ARTICLE
Auteur(s) : Celine
Auxenfans1, Julie Fradette2, Charlotte
Lequeux1, Lucie Germain2, Beste
Kinikoglu1, Nicolas Bechetoille3, Fabienne
Braye1, François A Auger2, Odile
Damour1
1Laboratoire des substituts cutanés, Hospices civils
de Lyon, Hôpital E. Herriot 69437 Lyon, Rhône, France
2Laboratoire d’organogénèse expérimentale (LOEX), Centre
de recherche FRSQ de l’Hôpital du Saint-Sacrement du CHA
and Département de Chirurgie, Université Laval, Québec,
Canada, G1S 4L8
3BASF – Beauty Care Solutions – Lyon, Rhône, France
accepté le 28 Septembre 2008
Since the culture of keratinocytes on feeder layers and their
use as epidermal sheets for burn treatment [1, 2], research,
motivated by the challenge in treating large burns and chronic
wounds, has been applied to the production of reconstructed skin
models comprising living dermis and epidermis. These models of
reconstructed skin have now come to the fore, following European
regulations which on one hand require proof of the innocuousness
and the effectiveness of cosmetic products, and on the other hand,
ban animal experimentation of active and finished products in
cosmetics. From 2009, the ingredients and the finished products
cannot be commercialized in Europe if they have been tested on
animals, regardless of the advances in the development of
substitution methods. With respect to other existing models, skin
equivalents are of great interest to test the effectiveness of
these molecules at the same time on the epidermis and dermis,
taking into consideration dermal-epidermal interactions. In
addition, they can be used to elucidate physiological cutaneous
mechanisms, and also to better understand their aging or different
pathological states. Hence, these models have applications in
clinical dermatology, in dermocosmetology and in fundamental
research. The dynamism of these three areas has led to the
evolution and complexification of reconstructed skin models.
Nowadays, clinicians in burn care are interested in adding dermal
reconstruction to the epidermis for a better functionality of the
skin. Development in dermocosmetology needs the most physiological
models possible to prove the innocuousness and the effectiveness of
their active principles or finished products. Finally, fundamental
research benefits from new tools that can be applied to elucidate
at the same time physiological mechanisms and intercellular
interactions taking place in the skin, or to better understand
their evolution during aging or various pathological processes. In
this paper we consider only skin equivalents comprising
pluristratified and differentiated epidermis laid over a living
dermal equivalent. The dermal compartment is recreated by culturing
fibroblasts i) either in combination with natural or synthetic
biomaterials such as collagen gels or sponges, ii) or by
stimulating the innate capacity of fibroblasts to secrete and
organize extracellular matrix elements (ECM) or iii) by the
self-assembly approach without any scaffolds.
The present review will mainly concentrate on the latest
developments in skin engineering and will mostly concern the
studies carried out by our groups.
Skin reconstructed from cells in combination
with scaffolds
Bell’s model – collagen gel
The first model of skin equivalent (SE) described, uses a collagen
gel [3, 4]. It was proposed by Karasek and Charlton in 1971, and
developed by Bell et al. in 1979. The resistance and insolubility
of collagen is obtained by retraction of the gel by fibroblasts. It
gives birth to a living dermal equivalent (DE) whose final size is
proportional to the number of cells and inversely proportional to
the collagen concentration. In this model, the proliferation of
fibroblasts is inhibited, probably by a retro-control and
biochemical confinement, the fibroblasts being restricted in an
environment of retracted collagen [5]. The second stage is the
seeding of keratinocytes onto the surface of the dermal equivalent
[6-8]; the keratinocytes multiply and differentiate, resulting in
the formation of epidermis. This multilayered epidermis encloses
the necessary elements for strength, i.e. desmosomes, and
tonofilaments [7, 9]. Epidermal differentiation is maximised by the
elevation of this skin equivalent to the air-liquid interface [10].
