More recently, other two lipid-based models have been reported as skin
mimetic systems and they were used for the study of the effect of a set
of synthetic surfactants on the skin. The new models proposed contain
DPPC and cholesterol in a molar ratio of 7:3 or a mixture of ceramide,
stearic acid and cholesterol in a molar ratio of 14:14:10 (Jurek et al.,
2019).

Recently, our research group recently developed and characterized a
cheaper and simpler alternative \emph{SC} mimetic model (Shakel et al.,
2019) that simulates this human skin layer and can allow the screening
of drug candidates. The design of this new model was inspired on PVPA
approaches and comprises a lipid composition which closer resembles that
found in the human \emph{SC} layer, namely in the percentage of
ceramides considered in its constitution. Thus, the novel human SC PVPA
model is made of liposomes composed by ceramide (50\% w/w), EPC (25\%
w/w), cholesterol (12.5\% w/w), free fatty acids (10\% w/w) and
cholesteryl sulphate (2.5\% w/w). This model presents some advantages
since it can be stored up to 2 weeks at -20 °C, without losing their
integrity.

\textbf{Cell-based skin models}

The complexity of the skin mimetic models has been increasing along the
time, starting from \emph{in vitro} non-lipid-based to lipid models and
later to models comprising simple or more complex cell cultures. Single
or multi-cell type 2D cultures, in which cells are grown as a monolayer
disposed on solid flat surfaces, such as polystyrene or glass, are some
of the mostly used cell-based approaches due to their relative
simplicity and cost-effectiveness (reviewed in (Randall et al., 2018)).

The first studies regarding 2D skin cell mimetics were performed by
Rheinwald and Green and consist in the growing of human keratinocytes
monolayer culture (obtained from foreskin of newborns) deposited on
plastic culture plates (Rheinwald and Green, 1977). As a further
advance, the authors included a primary fibroblast cell line (3T3) in
the keratinocyte cultures in order to close resemble the skin.
Furthermore, fibroblasts are important for the growth of keratinocyte
cultures, namely by the secretion of extracellular matrix components,
like collagen (Rheinwald and Green, 1975).

The successfully use of 2D skin mimetic models has been reported in many
studies for skin irritation and drug development, as referred in (Ponec,
1992, Amelian et al., 2017). Silva and co-workers established an\emph{in
vitro} epidermal monolayer using human keratinocytes (HaCaT cells)
differentiated in a high calcium concentration medium, and applied it in
the evaluation of drug delivery systems, namely lipid-based nanocarriers
(Silva et al., 2017).

Despite the good performance of 2D skin mimetic models, 3D cellular
models represent more precisely the human skin due to complexity and
stiffness. Bioengineered skin can be obtained by the building of
reconstructed skin models which constitutes artificially fabricated skin
mimetics. 3D (single or multi-cell type) reconstructed skin models
consist in the deposition of different layers of human cells in culture
on a polymeric matrix, thus conducing to the production of a more
complex and interactive system. In some recent studies, the
incorporation of melanocytes (Min et al., 2018, Dai et al., 2018a),
adipocytes (Klar et al., 2017) and endothelial cells (Dai et al., 2018b)
in 3D skin mimetic models has already been reported.

The main differences between 2D and 3D skin models and the advantages of
each class of models are depicted in Table 2.

\textbf{Table 2} -- Main characteristics of 2D and 3D cell-based skin
mimetic models.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& 2D cultures & 3D cultures\tabularnewline
\midrule
\endhead
Structure & Single or co-cultured monolayers & Multi-layered cultures /
stratification\tabularnewline
Application & Initial studies on drug-cell interaction & Studies on
complex drug-tissue interactions\tabularnewline
Air-liquid interface & No & Yes, required for
stratification\tabularnewline
Preparation/ cultivation time & Days & Weeks\tabularnewline
Costs & Low & High\tabularnewline
\bottomrule
\end{longtable}

Adapted from (L\selectlanguage{ngerman}öwa et al., 2018).

According to the type of layer from the human skin that each model can
mimic, two main types of reconstructed skin models are described:
reconstructed human epidermis (RHE) models which mimic the epidermis and
the living skin equivalents (LSEs) which aim to simulate the full human
skin (Kuchler et al., 2013, Flaten et al., 2015, Randall et al., 2018).
Nowadays, there are some commercially available models of the human
epidermis (EpiSkin\textsuperscript{®}, EpiDerm\textsuperscript{®},
SkinEthic\textsuperscript{®}) or full thickness skin
(Phenion\textsuperscript{®}) (Van Gele et al., 2011, Netzlaff et al.,
2005).

The construction of the more complex 3D models usually include
fibroblasts cultures which are embedded in a 3D matrix to mimic the
dermis layer; then, keratinocytes can be seeded on top of the latter
layer to simulate the epidermis. An air-liquid interface is established
in order to allow the differentiation keratinocytes and formation of the
different epidermal sublayers. The artificial matrix may include in
their composition collagen or fibrin fibbers or even alginate and
chitosan or different synthetic polymers (reviewed in (Sarkiri et al.,
2019, Yu et al., 2019)).

Skin engineered substitutes may be used not only as alternatives
to\emph{ex vivo} and \emph{in vitro} non-cell-based models for testing
drugs in healthy or pathological conditions but they can also be applied
in patients for regeneration of damaged skin (reviewed in (Yu et al.,
2019)). A diagram summarizing the different applications of skin
engineered substitutes is depicted in Figure 4.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/304989/articles/435563-mimicking-skin-healthy-and-disease-models-for-transdermal-permeation-studies}

\textbf{Figure 4} - Diagram of the different applications of skin
engineered substitutes. Adapted from (Sarkiri et al., 2019).

3D skin equivalents have been described in the literature such as a
model composed by fibroblasts and keratinocytes grafted in a viscose
rayon support, which was created to test potential skin irritants
(Canton et al., 2010). Other model comprises the use of sucrose
co-polymers and fibroblasts, thus leading to the formation of a
macromolecular assembling which potentiate collagen deposition (Au -
Benny et al., 2016). Uchino and co-workers developed a 3D human skin
model containing vitrified collagen that supported the culture of
dendritic cells, keratinocytes and fibroblasts in a layered construct
(Uchino et al., 2009). Other three-layered constructs featuring a
hypodermis-like layer have been reported as full thickness \emph{in
vitro} models of human skin (Trottier et al., 2008, Monfort et al.,
2013).

Some reconstructed skin models are produced in the laboratory, for
particular research purposes, however other models are already
commercially available. Amongst these, there are different classes of
systems, namely RHEs (eg, EpiSkin®, SkinEthic®, and EpiDerm®) and LSEs
(eg, GraftSkin®, EpiDermFT®, and Pheninon®) models (reviewed in (Abd et
al., 2016, Yun et al., 2018). Some of these models have been validated
according to European Union (EU) guidelines and implemented into the EU
and Organisation for Economic Cooperation and Development (OECD)
guidelines for testing dangerous ingredients for the skin (Kandarova et
al., 2004, OECD, 2015, Fentem, 1999, Fentem and Botham, 2004, Worth et
al., 1998).

SkinEthic®, EpiDerm® and EpiSkin® are probably the most used models and
their use was approved by the European Union Reference Laboratory for
alternatives to animal testing (EURL -- ECVAM) (OECD, 2011). The
SkinEthic® and EpiDerm® consist of epidermal keratinocytes cultured on
polycarbonate membrane whereas EpiSkin® is composed by stratified human
keratinocytes cultured on collagen-based matrix (Yun et al., 2018).