This optimization seems to be at least, in part, due to a retinoic
acid gradient, created by diffusion of the culture medium through
the dermal layer [11]. Skin equivalents produced with this model
are constantly being improved. In fact, it was completed by the
addition of melanocytes [12] and a hypodermis made up of
pre-adipocytes and mature adipocytes cultured in the inferior part
of the gel [13] ; in addition, this collagen source can be used to
support the growth of hair follicles [14, 15]. The decrease of the
final surface area of the skin equivalent can be controlled with a
peripheral anchorage [16].
Organogenesis Inc. (83 Rogers street, Cambridge, MA 02142, USA)
develops Apligraf®, a SE combining living human foreskin
fibroblasts with a collagen gel extracted from calf tendon, then
epidermalized with cultured allogenic keratinocytes. Growth factors
released by the fibroblasts and keratinocytes are powerful
activators of healing of venous ulcers [17].
Models using scaffolds
Different synthetic or biological materials in the form of sponges
or scaffolds can be used for fibroblast culture.
Many synthetic biocompatible polymers, biodegradable or not, can
be used as supports for fibroblast culture during the production of
dermal equivalents. They differ by their structure (porous, net or
layer), or by their composition (nylon, polyglycolic acid [18, 19]
poly-L-lactid, depolyethylene oxide, polybutylene teraphthalate)
[20]. After adhesion and proliferation, cells synthesize and
deposit an extracellular matrix thus forming a tri-dimensional
tissue closely resembling normal human dermis.
We will focus on collagen based scaffolds, the essential
compound of connective tissue, the first to be used to reconstitute
in vitro a dermal compartment [21-23]. This acellular dermal
scaffold (DS) is obtained by lyophilization of a composite matrix
made by co-precipitating bovine type I and III collagens and
chondroitin sulphate. The whole is cross-linked by covalent bonds,
introduced by the intermediary of glutaraldehyde, then covered by a
silicon film, acting as a temporary pseudo-epidermis. The addition
of glycosaminoglycans (chondroitin sulphate) increases the
resistance of the matrix to degradation by bacterial collagenases
and reduces the immunogenicity of collagen.
The cross-linking of collagen by glutaraldehyde is the most
common procedure [21-24]. Other methods of cross-linking have been
developed, such as periodate [25], acyl azide [26], or a
dehydrothermic physical treatment, which, on completely dehydrating
the matrix, draws the constituent molecules closer together and
thus permits the creation of amide links [27]. In order to avoid
the introduction of any chemical product that could be secondarily
released, a collagen sponge, made from bovine collagen and
chondroitin sulphate has been insolubilized by chitosan [28].
Chitosan, obtained by deacetylation of chitin, forms ionic bonds
with carboxyl groups of collagen and sulphate groups of chondroitin
sulphate. After lyophilization, these polymers form an alveolar
structure with pores between 50 and 150 μm. The ionic bonds
formed are sufficiently strong to give this sponge good mechanical
properties [5, 29].
Fibroblasts synthesize, in this bovine collagen framework,
different types of collagen, glycoproteins, glycosaminoglycans of
human extracellular matrix and thus induce a remodelling of the
initial matrix [5]. This living dermal equivalent could be used
either to prepare the wound for epidermalization in the treatment
of burns, or as a bioactive tissue releasing growth factors in the
treatment of chronic wounds [29, 30].
In vitro, after three weeks of fibroblast culture into the
dermal substrate, this dermal equivalent is epidermalized by
keratinocytes, giving a skin equivalent (SE) that is a
dermo-epidermal assembly. After a week of immersed culture and two
weeks of culture at the air-liquid interface, the quality of the
underlying DE permits the development of a multi-stratified
epidermis (figure
1). This epidermis is characterized by a layer of organized
and adhering basal cells and by suprabasal layers expressing
filaggrin, tranglutaminase, involucrin as well as the keratins 10
and 14 [31-33].
Characterization of the dermo-epidermal junction of
reconstructed skin by immunohistochemical studies, reveals the
linear deposit of the constituents of basal membrane, such as type
IV and VII collagens, laminin. Ultrastructural studies also show
the presence of a continuous lamina densa and numerous
hemidesmosomes at the level at which the anchoring filaments are
localized, made up of laminin-5 and anchoring fibrils composed
mainly of type VII collagen. The participation of fibroblasts in
the formation of the dermo-epidermal junction has been confirmed by
molecular biology (RT-PCR). They favor the adhesion of
keratinocytes and the formation of a dermo-epidermal junction by
stimulating the secretion and organization of these components
[32].
Deeper in the dermis, fibroblasts, after proliferation and
colonization of the porous substrate, synthesized all the
components of the ECM. The histological and immunochemical results
show that fibroblasts express some of their morphogenic potential:
reorganization of the matrix with de novo synthesis of matrix
constituents. The composition of this neosynthesized ECM is close
to that of normal human dermis. In fact, immunohistochemical
analysis has shown evidence of type I and III collagens,
fibronectin and type XII and XIV collagens [34]. Transmission
electron microscopy has revealed a structured organization of the
neosynthesized ECM in which the collagen fibres are grouped
perpendicularly in relation to each other and between which an
abundant microfibrillar material is inserted.
All of the components of the elastic tissue are present in the
dermal equivalent: fibulin-5, fibrillin-1 [35], lysyl oxidases LOX
and LOXL [36]. The latter are in charge of the reticulation of
macromolecules of the ECM comprising lysine clusters (collagen and
elastin) and were observed by immunohistology not only in dermis
but also unexpectedly in the epidermis of the skin equivalent. We
were able to demonstrate that LOXL is preferentially associated
with the components of elastic tissue but its association with
collagen fibrils can not be ruled out; whereas, LOX is
preferentially associated with collagen fibrils but also with
components of elastic tissue. Thanks to this model, we were able to
show that the presence of these enzymes, which were strongly
expressed in the epidermis, might be related to cellular
differentiation, and that they might play a part in
post-transcriptional modification of epidermal substrates yet
unknown [36].
It is now recognized that the components of ECM, like growth
factors released by the fibroblasts, induce effects not only on the
behavior of fibroblasts but equally on the morphogenesis of
epithelial cells.
Animal experimentation has shown that SE or sponges slow down
and reduce wound contraction [22, 30, 37]. In this SE, containing
fibroblasts and autologous keratinocytes, there is also evidence of
the formation of a complete dermo-epidermal junction after just two
weeks and its complete maturation after three months [37].
Clinical application of autologous skin equivalents seems to be
the best treatment for chronic wounds [38] and the treatment of
deep and extensive burns [37]. Secretion of growth factors results
in faster healing of donor sites and in better aesthetic results
than with conventional therapeutics (Biobrane-L) [38, 39].
The model of Boyce has undergone clinical trials on burn
patients since 1989 [40]. It is developed under the name
Permaderm® and has recently been bought by Cambrex.
Surgical treatment takes place in two operative parts. In the first
part, burned tissues are excised and a dermal substrate
(Integra®) is placed, which reduces the need for cadaver
skin. This strategy reduces skin harvest for autograft [41].
A skin biopsy is performed for the preparation of keratinocyte
and fibroblast cultures. Culturing lasts from 20 to 30 days. In the
second operative part, the silicone layer which covers
Integra® is removed and the autologous reconstructed
skin is grafted on top of the dermal substrate [42]. However, in
order to ensure the nutrition of the epidermal cells when waiting
for vascularization of the dermal part, Boyce recommends the
addition of nutrients and growth factors by topical application
[38]. These constraints have implications for the clinical use of
SE, still requiring intensive nursing care. Recently, this
treatment has resulted in a graft take rate of 98% [42] in three
burned children.
Skin equivalent models are also the models of choice for
pharmacotoxicological applications [43]. The collagen-GAG-chitosan
(CGC) based model was developed in order to test active principles.
It is licensed and registered by Engelhard group BASF of Lyon,
under the name of Mimedisk® for the dermal substrate,
Mimederm® for the dermal equivalent, and
Mimeskin® for the reconstructed skin. Even in the
absence of serum, the properties of the reconstructed skin are
retained: ultrastructure of the dermis and epidermis, expression of
the main components of the extracellular matrix (type I, III and V
collagens, fibronectin, elastin and fibrillin-1), and the
dermo-epidermal junction (laminin, type IV collagen, α6
integrin).