Schäfer-Korting and co-workers have extensively reported a comparison of
the permeation of several hydrophilic and lipophilic compounds in human
epidermal membranes, porcine skin and three RHE models (SkinEthic®,
EpiDerm® and EpiSkin®). The results pointed out that the RHE models,
mostly SkinEthic®, were significantly more permeable than human
epidermis and pig skin, however the permeation of the compounds through
pig skin and the RHEs is similar to those obtained in human epidermis.
Interestingly, they did not observe the expected improvement in
reproducibility with the RHEs compared to the \emph{ex vivo} skin
(Schafer-Korting et al., 2008).

Other 3D models were designed and commercialized to mimic the \emph{SC}
, like Strat-M®, however, this model is not a cell-based system since it
is absent of cells. Strat-M® is a synthetic membrane comprising multiple
layers of different types of materials, as porous polyether sulfone and
polyolefin, enclosed by a combination of lipids (ceramides, cholesterol,
free fatty acids) and other components. Strat-M® was used to evaluate
the permeation efficiency of hydrophilic molecules and to study the
mechanism of passive transport (Uchida et al., 2015, Haq et al., 2018).
Even though this model lacks the capacity to mimic the complex
architecture of full human skin, it represents a valuable alternative
since its composition closely resemble that of the \emph{SC} layer and
the results obtained using Strat-M® are highly reproducible due to the
simple nature and standardized construction of this model (Yun et al.,
2018).

These commercially available skin models have been used for several
purposes, namely for the evaluation of the permeability of drug as well
for irritation and toxicological studies (Alépée et al., 2015, Alépée et
al., 2014, Kandarova et al., 2004, Kandarova et al., 2005, Kandarova et
al., 2006). In the literature, several studies have compared LSE and HRE
systems with animal and human skin models and the results pointed out
their applicability as skin mimetic systems for the evaluation of skin
absorption, testing of cosmetic formulations and for toxicological
studies (van Ravenzwaay and Leibold, 2004, Schafer-Korting et al., 2008,
Schreiber et al., 2005). Despite the fact that cell-based models can
simulate better the human skin, due to the presence of human cell in
their composition, these models present some disadvantages, namely due
to the lack of skin appendages, their high cost of production and the
extremely short shelf time (Flaten et al., 2015, Netzlaff et al., 2005).

A summary of the main advantages and disadvantages of the \emph{in
vitro} models reviewed in the last subsection is reported in Table 3.

\textbf{Table 3 --} Summary of the main advantages and disadvantages of
silicone model membranes, PAMPA, PVPA and cell-based skin equivalents
models.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Skin model\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Advantages\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Limitations\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Silicone model membranes\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Reproducibility Low cost Prolonged storage stability\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Non-lipid-based Low resemblance to SC Non-biological origin Lack of skin
appendages\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
PAMPA\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Reproducibility Low cost Prolonged storage stability\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Synthetic lipids/non-lipid-based Lipid organization not characterized
Low resemblance to SC Non-biological origin Lack of skin
appendages\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
PVPA\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Reproducibility Lipid composition easily modified Relatively low cost
Storage\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Lipid organization not characterized Non-biological origin Lack of skin
appendages\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Cell-based models\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Use of human cell cultures Similarity with cellular composition of human
skin Permeability similar to human skin\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
High cost Lack of skin appendages\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{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 \emph{in vitro} cell-based skin substituents and \emph{in
vivo}animal models, namely based on modified mouse or guinea pig
organisms. Research has been focus on the development and application of
\emph{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, Boche\selectlanguage{polish}ń\selectlanguage{english}ska et al., 2017,
Sarkiri et al., 2019, Coricovac et al., 2018). \emph{In vivo} and
\emph{in vitro}models developed for skin diseases will be assessed in
the following sections according to the type of disease.

\textbf{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 \emph{in vivo} models of
psoriasis (Boche\selectlanguage{polish}ń\selectlanguage{english}ska 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-1\selectlanguage{greek}α \selectlanguage{english}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 \emph{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 (Boche\selectlanguage{polish}ń\selectlanguage{english}ska et al., 2017,
Yun et al., 2018). An example is the work published by
Chiricozzi\emph{et al} in which a full-thickness skin model closely
resembling\emph{in 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).

\textbf{Atopic dermatitis models}

Regarding atopic dermatitis, one of the most frequently used \emph{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 \emph{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 (L\selectlanguage{ngerman}öwa 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 \emph{in vivo} models, some \emph{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).

\textbf{Dermatophytosis models}

A skin disease model for dermatophytosis was reported by Cambier\emph{et
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 \emph{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 \emph{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).

\textbf{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 \emph{et al} (Cozzi et al., 2013), Singh \emph{et al}(Singh
et al., 2015) and Wang \emph{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 \emph{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 \emph{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 \emph{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 \emph{in vivo} tumour environment,
and showed \emph{in vivo} -like responses (Vorsmann et al., 2013).

\textbf{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.

\textbf{New trends in skin models engineering}

Despite the great developments done concerning \emph{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 \emph{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.

\textbf{Conclusions}

In face to the strict legislation regarding human and animal tests and
in particular to the urgent need of development of efficient skin
mimetic models for the evaluation of new skin pharmaceutical drugs and
cosmetics, without using animal models, many different skin substituents
have been described in the literature. The numerous examples mentioned
in the present review highlighted the many different types of models
from the most simple non-lipid approaches to the lipid-based models or
even cell-based \emph{in vitro} systems or at least \emph{ex vivo} human
or animals skin mimetics. In a lesser extent, some skin disease models
have been reported aiming to mimic the morphological and functional
characteristics of compromised skin, namely for psoriasis, atopic
dermatitis or melanoma diseases.

Several improvements in the quality, complexity and mimetic properties
of the skin mimetic models have been accomplished as well as the use of
different technologies to produce the models and to simulate the highly
complex and stratified structure of the human skin. Yet, some big
challenges persist such as: a) the need of reproducibility in the
results obtained using these models; b) the capacity of the models to
better mimic the multitude of human skin structure and functions; c) the
applicability of the use of that models to the evaluation of drugs
exhibiting distinct physicochemical properties d) the improvement of
storage conditions and shelf time and e) to guarantee the production of
cost-effective skin membrane models.

With the last advances promoted by the use of advanced skin engineering
technologies, the incorporation of various skin highly complex
components such as vascularization, innervation, pigments and immune
cells and other complex structures like glands or hairs in these models
is started to be possible.

In the future it is expected that the models can evolve towards the
``perfect'' skin mimetic model, overcoming the drawbacks of the already
described and allowing the efficient screening of new drugs and
cosmetics, in a heathy skin or in a disease scenario. However, the
selection of the most useful and adequate model(s) will probably be
always a crucial point in the design of a study to test skin agents
since the nature and complexity of the different described systems can
be largely distinct. In the selection process of the best skin mimetic
model several aspects may be considered as for example the main aim of
the case study, the physicochemical properties of the drugs of interest,
the type of biological effect expected for the compounds, amongst other
important factors, namely those regarding the characteristics of the
existent mimetic skin models such as their availability in the market,
quality, complexity, stability, price and mimetic properties of each
skin alternative.

In sum, perhaps the concomitant use of different skin mimetic models can
allow a superior understanding of each case of study, thus revealing
many different conclusions which probably cannot be obtained if a unique
skin mimetic model was considered.

\section*{Acknowledgements}

{\label{acknowledgements}}

This work received financial support from PT national funds (FCT/MCTES,
Fundação para a Ciência e Tecnologia and Ministério da Ciência,
Tecnologia e Ensino Superior) through grant UID/QUI/50006/2019 and from
the European Union (FEDER funds through COMPETE
POCI-01-0145-FEDER-030834) and National Funds (FCT, Fundação para a
Ciência e Tecnologia) through project PTDC/QUI-COL/30834/2017. TM thanks
her funding from this project. SCL thanks funding from FCT/MEC
(CEECIND/01620/2017) financed by national funds. To all financing
sources, the authors are greatly indebted.

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