This model has been used to evaluate cutaneous toxicity,
phototoxicity [44, 45] for the screening of new molecules with
photo-protective properties against UVA and UVB, and to assess the
effectiveness of cosmetic products administered topically or
systemically. The effectiveness was demonstrated on the
proliferation and the differentiation of the keratinocytes, on the
thickness of the epidermis, as well as on the synthesis of the
components of the ECM: type I collagen, fibrillin-1 and
elastin.
This model also allowed the production of skin subtitutes with
pathological cells. A Cutis Laxa (CL) skin equivalent (CL-SE)
model prepared with fibroblasts from CL patients compared with
control SE was successfully developed to define the behavior of CL
fibroblasts in a three-dimensional model. There was increased cell
death and a global extracellular matrix deficiency in the dermis of
this CL-SE model, and a low level of the main elastic fiber
expression. There was no basement membrane evident at the
ultrastructural level, and type-VII collagen could not be detected
at the histological level. This model reproduced some defects of
the extracellular matrix and highlighted other defects, which
occurred at the time of the basement membrane formation, which were
not evident in skin from patients [46]. This CL-SE model could be
adapted to screen for therapeutically active molecules.
Skin reconstructed by the self-assembly approach
This model features a dermal compartment devoid of exogenous
scaffolding elements. Fibroblasts are cultured on plastic
substratum until they form living tissue sheets. The presence in
the culture media of serum and ascorbic acid (vitamin C), a
co-activator of the enzyme prolyl hydroxylase, favors the secretion
and organization of endogenous human extracellular matrix
components such as collagens. After typically 28 days of culture,
fibroblasts are embedded into their own ECM, forming a flexible
tissue sheet which can be manipulated. A defined number of
tissue sheets can be superposed, typically two to four, to form a
dermis of a desired thickness. After one additional week of
maturation, the keratinocytes are seeded onto the reconstructed
dermis, and the substitutes are then cultured at the air-liquid
interface, a condition that induces epidermal cell differentiation
[47-49].
These reconstructed human skins have many histological
characteristics that are close to those of normal human skin (figure 2A). They
exhibit a stratified epidermis comprising a cuboidal basal layer,
suprabasal layers, a granular layer expressing filaggrin and
transglutaminase as well as a stratum corneum [47]. Keratinocytes
from the basal layer express keratin 14 while keratin 10 is
expressed in the suprabasal layers. A subpopulation of the
basal keratinocytes express keratin 19, indicating that the stem
cells are preserved in culture and located at their expected
position in this skin substitute [50]. The epidermis lies on a
complete basement membrane comprising hemidesmosomes, lamina lucida
and lamina densa (figure
2B). The underlying dermis contains a dense network of
collagen fibers. Type I and III collagens are present in the
reconstructed dermis whereas type IV and VII collagens as well as
laminin are deposited at the dermal-epidermal junction [51]. The
inclusion of hair follicles into the construct results in
reconstructed skin with hairs that can be used for
pharmacotoxicological in vitro studies [47]. The reconstructed skin
can easily be manipulated and results in skin with excellent
mechanical strength, cohesion and suppleness after grafting on
athymic mice [49]. Producing skin using the self-assembly approach
results in substitutes that are entirely autologous, thereby
avoiding potential immunogenicity or inflammation reactions after
transplantation in vivo.
Skin reconstructed using the self-assembly approach has also
been used for the treatment of chronic venous leg ulcers on a
limited number of patients. These substitutes favour the closure of
wounds previously unable to heal despite conventional treatment. In
this case of chronic wounds, the enhancement of the healing process
by skin substitutes is attributed to their signalling to the
autologous keratinocytes surrounding the wound that then migrate
and reepithelialize the defect [52, 53].
The reconstructed skin model is also useful to modelize various
pathologies with the advantage of using native human affected
cells. Skin reconstructed with cells originating from hypertrophic
scars has been used to better define the molecular changes
occurring as dermal fibroblasts acquire the characteristics of
myofibroblasts [54]. Using the self-assembly approach, it has been
possible to highlight the role of epidermis during fibrosis.
Finally, the same team is presently taking advantage of this useful
reconstruction model to study cellular events occurring over time
during fibrotic progression of the scleroderma pathology [55].
Another example of skin substitutes re-created from pathological
cells include the use of lesional and non-lesional psoriatic
fibroblasts and keratinocytes [56]. The self-assembly approach also
allowed the production of skin substitutes with distinct
phenotypes. The analysis of the stratum corneum helped uncover
specific modifications in these psoriatic substitutes compared to
those produced from normal skin.
Evolution of complex reconstructed skin models
Reconstructed skin models have improved over the years by the
incorporation of different cell types.
Pigmented skin equivalent model
Numerous models of pigmented epidermis have been described. The
first pigmented reconstructed skin dates back to 1986 [57]. Indeed,
melanocytes are one of the first cell types introduced in a
reconstructed skin.
In monolayer culture, mitogenic agents such as
12-0-tetradecanoylphorboll3-acetate (TPA) and bFGF are
indispensable for the proliferation of melanocytes. In addition, in
culture, melanocytes in monolayers lose their characteristic
morphology with numerous dendrites to become bipolar cells.
However, in the presence of keratinocytes, not only do they
proliferate without mitotic agents, but they also retain their
morphology and disperse their dendrites towards keratinocytes.
Hence, the skin equivalent models are of interest to better
understand the mechanisms implicated in melanogenesis and
pigmentation. In these models, melanocytes regain their
physiological localization at the level of the basal layer in the
epidermis. They preserve all their functionality, since in response
to UV rays they proliferate, synthesize and secrete melanin.
These models are of interest in pharmacotoxicology to test the
effect of solar protection products. Thereby, the coloration of the
reconstructed skin is compared with samples exposed to UVB,
previously treated or not with a photo-protector. Pigmented
reconstructed skin has been used for the investigation of
congenital disorders of pigmentation [58] by producing them with
fibroblasts and keratinocytes obtained from café-au-lait
macules.
Endothelialized skin equivalents
Under conditions that we optimized, human endothelial cells from
umbilical vessels, seeded together with fibroblasts, organise
themselves into tubular structures with a well-defined lumen
resulting in an endothelialized skin equivalent [59]. Contrary to
other in vitro models reconstructed using human endothelial cells,
this angiogenesis process was obtained without the addition of
specific growth factors or carcinogenic agents such as phorbol
12-myristate 13-acetate. Interestingly, the seeding of
keratinocytes on this endothelialized dermis results in the
formation of capillary structures with diameters near to those of
physiological capillaries. Moreover, the formation of capillaries
depends on the presence of ECM by the fibroblasts, which requires
vitamin C. The absence of vitamin C in the culture medium resulted
in a significant decrease (> 10-fold) in the number of
capillaries [60].
The endothelialized skin equivalent has applications in
pharmocotoxicology which allow evaluation of the effectiveness of
angiogenic and angiostatic molecules, both quantitatively and
qualitatively, and, in the long run, on angiogenesis [61]. By image
analysis, it is possible to quantify the tubular structures of the
endothelialized skin equivalent revealed by specific markers of
endothelial cells, and to evaluate the surface they occupy.
The endothelialized skin equivalent presents hope for the
treatment of large burns. It would allow acceleration of the
microcirculation in the reconstructed skin, which is essential for
the survival of the epidermis. Indeed, on nude mice, the
vascularization of skin equivalents takes 15 days; whereas,
vascularization takes only 4 days with endothelialized skin
equivalents. Early vascularization of endothelialized skin
equivalents is presumably due to the inosculation of the tubular
structures of the endothelialized skin equivalents with the
capillaries of the host [62]. These endothelialized skin
equivalents would not need nutrition of the epidermis by local
application of culture medium, with a consequent increased risk of
infection for the patient.
Immunocompetent skin equivalent model
Immunocompetent skin equivalent models represent new generation
products allowing evaluation of the allergenic/sensitizing
potential of novel molecules administered topically and/or
systemically. The first generation of immunocompetent skin
equivalent models was obtained with immunocompetent cells
differentiated from CD34+ precursors isolated from umbilical cord
blood, cultured in two-dimensions for six days with GM-CSF, TGFβ1
and TNFα. Then, the cells were seeded in the endothelialized skin
equivalents at different phases of differentiation of the
epidermis, to promote either the dermal or the epidermal
integration [63]. Under these conditions, endothelialized skin
equivalents constitute an environment favourable for the
differentiation of the precursors of dendritic cells (DC) into
Langerhans cells (Langerine+, CD1a+, HLA-DR+) in the epidermis and
into dendritic dermal cells (DC-SIGN+, HLA-DR+). Transmission
electron microscopy revealed scarce Birbeck granules in the
epidermal Langerhans cells of the reconstructed skin. The second
generation made use of CD 34+ cells isolated from circulating blood
and predifferentiated in a medium supplemented with GM-CSF, TGFβ1,
IL13 for six days, then stimulated by TNF for 18 h before
seeding onto skin equivalent models. This source of stem cells,
besides being easier to obtain, is also effective since it allows
having Langerhans cells in the epidermis, and the dendrocytes in
the dermis (figure
1). The functionality of this model has been proven in
relation to the effect of UV rays on Langerhans cells and dendritic
cells in their cutaneous microenvironment.
Reconstructed skin comprising a hypodermis
Recent developments in skin reconstruction include the production
of a substitute comprising a hypodermis in order to reconstruct all
the three layers of skin. Recently, the production of a human
tridimensional hypodermis has been reported using the self-assembly
model [64] and the CGC sponge.
Using an adapted self-assembly approach, adipose-derived
stem/stromal cells (ASCs) were differentiated in vitro towards the
adipogenic pathway, leading to a tissue devoid of exogenous
biomaterial but rich in adipocytes and extracellular components
such as collagens I, V and fibronectin [65]. A trilayered skin
including a hypodermis was therefore produced using this
methodology, highlighting the usefulness of ASCs for autologous
reconstruction of a more complete skin substitute [50]. We have
also developed human skin substitutes featuring keratinocytes
directly seeded on a stromal compartment made of adipose-derived
stem/stromal cells or on a stroma comprising adipocytes
differentiated in vitro from these ASCs. All these combinations
resulted in skin substitutes displaying histological features,
keratinocyte differentiation program and expression of basement
membrane components similar to reconstructed skin featuring a
stromal compartment made of dermal fibroblasts [50].
Thus, those models should prove to be useful tools for active
ingredients testing to determine their influence on the metabolic
activity of the cells involved, either leading to a stimulation or
an inhibition of adipogenesis, which could help uncover mechanisms
related to obesity or lipodystrophies, respectively. Moreover,
autologous substitutes comprising a hypodermis could prove useful
in reconstructive surgery.
Conclusion
Fibroblast stimulation with or without scaffolds induces the
neosynthesized ECM containing all the desired macromolecules
forming an ultrastructurally organised architecture. The quality of
the dermis generated favours the development and regeneration of a
pluristratified and differentiated epidermis firmly anchored by an
organised dermo-epidermal junction. Evolution of skin equivalent
models which are more and more complete and similar to the
physiological skin, are promising in the treatment of burns and
chronic wounds, and bring to research, as well as to
dermocosmetology and to the pharmaceutical industry, a wide range
of products such as pigmented, endothelialized, immunocompetent,
and now adipose skin equivalent models. They have become essential
tools to better understand the mechanisms of action of active
substances and to test the innocuity as well as the efficacy of
finished products.
Acknowledgements
Financial support: none. Conflict of interest: none. LG is the
holder of the Canadian Research Chair on Stem Cells and Tissue
Engineering from the CIHR. JF is Scholar from the FRSQ.
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